| Objective:The aim of this study is to explore the method of periodontal bone defect repair,and establish a tissue-engineered bone based on stem cells to reconstruct periodontal bone defects.Methods: The rabbit periodontal limit bone defects were reconstructed using h UC-MSCs loading on nano-hydroxyapatite/collagen/poly(L-lactide)(n HAC/PLA)regulated by recombinant human bone morphogenetic protein-7(rh BMP-7).The human umbilical cord mesenchymal stem cells(h UC-MSCs)were isolated from discarded Wharton’s Jelly of the human umbilical cord using the explant method.And then their growth curve and surface marker were evaluated using CCK8 kit and flow cytometry,and adipogenic,osteogenic and chondrogenic differentiation of cells were detected by oil red O,alizarin red and alizin blue staining.The conventional “gold standard” osteogenic media(OMD)as control,the effects of rh BMP-7 and/or OMD on the proliferation,osteogenic differentiation and ectopic bone regeneration capability of h UC-MSCs loading on n HAC/PLA were compared.The critical-size periodontal bone defects in New Zealand rabbit were treated with h UC-MSCs+n HAC/PLA+rh BMP-7.X-ray were performed post-operatively and the animals were sacrificed 3 months after operation for histological observation and histomorphometric analysis.Results:1.A complete set of h UC-MSCs culture system was established.HUC-MSCs were cultured using the explant method with serum-free medium.The cultured cells were h UC-MSCs with mesenchymal stem cell characteristics and low immunogenicity.Flow cytometry showed that cells expressed CD73,CD90 and CD105 of mesenchymal stem cells.They did not express surface markers of hematopoietic stem cells,such as CD34,CD45,CD11 a and human leukocyte antigen II class HLA-DR.After conditioned induction,the cells were differentiated into adipocytes,osteoblasts and chondrocytes.2.The effects of rh BMP-7 and/or OMD on the proliferation,osteogenic differentiation and ectopic bone formation of h UC-MSCs loading n HAC/PLA were studied.The cell proliferation,Ca2+ concentration,PO43-concentration,ALP activity,OCN concentration,mineral formation and the ability of subcutaneous ectopic bone formation in nude mice were respectively investigated.The results are as follows:(1)Cell proliferation Rh BMP-7 had no significant effect on the proliferation of h UC-MSCs cells loading on n HAC/PLA,while rh BMP-7+OMD had a significant interaction effect on the proliferation of cells.During the logarithmic growth period,rh BMP-7 and rh BMP-7+OMD had no effect on the proliferation of h UC-MSCs loading on n HAC/PLA;In the non logarithmic growth period,rh BMP-7 and rh BMP-7+OMD significantly inhibited the proliferation of cells.In the whole growth period of h UC-MSCs,besides the initial stress response,OMD had significant effect on cell proliferation,significantly inhibited cell proliferation,and the ability to significantly inhibit cell proliferation from large to small: OMD> rh BMP-7+OMD> rh BMP-7.(2)Ca2+ concentration Ca2+ concentration of h UC-MSCs loading on n HAC/PLA gradually increased with the prolongation of culture time.In different culture period of h UC-MSCs,the effects of rh BMP-7,OMD and rh BMP-7+OMD on Ca2+ concentration of h UC-MSCs loading on n HAC/PLA had the significant effect and interaction effect,and Ca2+ concentration of h UC-MSCs loading on n HAC/PLA was significantly promoted.The ability to significantly promote Ca2+ concentration from large to small: rh BMP-7> OMD> rh BMP-7+OMD.(3)PO43-concentration PO43-concentration of h UC-MSCs loading on n HAC/PLA gradually increased with the prolongation of culture time.In different culture period of h UC-MSCs,the effects of rh BMP-7,OMD and rh BMP-7+OMD on PO43-concentration of h UC-MSCs loading on n HAC/PLA had the significant effect and interaction effect.Irrespective of the factor Time,the effects of rh BMP-7+OMD on PO43-concentration of h UC-MSCs loading on n HAC/PLA had no the significant interaction effect,but rh BMP-7,OMD and rh BMP-7+OMD all significantly promoted PO43-concentration of h UC-MSCs loading on n HAC/PLA.The ability to significantly promote PO43-concentration from large to small: OMD> rh BMP-7> rh BMP-7+OMD.(4)ALP activity ALP activity of h UC-MSCs loading on n HAC/PLA gradually increased with the prolongation of culture time.In consideration of the factor Time,the effects of rh BMP-7,OMD and rh BMP-7+OMD on ALP activity of h UC-MSCs loading on n HAC/PLA had the significant effect and interaction effect.The effect of OMD on ALP activity of h UC-MSCs had the significant effect on the cuture of 14 day,but not 7day.Rh BMP-7,OMD and rh BMP-7+OMD all significantly promoted ALP activity of h UC-MSCs loading on n HAC/PLA.The ability to significantly promote ALP activity from large to small: OMD>rh BMP-7>rh BMP-7+OMD.(5)OCN concentration OCN concentration of h UC-MSCs loading on n HAC/PLA gradually increased with the prolongation of culture time.In consideration of the factor Time,the effects of rh BMP-7,OMD and rh BMP-7+OMD on OCN concentration of h UC-MSCs loading on n HAC/PLA had the significant effect and interaction effect,but the effect of Time on OCN concentration of h UC-MSCs had no the significant effect.The effect of OMD on OCN concentration of h UC-MSCs had the significant effect on the cuture of 14 day,but not 7day,however,rh BMP-7 and rh BMP-7+OMD had the significant effect on the cuture of both 7 and 14 day.Rh BMP-7,OMD and rh BMP-7+OMD all significantly promoted OCN concentration of h UC-MSCs loading on n HAC/PLA.The ability to significantly promote OCN concentration from large to small: rh BMP-7>rh BMP-7+OMD> OMD.(6)Mineral formation On the cell culture of 14 day,rh BMP-7 and OMD had significant effect on mineral formation of h UC-MSCs loading on n HAC/PLA,and rh BMP-7+OMD had an interaction effect on mineral formation of h UC-MSCs.OMD,rh BMP-7 and rh BMP-7+OMD significantly promoted mineral formation of h UC-MSCs loading on n HAC/PLA,and the ability to significantly promote mineral formation from large to small: rh BMP-7>rh BMP-7+OMD> OMD.(7)Ability of ectopic bone formation The pure n HAC/PLA and uninduced h UC-MSCs loading on n HAC/PLA had no subcutaneous ectopic bone formation after three months of implantation,rh BMP-7 and/or OMD all could induce h UC-MSCs loading on n HAC/PLA to subcutaneous ectopic bone formation in nude mice.The ability of subcutaneous ectopic bone formation from large to small: rh BMP-7>rh BMP-7+OMD> OMD.3.Reconstruction of rabbit’s periodontal bone defect The h UC-MSCs uninduced in vitro loading on n HAC/PLA had better bone regeneration in the rabbit periodontal bone defect(in situ bone environment)after the transplantation of three months,suggesting that there were some inducing bone regeneration factors in the orthotopic bone environment,while h UC-MSCs induced in vitro by rh BMP-7 loading on n HAC/PLA had best bone regeneration,indicating h UC-MSCs+n HAC/PLA+rh BMP-7 tissue engineered bone could replace autogenous bone to reconstruct periodontal bone defects in rabbits.Conclusion:1.The results showed that the tissue-engineered bone complex with n HAC/PLA,rh BMP-7 and allogeneic h UC-MSCs might be a better alternative to autologous bone for the clinical repairment of periodontal bone defects.2.The cells were cultured using a complete set of h UC-MSCs culture system established in this study,and the cultured cells were h UC-MSCs with mesenchymal stem cell characteristics and low immunogenicity. |
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