Application Of Three-layer Integrated High Bionic Scaffold To Repair Osteochondral Defects

Section ?Repair of articular osteochondral defects using an integrated,highly biomimetic,trilayer scaffoldObjectiveRepair of articular cartilage defects using bilayer scaffolds is problematic because tissue-engineered cartilage is prone to overgrowth toward subchondral bone.A"twice freeze-drying" technique was used to construct a dense isolation layer between the cartilage and subchondral bone layers in an integrated bilayer scaffold to prevent cartilage from excessive downgrowth and improve the efficacy of repair.Method(1)Determination of the chitosan/gelatin solution concentration used for the isolation layer and cartilage layer:A chitosan/geltatin solution[0.25%,0.5%,1%,or 2%(w/v)]was formed by mixing chitson and gelatin at a ratio of 2:5(w/w).Four kinds of scaffolds were prepared with four concentrations of mixed solution by a freeze-drying method.Then the pore size,porosity,water absorption,biomechanical properties,cell seeding rate and cell adhesion rate were tested to determine the suitable concentration of the mixed solution used for isolation and cartilage layer.(2)Production and characteristics of the integrated trilayer scaffold:?-TCP was used for the subchondral bone layer of the scaffold,high-concentration chitosan/gelatin solution for the dense isolation layer,and low-concentration chitosan/gelatin solution for the cartilage layer.After preparation,gross observation,transmission of high-intensity visible light,SEM scanning,interfacial adhesion strength test and cell proliferation,migration and distribution test in the scaffold were used to determine whether the three-layer structure of the scaffold and the boundary of each layer were clear,the connection between the layers was tight.Likewise,an integrated bilayer scaffold without a dense isolation layer was produced.(3)In vitro experiments:Chondrocytes were seeded on the cartilage layer of the integrated trilayer scaffold and bilayer scaffold.The scaffold with the cells was incubated for 28 days to construct tissue-engineered cartilage.Histological examination and biochemical analysis were used to confirm the quality of the tissue-engineered cartilage.(4)In vivo experiments:A standard osteochondral defect of 6 mm in diameter and 9 mm in depth was drilled in the medial femoral condyle using a standard 6-mm hollow drill.The animal experiments were conducted using four groups of sixteen randomly assigned goats each:? the mosaicplasty group,?the bilayer scaffold group,?the trilayer scaffold group,? the cell-free scaffold group.In addition,two goats comprised the normal group and eight goats that received no treatment for osteochondral defects were used as the control group.The repair effect was assessed by macroscopic observation,histological examination,immunohistochemical analysis,biomechanical testing,biochemical analysis.Results(1)Determination of the chitosan/gelatin solution concentration used for the isolation layer and cartilage layer:Based on the measure of the pore size,porosity,water absorption,biomechanical properties,cell seeding rate and cell adhesion rate,we found that 0.25%and 0.5%chitosan/gelatin scaffolds were suitable for the cartilage layer of the integrated trilayer scaffold.(2)Characteristics of the integrated trilayer scaffold:Interfacial adhesion strength testing and observation by high-intensity visible light transmission showed that the three-layer structure was clear and the boundaries between different layers were clear.The SEM results suggested that the properties of the scaffolds did not change after freeze-drying two times.The results of the detection of cell proliferation,migration and distribution in the scaffold indicated that the intermediate layer prevented the chondrocytes downgrowth into the bone layer.(3)In vitro experiments:The results of histological examination and biochemical analysis showed that there was no obvious difference between the tissue engieered cartilage constructed by the bilayer scaffold and trilayered scaffold in vitro.(4)In vivo experiments:The results of macroscopic observation,histological examination,immunohistochemical analysis,biomechanical testing,biochemical analysis at 3 months and 6 months showed the trilayer scaffold prevent the cartilage overgrowth into the subchondral bone effectively with better cartilage repair.ConclusionIn this study,a trilayer integrated composite scaffold using chitosan,gelatin,and?-TCP was successfully constructed by the freeze-drying method.The scaffold consisted of a cartilage layer,isolation layer,and bone layer,with an average effective pore size of 21.3 ± 3.8 ?m in the isolation layer.The results showed that the trilayer scaffold effectively prevented excessive cartilage downgrowth and better simulated the multi-layer structure of cartilage and subchondral bone under normal physiological conditions,thus its repair effect was superior to that of a traditional bilayer scaffold.Section ?To investigate a "Titanium-Mesh-Cage-based"Multilayered monolithic scaffoldObjectiveTo evaluate the biological safety of the " Titanium-Mesh-Cage-based"scaffold.The scaffolds mixed with rat articular chondrocytes were used to construct a tissue-engineered cartilage.Method(1)The scaffold was prepared by the novel " twice frzee-drying" method and 3D printing technology to prepare the trilayer scaffold.The" Titanium-Mesh-Cage-based" was used for the subchondral bone layer of the scaffold,2%concentration chitosan/gelatin solution for the dense isolation layer,and 0.5%concentration chitosan/gelatin solution for the cartilage layer.(2)The scaffolds were assessed via macroscopic observation,SEM scanning.And then the pore size and porosity was observed.(3)The biological safety experiments included system actute toxicity test,sub-acute toxicity test,blood compatibility test and CCK-8 test.(4)The rat articular chondrocytes were seeded into the cartilage layer of the scaffolds in vitro by negative pressure suction technique.The graft rate and attachment rate of the cells were calculated after seeding.Four weeks after culture in vitro,the tissue-engineered cartilage of the scaffolds were evaluated via macroscopic observation,SEM scanning,safranin O and Masson staining.Results(1)The diameter and the height of the scaffold were 6mm and 12mm,respectively.The pore size and porosity of the different parts:the cartilage layer was 50?100?m and 995.72±0.90%,the intermediate layer was 20?30?m and 93.18±0.49%.The bone layer was made up of a " Titanium-Mesh-Cage-based"which was 6mm in height,6mm in diameter,3mm in inner diameter.(2)After injection and implantation of extracts and scaffolds,the general condition of experimental animals remained normal.Weight,red and white blood cells of the animals altered slightly after two weeks.Gross observation of liver,heart and kidney remained normal.Hemolytic rate of the scaffold is 2.37%,which meet the requirement.CCK-8 test showed that the scaffold did not induce any cytotoxicity.(3)The graft rate and attachment rate of the biomimetic trilayer scaffold were above 80%and above 70%,respectively.(4)HE,Safranin O and Masson staining showed that the chondrocytes have good adherent and proliferative ability in the scaffolds.ConclusionThe "Titanium-Mesh-Cage-based" Multilayered monolithic scaffold has good biological safety,and the biomimetic trilayer scaffold was suitable for tissue engineering.