Biological Effects And Cellular Mechanism Of Low-intensity Pulsed Ultrasound On BMSCs Of Rabbit Cultured In Lginate Beads

The avascularity of the articular cartilage and lack of access to the subchondral vascular supply present two of the factors that limit the ability for spontaneous healing of cartilage defects that do not penetrate the subchon -dral bone plate.This has led to the development of surgical techniques such as abrasion,microfractures and drilling of the subchondral bone plan that introduce a vascular-mediated healing response to the injured cartilage by creating access to the subchondral bone marrow vascular system.These socalled marrow stimulation techniques introduce the classic elements of vascular healing by producing a fibrin-rich clot that contains pluripotential blood and bone marrow mesenchymal stem cells(BMSCs),cytokines,and growth factors.Laboratory investigations have shown that microfracture penetration of the subchondral bone results in the formation of a hybrid fibrohyaline repair cartilage tissue.Although unable to produce predictable and durable clinical improvement,this method indicated articular cartilage had the ability for spontaneous healing.Low-intensity pulsed ultrasound accelerates bone healing via upregulation of cartilage formation and maturation phases of endchondral bone formation.In the animal model,daily low-intensity pulsed ultrasound had a significant positive effect on the healing of osteochondral defects.Although a great deal of research has been conducted on the biologic and biochemical regulation of the behavior of these cells,very little is known about the biological effects and cellular mechanisms of low-intensity pulsed ultrasound on BMSCs.Object:To explore the effects of micro-enviroment of chondrocyte culture in alginate bead on BMSCs differentiation.To explore the biologic effects and cellular mechanism of low-intensity pulsed ultrasound(LIPU) on BMSCs viability,proliferation,phenotype,function and gene expression of collagen typeⅡ,aggrecan,TGF-β1,IGF-Ⅰ,bFGF mRNA,which control and/or regulate BMSC to differentiate directionally towards chondrocytes.Method:1.The chondrocytes were isolated from the articular cartilage of rabbits by mechanical smash and enzyme digestion,and proliferated through monolayer culture in vitro.2.The bone marrow mesenchymal stem cells of rabbit were isolated from bone marrow by flowing bone marrow cavity combining with the density gradient centrifugation,purified and proliferated through monolayer culture.3.BMSCs and chondrocytes were co-cultured in alginate beads in vitro, whose morphology,viability and proliferation was observed.4.LIPU was applied to BMSCs and chondrocyte co-culture system which were divided into 3 groups randomly:2mW/cm~2,10mW/cm~2 and 30mW/cm~2.The cell morpho -logy,viability and proliferation was observed periodically.The synthesis and secretion of collagen typeⅡ,collagen typeⅠ,collagen typeⅩand glycosaminoglycans were tested through immunohistochemistry and toluidine blue method,while the gene mRNA and protein expression of aggrecan,Collagen typeⅡ,Collagen typeⅠ,Collagen typeⅩ,TGF-β1,bFGF and IGF-Ⅰwere determined through RT-PCR and Western-bloting.Results:1.A large number of seeding cells with high proliferation ability can be harvested by enzyme digestion and monolayer culture,and cells of 1st-3rd generation chondrocytes have the same morphologic features as the primary source,that can be confirmed by immunocytochemical staining of collagen typeⅡ.2.Purified BMSCs can be obtained through the method of density gradient acentrifugation combing monolayer culture in vitro,whose phenotype can be examined by flow cytometry.3.BMSC and chondrocytes cultured in alginate bead can keep good prolife -ration,and BMSCs which co-cultured with chondrocytes differentiated into chongdrocytes,that was confirmed by positive staining of typeⅡcollagen immunocytochemical stain.4.LIPU had significant effect in accelerating BMSCs proliferation but did not affect the cell viability.The biological effects of LIPU varied with its intensity.LIPU of 2mW/cm~2,10mW/cm~2 can induce BMSC to differentiate into chongdrocytes,that were charactered of improvement of the synthesis and secretion of collagen typeⅡand glycosaminoglycans(GAG),which might be realized by upregulated the gene mRNA expression TGF-β1 and IGF-Ⅰ.LIPU of 30mW/cm~2 can induce BMSC to differentiate into hypertrophic chongdrocytes, that were charactered of improvement of the synthesis and secretion of typeⅩcollagen,which might be realized by upregulated the gene mRNA expression of bFGF.Conclusion:1.The method of mechanical smash combining enzyme digestion and monolayer cultured in vitro can acquire pure and enough isolated rabbits chondrocytes.2.Purified BMSCs can be obtained through the method of density gradient acentrifugation combining monolayer culture.Cells of 1st-3rd generation with high proliferation were fit to the further experiment3.Alginat scaffold appears to provide the satisfactory 3-dimensional support for BMSCs and chondrocytes,and the micro-enviroment of chondrocyte culture in alginate bead can induce BMSCs differentiated into chongdrocytes without adding cytokine in vitro.4.LIPU had significantly positive effects on remaining BMSCs viability, promoting proliferation.The biological effects of LIPU varied with its intensity.LIPU of 2mW/cm~2,10mW/cm~2 can induce BMSC to differentiate direction -ally towards chongdrocytes in vitro,while LIPU of 30mW/cm~2 can induce BMSC to differentiate into hypertrophic chongdrocytes,wbich may differentiate into osteoblasts automatically when cultured for longer time.5.Low-intensity pulsed ultrasound,a form of mechanical energy as high frequency acoustic pressure waves can produce micromechanical stresses on cells,that may plays a role in the modulation of cytokine synthesis and parathyroid hormone response in mesenchymal cells and prechondrocytes,which can influence the synthesis and secretion of collagen typeⅡand collagen typeⅩ.