| The present study was aimed to develop a novel b FGF/Chitosan derivatives/collagen composite hydrogel scaffold and to test the physical and chemical properties. By in vitro cell culture,the biocompatibility and odontoblast differentiation of this hydrogel to mouse dental papilla cells(m DPC6T) are evaluated. Furthermore, to detect the synergy between b FGF and Chitosan derivatives/collagen composite hydrogel as well as the potential of the scaffold used for regeneration in the dentin-pulp complex. The object is made up of two experiment as follows.PartⅠFabrication of the b FGF/Chitosan derivatives/collagen hydrogel scaffold and the physical and chemical properties testObjective By chemical modification of Chitosan to obtain its water soluble derivatives;Further, the well-physicochemical properties b FGF/Chitosan derivatives/collagen hydrogel scaffold is fabricated.MethodsBy chemical modification of Chitosan, corresponding Chitosan derivatives are obtained, including sulfonated Chitosan(SCS), phosphorylated Chitosan(PCS) and sulfonated and phosphorylated Chitosan(PSCS). Then the protein solubility of them are tested and their chemical structures and grafting rate are verified by fourier transform infrared spectroscopy(FTIR).Moreover, to fabricate of the b FGF/Chitosan derivatives/collagen hydrogel scaffold and to detect their physicochemical properties consisting of p H with β-glycerol phosphate(β-GP), gelation time.Results1.Three kinds of Chitosan derivatives had favorable water-soluble. At same time, sulfonated Chitosan was superior to the other two. 2. Characteristic peaks of sulfonated groups and phosphorylated groups were verified by FTIR and MNR. 3. As 8-10 %β-GP of total hydrogel scaffold, p H value situated 8-9.5. 4. Gelation times of different kinds of hydrogel were in the 8 to 9.5 minutes range.Conclusions1. Sulfonated Chitosan had remarkable solubility property. 2. Composite hydrogels were suitable to be used as a cell carrier apply for tissue engineering whenβ-GP of total hydrogel scaffold reaching 8-10 %.PartⅡ Biological detection of b FGF/Chitosan derivatives/collagen hydrogelObjectiveTo detect biocompatibility and morphological features of this scaffold to m DPC6 T. The potential of b FGF/Chitosan derivatives/collagen hydrogel used for odontoblastic differentiation and reparation.Methods1.To screen effective concentration of b FGF acting on m DPC6 T 2.m DPC6 T are seed into the surface of hydrogel, then the adhesion and biocompatibility of cell in the scaffold are observed by cytoskeleton staining. 3. The proliferation ability of cell seed into the surface of different hydrogel was detected by Cell Counting Kit-8( CCK-8). 4. To explore whether there exists synergy between b FGF and Chitosan derivatives/collagen composite hydrogel by comparing differences of differentiation and mineralization.Results1. 12.5~25 ng/ml b FGF was active to proliferation of m DPC6 T. 2. It was found that m DPC6 T were growth and expanded well in the both collagen hydrogel and Chitosan derivatives/collagen hydrogel. Filamentous cytoskeleton and expression of cytoskeleton were observed. 3. b FGF/PSCS/ collagen hydrogel group to cell proliferation was superior to the others. Meanwhile the proliferation of b FGF/SCS/ collagen hydrogel and b FGF/PSCS/ collagen hydrogel groups become stronger followed with the time. 4. Alkaline Phosphatase(ALP) staining and activity all discovered that b FGF/PSCS/ collagen hydrogel group in promoting differentiation effect was more obvious than the other group. 5. b FGF participating in group generated more red mineralized nodules in 7 d, while they generated less ones in 14 d.Conclusions1. 12.5~25 ng/ml bFGF to m DPC6 T were most significant. 2. Chitosan derivatives/collagen hydrogel had well biocompatibility and adhesion effect. 3. Introduction of sulfonated group can effectively improve the biological activity of composite hydrogel scaffold. |
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