Role of stem cells and bioactive scaffold in chronic joint injury: Working towards a regenerative medicine approach to stop osteoarthritis progression

Abstract

Purpose: Osteoarthritis is a leading cause of disability in aging individuals. Although commonly considered an age-related condition, a major predisposing factor is sports or trauma related joint injury sustained during young adulthood. Current treatments for joint injuries may help to relieve symptoms, but have little effect in preventing the development of chronic joint disease and progression to osteoarthritis later in life. Despite this being a significant clinical problem, the mechanism of impaired healing following joint injury, even when there is surgical intervention, remains poorly understood. This study tests the hypotheses that (1) impaired healing following joint injury is due to the inflammatory and highly inhibitory environment created by diseased cells, (2) this inhibitory environment can be reversed by ‘priming’ the diseased cells using human mesenchymal stem cells (hMSCs), which have documented anti-inflammatory and trophic properties, and (3) a bioactive scaffold can further promote regeneration in the injured joint. This study will aid the development of a novel regenerative therapy for joint injury, by restoring a joint environment that is conducive to healing and reducing the long-term risk of developing osteoarthritis. Methods: An in vitro model mimicking chronic joint injury was developed by co-culturing bone marrow-derived hMSCs with human synovial fibroblasts (HSFs) isolated from osteoarthritic joint tissues. To test hypothesis (1), the effects of HSFs on hMSCs were examined for 3, 7, 10 and 21 days in growth, osteogenic and chondrogenic media, which simulated the types of conditions present in the post-injury joint environment. To test hypothesis (2), the effects of hMSCs on HSFs were examined for 3 and 7 days in growth medium. To test hypothesis (3), the same set-up was used as for (1), with or without the addition of a bioactive scaffold composed of ‘Sr-HT-Gahnite’ ceramic (AU patent #2012262674; co-invented by J. Li). Quantitative RT-PCR was used to evaluate inflammatory response in the hMSCs and HSFs (IL-6, IL-8, MMP-1, MMP-2, MMP-13, ADAMTS4, ADAMTS5, CD44, TLR4, COX-2, CCL2), as well as differentiation in the hMSCs (RUNX2, BSP, SPP1, BGLAP for osteogenesis; SOX9, COL2A1, ACAN, TGFb1 for chondrogenesis), at all time points. Histology and histochemistry were performed at 21 days to compare osteogenesis and chondrogenesis in hMSCs between groups. Data was collected from 4 independent samples in each treatment group, and analysed using t-tests with p < 0.05 considered statistically significant. Results: The hMSCs showed significant upregulation of several inflammatory markers when co-cultured with diseased HSFs over a 21 day period. Notably, MMP-13 and TLR4 were upregulated at 7 days in all three media types, while IL-8, MMP-2, ADAMTS-5 and CD44 showed long-term upregulation at 21 days in at least one media type. Osteogenic and chondrogenic differentiation of hMSCs to form new tissues were both significantly impaired in the presence of diseased HSFs, as seen through reduced BSP and SPP1 expression and Alizarin red S staining for calcium (osteogenic markers), as well as reduced COL2A1 and ACAN expression and toluidine blue staining for proteoglycans (chondrogenic markers) at 21 days (Fig 1). When diseased HSFs were co-cultured with hMSCs, there was rapid downregulation of several inflammatory markers, including significant reduction in the levels of ADAMTS5, MMP-13 and TLR4, all of which are implicated in inflammation, matrix degradation and tissue degeneration. When a bioactive scaffold was added into the co-culture system, it showed simultaneous modulation of the inflammatory response in hMSCs and diseased HSFs through significant downregulation of ADAMTS-4, MMP-13 and CCL-2. Furthermore, the scaffold partly ‘rescued’ the impaired differentiation seen in hMSCs when co-cultured with diseased HSFs at 21 days, where the presence of the scaffold improved calcium (for osteogenesis) and proteoglycan (for chondrogenesis) deposition in the extracellular matrix (Fig 1). Conclusions: The findings suggest that diseased cells arising from joint injury can create a highly inhibitory environment that increases inflammation in stem cells normally involved in the repair of musculoskeletal tissues, which impairs their regenerative ability. This inhibitory environment can be reversed by ‘priming’ the injured joint prior to treatment, such as through an intra-articular injection of hMSCs, which can help modulate the pro-inflammatory state of the resident joint tissues. A Sr-HT-Gahnite bioactive scaffold may complement the effects of hMSCs to further promote regeneration in the joint. This novel regenerative medicine approach of combining stem cells and a bioactive scaffold may be useful as a new therapy to treat chronic joint injury and reduce the risk of osteoarthritis progression.