Mesenchymal stem cell derived extracellular vesicles as treatment for osteoarthritis in a rat high fat diet groove model

Abstract

Purpose: Mesenchymal stromal/stem cells (MSC) hold a great promise to control the imbalance between anabolic and catabolic processes in an OA joint due to their immunomodulatory and regenerative capacities. Recently, intra-articular injections with human MSC entered the clinical trials as a novel, low invasive and relatively cheap therapy for OA. Although, the results of this novel treatment are promising, it remains unclear how MSC exert their beneficial effects in inflammation and tissue (cartilage) repair. Increasing evidence suggests that the therapeutic efficacy of MSC depends on paracrine signaling. Recently we have shown that MSC-derived extracellular vesicles (MSC-EVs) are an important component of this signaling and have a major contribution to cartilage regeneration and inhibition of TNF-alpha stimulated inflammatory responses in chondrocytes in vitro. Extracellular vesicles (EVs) are secreted by all cell types and range in size from 40 - 100 nm (exosomes) and from 100 -1000 nm (microvesicles). They increasingly appear to play an important role in intercellular communication by transportation of important molecules (peptides, mRNA, miRNA, enzymes). MSC-derived EVs have a great potential to serve as a safer cell free therapy for OA. The purpose of this study was to investigate if MSC-derived EVs can inhibit inflammation and promote cartilage repair to a similar extent as MSCs in a rat high fat diet groove model of OA. Methods: 36 male Wistar-han (Crl:WI(Han) rats were fed a high fat diet for 24 weeks, starting at an age of 12 weeks old. In week 12 of the study the rats received unilateral surgery where local cartilage damage was induced at the right knee joint by making five longitudinal grooves on the femoral condyles. In week 13 the rats were randomized into 3 groups and given an intraarticular injection with either PBS, 2 million human bone marrow derived MSCs or EVs derived from 2 million MSCs. The extracellular vesicles group received 5 of these doses with 5 days interval. Before, during and after the injections pain behavior was monitored by dynamic weight bearing and via measuring the left and right hind paw withdrawal responses to the application of von Frey hairs. uCT scans were made at week 0 and week 24, and used to determine the number of osteophytes and cartilage mineralization in the tibia. Additionally, tibial subchondral plate thickness, trabecular bone thickness, and trabecular bone volume fraction (total volume/trabecular bone volume) were measured using ImageJ 1.47v. At endpoint animals were sacrificed and the knee joints were harvested. Joint tissue is currently under preparation for histological staining to determine the OARSI histopathological score for rats. Data is represented as mean +/- standard deviation (SD) and the difference between the means in the three groups was statistically tested using one way ANOVA. Results: Results from the von Frey tests showed a clear decrease in 50% withdrawal threshold in the right hind paw, which was expected as the right side was the grooved limb. However, no difference between the three treatment groups in either left or right paw was seen. The mean osteophyte number in the PBS group was 4.7 (+/- 0.65) versus 3.6 (+/-2.15) in the MSC group and 4.3 (+/-1.8) in the MSC-EV group (figure 1A). In the MSC and MSC-EV treatment groups the variation in osteophyte number and total osteophyte diameter between the animals was large compared to the PBS group. Cartilage mineralization were observed in 9 of 12 animals in the PBS and MSC group and in 5 of 12 animals in the MSC-EV group. Both the number and total diameter was lowest in the MSC-EV group, but there was no significant difference as compared with control groups (Figure 1B). Other parameters measured on uCT like bone volume fraction, subchondral plate and trabecular bone thickness were similar between the 3 groups. Results from the OARSI histopathological score for rats is currently under preparation. Conclusions: Preliminary results from this in vivo study show no significant differences between PBS, MSC or MSC-EV treatment on uCT data or von Frey test. However, we do observe a large spread in osteophyte score and total diameter in the MSC and MSC-EV treated groups, which could be a sign that individual animals are responding differently to the treatments. Data from the OARSI scoring will help us determine if the response really is variable for MSC and MSC-EV treatment groups. This would be in accordance with observations made in humans, as many clinical trials that test MSC-based therapies report a substantial nonresponder group. In summary, although osteophytes are one of the hallmarks of osteoarthritis, we feel a complete picture of the joint damage including cartilage and synovium is needed to draw conclusions on the effect of the treatments in this study.