Evaluations On Physical Property And Biological Compatibility Of A Novel Absorbable Chitosan-collagen Membrane For Guided Bone Regeneration

Objective:Guided bone regeneration (GBR) has been performed for reconstruction of alveolar bone defects around dental implants for almost20years. In the GBR process, a barrier membrane plays a key role by protecting the defect from invasion of epithelial and connective tissues, that may otherwise prevent the growth of new bone tissue. New compound biodegradable GBR membranes made by a combination of different biodegradable polymers or by adding bioactive inorganic, which improves the quality of reconstructed bone tissue in GBR and dental implant therapy better than previously used membrane types during the last decade. The purpose of this research is to fabricate a novel absorbable chitosan-collagen membrane which can be applied in guide bone regeneration in dental implant. Physical characteristics, biological compatibility and degradation were assessed by mechanical testing, in vitro cellular assessments and in vivo absorption tests.Methods:(1)2%chitosan glacial acetic acid solution (CHS-L) with low molecular weight(MW50,000-190,000) and0.3%collagen aqueous solution were mixed at volume ration by2:1(A),1:1(B) and1:2(C).1%NaOH solution was added to adjust the mixture to neutral solution. The mixtures were frozen in refrigerators at-20℃for24h and-80℃for12h. Chitosan-collagen membranes were fabricated by freeze-drying in the vacuum condition. Physical properties of A and B membrane were evaluated by elongation test and mercury injection experiments. Surface structure differences among A, B and Bio-Gide were observed and compared by scanning electron microscope(SEM).(2)2%chitosan glacial acetic acid solution (CHS-L) with low molecular weight,1%chitosan glacial acetic acid solution (CHS-M) with medium molecular weight (MW190,000-310,000) and0.1%collagen (MW300,000) glacial acetic acid solution (COL) were dialyzed to neutral solution respectively before mixed. Four solutions with chitosan and collagen at volume rations of2:1and4:1were made. The mixtures were frozen in refrigerators at-20℃for48h and-80℃for24h. Chitosan-collagen membranes were fabricated by freeze-drying in the vacuum condition. The films were sterilized individually by EO after cut to suitable size.(3) Sterilized membranes were soaked in culture solution for48h with membrane and solution at6cm2/ml ratio. MC3T3-E1cell proliferation activity was tested by MTT and comparing OD490values between experimental group and control group.(4) Sterile chitosan-collagen membranes were embedded in8rabbits subcutaneous tissue on back. The remaining membrane materials were drawn out in2rabbits after operation on1w,2w,4w and8w. Chitosan-collagen membranes degradation rate in vivo was analyzed by comparing the weight diminution before and after implantation.Results:(1) A, B, C, three kinds of freeze-dried membrane were made by CHS-L and collagen. Film-forming ability of group A and B are better than that of group C (not for physical performance evaluation). The mechanical properties of group A are better than group B. Porosity of group A (89.41%) and group B (83.92%) were tested by mercury injection experiments. The observation of membrane morphology by SEM showed that the median pore diameter of group A and group B was larger than Bio-Gide.(2) Both2%CHS-L and1%CHS-M can be mixed uniformly with0.1%COL. Mixture of chitosan and collagen exerted favorable film-forming characteristic by vacuum freeze-drying. Chitosan-collagen membrane made of CHS-M had lager thickness and density than which made of CHS-L. Freeze-dried film of CHS-L, due to poor physical performance, was not for biocompatibility evaluation.(3) CHS-M and COL freeze-dried membranes had no cytotoxicity to MC3T3-Elby MTT. From the4th day, especially, freeze-dried membrane made of chitosan and collagen solution at4:1volume ration showed significant differences of MTT values between experimental group and control group(P<0.01).However, freeze-dried membrane made of chitosan and collagen solution at2:1had no significant differences(P>0.05).(4) No obvious inflammatory response was caused by chitosan-collagen membrane made of CHS-M in subcutaneous tissue embedding. The degradation rate of absorbable chitosan-collagen membranes is26.45%(group G) and43.83%(group F) after embedding at8week, but the remaining membrane has been broken or swelling to slurry.Conclusion:(1) The freeze-dried chitosan-collagen membrane shows good physical characteristics and biocompatibility.(2) Freeze-dried compound absorbing film, made of dialysis CHS-M and COL glacial acetic acid solution, displays better film-forming ability, biocompatibility and biodegradability with volume ratio at4:1. These results suggest that the novel hybrid membrane made by chitosan and collagen in this research would have the potential as a GBR membrane.