The Repairment Of The Rabbit’s Oral Mucosa With Composite Of Bone Marrow Mesenchymal Stem Cells And Poly-letf-lactic Acid/silk Fibroin Scaffold

The research group has studied on the biocompatibility and physico-chemical propertiesof PLLA/SF fiber scaffolds. Compared with pure PLLA, pure SF and PLLA/SF fiberscaffolds of other mix proportions, the scaffolds of P70S30, P60S40and P50S50has betterbiocompatibility and physical and chemical performance. This experiment proposed tocomplicate BMMSCs and PLLA/SF fiber scaffolds, to study the repairing ability for rabbitoral mucosa wound in vitro and in vivo, and try to construct an ideal tissue engineering oralmucosa early.At the same time, this experiment try to evaluate the most potential mixedproportion of PLLA and SF for tissue engineering. The biocompatibility of PLLA/SFscaffold would be evaluated by systemic anaphylaxis test, acute toxicity experiment,hemolysis test, pyrogen test, cell growth and cell phenotype change on PLLA/SF scaffold,observing the structure of BMMSCs and PLLA/SF fiber scaffold complexes, subcutaneoustransplantation experiment and oral mucosa transplantation experiment.Then to discuss thefeasibility of PLLA/SF fiber scaffolds as oral mucosal tissue engineering scaffolds. In order toprovide the experimental basis for the feasibility of BMMSCs and PLLA/SF fiber scaffoldcomposite for repairing oral mucosa,we also evaluate the biocompatibility of the composite ofBMMSCs and PLLA/SF scafold by oral mucosa transplantation experiment and comparisonof wound healing rate. At the same time, the experimental try to evaluate the most idealproportion of PLLA and SF for tissue engineering.Materials and methods1. To evaluate the biocompatibility and biological safety of PLLA/SF scafold byexperiment in vitro and in vivo.1.1To evaluate the biocompatibility and biological safety of PLLA/SF scaffold by systemic anaphylaxis test, acute toxicity experiment,hemolysis test and pyrogen test with the threeroportion of70:30,60:40and50:50.1.2To culture BMMSCs on PLLA/SF fiber scaffold:Isolation, culture and identification ofrabbit BMMSCs, to culture BMMSCs on PLLA/SF fiber scaffold.To observation of growth ofBMMSCs on PLLA/SF fiber scaffolds by inverted microscope, to use CCK-8Kit(Cellcounting kit-8) to test cell proliferation.The control group has no scaffolds,to identify ofBMMSCs cell phenotype change by using immunofluorescent.Observing the structure of thecomplex by confocal laser scanning microscope and scanning electron microscope.2. To evaluate the biocompatibility of PLLA/SF scaffold and composite of BMMSCsand PLLA/SF fiber scaffold, to evaluate the healing effect of rabbit oral mucosal woundby implantation experiments in vivo.2.1PLLA/SF scaffoldimplantation experiments with guinea pigs subcutaneously and oralmucosa of New Zealand rabbit.2.2Composite of BMMSCs and PLLA/SF scaffold implantation experiments with oralmucosa of New Zealand rabbit.2.3To suture the complex of BMMSCs and PLLA/SF fiber scaffolds in New Zealand rabbitoral mucosa woundof,Dil staining is used for BMMSCs.Staining with Actin-Tracker Green,the experiment observed the fusion of BMMSCs with surrounding tissue by confocal laserscanning microscope.2.4Comparison of the wound healing rate of the sevevn groups which are natural repairmentgroup, P70S30scaffold group,P60S40scaffold group,P50S50scaffold group,composite groupwith BMMSCs and P70S30scaffold,composite group with BMMSCs and P50S50scaffold,composite group with BMMSCs and P70S30scaffold.Application of DP2-BSW microscopicimage control and analysis software,SPSS21.0statistical software for data analysis.Results1. The results of systemic allergic experiment: the extract of the PLLA/SF fiber scaffoldswith three ratio didn’t cause allergic symptoms such as nasal scratching, piloerection,difficulty in breathing, shock and death in guinea pig.According to the classification table of the allergic reaction degree, it belonged to level0.2. The results of pyrogen test: The increased numbers of every New Zealand white rabbits’body temperature was less than0.6℃.The sum of increased numbers of each group of NewZealand white rabbits’ body temperature was below1.4℃. It was in accordance withprovisions of the Pharmacopoeia of the detection of pyrogen test.3. The results of hemolysis test: After centrifugation,the upper stratum of each centrifugetubes in the experimental group and negative group was the colorless liquid,and the lowerstratum was erythrocyte precipitate. However,the upper stratum of each centrifuge tubes inthe positive group was the red liquid. The hemolysis rate was calculated according to thenumerical work of absorbance, and was compared with ISO standard. The result was that thehemolysis rate of the PLLA/SF fiber scaffolds with three ratio was less than5%.4. The results of acute toxicity test: All Kunming mice had normal food intake during theobservation period for72h, were in good condition, moved freely, and had no toxicityreactions of breathing difficulties and death. At the same time the mice had no significantchanges in weight before and after the experiment. The average daily increased weight ofeach group of mice had no significant difference (P>0.05).5. The results of the isolation, culture and identification of rabbit BMMSCs: Rabbits’BMMSCs was isolated and cultured successfully. The growth and proliferation condition ofcells is good.The expression of CD105and CD44on the surface of cells byimmunofluorescence staining confirmed the cells cultured were BMMSCs.6. BMMSCs proliferation and phenotype changes on PLLA/SF fiber scaffolds: The cellproliferation rate of P70S30scaffold was significantly higher than P60S40scaffold andP50S50scaffold,there had statistical significance on the difference in the fifth day and sixthday (p<0.01). The expression of CD105and CD44on the surface of cells confirmed that therewas no significant effect of PLLA/SF fiber scaffold material on cell phenotype of BMMSCs.7. The culture results of composite of BMMSCs and PLLA/SF fiber scaffold: BMMSCscould adhere to PLLA/SF fiber scaffolds in three ratio, cell growth status was good.8. Subcutaneous transplantation experimental results of PLLA/SF fiber scaffolds: Theresults showed that the inflammatory response caused by the three scaffolds were light, the inflammatory response caused by P70S30scaffold was the lightest.All animals were in goodcondition.9. Oral mucosa transplantation experimental results of PLLA/SF fiber scaffolds: Thefusion of PLLA/SF fiber scaffolds in three ratio was good after transplanted for a week, thescaffolds had degraded partly,most oral wound healed and PLLA/SF fiber scaffolds didn’tcaused obvious inflammation.All animals were in good condition.10. Oral mucosa transplantation experimental results of the complex of BMMSCs andPLLA/SF fiber scaffolds: The fusion of the complex of BMMSCs and PLLA/SF fiberscaffolds in three ratio was good after transplanted for a week, the scaffolds had degradedpartly, most oral wound healed and the complex of BMMSCs and PLLA/SF fiber scaffoldscaused lighter inflammation compared with the group of PLLA/SF fiber scaffolds. All animalswere in good condition. The results showed that BMMSCs on scaffolds in three ratio haddifferent degrees of fusion to the surrounding normal tissues. The group of the complex ofBMMSCs and P70S30PLLA/SF fiber scaffolds had the strongest fusion degree around thenormal tissue.11.The statistical results of healing rate: The complex of BMMSCs and P70S30PLLA/SFfiber scaffolds had the highest healing rate.Statistical significance on the difference wasshown between P70S30PLLA/SF fiber scaffolds and other groups (P<0.01).Conclusions1. PLLA/SF fiber scaffold material has good biocompatibility, and has the potential as ascaffold for tissue engineering oral mucosa.2. The complex composed of BMMSCs and PLLA/SF fiber scaffolds has betterbiocompatibility than pure PLLA/SF scaffolds, has better oral mucosal repairing ability thanpure PLLA/SF scaffolds and the body itself. The complex composed of BMMSCs andPLLA/SF fiber scaffolds has the potential as a scaffold for tissue engineering oral mucosa.3. The complex composed of BMMSCs and P70S30PLLA/SF fiber scaffolds is the mostbeneficial to cell growth and wound healing of oral mucosa,and has the most ideal potentialfor constructing tissue engineering oral mucosa.