Preparation Of Chitosan Scaffold And Its Preliminary Investigation As Adipose Tissue Engineering Material

Objectives:As a result of congenital malformation,trauma,tumor resection,infection and other factors,soft tissue defects often lead to changes in morphology and contour of human normal tissue,which affect the beauty of patients and cause certain physiological dysfunction,and then have a negative effect on their psychology.At present,dermis transplantation,autologous tissue flap transplantation,collagen injection and artificial synthetic material implantation are often used to repair the reconstruction defects,but they still can not meet the needs of all clinical plastic repair.Repair and reconstruction of soft tissue defects remains a major challenge for plastic surgery.Autograft adipose tissue transplantation is a hot technology.There are many disadvantages such as low survival rate,poor blood supply,easy absorption and repeated injection.Therefore,the use of adipose tissue engineering technology has become a hot spot of research.Adipose tissue engineering is about to inoculate ideal seed cells to three-dimensional scaffolds with good biological performance,and then transfer them to vivo after expansion,directional differentiation and culture.Finally,the scaffold gradually degraded,its degradation products was absorbed by the body,the seed cells differentiated into adipocytes,and the blood transport was established with the transplanted region,which was integrated with the recipient tissue and filled the tissue defect area completely to achieve the purpose of repairing and reconstructing the function.Therefore,in this experiment,we isolated the adipose tissue-derived mesenchymal stem cells(ADMSCs)from the mouse subcutaneous and groin adipose tissue as the seed cells of the adipose tissue engineering,and made the chitosan porous scaffold as the breakthrough point,to detect the porosity,microstructure,mechanical properties and biocompatibility of ADMSCs mice etc many index,in order to find the suitable scaffold for adipose tissue engineering,to accumulate experimental data for the further clinical application of tissue engineered fat.Methods:1.Selection of experimental animal:choosing some of health C57BL/6 mice as experimental animals.Each which weighs 20±1.5g is 4-8 weeks old.All is female.They were bought from experimental animals center of Kunming medical university.2.Primary isolation,culture and identification of adipose tissue-derived mesenchymal stem cells:using type I collagenase digestion method for separation and purification of ADMSCs from mouse adipose tissue,cultured and passaged fourth generations of adherent cells were prepared into single cell suspension,using the detection and identification of cell surface markers by flow cytometry,and ADMSCs differentiation into lipid and osteoblast cells.Oil red O and alizarin red were used to identify the cells differentiation ability.3.Preparation of chitosan scaffold and its porosity,mechanical properties and toxicity test:Three dimensional porous scaffolds of chitosan were prepared by lyophilization,and different crosslinking processes of chitosan scaffolds were explored.The cross-linking time was 2h,4h and 8h respectively.The scaffolds were observed and the microstructure of the scaffold was observed by scanning electron microscopy(SEM).The drainage method is used to detect the porosity of the scaffold.For ductile materials,the tensile properties of the materials are mainly investigated.The cytotoxicity test(MTT method)was done to identify the cytotoxicity of the material.4.Chitosan scaffold composite mouse adipose mesenchymal stem cells:Finally,the chitosan scaffold with crosslinked 8h was selected as the composite stem cell scaffold.The fourth generation mice were cultured with ADMSCs and chitosan scaffold,and the adhesion and growth of the cells on the scaffold were observed by scanning electron microscope.Results:1.The success of ADMSCs isolated from mouse adipose tissue in type I collagenase digestion method,adherent cells,with typical spindle shaped fibroblast like morphology;immunophenotype by flow cytometry showed that the cultured cells of the fourth generation of 98.78%+ 0.73%MSC marker CD44 expression,the expression of MSC marker CD90 98.31%+ 0.57%,however,hematopoietic stem cell markers CD34,CD45 negative expression,consistent with the characteristics of mesenchymal stem cells.After 14 days of ADMSCs adipogenesis,transparent round fat granules were seen in the cells.Orange red lipid droplets were seen after staining with oil red O.After 21 days of osteogenic induction,they had calcium deposition and red compact nodules stained by alizarin red.The results showed that ADMSCs was successfully isolated from the adipose tissue of mice.2.The chitosan scaffold is mostly milky white or yellowish,and the touch has a certain flexibility and softness.Scanning electron microscopy showed that the scaffolds were porous sponge like structures,and pores were connected to each other.The pore size was mainly distributed in the range of 25 to 260 ?m,and the porosity of scaffolds was 92.8%± 2.4%.The cross-linked chitosan scaffold has a three-dimensional porous structure,and the tensile strength increases with the increase of the crosslinking time.The tensile strength of the crosslinked 2h is(2.55 ± 0.89)MPa,the cross-linking 4h is(4.11 ±0.7)MPa,and the cross-linking 8h is(5.73±0.77)MPa.Compared with non crosslinked tensile strength(0.9 ± 0.27)MPa(p=0.04 t=3.08,p=0.002 t=7.38,p=0.0005 t=10.19),the difference was statistically significant(P<0.05).The tensile strength of the chitosan scaffold with cross linked 8h reached the maximum,which was(5.73 ±0.77)MPa.The cytotoxicity test showed that the cell proliferation degree(RGR)was(98.30%+ 5.93%)when concentration of material soaked fluid was 0.5mg/ml,and the proliferation degree(RGR)was(97.03%+ 4.12%)when concentration of material soaked fluid was 1.0mg/ml,and the proliferation degree(RGR)was(99.79%+ 1.82%)when concentration of material soaked fluid was 1.5mg/ml,and the cell proliferation(RGR)was(101.41%+ 3.54%)when concentration of material soaked fluid was 2.0mg/ml.Comparison between the experimental groups and the negative control group,There is no statistical significance in the difference(P>0.05).The cytotoxicity test showed that the cell proliferation was between 90 and 100,and the cytotoxicity was 0 or 1,indicating that the chitosan scaffold material was nontoxic.3.After cultured with adipose tissue derived mesenchymal stem cells and chitosan scaffolds,scanning electron microscopy showed that cells could adhere to and grow on the scaffolds and grow pseudopodium,indicating that chitosan had good cytocompatibility.Conclusions:1.ADMSCs can be successfully separated and purified by type I collagenase digestion.2.The chitosan scaffolds can be successfully prepared by lyophilization technology.It has good three-dimensional structure,namely higher porosity and suitable pore size.After cross-linking,the material has no toxicity to cells,and has good mechanical properties.lt can be used as a scaffold for tissue engineering.3.The cells proliferate and adhere well on the chitosan scaffold,and have good biocompatibility.Chitosan as an ideal scaffold material for tissue engineering is suitable for cell adhesion and proliferation,and it can accumulate experimental data for further clinical repair of soft tissue defects.