Effect Of Mechanical Loading On Behavior And Fate Of Bone Marrow Mesenchymal Stem Cells In Hyaluronic Acid-based Scaffolds

Background:Large bone defects caused by severe trauma,orthopedic diseases,tumor resection or infection are still a major challenge faced by orthopedics.Currently the clinical treatment strategies are mainly autograft,allograft and xenograft bone transplantation and so on,but there are some problems such as insufficient donors,immune rejection and infection risk.Tissue engineering strategy provides a new idea for bone defect repair.At present,tissue engineering strategy mainly studies bone regeneration by simulating intramembranous osteogenesis and endochondral osteogenesis.Compared with endochondral osteogenesis,the lack of vascularization in intramembranous osteogenesis easily leads to central necrosis.In addition,a number of studies have shown that the bone repair rate simulating the process of endochondral osteogenesis is faster.The strategy of endochondral osteogenesis in tissue engineering mainly includes scaffold materials,seed cells,biochemical factors or mechanical stimulation.Hyaluronic acid,alginate,polylactic acid,etc.are common sources of scaffold materials in tissue engineering.Previous studies have shown that chondrocytes,mesenchymal stem cells,induced pluripotent stem cells,etc.are seed cells that are widely used in osteochondral tissue engineering.In addition to scaffold materials and seed cells,tissue engineering also promotes bone defect repair by adding biochemical factors or applying mechanical stimulation.More and more studies have shown that mechanical stimulation plays an important role in regulating cell adhesion,proliferation and differentiation.The study confirm that cells can sense and respond to mechanical stimulation.However,the intervention and regulation mechanism of mechanical stimulation on cells in the bionic scaffold for endochondral osteogenesis have not been fully revealed.Methods: Firstly,different proportions of hyaluronic acid-based hydrogel scaffolds were made,and their physical characteristics(scanning electron microscope,hydration degree and swelling ratio,mechanical detection)and biological characteristics(living-death staining,H&E staining,KI67 staining)were tested and evaluated to obtain hydrogel scaffolds with appropriate proportions.Secondly,a periodic static pressure model is established,and mechanical stimulation was applied to hydrogel scaffolds in common culture conditions and induced culture conditions.The distribution,polarization,proliferation,chondrogenic differentiation,hypertrophy and maturation of rat bone marrow mesenchymal stem cells(r BMSCs)in the hydrogel scaffold are evaluated by H&E staining,immunofluorescence staining,scanning electron microscope,and nanoindentation.Results:(1)The physical characterization results show that the hydrogel scaffold with the ratio of 2%HAMA+2%Alg has suitable hydration degree and swelling ratio,suitable pore structure,good space maintenance ability and excellent mechanical properties.The results of biocompatibility evaluation showed that r BMSCs had good adhesion and extension in hydrogel scaffolds with the ratio of 2%HAMA+2%Alg,and had good survival and proliferation.(2)Under short-term periodic static compressive stress,the distribution of r BMSCs in hydrogel scaffolds is more uniform,and the statistical results of the polarity distribution of r BMSCs show that the cells in the polarity distribution state under mechanical loading obviously increase.In addition,the results of H&E and KI67 staining showed that the number of cells in proliferation state in the loading group and induced &loading group was significantly higher than that in the control group and induced group.(3)Short-term mechanical stimulation plays an important role in regulating the chondrogenic differentiation of r BMSCs.The results of immunofluorescence staining showed that the expression of cartilage markers(COLⅡ,ACAN,SOX9)in the loading group and induced & loading group were higher than those in the control group and induced group,respectively.(4)Under normal culture conditions,long-term mechanical stimulation promotes the hypertrophy and maturation of chondrocytes induced by r BMSCs;Long-term mechanical stimulation in culture condition inhibits the hypertrophy and maturation of chondrocytes induced by r BMSCs.The results of H&E and scanning electron microscope showed that there were larger vesicular cells in the induced group and the induced & loading group.The staining results of cartilage hypertrophy differentiation markers MMP-13 and COLⅩ showed that COL Ⅹ positive cells begin to appear in the induced group and induced & loading group after two weeks of mechanical loading,but the distribution range is small.After four weeks of mechanical loading,the expression of COLⅩ and MMP13 also appear in the loading group.The statistical results showed that COLⅩ and MMP13 in induced group is the most widely distributed.(5)Nano-indentation results showed that the induced group has the highest hardness,followed by the loading group,the induced & loading group and the control group.After that,the relative calcium ion deposition of scaffolds in each group was analyzed,and it is found that the induced group has the highest relative calcium ion deposition,followed by the loading group and induced & loading group,and the control group has the least,the results are consistent with those of immunofluorescence staining.Conclusion:(1)Hyaluronic acid-based hydrogel scaffold with the ratio of 2% HAMA+2% Alg has excellent physical properties and good biocompatibility.(2)Short-term static compressive stress promotes the uniform distribution,polarization and proliferation of r BMSCs in scaffold.(3)Periodic static compressive stress is beneficial to chondrogenic differentiation of r BMSCs.(4)Under normal culture conditions,long-term mechanical stimulation promotes the hypertrophy and maturation of chondrocytes induced by r BMSCs.Long-term mechanical loading inhibits hypertrophy and maturation of chondrocytes induced by BMSCs.(5)Under common culture conditions,long-term mechanical loading promotes ECM and mineral accumulation.However,in the induced culture conditions,long-term mechanical loading had no significant effect on mineral accumulation.