| Osteoarthritis?OA?is the most common and disabling rheumatic disease affecting millions of people worldwide.The disease involves inflammation,cartilage degradation and structural changes in the affected joints,leading to severe pain and functional disability,severely damaging the individual's ability to perform activities of daily living.The pathogenesis of osteoarthritis is complicated,and it is generally considered to be a chronic joint degenerative disease characterized by significant imbalance of cartilage matrix degradation and synthetic articular cartilage.Despite the high prevalence and social burden of OA,there is currently no cure for this disease.For joint damage or cartilage damage that has not yet developed a degenerative change,the current clinical treatments are mainly to relieve the symptoms of injury,such as cartilage transplantation,autologous chondrocyte transplantation and other repair techniques;for the appearance of degenerative changes or the presence of symptomatic bone and joint Inflammatory joints,a series of non-surgical treatments are used clinically,and non-surgical treatment can be divided into non-medical treatment and medical treatment.Non-pharmacological treatment mainly focuses on patients'access to information and education,weight loss and control of sports programs,etc.Drug treatment mainly involves analgesics and non-steroidal anti-inflammatory drugs.For patients whose symptoms cannot be controlled by other non-surgical treatments,they can be intra-articular.Inject corticosteroids or hyaluronic acid.Total joint replacement is the ultimate surgical procedure for patients with osteoarthritis who have failed non-surgical treatment.Although arthroplasty removes diseased joints and replaces their function with implants,the increased risk of these surgical and surgical complications and implant life is limited to approximately 20 years.Clearly,all currently available treatments for osteoarthritis have many limitations and,more importantly,have little effect on slowing disease progression.There is an urgent need for an alternative treatment that can meet these challenges.Mesenchymal stem cells?MSCs?are pluripotent adult stem cells,which are highly present in various tissue types.MSCs from different sources have functions such as tissue damage repair and inflammatory immune regulation,making MSCs ideal for cell therapy cell.In recent years,the use of MSCs in clinical trials for a variety of disease indications has grown exponentially.Human umbilical cord mesenchymal stem cells?hUC-MSC?have the regenerative potential and multi-directional differentiation ability,low immunogenicity,long-term amplification,and no ethical restrictions.Their cryopreservation has been commonly used for cell storage in clinical.This study focused on the comparison of the therapeutic effects of Fresh hUC-MSC and Frozen hUC-MSC on osteoarthritis in rats and some of its mechanisms.Objective:Rat model of OA was established by type II collagenase induction method.The therapeutic effects of Fresh hUC-MSC and Frozen hUC-MSC on osteoarthritis in rats were observed.Frozen hUC-MSC and fibroblast-like synoviocytes?FLS?were co-culture in vitro to observe the effect of Frozen hUC-MSC on FLS function,and explore its mechanism of action,provide experimental basis for the clinical use of Frozen hUC-MSC in the treatment of OA.Methods:1.Sprague-Dawley?SD?rats were used to establish rat osteoarthritis model by type II collagenase injection.The rats were randomly divided into normal group,model group and fresh human umbilical cord.Source mesenchymal stem cell group?Fresh hUC-MSC?,frozen human umbilical cord-derived mesenchymal stem cell group?Frozen hUC-MSC?and hyaluronic acid group?HA?,10 rats in each group,the first injection of type II collagen The drug was administered on the 7th day after the enzyme.Rats in the Fresh hUC-MSC and Frozen hUC-MSC groups were injected with Fresh hUC-MSC and Frozen hUC-MSC 1×106/50?l,respectively;rats in the HA group were given 50?l of HA;rats in the normal group and the model group were given equal volume.Sterile saline.The rats were sacrificed 6 weeks after administration,and various indexes were observed.The knee circumference difference,articular cartilage gross observation score,knee X-ray observation and scoring,knee joint pathological changes and matrix metalloproteinase-13?MMP-13?and knee joint were measured.Expression of Tissue inhibitor of metalloproteinase-1?TIMP-1?.2.In vitro experiments,cultured bronchial cells?fibroblast-like synoviocytes,FLS?were cultured by enzymatic digestion,and mRNA expression levels of IL-1?,IL-6 and MMP-13 were measured by co-culture with Frozen hUC-MSC;The FLS of OA rats were cultured and co-cultured with Frozen hUC-MSC to measure the effect of Frozen hUC-MSC on the migration ability of synoviocytes in OA rats.The effects of Frozen hUC-MSC on the expression of cytokines IL-6,IL-1?,MMP-13 and TIMP-1 were detected by ELISA assay.Results:1.Therapeutic effect of Fresh hUC-MSC and Frozen hUC-MSC on OA ratsThe rat model of OA was successfully established by intra-articular injection of type II collagenase.The results of the experiment showed that the difference in joint circumference and articular cartilage were significantly different between Fresh hUC-MSC and Frozen hUC-MSC group compared with the model group.The scores were significantly reduced.Fresh hUC-MSC and Frozen hUC-MSC could effectively improve the cartilage damage of OA rats.At the same time,the Kelgren-Lawrence grading score and the pathological Mankin's score of X-ray were significantly reduced.2.Effects of Fresh hUC-MSC and Frozen hUC-MSC on the expression of MMP-13 and TIMP-1 in articular cartilage of OA rats.The results of immunohistochemistry showed that the expression of MMP-13 in the articular cartilage of the model group was significantly increased,while the expression of TIMP-1 was significantly decreased.Fresh hUC-MSC and Frozen hUC-MSC could effectively reduce the joint of OA rats.The expression of MMP-13 in cartilage tissue-while significantly increasing the expression of TIMP-1,the ratio of MMP-13 and TIMP-1 was significantly reduced.There was no significant difference between the two groups of Fresh hUC-MSC and Frozen hUC-MSC.3.Effects of Frozen hUC-MSC on the expression of FLS inflammatory cytokines and MMP-13 and TIMP-1 in OA rats.The results of ELISA showed that TNF-?significantly decreased the expression of TIMP-1 in FLS of OA rats,significantly increased the expression of IL-6,IL-1?and MMP-13,and significantly increased MMP-13/TIMP-1 ratio.Frozen hUC-MSC significantly increased the expression of TIMP-1 in FLS of OA rats,and significantly decreased the expression of IL-6,IL-1?,MMP-13 and the ratio of MMP-13/TIMP-1.4.Effect of Frozen hUC-MSC on FLS migration ability in OA ratsThe Frozen hUC-MSC were pretreated with INDO or L-NAME for 48 h,and the effects of Frozen hUC-MSC on the migration ability of FLS in OA rats were detected by Transwell chamber method after co-culture of FLS with Frozen hUC-MSC for 24 h.The results showed that Frozen hUC-MSC significantly inhibited the migration of FLS in OA rats.Both COX-2 inhibitor INDO and iNOS inhibitor L-NAME significantly reversed the ability of Frozen hUC-MSC to inhibit FLS migration in OA rats.5.Effects of Frozen hUC-MSC on the expression of IL-1?,IL-6 and MMP-13 mRNA in FLS of OA patientsTNF-??20 ng/ml?was used to stimulate FLS for 48 h in OA patients.The expression of IL-1?,IL-6 and MMP13 mRNA in FLS of OA patients was detected by Transwell chamber method after co-culture of FLS with Frozen hUC-MSC for 72 h.Levels,the results showed that TNF-??20ng/ml?can significantly increase the mRNA expression of IL-1?,IL-6 and MMP-13 in FLS of OA patients,Frozen hUC-MSC can significantly reduce IL-1?,IL-6 and MMP-13 mRNA levels in FLS of OA patients.Conclusions:1.Rat model of OA can be successfully established by intra-articular injection of type II collagenase.Fresh hUC-MSC and Frozen hUC-MSC can effectively improve the joint damage and pathological score of OA rats,and there was no significant difference in the treatment of osteoarthritis between the two groups.2.Fresh hUC-MSC and Frozen hUC-MSC intra-articular injection can effectively reduce the expression of MMP-13 in cartilage tissue of OA rats,increase the expression of TIMP-1,and decrease the ratio of MMP-13/TIMP-1,and there were no significant differences between the two groups,Fresh hUC-MSC and Frozen hUC-MSC.3.Frozen hUC-MSC can significantly inhibit the migration ability of FLS.Its mechanism may be related to PGE2 and NO secreted by Frozen hUC-MSC.4.The therapeutic effect of Frozen hUC-MSC on OA may be related to inhibition of FLS migration,expression of inflammatory cytokines,and recovery of cartilage and MMP-13/TIMP-1 ratio in FLS. |
PDF Full Text Request