| Objective:To fabricate a novel absorbable membrane made by medium-molecular weight chitosan and collagen, and evaluate the physical and biological property for guided bone regeneration in dental implant.Methods:(1)2%and1%medium-molecular weight chitosan solution and0.1%collagen solution were mixed at volume ratio of2:1, then the mixtures were freezed-dried to make membranes before being frozen at4℃for12h,-20℃for24h and-80℃for24h. The chitosan-collagen membrane made by2%chitosan solution is called H membrane, while the membrane by1%chitosan solution is called E membrane. Surface structure of chitosan-collagen membrane was observed by SEM and compared to Heal-All collagen membrane (ZH-Bio company, China). The mechanical properties including tensile strength and elastic modulus were tested by a universal testing instrument.(2) The sterilized H and E membrane were put into culture medium at37℃for24h, then MC3T3-E1was cultured in the two solution. The cell proliferation level was measured by MTT assay.(3) Cut the sterilized H and E membrane into1×1cm2pieces, and allocated into subcutaneous pouches on the back of10rats randomly. After4w and8w, the remaining materials were collected and prepared for histologic analysis. (4) Two circular calvarial critical-sized defects (d=5mm) were made in SD rats, and covered by chitosan-collagen H membrane, Heal-all collagen membrane and no membrane randomly. At6w, all rats were sacrificed and the histology was performed. The paraffin sections were stained with haematoxylin-eosin and Masson Trichrome for histological evaluation and histomorphometric analysis. The bone regeneration ratio and defect closure was measured and compared among three groups.Results:(1) The chitosan-collagen membranes of group H and E were uniform, smooth, and had good strength and toughness. The H membrane is thicker, and better than E membrane on the strength and toughness. SEM shows that the surface of chitosan-collagen membrane is even and porous, and the average diameter of pores was12±2um. By mechanical testing, the elastic modulus is15.7±7.65MPa, and tensile strength is0.693±0.280MPa.(2) Compared to the normal culture medium, the leaching solutions of H membrane and E membrane have no cytotoxicity to MC3T3-E1cells. Especially, from4to7day, the cells in the H membrane showed higher proliferation level than other two groups.(3) H membrane and E membrane only caused mild inflammatory reaction when implanted subcutaneously. At4weeks, the two membranes were encapsulated by a fibrous connective tissue capsule and some inflammatory cells were infiltrated in the membrane. When compared at8weeks, the fibrous capsule became thinner and the inflammatory cell density reduced. The chitosan-collagen membrane was dissolved and absorbed a little, and when at8weeks, the two membranes still almost remained intact.(4) The rat calvarial model demonstrates that at6weeks, bone regeneration ratio of H membrane is29.18±3.78%, which is significantly higher than the control (9.09±3.56%)(P<0.01). The bone regeneration ratio of commercial collagen is34.15±3.33%, with no difference with H membrane. Conclusion:Chitosan-collagen membrane made by2%chitosan and0.1%collagen solution at volume ratio of2:1, shows good mechanical property and biocompatibility. It can be graded gradually, without severe inflammatory reaction, and keeps intact in vivo for at least8week. It can significantly enhance bone regeneration than control. These results suggest that the chitosan-collagen membrane prepared in this study would have the potential for use as a GBR membrane. |
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