Intra-articular (i.a.) drug delivery for local treatment of osteoarthritis remains inadequate due to rapid clearance by the vasculature or lymphatics. Local therapy targeting articular cartilage is further complicated by its dense meshwork of collagen and negatively charged proteoglycans, which can prevent even nano-sized solutes from entering. In a previous in vitro study, we showed that Avidin, due to its size (7 nm diameter) and high positive charge (pI 10.5), penetrated the full thickness of bovine cartilage and was retained for 15 days. With the goal of using Avidin as a nano-carrier for cartilage drug delivery, we investigated its transport properties within rat knee joints. Avidin penetrated the full thickness of articular cartilage within 6 h, with a half-life of 29 h, and stayed inside the joint for 7 days after i.a. injection. The highest concentration of Avidin was found in cartilage, the least in patellar tendon and none in the femoral bone; in contrast, negligible Neutravidin (neutral counterpart of Avidin) was present in cartilage after 24 h. A positive correlation between tissue sGAG content and Avidin uptake (R 2 ¼ 0.83) confirmed the effects of electrostatic interactions. Avidin doses up to at least 1 mM did not affect bovine cartilage explant cell viability, matrix catabolism or biosynthesis. Keywords: Avidin; intra-articular drug delivery; rat; glycosaminoglycans; cartilage Osteoarthritis (OA) affects individual joints, necessitating localized therapy. 1,2 Intra-articular (i.a.) injections allow for local and targeted delivery of drugs into the joint space, thereby reducing systemic toxicity. However, i.a. therapy often remains inadequate due to rapid clearance of drugs from the joint space; small molecules exit via the vasculature while larger macromolecules (e.g., hyaluronan) are cleared by the lymphatic system. 2,3 Mean half-lives of NSAIDs in the synovial fluid have been reported to be 1-4 h. 4,5 Solutes in synovial fluid with sizes similar to plasma proteins (albumin $67 kDa, globulin $150 kDa, fibrinogen $340 kDa) have equal permeability through the lymphatics. 2 Their clearance, however, is dependent on the rate of synovial fluid turnover and solute diffusivity; the latter is a function of solute size and molecular weight, viscosity of synovial fluid and temperature. For example, intraarticular half-lives in normal rabbit knee joints have been reported to range from 0.23 h for Acridine Blue (370 Da) to 1.23 h for Albumin (67 kDa) and 26.3 h for Hyaluronan (300 kDa). 2,3 Intra-articular injection of drug-encapsulating particles can increase half-lives of therapeutic drugs in the synovial fluid. 1,6,7 However, therapeutic efficacy depends on the ability of the drugs (or particle-bound drugs) to penetrate into specific target tissues and to be retained by those tissues over time. Entry of macromolecules into cartilage is hindered by its dense extracellular matrix (ECM) of collagen fibrils and aggrecan proteoglycans containing highly negatively charged glycosaminoglycan (GAG) chai...

Disease-modifying osteoarthritis drugs (DMOADs) should reach their intra-tissue target sites at optimal doses for clinical efficacy. The dense, negatively charged matrix of cartilage poses a major hindrance to the transport of potential therapeutics. In this work, electrostatic interactions were utilised to overcome this challenge and enable higher uptake, full-thickness penetration and enhanced retention of dexamethasone (Dex) inside rabbit cartilage. This was accomplished by using the positively charged glycoprotein avidin as nanocarrier, conjugated to Dex by releasable linkers. Therapeutic effects of a single intra-articular injection of low dose avidin-Dex (0.5 mg Dex) were evaluated in rabbits 3 weeks after anterior cruciate ligament transection (ACLT). Immunostaining confirmed that avidin penetrated the full cartilage thickness and was retained for at least 3 weeks. Avidin-Dex suppressed injury-induced joint swelling and catabolic gene expression to a greater extent than free Dex. It also significantly improved the histological score of cell infiltration and morphogenesis within the periarticular synovium. Micro-computed tomography confirmed the reduced incidence and volume of osteophytes following avidin-Dex treatment. However, neither treatment restored the loss of cartilage stiffness following ACLT, suggesting the need for a combinational therapy with a pro-anabolic factor for enhancing matrix biosynthesis. The avidin dose used caused significant glycosaminoglycan (GAG) loss, suggesting the use of higher Dex : avidin ratios in future formulations, such that the delivered avidin dose could be much less than that shown to affect GAGs. This charge-based delivery system converted cartilage into a drug depot that could also be employed for delivery to nearby synovium, menisci and ligaments, enabling clinical translation of a variety of DMOADs.

For evaluation of new approaches to drug delivery into cartilage, the choice of an animal model is critically important. Since cartilage thickness varies with animal size, different levels of drug uptake, transport and retention should be expected. Simple intra-articular injection can require very high drug doses to achieve a concentration gradient high enough for drug diffusion into cartilage. New approaches involve nanoparticle delivery of functionalized drugs directly into cartilage; however, diffusion-binding kinetics proceeds as the square of cartilage thickness. In this study, we demonstrate the necessity of using larger animals for sustained intra-cartilage delivery and retention, exemplified by intra-articular injection of Avidin (drug-carrier) into rabbits and compared to rats in vivo. Penetration and retention of Avidin within cartilage is greatly enhanced by electrostatic interactions. Medial tibial cartilage was the thickest of rabbit cartilages, which generated the longest intra-cartilage half-life of Avidin (t 1/2 ¼ 154 h). In contrast, Avidin half-life in thinner rat cartilage was 5-6 times shorter (t 1/2 $ 29 h). While a weak correlation (R 2 ¼ 0.43) was found between Avidin halflives and rabbit tissue GAG concentrations, this correlation improved dramatically (R 2 ¼ 0.96) when normalized to the square of cartilage thickness, consistent with the importance of cartilage thickness to evaluation of drug delivery and retention. ß

These results provide evidence that the use of MSC seeded in a collagen type I scaffold in the treatment of ACL injuries is associated with an enhancement of ligament regeneration. This MSC-based technique is a potentially attractive tool for improving the treatment of ACL ruptures.

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Chondrogenesis is critical to the development and repair of not only articular cartilage but also bone. Preclinical studies suggest that defects in both tissues can be repaired using culture-expanded chondroprogenitor cells, such as mesenchymal stem/stromal cells (MSCs), but directing efficient chondrogenesis by candidate cell populations is an ongoing bottleneck to their clinical application. The goal of this study was to describe a method for the molecular reporting of chondrogenic activity by primary stem/progenitor cells that can complement more labor-intensive destructive measures. A chondrogenesis-responsive promoter was generated, consisting of four repeats of a SOX9-binding enhancer sequence from the first intron of COL2A1 positioned upstream of the core COL2A1 promoter. This promoter was inserted into a lentiviral expression plasmid containing reporter genes copepod green fluorescent protein (copGFP) and firefly luciferase (fLuc), and the resulting lentiviral vector (LV) was used to transduce human MSCs derived from intramedullary reamings. To determine the specificity and stability of reporter expression, MSCs were differentiated in pellet culture for up to 4 weeks. To assess the sensitivity of reporter detection in vivo, undifferentiated and predifferentiated MSC pellets were implanted into osteochondral defects made in immune-suppressed rats. Chondrogenic differentiation of LV-transduced MSCs in pellet culture led to a strong upregulation of both copGFP and fLuc. Robust reporter activity was achieved using LV doses that did not compromise MSC chondrogenesis. Specific reporter induction was sustained over several passages post-transduction. Reporter expression levels were dependent on both pellet culture duration and TGF-b1 dose. When predifferentiated pellets were implanted into rat osteochondral defects, reporter activity was initially diminished but recovered over the following 2 weeks, suggesting acute postsurgical inflammation suppressed reporter expression. This hypothesis was supported by potent cytokine inhibition of reporter levels and glycosaminoglycan deposition within additional pellets in vitro. When combined with lentiviral transgene integration, the COL2A1-based promoter allowed specific, sensitive, and stable reporting of MSC chondrogenic activity. This promoter can be used with the extensive selection of reporter vectors now available to study different chondroprogenitor cells with promise for cartilage and bone tissue engineering and regenerative medicine.

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