The Chondrogenic Inductive Effect Of Cyclic Compression On The Composite Of Bone Marrow Derived Mesenchymal Stem Cells And Chitosan/Silk Fibrin/nano-Hydroxyapatite Multilayer Scaffold In A Three-Dimensional Culture System

Objective To observe the structure,biomechanical properties,cell compatibility and degradability in vivo of novel CS/SF/n-HA multilayer composite scaffold To survey the chondrogenic inductive effect of cyclic compression on BMSCs/scaffold composite in three-dimensional culture system To observe the reparative effect of BMSCs/scaffold composite on unilateral femoral condyle cartilage defects of mini bigsMethods Ca?NO₃?₂·4H₂O and?NH₄?₂HPO₄ were used as raw materials to synthesize n-HA by precipitation at room temperature.After extraction of SF,CS/SF mixture was prepared with CS:SF concentration ratio of 1:3,2:2,3:1 to fabricate the upper three layers of the scaffold.CS/n-HA complex was obtained by in situ precipitation in a CS:HA ratio of 3:7to prepare the bottomlayer of the scaffold.Low temperature freeze molding method was applied to mold and freeze-dried layer by layer.After soaked in 2%NaHCO₃ to neutralize and cross-linked by sodium tripolyphosphate?TPP?,the scaffold mechanical strength was further enhanced.Porosity of the scaffold was measured by liquid displacement method.The internal structure of the scaffold was observed by HE staining,and 20 pores w ere randomly selected from each layer of the scaffold.The average pore size of each layer was measured and calculated by ImagePro Plus 5.0 image processing software.The scaffold compression modulus in dry and wet state was tested using a mechanical tester.The scaffold microstructure was observed using Micro-CT and scanning electron microscopy?SEM?.SD rat bone marrow mesenchymal stem cells?BMSCs?were harvested for osteogenic,adipogenic and chondrogenic differentiation and passage.Scaffold cytotoxicity was examined by CCK-8 assay.Cell/scaffold co-culture was observed by HE staining,calcein staining and SEM.The scaffold was subcutaneously embedded under the back skin of SD rats to observe its degradation in vivo.Mini pig BMSCs were harvested,purified and amplified.Metabolism and viability of cells seeded on the scaffold under different culture conditions were examined by CCK-8 and live/dead cell staining.Compressive modulus of the cell/scaffold complex in the dynamic culture system was tested by mechanical tester.Chondrogenesis of cells seeded on the scaffold under different culture conditions was observed by toluidine blue staining,Western Blotting,RT-qPCR and Elisa methods.Detection of the phosphorylation of related signaling pathways?Erk1/2,p38,Smad1/5,Smad2/3,BMP2?under different culture conditions was performed by Western Blotting and RT-qPCR.Model of mini pig with unilateral femoral condyle cartilage defect was used to observe the reparative effect of cell/scaffold complex cultured under different conditions on cartilage defects by magnetic resonance,naked eyes,HE staining and safranin O-solid green stainingResults The prepared CS/SF/n-HA scaffold possessed a diameter of 4cm and a thickness of 4mm,presenting white round cake-shape.The scaffold contained four layers linking to adjacent layers closely with connective pores with a porosity of up to 92.2±1.3%and the average pore size of each layer was much larger than the size of BMSCs.The compressive modulus of the scaffold in wet state?medium saturated?was significantly higher than that in dry state.The scaffold was non-toxic and cells/scaffold co-culture showed that cells adhesion,metabolism and migration were good.Scaffold in vivo degraded with an appropriate degradation rate and was finally absorbed.By setting the parameters of the dynamic culture system?bioreactor?,it is possible to simulate the squeeze effect of the self-gravity on the articular cartilage when a person normally walking.Cyclic compression can promote BMSCs metabolism,enhance its vitality and reduce its apoptosis and increase the compression modulus of the cell/scaffold complex.Cyclic compression can promote the chondrogenesis of BMSCs on the scaffold.The MAPK?p38 and Erk?pathway,BMP2,and Smads-related pathway factors are involved in the chondrogenic differentiation of BMSCs under cyclic stress.During this process,phosphorylation of p38,BMP2,Smad1/5,and Smad2/3 was activated,while Erk phosphorylation was inhibited.Conclusion The CS/SF/n-HA multilayer composite bionic scaffold possesses good mechanical properties,cell compatibility and biodegradability in vivo.Cyclic compression on the BMSCs/scaffold complex can promote the metabolism of cells on the scaffold,maintain cell viability,inhibit its apoptosis,enhance the compressive modulus of cell/scaffold complex,promote chondrogenesis of the cells on the scaffold,activate phosphorylation of p38,BMP2,Smad1/5,Smad2/3 and inhibit Erk phosphorylation.The BMSCs/scaffold complex under cyclic compression repaired cartilage defects well...