StemCell Therapy

4 Reasons To Save Baby Teeth And Ways To Preserve Them  MomJunction

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4 Reasons To Save Baby Teeth And Ways To Preserve Them - MomJunction

Stem Cells May Help In Treatment of Tuberculosis, But Challenges Remain: Study  News18

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Stem Cells May Help In Treatment of Tuberculosis, But Challenges Remain: Study - News18

Adipose tissues hold great promise in bone tissue engineering since they are available in large quantities as a waste material. The buccal fat pad (BFP) is a specialized adipose tissue that is easy to harvest and contains a rich blood supply, and its harvesting causes low complications for patients. This review focuses on the characteristics and osteogenic capability of stem cells derived from BFP as a valuable cell source for bone tissue engineering. An electronic search was performed on all in vitro and in vivo studies that used stem cells from BFP for the purpose of bone tissue engineering from 2010 until 2016. This review was organized according to the PRISMA statement. Adipose-derived stem cells derived from BFP (BFPSCs) were compared with adipose tissues from other parts of the body (AdSCs). Moreover, the osteogenic capability of dedifferentiated fat cells (DFAT) derived from BFP (BFP-DFAT) has been reported in comparison with BFPSCs. BFP is an easily accessible source of stem cells that can be obtained via the oral cavity without injury to the external body surface. Comparing BFPSCs with AdSCs indicated similar cell yield, morphology, and multilineage differentiation. However, BFPSCs proliferate faster and are more prone to producing colonies than AdSCs.

Mesenchymal stem cells (MSCs) derived from bone marrow aspirates have been frequently used as a cell source in bone tissue engineering [1]. However, several problems are associated with the clinical application of bone marrow stem cells (BMSCs) [1]. The harvesting procedure is associated with pain and discomfort for patients, and their differentiation capability is dependent on the donor age [2].

Adipose tissues have been introduced as a promising source of MSCs that can be obtained with minimal discomfort for patients, since subcutaneous adipose tissues are usually discarded after aesthetic surgical procedures. In addition, several studies have shown that the cell yield from adipose tissues is 100 to 500 times greater than that from bone marrow aspirates [35]. Therefore, minimally invasive procedures can be used to obtain a high number of MSCs with similar multilineage capabilities [68]. However, not all patients undergo liposuction, and fat distribution is dependent on body weight.

Recently, Farre-Guasch et al. isolated adipose-derived stem cells (AdSCs) from a mass of fatty tissue in the oral cavity called Bichats fat pad or the buccal fat pad (BFP). These cells have a similar phenotype to AdSCs from abdominal subcutaneous adipose tissue [9]. Under appropriate conditions, AdSCs derived from BFP (BFPSCs) have been shown to differentiate into chondrocytes, osteoblasts, or adipocytes in vitro [9]. Moreover, Shiraishi et al. reported that BFPSCs can form engineered bone in the back subcutaneous pockets of nude mice [10]. Khojasteh and Sadeghi recently used BFPSCs in conjunction with iliac bone block grafts and showed an increase in the amount of new bone formation and a decrease in secondary bone resorption in extensively atrophic jaws [11].

In addition to BFPSCs, dedifferentiated fat cells (DFAT) derived from BFP (BFP-DFAT) can be produced from mature adipocytes by a convenient method called ceiling culture technique. These cells possess high potential for regeneration of the bone and periodontal tissues [10, 12]. Therefore, BFP could be considered as a potential cell source for bone engineering in oral and craniofacial areas since it is easy to harvest and provides a proper quantity of tissue for cell isolation. The present study reviews research on the characteristics and osteogenic capability of stem cells derived from BFP as a promising cell source for bone tissue engineering in the oral and craniofacial regions.

This systematic review has been organized according to the preferred reporting items for systematic reviews and meta-analysis (PRISMA) statement.

This review included all in vitro and in vivo studies that used BFPSCs and BFP-DFAT cells from human or animal sources for bone regeneration. Abstracts, reviews, letters, and theses were excluded. Studies were excluded if they used the BFP flap or mass (i.e., without cells) and if they did not focus on bone formation or differentiation towards the osteoblast lineage.

The PubMed/MEDLINE, EMBASE, Web of Science, and Cochrane electronic databases were searched for relevant studies published between January 2010 and November 2016. No limitation in language was applied in the search process. The following search terms were used, in which mh represents the MeSH terms and tiab represents the title or abstract: (buccal fat pad (mh) or buccal fat pad (tiab) or BFP (mh) or BFP (tiab)) and (cell (tiab) or stem cell (tiab) or tissue engineering (tiab) or adipose tissue stem cell (tiab)). Additionally, a manual search was also performed in the following journals in the given time periods: Stem Cells, Stem Cell Research, Journal of Stem Cells, and Regenerative Medicine.

Initial screening of titles and abstracts was carried out based on the inclusion and exclusion criteria, and full texts of all eligible studies were obtained. Two independent reviewers extracted and processed data for analysis according to the predefined eligibility criteria. In case of any disagreement, agreement was obtained following a discussion with the third reviewer. The fourth and fifth reviewers contributed to discussion section.

The results and data were extracted from the full text of the articles. The studies were then classified and summarized as in vitro and in vivo studies. In in vitro studies, the following outcomes were assessed: alkaline phosphate (ALP) activity, alizarin red staining, osteocalcin (OCN) content, and gene expression using reverse transcription polymerase chain reaction (RT-PCR). The results of histological evaluation and radiographic evaluation were also assessed for in vivo studies. Since the focus of the present review is the BFP stem cells derived from both human and animal origins, the sources of cells were also identified (Tables 1 and 2).

As illustrated in the PRISMA flow diagram in Figure 1, the initial search retrieved a total of 406 studies. Following the initial screening of titles and abstracts, 16 studies were selected for final screening of the full texts. A total of 10 articles met the inclusion criteria and were included in the analysis (Figure 1). Of these 10 studies, seven were conducted in vitro [9, 1217], and the other three were conducted both in vitro and in vivo [10, 11, 18].

All in vitro studies except for two compared BFPSCs with other cell sources, including AdSCs, BMSCs, unrestricted somatic stem cells (USSCs), and subcutaneous adipose stem cells (SC-AdSCs) [9, 1214, 17]. All studies derived stem cells from human volunteers except for a study by Niada et al., where BFPSCs were derived from swine and compared to SC-AdSCs [13]. Three of the 10 studies focused on BFP-DFAT cells [1416]. One experiment focused on the size of DFAT cells and compared small cells (<40m) with large cells (40100m) based on the characteristics for MSCs [16].

Three studies reported in vivo results in addition to in vitro assessment [10, 11, 18]. Two studies reported bone formation results after the application of BFPSCs in animal models [10, 11], and one study performed human bone regeneration. Shiraishi et al. used recombinant human bone morphogenetic protein 2 (rhBMP2) with cells [10], and Nagasaki et al. combined low-intensity pulsed ultrasound and nanohydroxyapatite scaffolds for transplanting BFPSCs into the calvarial defects of mice [18]. Khojasteh and Sadeghi loaded BFPSCs on an allograft and implanted that in combination with autogenous iliac bone in severely atrophic jaws [11].

Data extracted on the characteristics of BFPSCs and SC-AdSCs are compared in Table 3. Tissue volume, number of cells, collagen deposition, ALP activity, and glycosaminoglycan (GAG) content have been shown to be greater in SC-AdSCs, but cell proliferation, morphology, size, adipogenic differentiation, and expression of MSCs markers are similar. In addition, unlike SC-AdSCs, BFPSCs are capable of producing colony-forming units [9, 12, 13].

Adipose tissue contains two different fractions: (1) stromal vascular fraction (SVF), which includes MSCs (preadipocytes), fibroblasts, and erythrocytes and (2) mature adipocytes [9]. AdSCs isolated from the SVF were considered to be the key MSCs within this tissue and can be induced towards adipocytes, osteocytes, chondrocytes, myocytes, and neurons [1921]. Since the SVF has a complex structure and cellular composition, AdSCs derived from SVF, particularly in early passages, are heterogeneous populations composed of cells with various characteristics and behaviors [9, 2227].

Mature adipocytes are another abundant type of cells in fat tissue, and they have also shown dynamic plasticity to be converted into DFAT cells by a ceiling culture technique [2830]. Unlike terminally differentiated adipocytes, DFAT cells have significant and steady proliferation capability [3033]. In contrast to AdSCs, DFAT cells have been shown to have a more homogeneous cell population [15, 16, 30]. In addition, a greater number of DFAT cells can be produced from a given amount of fat tissue [34, 35].

The cellular nature and differentiation stage of DFAT cells have not been fully clarified. However, several studies have suggested that DFAT cells are in the late stage of the differentiation process and classified them into preadipocytes [36, 37]. Similar to preadipocytes, DFAT cells could be redifferentiated into lipid-filled adipocytes under proper induction [29, 30, 38, 39]. Evaluations of stem cell-related markers and multilineage differentiation assays (i.e., adipogenesis, chondrogenesis, and osteogenesis) have also suggested that DFAT cells are similar to AdSCs [30, 38]. Poloni et al. also produced neurospheres from DFAT cells [40, 41], indicating that their multipotency might extend beyond the mesodermal lineages [40, 42]. Kishimoto et al. demonstrated that DFAT cells also showed proliferation and differentiation towards osteoblasts when they were cultured on self-assembling peptide or titanium fiber mesh scaffolds [14, 43, 44]. These findings highlight the hypothesis that DFAT cells are multipotent cells and have potential for use in tissue engineering.

One major goal of tissue engineering is to find a source of stem cells that can provide an adequate number for clinical application with minimal morbidity, maximal proliferation rate, and high differentiation potential [9, 17]. The BFP in the oral cavity is a mass of specialized fatty tissue that is distinct from subcutaneous fat and is located on either sides of the face between the buccinator muscle and other superficial muscles (Figure 2(a)) [4548]. The BFP has a rich vascular supply [4952] and can be harvested easily by an intraoral flap with minimal discomfort and complications for patients (Figure 2(b)) [9, 5254]. In addition, BFP is a discarded tissue in plastic surgery for cheek reduction [13]. Furthermore, it is routinely administered in the treatment of the bone, periodontal defects [49, 5559], congenital oroantral diseases, oronasal diseases [60], congenital cleft palate repair [61], oral submucous fibrosis [62, 63], intraoral malignant defects [64], and cheek mucosa defects [65].

Another advantage of BFP over subcutaneous fat is that its size appears to be similar among different people, independent of body weight and fat distribution [66]. Patients with little subcutaneous fat have BFP with normal weight and volume [9]. Recent studies have shown that both AdSCs [9, 10] and DFAT cells [14] isolated from human BFP (i.e., BFPSCs and BFP-DFAT cells) are similar to SC-AdSCs and possess high potential for regeneration of the bone and periodontal tissues [10, 12]. These properties may make the BFP a desirable cell source for tissue engineering and cell isolation.

Since BFP is easily accessible via the oral cavity without injury to the external body surface, several research groups have recently evaluated the behavior of isolated AdSCs from the BFP as a proper source of adult cells for clinical applications [13].

BFPSCs have been reported to remain in a quiescent state during 24 days of culture and showed spindle-shaped morphology similar to AdSCs, BMSCs, and USSCs [17]. Afterward, they began to multiply rapidly, formed a monolayer of large flat cells, and exhibited a more fibroblast-like morphology characteristic of AdSCs [9].

Cell surface markers on BFPSCs were characterized by immunofluorescence combined with flow cytometric analysis [10, 12, 67]. BFPSCs expressed MSC-defined markers, including CD73, CD90, and CD29, whereas they did not express lymphocyte or leucocyte antigens [12] and hematopoietic markers such as CD14, CD31, CD34 [9, 12], CD45 [10], CD19, and HLADR [9]. In addition, Traktuev et al. reported that BFPSCs showed some expression of CD34, which is characteristic of fresh AdSCs [68]. However, this marker declined with passage in AdSCs [9]. CD34+ cells have been shown to stimulate angiogenesis, and they are involved in neovascularization processes that facilitate healing of damaged tissues [69, 70].

Similar to other AdSCs, freshly isolated BFPSCs lack expression of CD105, but expression of this marker increases rapidly after seeding [20, 71]. AdSCs also usually lack expression of CD146, a characteristic marker of endothelial cells as well as vascular smooth muscle cells. However, Farr-Guasch et al. found a small population of CD146 cells in the first passages of BFPSCs [9]. The presence of this CD146-contaminated population, as well as the presence of CD34 cells, might be due to the highly enriched blood vessel supply in BFP [72]. This could be related to the excellent wound-healing properties of BFP as a pedicled graft in oral surgery for treatment of oroantral communications [55, 73], maxillary defects [51], oral submucous fibrosis [74], and vocal cord defects [75].

Several researchers have shown that BFPSCs are multipotent and differentiate towards osteogenic, adipogenic, and chondrogenic lineages in the presence of inductive stimuli [9, 12, 76, 77]. Broccaioli et al. showed that after 14 days of osteogenic induction, BFPSCs showed a significant upregulation of two specific markers, ALP activity and collagen deposition [12]. Farr-Guasch et al. also showed that after 1 week of culture in osteogenic medium, BFPSCs changed their morphology from spindle shaped to more polygon shaped, which was accompanied by an increase in ALP activity [9]. They reported that BFPSCs cultured in osteogenic medium showed osteocalcin expression. In addition, Niada et al. demonstrated the expression of core-binding factor alpha subunit 1 (CBFA1) and osteonectin. They also showed that compared with undifferentiated cells, osteogenic-differentiated BFPSCs derived from swine significantly increased the production of bone-specific markers, such as collagen, calcified extracellular matrix (ECM), ALP, and osteonectin [13].

After adipogenic induction of BFPSCs, the classic fibroblast-like shape of human AdSCs changed, and BFPSC populations showed intracellular lipid vacuoles that increased in size and number during culture [9, 12, 13]. Farr-Guasch et al. showed increased expression levels of a specific adipocyte marker during culture, peroxisome-proliferating receptor gamma (PPAR), reaching approximately four times higher induction compared to those of undifferentiated BFPSCs [9]. Some studies also showed that BFPSCs synthesized cartilage matrix molecules and produced an extracellular matrix characteristic for chondrocytes when grown in chondrogenic medium. Farr-Guasch et al. observed that after five days of chondrogenic induction, BFPSC morphology became more spheroid shaped [9]. Immunohistochemistry in differentiated BFPSCs indicated Toluidine blue-stained nodules indicative of a proteoglycan matrix characteristic for cartilage and expression of collagen II, a marker believed to be specific for articular cartilage. In addition, increased expression of the master chondrogenic factor, SOX9, was observed in BFPSCs, followed by decreased expression of the adipogenic marker PPAR when they induced towards the chondroblast lineage [9]. Niada et al. also observed GAG content in chondrogenic differentiated BFPSCs following three weeks of induction [13].

It is well known that rhBMP-2 enhances osteogenic differentiation of BMSCs [78]. However, it has been suggested that rhBMP-2 may not influence the osteogenic differentiation of AdSCs [25, 79]. Shiraishi et al. analyzed the capability of rhBMP-2 to induce osteogenesis on BFPSCs cultured in different culture conditions (i.e., osteoinductive reagents (OS), rhBMP2 (BMP), and the combination of BMP and OS (BMP-OS)) [10]. Their results indicated that rhBMP-2 strongly induced the osteogenic differentiation of human BFPSCs. After 1014 days in culture, rhBMP-2 treatments (BMP and BMP-OS) induced distinct and substantial calcified-nodule formation and the expression of osteogenic markers in BFPSCs. In addition, they showed that BFPSCs pretreated with BMP-OS generated abundant bone tissue upon in vivo transplantation to the back subcutaneous pockets of nude mice [10].

Amelogenin (AM) is the most abundant enamel matrix protein, and it is favored for the repair of periodontal defects. Broccaioli et al. indicated that osteogenic differentiation of BFPSCs is specifically induced and upregulated by AM with a synergic effect with other osteoinductive factors [12], which was also reported for bone marrow MSCs [8082]. This effect is more evident for BFPSCs than for SC-AdSCs, possibly due to the natural localization of BFPSCs, which could make them more prone to responding to stimuli naturally secreted in the same area of the body [12].

Several research groups compared AdSCs derived from different areas of the body and evaluated their behavior in vitro to identify a convenient source for future preclinical studies (Table 1).

Several studies showed that the number of SC-AdSCs is similar to the number of BFPSCs, despite the different amounts of raw adipose tissues [12, 32]. However, Farr-Guasch et al. observed that the number of BFPSCs was two times higher than the number of SC-AdSCs after one week of culture. They admitted that this difference might be due to the differences in age, intrinsic characteristics of the patients, or particular properties of the adipose source [9].

Several studies reported that both BFPSCs and SC-AdSCs showed similar morphology (fibroblast-like morphology) [9, 10, 13]. However, Broccaioli et al. observed that BFPSCs appeared slightly smaller and rounder than SC-AdSCs [12].

Both types of cells similarly expressed defined MSC markers. Farr-Guasch et al. reported that the expression of CD34 was much higher in BFPSCs than in SC-AdSCs [9]. They also found the expression of CD146 in a small population of CD146 cells in the first passages of BFPSCs, but not in SC-AdSCs.

Broccaioli et al. showed that SC-AdSCs proliferated faster than BFPSCs, with an average doubling time of 73h compared to 126h. This was also confirmed through a viability test, in which MTT incorporation by SC-AdSCs was mildly higher than that by BFPSCs [12]. However, similar doubling times were observed for BFPSCs and SC-AdSCs derived from swine [13].

Broccaioli et al. showed that BFPSCs were more prone to producing colonies than SC-AdSCs [12]. Interestingly, BFPSCs showed a significant increase in colony formation in late passages (from passages 7 to 9), suggesting a delayed selection of progenitor cells [12]. However, no significant difference has been observed between colony formation of porcine SC-AdSCs and BFPSCs from passages 1 to 4 [13].

Several researchers showed that both cell types are multipotent and differentiate into different cell lineages in the presence of inductive stimuli [13, 76, 77]. Broccaioli et al. showed that after 14 days of osteogenic induction, both BFPSCs and SC-AdSCs showed a significant upregulation of two specific markers, ALP activity, and collagen deposition [12]. Niada et al. also showed that osteogenic-differentiated SC-AdSCs and BFPSCs derived from swine significantly increased the production of bone-specific markers compared with undifferentiated cells, such as collagen, calcified ECM, ALP, and osteonectin. The greatest difference was observed in the collagen level of 7-day-osteoinduced BFPSCs, which was 7 times higher than that of osteoinduced SC-AdSCs [13]. Similar capabilities in adipogenic and chondrogenic differentiation have been observed in both BFPSCs and SC-AdSCs [9, 13, 76].

Considering possible clinical applications, Broccaioli et al. studied the ability of BFPSCs and SC-AdSCs to grow in a medium supplemented with human serum [12]. Both populations were cultured in the presence of autologous serum (HAS) or heterologous serum (HHS), and their growth was compared to that of cells maintained in standard conditions (FBS). No differences in morphology were observed. In all the growth conditions, the AdSC populations maintained the fibroblast-like shape, and HAS induced a prompt increase in both BFPSC and SC-ASC numbers compared to other serums within 7 days. They noticed that the presence of human serum enhanced the proliferation rate of both cells types. This effect has not been previously observed in AdSCs, but similar heterogeneity in the response to autologous serum has also been described for BMSCs [26, 8385] and could be explained by the differences in serum from the donors. Growing AdSCs in the presence of autologous serum could be a convenient and safe procedure in future cellular therapy, which would eliminate concerns about contact with animal proteins [12].

Several research groups evaluated the behaviors of BFPSCs (e.g., adhesion, growth, and differentiation) on natural and synthetic biomaterials and compared them with other stem cells. Broccaioli et al. showed that both SC-AdSCs and BFPSCs can adhere to the autologous alveolar bone and periodontal ligament [12]. Moreover, they reported that these cells efficiently adhered to a collagen membrane. However, BFPSCs have not shown tight bonding to suture filaments of polyglycolic acid compared to AdSCs [12].

Niada et al. evaluated the ability of porcine AdSCs derived from SC and BFP to grow and differentiate on two synthetic scaffolds: titanium and plasma-treated silicon carbide [13]. They showed that both porcine AdSCs adhered and differentiated on these scaffolds. Ardeshirylajimi et al. assessed the osteogenic differentiation potential of MSCs on surface-modified poly(L-lactide) acid (PLLA) nanofibers. The MSCs were derived from four different sites: BFP, the bone marrow, subcutaneous adipose tissue, and unrestricted somatic stem cells [17]. No significant difference was observed in the proliferation rates. All four types of stem cells were demonstrated to differentiate efficiently into osteoblast-like cells on nanofibrous scaffolds in osteogenic medium. The highest ALP activity and calcium content were observed in BMSCs cultured on PLLA. Interestingly, BFPSCs resembled BMSCs in both ALP and calcium content. In addition, the highest expression of bone-related gene expression (i.e., Runx2, osteonectin, and osteocalcin) was observed in BFPSCs and BMSCs compared to that in other stem cell types [17].

AdSCs are used extensively for tissue engineering, and various studies have reported their utility [2224]. However, AdSCs at passage 0 include contaminating endothelial cells, smooth muscle cells, and pericytes [86]. In contrast to AdSCs, mature adipocytes are the most abundant cell type in adipose tissue and have dynamic plasticity to be converted into DFAT cells [31]. Compared with ASCs, a relatively homogeneous cell population of DFAT cells has been revealed by flow cytometric analysis [31]. DFAT cells could not only redifferentiate into lipid-filled adipocytes in the same way as preadipocytes but they can also transdifferentiate into other cell types under proper conditions in vitro, including osteoblasts [14, 30, 38], chondrocytes [30], and myocytes [8790]. In vivo studies also suggested that DFAT cells could regenerate fat pads, ectopic osteoid tissue, or muscle tissue [30, 3638, 88, 90].

BFP-DFAT cells have been shown to be positive for CD90, CD105 [14], CD13, CD29, and CD44 [15] but negative for CD11b (monocyte marker), CD34 (hematopoietic progenitor cell marker), CD45 (leukocyte common antigen) [14], CD31, CD309, CD106, and alpha-smooth muscle actin [15, 30].

Kou et al. showed that after 3 weeks of osteogenic culture, the DFAT cells demonstrated limited mineralized matrix indicated by alizarin red S staining, while a relatively large portion of cells assumed a multilocular appearance and they were positive for adipose staining [15]. Previous reports confirmed the expression of osteogenic transcription factors in DFAT cells, including Runx2, osteopontin, osteorix, and osteocalcin [22, 29, 30]. However, Kou et al. showed that DFAT cells exhibited lower potential of differentiating towards osteoblasts than an adipocyte lineage [15]. This could be due to the fact that the multipotent capacity of DFAT cells is tissue specific [15].

Tsurumachi et al. divided adipocytes into two groups based on their size: those with cell diameters less than 40m (small adipocytes: S-adipocytes) and those with diameters of 40100m (large adipocytes: L-adipocytes). They investigated the influence of the adipocyte size on the dedifferentiation efficiency into DFAT cells and compared the S- and L-DFAT cells based on the characteristics for MSCs. They showed that the S-adipocytes contained more juvenile adipocytes than the L-adipocytes. The results suggested higher rates of dedifferentiation for S-DFAT cells compared to those for L-DFAT cells and that the adipocyte size is positively associated with dedifferentiation. However, more studies are needed to reveal how the cell size could influence the efficiency of mature adipocyte dedifferentiation [16].

Tsurumachi et al. conducted flow cytometry and revealed higher CD146 expression in S-DFAT cells compared to that in L-DFAT cells, although both cells showed high expression levels of CD13, CD44, CD73, CD90, and CD105 [16]. S-DFAT cells showed higher osteogenic potential in particular compared to the L-DFAT cells. Similarly, a comparison between AdSCs and DFAT cells obtained from BFP demonstrated more effective induction of osteoblasts from DFAT cells than from AdSCs [14]. S-DFAT cells also exhibited higher osteogenic potential than L-DFAT cells. The results suggested that S-DFAT cells have an advantage over L-DFAT cells and AdSCs in bone tissue engineering.

Matsumoto et al. reported that human AdSCs at passage 1 are 13.3% positive for CD11b (monocyte marker) and 12.8% positive for CD45 (leukocyte common antigen). However, human DFAT cells at passage 1 are negative for these markers, indicating greater homogenicity of DFAT cells compared with AdSCs [30]. Kishimoto et al. showed that the expression of osteoblastic differentiation markers (BAP, OCN, and calcium) in BFP-DFAT cells was more prevalent than that in BFPSCs [14]. However, they indicated that the difference in the osteoblastic differentiation ability of BFPSCs and BFP-DFAT cells was not because of the difference of purity in the cell populations [14]. The same group also reported various osteoblastic differentiation abilities between human DFAT cells derived from the submandibular and human BMSCs [91]. Gene expression of Runx2, ALP, OCN expression, and calcium deposition was higher in DFAT cells than in bone marrow MSCs. More studies are needed to come to a general conclusion regarding the osteogenic capability of BFP-DFAT.

This study has reviewed the characteristics and osteogenic capability of AdSCs derived from BFP. This source of cells was also compared with other AdSCs from other parts of the body. BFP is an easily accessible source of stem cells that can be obtained easily via the oral cavity without injury to the external body surface. Its size is similar between people and independent of body weight and fat distribution. Comparing BFPSCs with other AdSCs showed similarities in cell yield, morphology, and multilineage differentiation. However, BFP has been shown to proliferate faster and is more prone to producing colonies. Limited studies have been conducted on the osteogenic capability of BFP-DFAT cells, which makes conclusions infeasible.

The authors declare that there is no conflict of interest regarding the publication of this paper.

This research was supported by the Research Deputy, Dental School, Shahid Behehsti University of Medical Sciences.

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Buccal Fat Pad as a Potential Source of Stem Cells for Bone ... - Hindawi

The key difference between adult and embryonic stem cells is that adult stem cells are multipotent while embryonic stem cells are pluripotent.

Stem cells are a category of cells with the ability to divide and develop into different types of cells in the body. They are distinguished from the normal cells since they divide and renew themselves over a longer period of time. Moreover, they are unspecialized cells with no specific cellular function in the body. They have the potential to differentiate and become specialized cells in the body such as brain cells, blood cells, and muscle cells. Adult stem cells and embryonic stem cells are two types of stem cells.

1. Overview and Key Difference2. What areAdult Stem Cells3. What are Embryonic Stem Cells4. Similarities Between Adult and Embryonic Stem Cells5. Side by Side Comparison Adult vs Embryonic Stem Cells in Tabular Form6. Summary

Adult stem cells are present in differentiated tissues of the body. These tissues include skeletal muscle, liver, pancreas, brain, eye, dental pulp, skin, bone marrow, blood and lining of the gastrointestinal tract. Moreover, adult stem cells remain in these tissues undifferentiated, with continuous self-renewal and producing identical copies of cells throughout the lifetime of the organism. They undergo differentiation into specialized cells of their tissues of origin when needed.

Figure 01: Adult Stem Cells Repair

Hematopoietic stem cells are a type of adult stem cells present in the bone marrow. They are considered multipotent stem cells as they give rise to different types of blood cells from a single type of cells. Regulated gene expression is responsible for these variations in differentiated cells. It is controlled by special types of transcription factors. Also, stem cells present in the brain are multipotent. They give rise to both muscle and blood cells.

Embryonic stem cells are the undifferentiated cells present in the inner cell mass of the blastocyst a hollow ball of cells developed from the zygote after rapid mitosis. Hence, these stem cells are categorized as stem cells present in the early stages of embryonic development.

Embryonic stem cells are pluripotent. Therefore, they give rise to cells of three germ layers endoderm, ectoderm, and mesoderm except the placenta and umbilical cord. Pluripotency distinguishes embryonic stem cells from adult stem cells.

Figure 02: Embryonic Stem Cells

Embryonic stem cells provide valuable assistance as a renewable resource in the study of diseases and for testing of potential therapeutics and drugs. Under defined conditions, embryonic stem cells possess the ability to divide indefinitely.

Adult stem cells and embryonic stem cells are the two main types of stem cells. The key difference between adult and embryonic stem cells is that adult cells are multipotent as they have a limited ability to differentiate while embryonic stem cells are pluripotent as they have the ability to differentiate into any cell type. Also, a further difference between adult and embryonic stem cells is that the embryonic stem cells can readily grow in cell cultures while the growth of adult stem cells in cell cultures is very challenging.

Moreover, a significant difference between adult and embryonic stem cells is that the adult stem cells are present in adult tissues while embryonic stem cells are present in the early development at the blastocyst stage.

The key difference between adult and embryonic stem cells lies in their potency. That is; the adult stem cells are multipotent while embryonic stem cells are pluripotent. Adult stem cells are present in differentiated tissues of the body such as liver, pancreas, skeletal muscle, etc. Regulated gene expression is responsible for variations in differentiated cells derived from adult stem cells. On the other hand, embryonic stem cells are present in the inner cell mass of the blastocyst. These stem cells give rise to the cells of ectoderm, endoderm, and mesoderm. Furthermore, embryonic stem cells divide indefinitely under defined conditions. This is the summary of the difference between adult and embryonic stem cells.

1. The Adult Stem Cell. National Institutes of Health, U.S. Department of Health and Human Services, Available here.2. Stem Cells. A Closer Look at Stem Cells, Available here.

1. Stemcellheartrepair By US gov -(Public Domain) via Commons Wikimedia2. Human embryonic stem cells only A : Human_embryonic_stem_cells.png: (Images: Nissim Benvenisty)derivative work: Vojtech.dostal (talk) Human_embryonic_stem_cells.png (CC BY 2.5) via Commons Wikimedia

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Difference Between Adult and Embryonic Stem Cells

Nicklas Brendborg: Keeping your mouth clean is one of the few easy things you can do to extend your life  EL PAS USA

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Nicklas Brendborg: Keeping your mouth clean is one of the few easy things you can do to extend your life - EL PAS USA

Reparative techniques are vital in endodontics, andconservative measureshelp preserve the vitality of teeth and ensuring they are in good health [1,2]. Mineral trioxide aggregate(MTA) is a biocompatible compound that has found widespread usage in clinical endodontic therapy because of its low cytotoxicity and high biocompatibility and ability to stimulate new dentin development. It has been the material of paramount importance since its introduction in the 1900s [3]. Uses of MTAinclude conservative management of root fractures, perforation repair [4], pulp capping agent [5], apexification [6], retrograde filling material in apical microsurgeries [7], and revascularization measures as a coronal barrier[2]. The above procedures involve close contact with the body fluids and vital tissues, favoring physical alterations and chemical/biological communications with the material [8].

Various properties of MTA, such as physical, chemical, and biological, have been explored for extended periods, leading to discoveries of its efficient substitutes. However, improvisations are still needed to arrive at an ideal composition of the constituents of the cement. The development of a model, unflawed restorative material is still long due. To achieve thisit should possess the following characteristic properties: sealing ability, dimensional [9] and color stability [10], radio-opacity [11], insolubility when in contact with body fluids, and ability to flow with easy insertion. It should also possess biological and chemical properties such as alkaline pH, calcium (Ca) ion release, bioactivity, cell attachment, and biocompatibility[12]. Mineral trioxide aggregate owns most of the mentioned ideal properties but lacks a few, primarily color and consistency which require the most improvisations[13]. Therefore, materials with newer innovations have been launched commercially to overcome these shortcomings. This review intends to highlight the properties of MTAwith their limitations and to arrive at the developments in innovative Ca silicate-based cements (CSCs).

The main emphasis should be on the clinical facet of these CSCs, as the site of placement directly influences and determines materialproperties[14]. Mineral trioxide aggregateis a dynamic, active material as its application and placement result in constant contact of the cement with tissues and fluids. It persists for years after its insertion[15]. Its mechanism comprises Ca hydroxide leaching out of the hydrated MTA, thereby highlighting the bioactivity of MTA, which relates to the calcium ion(Ca2+) release. Placement of MTAis usually required at the site where there is a presence of blood that contaminates it, affecting the structure of the set material and reducing the Ca2+ release[6,16]. The principal limitations of MTA include a delayed setting time, lack of good handling features, and the disadvantage of discoloration.

Also, the contact of MTAwith blood can alter the color of the material andinterferes with radiopacity over time[17]. Moisture drastically affects the time of setting and the material solubility. Excessive water results in increased solubility and setting time of MTA. During the setting process of MTA, it chemically interacts with tissues making the environment alkaline by releasing Ca2+ ions, which are linked to the development of portlandite (calcium hydroxide) by tricalcium silicate (C3S) and dicalcium silicate (C2S)[18].

In vitro studies done with MTAAngelus and ProRoot MTArevealed Ca2+ ion discharge andalkalization of the environment when the material was submerged in water. The release of Ca2+ ions was detected by von Kossa staining of subcutaneous tissues of rats[19]. These properties lead to mineralization on the surface of the set MTAin pulpotomy procedures. This is proven by studies where hard tissue was formed apically in a dog's teeth which were seen along with the sealing ability in cases of furcation perforation[20].

In an in vivo study by Han et al., the odontogenic potential of osteostatin (OST) and the combined effect with bioceramic materials on human dental pulp stem cells (hDPCs) were investigated, and it was discovered that the combination of MTAand OSThad a synergistic odontogenic differentiation of hDPCs when compared to MTAalone[21]. Micro-CT research demonstrated that OSTwith ProRoot MTAgroups formed more mineralized dentin bridges[22].

During dental operations, the most significant property of observation is color. Tooth discoloration damages the tooth's aesthetic appearance. The complex response between filling materials and coronal dentine of the pulp chamber, which modifies the crowns appearance, is a significant cause of tooth discoloration.

Initially, when developed, MTAhad a grey color owing to the presence of tetra Ca aluminoferrite, making it unsuitable for its application on anterior teeth. Therefore, this led to the establishment of white MTA which is devoid of iron to prevent the discoloration of the tooth. On the contrary, many studies have proven the alteration in color even with white MTA[23]. The composition of white MTAincludes C2S and C3S silicate with 20% of bismuth oxide. According to reports, the amount of bismuth oxide added to MTA to increase its radio-opacity was only 8.4% in the set material compared to 21.6% in the unset material [8]. When reduction of bismuth oxide occurs along with its contact with the tooth structure, it alters the color of the cement and the color of the adjacent tooth structure. The cause of color change has been identified and attributed to the loss of stability of the bismuth oxide molecules, which occurs as they come in close contact with a potent oxidizing agent[24]. Hence, it is suggested that if the radiopacifier agent is changed, it can help prevent the discoloration of the tooth. Two materials have been lab tested to replace bismuth oxide, namely zirconium oxide and Ca tungstate. However, large amounts are necessary to match the radiopacity ofbismuth oxide. Adding such large amounts can negatively impact the chemical and physical properties of the dental material[25]. Newer CSCs such as Biodentine and Bioceramic (BC) sealer, and MTA high plasticity (HP) can alter the radiopacifier agent into Ca tungstate or zirconium oxide.These constituents caused no alteration in color[26]. The second substitute is the addition of 5% zinc oxide (ZnO) to MTAas this ZnO converts bismuth oxide into bismite, a product that helps prevent the change of color[10].

There is a difference in opinion regarding the consistency of MTA. The ratio of powder to water is essential as increasing the quantity of water reduces radiopacity. The particle size is vital here as the newer advances in silicate types of cement have been developed using nanoparticles of Ca silicate (CS). The BCsealer and biosealer containing nanoparticles of CSwith the addition of a polymer help in easy handling and givean ideal material consistency. Adding propylene glycol to MTAcaused no interference in its biological properties. Propylene glycol was tested using different ratios for chemical and physical properties in which 20% propylene glycol was mixed with 80% distilled H2O,which led to efficient handling of MTA, pH, enhanced Ca release, and flowability. However, it caused slight alterations in setting time[27]. Few studies have proven that propylene glycol caused improved adhesion of MTA.

The advances which lead to enhanced flow ability comprise MTA HP, MTA Flow, Biodentine (Septodont, Saint-Maur-des-Fosss, France),and ones having ceramic complexes incorporated with Biodentine, EndoSequence (Brasseler, Savannah, GA, USA), and BioAggregate (Verio Dental Co. Ltd., Vancouver, Canada).

In 2009, Biodentine, a Ca silicate-based product, was introduced. Zirconium oxide is used instead of bismuth oxide in Biodentine. Zirconium oxide is a bioinert substance with good mechanical qualities & corrosion resistance. Dettwiler et al. 2016 observed this closely in an experiment [28]. Biodentine had a minor discoloration, higher solubility than MTA, and a significantly faster setting time. In as little as 12 minutes, Biodentine can begin to block blood components, becoming denser and packed as it sets. As a result, erythrocyte penetration is reduced, resulting in less tooth discoloration during the pulpotomy operation. Because it comprises more powder with a water-reducing agent and less porosity, the Biodentine material significantly impacts various factors such as absorption, strength, and density[29]. Biodentine and zinc oxide-eugenolcement (IRM) had the lowest level or degree of porosity and the least amount of tooth discoloration, according to Camilleri et al. in 2013[14].

Endosequence root repair material (ERRM), is available as a premixed putty with uniform consistency and easier handling and application. According to the manufacturer, the setting begins with the presence of moisture in the dentinal tubules. When pulp cells were exposed to ERRMor ProRoot MTA, survival and proliferation were identical, suggesting that it could be a good choice for pulp capping treatments[30].

BioAggregate (BA)contains monobasic Ca phosphate, amorphous silicon dioxide, and tantalum pentoxide for radiopacity. And due to its Ca phosphate content,it is classified as a biphasic material (one that contains two cementitious ingredients)[31]. It is more acid resistant than MTA, has a longer-lasting strengthening effect on weaker teeth, and has a lower risk of discoloration[31]. In the treatment of immature teeth, it has demonstrated similar results as MTA.

The main composition of MTAis CS. Bioactivity is one characteristic feature of Ca silicate-based types of cement[27]. Newer CS-based restorative types of cement have been launched to substitute bismuth oxide like Biodentine, Neo MTAPlus (Avalon Biomed Inc.Bradenton, FL, USA), and MTARepair HP (Angelus, Londrina, PR, Brazil). Others include MTAFillapex (Angelus, Londrina, PR, Brazil), Neo MTAPlus, iRoot SP (Innovative BioCreamix Inc, Vancouver, BC, Canada), and TotalFill BC (Davis Schottlander & Davis Ltd. Letchworth, Herts, UK) sealer.

The MTAFillapex cement comes in a paste-paste form which comprises salicylate and natural resin, infused silica nanoparticles, MTA, and Ca tungstate which acts as radiopacifier.There is a newly introduced C2S silicate-based system with a powder-gel formulation named Neo MTA, a remarkable restorative and endodontic cement that can be used with various proportions of powder gel ratios. The composition of iRoot SPis zirconium oxide, CS, Ca phosphate, Ca hydroxide, and thickening agents, which are commercially accessible and is used as a root canal filling material. On the other hand, EndoSequence BC sealer and TotalFill BC sealer comprises zirconium oxide, CS, monobasic Caphosphate, Ca hydroxide, and thickening agents. This latter cement is advantageous as it sets in the presence of dentin moisture and hence was used as canal filling material.

A study on iRoot SPendodontic cement advocated the absence of cytotoxicity to fibroblasts when tested in rats[32]. Alternative research by Zoufan et al., checked the cell compatibility of iRoot SPcement at two stages: after the cement was freshly mixed, and after the cement had been set[33]. It was found that this cement had a greater induction capacity of osteoblastic differentiation and decreased inflammatory response with the periodontal ligament cells compared to Sealapex[34].

The MTAand iRoot SPtypes of cement have been proven to induce differentiation in osteoblastic cells in the tooth germ. The iRoot SPsignificantly showed its antibacterial activity against Enterococcus faecalis[35]. Zhu et al. found evidence of the ability of BioAggregate cement to promote cell adhesion to each other, migration, and fixation of human dental pulp cells, thus proving its cytocompatibility[36].

Bioceramic endodontic cement-like Endosequence BC sealerhas displayed promoting superior cell viability than AHPlus sealer and also offered an increased level of biocompatibility when compared with newly handled AHPlus and MTAFillapex,when freshly mixed and after the setting. Bioceramic sealer has shown satisfactory adhesion to fibroblasts[37]. Upon contact with the biological solution, discharge of Ca and development of the Ca phosphate phase was seen. Antibacterial activity against biofilm formed on dentin was greater when Endosequence BC sealer was used along with 5% sodium hypochlorite than the irrigation solution alone[38]. In a study using confocal laser microscopy, Wang et al. concluded that in 30 days, a BioCeramic sealer could eliminate 45% of E. faecalis from the dentinal tubules, indicating the antibacterial action of theBioceramic sealer lasted even after the setting of the material [39]. Total fill BioCeramic sealer is identical to Endosequence BC sealer. The only difference is that the former promotes extensively higher proliferation of cells compared to AHPlus and MTA Fillapex.The structure of cells embedded on Total Fill BioCeramic Sealer and AHPlus showed similar physiognomies, along with the assembly of the extracellular matrix. In contrast, limited fixation of cells was seen on discs of MTAFillapex, with decreased number of cells on the material surface[40].

The MTA Angelus, MTA HP, and Neo MTA P presented viability of cells and a higher degree of cellular proliferation along with adhesion. Using HDPCs, greater viability was seen with MTAplus compared to MTAFillapex and Fillcanal; increased phosphates activity was observed with MTAPlus[41,42]. No cytotoxic effect was seen with Neo MTAPlus, MTAAngelus, and experimental C3S silicate-based cement with tantalum oxide (TSC/Ta205). According to the alizarin red assay, the three materials were proven to induce the formation of mineralized nodules; on the other hand, NEOproduced a considerable quantity of mineralized nodules compared to MTAand TSC[43]. Following subcutaneous implantation in rats, histological analysis established that MTA HPshowed similar biomineralization and biocompatibility potentials to MTA Angelus[43]. The MTAAngelus and MTAPlus showed no presence of cytotoxicity and induced mineralized nodule formation. When PCR was used, the authors concluded that when HDCPs were exposed to extract the two types of cement, it increased the expression of osteogenic markers of the cell[44].

According to Petrovic et al., materials based on CSand hydroxyapatite (HA-CS) showed a superior grade of biocompatibility compared to MTAAngelus [45]. Also, improved outcomes were seen for CS and HA-CS when subcutaneous implants were placed in rats. In the assessment of the biocompatibility of three Ca silicate-based types of cement, which include Bioroot BC sealer (BR), Endoseal MTA(ES) & Nanoceramic sealer (NCS), along with human periodontal ligaments stem cells (hPDLSCs), BRand NCSshowed superior cytocompatibility as compared to ES[46]. The BCsealer was proficient in hindering the release of immunoreactive calcitonin gene-related peptide (iCGRP) from trigeminal ganglion neurons and excellent biocompatibility, thereby reducing the symptomatology level after extravasation of the cement in ongoing treatment[47].

In a study by Almedia et al., a comparison of physiochemical and biological properties of already mixed Ca silicate-based endo sealers with routinely used root canal (RC) filling materials by thoroughly revising lab investigations [48]. Calcium silicate-based endodontic sealers followthe ISO 6876:2012 standard for most physicochemical properties, except solubility. The target sealers depicted commendatory biological traits in comparison to conventional sealers. Despite failing to test the target premixed Ca silicate-based sealers in long-term experimental clinical trials, they presented with good physicochemical and biological traits in vitro.

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A Breakthrough in the Era of Calcium Silicate-Based Cements: A Critical Review - Cureus

Introduction

As scholars have been studying tissue engineering more and more, oral bone regeneration which is of fundamental importance in the dentistry field has become a hot research topic.1 Mesenchymal stem cells (MSCs) are undifferentiated cells known for their self-renewal and differentiation properties, and they can secrete immunomodulatory factors, leading to the creation of a regenerative microenvironment, and trans-differentiate into cells of the different germ layers: mesoderm lineage cells, as well as ectoderm and endoderm lineage cells.2 The capacity of MSCs is useful for osteogenic differentiation and tissue regeneration.3 Some clinical studies have demonstrated that MSCs from different sources may have the ability to repair, replace, and regenerate cells, tissues, and bones.4 MSCs can be extracted from different tissues such as bone marrow, skeletal muscle, cartilage, dental organ, adipose tissue, synovium, and cardiac tissue.5 BMSCs were the first to be discovered.6 Bone is formed via endochondral and intramembranous ossification.7 MSCs play a vital role in bone formation. On the one hand, MSC-driven condensation occurs firstly, and then, MSCs differentiate into chondrocytes during the process of formation of growth plates, which are replaced by new bone in longitudinal-endochondral bone growth.8 On the other hand, MSCs can also directly differentiate into osteoblasts in bone formation such as skull, facial bones, and pelvis, generated by intramembranous ossification without a cartilaginous template.9,10

Transverse maxillary constriction often manifests a typical vertical skeletal pattern, with long anterior lower facial height, high palatal vault, low tongue posture, incompetent lip muscles, and mouth-breathing.11 Previous studies indicated that approximately 18% of mixed-dentition patients had a transverse maxillary constriction,12 which led to dentofacial deformities such as anterior deep overbite, posterior reverse overbite, and dental crowding. In general, the mid-palatal suture can be disrupted and separated by exerting a rapid transverse force on the maxillary dentition which surpasses the limit of orthodontic movement; continuous force increases cellular activity in the area and induces bone remodeling13 in a process called rapid maxillary expansion (RME). Since mid-palatal suture opening was first reported by Angell, RME has become a widely performed procedure by orthodontists. RME is also considered crucial for remedying maxillary constriction in children and growing adolescents, as skeletal component rigidity limits expansion extent and stability as the patient matures. Some orthodontists suggest that early treatment to correct transverse discrepancy may avoid future extractions.14 Of note, although the mid-palatal suture can be successfully opened, relapse of the posterior dentition width has been frequently reported;15,16 forces that induce relapse continue to act for up to six weeks after active expansion.17 A major reason for early relapse is inadequate bone formation in the suture. Consequently, a long retention period using a fixed retainer is often used to lessen the relapse. It was previously reported that the extent of relapse was related to the retention procedure after expansion, and thus a fixed retainer was required for at least two months.18 However, the discomfort caused by the considerable volume of a fixed retainer may reduce patient self-discipline to maintain the effectiveness of a previous RME, and similarly, the fixed retainer may increase the risk of caries due to accumulated dental plaque. Therefore, many RME studies have focused on different approaches to enhance new bone formation, strengthen post-treatment width, ensure enough stability, and shorten the retention period.1923

The pueraria plant is believed to be one of the earliest traditional herbs used in ancient Chinese medicine. Puerarin is a phytoestrogen first isolated from the pueraria root in the late 1950s and is one of the main isoflavone components in the root.24 In 2005, the pueraria plant was identified as the 6th most important food crop by the World Food and Agriculture Organization. The pharmacological activity of puerarin has been extensively investigated since its isolation, with activities including neuroprotective effects,25 vasodilatory activity,26 cardioprotective activity,27 anti-diabetic activity and the inhibition of diabetic complications,28 anti-Parkinsons disease activity,29 anti-Alzheimers disease activity,30 anti-osteoporotic activity,31 antioxidant activity,32 and others.24 Furthermore, evidence has suggested that puerarin dissolved in collagen matrix increases new bone formation in bone graft defect sites and may be used for bone grafting and bone regeneration after surgery.33,34 Therefore, it is reasonable to hypothesize that pueraria treatment may promote bone regeneration in the mid-palatal suture. As no reports on the puerarin stimulation of bone formation in the mid-palatal suture have been published, our objective was to investigate the effects of puerarin on osteogenesis in vitro and bone regeneration in vivo in the expanding mid-palatal suture and provide a theoretical foundation for its therapeutic effects toward RME and relapse prevention.

The study was complied with the ARRIVE guidelines and carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU. The study was approved by the Animal Research Committee of School of Stomatology, Shandong University (Protocol No.: 20210121). All efforts were made to minimize the number of animals used and their suffering.

Rat bone marrow-derived mesenchymal stem cells (BMSCs) were accessed from bilateral femora and tibiae of two-week-old Wistar rats from the Laboratory Animal Center of Shandong University. Euthanized rats were soaked in and sterilized with 75% alcohol; bilateral femurs and tibiae were separated under aseptic conditions within 15 min to ensure the cell activity. After washing the long bones, the metaphyses were removed, and bone marrow was harvested out from the cavity with -minimum essential medium (-MEM; Hyclone, Logan, UT, USA), complemented with 15% fetal bovine serum (FBS; Biological Industries, Israel) and 1% penicillin/streptomycin (Hyclone; GEHealthcare Life Sciences, Logan, UT, USA). BMSCs were collected after suspension and cultured in the incubator in a humidified atmosphere of 95% air and 5% CO2 at 37 , the medium was renewed every 3 days. Once the cells reached 80%, they were rinsed with phosphate-buffered saline (PBS), digested with a 0.25% trypsin-EDTA solution (Thermo Fisher Scientific Inc) and sub-cultured with complete medium. BMSCs at passage 3 were used in subsequent experiments. Eventually, the expressions of cell surface molecular markers (CD 34, CD 44, CD 45, and CD 90) were analyzed by flow cytometer (Beckman Coulter, Franklin Lakes, NJ, USA) to identify the stem cell properties of the collected BMSCs.

To identify the multi-directional differentiation potential of BMSCs, they were induced by osteogenesis and adipogenesis. The cells in passage 3 were seeded in 6-well plates at a density of 1.0 105 cells per well and cultured to 90% confluence with complete medium, and then, the medium was changed to osteogenic inducing medium (-MEM containing 8% FBS, 50 g/mL ascorbic acid, 10 mM -glycerophosphate and 0.01 M dexamethasone) (Sigma-Aldrich) or adipogenic inducing medium (-MEM containing 8% FBS, 500 M 3-isobutyl-1-methylxanthine, 200 M indomethacin, 1 M dexamethasone and 10 g/mL insulin) (Sigma-Aldrich). After culturing for 21 days, BMSCs were fixed with 4% paraformaldehyde, stained with Oil Red O and Alizarin Red S (Cyagen Bio-Sciences, Guangzhou, China).

The Cell-counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was used to determine the effect of puerarin on the proliferation of BMSCs. BMSCs were placed in 96-well plates with complete medium at a density of 5000 cells per well for 24 h. Next, the medium was replaced by complete medium supplemented with puerarin (GN10680; GlpBio, American) (Figure 1A) at different concentrations (0, 103, 104, 105, 106, 107 and 108 mol/L), five duplicate wells were set for each concentration group. 1, 3, 5 day(s) later, the medium was aspirated, and then 100 L of Cell-counting Kit-8 solution (-MEM and CCK-8 reagent mixing in a ratio of 9 to 1) was added into every tested well. Wells containing 100 L CCK-8 solution without seeding cells were used as blank control. The absorbance of samples was measured by a microplate reader (SPECTRAstar, Nano, BMG Labtech, Ortenberg, Germany) at 450 nm after incubation for 2 h at 37 in a darkroom.

Figure 1 Model of rapid maxillary expansion (RME) and three-dimensional reconstruction of the occlusal view of the rat maxilla. (A) Chemical Structure of Puerarin (C21H20O10). (B) Plaster model of rat maxillary. (C) Expansion appliance. (D) Inserted expansion appliance. (E) Bonded expansion appliance. (F) Rat maxillae after carefully dissected. (G) Rat head in the occlusal view, the vertical red line marking the occlusal position of the mid-coronal plane of the upper first molar, the horizontal red line marking the position of mid-palatal suture. (H) Rat head in the sagittal view, the red line marking the sagittal position of the mid-coronal plane of the upper first molar. (I) The distance of the mid-palatal suture in one of the rats in group 1 is 0.13mm. (J) The position and length of the ROI (2.0mm*1.0mm*0.8mm) in the coronal plane, horizontal plane, sagittal plane respectively. (K) 3D position view of ROI. (L) Location and plane of sectioning in the mid-palatal region.

To determine the ability of clone formation, BMSCs were seeded in 6-well plates at a density of 600 cells per well for 10 days, after fixing with 4% paraformaldehyde, cells were stained with crystal violet (Solarbio, Beijing, China). Cell colonies (clusters with 50 or more cells originated from the same cell) were counted to determine the ability of BMSCs to proliferate and form colonies.

The ALP activity assay is widely used to estimate the early osteogenesis ability of stem cells. BMSCs were plated in 6-well plates at a density of 1.0 105 cells per well and treated with osteogenic inducing medium containing different concentrations of puerarin (0, 104, 105, 106 and 107 mol/L). After 7 and 14 days of induction, cells were rinsed three times with PBS and solubilized in the lysis solution (ripa buffer and PMSF mixing in a ratio of 99 to 1) (Solarbio, Beijing, China) for 15 min on ice. Lysed the collected solution under ultrasound for 10 cycles (Bioruptor Pico, Diagenode, Belgium), and then, cell lysates were centrifuged at 12,000 g for 5 min at 4 . The supernatant was collected to obtain protein. Following the instructions of the manufacturer, an ALP activity assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to measure the absorbance of the samples using a microplate reader at 520 nm. ALP activity was normalized to the respective total protein concentration detected by the bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China).

BMSCs were plated in 6-well plates at a density of 105 cells per well and treated with osteogenic inducing medium containing different concentrations of puerarin (0, 104, 105, 106 and 107 mol/L) for 21 days. After fixed with 4% paraformaldehyde for 30 min, cells were stained with Alizarin red S (pH 4.1, Sigma-Aldrich) for 15 min, the stained plates were scanned to evaluate mineralized matrix deposition. Then, 10% cetylpyridinium chloride (CPC; Solarbio, Beijing, China) was added to the stained plates to dissolve the mineral nodules. The absorbance of the solution used to quantify the mineral nodules was measured by a microplate reader at 562 nm.

BMSCs were cultured in osteogenic inducing medium containing different concentrations of puerarin (0, 105, 106 mol/L) for 7 and 14 days. According to the manufacturers instructions, the Evo M-MLV RT Kit with gDNA Clean for qPCR II (AG11711; Accurate Biology, Hunan, China) was used to isolate total mRNA and prepare cDNA. The SYBR Green Premix Pro Taq HS qPCR Kit (AG11701; Accurate Biology, Hunan, China) and a Roche Light Cycler 480 Sequence Detection System (Roche Diagnostics GmbH, Mannheim, Germany) were used to perform reverse transcriptase polymerase chain reaction (RT-PCR), and a reaction system of 10 L volume was adopted. Every RNA sample was tested in triplicate, and each experiment was repeated at least 3 times, the mRNA expression levels were calculated by the 2Ct method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. The primer sequences used in the present study were as follows: ALP (+): 5-AGTGTGGCAGTGGTATTGTAGG-3 and 5-CACACACAAAGCACTCGGGG-3; SP7 (-): 5- GGTCCTGGCAACACTCCTAC-3 and 5-AAGAGGTGGGGTGCTGGATA-3; BSP (-): 5-AGCTGACCAGTTATGGCACC-3 and 5-TTCCCCATACTCAACCGTGC-3; OCN (+): 5-TGACAAAGCCTTCATGTCCAAG-3 and 5-GAAGCCAATGTGGTCCGCTA-3; GAPDH (+): 5- ACTCCCATTCTTCCACCTTT-3 and 5-CCCTGTTGCTGTAGCCATATT-3. The plus sign (+) indicates that the primers cross exon boundaries.

After culturing in osteogenic inducing medium containing different concentrations of puerarin (0 and 105 mol/L) for 14 days, BMSCs were lysed with RIPA lysis buffer containing 1% PMSF (Solarbio, Beijing, China). The total collected protein concentrations were quantified by a BCA protein assay kit. All protein samples (20g) were denatured and separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto 0.45-m polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). Afterwards, the membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with primary antibodies that recognized -catenin (1:800; Cell Signaling Technology, Danvers, MA, USA), GAPDH, ALP, Runx2, Collagen I (Col I) (1:1000; Abcam, Cambridge, MA, USA) overnight at 4 . After washing in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with a secondary antibody (Absin, Shanghai, China) solution at 37 for 1 h. Secondary antibodies were selected based on the source of primary antibodies. An enhanced chemiluminescent substrate kit (Millipore) and a chemical imaging system (Amersham Imager 600; GE Healthcare, Little Chalfont, UK) were used to detect immunoreactive proteins. GAPDH was used as internal reference.

The rats were pair-housed in standard plastic cages in a specific pathogen-free animal laboratory of School of Stomatology, Shandong University, under controlled temperature (22 1), humidity (55 10%), interior noise (below 60dB) and a 12-h light/dark cycle. They were provided with a powder diet and water ad libitum. All the animals were acclimated for 1 week before the experiment started. The general condition and weight of each rat were monitored daily during the experiment.

Eighteen 6-weeks-old male Wistar rats (mean weight 200~220 g) were adopted in the present research. The animals were randomly divided into three groups as follows: group 1, six control rats without any treatment; group 2, six rats received rapid maxillary expansion and saline solution (15mg/kg/day) containing 2% DMSO; group 3, six rats received rapid maxillary expansion and puerarin (15mg/kg/day) dissolved in 2% DMSO and then diluted with saline. Based on the width of the dental arch from the rat maxillary plaster model (Figure 1B), a 0.014-inch Australian wire (TP Original Premier Wire, TP Orthodontic Appliance Co. Ltd, Wuxi, China) was used to bend the expansion appliances with two helices and two arms (Figure 1C). Rats in groups 2 and 3 are anesthetized by an intramuscular injection of 3 mg/kg xylazine hydrochloride and 35mg/kg ketamine hydrochloride to ensure the smooth progress of the maxillary expansion surgery. After calibrating the expansion force between the two arms to 100 5g, the appliance was inserted into the bilateral first and second maxillary molars (Figure 1D) and the stability of the RME system was enhanced with the addition of light-cured adhesives (Gluma Comfort Bond, Heraeus Kulzer GmbH, Hanau, Germany) (Figure 1E). The injection solution of puerarin was freshly prepared by solubilizing in 2% dimethyl sulfoxide (DMSO) before diluted in saline, and it must be applied within 15 min in the case of puerarin precipitate. The injection sites were located in the space between the frontal periosteum and the maxillary suture, and a disposable sterile insulin syringe with a 29G, 0.33*13 mm needle (Kindly Medical Devices, Shanghai, China) was used to minimize tissue damage. On day 14 after installation, all animals were generally anesthetized and then perfused transcardially with 4% paraformaldehyde (pH 7.2~7.6) for fixing, and their maxillae were carefully dissected (Figure 1F) for micro-CT analyses and histological examinations.

The maxillae of the rats were scanned using high-resolution scan mode (Quantum GX2 micro-CT, PerkinElmer, American) at the condition of 90 kV and 88 A, the 72*72mm FOVs was chosen with an effective pixel size of 9.0 m. The digital image was analyzed with Materialises interactive medical image control system V20.0 (MIMICS V20.0) and its accompanying software 3-MATIC. We imported the scanned data from micro-CT into MIMICS to build a 3D model of rat and placed the maxillary bones in the same orientation by calibrating the red reference line on the figure (Figure 1G and H). The width of the mid-palatal suture was obtained by measuring the expanded distance at the level of the mid-coronal plane of the upper first molar (Figure 1I). Meanwhile, the region of interest (ROI) (2.0mm*1.0mm*0.8mm) builded through 3-MATIC included the mid-palatal suture and the bilateral bone, the position of the ROI was shifted to ensure that the intersection of the red dotted lines (Figure 1J) was in the center of the ROI (Figure 1K). The osteogenesis ability of puerarin during RME was investigated by measuring the changes of the bone volume in ROI.

The specimens were decalcified in 10% ethylenediaminetetraacetic acid/phosphate-buffered saline for 8 weeks, then dehydrated through the ethanol series, rendered transparent by xylene, embedded in paraffin wax. Serial sections with a thickness of 5 m were prepared through bilateral maxillary first molars on the coronal plane (Figure 1L). Hematoxylin and eosin (HE) staining and Masson staining for histologic observation were performed following manufacturers instruction.

Sections were dewaxed in xylene and rehydrated in graded ethanol baths, then enzyme-treated with 0.1% (w/v) trypsin at 37 for 10 min to antigen retrieval, blocked with 3% hydrogen peroxidase for 30 min to inhibit endogenous peroxidase activity, preincubated in normal goat serum for 35 min to blocked nonspecific binding. Next, we incubated rabbit polyclonal antibody (Abcam Inc., MA, USA) against BMP2 (working dilution, 1:200) and ALP (working dilution, 1:150) in humid chamber overnight. Subsequently, sections were rinsed in PBS, and the immune reaction was detected according to the 2-step DAB detection kit (Zhongshan Golden Bridge Biotechnology, Beijing, China). All sections were counterstained with hematoxylin for 3 min, followed by running water for 10 min. Under 400 magnification, the average optical density (AOD) value of the immunohistochemical images was analyzed by ImageJ (National Institutes of Health). The process was performed in five randomly selected visual fields per animal, and the average values were calculated by one person repeating at least three times.

All data were obtained from at least three replicates of each experiment. Statistical analyses were performed with GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA, USA) and Microsoft Excel 2020 (Microsoft Corporation, Redmond, WA, USA). A one-way or two-way analysis of variance (ANOVA) was performed to analyze statistical calculations. All the above results were shown as the means standard deviation. All data were considered statistically significant when P < 0.05.

Rat BMSCs were harvested, purified, and cultured through the whole bone marrow wall-adherence method in vitro. Generally, primary BMSCs exhibited colony growth after 3~5 days with a typical longspindlelike and a number of protruding formations (Figure 2A). After 3 or more passages in culture, they tended to be more morphologically heterogeneous (Figure 2B). Following 3 weeks of osteogenic and adipogenic induction, the formation of Alizarin Red mineralized nodules showed the osteogenic potential of BMSCs (Figure 2C), and the Oil Red O lipid droplets indicated their adipogenic potential (Figure 2D). Furthermore, flow cytometry analysis was performed to identify the phenotypic characteristics of these mesenchymal stem cells (MSC). The results showed that BMSCs had high expression of MSC-specific markers CD44 and CD90 but negative for CD34 and CD45 (Figure 2EH). Collectively, the above conclusions indicated that the isolated adherent cells were phenotypically and functionally equivalent to typical MSCs.

Figure 2 Cultivation and characterization of BMSCs. (A) Cell morphology of primary BMSCs. Scale bar: 100 m. (B) Cell morphology of passage 3 BMSCs. Scale bar: 100 m. (C) BMSCs were stained with Alizarin red S after osteogenic differentiation induction. Scale bar: 50 m. (D) BMSCs were stained with oil red O after adipogenic induction. Scale bar: 100 m. (EH) Analysis of BMSCs surface markers expression by flow cytometry. The high expression is on the right side of the central axis. The expression of CD34 and CD45 were negative, while CD90 and CD44 were highly expressed.

The results of the cell proliferation are analyzed by the CCK-8 assay (Figure 3A and B). On day 3, compared with the control group, proliferative capacity of BMSCs at the puerarin concentrations of 106 mol/L was significantly higher (P < 0.01), the 104, 105 and 107 mol/L group also showed a clear increase (P < 0.05), the 108 mol/L group showed a slight increase, but there was no statistically significant (P > 0.05). As time gone by, the trend of the effect of puerarin on proliferation of BMSCs became pronounced. Conversely, the 103 mol/L group markedly inhibited the proliferation of BMSCs (P < 0.0001). Considering the cytotoxic effects, 104, 105, 106 and 107 mol/L groups were chosen for the following assays. The colony formation assay showed that after culturing for 10 days, the cell colonies of the 106 mol/L group were obviously larger and more numerous (P < 0.01) than those of the control group (Figure 3CE).

Figure 3 Effects of puerarin on the proliferation of BMSCs. (A) The growth curves of puerarin-treated groups at different concentration were drawn according to the results of CCK-8 analysis. (B) CCK-8 analysis for the proliferation of BMSCs in various concentration of puerarin on day 3 and 5 (two-way analysis of variance). (C and D) Colony formation assay was performed to test the colony forming capacity of BMSCs. After 10 days, more and larger cell colonies were observed in the experimental group (D) than those in the control group (C). (E) The colony forming efficiency of 106 mol/L puerarin group (one-way analysis of variance). Scale bar: 100 m. The columns represent the means. Error bars represent standard deviations. *P< 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

This research measured the ALP activity level of BMSCs cultured with different concentrations of puerarin (0, 104, 105, 106 and 107 mol/) in two periods (Figure 4A). It was found that compared with the control group, ALP activity level at the various concentrations of puerarin measurably increased to different degrees (P < 0.05) on day 7 and day 14 with the similar trend; clearly, 106 mol/L group obtained the best effects. For the alizarin red S staining assay (Figure 4BE), the 106 mol/L group showed the strongest capacity of matrix mineralization (P < 0.01), more and larger calcified nodules were observed in the 105 and 106 mol/L groups. Based on the above measurements, we conclude that 106 mol/L is the optimal concentration for the proliferation and osteogenesis of BMSCs. Besides, it is worth noting that 105 mol/L has the same positive effect on BMSCs which is only slightly weaker than the optimal concentration, so the concentrations of 105 and 106 mol/L were used in the real-time PCR analysis to enhance the reliability of the experiment, and the concentrations of 106 were used in the Western blot analysis. The mRNA expression levels of the osteogenesis-related genes (ALP, SP7, BSP and OCN) and the protein expression levels of the osteogenesis-related proteins (Col I, -catenin, Runx2, and ALP) were evaluated to assess the osteogenic promotion effect of puerarin. Compared with the control group, the two puerarin-treated group significantly enhanced the expression of the above-mentioned genes on day 7 and day 14 (P < 0.05; Figure 5AD). Furthermore, compared to the control group, the protein expression levels of Col I, -catenin, ALP, Runx2 were showed an upregulated trend on day 14 (Figure 5E). These data indicated that puerarin might play a positive role in the osteogenic differentiation of BMSCs.

Figure 4 Effects of puerarin on ALP activity and mineralized nodule deposition of BMSCs. (A) ALP activity quantification of BMSCs stimulated with puerarin for 7 and 14 days (two-way analysis of variance). (B) Quantitative analysis of Alizarin red S staining of BMSCs stimulated with puerarin for 4 weeks (one-way analysis of variance). (CE) Alizarin red S staining of control group (C), 105 mol/L (D) and 106 mol/L (E) puerarin group. Scale bar: 200 m. The columns represent the means. Error bars represent standard deviations. *P < 0.05, **P < 0.01.

Figure 5 Effect of puerarin on the ALP (A), BSP (B), OCN (C), SP7 (D) expression of BMSCs at 7 and 14 days (two-way analysis of variance). The mRNA expression level of GAPDH was used as internal reference. The columns represent the means. Error bars represent standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

The body weights decreased of the rats in group 2 and group 3 at day 1~5, due to the initial in adaptation to the maxillary expansion appliances, which were significantly different from the steady increase in group 1 (P < 0.05; Figure 6A). However, from day 6, the weight gradually gained among all rats with no significant differences. In addition, there is no significant difference in weight between group 2 and group 3 during the study (P > 0.05). The results showed that the rats recovered quickly from the surgery and were well tolerated to the experimental condition. Micro-CT analysis revealed that compared with group 1, the mid-palatal sutures of rats in groups 2 and 3 were expanded after expansion surgery at day 14 (P < 0.01; Figure 6B), indicating that the RME animal models were successfully established. However, there was no statistical significance (P > 0.05) between group 2 and 3 in terms of the expanded width of the mid-palatal suture. The data of bone volume in the fixed region was measured for evaluating new bone formation in the mid-palatal suture. Compared with group 1, the bone volume in groups 2 and 3 showed significantly reduce (P < 0.01; Figure 6C). Moreover, the bone volume in group 3 was higher than that in group 2 (P < 0.01), implied the application of puerarin had a positive effect on mid-palatal suture osteogenesis.

Figure 6 Animal weight and changes of mid-palatal suture response to the expansion force (n=6). (A) Changes of body weight during experimental period (two-way analysis of variance). (B) Width of the mid-palatal suture (one-way analysis of variance). (C) Bone volume of the mid-palatal suture (one-way analysis of variance). The columns represent the means. Error bars represent standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001.

HE-stained (Figure 7AC) showed the mid-palatal suture in rat of group 1 is made up of a thin band cellular fibrous tissue in the middle and bilateral cartilage with chondrocytes covering the edges of palatal bones. After the mechanical stimulation, the width of the mid-palatal suture is significantly enlarged in response to external force, and two layers of secondary cartilage expand towards the reddish widened fibrous tissue following the same direction as the expansive force, concomitant with the chondrocytes proliferated and differentiated into hypertrophic chondrocytes. Masson staining (Figure 7DF) showed that the cartilage and the collagen fibers in the area of the expanded mid-palatal suture were stained blue, and more hypertrophic chondrocytes were found in group 3 compared with group 2, implying more active endochondral ossification was in progress in group 3.

Figure 7 Histological alterations and immunohistochemistry analyses in the mid-palatal sutures. (AC) HE staining showed the changes of the mid-palatal suture in histological sections. (DF) Masson staining showed the changes of the mid-palatal suture in histological sections. b, maxillary bone; c, cartilage; f, fibrous tissue; black arrow: the direction of stretch force; black triangle: the chondrocyte; black pentagram: the capillary; black dotted line: expanded region. Scale bar: 50 m. (GI) ALP was detected by immunohistochemistry analyses. (JL) BMP2 was detected by immunohistochemistry analyses. Yellow triangle: the positive signal. Scale bar: 20 m. (M and N) Quantification of the expression level of ALP and BMP2 (one-way analysis of variance). The columns represent the means. Error bars represent standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001.

The positive expressions for osteogenic markers ALP (Figure 7GI) and BMP2 (Figure 7JL) were the brownish-yellow stained particles that were mainly observed in the osteoblasts, chondrocytes and fibrous tissue around the mid-palatal suture. Low expression level of ALP and BMP2 was detected in the mid-palatal suture of group 1 accompanied by the absence of few positive cells, while strong signals of them were observed around the expanded suture in group 2 and group 3 which possess the characteristics of big volume and abundant amount of the positive cells, implying active new bone formation in the mid-palatal suture region. Furthermore, compared with group 2, more intense expression was recognized in group 3 according to the higher AOD value (P < 0.01; Figure 7M and N).

Our data suggested that puerarin upregulated the proliferation and osteogenic differentiation of BMSCs. Also, the local administration of puerarin enhanced new bone formation in our RME rat model. RME is a distraction osteogenesis (DO) surgical technique that generates new bone between separated bone segments via the application of continuous and stable force. The procedure is advantageous in terms of low surgical trauma, no requirements for bone grafting, and peripheral soft tissue can be expanded at the same time. Since its first introduction in 1969,35 the DO technique has been widely used to enhance bone regeneration in orthopedic and oral/maxillofacial disorders.36 However, a limitation of RME is that newly formed immature bone tissue requires a prolonged consolidation period to mature, mineralize, and achieve desired distances, which may sometimes trigger oral complications or be often ignored by patients. To ensure its therapeutic efficacy, numerous methods have been investigated, including low-power laser therapy,19 LED (light-emitting diode) phototherapy,37 vitamin supplementation,20 isoquercitrin administration,21 sex steroids,22 curcumin and melatonin,23 and strontium ranelate.38

Based on the data from in vitro and in vivo studies, puerarin was effective in inhibiting bone resorption and improving bone structure. Previous studies showed puerarin decreased receptor activator of nuclear factor -B ligand (RANKL) expression and increased osteoprotegerin (OPG) expression to stimulate osteoblastic proliferation,39 which induced the upregulation of miR1553p,33 BMP2 expression and nitric oxide (NO) synthesis40 to promote cell differentiation and bone formation. Furthermore, puerarin can promote osteogenic differentiation which involved ERK1/2 and p38-MAPK pathway,41 ER, p38 MAPK, and Wnt/-catenin pathways,42 and PI3K/Akt pathway.43 Also, puerarin prevented osteoclastogenesis by inhibiting Akt activation in RAW264.7 cells44 and blocking monocyte chemotactic protein-1 (MCP-1) production.45 In our study, puerarin dose-dependently enhanced osteogenic differentiation and mineralization, and upregulated ALP, SP7, BSP and OCN mRNA levels, suggesting positive stimulatory effects on osteogenic differentiation. Moreover, increased ALP activity after treatment with puerarin was observed in other studies46 in agreement with our findings. ALP has important roles in osteoid formation and mineralization,46 therefore ALP activity is an early osteoblast differentiation marker; BSP is a phosphorylated glycoprotein mainly expressed in mineralized tissue such as bone, and was shown to be the main synthetic product of active osteoblasts;47 OCN is an important component in bone endocrinology and is secreted solely by osteoblasts;48 and SP7 is a critical regulator of osteoblast differentiation and bone formation and induces pre-osteoblast differentiation into fully functional osteoblasts.49 A lot of growth factors, hormones and proteins participate in osteoblast differentiation of MSCs. The Wnt/-catenin signaling pathway is known as one of the important and typical molecular cascades that regulate osteogenic throughout lifespan. Studies have shown that activation of Wnt/-catenin pathway promotes BMSC osteogenic differentiation and osteogenesis.50,51 The protein -catenin is the central target and an essential component of the Wnt/-catenin signaling pathway.52 -catenin also can preserve the stem state of BMSCs through activation of EZH2.53 Collagen type I (Col I), a protein abundantly found in the extracellular matrix, has been broadly shown to promote proliferation, survival, adhesion and osteogenesis in bone marrow MSCs.54 There is evidence that Col I promotes osteogenic differentiation of amniotic membrane-derived mesenchymal stromal cells in basal and induction media.55 It has been reported that growth on a remodeled Col I matrix by MMP13 stimulates osteogenic differentiation and self-healing of bone tissue via an MMP13/ITGA3/RUNX2 positive feedback loop.56 Runx2 is a key transcriptional modulator for osteoblast differentiation that plays a fundamental role in osteoblast maturation and homeostasis;57 it is considered as the master osteoblast-specific transcription factor even if many other factors coordinate bone remodeling. It is crucial in regulating bone differentiation of MSCs and is a key protein for bone formation.58,59 Due to the vital role of MSCs in osteogenic differentiation, we conclude from the above study that puerarin can promote bone regeneration in vitro. Furthermore, the optimal puerarin concentration for BMSC proliferation and osteogenesis was 106 mol/L.

Several animal models, including cynomolgus monkeys, miniature pigs, beagles, rabbits, and rats have been used to study bone regeneration; however, the rat model is widespread due to low costs, wide access to animals, simple model operation, and minimally invasive procedures. The fall-off rate for expansion devices during rat studies is relatively low, which in turn decreases experimental steps and ensures the accuracy of experimental results. This model is similar to clinical RME, using bilateral maxillary first molar as the anchorage teeth, and the mid-palatal suture separated accompanied by the buccal movement of the bilateral maxillary first molar. A previous study reported that the ideal time for RME was the prepubertal or pubertal period, as ongoing growth and development usually generated more stable orthopedic results,22 therefore 6-week-old rats were selected for this study. The experimental period was designed for 14 days. On the one hand, there would be a greater likelihood that the orthodontic force would decay to the point that it would not provide sufficient expansion of the mid-palatal suture after 2 weeks, thus, continuing the experiment may have less effect on the results. On the other hand, studies have shown that 7 and 10 days of RME already allow for an effective expansion of the mid-palatal suture.21,38 As it is medically unethical to systemically administer extrinsic medicines to growing healthy patients, we used local injections to minimize adverse systemic effects and support bone formation at regular time intervals in a particular area of rats in this study. Moreover, assessing direct responses to puerarin in mid-palatal suture bone formation may limit its systemic-administration due to the aforementioned multiple puerarin interactions with various organs or tissues. To the best of our knowledge, ours is the first study to investigate the effects of this locally administered traditional herb and observe no degenerative changes around injection sites.

In recent years, micro-computed tomography has rapidly gained recognition as a standard scanning and analytical tool for bone structures due to its ability to gather key bone structural parameters, and accurately visualize structures in three dimensions.60 In our study, the expanded distance of the mid-palatal suture was wider in groups 2 and 3 when compared with group 1, demonstrating a significant efficacy for RME in rats, in agreement with a previous study.21 However, we observed no significant differences between groups 2 and 3 in terms of the expanded width of the mid-palatal suture, probably because DO can be divided into three temporal phases: a latency period of 510 days, a distraction phase, and a consolidation phase. The mid-palatal suture was in the early stage of distraction at day 14, forming a central fibrous zone as the primitive callus was stretched; this phase was rich in chondrocyte-like cells, fibroblasts, and oval cells which were morphological intermediates between fibroblasts and chondrocytes.61,62 At this time, the puerarin effects on bone remodeling were at initial microscopic stages and were not yet reflected at the macroscopic level. Notably, a significant increase in bone volume was identified in group 3 in the expanded mid-palatal suture, suggesting accelerated new bone deposition and formation in response to puerarin.

Our immunohistochemical analyses showed that BMP2 and ALP expression increased in the expanded mid-palatal suture. BMPs are growth factors which belong to the transforming growth factor-superfamily; they induce endochondral bone formation63 and are involved in bone regeneration during osteoblast differentiation, and their increased expression enhances new bone formation.64 Similarly, ALP is a reliable biochemical marker of bone formation.65 The AOD value showed puerarin increased ALP and BMP2 expression levels during RME, thereby upregulating bone regeneration. Heterotopic ossification (HO) is one of the hot spots of research on post-traumatic complications,66,67 and it has been reported that elevated BMP2 is positively correlated with the occurrence of HO.68 It is noteworthy that though puerarin upregulates BMP2 level when it is used to stimulate osteogenesis in mid-palatal suture, we presume that HO is less likely to occur in the context of safe, physiological, controlled RME. The limitation of the study was that the precise osteogenic mechanism of puerarin towards BMSCs was not fully elucidated as RME is a complex process; the effect of puerarin on osteoblast differentiation is an area worthy of further exploration, therefore future research must elucidate more biological effects of puerarin on bone regeneration.

We demonstrated that puerarin promoted BMSCs proliferation and osteogenic differentiation in vitro and enhanced new bone regeneration in vivo. Our research may serve as an experimental paradigm for the appropriate utilization of puerarin in clinical studies to accelerate bone formation and prevent relapse for RME.

BMSCs, bone marrow-derived mesenchymal stem cells; RME, rapid maxillary expansion; CCK-8, cell-counting kit-8; ALP, Alkaline phosphatase; BSP, bone sialoprotein; OCN, osteocalcin; Micro-CT, micro-computed tomography; HE, hematoxylin and eosin; BMP2, bone morphogenetic protein 2; MSCs, mesenchymal stem cells; -MEM, -minimum essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BCA, bicinchoninic acid; CPC, cetylpyridinium chloride; RT-PCR, reverse transcriptase polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Col I, Collagen I; DMSO, dimethyl sulfoxide; AOD, average optical density; DO, distraction osteogenesis; LED, light-emitting diode; RANKL, receptor activator of nuclear factor -B ligand; OPG, osteoprotegerin; MCP-1, monocyte chemotactic protein-1; NO, nitric oxide.

This work was supported by the Natural Science Foundation of Shandong Province, China (No. ZR2021QH340).

The authors report no conflicts of interest in this work.

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54. Linsley C, Wu B, Tawil B. The effect of fibrinogen, collagen type I, and fibronectin on mesenchymal stem cell growth and differentiation into osteoblasts. Tissue Eng Part A. 2013;19(1112):14161423. doi:10.1089/ten.tea.2012.0523

55. Akhir HM, Teoh PL. Collagen type I promotes osteogenic differentiation of amniotic membrane-derived mesenchymal stromal cells in basal and induction media. Biosci Rep. 2020;40(12). doi:10.1042/BSR20201325

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Effect of Puerarin on New Bone Formation In Vivo | DDDT - Dove Medical Press

image:Autologous intestinal organoids of ulcerative colitis patients are delivered for transplantation by GI endoscopists. view more

Credit: Department of Gastroenterology and Hepatology, TMDU

Tokyo Medical and Dental University (TMDU) research team announced on July 7 that it has succeeded in the worlds first clinical transplantation of a mini organ (also called Organoid) into a patient with Ulcerative Colitis (UC). UC causes inflammation and ulcers (sores) in your digestive tract. It can be debilitating and can sometimes lead to life-threatening complications. UC belongs to a group of conditions called Inflammatory Bowel Disease (IBD). The number of patients is increasing in Japan and in the world is estimated to be about 220,000 and 5,000,000. The common treatment is to suppress inflammation with drugs, but in severe cases, the entire colon may be removed.

Dr. Mamoru Watanabe, vice president and distinguished professor of Tokyo Medical & Dental University said, If our first-in-human research using organoids transplantation yields good results, we expect that the development of organoid medicine for intractable diseases of the digestive tract such as Crohn's disease will progress.

Dr. Ryuichi Okamoto, a professor of the Department of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences said, We embarked on the path of developing new methods for treating intractable diseases. This treatment should establish the efficacy and safety as soon as possible and deliver to the patients. If the team's effort is successful, the mucous membrane may regenerate and lead to a radical cure of UC.

The clinical research started with collecting from the patients vicinity of a healthy colonic mucosa and culturing them for about one month to form spherical organoids with a diameter of about 0.1 to 0.2 mm. On July 5, an organoid was transplanted into the colon of the same patient using a colonoscopy. The patient did well and was discharged July 6.

In previous experiments using mice models, the team confirmed that the cells were cultured in organoids and then transplanted, the mucous membranes regenerated in about a month and the clinical course improved, while the stem cells alone did not transplant because they were not able to culture in vitro.

In this clinical study, since the patient's own cells are used, there is an advantage that transplant rejection does not occur. In addition, since colonoscopy is used for collection and transplantation, there is no need for laparotomy, and the treatment can be performed in a minimally invasive method.

After this transplantation, medical examination will be conducted at the time after 4 weeks and 8 weeks. The patient will be monitored for up to a year to verify safety and efficacy. A further organoids transplantation is to be performed for up to eight patients.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

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The Tokyo Medical and Dental University (TMDU) team succeeded with the world's first Mini Organ transplantation to a patient with Ulcerative Colitis...

Trends Cell Biol. 2010 Dec; 20-206(12-6): 715722.

1Department of Craniofacial Development and MRC Centre for Transplantation, Kings College London; NIHR comprehensive Biomedical Research Centre at Guys and St Thomas NHS Foundation Trust and Kings College London, London, UK

1Department of Craniofacial Development and MRC Centre for Transplantation, Kings College London; NIHR comprehensive Biomedical Research Centre at Guys and St Thomas NHS Foundation Trust and Kings College London, London, UK

2Advanced Centre for Biochemical Engineering, University College London, London, UK

1Department of Craniofacial Development and MRC Centre for Transplantation, Kings College London; NIHR comprehensive Biomedical Research Centre at Guys and St Thomas NHS Foundation Trust and Kings College London, London, UK

1Department of Craniofacial Development and MRC Centre for Transplantation, Kings College London; NIHR comprehensive Biomedical Research Centre at Guys and St Thomas NHS Foundation Trust and Kings College London, London, UK

2Advanced Centre for Biochemical Engineering, University College London, London, UK

Teeth exhibit limited repair in response to damage, and dental pulp stem cells probably provide a source of cells to replace those damaged and to facilitate repair. Stem cells in other parts of the tooth, such as the periodontal ligament and growing roots, play more dynamic roles in tooth function and development. Dental stem cells can be obtained with ease, making them an attractive source of autologous stem cells for use in restoring vital pulp tissue removed because of infection, in regeneration of periodontal ligament lost in periodontal disease, and for generation of complete or partial tooth structures to form biological implants. As dental stem cells share properties with mesenchymal stem cells, there is also considerable interest in their wider potential to treat disorders involving mesenchymal (or indeed non-mesenchymal) cell derivatives, such as in Parkinson's disease.

Teeth are complex organs containing two separate specialized hard tissues, dentine and enamel, which form an integrated attachment complex with bone via a specialized (periodontal) ligament. Embryologically, teeth are ectodermal organs that form from sequential reciprocal interactions between oral epithelial cells (ectoderm) and cranial neural crest derived mesenchymal cells. The epithelial cells give rise to enamel forming ameloblasts, and the mesenchymal cells form all other differentiated cells (e.g., dentine forming odontoblasts, pulp, periodontal ligament) (Box 1). Teeth continue developing postnatally; the outer covering of enamel gradually becomes harder, and root formation, which is essential for tooth function, only starts to occur as part of tooth eruption in children.

Tooth development

Tooth development is traditionally considered a series of stages that reflect key processes (). The first step is induction, in which signals from the epithelium to the mesenchyme initiate the developmental process. As localized proliferation of the dental epithelial cells takes place, the cells form a bud around which the mesenchymal cells condense. Differentiation and localized proliferation of the epithelial cells in the bud leads to the cap stage. It is at this stage that crown morphogenesis is initiated by the epithelial signalling centre, an enamel knot regulating the folding of the epithelium. By the bell stage, the precursors of the specialized tooth cells, ameloblasts, coordinate enamel deposition, and odontoblasts, which produce dentine, are formed. Tooth eruption involves the coordination of bone resorption and root development, and occurs postnatally.

Throughout tooth development, signals are exchanged between epithelial and mesenchymal cells to coordinate each process. The key initial signals occur at induction (epithelium) and bud formation (mesenchyme). Once the mesenchymal cells receive signals from the epithelium, the mesenchyme sends reciprocal signals back to the epithelium. Strategies for biological replacement teeth aim to utilize these first signal exchanges by identifying either epithelial cells that can induce a naive mesenchyme or mesenchymal cells that can induce a naive epithelium to stimulate tooth development.

Repair, restoration and replacement of teeth is unique among clinical treatments because of the huge numbers of patients involved. Paradoxically, although teeth are nonessential for life and thus not considered a major target for regenerative medicine research, in comparison with neural or cardiac diseases, for example, this very fact makes teeth ideal for testing new cell-based treatments. Because the patients are not usually ill, if anything goes wrong it is far less life threatening, and the accessibility of teeth means that treatment does not require major surgery. Added to this is the existence of highly proliferative stem cell populations in teeth, which can be easily obtained from naturally lost or surgically removed teeth. These stem cells can be used for tooth repair, restoration and regeneration and, significantly, non-dental uses, such as developing stem cell-based therapies for major life-threatening diseases. An important but often overlooked advantage of teeth as a source of stem cells is that postnatal root formation (a rich source of dental stem cells) is a developmental process, and thus cells involved in root formation are more embryonic-like than other sources of dental stem cells. The humble tooth clearly has a very important role to play in future developments in regenerative medicine.

In this review, we outline the important biological properties of dental stem cells and illustrate examples of research showing the rapid progress being made in using these cells for tooth repair. We also highlight the major obstacles that need to be overcome before any form of usable, cell-based tooth replacement becomes available to practising dentists.

Several populations of cells with stem cell properties have been isolated from different parts of the tooth. These include cells from the pulp of both exfoliated (children's) and adult teeth, from the periodontal ligament that links the tooth root with the bone, from the tips of developing roots and from the tissue (dental follicle) that surrounds the unerupted tooth. All these cells probably share a common lineage of being derived from neural crest cells and all have generic mesenchymal stem cell-like properties, including expression of marker genes and differentiation into mesenchymal cell lineages (osteoblasts, chondrocytes and adipocytes) in vitro and, to some extent, in vivo. The different cell populations do, however, differ in certain aspects of their growth rate in culture, marker gene expression and cell differentiation, although the extent to which these differences can be attributed to tissue of origin, function or culture conditions remains unclear.

The possibility that tooth pulp might contain mesenchymal stem cells was first suggested by the observation that severe tooth damage that penetrates both enamel and dentine and into the pulp stimulates a limited natural repair process, by which new odontoblasts are formed, which produce new dentine to repair the lesion [1,2]. Putative stem cells from the tooth pulp and several other dental tissues have now been identified (Box 2) [38].

Human third molar as a source of dental stem cells

Human third molars (wisdom teeth) start their development postnatally, during childhood (ages of 56 years) and begin their calcification process from the age of 710 years. By the age of 1825 years, the roots of the third molars have completed their development. These teeth are most commonly extracted and discarded in the dental clinic, but because they are still undergoing root development, they provide an excellent source of dental stem cells including DPSC, PDL cells and SCAP cells ().

The first stem cells isolated from adult human dental pulp were termed dental pulp stem cells (DPSCs) [3]. They were isolated from permanent third molars, and exhibited high proliferation and high frequency of colony formation that produced sporadic, but densely calcified nodules. Additionally, in vivo transplantation into immunocompromised mice demonstrated the ability of DPSCs to generate functional dental tissue in the form of dentine/pulp-like complexes [4]. Further characterization revealed that DPSCs were also capable of differentiating into other mesenchymal cell derivatives in vitro such as odontoblasts, adipoctyes, chondrocytes and osteoblasts [912]. DPSCs differentiate into functionally active neurons, and implanted DPSCs induce endogenous axon guidance, suggesting their potential as cellular therapy for neuronal disorders [1315].

Stem cells isolated from the pulp of human exfoliated deciduous (children's milk) teeth (SHED) have the capacity to induce bone formation, generate dentine and differentiate into other non-dental mesenchymal cell derivatives in vitro[1620]. In contrast to DPSCs, SHED exhibit higher proliferation rates [21], increased population doublings, osteoinductive capacity in vivo and an ability to form sphere-like clusters [16]. SHED seeded onto tooth slices/scaffolds and implanted subcutaneously into immunodeficient mice differentiated into functional odontoblasts capable of generating tubular dentine and angiogenic endothelial cells [18].

Studies using SHED as a tool in dental pulp tissue engineering in vivo, where pulp removed because of infection is replaced with stem cells, have revealed that the tissue formed has architecture and cellularity closely resembling that of dental pulp, a tissue important for tooth vitality [19]. Another interesting clinical application has been suggested by investigations of the therapeutic efficacy of SHED in alleviating Parkinson's disease (PD) [20]. Transplantation of SHED spheres into the striatum of parkinsonian rats partially improved the apomorphine evoked rotation of behavioural disorders. The results of this study indicate that SHED might be a useful source of postnatal stem cells for PD treatment. SHED are isolated from children's exfoliated teeth, however, so autologous stem cell therapy for a disease such as PD would require that these cells be stored from childhood. DPSCs, which are obtained from adult tooth pulp, might well have similar properties, however, and collection and expansion of these autologous cells would simply require removal of a tooth from the patient.

SHED and other dental stem cells are derived from cranial neural crest ectomesenchyme, and so developmentally and functionally would appear identical, but studies have shown that they do differ and have different gene expression profiles. SHED have significantly higher proliferation rates compared with DPSC and bone marrow-derived mesenchymal stem cells [21]. Comparison of the gene expression profiles showed 4386 genes that are differentially expressed between DPSC and SHED by two-fold or more. Higher expression in SHED was observed for genes that participate in pathways related to cell proliferation and extracellular matrix formation, including several growth factors such as fibroblast growth factor and transforming growth factor (TGF)- [21]. TGF- in particular is important, because it is released after damage to dentine and might act to mobilize pulp stem cells to differentiate into odontoblasts [1,22].

DPSC are highly proliferative and retain their stem cell characteristics after prolonged culture [23]. They could therefore be used as a generic allogeneic source of mesenchymal stem cells. Their use as autologous cells, however, is currently restricted to children who have not yet lost all their deciduous teeth. Commercial banking of these cells is thus becoming widespread to enable them to be used once the child becomes an adult. Limited studies have shown that frozen SHED cells do maintain their properties after cryopreservation for 2 years [24], but one caveat is that the effects of long-term storage (10 years, plus) have not yet been assessed. Because children naturally lose 20 deciduous teeth, there are multiple opportunities to bank these cells, unlike cord blood, for example.

The periodontal ligament (PDL) is a fibrous connective tissue that contains specialized cells located between the bone-like cementum and the inner wall of the alveolar bone socket that acts as a shock absorber during mastication (Box 2). The PDL has long been recognized to contain a population of progenitor cells [25] and recently, several studies [26] identified a population of stem cells from human periodontal ligament (PDLSC) capable of differentiating along mesenchymal cell lineages to produce cementoblast-like cells, adipocytes and connective tissue rich in collagen I in vitro and in vivo[2629].

The periodontal ligament is under constant strain from the forces of mastication, and thus PDLSC are likely to play an endogenous role in maintaining PDL cell numbers. This might explain why they are better than other dental stem cell populations at forming PDL-like structures [17].

A unique population of dental stem cells known as stem cells from the root apical papilla (SCAP) is located at the tips of growing tooth roots (Box 2). The apical papilla tissue is only present during root development before the tooth erupts into the oral cavity [30]. SCAP have the capacity to differentiate into odontoblasts and adipocytes [27]. These cells are CD24+ but expression is downregulated upon odontogenic differentiation in vitro coincident with alkaline phosphatase upregulation. SCAP cells exhibit higher rates of proliferation in vitro than do DPSC [27]. By co-transplanting SCAP cells (to form a root) and PDLSC (to form a periodontal ligament) into tooth sockets of mini pigs, dentine and periodontal ligament was formed. These findings suggest that this population of cells, together with PDLSC, could be used to create a biological root that could be used in a similar way as a metal implant, by capping with an artificial dental crown. Most human tissues from early in their development are not clinically available for stem cell isolation; however, because roots develop postnatally, the root apical papilla is accessible in dental clinical practice from extracted wisdom teeth. Thus, a very active source of stem cells with embryonic-like properties (i.e., in the process of development) can be readily obtained. Further experiments on the properties of these cells obtained from human teeth following expansion in culture are needed.

The dental follicle is a loose ectomesenchyme-derived connective tissue sac surrounding the enamel organ and the dental papilla of the developing tooth germ before eruption [31]. It is believed to contain progenitors for cementoblasts, PDL and osteoblasts. Dental follicle cells (DFC) form the PDL by differentiating into PDL fibroblasts that secrete collagen and interact with fibres on the surfaces of adjacent bone and cementum. DFC can form cementoblast-like cells after transplantation into SCID mice [32,33].

Dental follicle progenitor cells isolated from human third molars are characterized by their rapid attachment in culture, expression of the putative stem cell markers Nestin and Notch-1, and ability to form compact calcified nodules in vitro[34]. When DFC were transplanted into immunocompromised mice, however, there was little indication of cementum or bone formation [34]. DFC, in common with SCAP, represent cells from a developing tissue and might thus exhibit a greater plasticity than other dental stem cells. However, also similar to SCAP, further research needs to be carried out on the properties and potential uses of these cells.

There are several areas of research for which dental stem cells are currently considered to offer potential for tissue regeneration. These include the obvious uses of cells to repair damaged tooth tissues such as dentine, periodontal ligament and dental pulp [1619,3236]. Even enamel tissue engineering has been suggested [37], as well as the use of dental stem cells as sources of cells to facilitate repair of non-dental tissues such as bone and nerves [1215,20,38,39].

The periodontium is a set of specialized tissues that surround and support the teeth to maintain them in the jaw. Periodontitis is an inflammatory disease that affects the periodontium and results in irreversible loss of connective tissue attachment and the supporting alveolar bone. The challenge for cell-based replacement of a functional periodontium is therefore to form new ligament and bone, and to ensure that the appropriate connections are made between these tissues, as well as between the bone and tooth root. This is not a trivial undertaking, as these are very different tissues that form in an ordered manner (spatially and temporally) during tooth development. One aim of current research is to use different populations of dental stem cells to replicate the key events in periodontal development both temporally and spatially, so that healing can occur in a sequential manner to regenerate the periodontium [34].

A conceptually simpler approach to periodontal regeneration methods involves engineered cell sheets to facilitate human periodontal ligament (HPDL) cell transplantation [35]. Periodontal ligament cells isolated from a human third molar tooth were cultured on poly(N-isopropylacryl-amide) (PIPAAm)-grafted dishes that induce spontaneous detachment of the cells as viable cell sheets upon low temperature treatment. HPDL cells sheets were implanted into athymic rats that had the periodontium and cementum removed from their first molars. Fibril anchoring resembling native periodontal ligament fibres, together with an acellular cementum-like layer, was observed, indicating that this technique could be applicable to future periodontal regeneration. Although promising, this approach does not take into account any replacement of bone that might be required.

The outstanding issue with these approaches is the extent to which any reconstituted periodontium can maintain integrity and function during mastication over long periods of time. Current treatments for severe periodontitis are poor, however, and thus, despite their flaws, any new dental stem cell-based treatments are likely to be the subject of intensive clinical research in the near future.

Dental pulp needs to be removed when it becomes infected, and this is particularly problematic for root pulp that requires endodontic (root canal) treatment. The restoration of tooth pulp is thus a much sought after goal in dentistry because the current practice of replacing infected pulp with inorganic materials (cements) results in a devitalized (dead) tooth. A recent study demonstrated de novo regeneration of dental pulp in emptied root canal space using dental stem cells [36]. DPSC and SCAP isolated from the human third molars were seeded onto a poly-D,L-lactide/glycolide scaffold and inserted into the canal space of root fragments, followed by subcutaneous transplantation into SCID mice. Subsequent histological analysis of the tooth fragments 34 months after surgery indicated that the root canal space was completely filled with pulp-like tissue with well established vascularization. Moreover, a continuous layer of mineralized tissue resembling dentine was deposited on the existing dentinal walls of the canal [36]. Recent studies using genetically marked cells in mice have suggested that adding stem cells makes little difference to the extent to which an empty pulp cavity regenerates because the majority of cells are provided by the vasculature (Sharpe P.T, unpublished data). Stem cell pulp restoration might therefore not be a problem of providing exogenous stem cells but one of surgically ensuring that an adequate blood supply is maintained after pulp removal.

The current state of the art in tooth replacement is a dental implant that involves screwing a threaded metal rod into a predrilled hole in the bone, which is then capped with a plastic or ceramic crown. Implant use requires a minimum amount of bone to be present. Because these implants attach directly to the bone without the PDL shock absorber, the forces of mastication are transmitted directly to the bone, which is one reason implants can fail. In cases where there is insufficient bone for implants, such as tooth loss as a consequence of the bone loss that occurs in postmenopausal osteoporosis, implants have to be preceded by bone grafts. The ultimate goal in dentistry is to have a method to biologically replace lost teeth; in essence, a cell-based implant rather than a metal one. The minimum requirement for a biological replacement is to form the essential components required for a functional tooth, including roots, periodontal ligament, and nerve and blood supplies. Paradoxically, the visible part of the tooth, the crown, is less important because, although essential for function, synthetic tooth crowns function well, and can be perfectly matched for size, shape and colour. The challenge, therefore, for biological tooth replacement is ultimately one of forming a biological root.

Currently, the major challenges in whole tooth regeneration are to identify non-embryonic sources of cells with the same properties as tooth germ cells and to develop culture systems that can expand cells that retain tooth forming potential (). This is even more challenging when considering the fact that tooth development requires two cell types, epithelial and mesenchymal [4042].

Tooth formation in vitro from combinations of mouse epithelial and mesenchymal cells. The epithelium (red arrow) and mesenchyme (black arrow) are separated from pre-bud stage tooth primordia,and cells dissociated in single cell populations. (a) The cells are recombined (as shown in this figure) and grown in vitro for 6 days. (b) Gross appearance after 9 days in culture with higher magnification of a tooth primordium. (c) Sections of tooth primordia from (a), showing development to the bell stage.

The induction of odontogenic potential lies in the dental epithelium [4345]. Dental epithelium from pre-bud stages can induce tooth formation when combined with nonodontogenic mesenchyme as long as the mesenchymal cells have stem cell-like properties in common with neural crest cells [46]. After epithelial induction of the mesenchyme, this becomes the inductive tissue and reciprocates inductive signals back to the now noninductive epithelium. Tooth regeneration can thus be approached in one of two ways; identification of either epithelial or mesenchymal cells than can induce tooth formation in the other cell type.

No sources of epithelial cells capable of inducing odontogenesis have been identified to date, other than the endogenous dental epithelium of early stage embryos. The main limitation for identifying sources of epithelial cells that can be grown in culture and form teeth after association with inducing mesenchymal cells is that these epithelial cells retain an immature state.

The epithelial rests of Malassez (ERM) are a group of cells that remain during root formation; thus, these cells are present in adult teeth and can be isolated and cultured [5155]. When ERM cells are maintained in vitro on feeder layers, they can be induced to form enamel-like tissues following recombination with primary (uncultured) dental pulp cells [55].

Oral mucosa epithelial cells from embryos and adults have been used in recombination experiments and shown to give rise to complex dental structures, but not whole functional teeth, when combined with embryonic dental mesenchyme [56,57]. Some evidence of tooth formation was seen when oral epithelial lines established from p-53-deficient mouse embryos at embryonic day (E)18 were combined with fetal E16.5 molar mesenchymal tissues and transplanted for 23 weeks [56]. Postnatal oral mucosal epithelium might also offer some potential as a replacement for embryonic dental epithelium, because cells isolated from young animals, grown as cell sheets and re-associated with dental mesenchyme from E12.5 embryos, can give rise to tooth-like structures [57].

There are sources of epithelial cells that can contribute to tooth formation following culture, suggesting that exogenously adding factors to these cells could make them inducible. Such factors, include signalling proteins of the fibroblast growth factor bone morphogenetic protein and Wnt families, but the issue of reproducing the temporal, spatial and quantitative delivery of these, as seen in vivo, is daunting. Identification of key intracellular factors (e.g., kinases, transcription factors.) is likely to be a more fruitful direction because these are more easily manipulated.

The ability of non-dental mesenchymal cell sources to respond to odontogenic epithelial signals following in vitro expansion was demonstrated when it was shown that expanded adult bone marrow stromal cells would form teeth in vitro when combined with inductive embryonic oral epithelium [46]. This study also showed that embryonic tooth primordia could develop into complete teeth, following transplantation into the adult oral cavity. Such transplants, when left for sufficient time, will form roots and erupt [47,58]. The issues with producing inductive epithelium in vitro illustrated in the above section suggest that the alternative approach of identifying mesenchymal cells with inductive capacity might be more fruitful. The cells that have this capacity in vivo are the early embryonic neural crest-derived ectomesenchyme cells that have already received the first inductive signals from the dental epithelium (Box 1). Bone marrow mesenchymal cells, although able to respond to odontogenic signals from the epithelium, are only able to induce tooth formation after receiving these epithelial signals. Such priming of bone marrow mesenchymal cells by inducing factors or embryonic dental epithelium is possible, but in reality too laborious and difficult to be of any clinical value.

If ectomesenchyme cells have odontogenic-inducing capacity, can this be maintained in vitro? Embryonic tooth primordia mesenchymal cells from mice have been shown to retain their potential to respond to odontogenic signals following in vitro culture after immortalization but it is uncertain if cells with inducing capacity can retain this following culture (Jung H.-S., personal communication). Similarly, equivalent cells from human embryos have been isolated and shown to form teeth in re-association experiments (Volponi A.A. and Sharpe P.T., unpublished data).

Adult dental pulp mesenchymal stem cells are an obvious source of cells to replace embryonic ectomesenchyme because they are derived from cranial neural crest and are dental cells. Indeed, these cells retain expression of many genes expressed in neural crest, in addition to a number of stem cell marker genes. However, it has yet to be shown that adult dental pulp mesenchymal stem cells retain any odontogenic inductive or responsive capacity. One interesting direction is to identify the factors expressed by ectomesenchyme cells (embryonic dental mesenchyme) that render them capable of forming teeth that are not expressed by adult dental stem cells. Approaches similar to those developed for producing induced pluripotent stem cells can be used to convert adult dental stem cells into ectomesenchyme cells that can form teeth.

Functional teeth can be experimentally bioengineered in mice by re-association of dissociated tooth cells [4851]. These experiments actually demonstrate the ability of dissociated cells to re-aggregate, however, rather than the bioengineering of whole teeth. The cells used are obtained from embryonic tooth primordia, many of which are required to produce one tooth. When tooth germs are dissociated and allowed to re-associate in an extracellular matrix (scaffold), they sort out and re-aggregate to reform the tooth germs [48,51]. The re-aggregation produces multiple small toothlets, whose shape bears no resemblance to that of the scaffold used (). Similarly, the tooth germ epithelial and mesenchymal cell components can be physically separated, the cells dissociated and recombined, whereupon they sort and re-aggregate to reform the tooth germ [48,51]. In this case, 5104 cells dissociated from multiple tooth germs are required to generate a single new tooth germ [48]. The large cell numbers required necessitate in vitro expansion of epithelial and mesenchymal cells that will retain their odontogenic properties.

Diagrammatic representation of the generation of biological replacement teeth. Suitable sources of epithelial and mesenchymal cells are expanded in culture to generate sufficient cells. The two cell populations are combined to bring the epithelial and mesenchymal cells into direct contact, mimicking the in vivo arrangement. Interaction between these cell types leads to formation of an early stage tooth primordium, equivalent to a tooth bud or cap, around which the mesenchyme cells condense (dark blue dots) (see also Box 1). The tooth primordium is surgically transplanted into the mouth and left to develop.

Despite some progress, there remain major obstacles to formulating safe, simple and reproducible cell-based approaches for tooth repair and regeneration that could be used on patients. It is clear that there is both a clinical need for such treatments and a vast patient resource. Dental stem cells have many advantages, and results to date suggest that teeth are a viable source of adult mesenchymal stem cells for a wide range of clinical applications. Ultimately, the use of these dental stem cells over other sources of mesenchymal stem cells for therapeutic use will not only depend on ease of use and accessibility, but also on the efficiency and quality of repair in relation to cost. Dental pulp cells grow well in culture and, unusually, the proportion of cells with stem cell properties appears to increase with passage. The molecular basis of this phenomenon needs to be investigated because it might provide a paradigm for increasing stem cell numbers in cultures of other cell types.

For whole tooth regeneration, there remain many major issues that will take considerable time to resolve. Most immediate is the identification of epithelial and mesenchymal cell populations that can be maintained and expanded in culture to provide the large numbers needed to make a tooth. Related to this is the issue of whether the cells will need to be autologous (expensive, but safe) or allogeneic (cheaper, but with possible rejection problems). Finally, an additional fundamental issue that needs to be considered is that human tooth development is a much slower process than in mice. Human tooth embryogenesis is approximately eight times slower, and postnatal development lasts several years. Thus, whereas growth, implantation and eruption of bioengineered mouse teeth might take a few weeks, the equivalent time to create a functional human tooth might be many months or even years. Research thus needs to be done to investigate ways of possibly accelerating human tooth development.

Diagrammatic representation of tooth development.

Photograph and diagram of a human third molar tooth following extraction. A hemisected tooth showing the internal tissues is shown on the right. Because the tooth was in the process of erupting, root growth is incomplete, and the apical papilla is visible. A diagrammatic representation of this tooth is shown on the left.

Research in the author's laboratory is supported by the MRC, Wellcome Trust and the Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guys. YP is supported by the UK Stem Cell Foundation. We are grateful to Han-Sung Jung for his permission to cite unpublished work and to Andrea Mantesso for comments on the manuscript.

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Stem cell-based biological tooth repair and regeneration - PMC

Stem cells are found throughout the body

The term stem cell may conjure up thoughts of some rare type of cell that can only be found in very specific locations. The opposite is really true. Stem cells are pretty ubiquitous in the body, appearing in many different organs and tissues including the brain, blood, bone marrow, muscle, skin, heart, and liver tissues. In these areas, they lie dormant until needed to regenerate lost or damaged tissue. They can do this because of their unique abilities to become many different types of cells and to replicate rapidly. (You can read more about the unique traits of stem cells here.)

As stem cells can be found throughout the body, it may seem as though they can easily be harvested for transplantation and regenerative medicine, but its the volume and the age of stem cells that are the main driving factors in where they are collected. Volume is important because there is no conclusive way to spur replication, so as of now, what you get is what you get. Age is also a factor because as stem cells age, they lose their ability to reproduce and differentiate into other cell types, they may become contaminated with a latent virus or affected by a disease, or they may have been exposed to toxins and have undergone mutation. They are also more likely to cause an autoimmune response, which is when your body attacks itself.

Found in large numbers during gestation, embryonic stem cells are by far the youngest stem cells and have the unique ability to become any type of cell in the body. There is a lot of controversy and ethical considerations concerning the embryonic stem cell. Thankfully, we can also acquire stem cells that form just a little bit later down the road and can be found in the umbilical cord blood and cord tissue. These stem cells are more limited in the types of cells they can become, something known as being tissue-specific, and they stay with us throughout our lives, which is why they are referred to as adult stem cells.

Extracting the cord blood is painless and risk-free

The second to youngest stem cells are still called adult stem cells even though they can be collected at the time of delivery. Cord blood stem cells were discovered in 1978, and after the first cord blood transplant in 1988, the cord blood banking industry was formed. Cord tissue stem cells were discovered in the late '90s, and this discovery spurred cord tissue banking for many cord blood banks. Cord blood and cord tissue stem cells have the special quality of being the purest and youngest tissue-specific stem cells you can collect and function more quickly and effectively than adult stem cells from other sources. They are also easily collected at the time of birth. (Dive into the differences between cord blood and cord tissue.)

Placental tissue can also be easily collected at the time of birth

The placenta and other amniotic tissues are also a rich source of the same type of stem cells found in cord tissue, and as with cord tissue, they can be easily collected at the time of birth. Despite these similarities to cord tissue, the major difference is that the placental tissue has a mix of the baby's and the mother's stem cells, and in order for these to be properly utilized in a stem cell treatment, they need to be separated. As the mother's stem cells often replicate more quickly than the fetal stem cells, placental stem cells are more likely to be preserved for the exclusive use of the mother.

A bone marrow draw requires the use of anesthesia and usually takes 20 days to fully recover

There are also areas where stem cells can be collected later in life. Bone marrow is rich in the blood-forming stem cells like those found in cord blood. To collect bone marrow stem cells, a needle is inserted into the soft center of the bone and requires the donor to undergo anesthesia. While it would be best to obtain bone marrow stem cells right from the person who needs them, the bone marrow procedure could be too much for the patient or the patients bone marrow could be too diseased. If this is the case, a matching donor must be found. A matching donor may be hard to obtain, and unfortunately, all non-related stem cell transplants come with a high degree of risk for an autoimmune response like graft-versus-host disease. (Read more about how cord blood and bone marrow compare.)

The procedure for capturing peripheral blood stem cells is like a long blood donation

As noted earlier, blood contains stem cells, just not too many. To gather a large number of stem cells from blood, the blood- and immune systemforming stem cells in bone marrow need to be coaxed out and collected. The non-surgical procedure is called apheresis. It begins days before the stem cell transplant with injections to get the stem cells in the bone marrow to enter the blood stream. The blood is then drawn from one arm and filtered through a machine to catch the stem cells from the peripheral blood. The rest of the blood is returned to the donor's other arm through another needle. Unfortunately, these stem cells have proven less effective compared with cord blood and bone marrow stem cells. In a meta-analysis of 9 trials totaling 1,111 patients, researchers found time to engraftment was slower and the frequency of graft-versus-host disease was greater in transplantations using peripheral blood than bone marrow. Researchers believe this has something to do with the removal of the stem cells from their bone marrow environment although the exact reason is not clear.

Obtaining adipose-derived stem cells requires a liposuction-like procedure that may itself take weeks of healing

Adipose stem cells are collected from fat tissue by way of an invasive liposuction-like procedure and are not the same as those found in cord blood or bone marrow. This means they are not used to treat the blood cancers and diseases that cord blood or bone marrow treat. The adipose tissue is more abundant in the same kind of stem cells found in cord tissue. These stem cells show promise for heart and kidney disease, ALS, wound healing and some autoimmune diseases.

Because stem cells taken from the patient and re-infused within 48 hours fall under different guidelines than stem cells collected through other methods, a market has sprung up for adipose stem cells, with many clinics touting their benefits in treatments that go well beyond current research. There is an inherit risk in using stem cell therapies neither approved by the FDA nor a part of an FDA-approved clinical trial.

Dental pulp can be collected as a child loses his or her baby teeth

A relatively new discovery is the stem cells in dental pulp. Teeth contain the same type of stem cells as adipose tissue and umbilical cord tissue, so once again, they are not used to treat the blood cancers and diseases that cord blood or bone marrow treat. Like cord tissue, however, dental pulp could hold future potential for heart and kidney disease, ALS, wound healing and some autoimmune diseases, and collection could involve simply saving all the teeth that fall out as the child grows.

Its too early to know if dental pulp will prove to be an quality source of these types of stem cells, and the volume of stem cells is known to be small. As cord tissue stem cells are plentiful and have been being used in clinical trials for the past 20 years, comparing its progress to that of dental pulp is akin to comparing a great 9-year-old tee ball player to the upcoming major league superstar. Dental pulp as a source of stem cells is a new idea, and maybe it has potential, but it's still has to undergo years of trials, data collection and analysis before it will be a proven science.

Stem cells can be found throughout the body, but the volume of the stem cells, the age and purity of the stem cells, the ease of collecting, the degree to which they have proven successful in transplants and clinical data and any ethical considerations are all major factors as to which is the preferred source. These are all factors where cord blood and cord tissue prove superior.

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Where Stem Cells Are Found, & the Difference That Makes | Cryo-Cell

Research Article

24 Jun 2022

Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying MicroRNA-181c-5p Promote BMP2-Induced Repair of Cartilage Injury through Inhibition of SMAD7 Expression

Qiang Zhang|Le Cao|...|Yongping Wu

The therapy role of mesenchymal stem cell- (MSC-) derived extracellular vesicles (EVs) in cartilage regeneration has been well studied. Herein, we tried to analyze the role of human umbilical cord MSC- (hUCMSC-) EVs carrying microRNA- (miR-) 181c-5p in repair of cartilage injury. After successful isolation of hUCMSCs, the multidirectional differentiation abilities were analyzed. Then, the EVs were isolated and identified. After coculture of PKH26-labled EVs with bone marrow MSCs (BMSCs), the biological behaviors of which were detected. The relationship between the predicted early posttraumatic osteoarthritis-associated miRNA, miR-181c-5p, and SMAD7 was verified. Gain- and loss-of functions were performed for investing the role of miR-181c-5p and SMAD7 in BMP-induced chondrogenesis in vitro and in vivo. hUCMSC-EVs could be internalized by BMSCs and promote the proliferative, migratory, and chondrogenic differentiation potentials of BMSCs. Additionally, miR-181c-5p could target and inhibit SMAD7 expression to promote the bone morphogenic protein 2- (BMP2-) induced proliferative, migratory, and chondrogenic differentiation potentials of BMSCs. Also, overexpression of SMAD7 inhibited the repairing effect of BMP2, and overexpression of BMP2 and miR-181c-5p further promoted the repair of cartilage injury in vivo. Our present study highlighted the repairing effect of hUCMSC-EVs carrying miR-181c-5p on cartilage injury.

Research Article

22 Jun 2022

Human Placental Mesenchymal Stem Cells for the Treatment of ARDS in Rat

Zurab Kakabadze|Nicholas Kipshidze|...|David Chakhunashvili

The acute respiratory distress syndrome (ARDS) is one of the main causes of high mortality in patients with coronavirus (COVID-19). In recent years, due to the coronavirus pandemic, the number of patients with ARDS has increased significantly. Unfortunately, until now, there are no effective treatments for ARDS caused by COVID-19. Many drugs are either ineffective or have a low effect. Currently, there have been reports of efficient use of mesenchymal stem cells (MSCs) for the treatment of ARDS caused by COVID-19. We investigated the influence of freeze-dried human placenta-derived mesenchymal stem cells (HPMSCs) in ARDS rat model. All animals have received intratracheal injection of 6mg/kg of lipopolysaccharide (LPS). The rats were randomly divided into five groups: I: LPS, II: LPS+dexamethasone, III: LPS+HPMSCs, IV: HPMSC, and V: saline. ARDS observation time was short-term and amounted to 168 hours. The study has shown that HPMSCs are able to migrate and attach to damaged lung tissue, contributing to the resolution of pathology, restoration of function, and tissue repair in the alveolar space. Studies have also shown that the administration of HPMSCs in animals with ARDS model significantly reduced the levels of key cytokines such as IL-1, IL-6, and TNF-. Freeze-dried placental stem cell is a very promising biomaterial for the treatment of ARDS. The human placenta can be easily obtained because it is considered as a medical waste. At the same time, a huge number of MSCs can be obtained from the placental tissue, and there is no ethical controversy around their use. The freeze-dried MSCs from human placental tissue can be stored sterile at room temperature for a long time before use.

Research Article

20 Jun 2022

GMP Compliant Production of a Cryopreserved Adipose-Derived Stromal Cell Product for Feasible and Allogeneic Clinical Use

Mandana Haack-Srensen|Ellen Mnsted Johansen|...|Annette Ekblond

The emerging field of advanced therapy medicinal products (ATMP) holds promise of treating a variety of diseases. Adipose-derived stromal cells (ASCs) are currently being marketed or tested as cell-based therapies in numerous clinical trials. To ensure safety and efficacy of treatments, high-quality products must be manufactured. A good manufacturing practice (GMP) compliant and consistent manufacturing process including validated quality control methods is critical. Product design and formulation are equally important to ensure clinical feasibility. Here, we present a GMP-compliant, xeno-free, and semiautomated manufacturing process and quality controls, used for large-scale production of a cryopreserved off-the-shelf ASC product and tested in several phase I and II allogeneic clinical applications.

Research Article

18 Jun 2022

Human Umbilical Cord Mesenchymal Stem Cells Encapsulated with Pluronic F-127 Enhance the Regeneration and Angiogenesis of Thin Endometrium in Rat via Local IL-1 Stimulation

Shuling Zhou|Yu Lei|...|Jiang Gu

Thin endometrium (< 7mm) could cause low clinical pregnancy, reduced live birth, increased spontaneous abortion, and decreased birth weight. However, the treatments for thin endometrium have not been well developed. In this study, we aim to determine the role of Pluronic F-127 (PF-127) encapsulation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in the regeneration of thin endometrium and its underlying mechanism. Thin endometrium rat model was created by infusion of 95% ethanol. Thin endometrium modeled rat uterus were treated with saline, hUC-MSCs, PF-127, or hUC-MSCs plus PF-127 separately. Regenerated rat uterus was measured for gene expression levels of angiogenesis factors and histological morphology. Angiogenesis capacity of interleukin-1 beta (IL-1)-primed hUC-MSCs was monitored via quantitative polymerase chain reaction (q-PCR), Luminex assay, and tube formation assay. Decreased endometrium thickness and gland number and increased inflammatory factor IL-1 were achieved in the thin endometrium rat model. Embedding of hUC-MSCs with PF-127 could prolong the hUC-MSCs retaining, which could further enhance endometrium thickness and gland number in the thin endometrium rat model via increasing angiogenesis capacity. Conditional medium derived from IL-1-primed hUC-MSCs increased the concentration of angiogenesis factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGF), and hepatocyte growth factor (HGF)). Improvement in the thickness, number of glands, and newly generated blood vessels could be achieved by uterus endometrium treatment with PF-127 and hUC-MSCs transplantation. Local IL-1 stimulation-primed hUC-MSCs promoted the release of angiogenesis factors and may play a vital role on thin endometrium regeneration.

Review Article

18 Jun 2022

Role of Primary Cilia in Skeletal Disorders

Xinhua Li|Song Guo|...|Ziqing Li

Primary cilia are highly conserved microtubule-based organelles that project from the cell surface into the extracellular environment and play important roles in mechanosensation, mechanotransduction, polarity maintenance, and cell behaviors during organ development and pathological changes. Intraflagellar transport (IFT) proteins are essential for cilium formation and function. The skeletal system consists of bones and connective tissue, including cartilage, tendons, and ligaments, providing support, stability, and movement to the body. Great progress has been achieved in primary cilia and skeletal disorders in recent decades. Increasing evidence suggests that cells with cilium defects in the skeletal system can cause numerous human diseases. Moreover, specific deletion of ciliary proteins in skeletal tissues with different Cre mice resulted in diverse malformations, suggesting that primary cilia are involved in the development of skeletal diseases. In addition, the intact of primary cilium is essential to osteogenic/chondrogenic induction of mesenchymal stem cells, regarded as a promising target for clinical intervention for skeletal disorders. In this review, we summarized the role of primary cilia and ciliary proteins in the pathogenesis of skeletal diseases, including osteoporosis, bone/cartilage tumor, osteoarthritis, intervertebral disc degeneration, spine scoliosis, and other cilium-related skeletal diseases, and highlighted their promising treatment methods, including using mesenchymal stem cells. Our review tries to present evidence for primary cilium as a promising target for clinical intervention for skeletal diseases.

Research Article

18 Jun 2022

Global Research Trends in Tendon Stem Cells from 1991 to 2020: A Bibliometric and Visualized Study

Huibin Long|Ziyang Yuan|...|Ai Guo

Background. Tendinopathy is a disabling musculoskeletal disorder affecting the athletics and general populations. There have been increased studies using stem cells in treating tendon diseases. The aim of this bibliometric and visualized study is to comprehensively investigate the current status and global trends of research in tendon stem cells. Methods. Publications related to tendon stem cells from 1991 to 2020 were retrieved from Web of Science and then indexed using a bibliometric methodology. VOSviewer software was used to conduct the visualized study, including coauthorship, cocitation, and cooccurrence analysis and to analyze the publication trends of research in tendon stem cells. Results. In total, 2492 articles were included and the number of publications increased annually worldwide. The United States made the largest contribution to this field, with the most publications (938 papers, 37.64%), citation frequency (68,195 times), and the highest -index (103). The most contributive institutions were University of Pittsburgh (96 papers), Zhejiang University (70 papers), Shanghai Jiao Tong University, and Chinese University of Hong Kong (both 64 papers). The Journal of Orthopaedic Research published the most relative articles. Studies could be classified into five clusters: Animal study, Tissue engineering, Clinical study, Mechanism research, and Stem cells research, which show a balanced development trend. Conclusion. Publications on tendon stem cells may reached a platform based on current global trends. According to the inherent changes of hotspots in each cluster and the possibilities of cross-research, the research in tendon stem cells may exist a balanced development trend.

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Stem Cells International | Hindawi

However, breast milk is a constantly changing fluid so in a way it's a moving target, with some components still not fully understood, says Fewtrell, the professor of paediatric nutrition at University College London.

"We can quite successfully produce formulas to provide adequate and safe nutrition so the baby grows and develops as expected," she says. "Indeed, there have been improvements to the composition of formulas in recent years so that they can more closely reproduce the growth patterns and some outcomes seen in breast-fed infants. However I think it would be impossible to ever mimic the 'non-nutrient' components in this complex fluid."

As for my investigation into my own body's toxic load, and the harmful chemicals that were perhaps present in my breastmilk, Bloxam, the dietician, reassures me: "I'd encourage breastfeeding wherever possible as the benefits for mother and baby would far outweigh any risks [from contamination]."

Still it appears I'm not the only one wondering about the ingredients in my own milk. Stephanie Canale, previously a family medical doctor, is the founder of Lactation Lab in California, a private company that analyses breast milk for nutritional content as well as environmental toxics.Mothers send in frozen samples of their breast milk to check the levels of various ingredients including minerals and vitamins. The idea is that they can then adapt their diet accordingly.

Canale says that when we look at a baby's nutrition, we need to include everything from prenatal vitamins to the food a breastfeeding mother consumes and the meals a weaning baby eats. Formula may be one part of that mosaic, in families where it is used.

"It's this holistic approach," says Canale who would like to see stricter regulations in the US about the contents of formula. "I'm from Canada and it still surprises how much high-fructose corn syrup is present in US products, including formula. Moms are going to drive this change by saying we need to be better aware of what is going into these products, especially formula because that child is eating the same thing every single day there's no variation [like there is naturally with breast milk]."

In the case of the toxic chemicals whether they find their way into breast milk or into formula the question is clearly not just about how we can provide our children with safe nutrition. It is also about how we can provide them and future generations with a safe, liveable environment, and reduce pollution along the entire food chain. One answer, surely, is to start by using fewer harmful chemicals in the first place.

* Listen toMy Toxic Cocktail, Anna Turns's investigation for BBC Radio 4's Costing the Earth series on BBC Sounds.Go Toxic Free: Easy and Sustainable Ways to Reduce Chemical Pollutionby Anna Turns is out now

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The surprising science of breast milk - BBC

The new multi-organ chip has the size of a glass microscope slide and allows the culture of up to four human engineered tissues, whose location and number can be tailored to the question being asked. These tissues are connected by vascular flow, but the presence of a selectively permeable endothelial barrier maintains their tissue-specific niche. Credit: Kacey Ronaldson-Bouchard/Columbia Engineering

Major advance from Columbia Engineering team demonstrates the first multi-organ chip made of engineered human tissues linked by vascular flow for improved modeling of systemic diseases like cancer.

Engineered tissues have become an essential component for modeling diseases and testing the efficacy and safety of drugs in a human context. A key hurdle for researchers has been figuring how to model body functions and systemic diseases with multiple engineered tissues that can physiologically communicate just like they do in the body. However, it is essential to provide each engineered tissue with its own environment so that the specific tissue phenotypes can be maintained for weeks to months, as required for biological and biomedical studies. Making the challenge even more complex is the necessity of linking the tissue modules together to facilitate their physiological communication, which is required for modeling conditions that involve more than one organ system, without sacrificing the individual engineered tissue environments.

Up to now, no one has been able to meet both conditions. Today, a team of researchers from Columbia Engineering and Columbia University Irving Medical Center reports that they have developed a model of human physiology in the form of a multi-organ chip consisting of engineered human heart, bone, liver, and skin that are linked by vascular flow with circulating immune cells, to allow recapitulation of interdependent organ functions. The researchers have essentially created a plug-and-play multi-organ chip, which is the size of a microscope slide, that can be customized to the patient. Because disease progression and responses to treatment vary greatly from one person to another, such a chip will eventually enable personalized optimization of therapy for each patient. The study is the cover story of the April 2022 issue of the journal Nature Biomedical Engineering.

In our study, we cultured liver, heart, bone, and skin, connected by vascular flow for four weeks. These tissues can be generated from a single human induced pluripotent stem cell, generating a patient-specific chip, a great model for individualized studies of human disease and drug testing. Credit: Keith Yeager/Columbia Engineering

This is a huge achievement for usweve spent ten years running hundreds of experiments, exploring innumerable great ideas, and building many prototypes, and now at last weve developed this platform that successfully captures the biology of organ interactions in the body, said the project leader Gordana Vunjak-Novakovic, University Professor and the Mikati Foundation Professor of Biomedical Engineering, Medical Sciences, and Dental Medicine.

Taking inspiration from how the human body works, the team has built a human tissue-chip system in which they linked matured heart, liver, bone, and skin tissue modules by recirculating vascular flow, allowing for interdependent organs to communicate just as they do in the human body. The researchers chose these tissues because they have distinctly different embryonic origins, structural and functional properties, and are adversely affected by cancer treatment drugs, presenting a rigorous test of the proposed approach.

The tissues cultured in the multi-organ chip (skin, heart, bone, liver, and endothelial barrier from left to right) maintained their tissue-specific structure and function after being linked by vascular flow. Credit: Kacey Ronaldson-Bouchard/Columbia Engineering

Providing communication between tissues while preserving their individual phenotypes has been a major challenge, said Kacey Ronaldson-Bouchard, the studys lead author and an associate research scientist in Vunjak-Novakovics Laboratory for Stem Cells and Tissue Engineering. Because we focus on using patient-derived tissue models we must individually mature each tissue so that it functions in a way that mimics responses you would see in the patient, and we dont want to sacrifice this advanced functionality when connecting multiple tissues. In the body, each organ maintains its own environment, while interacting with other organs by vascular flow carrying circulating cells and bioactive factors. So we chose to connect the tissues by vascular circulation, while preserving each individual tissue niche that is necessary to maintain its biological fidelity, mimicking the way that our organs are connected within the body.

The group created tissue modules, each within its optimized environment and separated them from the common vascular flow by a selectively permeable endothelial barrier. The individual tissue environments were able to communicate across the endothelial barriers and via vascular circulation. The researchers also introduced into the vascular circulation the monocytes giving rise to macrophages, because of their important roles in directing tissue responses to injury, disease, and therapeutic outcomes.

All tissues were derived from the same line of human induced pluripotent stem cells (iPSC), obtained from a small sample of blood, in order to demonstrate the ability for individualized, patient-specific studies. And, to prove the model can be used for long-term studies, the team maintained the tissues, which had already been grown and matured for four to six weeks, for an additional four weeks, after they were linked by vascular perfusion.

The researchers also wanted to demonstrate how the model could be used for studies of an important systemic condition in a human context and chose to examine the adverse effects of anticancer drugs. They investigated the effects of doxorubicin a broadly used anticancer drug on heart, liver, bone, skin, and vasculature. They showed that the measured effects recapitulated those reported from clinical studies of cancer therapy using the same drug.

The team developed in parallel a novel computational model of the multi-organ chip for mathematical simulations of drugs absorption, distribution, metabolism, and secretion. This model correctly predicted doxorubicins metabolism into doxorubicinol and its diffusion into the chip. The combination of the multi-organ chip with computational methodology in future studies of pharmacokinetics and pharmacodynamics of other drugs provides an improved basis for preclinical to clinical extrapolation, with improvements in the drug development pipeline.

While doing that, we were also able to identify some early molecular markers of cardiotoxicity, the main side-effect that limits the broad use of the drug. Most notably, the multi-organ chip predicted precisely the cardiotoxicity and cardiomyopathy that often require clinicians to decrease therapeutic dosages of doxorubicin or even to stop the therapy, said Vunjak-Novakovic.

The development of the multi-organ chip began from a platform with the heart, liver, and vasculature, nicknamed the HeLiVa platform. As is always the case with Vunjak-Novakovics biomedical research, collaborations were critical for completing the work. These include the collective talent of her laboratory, Andrea Califano and his systems biology team (Columbia University), Christopher S. Chen (Boston University) and Karen K. Hirschi (University of Virginia) with their expertise in vascular biology and engineering, Angela M. Christiano and her skin research team (Columbia University), Rajesh K. Soni of the Proteomics Core at Columbia University, and the computational modeling support of the team at CFD Research Corporation.

The research team is currently using variations of this chip to study, all in individualized patient-specific contexts: breast cancer metastasis; prostate cancer metastasis; leukemia; effects of radiation on human tissues; the effects of SARS-CoV-2 on heart, lung, and vasculature; the effects of ischemia on the heart and brain; and the safety and effectiveness of drugs. The group is also developing a user-friendly standardized chip for both academic and clinical laboratories, to help utilize its full potential for advancing biological and medical studies.

Vunjak-Novakovic added, After ten years of research on organs-on-chips, we still find it amazing that we can model a patients physiology by connecting millimeter sized tissues the beating heart muscle, the metabolizing liver, and the functioning skin and bone that are grown from the patients cells. We are excited about the potential of this approach. Its uniquely designed for studies of systemic conditions associated with injury or disease, and will enable us to maintain the biological properties of engineered human tissues along with their communication. One patient at a time, from inflammation to cancer!

Reference: A multi-organ chip with matured tissue niches linked by vascular flow by Kacey Ronaldson-Bouchard, Diogo Teles, Keith Yeager, Daniel Naveed Tavakol, Yimu Zhao, Alan Chramiec, Somnath Tagore, Max Summers, Sophia Stylianos, Manuel Tamargo, Busub Marcus Lee, Susan P. Halligan, Erbil Hasan Abaci, Zongyou Guo, Joanna Jackw, Alberto Pappalardo, Jerry Shih, Rajesh K. Soni, Shivam Sonar, Carrie German, Angela M. Christiano, Andrea Califano, Karen K. Hirschi, Christopher S. Chen, Andrzej Przekwas and Gordana Vunjak-Novakovic, 27 April 2022, Nature Biomedical Engineering.DOI: 10.1038/s41551-022-00882-6

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Plug-and-Play Human Organ-on-a-Chip Can Be Customized to the Patient - SciTechDaily

MaxHggblom Distinguished Professor and ChairDepartment of Biochemistry and MicrobiologySchool of Environmental and Biological SciencesRutgers-New BrunswickHonored for distinguished contributions to understanding both the fundamental and application components of microbialbiotransformationsof pollutants, especially chlorinated aromaticcompoundsand metalloids.

MaxHggblomis a renowned research scientist and educator with a large body of microbial ecology and environmental biotechnology research that has expanded our understanding of how the biodegradation of environmental pollutants, such as dioxins and PCBs,impact our planet.

His research interests revolve around thebioexploration, cultivation and characterization of novel microbes.His research on bacteria has provided a foundation for applications that address the pollution problems facing impacted industrialized and urbanized environments.

Hggblomslab is also actively studying microorganisms that degrade pharmaceutical and personal care products in aquatic environments.

Over the past decadesthediverse chemicalsin pharmaceutical and personal care productshave emerged as a major group of environmental contaminants in numerous watersheds around the world; therefore, it is important to understand how microbes can degrade them.There is much to explore and learn,Hggblomadded.

Hggblomswork also touches climate change, particularly the roles and responses of microbes in rapidly changing environments, such as the Arctic.In his lab at Rutgers, students have the unique opportunity to exploreareas of research such asthe biodegradation and detoxification of anthropogenic pollutant chemicals, including certainpesticides;respiration of rare metalloids; or life in the frozen tundra soils.

For several years,my lab has worked on studying the microbial ecology of Arctic tundra soils to understand how the changing conditions impact microbial activity and turnover of soil organic matter, and consequently enhanced greenhouse gas flux,Hggblomsaid. This is an important area of research as the threat of microbial contribution to positive feedback of greenhouse gas flux is substantial.

His lab recently received funding from the National Science Foundation to studyhowdiverse microbial communitiesare established insoils.Hggblomwill work with an international research team of scientists from the U.S., China, South Africa and Finland to study soils from the three differentregionsacross Arctic, Tibetan Plateau and Antarctic habitats to expand our understanding of how soil ecosystems respond in critical polar regions.

Emily EversonLayden

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Twelve Rutgers Professors Named Fellows of the American Association for the Advancement of Science - Rutgers Today

While youre receiving treatment for cancer, some of the drugs you take can cause painful sores to develop inside your mouth. You can also get them if youve had a bone marrow (stem cell) transplant as part of your cancer care.

Although they often heal on their own, these mouth sores can make it uncomfortable to eat and talk. Well discuss what you can do to relieve the pain and prevent them from getting worse.

Mouth sores can be a common side effect of cancer treatment. The condition, known as stomatitis or mucositis, is an inflammation of the tissues inside your mouth.

Whitish, ulcer-like sores can form on your cheeks, gums, lips, tongue, or on the roof or floor of your mouth. Even if you dont develop mouth ulcers, you may have patches that feel inflamed and painful, as if theyve been burned.

Anyone who is receiving chemotherapy, radiation therapy, or a bone marrow (stem cell) transplant can develop mouth sores as a side effect of these treatments.

If you have dry mouth or gum disease, or if your teeth and gums are not well taken care of, you may be at a higher risk of getting mouth sores during your treatment. Women and people who smoke or drink alcohol are also at a higher risk, according to the Oral Cancer Foundation.

If youre receiving chemotherapy, the sores could begin forming anywhere from 5 days to 2 weeks after your treatment. Depending on the specific cause, the sores could go away on their own in a few weeks, or they could last longer.

Its important to find ways to manage your pain and to watch for signs of an infection. Cancer-related mouth sores can lead to weight loss, dehydration, and other serious complications.

Cancer cells can grow very quickly. The aim of cancer treatment is to stop or slow down that growth. The cells in the mucous membranes lining your mouth are also fast-growing cells, so cancer treatments affect them, too.

Cancer treatments also keep the cells in your mouth from being able to repair themselves efficiently when theyre damaged.

Radiation therapy can also damage the glands in your mouth that make saliva. A dry mouth is more susceptible to infections that cause mouth sores.

Chemotherapy and radiation can both change the microbiome in your mouth, upsetting the balance between good and bad bacteria. The growth of harmful bacteria in your mouth can also lead to mouth sores.

Sometimes cancer treatments suppress your immune system, which may make it more likely that youll get a bacterial, viral, or fungal infection that causes mouth sores. An older infection (such as the herpes simplex virus) can also suddenly flare up again.

If youve had a bone marrow (stem cell) transplant, sores may be a sign that youve developed a condition known as graft-versus-host disease (GVHD).

When this happens, the cells in your body are attacking the transplanted cells as though they were an unhealthy invader. According to research published in Journal of Clinical and Experimental Dentistry, short-term (acute) GVHD occurs in 50 to 70 percent of stem cell transplant cases and longer-term (chronic) GVHD is seen in 30 to 50 percent of cases.

The form of GVHD that causes mouth sores is usually mild, and doctors often treat it with corticosteroid medications.

Its important to talk with your doctor if you develop mouth sores after a stem cell transplant, as some kinds of GVHD can turn serious if left untreated.

There is a good chance that youll experience mouth sores at some point during your cancer treatment. Researchers estimate that 20 to 40 percent of those who have chemotherapy and 80 percent of those who have high-dose chemotherapy will develop mucositis afterward.

Still, there are steps you and your cancer care team can take to lower your risk, reduce the severity of the sores, and promote faster healing.

About a month before your cancer treatment begins, schedule an appointment with your dentist to make sure your teeth and gums are healthy. If you have cavities, broken teeth, or gum disease, its important to come up with a dental treatment plan to take care of these conditions so they dont lead to infections later, when your immune system may be vulnerable.

If you wear braces or dentures, ask your dentist to check the fit and remove any part of the device you dont need during your treatment.

Its very important to maintain good oral hygiene practices throughout your treatment to lower your risk of infection. Brush and floss gently but regularly, avoiding any painful areas. You can also ask your dentist whether a mouth rinse with fluoride is advisable in your case.

For certain kinds of chemotherapy (bolus 5fluorouracil chemotherapy and some high-dose therapies), your healthcare team may give you ice chips to chew for 30 minutes before your treatment. This type of cold therapy can lower your risk of getting mouth sores later.

During treatment of some blood cancers, doctors may give you injections of palifermin, also known as human keratinocyte growth factor-1 (KGF-1), to prevent mouth sores.

If youre scheduled to receive high-dose chemotherapy or radiotherapy, your cancer care team may prepare your mouth using low-level laser therapy beforehand to keep you from getting mouth sores.

For people who have radiation therapy for head and neck cancers, doctors may prescribe this medicated mouthwash to minimize mouth sores.

The length of time your mouth sores may last depends on the specific cancer treatment youve had. Here are some estimates broken down by treatment:

You may notice symptoms anywhere between a few days and a few weeks after your cancer treatment. Heres what you may see and feel as mucositis develops:

You may notice that the sores become slightly crusty as they heal. Its important to keep track of your symptoms and let your oncologist know if the sores arent healing on their own.

Contact your doctor right away if you:

Untreated mouth sores can lead to malnutrition, dehydration, and life-threatening infections.

There are a few different ways that you can help mouth sores heal and avoid prolonger pain or an infection.

While the sores are healing, its very important to keep the inside of your mouth clean to prevent an infection from developing.

The National Cancer Institute recommends that you gently clean your teeth every 4 hours and just before you go to sleep at night. Here are a few tips to consider:

If the pain from mouth sores is interfering with your ability to eat and drink, your doctor may treat the condition with a opioid mouthwash or one containing doxepin or lidocaine.

To ease discomfort and keep your mouth from feeling dry, you may want to try rinsing with a mild saltwater or baking soda solution. Heres how to make each of them:

Your cancer care team may recommend that you use a lubricating liquid (artificial saliva) to moisten the inside of your mouth if dryness is a problem. These liquids are usually gel-like. They coat your mouth with a thin film to help ease discomfort and promote healing.

Some people have found it useful to rinse with a blend of medications called the magic mouthwash. Formulas for this mouthwash vary, but most of them include a combination of medications to treat different symptoms, including:

Magic or miracle mouthwash solutions usually have to be prescribed by a doctor and prepared by a pharmacist, although some people mix up an over-the-counter version at home.

There isnt enough research to say for sure whether magic mouthwash works. If you think youd like to try it, talk with your oncologist or a healthcare professional about whether its a good idea for you.

Here are a few more things you can try at home that may help ease pain from mouth sores:

Mouth sores are one of the most common side effects of cancer treatment. Shortly after chemotherapy, radiation, or transplant treatments, painful, ulcer-like sores can form on the inside of your mouth.

These sores may go away on their own. If they dont, its important to seek medical treatment for them because they can lead to very serious complications.

Before you start cancer treatments, visit a dentist to make sure your teeth and gums are healthy. Keeping up good dental hygiene practices during and after cancer treatment will help limit mouth sores.

If the sores are keeping you from eating and drinking, talk with your oncologist about medications could relieve the pain and speed up the healing process, so you can enjoy a better quality of life during treatment.

Its really important to keep track of any sores in your mouth so you can reach out to your healthcare team if they dont improve. Sores that deepen or worsen can lead to serious even life-threatening complications.

See the article here:
Mouth Sores from Chemo: Symptoms, Causes, and Treatments - Healthline

The report provides study at global and regional level to provide comprehensive value market analysis for the years (2017 & 2018 Historic Years, 2019 Base Year and 2020-2027 Forecast Period). The Tooth Regeneration Market research report is a wide-ranging study of current trends, market growth drivers, and restraints. Each market segment is broadly analyzed at a powdered level by region (North America, Europe, Asia Pacific, Middle East & Africa, and South & Central America) to provide thorough information on the global and regional level.

Tooth regeneration is a regenerative surgical technique based on stem cells that is used in the fields of tissue engineering and stem cell biology. By developing it from autologous stem cells, the tooth regeneration procedure restores the weakened or missing tooth. With the aid of reabsorbable biopolymer, somatic cells are harvested and reprogrammed to stimulate pluripotent stem cells and dental lamina.

Some of the key players in this market include

Download Sample PDF Brochure of this research study at https://www.theinsightpartners.com/sample/TIPRE00018967/

The Tooth Regeneration Market is segmented on the basis of services and application. Based on services , the market is segmented as Medical Writing and Publishing, Medical Monitoring, Medical Science Liaisons (MSLs), Medical Information, Others. Based on application, the market is segmented as pharmaceutical, biopharmaceutical, medical devices.

The segmentation in this research study has been finalized post in-depth secondary research and extensive primary research. In addition, the market is also segmented on the basis of technology offered by the leading participants in the industry in order to understand widely used market specific terminologies. Thus, we have incorporated the segments of the research and have finalized the market segmentation.

The Insight Partners Tooth Regeneration Market Research Report Scenario includes:

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The report also covers a detailed chapter of the analysis on COVID-19 impact on this market at global and regional level in our final reports.

This research provides detailed information regarding the major factors influencing the growth of the Tooth Regeneration Market in Global and Regional Level (drivers, restraints, opportunities, and challenges), forecast of the market size, in terms of value, market share by region and segment; regional market positions; segment and country opportunities for growth; New product developments, strengths and weaknesses, brand portfolio; Marketing and distribution strategies; challenges and threats from current competition and prospects; Key company profiles, SWOT, product portfolio and growth strategies.

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Tooth Regeneration Market to Exhibit Steadfast Expansion by 2027 | Unilever, Ocata Therapeutics, Integra LifeSciences, CryoLife, BioMimetic...

AZoM speaks withDr. Iman Roohani from UNSW.Dr.Roohani is part of a team of researchersthat developed a technique referred to as Ceramic Omnidirectional Bioprinting in Cell-Suspensions (COBICS). This techniquecould allow surgeons to print structures that can be submerged in water and hardened within just minutes, resembling natural bone.Even more revolutionary, the structures contain living cells that continue to grow after they are implanted.

We have developed a technique (COBICS) that enables printing constructs with the same chemistry to native bone mineral at room temperature with living cells. These structures are the most accurate mimics of the bone tissue. COBICS can print complex and biologically relevant architecture constructs without the need for sacricial support materials, on-spot and laborious post-processing steps.

COBICS has two main components. A chemically cross-linked microhydrogel bath with optimized yield-stress properties that support the printing of the ceramic ink, the second component, in the presence of live cells. The ink is a calcium phosphate paste with a specific formulation that allows the material to be used directly in the aqueous environment, without the need for any post-processing steps, such as high-temperature treatments, that are required for other types of existing ceramic materials.

Once ink comes in contact with the microgel, nanocrystalization kicks off at the interface between ink and hydrogel, which further locks the filament in place. Since this ink can harden quickly without imposing adverse effects on living cells, COBICS enables printing within a suspension of living cells to achieve complex bone shapes, where the cells integrate to form natural bone tissue.

Traditional bone grafts, particularly synthetic ones, are mostly fabricated from ceramic materials at high temperatures, which disallows integration with cells and growth factors. Moreover, due to high temperature processing, the microstructure of such grafts does not resemble the native bone.

COBICS paves the way to fabricate autologous graft like structures in the laboratory, which significantly reduces the risks and drawbacks involved in harvesting these grafts from the patient in the clinical setting by using only cells from the patient or other sources of regenerative cells. This also could enable patient-specific real-time bone reconstruction where the bioprinter could directly print new bone into the resected space.

You could even isolate the patient's stem cells before surgery for inclusion with the ink to improve the integration of the new bone into the surgery site or in dental reconstruction. In another example, drugs could be integrated with the ink for sustained release over time to increase natural bone formation, combat bacteria, or influence the immune system (e.g., enhance wound healing).

The ink has an essential role in printing the constructs by the COBICS technique. The optimization process of formulating the ink took around 2 years since we had to ensure that ink has several properties that were mutually exclusive. Those properties included biocompatibility, being printable, proper setting time, adequate strength and firmness after printing, and printing in contact with the microgel bath.

We have an ongoing animal study at the moment, that will hopefully confirm our hypothesis that there should be no harmful components in our material.Thus far, all of our tests with human cells in the laboratory have confirmed high biocompatibility. We plan to scale up our production of bone-like grafts and test the regenerative properties of the printed grafts in large animal models before proceeding with humantrials and regulatory approval.If everything goes well and we findexternal funding support,we are optimistic the technique may be ready for the clinic within 5 years.

Readers can check out the full article at https://doi.org/10.1002/adfm.202008216.

From 2010 to 2014, Dr. Roohani studied and received his Ph.D. degree at the School of Aerospace, Mechanical and Mechatronic Engineering at the University of Sydney (USYD) in Sydney, Australia. From 2016 to 2020, he worked in the field of biomaterials and tissue engineering as the National Health and Medical Research Council (NHMRC) early career fellow, first in the biomedical engineering department at the University of Sydney, and then at the School of Chemistry in the University of New South Wales (UNSW).

Dr. Roohani is interested in the use of biomaterials as the bone substitute, drug delivery and instructive source for cells. More specifically, his interests comprise synthesis and development of a range of bioceramics, including calcium phosphates, understanding of the interaction between living cells and synthetic substrates, and translation of the application of these materials and concepts to clinical applications.

Dr. Roohani is the inventor of several patented products, including the COBICs techniques. He is the author of more than 60 peer-reviewed publications (h index of 23), book chapters, and 3 patent applications.

ResearchGate: https://www.researchgate.net/profile/Iman_Roohani

Twitter: @ImanRoohani

Email:[emailprotected]

Google Scholar:https://scholar.google.com.au/citations?user=NyzEeygAAAAJ&hl=en

Disclaimer: The views expressed here are those of the interviewee and do not necessarily represent the views of AZoM.com Limited (T/A) AZoNetwork, the owner and operator of this website. This disclaimer forms part of the Terms and Conditions of use of this website.

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Using 3D Printing to Develop Bone-Like Structures that Contain Living Cells - AZoM

The new health guidelines have allowed private dental clinics to start functioningNew Delhi : Observably, the Covid-19 pandemic hit a pause button on many of our routine activities, including regular visits to the dentist. When the pandemic peaked, health authorities put restrictions in place for dental care, and dental clinics saw only emergency cases and advised patients to wait when it came to non-essential or non-urgent procedures including regular check-ups, cleaning, fillings, etc., which resulted in a substantial drop in the preventive dental care visits of oral patients across India.

Lt Gen. Dr Vimal Arora, Chief Clinical Officer, Clove Dental said, Oral Health data in India says that 8 out of 10 Indians suffer from some or the other kind of dental diseases which clearly reflects that Oral & Dental Health has always been deprioritized even in pre-Coovid times. However, with this pandemic people have further delayed their dental visits for past 10 months, the result of which is that the oral health conditions which could have been handled with simple cavity filling, now need RCT & Crowning and even extraction in some cases leading to loss of tooth. Ministry of Health and Family Welfare estimates that currently (2019), about 60% of Indias adult population and 70% of its school-going children are affected by dental caries (cavities) or tooth decay. And, periodontal disease infections of the tissue around the teeth has ended up affecting at least 85% of the population. The country is also considered the world capital for oral cancer. Of the total body cancers, Oral cancers accounts for over 30% of all body cancers.

Drop in Dental Care

Dr Anirudh Singh, General Manager, Clove Dental, South West India said, Traditionally, check-ups are recommended every six months. But people often ignore this, which leads to the severity of the dental problems demanding expensive treatments and care. According to the draft National Oral Health Policy, the proportion of untreated caries of permanent teeth and severe periodontitis is the maximum compared to other oral disorders. Yet, only 12.4% of adults have ever had their mouth examined by a dentist. Routine check-ups are the part of preventive treatments. However, due to COVID, preventive treatments have gone down by 63.7%, as the patient visits have declined. As COVID has prompted the delay in treatments, we see the need for more restorative treatments now. Therefore, prioritising dental care is as important as any other healthcare problems.

A fundamentally different approach is required to effectively tackle the global burden of oral diseases. The public health problems associated with oral diseases are a serious burden in every nation around the globe. What is lacking is the awareness to the disease and the information as to what all poor oral health can create in the body. Excellent dental health is the gateway for the overall body health; is the true slogan.

The rest is here:
Fear of Covid keeps patients away from dental clinics resulting in an increased need of treatment. - ETHealthworld.com

3d printing human body. 3d printed implants on white background.

Back in the 1970s, there was a television show called The Six-Million-Dollar Man about a fictional astronaut severely injured in a test crash, and then having several body parts replaced by robotics. The shows opening narration promised that he would be better than he was before. Better. Faster. Stronger.

Life is now imitating art, and then some. 3D printers are currently being used to construct not only customized prosthetics for patients in need, but a wide variety of other medical items foremost among them tissues and organoids, as well as surgical models and tools. It is estimated that 13 percent of 3D printing revenue comes courtesy of the medical field, and that some $3.19 billion will be spent on technology in that sector by 2025. Thats over $2 billion more than in 2018 ($1.13 billion), a compound annual growth rate (CAGR) of 15.89 percent.

There are many reasons for this uptick, not the least of which are that these items can be produced quickly and inexpensively. But there is none bigger than the ever-increasing demand for prosthetics. Some 30 million people around the world, including 1.9 million in the U.S., are in need of such devices, and as of 2018 only 20 percent of them had been provided for.

Factor in other devices and an aging population with a consequent increase in demand for personalized treatment as noted by Tim Deng, Principal Medical Devices Analyst at GlobalData, in a report on the website Express Healthcare and the overwhelming need becomes that much clearer.

So too are the benefits of meeting that need. Better, faster, stronger? Well, that part might be a bit of a stretch, at least for now. But certainly it appears that 3D printers are making it possible for patients to be as good, as fast and as strong as they were before illness or injury left them a shell of their former selves.

Serendipity helped galvanize the 3D prosthetics industry. In 2011 an artist named Ivan Owen developed a puppet hand for a steampunk event i.e., an event where modern technology melds with elements of Victorian-era history and fashion. He circulated a video of his creation, which was seen by Richard Van As, a South African carpenter who, having just lost four fingers in an accident, was looking for a prosthetic hand that would enable him to return to work. The two of them collaborated to construct just such a device, then used a 3D printer to develop another for a five-year-old boy who was born without fingers.

Owen, instead of patenting his invention, elected to open-source it. That led in 2013 to the formation of the nonprofit organization e-NABLE, an online community enabling people to collaborate on the design of 3D prosthetic limbs. Another nonprofit, Limbitless Solutions, came along a year later, with the mission of providing 3D-printed arms for children.

Then there is the startup Unlimited Tomorrow. In 2020, some six years after its founding, the Rhinebeck, N.Y.-based company also rolled out a prosthetic arm courtesy of a 3D printer, while emphasizing its affordability (as low as $7,995.) compared to prosthetics produced by other means (over $50,000).

3D printers are getting ever closer to being able to produce organs, and progress is being made on other fronts as well. In 2018, researchers at the University of Utah became the first to produce ligaments and tendons in that fashion, by extracting stem cells from a patients body fat, printing them onto a layer of hydrogel, allowing time for the cells to form the required connective tissue outside the body and then implanting it where needed.

This is a particularly important breakthrough, since injuries to tendons and ligaments had in the past proven to be difficult to treat. Most commonly, tissue from cadavers was used, but there was the risk of rejection, or that the connective tissue wouldnt perform as expected.

In 2019, there was another promising development when a team at Rice University made promising strides toward producing cardiovascular networks and lung-like air sacs through the use of bioprinting technology called stereolithography apparatus for tissue engineering, or SLATE. The team hopes to commercialize that technology in the future, which could have obvious benefits for those suffering from heart or lung disease.

Truly there seem to be no bounds to what 3D printers might mean for healthcare. The possibility exists of dental professionals using the technology to create customized dental implants, prosthetics and braces that printing could be done in-office without long wait times. Once the patients teeth have been scanned, their dental treatment will be printed in-office. This allows for a better fit and even time for immediate troubleshooting. Compared to taking a mold and relying on an outside source, this is more convenient for both patients and professionals.

On the other side of the equation, a 3D printer can be used to create customized instruments for use in complex surgeries and the process can be done far more quickly than is the case by other means. It is also possible to create three-dimensional models of patients internal systems before surgical procedures, giving doctors a clear understanding of the challenges they face.

Also of interest is the production of customized medication via 3D printer, a process begun in 2015 to counter the trend toward producing dosages best-suited for white adult men, meaning women and children were receiving more than was necessary. Customizing the dosage, Multiply Labs CEO Fred Paretti told the website 3D Natives, goes a long way toward highlighting the individuality of each patient, since the error in dosage of certain active ingredients can even lead to the malfunctioning of some treatments.

The bottom line is that 3D printers will be making an even greater impact on the medical field in the years ahead, as evidenced by the fact that the number of U.S. hospitals featuring the technology grew from three in 2010 to over 100 in 2019. The need is there, and the evolution will certainly only continue. And someday, maybe there really will be a Six-Million-Dollar Man.

Photo: belekekin, Getty Images

Excerpt from:
3D medical printing making strides, and helping patients do the same - MedCity News

DUBLIN, Jan. 13, 2021 /PRNewswire/ -- The "Global Cord Blood Banking Industry Report 2021" report has been added to ResearchAndMarkets.com's offering.

From the early 1900s through the mid-2000s, the global cord blood banking industry proliferated with cord blood banks emerging in all major healthcare markets worldwide. From 2005 to 2010, the market reached saturation and stabilized. From 2010 to 2020, the market began to aggressively consolidate, creating both threats and opportunities within the industry.

Serious threats to the industry include low rates of utilization for stored cord blood, expensive cord blood transplantation procedures, difficulty educating obstetricians about cellular therapies, and an increasing trend toward industry consolidation. Opportunities for the industry include price efficiencies associated with scale and consolidation, accelerated regulatory pathways for cord blood and tissue-based cell therapies, and progress with ex vivo cellular expansion technologies.

Cord Blood Industry Trends

Within recent years, new themes have been impacting the industry, including the pairing of stem cell storage services with genetic and genomic testing services, as well as reproductive health services. Cord blood banks are diversifying into new types of stem cell storage, including umbilical cord tissue storage, placental blood and tissue, amniotic fluid and tissue, and dental pulp. Cord blood banks are also investigating means of becoming integrated therapeutic companies. With hundreds of companies offering cord blood banking services worldwide, the maturation of the market means that each company is fighting harder for market share.

Growing numbers of investors are also entering the marketplace, with M&A activity accelerating in the U.S. and abroad. Holding companies are emerging as a global theme, allowing for increased operational efficiency and economy of scale.Cryoholdco has established itself as the market leader within Latin America. Created in 2015, Cryoholdco is a holding company that controls over a quarter of a million stem cell units (approximately 270,000). It owns a half dozen cord blood banks, as well as a dental stem cell storage company.

Globally, networks of cord blood banks have become commonplace, with Sanpower Group establishing its dominance in Asia. Although Sanpower has been quiet about its operations, it holds 4 licenses out of only 7 issued provincial-level cord blood bank licenses in China. It has reserved over 900,000 cord blood samples in China, and its reserves amount to over 1.2 million units when Cordlife's reserves within Southeast Asian countries are included. This positions Sanpower Group and it's subsidiary Nanjing Cenbest as the world's largest cord blood banking operator not only in China and Southeast Asia but in the world.

The number of cord blood banks in Europe has dropped by more than one-third over the past ten years, from approximately 150 to under 100. The industry leaders in this market segment include FamiCord Group, which has executed a dozen M&A transactions, and Vita34, which has executed approximately a half dozen. Stemlab, the largest cord blood bank in Portugal, also executed three acquisition deals prior to being acquired by FamiCord. FamiCord is now the leading stem cell bank in Europe and one of the largest worldwide.

Cord Blood Expansion Technologies

Because cord blood utilization is largely limited to use in pediatric patients, growing investment is flowing into ex-vivocord blood expansion technologies. If successful, this technology could greatly expand the market potential for cord blood, encouraging its use within new markets, such as regenerative medicine, aging, and augmented immunity.

Currently, Gamida Cell, Nohla Therapeutics, Excellthera, and Magenta Therapeutics have ex vivo cord blood expansion products proceeding through clinical trials. Growing numbers of investors have also entered the cord blood banking marketplace, led by groups such as GI Partners, ABS Capital Partners & HLM Management, KKR & Company, Bay City Capital, GTCR, LLC, and Excalibur.

Key questions answered in this report are:

Profiles of Select Cord Blood Banks and Companies

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Global Cord Blood Banking Industry Report 2021: Industry Trends, Expansion Technologies, Profiles of Select Cord Blood Banks and Companies -...