| Objective:Bone defects,such as trauma,degeneration or tumor,remain the major challenges of bone reconstruction.Bone allograft and autograft are currently the criteria for the treatment of bone defects.There are limits to both treatments,such as the donor organization is limited,high donor site morbidity,complex operation and allogeneic grafts the spread of disease or the possibility of immune rejection,and not to shape implants into the accurate shape of bone defect that lack of alternative availability.How to realize the functional reconstruction of bone tissue has always been a difficult problem for the medical community,and it is a good idea to organize engineering scaffolds to solve this problem.The aim of this experiment was to prepare the scaffold of collagen/chitosan/hydroxyapatite 3d bionic bone tissue engineering by freeze-drying method,and observe the proliferation and differentiation of bone marrow mesenchymal stem cells in 3d composite scaffold of collagen/chitosan/hydroxyapatite.Methods:1.Isolation culture and differentiation of mesenchymal stem cells in rat bone marrow: The bone marrow of healthy male SD rats was extracted,A single nucleus cell was isolated by density gradient centrifugation,and the mesenchymal stem cells were isolated by pasting.The cell growth curve was detected by cck-8 method.After osteoinduced differentiation culture,the mineral nodules were performed to evaluate the osteogenic differentiation.2.Preparation of collagen/chitosan/ hydroxyapatite composite scaffolds: The scaffold of collagen/chitosan/hydroxyapatite 3d bionic bone tissue engineering was prepared by freeze-drying method.The general form of the composite scaffold is observed by the naked eye,the external morphological characteristics of the internal structure and the surface of composite materials were observed under scanning electron microscope.The porosity of stents was determined by liquid displacement method.The degradation rate and pH value of the degradation liquid were detected in the in vitro simulation fluid.To test the mechanical properties of the stents using the unic 5865 mechanical test machine.3.The effect of composite stents on the adhesion,colonization,migration and differentiation of mesenchymal stem cells: Using BMSCs as seed cells,the scaffold cells were cultured with collagen/chitosan/ hydroxyapatite composite scaffolds as carriers.The cell adhesion rate and cell migration were detected by laser holographic cell analysis and imaging system.The growth of cells in composite scaffolds was observed using an inverted microscope and scanning electron microscope.CCK-8 method was used to detect cell activity after compound culture.The cytoskeleton was observed by the stylus cyclopeptide staining of methyl rhodamine.qRT-PCR method was used to detect the expression of osteocalcin(OCN),osteocalcin(OPN)and type I collagen(COL-I)in the 2W after cell inoculation.Results:1.The concentration of bone marrow mesenchymal stem cells was obtained by separating the marrow cavity of male SD rats.Under the inverted microscope,the cells were observed to be round,the cytoplasm was plump,the size was uneven,and the growth was good.Over time,the number of cells increases,and most cells form long fusiform.The growth curve of bone marrow mesenchymal stem cells was detected by cck-8 method,and the "S" trend was presented in the whole,which reflected the growth cycle characteristics of mesenchymal stem cells.When the bone marrow mesenchymal stem cells were cultured in 2W,alizarin red staining and alkaline phosphatase staining were positive,indicating that bone marrow mesenchymal stem cells were differentiated and secreted by the osteoblast cells.2.It is observed that the surface of the stent is smooth and the pore distribution is uniform,and it has a three-dimensional porous communication structure.Scanning electron microscopy observation showed how aperture bracket internal three-dimensional structure,the average is 200-250 microns in diameter,hole wall thickness in 6 microns,the aperture between suitable bone marrow mesenchymal stem cell migration and the transport of nutrients.In addition,the surface of the material also has the laminated shape pore,which is beneficial to the adhesion and extension of seed cells.The porosity of the shelf was(71.1 plus or minus 2.2)%,the degradation rate of the 8th week was(26.3+1.8)%,and the fluctuation of the stent degradation liquid p H was between 6.5 and 7.5.The mechanical property test results confirmed that the stent was tough and soft after soaking in PBS,with the shape of the elastic modulus(26.14892±6.53726)MPa.3.The results showed that the adhesion rate of NSCs on scaffolds increased with the prolongation of culture time and reached 85.3 ± 10.1% after 24 h of planting.Under the microscope,the BMSCs were adhered to the surface of the scaffold,and the scaffolds were staggered with each other.CCK-8 test results confirmed that collagen/chitosan/ hydroxyapatite threedimensional composite scaffold material is non-toxic,and can be seen in the stent surface adsorption of BMSCs,was beaded,Promote the growth and proliferation of BMSCs.The expression of osteocalcin(OCN),osteopontin(OPN)and type I collagen(COL-I)in the co-culture group were higher than those in the basal medium group after 7 days and 14 days of induction.The difference was statistically significant(P <0.05).Conclusions:1.This experiment was conducted with a simple and no special condition,the whole bone marrow adherent wall culture method can successfully isolate the BMSCs of the SD rats with high biological activity,high purification degree and multi-directional differentiation potential.2.The low temperature freeze drying technology can successful preparation of collagen/chitosan/ hydroxyapatite composite scaffolds,stent aperture crisscross each other,a multi-layer three-dimensional structure,high specific surface area,dense micro porosity,mechanical performance is good.3.Collagen/chitosan/hydroxyapatite 3d composite scaffolds have good biocompatibility and can promote the proliferation and differentiation of bone marrow mesenchymal stem cells. |
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