SHED repair critical-size calvarial defects in mice

Abstract

Objective: Stem cells from human exfoliated deciduous teeth (SHED) are a population of highly proliferative postnatal stem cells capable of differentiating into odontoblasts, adipocytes, neural cells, and osteo-inductive cells. To examine whether SHED-mediated bone regeneration can be utilized for therapeutic purposes, we used SHED to repair critical-size calvarial defects in immunocompromised mice.

Materials and methods: We generated calvarial defects and transplanted SHED with hydroxyapatite/tricalcium phosphate as a carrier into the defect areas.

Results: SHED were able to repair the defects with substantial bone formation. Interestingly, SHED-mediated osteogenesis failed to recruit hematopoietic marrow elements that are commonly seen in bone marrow mesenchymal stem cell-generated bone. Furthermore, SHED were found to co-express mesenchymal stem cell marker, CC9/MUC18/CD146, with an array of growth factor receptors such as transforming growth factor beta receptor I and II, fibroblast growth factor receptor I and III, and vascular endothelial growth factor receptor I, implying their comprehensive differentiation potential.

Conclusions: Our data indicate that SHED, derived from neural crest cells, may select unique mechanisms to exert osteogenesis. SHED might be a suitable resource for orofacial bone regeneration.

Figures

Figure 1

Histology of SHED-mediated bone regeneration for repairing parietal defects in immunocompromised mice. Cross sections of hydroxyapatite/tricalcium phosphate carrier transplant (a, d) as negative control, SHED transplant (b, e), and bone marrow mesenchymal stem cell transplant (c, f) as positive control, at 8 weeks posttransplantation stained with hematoxylin and eosin. There was no mineralized tissue regeneration found in the negative control group as shown in low (a) and selected area with high (d) magnification. Only connective tissue (CT) was found around hydroxyapatite/tricalcium phosphate (HA) carrier. However, bone (B) formation presented on the surface of hydroxyapatite/tricalcium phosphate in SHED and bone marrow mesenchymal stem cell transplants as seen in low (b, c) and selected areas with high (e, f) magnification. Clearly, bone marrow elements (BM) were generated in bone marrow mesenchymal stem cell transplants (f), but absent in SHED transplants (e). Bar: 500 μm in (a)–(c), 100 μm in (d)–(f). Semi-quantitative analysis showed that bone regeneration capacity of SHED was similar to that of bone marrow mesenchymal stem cells when transplanted into immunocompromised mice (g) using Scion Image analysis (Scion Image, Rockville, MD, USA). Error bars represent the mean ± s.d. However, control hydroxyapatite/tricalcium phosphate transplant lacked bone formation (g)

Figure 2

Characterization of SHED-mediated bone formation. After 6 months of transplantation, SHED were capable of maintaining bone structure (B) on the surfaces of hydroxyapatite/tricalcium phosphate (HA) along with connective tissue (CT, a). Same field of polarized picture showed dense collagen fibers (b). In contrast, bone marrow mesenchymal stem cells maintained both bone (B) and bone marrow elements (BM) after 6 months posttransplantation (c). Same field of polarized microscopy view showed dense collagen fibers (d). In situ hybridization studies showed the murine-specific pf1 DNA probe reacting with recipient osteoblasts and osteocytes associated with the new bone formation (B, black arrows in e). Mouse tissue (MT) reacted positive for pf1 probe in SHED transplant (open arrows in e). Human-specific alu in situ hybridization showed that SHED (black arrows in f) were associated with bone formation (B). Mouse tissue (MT) was negative for alu in situ hybridization. Immunohistochemical staining showed that SHED generated bone (B) and differentiated into osteocytes that were positive for anti-human-specific mitochondria antibody staining (open arrows in g). Mouse tissue (MT in g) and preimmunoserum control (h) were negative for anti-human-specific mitochondria antibody staining. Single colony-derived SHED were also capable of forming bone (B) on the surface of hydroxyapatite/tricalcium phosphate (HA) to repair critical size of calvarial defects (black line in i) in immunocompromised mice (i, j) same as seen in mixed population of SHED. CB indicates preexisting calvarial bone. The mRNA isolated from two different SHED transplants (T1 and T2) was applied for RT-PCR analysis (k). Both human (h) and mouse (m) BSP and OSC were positively detected on both transplants, suggesting that both human and mouse osteogenic cells involved in the bone formation in the SHED transplants. The mRNA extracted from human (H) and mouse (M) intact bones were used as positive and negative controls for RT-PCR amplification. GAPDH was used for internal control

Figure 3

Characterization of SHED in vivo. After 8 weeks transplantation in immunocompromised mice, SHED were able to form bone (B) on the surface of hydroxyapatite/tricalcium phosphate (HA). Osteogenic cells were positive for anti-ALP (open arrows in a), BSP (open arrows in b) and type I collagen (open arrows in c) antibody staining. BSP and type I collagen showed a positive staining on the cells in connective tissue (CT) compartment. (d) Negative control of immunohistochemical staining on SHED transplant with preimmuno serum. The expressions of DSP as odontogenic marker were not detected in both bone marrow mesenchymal stem cells (e) and SHED (f). On the other hand, the expressions of BSP were positive in both bone marrow mesenchymal stem cell-mediated bone (g) and SHED-mediated bone (h). BM: bone marrow, HA: HA/TCP

Figure 4

Immunocytochemical characterization of SHED. Osteogenic associated growth factor receptors including TGFβR-I/-II, FGFR-I/-III and VEGFR-I may co-express with CC9/MUC18/CD146, an early marker of mesenchymal stem cells. All those growth factor receptors may also not co-express with CC9/MUC18/CD146, implying a heterogenic property of SHED. Bars in merged images: 50 μm