| BackgroundBecause of the lack of blood vessels in the articular cartilage,its self-healing ability is poor.If the articular cartilage is damaged and not treated,the cartilage defect will continue to increase,resulting in the disorder of cartilage anabolism and catabolism,osteoarthritis,and even amputation when the disease is serious.At present,the main methods of cartilage defect repair reported in the literature are osteochondral transplantation,micro fracture after debridement,chondrocyte transplantation and so on.Osteochondral transplantation is a good method to repair cartilage defects,but it will cause damage or functional degradation of the donor area,and the donor area is limited,so it can not repair serious cartilage defects,which has great limitations.Microfracture has been reported to be effective only in the treatment of small cartilage defects.Chondrocyte transplantation has a long-term problem that has not been solved,that is,the difficulty of obtaining chondrocytes and dedifferentiation after implantation of damaged sites.There is an urgent need to find a better treatment and improve the effect and efficiency of treatment.The construction of functional tissue-engineered cartilage in vitro has gradually become an important part of cartilage tissue engineering research.The first problem faced by cartilage tissue engineering is the selection of seed cells,which are mainly chondrocytes or mesenchymal stem cells.It is difficult to obtain chondrocytes,which are reported to face the problem of rapid dedifferentiation in many literatures.Mesenchymal stem cells such as adipose mesenchymal stem cells,bone marrow mesenchymal stem cells,cord blood stem cells have been reported to have the potential of chondrogenic differentiation.Adipose stem cells are one of the most widely used adult stem cells in the field of tissue engineering.Compared with stem cells from bone marrow,the number of stem cells in adipose tissue with the same volume is hundreds of times of that in bone marrow,so adipose stem cells have a broad prospect in cartilage tissue engineering.There are different opinions on the concentration of several components such as fetal bovine serum,its,TGF-β3 and so on.In vitro,the differentiation of adipose stem cells into chondrocytes is affected by many factors,especially the mechanical effect.Many studies have found that mechanical stimulation can affect the proliferation,differentiation and extracellular matrix changes of chondroblasts.ECM hardness(usually expressed by young’s modulus or elastic modulus)is an important external stimulation of cells,which plays a key role in chondrogenic differentiation of MSCs.In order to observe the effect of ECM hardness on chondrogenic differentiation,it is necessary to prepare materials with different stiffness and good cell affinity.Generally,the way to change the matrix stiffness is to change the proportion of its polymerization components,so as to make materials with different mechanical strength.However,the change of the surface chemical composition will inevitably result in the change of the matrix polymerization composition.Therefore,the core problem of studying the effect of ECM hardness on the inoculated cells is to find out the chemical composition of the surface without changing the matrix stiffness.The molecular mechanism of mechanical transduction in the regulation of cartilage formation of mesenchymal stem cells(MSc)has not been fully understood.Integrin is located at the beginning of the sensing pathway,which is considered as the mechanical sensor of cells.Integrins are heteropolymerized transmembrane receptors composed of 18 a subunits and 8 β subunits.α subunits are usually responsible for binding to extracellular proteins,while β subunits are usually responsible for intracellular recruitment of regulatory proteins.The mechanism of mechanosensitive components and functions of the integrin subunits is not clear.At present,it is necessary to use appropriate animal models for more thorough research to clarify the therapeutic potential of downstream mechanosensitive signaling events in various diseases and tissue regeneration.Purpose and meaningFirst of all,the proper concentrations of fetal bovine serum,ITS,TGF-injection 3 and other components in hASCs chondroblast induction medium were explored,which not only verified the excellent potential of hASCs chondroblast differentiation,but also laid a foundation for subsequent experiments.A polyacrylamide gel with adjustable stiffness was prepared to observe the effect of ECM with different stiffness on hASCs proliferation and chondrogenic differentiation.Finally,the expression of hASCs integrin on ECM with different stiffness was detected,providing a clue for integrin to sense the mechanism of external mechanical action.The research methods1.The extraction and culture of fat stem cells and the comparison of three kinds of chondrogenic media.2.Preparation,mechanical properties and cell proliferation of polyacrylamide hydrogels with different stiffness.3.Chondrogenic differentiation of hASCs on polyacrylamide gel with different stiffness.Results1.The addition of 10 ng/ml of TGF-mash 3 into the chondrogenic medium in single-layer culture or microsphere culture and the substitution of ITS for FBS as the cell medium additive could promote the chondrogenic differentiation of hASCs.Meanwhile,we also verified the excellent potential of chondrogenic differentiation of hASCs.2.Three groups of polyacrylamide gels with different mechanical properties were successfully prepared and tested for mechanical strength.The average stiffness of the gels in group 1 was 0.67kpa,the average stiffness of the gels in group 2 was 13.44kpa,and the average stiffness of the gels in group 3 was 99.3 1kpa.The hASCs cells on the gel in group 1 were smaller and proliferated more slowly in group 1.3.Chondrocyte markers were detected 3 weeks after induction of hASCs chondroblast on gel in group 3,and the highest level of chondroblast differentiation was observed in hASCs on gel in group 1.ConclusionHASCs has good chondrogenic differentiation potential.The selection of ITS instead of FBS as cell additive in hASCs chondroblast induced medium is more conducive to chondroblast differentiation.HASCs on the less rigid extracellular matrix proliferated more slowly but showed higher levels of chondrogenic differentiation. |
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