2 Characterization of regenerated tissue after extracted tooth transplantation. extracts together with or without MDPSC conditioned medium (CM) were reconstituted systematically with autoclaved teeth in which the chemical components were completely inactivated and only the physical microenvironment was preserved. Their pulp/dentin regenerative potential and angiogenic potential were compared 28 days after ectopic tooth transplantation by histomorphometry and real-time RT-PCR analysis. Results Expression of Monensin sodium an odontoblastic marker, in the regenerated tissues from each four distinct teeth 28 days after transplantation ((Table?1), in the cells from each of three dishes (base pair, dentin sialophosphoprotein To analyze the enhanced endothelial differentiation, human umbilical vein endothelial cells (HUVEC) were cultured in DMEM containing 2 % FBS, 1 g/ml heparin (Lonza, Muenchensteinerstrasse, Switzerland), Monensin sodium 1 g/ml ascorbic acid (Lonza), and 0.4 g/ml hydrocortisone (Lonza) supplemented with the EDTA extracts alone or together with the CM for 14 days. Vascular endothelial growth factor (VEGF) (Lonza), basic fibroblast growth factor (b-FGF) (Lonza), and insulin-like growth factor (IGF) (Lonza) at a final concentration of 1 g/ml, respectively, was used as a positive control. Immunocytochemical analyses were performed for anti-vascular endothelial (VE)-cadherin (primary antibody, 1:50; Acris, Herford, Germany), and the positive cells were observed on a BZ-9000 BIOREVO fluorescence microscope after counterstaining with Hoechst 33342. Statistical analyses Data are reported as means??SD. values were calculated using the Students test and Tukeys multiple comparison test in SPSS 21.0 (IBM, Armonk, NY, USA). Results Pulp/dentin regeneration after tooth transplantation The regenerative potential of the three distinct types of extracted teeth was compared with control nonextracted tooth in an ectopic tooth transplantation assay of SCID mice. Pulp-like tissue with well-organized vasculature was regenerated in the teeth 28 days after MDPSC transplantation as a positive control (Fig.?1a, e). Similar pulp-like loose connective tissue was observed in the transplants of the teeth extracted with HCl, GdnHCl, and EDTA (Fig.?1bCd, fCh) and in the transplant of nonextracted teeth (Fig.?1a, e). The regenerated tissue in the EDTA-extracted tooth transplant (Fig.?1m) had fewer Hoechst 33342-stained cells compared with those in the nonextracted, HCl-extracted, and GdnHCl-extracted tooth transplants (Fig.?1jCl). The histomorphometric analysis confirmed that the regenerated pulp area and cell density of the GdnHCl-extracted tooth transplants and the EDTA-extracted tooth transplants were significantly lower than those of the nonextracted tooth transplants on day 28 (Fig.?1n). The histomorphometric analysis confirmed that the regenerated pulp area in the tooth transplants of the three types of treatment was significantly lower than that of the nontreatment on day 28 (Fig.?1i). There were Monensin sodium no significant Monensin sodium differences in the regenerated area between the HCl-extracted tooth transplant and the GdnHCl-extracted tooth transplant. Transplantation of the EDTA-extracted teeth yielded significantly less regenerated tissue compared with those of the other three teeth on day 28 (Fig.?1i). These results suggest that chemical components extracted by EDTA may mainly generate an inductive microenvironment for pulp regeneration. Immunostaining with a RECA1 antibody revealed neovascularization in the regenerated tissues by nonextracted tooth transplantation and the other three types TSPAN16 of tooth transplantation (Fig.?1oCr). Histomorphometric analysis demonstrated that neovascularization in the nonextracted tooth transplant was significantly higher than that in the HCl-extracted, GdnHCl-extracted, and EDTA-extracted tooth transplants on day 28. There was no significant difference in neovascularization between the HCl-extracted and GdnHCl-extracted tooth transplants, and a significant difference between the EDTA-extracted tooth transplant and others (Fig.?1s). These results suggest that chemical components extracted by EDTA may mainly generate an inductive microenvironment for pulp regeneration and neovascularization. Open in a separate window Fig. 1 Pulp regeneration after ectopic tooth root transplantation. Pulp regeneration after ectopic tooth root transplantation in SCID mice. Twenty-eight days after transplantation of MDPSCs with (a, e, j, o) nonextracted tooth, (b, f, k, p) HCl-extracted tooth, (c, g, l, q) GdnHCl-extracted tooth, and (d, h, m, r) EDTA-extracted tooth. aCh H & E staining. Pulp-like cells (mRNA in the regenerated tissue of the nonextracted, HCl-extracted, and GdnHCl-extracted tooth transplants to that in normal pulp tissue, which was significantly higher than that of the EDTA-extracted tooth transplant (Table?2). Open in a separate window Fig. 2 Characterization of regenerated tissue after extracted tooth transplantation. Twenty-eight days after transplantation of (a, e, j, n) nonextracted tooth, (b, f, k, o) HCl-extracted tooth, (c, g, l, p) GdnHCl-extracted tooth, and (d, h, m, q) EDTA-extracted tooth. aCd In-situ hybridization analysis of mRNA expression of thyrotropin-releasing hormone degrading enzyme (as an odontoblast marker using an anti-sense probe reactive to both porcine and mouse genes..