The Role Of FGFs/FGFRs Signaling In The Development And Injury Repair Of Mice Meniscus

Part I:Expression pattern of FGFRs in the meniscal development of mice after birth.The meniscus is a fibrocartilage tissue located between the tibia and the femoral articular surface of the knee joint.It plays a key role in maintaining joint stability,protecting articular cartilage,cushioning shock absorption,and transmitting body sensation.Meniscus injury is the most common sports injury in the clinic.However,due to its special blood supply characteristics,its self-repair ability is poor when it occurs in the bloodless area(white area).There is currently no effective treatment in clinical practice and surgical resection is always required at the end.However,long-term follow-up results showed that the incidence of osteoarthritis was significantly increased after meniscus resection so that exploring new treatment methods after meniscus injury,especially the biotherapy method to promote self-repair is urgent to be solved.There are certain similarities between the process of development and regeneration.Therefore,we study the development process of the meniscus and interpret its related molecular mechanisms,which can provide clues for the study of the treatment of meniscus injury.Fibroblast growth factors(FGFs)and fibroblast growth factor receptors(FGFRs)are important signaling pathways in vivo.FGFRs participate in the regulation of life activities such as development,degeneration and regeneration of organisms by binding to their specific FGFs ligands,especially in the bone and cartilage.As a kind of fibrocartilage,the meniscus has a certain similarity with the articular cartilage in the developmental origin.We speculate that the FGFs/FGFRs signal may play an important role in the development and disease of the meniscus.Therefore,we use the most widely used animal model in bone research-mice,to study the development of meniscus and the expression of several types of FGFRs,which will lay the foundation for further study of the specific role of FGF signal in meniscus.Methods:1.Male wild-type C57BL/6J mice with good health were selected and grouped according to age:1 day old,1,2,4,6 and 12 weeks old.Take the lower limbs and slice the regular paraffin specimens.2.Sections of the knee joints were stained with Safranin O/Fast Green to assess the changes in cartilage matrix during meniscus development.3.Immunohistochemical staining(SP method)was performed to observe the expression patterns of type I,type II collagen and three kinds of FGFR during meniscus development.Results:1.The results of Safranin O/Fast Green staining showed that the mice had a clear inner-outer partition after birth.In the inner 2/3 area,the Safranin O stained darker and the cartilage matrix was richer;the outer 1/3 area was hardly Safranin O stained.2.The results of collagen immunohistochemical staining showed that the collagen expression of mouse meniscus had obvious specificity:type I collagen was mainly expressed in the outer anterior horn of the meniscus and the entire posterior horn while type II collagen Expressed in the inner area of the anterior and posterior horn.3.The expression of FGFRs in mouse meniscus has obvious spatio-temporal specificity:no expression of three FGFRs was detected in meniscus of 1 day old mice.At1-2 weeks,the expression of three FGFRs was significantly increased but subsequently decreased until 12 weeks old.The expression position also changed from uniformly distributed in the inner region of meniscus to the edge.and until 12 weeks,it is only distributed in the surface of 3-4 cells.However,the three FGFRs in the posterior horn of the meniscus are evenly distributed in the inner zone,and the expression level did not change significantly.Conclusion:The expression of cartilage matrix,collagen and three FGFRs is temporally and spatially specific during the development of meniscus in mice,suggesting that FGFRs play an important role in meniscus.Part II:The role of FGFs/FGFRs signaling in meniscus injury of mice treated with LIPUSLow-intensity pulsed ultrasound(LIPUS)is a clinical therapeutic ultrasound with low intensity levels between 5 to500 mW/cm~2.In tissue,LIPUS hardly produces thermal effects,but affects cell activities such as cell proliferation,migration,and differentiation through mechanical mechanics.LIPUS was approved by the FDA in 1994 to promote fracture healing.In addition,many studies have confirmed that LIPUS can also promote the healing of nerve,muscle,skin and other soft tissues,but the specific mechanism is still unclear.Meniscus injury is a common clinical disease,and the treatment is relatively simple.However,there is no research on LIPUS treatment of meniscus injury.Based on the important role of LIPUS in the field of regeneration and repair,we speculate that LIPUS may contribute to healing after meniscus injury.FGFRs are important regulators in the process of fracture healing and bone formation.In the first part,we also found that a temporio-spatial expression pattern of three FGFRs in mouse meniscus development,suggesting an important role of FGFs/FGFRs signaling in the process of meniscus regeneration.Therefore,we intend to explore the effects of LIPUS on meniscal injury repair and the role of FGFs/FGFRs signals,especially FGFRs,during this process.We established a half-cut model of the medial meniscus in mice to compare the meniscus regeneration process,matrix and collagen content between LIPUS treatment group and control group.We also conducted a series of in vitro experiments to compare the effect of LIPUS treatment on meniscal cell migration behavior and matrix-related gene expression and three FGFRs and further used FGFR inhibitor to confirm the important role of LIPUS in the repair process of meniscus injury in mice.The results showed that LIPUS can promote the regeneration and repair of mouse meniscus injury,promote matrix and collagen secretion,promote the expression of FGFRs,especially FGFR1,3,as well as promote the migration of meniscus cells and expression of matrix related genes in vitro and the effect of LIPUS is significantly weakened after blocking the FGFR signal.The experiment lay a foundation for the further study of the relationship between LIPUS and meniscus repair.Methods:1.10 weeks old male wild type C57BL/6J mice were treated with LIPUS after medial meniscus half-cutting,(5 days per week,20 minutes each time for two consecutive weeks),and the knee joints were taken at 2,4,and 6 weeks respectively.2.The regeneration of meniscus was observed by Safranin O/Fast Green staining.The expression of type I,type II collagen and three FGFRs in meniscus was observed by immunohistochemical staining.in the LIPUS-treated group and control group.3.Cell scratch assay to determine the effect of LIPUS on the migration of mouse meniscus cells.4.RT-PCR was used to detect the effect of LIPUS on the expression of cell-related matrix genes and FGFRs in mouse meniscus.Results:1.LIPUS can accelerate the healing of mouse meniscus injury,promote matrix secretion and morphological remodeling,and promote the expression of FGFRs,especially FGFR1,3.2.LIPUS promotes migration of meniscus cells and expression of matrix-related genes.3.FGFR inhibitor can reduce the promotion effect of LIPUS on the expression of cartilage and matrix differentiation genes.Conclusion:LIPUS can promote healing process of meniscus injury in mice by affecting FGFs/FGFRs signaling.