The Experimental Research On Cyclic Axial Compression Stress Promoting The Bone Regeneration Ability Of Matrix-based Tissue Engineering Bone

BackgroundApproximately 6 million people in China suffer from bone defects or bone dysfunction every year,of which approximately 4 million require bone transplantation treatment.At present,there is a common problem of insufficient osteogenic activity in bone transplant materials.To address this challenge,we previously proposed a novel tissue engineering bone strategy —— matrix-based tissue engineering bone(M-TEB).This strategy involves using freeze-drying and other methods to remove the activity of seed cells cultured on bone scaffold materials,while retaining the matrix components derived from the seed cells,to construct tissue engineering bone materials with excellent bone-inducing biological activity.M-TEB has the advantages of easy material extraction,convenient storage and transportation,low immunogenicity,and good bone induction ability.However,it also faces problems such as insufficient blood supply and low osteogenic ability in the early stage of implantation.Bone marrow mesenchymal stem cells(BMSCs),as the most commonly used seed cells in bone tissue engineering,are often accompanied by the expression and secretion of various cytokines and growth factors during osteogenic differentiation.These factors play an essential role in maintaining bone homeostasis and bone remodeling.BMSCs are sensitive to mechanical stimuli,and specific external mechanical loads can guide their osteogenic lineage differentiation.Therefore,the osteogenic lineage differentiation of BMSCs can be regulated by loading specific external mechanical loads,thereby altering the expression and secretion of particular factors in BMSCs.Cyclic axial compression stress(CACS)is a kind of compressive stress with cyclic changes in amplitude and axial loading,which has been partially studied to guide the differentiation of MSCs lineage.This study selected CACS as the external mechanical load on the growth of BMSCs and proposed a strategy to enhance the bone regeneration ability of M-TEB by introducing this stress during the M-TEB construction process.Furthermore,we optimized the CACS loading scheme suitable for the osteogenic differentiation of BMSCs and explored the specific mechanism by which CACS functioned.Osteoclast precursors(POCs)are immature osteoclasts,and the PDGF-BB secreted by them has been proven to promote neovascularization and new bone formation during bone remodeling.The previous research of our group found that BMSCs and POCs were used as seed cells in a 10:1 ratio,and the M-TEB constructed under static conditions showed better bone defect repair ability compared to the M-TEB based on BMSCs.Osteoclast is also proven to be sensitive to external mechanical stimulation,which will affect its growth and differentiation.Therefore,we further constructed M-TEB derived from BMSCs/POCs under3 D dynamic culture conditions,investigated the bone regeneration ability of this material,and studied the roles which CACS played in the construction of this material.Methods(1)We optimized a stress loading scheme which is suitable for the differentiation of BMSCs in the osteogenic lineage by observing the effects of stress frequency,amplitude,single continuous action time,and continuous cultivation days of CACS on the expression levels of osteogenic-related genes in BMSCs.(2)This study constructed M-TEB based on BMSCs under 3D static or 3D dynamic cultivation conditions,and denoted as B＿M-TEB,B(+)＿M-TEB;Constructing a critical bone defect model of the rat femur to evaluate the bone regeneration ability of each group of M-TEB;The effect of CACS on BMSCs cultured in 3D conditions was analyzed by transcriptome sequencing and explored the potential mechanism of B(+)＿M-TEB bone regeneration ability superior to B＿M-TEB.(3)We constructed M-TEB based on BMSCs and POCs in a 10:1 ratio under 3D dynamic or 3D static culture conditions,denoted as B/P(+)＿M-TEB and B/P＿M-TEB;Evaluated the bone regeneration ability of B(+)＿M-TEB,B/P＿M-TEB and B/P(+)＿M-TEB by measuring the differences in VEGFA and PDGF-BB protein concentrations and the repair situation of femoral defects in rats after implantation of the three groups of M-TEB;Using RT-q PCR assay,immunoblotting assay,and enzyme-linked immunosorbent assay to study the specific mechanism of CACS enhancing the bone regeneration ability of B/P(+)＿M-TEB was verified through corresponding blocking experiments.Results(1)The CACS loading scheme suitable for the directional differentiation of bone formation lineages in BMSCs is CACS with a frequency of 1.0 Hz and a strength of 10 k Pa,which is loaded at a rate of 4 h/d for 14 days.(2)B(+)＿M-TEB showed better bone defect repair ability compared with B＿M-TEB in the critical bone defect model of the rat femur;After being exposed to CACS,the expression of phosphorylated p38 protein in BMSCs was significantly upregulated(P<0.001),thereby enhancing the expression of p38 MAPK pathway-dependent VEGFA protein in BMSCs;Compared with the other two groups of conditioned medium,the conditioned medium derived from B(+)＿M-TEB showed a stronger promoting effect on the migration,proliferation of EPCs,and osteogenic differentiation of BMSCs.(3)The concentrations of VEGFA and PDGF-BB in B/P(+)＿M-TEB were significantly higher than those in B(+)＿M-TEB and B/P＿M-TEB(P<0.05,P<0.001;P<0.001,P<0.001),and showed a better promoting effect on bone defect repair in vivo;During the B/P(+)construction process,CACS inhibited osteoclast differentiation of POCs,but upregulated the expression of PDGF-BB gene and protein,and enhanced VEGFA expression in cells by activating the p38 MAPK pathway in BMSCs;The PDGF-BB secreted by POCs acted on BMSCs and synergistically promoted VEGFA expression in BMSCs with CACS.ConclusionThis study optimized the CACS loading scheme suitable for osteogenic lineage differentiation of BMSCs.For BMSCs derived from M-TEB,CACS enhanced the bone regeneration ability of the material by upregulating the expression of p38 MAPK pathwaydependent VEGFA in BMSCs.For BMSCs/POCs derived from M-TEB,CACS enhanced the expression of PDGF-BB in POCs and promoted the expression of VEGFA protein in BMSCs together with PDGF-BB from POCs;Animal experiments confirmed that this material had better bone defect repair effects.This study proves that it is feasible to guide the differentiation of seed cells’ lineage and optimize the bone regeneration ability of M-TEB by regulating the stress microenvironment during the construction of M-TEB.