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Figure 4. Characterization of DPSC-mediated dentinogenesis in vivo. (A) Odontoblasts of human dental pulp show a positive immunostaining for DSP antibody (black arrows). Other cells and blood vessels (open arrows) within the dental pulp are negative to DSP antibody staining. (B) There is no DSP antibody immunostaining on human bone (B), osteoblasts (black arrows), and hematopoietic marrow elements (HM). (C) DPSC transplant at 8 wks post-transplantation. There is DSP-positive immunostaining on dentinogenic cells (open arrows) and tubular dentin (black arrows). Pulp-like tissue (P) contains blood vessel (BV) and connective tissue. Dashed line shows the interface between the hydroxyapatite carrier (HA) and the newly generated dentin (D). (D) At 8 wks post-transplantation, BMSSCs differentiated into osteoblasts (black arrows) to generate bone (B) and induced hematopoietic marrow formation (HM). There was no DSPP antibody immunostaining on newly formed bone and osteogenic cells. (E) BMSSCs were loaded onto the acid-treated dentin and cultured for 12 hrs. Then BMSSC/dentin complexes were transplanted into immunocompromised mice. There was no mineralized tissue regeneration after 8 wks’ transplantation. (F) Newly formed reparative dentin-like structure (ND) attached to the surfaces of human dentin in DPSC/dentin transplants. There were blood vessels (BV) and connective tissue (CT) associated with dentinogenic cells (black arrows). DPSCs were unable to generate organized tubular dentin structure; instead, they formed reparative dentin-like structure containing entrapped cells (triangle open arrows). (G) In DPSC/dentin transplants, dentinogenic cells (open arrows) and trapped cells (open triangles) within the newly formed reparative dentin-like structure (ND) were immunoreactive to human DSP antibody. Human dentin scaffold (Dentin) showed a positive DSP immunostaining on peritubular structures (black arrows). Connective tissue (CT) contained blood vessels (BV). (H) DSP immunohistochemical staining of BMSSC/dentin transplant showed a positive staining on the peritubular dentin (black arrows). Connective tissue (CT) showed a negative immunostaining for DSP antibody. (I) Immunohistochemical staining of human-specific anti-mitochondria antibody showed that human DPSCs differentiated into dentinogenic cells lining the surfaces (open arrows) or trapped within (open triangles) the newly generated reparative dentin-like tissue (ND). DPSCs were also found within the connective tissue compartment (CT) at 8 wks post-transplantation. (J) Negative control of immunohistochemical staining on DPSC/dentin transplant with pre-immuno serum. Bar, 40 µm in panels A and B, 20 µm in C-J. E and F were stained with hematoxylin & eosin.
