| BackgroundGuided bone regeneration (GBR) membrane which used as barrier membrane to keep bone orientative matrix in the defect zone, can shield fibroblast cells and other nonosteoblastic cells grow into the defect area, allowing bone tissue regenerate in the remaining space under the membrane, in order to reduce the loss of bone graft. Collagen membrane which has good biocompatibility and biodegradability,can promote cell proliferation and adhesion, and has been widely used as guide bone regeneration membrane in clinical. But pure collagen membrane has week mechanical performance, fast degradation and poor waterresistance, that can not match tissue growth speed. Chitosan is a kind of biodegradable natural gather cationic biological polysaccharide. Its degradation products are glucosamine and small molecules, which are non-toxic, no stimulation to the human body, and has a good histocompatibility. Chitosan molecules take a positive charge, have negative interactions with bacterial membrane and interfere bacteria membrane function. Chitosan has antibacterial function.This research use collagen type I from bovine tendons and chitosan to prepare guide bone regeneration membrane with perfect mechanical strength, biocompatibility and antibacterial properties, through crosslinking, in order to improve insufficient of pure collagen guide bone regeneration membrane Considering the postoperative complications of GBR surgery such as the soft tissue burst, the membrane exposure and infection, to make GBR membrane has antibacterial properties, through add chitosan in collagen, will reduce infections caused by membrane exposure, promote bone ossify,and promote the healing of soft tissue at the same time, improving the success rate of GBR surgery.This experiment use collagen type I from bovine tendons (collagen, COL) and chitosan(CS) of85%deacelation degree, according to mass ratio(COL:CS=3:1), to produce Chitosan-collagen I type composite membrane. Comperaed with pure collagen type I membrane, to test the physico-chemical properties of Chitosan-collagen type I composite membrane, inoculate HGF-1cells and MG63osteoblasts-like cells in this two membranes, and observe cells proliferation on the them, in order to evaluate the physico-chemical properties and biocompatibility of Chitosan-collagen type I composite membrane. At the same time the antibacterial properties of chitosan-collagen type I composite membrane was tested, in order to evaluate its antibacterial function as the barrier membrane of guided bone regeneration.ObjectiveTo prepare Chitosan-collagen type I composite membrane and investigate its physico-chemical properties, testing its biocompatibility with HGF-1cells and MG63osteoblast-like cells, in order to evaluate the feasibility of Chitosan-collagen type I composite membrane as the barrier membrane of guided bone regeneration. Meanwhile to investigate and evaluate its antibacterial properties, in order to provide the reference for its application as guided bone regeneration membrane.Methods1. To extract collagen from bovine tendons and prepare Chitosan-collagen type I composite membrane, through lyophilization, thermal cross-linking and chemical crosslinking with EDC-NHS. We use Chitosan-collagen type I composite membrane as experimental group and collagen type I membrane as control group. Properties of collagen type I from bovine tendons was examined through Amino acid content analysis and DSC. To analyse the physical and chemical properties of group and control group with the infrared spectroscopy(FTIR), scanning electron microscopy(SEM), bibulous rate test and mechanics performance testing.2. To inoculate HGF-1cells with inoculation density of5x104cell/per hole in experimental group membranes and the control group membranes, both of which is with the size of0.5cm x0.5cm(total24pieces in each group). The biocompatibility with HGF-1cell was checked by MTS, which is measured by the absorbency value (OD) in Day1,3,5,7, comparing the proliferation of HGF-1cell in this two groups.3. To inoculate MG63osteoblast-like cells with inoculation density of5x104cell/per hole in experimental group membranes and the control group membranes, both of which is with the size of1.0cm x1.0cm(total12pieces in each group). The biocompatibility with MG63osteoblast-like cells was checked by MTS,which is measured by the absorbency value (OD) in Day1,3,5,7, comparing the proliferation of MG63osteoblast-like cellsin this two groups.4. To inoculate HGF-1cells with inoculation density of5x104cell/per hole in experimental group membranes and the control group membranes, and use SEM to observe the morphological change of cells adhesion, stretching and differentiation in 8h, day1, day3.5. To inoculate MG63osteoblast-like cells with inoculation density of5x104cell/per hole in experimental group membranes and the control group membranes, and use SEM to observe the morphological change of cells adhesion, stretching and differentiation in4h and day1.6. The experimental group membranes and the control group membranes, both of which is with the size of1.0cm x1.0cm(total3pieces in each group), were prepared. Porphyromonas gingivalis(P.g), Actinobacillus actinomycetemcomitans (A.a), Staphylococcus aureus(S.aureus), Escherichia coli (E.coli) were utilized to test the antibacterial effect using oscillametric method.The colonies count were measured by plate count method.7. The experimental group membranes and the control group membranes, both of which is with the size of1.0cm×1.0cm(total3pieces in each group), were immersed in of P.g and bacterium suspension of A.a respectively (the inoculation density bacterium suspension is of108CFU/mL). Use SEM to observe the bacterial adhesion on this two membranes’ surface in24h.8. Statistieal analysis software is SPSS13.0. Factorial analysis of variance were used to detect the interaction effect between experimental group and control group. One-way ANOVA and independent sample t-test were used for comparison. Levene ’test and Melch test were used for the homogeneity of variance.If equal variance, use LSD to do multiple comparison; if not, use Dunnett’ s T3. Hypothesis test is for bilateral inspection, testing standard0.05, when P<0.05, difference is statistically significant.Results1. The amino acid composition analysis and DSC results showed that the three helical structure of collagen type I from bovine tendons has been retained completely. Its component,proportion and the properties accord with the characteristic of collagen type. FTIR results showed that the experimental group has characteristic peak of the collagen and chitosan; hydrogen bond formed between this two materials,meaning the binding force between molecules is storng. The SEM results of experimental group and control group found that surface and cross section of this two membrane has porous structure; the pore diameter of experimental group is bigger, for10-100μm. The hydroscopicity of experimental group was8.38, the control group was9.19. The tensile strength of experimental group was3.75±0.32MPa, significantly higher than those in the control group of which tensile strength was1.54±0.27MPa.2. The MTS results showed that the OD value of experimental group and control group both increased with times, signifying the growth trend was obvious. The biocompatibility with HGF-1cell and MG63osteoblast-like cells in this two group did not have significant difference.3. At8h, there were numbers of HGF-1cells inoculating in both experimental group and control group. Those cells had long spindle shape, differed in size, had rich cytoplasm and thin cytoplasmic process, which was radial pattern. The HGF-1cells inoculating in control group had become flat, and have intercellular contact; HGF-1cells inoculating in experimental group also had intercellular contact, however less than the control group. At24h, HGF-1cells adhered to both experimental group and control group had fully extended, with cytoplasmic process stretching to different direction; cells had established intercellular contact, and joined together with the surface of membranes. At72h HGF-1cells adhered to both experimental group and control group had grown to cover the membrane surface completely, and breeding new cells.4. At4h, the MG63cells adhered to both experimental group and control group had begun to extend, with some cytoplasmic process. At24h, MG63cells adhered in the control group has spread, with cytoplasmic process stretching to different direction; cells had established intercellular contact, and joined together with the surface of membranes, on which a large number of cells matrix secretion can be saw. MG63cells adhered to both experimental group had become flat, and grew between the pore.5. Antibacterial properties test results showed that the bacteria assay reflected to the antibacterial effect,99.5%of P.gingivalis,99.97%of A.a,99.80%of S.aureus, and99.93%of E.coli. Pure collagen type-I composite film do not have antibacterial properties.6. The SEM results showed control group membrane immersed in of P.g and bacterium suspension and bacterium suspension of A.a for24h had a large number of bacterial adhesion, bacteria distributed like crumb; and the bacteria adhered to experimental group was less than that in control group, meanwhile the bacteria was scattered in the distribution and some bacteria shrank, dissolved, even broken.ConclusionChitosan-collagen type I composite membrane has no growth inhibition with HGF-1cells and MG63osteoblast-like cells, and has favorable structure and biological performance, showing wide applications prospects as GBR membrane. Chitosan-collagen type I composite membrane has good physico-chemical properties and surface morphology, no cell toxicity, good surface compatibility, good structure compatibility,which expected to be favorable GBR membrane. Chitosan-collagen type I composite membrane also has strong antibacterial properties, to prevent GBR postoperative infection, having positive significance. |
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