Enhancement Of Acellular Cartilage Matrix Scaffold By Wharton's Jelly Mesenchymal Stem Cell-derived Exosomes To Promote Osteochondral Regeneration

Objective: Articular cartilage is hyaline cartilage,lacking blood vessels,lymphatic vessels and nerves.It only relies on the infiltration of joint synovial fluid to provide nutrition,and its ability to heal itself after injury is very limited.If articular cartilage damage is not treated in time,it is very easy to cause osteoarthritis(OA).OA not only seriously endangers the physical and mental health of human beings,but also brings a heavy economic burden to society and families.Unfortunately,it is difficult to regenerate hyaline cartilage,whether it is medical treatment or traditional surgical treatment.With the gradual in-depth exploration of stem cells by researchers,mesenchymal stem cell(MSC)therapy has shown the potential to regenerate articular cartilage,and MSCs-based tissue engineered cartilage has achieved encouraging results.MSCs have strong self-renewal ability and the potential to differentiate into bone,cartilage and fat,and have a wide range of sources,making them ideal seed cells for tissue engineering.A large number of studies have promoted the regeneration and repair of articular cartilage or osteochondral by combining MSCs with different forms of tissue engineering scaffolds or simply injecting MSCs into the joint cavity.However,MSCs' tumorigenesis risk,disease transmission risk,higher standards for cell storage and transportation,and strict requirements for stem cell applications in related regulatory policies restrict the further development of MSCs-based tissue engineered cartilage.Therefore,there is an urgent need for a cell-free cartilage regeneration method that can not only exert the functions of MSCs but also avoid the limitations of MSCs as much as possible.In recent years,more and more studies have shown that the process of MSCs promoting tissue repair and regeneration may not be caused by the direct differentiation of MSCs,but by their paracrine exosomes(Exos).Exos are important communication carriers between cells,containing a variety of proteins,nucleic acids and lipids,and have biological functions similar to the source cells.Studies have shown that MSC-Exos have a beneficial effect on the regeneration of articular cartilage and can inhibit the progression of OA.In addition,we have successfully prepared the acellular cartilage extracellular matrix(ACECM)scaffold with dual bionic properties of composition and structure.Animal experiments show that the ACECM scaffold can promote the regeneration of articular cartilage after compounding with cells.The purpose of this study is to construct a "cell-free" tissue engineered cartilage based on human wharton's jelly mesenchymal stem cell-derived exosomes and the ACECM scaffold,and to explore whether hWJMSC-Exos can enhance the repair effect of the ACECM scaffold and promote osteochondral regeneration in rabbits.In addition,this study innovatively explored the possible mechanism of hWJMSC-Exos to promote osteochondral regeneration from the perspective of joint cavity microenvironment,which provides a theoretical reference for the construction and further promotion of the "cell-free" tissue engineered cartilage.Methods: The tissue fragments adherence method was used to isolate primary hWJMSCs,flow cytometry was used to identify their markers,and three-lineage induced differentiation culture was used to identify their potential to differentiate into bone,cartilage and fat.The culture supernatant of hWJMSCs of P3-P5 generation was collected,and Exos were extracted by differential ultracentrifugation.The morphology of Exos was observed by transmission electron microscope(TEM),the particle size of Exos was detected by nanoparticle tracking analysis(NTA),and the protein markers of Exos were identified by western blot(WB).Fresh porcine articular cartilage was used to prepare the ACECM scaffolds,which were then characterized and their cell compatibility was verified.Cell proliferation experiments,cell migration experiments and RT-PCR were used to verify the effects of hWJMSC-Exos on the proliferation of bone marrow mesenchymal stem cells(BMSCs)and chondrocytes,BMSCs migration and macrophage polarization in vitro.A rat knee joint osteochondral defect model was established.The intra-articular injection of PBS was used as the control group,and the intra-articular injection of hWJMSC-Exos was used as the experimental group.Through immunohistochemical staining of anti-inflammatory cytokine IL-10 and proinflammatory cytokine IL-1 and TNF-?,the effect of hWJMSC-Exos on the inflammatory response of the joint cavity microenvironment was explored.Then,immunohistofluorescence staining was performed on the total macrophage marker CD68,M1 macrophage marker CD86 and M2 macrophage marker CD206 to evaluate the effect of hWJMSC-Exos on macrophage polarization in vivo.The effect of hWJMSC-Exos on the migration of endogenous stem cells in vivo was evaluated by immunofluorescence staining of MSC markers CD73 and CD105.The osteochondral defect model of rabbit knee joint was established.The control groups were PBS intraarticular injection group(PBS group),PBS intra-articular injection combined with ACECM scaffold implantation group(PBS + S group),Exos intra-articular injection group(Exo group)and sham operation without osteochondral defect group(Sham group).The experimental group was Exos intra-articular injection combined with ACECM scaffold implantation(Exo + S group).The intra-articular injection of PBS or hWJMSC-Exos was repeated on the day after the operation and every 7 days after the operation for 4 weeks.Half of the rabbits were sacrificed at 3 and 6 months after the operation,and the knee joints were collected.Through macroscopic manifestations,imaging manifestations,histomorphological manifestations,biomechanics and biochemical tests,it was evaluated whether hWJMSC-Exos can enhance the repair effect of ACECM scaffold and promote osteochondral regeneration in rabbit knee osteochondral defect models.Finally,through miRNA sequencing and pathway enrichment analysis,the miRNA and its mechanism that hWJMSC-Exos may play a role in osteochondral regeneration were preliminarily explored.Results:(1)hWJMSC-Exos had typical morphological characteristics of Exo.(2)The ACECM scaffold had a loose porous structure and a vertical orientation structure,and had good cell compatibility.(3)hWJMSC-Exos could promote the proliferation of BMSCs and chondrocytes in vitro.(4)hWJMSC-Exos could promote the migration of BMSCs in vitro,and this effect was dose-dependent.(5)hWJMSC-Exos could promote the polarization of macrophages to the M2 phenotype in vitro.(6)hWJMSC-Exos promoted the polarization of macrophages to the M2 phenotype in vivo,thereby promoted the increase of the anti-inflammatory cytokine IL-10 and inhibited the inflammatory response in the joint cavity microenvironment.(7)hWJMSC-Exos might promote the migration of endogenous stem cells in vivo to a certain extent,but compared with the control group(PBS),this effect had no significant difference.(8)hWJMSC-Exos enhanced the repair effect of the ACECM scaffold.The new cartilage in the Exo+S group had typical hyaline cartilage characteristics.(9)hWJMSC-Exos contained a variety of miRNAs that have been proven to promote osteochondral regeneration.Through screening,20 miRNAs that may play a role in osteochondral regeneration were identified.Conclusion: hWJMSC-Exos can enhance the ability of the ACECM scaffold to repair osteochondral defects.The mechanism may be through the influence on the biological behavior of MSCs,chondrocytes and macrophages to regulate the microenvironment of the joint cavity,thereby promoting osteochondral regeneration.The "cell-free" tissue engineering strategy based on hWJMSC-Exos and the ACECM scaffold is expected to become an effective way to promote osteochondral regeneration.