The Role Of Mechano Growth Factor In Anterior Cruciate Ligament Injury Repair And Cartilage Degeneration Prevention

The knee joint is a major load-bearing joint of the human body and is subject to significant forces from daily activities.Cruciate ligament,a fibrous connective tissue which links tibia to the femur,is crucial to knee stability.Anterior cruciate ligament(ACL)injury,accounts for a noteworthy proportion of knee-joint injuries and result in disability and complication in patients worldwide.The relative avascularity and articular cavity microenvironment effect during injury restricts the healing of ACL.Additionally,ACL injury-induced mechanical system disorder always results in cartilage degradation in the knee joint.In turn,the cartilage degradation further weakens the ACL and this degradation process continues in a cyclic fashion.Clinically,ligament reconstruction mainly focuses on auto-/allo-grafts,xenografts,and prosthetic devices.However,these treatment methods are associated with complications and variable outcomes,resulting in restricted therapeutic effect.Ligament tissue engineering blends biomechanics,seed cells,growth factors and scaffolds together,provides a novel strategy for ligament injury repair and regeneration.Mechano growth factor(MGF)is one of the alternative splicing products of insulin-like growth factor 1(IGF-1)and plays a pivotal role in tissue engineering fields,such as musculoskeletal system regeneration and nervous system injury repair.The study demonstrates the role of MGF in regulating the microenvironment of an articular cavity by synovium to repair and regrowth the injured ligament.Additionally,the role of MGF in remits ligament injury induced cartilage degradation(osteoarthritis,OA)and in anti-inflammation was explored.The main results are shown as follows:The objective of this study is to detect the role of MGF-C24 E on regulating the knee-joint microenvironment via synovium after ACL injury.Fibroblast-like synoviocytes of osteoarthritis(OA-FLS)achieved from 5 different patients with ligament rupture induced OA.Cell proliferation,metabolism,response to stress and inflammation were detected to evaluate the effects of exogenous MGF-C24 E on OA-FLS.The present study finds that MGF-C24 E has no toxic effect on OA-FLS but inhibits the cell proliferation.On the other hand,MGF-C24 E promotes lysyl oxidase(LOX)-family genes expression,induces endoplasmic reticulum(ER)stress and down-regulates protein levels of tumour necrosis factor alpha(TNF-α)and interleukin-1 beta(IL-1β)in OA-FLS.These results imply that MGF-C24 E can inhibits synovial hyperplasia,activates synovial metabolism for tissue repair/reconstruction and possesses anti-inflammatory effects.Therefore,MGF-C24 E may have therapeutic potential in knee-joint microenvironment with injured ligament.Based on above study,we have already understood that MGF-C24 E can provide a positive microenvironment for ACL injury repair.To further prove the potential of MGF-C24 E in ACL treatment,a synoviocytes supernate-ACL fibroblasts co-culturing system was set for detect the role of MGF-C24 E in ACL injury repair.Fibroblast-like synoviocytes(FLS)and ACL fibroblasts(ACLFs)achieved from human normal synovium and ACL.To detect the effect of MGF-C24 E on synovium injury,FLS were seeded uniformly into flexible-bottom 6-well culture plates and undertook static stretching with 12% strain for 12 h to mimic the synovium injury,during the stretching,FLS were treated with or without MGF-C24 E.At the same time,all the FLS supernates were collected to mimic the synovial fluid(SF)from injured and MGF-C24E-treated synovium.And then,ACLFs were co-cultured with FLS supernates and undertook the mechano-injury by static stretch,to investigate the role of MGF-C24 E on repairing injured ACL via regulates articular cavity microenvironment.The present study demonstrated that MGF-C24 E treated-FLS subjected to the machano-stretching were experienced significant lower oxidative stress,ER stress and deformation.Also,MGF-C24 E regulates the LOX family genes expression and down-regulates the matrix metalloproteinase-2(MMP-2)activity in FLS after mechano-injury.In addition,ACLFs were stretched and then co-cultured with MGF-C24E-FLS supernates,the ACLFs exhibited increased proliferation,migration,collagen synthesis and decreased expression of MMPs to injuried ACLFs,the viscoelasticity of injured ACLFs were also enhanced to normal ACLFs.The present study suggested that MGF-C24 E interacts with injured-synovium and regulates the microenvironment of knee-joint,which is benefit to ACL reconstruction.It has been demonstrated that MGF-C24 E potentially regulates the microenvironment of knee joint cavity,and provide advantages for ACL reconstruction.However,in clinical therapy,MGF-C24 E inject or deliver in knee-joint,it will not only interact with adult cells but also effect the stem cells in synovium.To investigate stem cells in synovium contribute to ligament natural repair,human knee joint synovium were digested into type Ⅰ collagenase and achieved single-cell suspensions.After inoculating cells derived from synovium at relative low density,colonies were detected with different types in morphology.Cell-spheres formed at the first passage after culture the synovium derived cells on low-attachment dishes,and continuous cell culturing the cell-spheres formed again.To detect the differentiation potentials,synovium-derived cells were cultured with specific induction culture medium,and demonstrate that the cell can differentiate into osteoblasts,chondrocytes and adipocytes.In sum,cells derived from human synovium are clonogenicity,self-renewable and pluripotency,which can be initiatory defined as a pluripotent-stem cell.Flow cytometry and immunofluorescence further demonstrate that the pluripotent-stem cells were positive for general surface antigens of mesenchymal stem cells(MSCs),such as CD29,CD44 and CD 90,and the cells were positive for a series markers of MSCs,such as Vimentin、SSEA-1 、 NG2 、 Sox10 、 Snail and Slug.These results demonstrated that the synovium-derived stem cell is a subline of MSCs.Herein,we designated the cell as synovium mesenchymal stem cell(SMSC).After 7 days MGF-C24 E induction,the gene expression of markers on ligament fibroblast/tenocytes,osteoblasts,chondrocytes and adipocytes were detected by qRT-PCR in SMSCs.The result finding that the MGF-C24 E down-regulates the gene level of markers on osteoblasts and adipocytes but up-regulates the expression of markers on ligament fibroblast/tenocytes in SMSCs,and there is no capibality of MGF-C24 E to inducing SMSCs different into chondrocytes.In addition,after 21 days MGF-C24 E induction,the Youngs’ modulus of MGF-C24E-treated SMSCs were increased which is similar with normal ligament fibroblasts.,immunofluorescence revealed the positive expression of protein markers on ligament fibroblast/tenocytes in SMSCs,such as tenascin-C,type I collagen,scleraxis and tenomodulin.In sum,MGF-C24 E has potential to directs SMSCs differentiation in to ligament/tendon.To further detect the contribute of MGF-C24 E and SMSCs to ligament regeneration,a rabbit external collateral ligament defected model was employed.Rabbit synovium mesenchymal stem cells(SMSCs)were isolated form rabbit synovium and mixed with Matrigel and MGF-C24 E together as a compound to fill the defection,compounds were implant into ligament defected area for 4 weeks and 8 weeks.H&E stain demonstrated that the MGF-C24E/SMSCs/Matrigel-implanted group has better ligament regenerative capability than the MGF-C24E/Matrigel-,SMSCs/Matrigel-and only Matrigel implanted groups or SMSCs injection group.Overall,these results imply that MGF-C24 E is capable to inducing SMSCs ligament differentiation.Additionally,we explored the role of MGF-C24 E in regulating SMSCs migration.Transwell assay was used to detect the SMSCs pass through the screen,with or without MGF-C24 E treatment.The result suggested that SMSCs in MGF-C24E-treated group have superior capability to pass through the transwell membrane,which is regulated by FAK-ERK cell signaling pathway.All in all,MGF-C24 E has potential to inducing SMSCs differentiation into ligament and regulates the cell pass through matrix screen,which provide a novel strategy for ligament regenerative medicine.Ligament injury should induce imbalance of knee joint and result in osteoarthritis(OA),which is the main complication of ligament injury.Herein,the capability of MGF-C24 E to prevent OA-cartilage degeneration was evaluated in vitro and in vivo.Chondrocytes derived from 5 OA patients,and treated with MGF-C24 E for 12 h.Flow cytometry,MTS assay,EdU stain and western blotting were used to detected the cell proliferation and apoptosis.This study reveals that MGF-C24 E treatment can efficient promotes chondrocytes proliferation and inhibits OA induced cell apoptosis.F-actin structures stain and transwell assay explored that MGF-C24 E treatment are potentially induce filopodia formation in chondrocytes and promote the cell migration.Young’s modulus of chondrocytes was tested by AFM contact modal,and demonstrate that MGF-C24E-treated OA-chondrocytes have higher stiffness than untreated cells.In addition,qRT-PCR further demonstrate MGF-C24 E treatment can up-regulate the gene level of cartilage matrix markers,and down-regulate the fibrochondrogenesis related genes expression.The ER stress and the OA pathology relevant proteins,such as TGF-β,Smad3 and HIF-2α of OA chondrocytes,were also detected by western blotting.Furthermore,PERK was silenced via siRNA to illuminate the mechanism of MGF-C24 E prevent cartilage degeneration in OA.The results further demonstrate that the cartilage loss of OA can be delayed by MGF-C24 E treatment partially by PERK-regulated ER stress.In the rabbit hind legs’ knee joint OA model,cartilage degeneration was inhibited after two weeks of treatment with articular cavity MGF-C24 E injection.In sum,MGF-C24 E can effectively prevent OA deterioration via inhibit the pathological apoptosis of OA chondrocytes and promote the cell proliferation,migration and cartilage matrix synthesis,suggest a potential therapeutic application for OA therapy.During the process of in vivo experiment on MGF-C24 E knee joint injection,the results shown that MGF-C24 E has a capability either prevent the cartilage degradation or remit the inflammation of knee joint.Base on above finding,this study also detected the role of MGF-C24 E on regulate cell polarization of macrophages.RAW264.7 macrophages treated with MGF-C25 E,and different bio-assay were used to analysis cell polarization,such as qRT-PCT,immunofluorescence and cell morphology measurement.All above results finding that MGF-C25 E is capable to anti-inflammation by regulates the M1 macrophages transform to M2 macrophages.To avoid the short half-life of MGF-C25 E overspread the function of MGF-C25 E in vitro and in vivo,click-chemistry technique was used to modify a nanofibrous scaffold.Silk fibroin(SF)layer-by-layer self-assembled on polycaprolactone(PCL)nanofibrous scaffolds,and click chemistry reactions intended to bond the MGF-C25 E on the SF.In in vitro experiment,macrophages seeded on modified scaffolds for 24 h,qRT-PCT,immunofluorescence and cell morphology measurement demonstrated that the MGF-C25 E modified scaffold is superior to transform M1 macrophages to M2 macrophages.RNA-sequencing shown that the MGF-C25 E regulates macrophages polarization could cascade cell signaling pathway involving VEGF pathway,MAPK pathway,actin cytoskeleton regulation and fatty-acid oxidation.In vivo,rat-subcutaneous model was fabricated,and MGF-C25 E modified or unmodified scaffolds were subcutaneous implanted for 1 day,3 days,7 days,14 days and 28 days.Immunostaining further revealed that MGF-C25 E is capable to promote M2 phenotype macrophages formation to anti-inflammation.In sum,MGF-C25 E has potential to direct macrophages polarization,induce the M1 macrophages transform to M2 macrophages and result in inflammation relief.In conclusion,the present study discussed the possibility and mechanisms of MGF-C24 E regulates the articular cavity microenvironment and directs the synovium-derived stem cells’ differentiation for ACL regeneration.Also,this study preliminarily revealed efficiency and mechanism of MGF-C24 E in preventing ligament injury induced osteoarthritis,and further verified the role of MGF-C24 E in macrophages polarization for anti-inflammation.This study broadens the general understanding of MGF-C24 E in clinical therapy and biomedical study,provides a theoretical reference for repairing the injured soft tissue in articular system.