Regulation Of Chondrogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By Stiff Differential Hydrogel Combined With Compressive Stimuli

Background:The repair of cartilage injury is still a thorny problem in the clinic.The self-healing ability of cartilage tissue is limited.The damaged cartilage tissue usually degenerates gradually,which affects the structural integrity of the joint,and eventually leads to functional damage and degenerative lesions in the joint.There is no clinical method to completely restore the structural and functional integrity of cartilage at present.The development of tissue engineering technology provides an opportunity to construct ideal cartilage implants in vitro.In creating cartilage tissue engineering scaffolds,mechanical inducing factors have the same important role as biochemical inducing factors,gradually attracting attentions.In healthy cartilage tissue,the mechanical force generated by daily exercise is essential for maintaining the dynamic balance of extracellular matrix deposition and remodeling of cartilage.Moreover,appropriate mechanical stimulation is significant for maintaining normal chondrocyte phenotype and metabolic activity.However,how mechanical stimuli carry out transduction with cells and how mechanical signals affect cell proliferation and differentiation remain unknown.Objectives:In this research series,relevant exploration will be carried out to explore the effects of mechanical stimulation on the proliferation and differentiation of bone marrow mesenchymal stem cells(BM-MSCs)and seek an appropriate mechanical scheme for cartilage repair.Hyaluronic acid(HA)was selected as the primary material of the cell culture matrix.Methacrylate hyaluronic acid(Me-HA)was prepared by reacting methacrylic anhydride with HA.Through functional modification,Me-HA can be photo crosslinked under the catalysis of the photoinitiator.Me-HA hydrogels with different mechanical strengths were prepared using the different relative molecular weights of HA.In the first experimental system,BM-MSCs were cultured on the surface of hydrogels with different matrix stiffness.The effects of matrix stiffness on cell morphology and proliferation behavior were investigated to explore the mechanism by how cells sense external mechanical signals.Subsequently,BM-MSCs were loaded inside Me-HA hydrogels with different stiffness in vitro,and the effect of matrix stiffness on inducing chondrogenic differentiation of BM-MSCs was explored.After verifying the impact of matrix stiffness on BM-MSCs in vitro,the BM-MSCs loaded Me-HA hydrogel was implanted into animals to repair osteochondral defects,confirming its repair effect on osteochondral defects in vivo.In the second experimental system,we developed a suite of mechanical stimulation devices to apply compressive stress to BM-MSCs.The effects of stress stimuli to induce chondrogenic differentiation of BM-MSCs and possible mechanism were explored.It provides a reference for the construction of osteochondral scaffolds with biomimetic structure and function by mechanical stimulation in the future.Materials and methods:In this study,Me-HA was used as a hydrogel material,and lithium phenyl-2,4,6-trimethyl-benzoyl phosphorite(LAP)was used as a photoinitiator to catalyze Me-HA to crosslink photopolymerization.By choosing HA with different relative molecular weights,Me-HA was constructed with different relative molecular weights.The successful synthesis of the material was characterized by ~1H nuclear magnetic resonance and infrared radiation spectroscopy,and suitable photoinitiator concentrations were selected through cytotoxicity experiments.Subsequently,we prepared Me-HA hydrogels with different mass fractions characterized by the storage modulus and compressive strength of the hydrogels by rheometer and universal mechanical testing machine.The Me-HA hydrogels with suitable mechanical strength were selected as the carrier to study the effects of matrix stiffness on BM-MSCs proliferation and differentiation.The pore structure and biocompatibility of the hydrogels were verified by scanning electron microscopy,live/dead cell staining,and in vivo degradation experiments.In the in vitro experiments,we implanted BM-MSCs onto Me-HA hydrogel surface and into the hydrogels,respectively,verifying the effect of matrix stiffness on the cellular morphology,proliferation,and differentiation behavior of BM-MSCs,and explored the possible mechanisms.In the in vivo experiments,different matrix stiffness hydrogels loaded with BM-MSCs were used to repair osteochondral defects.The repair effects of different matrix stiffness on cartilage and bone tissues were verified by micro-computed tomography,histological evaluation,and so on.After investigating the impact of matrix stiffness on chondrogenic differentiation of BM-MSCs,we applied compressive stress to the hydrogel.We deliver stress stimuli to BM-MSCs to investigate the phenotype changes associated with chondrogenic differentiation with gene and protein levels,the expression of mechanical signal protein was also analyzedResults and conclusions:In this study,Me-HA hydrogels that can be rapidly photo crosslinked and have good biocompatibility were prepared by functional modification of HA.By adjusting the relative molecular weight of HA and the mass fraction of Me-HA,hydrogels with different matrix stiffness with different storage modulus and compressive strengths were prepared.After cultured BM-MSCs on the surface of hydrogels with different matrix stiffness,phalloidin staining for cellular actin revealed that cells had a larger spreading area on the surface of hydrogels with higher stiffness.The mechanical signal affected the configuration of actin after being introduced intracellularly.It leads to the reorganization of the cytoskeleton under the stress environment caused by the change of matrix stiffness.By immunofluorescence staining of Yes associated protein(YAP),we found that after cells sense the mechanical signal,the distribution of mechanical signal YAP in the cytoplasm and nucleus was affected.And culturing stiff hydrogel leads to more distribution of YAP in the nucleus,affecting the transcriptional behavior of cells.It is indicated that BM-MSCs can affect the distribution of YAP and cell behavior by changing actin conformation after sensing external mechanical signals.In the in vitro experiment to explore matrix stiffness on chondrogenic differentiation of BM-MSCs,the chondrogenic differentiation degree of BM-MSCs was low in soft hydrogel and gradually increased with the increase of matrix stiffness.But in the stiff hydrogel,BM-MSCs tended to differentiate into hypertrophic cartilage.It indicated that the increased stiffness of the matrix would benefit the differentiation of BM-MSCs toward the chondrogenic direction.Still,the excessive stiffness would induce the differentiation of BM-MSCs into hypertrophic cartilage.In the in vivo experiment on the effect of matrix stiffness on BM-MSCs differentiation,soft hydrogel loaded with BM-MSCs was less suitable for cartilage and subchondral bone repair.In contrast,the medium stiffness hydrogel had a better effect on repairing cartilage tissues than the other two groups.In contrast,the stiff hydrogel had a better impact on the repair of subchondral bone.In vivo experiments also demonstrated that hydrogels also enhanced the osteogenic differentiation of BM-MSCs with increasing matrix stiffness,and the stiff matrix hydrogels had a better effect of inducing osteogenic differentiation of BM-MSCs.In the second system,where external compressive stress was applied,BM-MSCs showed various differentiation performances in response to external stress stimuli.In soft matrix hydrogels,compressive stress stimuli activated downstream mechanotransduction pathways by elevating Rho-associated kinase(ROCK)expression in BM-MSCs They promoted their tendency toward chondrogenic differentiation but had no apparent effect on their trend toward hypertrophic chondrogenic differentiation.However,with the increased stiffness of the hydrogel matrix,the external stress stimulus is gradually reduced to improve the expression of ROCK protein in BM-MSCs,still has a promoting effect on enhancing the tendency of BM-MSCs to undergo chondrogenic differentiation in vivo.This study validated the pathway of cells sensing external mechanical signals.It investigated the role of matrix stiffness change for BM-MSCs proliferation,differentiation,and external applied compressive stress on BM-MSCs chondrogenic differentiation,leading to the conclusion that appropriate compressive stress on BM-MSCs under suitable conditions is helpful to improve the chondrogenic differentiation of BM-MSCs.The implementation of this study will provide a reference for the construction of osteochondral tissue engineering scaffolds with the application of mechanical stimuli in vitro.