The Osteoinductive Mechanism Of Konjac Glucomannan/ Nano-Hydroxyapatite/Chitosan Scaffolds

Objective Previous studies that have shown konjac glucomannan/nano-hydroxyapatite/chitosan (KHC) bone substitute scaffolds loaded vancomycin cationic liposome have good resistance to infection and bone repair activity for infected bone defects in rabbits. The problem that how to induce new bone formation for KHC bone substitute scaffolds in must be clear KHC bone substitute material development process.The osteoinductive mechanism of KHC bone substitute scaffolds was discussied in BMPs and FGFS/FGFR signaling pathway by the study that KHC effect of stem cells, osteoblast proliferation and differentiation biological behavior of cell.MethodsThe first part1.The C57BL mouse bone marrow mesenchymal stem cells were cultured and amplified by the method of bone marrow adherent.and identified by morphological and biological function.2. BMSCs that grow in the bone substitute scaffold was observed by the inverted phase contrast microscope, scanning electron microscopy in morphology, and adhesion situation.3. BMSCs growed in KHC bone substitute scaffold and cultured in 24-well plates,a simple BMSCs culture as a negative control group, with 0.64% phenol and BMSCs composite culture as a positive control group, at 24h,48h,72h collect samples by MTT assay BMSCs proliferation.4. Real-time PCR to detect the expression of osteocalcin (OCN)and Runx2 which BMSCs grow KHC bone substitute. Experimental groups were as follows:cells were cultured alone as a negative control group, people decalcified bone matrix(DBM) as a positive control, anegative control group:BMSCs+DMEM-F12; negative induction group:BMSCs+osteogenic medium; DBM group:BMSCs+DMEM-F12+DBM; DBM induction group:BMSCs+osteogenic medium+DBM; KHC group:BMSCs+ KHC+DMEM-F12; KHC induction group:BMSCs+KHC+osteogenic induction medium. By understanding osteogenic differentiation of KHC bone substitute scaffold for BMSCs in vitro studies the osteogenic mechanism.The second part1.MC3T3-E1 osteoblasts that grow in the bone substitute scaffold was observed by the inverted phase contrast microscope, scanning electron microscopy in morphology, and adhesion situation.2. MC3T3-E1 osteoblast growed in KHC bone substitute scaffold and cultured in 24-well plates,a simple BMSCs culture as a negative control group, with 0.64% phenol and MC3T3-E1 osteoblast composite culture as a positive control group, at 24h,48h,72h collect samples by MTT assay BMSCs proliferation.3.Real-time PCR to detect the expression of Runx2, BMP-4, BMPRI A, FGFR-1 which MC3T3-E1 osteoblasts growed in KHC bone substitute scaffolds. Experimental groups were as follows:cells were cultured alone as a negative control group, the same kind of DBM as a positive control, a negative control group:MC3T3-E1 osteoblast+a-MEM medium; negative induction group:MC3T3-E1 osteoblasts+ osteogenic medium; DBM Group:MC3T3-E1 osteoblast+a-MEM medium+DBM; DBM induction group:MC3T3-E1 osteoblasts+osteogenic medium+DBM; KHC Group:MC3T3-E1 osteoblasts+KHC bone substitute+a-MEM medium; KHC induction group:MC3T3-E1 osteoblasts+KHC bone substitute scaffold+osteogenic induction medium;ResultsThe first part1.The inverted phase contrast microscope to form uniform, active and passage of a large number of very capable fusiform adherent cells, osteogenic medium for 21 days, a large number of calcified nodules confirmed the extracted cells are BMSCs that have a goog ablitity of osteogenic differentiation.2.BMSCs grow in KHC bone substitute scaffold cultured cells grew well, inverted phase contrast microscope material around BMSCs vigorous growth, cells were spindle, arranged direction. Scanning electron microscopy to a large gap in the stretch BMSCs in the cradle and good adhesion between cells pseudopodium adhesion to KHC bone substitute bracket.3.MTT assay KHC bone substitute scaffold for the proliferation of BMSCs show: KHC bone substitute was statistically significant (P<0.05) between scaffold group and control group differences, KHC bone substitute scaffold and BMSCs co-cultured 24h,48h,72h cytotoxicity was 2,1,1.4. All osteogenic gene expression was higher than the negative control group, the chemical composition of bone induced gene expression was higher than non-induced group, KHC group Runx2 in the first 14 days upregulated 1.8 fold, OCN in the first 14 days upregulated 2-fold, osteogenic negative control cells cultured under conditions with the highest level of expression.The second part1. MC3T3-E1 and KHC bone substitute scaffold cultured cells grew well, inverted phase contrast microscope material around MC3T3-E1 vigorous growth, fusiform cells arranged sexual orientation. Scanning electron microscopy to KHC bone substitute scaffold porosity between a large number of MC3T3-E1 osteoblast ingrowth, cell pseudopodia interconnected, adhesion to KHC bone substitute bracket.2. MTT assay KHC bone substitute scaffold for the proliferation of MC3T3-E1 show: KHC bone substitute was statistically significant (P<0.05) between scaffold group and control group differences, KHC bone substitute scaffold and MC3T3-E1 co-cultured 24h,48h,72h cytotoxicity was 2,1,1.3. The expression of BMP-4 in each group reached the highest level at 2w, BMP-4 in KHC osteoinductive group was significantly higher than other groups,3w all groups BMP-4 expression levels decreased slightly. Over time, the expression of KHC group BMPRI A gradually increased up to 3.2 times the 3w. Runx2 expression of negative induction group reached the highest at 2w, KHC group at 1w and 3w expression inhibited the expression reached the highest at 2w, DBM group reached the highest expression of Runx2 at 3w. FGFR 1-at 1w,2w suppressed when expressed in each group reach the peak expression at the 3w.Conclusion1. The method of whole bone marrow adherence in the volume fraction of 5%CO2 37℃ in a humidified incubator with 10% FBS-DMEM-F12 medium can be cultured in vitro amplification of mouse BMSCs.2.BMSCs in KHC bone substitut scaffold spread well on the stand, KHC bone substitute scaffold may stimulated the expression of Runx2 OCN, activate the BMPs, MAPK, Wnt signaling pathways and other osteogenic promote new bone formation. On the other hand, KHC bone substitute scaffold cell adhesion by changes in cell morphology, changeing in intracellular mechanical conduction effect, activating osteoblast signaling pathways, thereby promoting osteoblast differentiation.3.KHC bone substitute scaffold through induction of BMP-4, BMPR I A BMPs signaling pathway activation, which results in activation Runx2, promotes osteoblast maturation. Besides, KHC inducing the expression of FGFR-1, activating FGFs/ FGFRs system to further activate Runx2, BMPs, osteogenic differentiation regulation, to promote new bone formation.