The Experimental Research On Osteoclast-derived Exosomal Let-7a-5p Promoting Chondrocyte Hypertrophy

BackgroudMost bones develop by the manner named endochondral ossification,such as limbs and spinal columns.Endochondral ossification oucurs from fetal gestation to early adulthood,which growth ceases.Firstly,mesenchymal cells condense and then differentiate into chondrocytes.After chondrocyte hypertrophy,osteoclasts,and other bone-related cells following with blood vessels invade the cartilage,and residual cartilage is gradually replaced by bone cells.Endochondral ossification is a process depending on the collaboration of blood vessels,chondrocytes and osteoclasts.Chondrocytes are the key cells in endochondral ossification.Besides,osteoclasts absorb extracellular matrix so as to osteoblasts replace remaining cartilage.So osteoclasts also play an important role in endochondral ossification.It is reported that chondrocytes secretes vascular endothelial growth factor to regulate angiogenesis and enhance the activity of osteoclasts.And aggrecanases and metalloproteinases expressed by hypertrophic chondrocytes also regulates osteoclasts survival.These studies revealed that interaction between cells in endochondral ossification,which contributes to better understand bone growth and turnover.So our research speculates that osteoclasts may have an effect on chondrocytes.However,there has been few relating reports.Exosomes are a new method to interpret intercellular communication,which have aroused extensive attention.The diameter of exosomes is 40-100 nm,and they have subcellular bilayer membrane.Moreover,exosomes are formed through endocytosis,fusion,and secretion.Biologically active substances in exosomes,for example,lipids,proteins,and coding or noncoding RNAs,are derived from source cells.Exosomes release these substances into target cells to make functions.It is reported that,osteoclast precursors,osteoclasts and osteoblasts secrete exosomes.And osteoclast-derived exosomes are involved in regulation of bone metabolism.In addition,microRNAs in several exosomes can play a role in bone formation.Therefore,our research intends to study the effect of osteoclasts on chondrocytes from the perspective of exosomes.In chondrogenesis,chondrocytes extensively express the Smad proteins.Transforming growth factor-β signaling is specifically transmitted by Smad2 and Smad3,and inhibits the chondrocyte hypertrophy,which is of benefit to the maintenance of articular cartilage.The disorder of the interaction between cells in endochondral ossification leads to skeletal dysplasias.However,valid treatment remains to be found.So it is important to expore the role of osteoclasts in growth plate chondrocytes.It may provide experimental basis for the new methods to cure the skeletal dysplasias and repair bone defects.Materials and methods(1)Firstly,a mouse fetal developmental model was established,and samples were made into sections for immunofluorescence staining.Col10a1 and TRAP were selected to represent chondrocytes and osteoclasts,respectively,and their respective localizations were observed.BMMs were isolated and an osteoclast induction model was established in vitro.BMMs were cultured with α-MEM containing 100 ng / ml RANKL and 30 ng / ml M-CSF for 24 h and 120 h to generate osteoclast precursors and osteoclasts,respectively.And the mode was verified by TRAP staining.ATDC5 cells and MSCs were induced with chondrogenic differentiation-induction medium for 7 d and 14 d,respectively,to generate chondrocytes.Transwell was used to establish an indirect co-culture system of osteoclast precursors,osteoclasts and chondrocytes in vitro,and co-cultured using hypertrophic differentiation-induction medium.After 7 d,the RNA and protein of chondrocytes were extracted,and qRT-PCR and western blot were performed to detect markers related to chondrocyte differentiation.We also established a conditioned culture system.The conditioned medium of osteoclast precursors and osteoclasts were extracted,and the conditioned medium was mixed with an equal volume of the hypertrophic differentiation-induction medium to culture chondrocytes.After 7 d or 14 d,samples were collected for detection of key markers.(2)In our research,GW4869 was used to inhibit exosomes in osteoclast conditioned medium.Exosomes were also directly isolated from osteoclasts,treating chondrocytes to observe whether osteoclast-derived exosomes have an effect on chondrocytes.Exosomes of BMMs,osteoclast precursors,and osteoclast were isolated,and microarrays were used to detect the expression of micro RNAs.Differentially expressed micro RNAs were selected through analysing.let-7a-5p inhibitor or corresponding negative control was used to transfect osteoclasts,so that the expression of let-7a-5p in osteoclast-derived exosomes is inhibited or not affected.Chondrocytes were treated with osteoclast-derived exosomes after transfection for 7 d,and chondrocytes were detected.At the same time,potential target genes of let-7a-5p were predicted and verified with dual-luciferase reporter assay.Results(1)In mouse fetal developmental model,there were regions where chondrocytes and osteoclasts were adjacent or even co-localized.In co-culture system,osteoclast precursors and osteoclasts did not significantly affect the expression of Col2a1 and Sox9,but both of them up-regulated the expression of Col10a1,Runx2,and Mmp13 in ATDC5 cells.Conditioned medium of osteoclast precursor and osteoclast up-regulated the expression of Col10a1,Runx2,and Mmp13 in ATDC5,MSCs cells.Moreover,the relative expression levels of these genes were higher in chondrocytes treated with osteoclast conditioned medium.(2)The conditioned medium of osteoclasts without exosomes had no effect on the expression of hypertrophic differentiation-specific markers,while osteoclast-derived exosomes up-regulated the expression of Col10a1,Runx2,and Mmp13 in MSCs cells.Osteoclast-derived exosomal let-7a-5p was selected according to analysis of microarrays.After inhibiting let-7a-5p,osteoclast-derived exosomes had no effect on the expression of hypertrophic differentiation-specific markers.Moreover,let-7a-5p targeted Smad2 to reduce its activation,thus inhibiting Smad2-mediated signaling.ConclusionOsteoclast precursors and osteoclasts promote chondrocyte hypertrophy,thus promoting endochondral ossification,and the promotion effect of osteoclasts was strong er than that of osteoclast precursors.Osteoclast-derived exosomes play an indispensable role in the promotion effect of osteoclasts.Osteoclast-derived exosomal let-7a-5p targets Smad2 to suppress the the inhibitory effect of the TGF-β signaling on chondrocyte hypertrophy,thus promoting chondrocyte hypertrophy.Understanding of endochondral ossification from the perspective of exosome-based intercellular communication may provide new ideas for bone regeneration and bone tissue engineering to repair bone defects.