StemCell Therapy

Early studies

Maintenance of dental pulp function is critical for the homeostasis of teeth; loss of dental pulp is often followed by tooth fracture and/or periapical disease and, finally, loss of teeth. When dental pulp is infected it is difficult for the immune system to eradicate the infection, due to lack of blood supply to the pulp. Partially removing the infected pulp, termed partial pulpectomy, has proved to be ineffective, as infecting organizms may be left behind (Huang et al., 2009a, 2009b). Thus infection of adult pulp by trauma or caries often necessitates root canal therapy, in which the entire pulp is removed and the pulp cavity disinfected and filled with an artificial material. Biological alternatives to root canal therapy have inspired regenerative endodontics, whereby the diseased or necrotic pulp tissues are removed and replaced with regenerated pulp tissue, capable of revitalizing teeth (Sun et al., 2011). For recent reviews of dental pulp regeneration, the reader is referred to a number of excellent papers (Sloan and Smith, 2007; Sun et al., 2011; Huang, 2011; Nakashima and Iohara, 2011).

Whilst the volume of mature pulp tissue is very small (ca. 10100l) it is a difficult task to engineer and regenerate this tissue, due to its anatomical location, unique microstructure with different cell types and complex innervations, specific location of dentin and the highly organized structure of dentinal tubules (Huang et al., 2009a, 2009b). Although dental pulp tissue engineering was investigated in the late 1990s (Mooney et al., 1996; Bohl et al., 1998), it was the identification of dental pulp stem cells capable of generating dentin that rendered dentin pulp regeneration possible (Gronthos et al., 2000).

Human DPSCs were transplanted in conjunction with hydroxyapatite/tricalcium phosphate (HA/TCP) powder into immunocompromised mice. After 6weeks DPSCs generated a dentin-like structure lining the surfaces of the HA/TCP particles, comprised of a highly ordered collagenous matrix deposited perpendicular to the odontoblast-like layer (Gronthos et al., 2000). The aligned odontoblast-like cells expressed the dentin-specific protein DSPP and extended as tubular structures within newly generated dentin. The collagen matrix mimicked the structure of primary dentin with ordered perpendicular fibres, rather than reparative dentin, which usually consists of a disorganized matrix. In addition, the DPSC transplants contained a fibrous tissue containing blood vessels, similar to the arrangement found in the dentinpulp complex in normal human teeth. To assess the self-renewal characteristics of DPSCs, Gronthos et al. (2002) re-isolated stromal-like cells from the 3month-old primary DPSC transplants. After in vitro expansion, human cells were re-transplanted into immunocompromised mice. These secondary transplants produced human alu-positive odontoblasts within a dentinpulp-like complex containing organized collagen fibres, thus showing that the human DPSCs were able to self-renew in vivo.

In these early studies, transplantation of expanded DPSCs formed a dentinpulp complex and transplantation of expanded bone marrow mesenchymal stem cells (BMMSCs) formed ectopic bone. The tissue regeneration capability of BMMSCs and DPSCs was further examined by transplantation using human dentin as a carrier (Batouli et al., 2003). Although BMMSCs failed to form mineralized tissue on the surface of dentin or a pulp-like connective tissue, DPSCs generated a reparative dentin-like structure directly on the surface of human dentin, indicating the possibility of using DPSCs in tooth repair.

This isolation and characterization of dental pulp stem cells, combined with increased understanding of tooth development, has led to two major strategies in tooth tissue engineering: in vivo transplantation of stem cells and in vitro culture of stem cells on biodegradable scaffolds and subsequent transplantation in vivo (Galler et al., 2011). Both strategies have found application in pulp regeneration utilizing DPSCs.

A number of studies have indicated that the DPSCs may be used to regenerate partially lost pulp and dentin. Nakashima's group were able to demonstrate partial regeneration of pulp using porcine pulp cells, cultured as a three-dimensional (3D) pellet (Iohara et al., 2004). The expression of dentin sialophosphoprotein (DSPP) confirmed the differentiation of DPSCs into odontoblasts. Additionally, autogenous transplantation of a bone morphogenetic protein-2 (BMP-2) treated pellet culture onto the amputated pulp of a dog stimulated reparative dentin formation. Similar results were achieved with a 3D pellet culture system of pulp cells electrotransfected with growth/differentiation factor 11 (Gdf11) (Nakashima and Akamine, 2005).

Iohara et al. (2006) continued their investigations of dental pulp regeneration by isolating a side population (SP) of cells from dental pulp based on the efflux of fluorescent dye Hoechst 33342. These SP cells, derived from porcine dental pulp, differentiated into odontoblasts in response to BMP-2. Furthermore, autogenous transplantation of BMP-2-treated canine SP cells induced osteodentin formation in surgically created defects on amputated canine dental pulp Two further fractions of SP cells were isolated from canine dental pulp: CD31/CD146 and CD31+/CD146+ SP cells were separately cultured as pellets with collagen type I and collagen type III and autogenously transplanted into amputated pulps (Iohara et al., 2009). Pulp-derived CD31/CD146 SP cells induced a strong vasculogenic response; cells differentiated into odontoblasts only at the periphery of dentin and thus produced a physiologically normal regenerated pulp tissue.

Complete pulp regeneration with neurogenesis and vasculogenesis occurred in an adult canine model of pulpectomy with autogenous transplantation of pulp CD105+ SP cells with stromal cell-derived factor-1 (SDF-1) (Iohara et al., 2011). Side population CD105+ cells formed pulp-like tissue by day 14 when transplanted with SDF-1 and induced complete apical closure, whereas transplantation of CD105+ cells alone or SDF-1 alone yielded less pulp. This seminal work by Nakashima's group was the first demonstration of complete in situ pulp regeneration.

A recent study from this group has compared the biological characteristics and regenerative potentials of dental pulp, bone marrow and adipose stem cells taken from the same individual (Ishizaka et al., 2012). In this investigation SP cells were further sub-fractionated into CD31 cells, previously shown to stimulate angiogenesis/vasculogenesis in vitro and in vivo (Iohara et al., 2008). The differential potentials of pulp regeneration of the three SP fractions were determined using an in vivo model previously described (Huang, 2011), whereby the three CD31 SP populations were injected into porcine tooth root fragments prior to transplantation into immunocompromised mice. Whilst pulp-like tissue was observed after transplantation of all three SP fractions, the total volume of regenerated tissue was significantly higher with the dental pulp SP and the density of vasculature and innervations was also higher (Ishizaka et al., 2012).

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