Exploration Of The Modulatory Effects Of Mesenchymal Stem Cells On The Biological Behavior Of Articular Chondrocytes

Cartilage has limited self-repair capacity and traditional clinical treatments are ineffective in long term.In recent years,cell-based strategy including tissue engineering and cell therapy become very promising to regenerate cartilage defects.In this strategy,cells are the most essential component and currently,the most potential candidate cells are mesenchymal stem cells(MSCs)and articular chondrocytes(ACs).However,both cell types bear their own advantages and limitions.Therefore,a novel idea has been proposed to combine the two cell types for therapeutic applications.Thus far,a series of studies concerning coculturing MSCs and ACs have been carried out to gain a thorough understanding of their interactions,which would lead to the development of methodologies for regulating these interactions.Such that,a potential route can be envisioned for the cell-based strategy in cartilage regeneration.A recent clinical trial to test the application of mixture of MSCs and ACs in treating cartilage damage is especially inspiring.However,due to the complexity in coculture parameters,for example,coculture setup,culture medium and origin of cells,which may vary in different studies,most findings are essentially inconclusive.Hence,a comprehensive understanding of the interactions between MSCs and ACs remain elusive.Moreover,only the effects of ACs on MSCs have been paid attention to in most of studies and whether MSCs can modulate the biological behaviors of ACs is barely paid attention to.In our present work,based on our previous finding on the potential modulatory effects of rabbit bone-marrow-derived MSCs(rbBMSCs)on rabbit ACs(rbACs),a systemic investigation was performed by validating its universality,unraveling the molecular mechanism,characterizing ACs comprehensively and re-evaluaing the interactions between MSCs and ACs in complex coculture settings.The major findings are described as followed.First,in the Transwell-based indirect coculture system,it was found that rbBMSCs could regulate the differentiated phenotype of rbACs,by inducing rbACs to transform from the typical rounded shape into a fibroblast-like spindle shape,downregulating the production of cartilaginous matrix components,and provoking cell proliferation,and this is owing to the paracrine factors of rbBMSCs.To further demonstrate whether this phenomenon is independent of the origin of cells,different MSCs,including human bone marrow-derived MSCs(hBMSCs),human adipose-derived MSCs(hAMSCs)and rat bone marrow-derived MSCs(rBMSCs),were exploited to coculture with different chondrocytes,including rbACs,bovine ACs(bACs)and a mouse teratocarcinoma fibroblastic chondroprogenitor cell line ATDC5,repsectively.It was demonstrated that the three different MSCs could induce similar changes in rbACs,bACs and ATDC5 as rbBMSCs did to rbACs albeit different extents of changes were for different cell types.These findings confirm the universality of the modulatory effects of MSCs on chondrocytes.Next,how MSCs modulate ACs was investigated by coculturing rbBMSCs and rbACs in the Transwell system.By coculturing rbACs with different amounts of rbBMSCs(2×104,2.5×105,5×105 and 1×106 Cells/well),it was demonstrated that both shape transformation and stimulation of cell proliferation of rbACs were positively correlated with the seeding amount of rbBMSCs,indicating the paracrine effects by rbBMSCs.In addition,in the Transwell coculture system,a Rho-associated protein kinase(ROCK)inhibitor Y27632 was supplemented.It was found that Y27632 could refrain rbACs from shape transformation in coculture with rbBMSCs.Importantly,rbACs retained partially cartilaginous matrix production in coculture,especially with the lowest seeding density of rbBMSCs(2×104 cells/well),and showed similar gene expression as those in monoculture control,while cell proliferation was not changed in coculture.These implicate the involvement of RhoA/ROCK signalling pathway in the transformation of rbACs in response to rbBMSCs.Additionally,when an FGFR1/2/3,VEGFR1/2/3 and PDGFR?/? inhibitor BIBF1120 in coculture,the effects of rbBMSCs on rbACs were restricted.By adding FGF-1,VEGF-A and PDGFbb in monocultured rbACs,it was found that a combination of FGF-1,VEGF-A and PDGFbb could potentially recapitulate the major effects of rbBMSCs on rbACs.These results together would support the postulation that rbBMSCs secreted FGF-1,VEGF-A and PDGFbb and mediated the modulatory effects on rbACs.Then,to gain an insight understanding of the nature of changes in rbACs upon coculture with rbBMSCs,rbACs after coculture were subjected to comprehensive characterizations concerning three aspects.(1)Cocultured rbACs were subcultured and demonstrated better proliferative capacity and chondrogenic potential than monocultured rbACs.(2)Flow cytometry was applied to analyze the surface molecules including CD 14,CD81,CD49f,CD51/61,CD49e,CD166,CD90,CD44 and CD29.It was found that cocultured rbACs expressed a differential surface molecular patterne from both dedifferentiated rbACs and rbBMSCs,implicating the different phenotypes.(3)RNA Sequencing was applied to analyze the global gene expression profile of cocultured rbACs,monocultured rbACs and dedifferentiated rbACs.It was shown that genes related to cytokines/receptors,extracelluar matrix,integrins,inflammation,cell proliferation and signaling molecues were differentially expressed among these cells.In particular,compared to monocultured cells,cocultured rbACs showed upregulated cytokine receptor genes(e.g.,IGF1R),proliferation related genes(e.g.,KSR1),cell adhension related genes(e.g.,PECAM1 and VCAM1),cartilaginous matrix synthesis and degradation related genes(e.g.,MMP1,MMP3,HS3ST5 and CHST1),inflammation related genes(e.g.,NOS2,IL4R,IL7,IL18 and IL6)and downregulated key signaling molecules(e.g.,SFRP5).These changes are in consistent with those in both proliferation and matrix production of rbACs in coculture.In the end,coculture was performed in more complex settings.(1)The mixed coculture was carried out by seeding rbBMSCs and rbACs together in culture plates in growth medium.It was found that coculture with direct cell-cell contacts stimulated cell proliferation.However,GAG production was promoted at rbACs:rbBMSCs of 1:1 and inhibited at a ratio of 1:2,indicating that both direct contact and paracrine serection might be involved in the mixed coculture.(2)Alginate gel beads were applied to encapsulate rbBMSCs and rbACs,respectively,which were then cocultured in either growth medium or chondrogenic medium using the Transwell system.It was shown that in growth medium,rbBMSCs could downregulate the differentiated phenotype of rbACs,while in chondrogenic medium,rbBMSCs could further promote the differentiation of rbAC.This indicates that it is medium composition,rather than the three-dimensional microenvironment,that can significantly affect the interactions between MSCs and ACs.Additionally,rbBMSCs did not show significant changes in coculture in growth medium and displayed stimulated chondrogenic differentiation in coculture in chondrogenic medium.Hence,this implicates that the differential statuses of rbBMSCs in different culture media possibly affects their impacts on rbACs in coculture.Collectively,this study performed a comprehensive investigation on the modulatory effects of MSCs on ACs.MSCs can downregulate the differentiated phenotype of ACs,potentially by secreting protein factors such as FGF-1,VEGF-A and PDGFbb.Importantly,cocultured rbACs show superior proliferation and chondrogenic potential than monocultured ones and are distinct from both dedifferentiated rbACs and rbBMSCs.These findings may have great significance to applying coculture of MSCs and ACs as the novel cellular source in cell-based cartilage regeneration.