Umbilical cord derived mesenchymal stem cells attenuate rodent Osteoarthritis progression via preserving articular cartilage superficial layer cells and inhibiting synovitis

Abstract

Purpose: Osteoarthritis (OA) is one of the most common joint diseases, which is characterized by pain and joint dysfunction. Inhibition of synovial inflammation and promotion of cartilage regeneration is considered to effectively attenuate the manifestation. Mesenchymal stem cells showed tissue regeneration effects mainly through tissue replacement effects, inflammation and immune response modulation, and initiation of endogenous repair. Umbilical cord derived MSCs (UCMSCs) are becoming increasingly popular as it is highly proliferative and could be noninvasively harvested compared with other MSCs. Therefore, we aim to test the effects of UCMSCs on rodent inflammatory arthritis induced by Monosodium iodoacetate (MIA) and posttraumatic OA (PTOA) models induced by DMM surgery. Methods: Intra-articular injection of 0.3 mg Monosodium iodoacetate (MIA) in 50 μL saline was injected into the left knee of the 200g weight male SD rat to induce rat knee inflammatory arthritis, and saline as veh group on contralateral right knees. 2.5× 105 UCMSCs or chondrocytes (CHO) in 50 μL normal saline were then given on day1, day 7, and day 14 respectively two weeks after MIA injection. Joints were harvested 4 weeks after MIA injection for paraffin section and then Safranine-O, H&E and immunohistochemistry staining were performed to observe cartilage erosion and synovium. In addition, PTOA mouse model was established on 3 months old C57 male mice by DMM (destablization of medial meniscus) surgery. 2.5× 105 UCMSCs were injected in to the DMM knees during the surgery, and joints were harvested 3 months post surgery for histology. For invitro studies, migration ability of SFCs by UCMSCs were accessed by transwell assay. Furthermore, catabolism change of MIA induced SFCs by UCMSCs is performed by real-rime PCR of Col-X and BCL-2 genes. CCK-8 assay was done to check proliferation ability of SFCs by UCMSCs conditioned media. Results: First, we performed Safranine-O staining and found delayed OA progression by UCMSCs treatment, compared with vehicle (Fig. 1 A). Further Mankin Score confirmed a significant decrease in UCMSCs treatment (4±0.45 VS 12.5±0.34 in vehicle, P<0.0001) (Fig. 1 B). Based on histology, we quantified the cartilage thickness, and found a 125% increase in UCMSCs treatment compared with vehicle (Fig. 1 C). The ratio of uncalcified zone to total thickness shows similar result (Fig. 1 D). To understand the molecular mechanism, we performed immunohistochemistry and found that UCMSCs treatment had more type 2 collagen staining, less ADAMTS-5 and less MMP-13 staining compared with vehicle group (Fig. 2 A C D). In addition, increased hypertrophic chondrocytes, another cartilage catabolism marker, by MIA injection is attenuated by UCMSCs (Fig. 2 E). Those data suggested that UCMSCs are capable of switching the catabolism to anabolism of chondrocytes during OA. Interestingly, we found that UCMSCs treatment moderately reserve the loss of SFCs after MIA treatment (Fig. 3 A B), possibly due to elevated proliferation (Fig. 3 C), decreased catabolism gene expression: Col-X and BCL-2 gene expression (Fig. 3 D E). by UCMSCs conditioned media. Interestingly, we observed that SFCs may accumulate in cartilage erosion area during the process OA (Fig 4 A), we performed a transwell assay and revealed that that SFCs were more likely to migrate to injured chondrocytes by serum starvation compared to normal chondrocytes (Fig 4 B D). Moreover, UCMSCs conditioned media could promote the migration of SFCs compared to vehicle (Fig 4C E). Combining those data, we hypothesized that migration of SFCs is crucial to initiate cartilage repair and UCMSCs could effectively promote this capacity, which may attribute to its rescue effects on OA progression. As synovitis is also a major manifestation of OA and main cause of pain, we performed H&E staining on synovium and revealed that MIA induced obvious inflammatory cells infiltration and obvious hyperplasia (Fig. 5A). On the contrary, UCMSCs treatment could greatly diminish these effects and significantly bring the synovitis score down (1.60 vs vehicle 5.33, P=0.0002, Fig 5 A B). To further identify the identity of those infiltrated inflammatory cells in the synovium, we did IHC staining of CD68 and CD4. Interestingly, both CD4+ Th cells and CD68+ macrophages were elevated by MIA, and obviously attenuated by multiple UCMSCs treatment (Fig. 5 C D). As MIA induced arthritis is an inflammatory arthritis model, so we introduced another typical OA model by DMM surgery, and we also found that UCMSCs could effectively attenuate cartilage erosion (Fig 6 A) and thus delay OA progression by significantly bringing down the Mankin Score (5.75±0.37 VS 12.20±0.84, N=9, P<0.0001, Fig 6 B). Further H&E staining also showed preserved SFCs numbers by UCMSCs treatment after DMM surgery (2.57±0.98 VS 0.83±0.75, N=9, P<0.05, Fig 6 C D) Conclusions: This study highlights the alleviating effects on OA progression by UCMSCs on two arthritis models. Furthermore, the effects are possibly via inhibited synovial inflammation and preserved SFCs on the articular cartilage. However, detailed celluar and molecular mechanisms are needed in future studies.