Self-Healing Hyaluronic Acid as a Viscosupplement and Biomolecule Carrier to Mitigate Post-Traumatic Osteoarthritis

Abstract

Post-traumatic osteoarthritis (PTOA), a form of osteoarthritis which results from injury to the joint, is estimated to affect 5.6 million people in the United States. Pathological features of PTOA include cartilage degeneration, osteophyte formation, synovial inflammation, and altered synovial fluid composition. In particular, OA synovial fluid has a reduced concentration and a lower molecular weight (MW) of the component hyaluronic acid (HA), which is responsible for its viscoelastic and lubrication properties. Viscosupplementation, or intra-articular injection of exogenous high MW HA, is therefore a common intervention used in the clinic to attenuate OA symptoms. Although there are many HA-based viscosupplements currently on the market, their effectiveness has been widely debated. Clinical studies of viscosupplements have revealed mixed results, with many showing little-to-no effect on disease outcomes compared to placebo. There are many formulations which differ in terms of the source, MW, concentration, and crosslinking of HA. The rapid clearance of HA from the joint is one of the greatest drawbacks that diminishes viscosupplements’ effectiveness. Some products like Synvisc® use chemically crosslinked HA to achieve a longer residence time and hence require fewer intra-articular injections. However, the crosslinking agents used can be inflammatory and lead to pseudoseptic reactions in the joint. Furthermore, irreversibly crosslinking HA can make these formulations difficult to inject. To overcome the limitations associated with current viscosupplements, we have developed a self-healing HA through incorporation of quadruple hydrogen bonding ureidopyrimidinone (UPy) moieties. UPy-mediated hydrogen bonding can be disrupted in response to shear stress, enabling improved injectability through small-diameter needles. Within the joint, self-healing HA polymer chains form a dynamic network for enhanced retention and improved viscoelastic properties. The overarching goal of this dissertation work was therefore to assess whether the self-healing HA could be used as a viscosupplement to slow the progression of PTOA and whether its therapeutic effect can be augmented through incorporation of chondroanabolic and anti-catabolic bioactive agents. As a proof of concept, we first examined the effects of UPy modification on the retention and chondroprotection of low MW HA. Using low MW HA allows us to distinguish the role of UPy-mediated hydrogen bonding without confounding contributions of high MW polymer chain entanglement. Towards this end, in Chapter 3, we modified 200 kDa HA with UPy groups and compared its rheological and biological functions to corresponding unmodified HA. HA-UPy exhibited shear-thinning and self-healing properties, as well as improved viscoelasticity, lubrication, and free radical scavenging capacity as compared to unmodified HA. In addition, we examined the in vivo retention and chondroprotective function of self-healing HA in a minimally invasive anterior cruciate ligament transection (mi-ACLT) rat model. This PTOA model, described and characterized in Chapter 2, is a novel, non-surgical method of destabilizing the joint that uses a needle to sever the ACL. We observed that HA-UPy was retained for nearly a month post-injection in both healthy and injured rat knees, whereas unmodified HA was cleared in one week. Furthermore, intra-articular injections of self-healing HA reduced the extent of cartilage degeneration post-injury as compared to injections of unmodified HA. Together, these results showed that modifying HA with UPy groups can improve its viscoelastic and biological properties without affecting its biocompatibility, allowing it to function as a viscosupplement. In Chapter 4, we imparted UPy-mediated self-healing functionality to high MW HA to create a formulation comparable to existing viscosupplements. We similarly modified 1 MDa HA with UPy groups, which enhanced its rheological and free radical scavenging properties, albeit to a less considerable extent than was observed with low MW HA. We then tested the ability of 1 MDa HA-UPy to slow the progression of PTOA in a surgical rat model of combined ACLT and destabilization of the medial meniscus (ACLT+DMM). Rats received monthly intra-articular injections of HA-UPy, saline, or Orthovisc® , a non-crosslinked HA viscosupplement that is used in the clinic, following surgery. Von Frey testing revealed that rats treated with HA-UPy showed reduced mechanical allodynia, a surrogate pain measure, as compared to those treated with saline in the third month of testing. Histological and microCT analysis of rat joints 12 weeks post-surgery revealed joint degeneration in all ACLT+DMM groups, with a lack of statistically significant differences observed between treatments in all outcome measures. Nonetheless, we observed some trends in terms of osteophyte formation; rats treated with HA-UPy had smaller osteophytes on average as compared to those treated with saline (via microCT) and Orthovisc® (via histology). However, these results overall indicate that self-healing HA alone may not have a significant disease-modifying effect. To improve the therapeutic potential of self-healing HA, we examined whether it could be used as a carrier for chondroanabolic and anti-catabolic biomolecules (Chapter 5). We used insulin growth factor-1 (IGF-1), as it has been shown to reduce catabolic enzyme expression and promote proteoglycan synthesis in chondrocytes. We observed that physically incorporating IGF-1 into HA-UPy maintained its activity or promoted its stability for at least 72 hours at 37 °C, as measured by its ability to stimulate cell proliferation and to be detected via ELISA. We then used cytokine-challenged in vitro and ex vivo OA models to assess its effect to mitigate catabolic enzyme activity and proteoglycan loss. Although treatment of healthy chondrocytes with HA-UPy showed no catabolic effect, chondrocytes treated with IL-1β in the presence of HA-UPy showed a greater expression of catabolic enzymes compared to IL-1β treatment alone, possibly due to HA-UPy maintaining the activity of IL-1β. This IL-1β-induced upregulation of catabolic markers was reduced, however, in chondrocytes treated with HA-UPy containing IGF-1. Furthermore, cartilage explants stimulated with IL-1β that were subsequently treated with HA-UPy+IGF-1 showed less proteoglycan loss as compared to other IL-1β-stimulated groups. These results indicate that incorporation of IGF-1 into HA-UPy can enhance the stability and activity of IGF-1 and that this combined treatment can have a chondroprotective effect. Future studies will examine the disease-modifying effect of this combined treatment in an animal model of PTOA.