Spatial and Temporal Mapping of Articular Cartilage Poro-Viscoelastic Material Properties Using Indentation.

Biphasic poro-viscoelastic constitutive material model (BPVE) captures both the fluid flow dependent and independent behavior of cartilage under stress relaxation type indentation. A finite element model based on BPVE formulation was developed to explore the sensitivity of the model to Young's modulus, Poisson's ratio, permeability, and viscoelastic constitutive parameters expressed in terms of Prony series coefficients. Then we fit the numerical model to experimental force versus time curves from stress relaxation indents on bovine tibial plateaus to extract the material properties. Measurements were made over the period of two days to capture the material property changes that resulted from trypsin-induced degradation. We measured spatial and temporal changes in mechanical properties in the cartilage. The areas of degradation were characterized by an increase in both permeability and summation of Prony series shear relaxation amplitude constants. These findings suggest that cartilage degradation reduces the intrinsic viscoelastic properties of the solid phase of the tissue in addition to impairing its capacity to offer frictional drag to the interstitial fluid flow (permeability). The changes in material properties are measurable well before structural degradation occurs.

Resveratrol and Curcumin Attenuate Ex Vivo Sugar-Induced Cartilage Glycation, Stiffening, Senescence, and Degeneration.

OBJECTIVE: Advanced glycation end-product (AGE) accumulation is implicated in osteoarthritis (OA) pathogenesis in aging and diabetic populations. Here, we develop a representative nonenzymatic glycation-induced OA cartilage explant culture model and investigate the effectiveness of resveratrol, curcumin, and eugenol in inhibiting AGEs and the structural and biological hallmarks of cartilage degeneration. DESIGN: Bovine cartilage explants were treated with AGE-bovine serum albumin, threose, and ribose to determine the optimal conditions that induce physiological levels of AGEs while maintaining chondrocyte viability. AGE crosslinks, tissue stiffness, cell viability, metabolism and senescence, nitrite release and loss of glycosaminoglycans were assessed. Explants were cotreated with resveratrol, curcumin, or eugenol to evaluate their anti-AGE properties. Blind docking analysis was conducted to estimate binding energies of drugs with collagen II. RESULTS: Treatment with 100 mM ribose significantly increased AGE crosslink formation and tissue stiffness, resulting in reduced chondrocyte metabolism and enhanced senescence. Blind docking analysis revealed stronger binding energies of both resveratrol and curcumin than ribose, with glycation sites along a human collagen II fragment, indicating their increased likelihood of competitively inhibiting ribose activity. Resveratrol and curcumin, but not eugenol, successfully inhibited AGE crosslink formation and its associated downstream biological response. CONCLUSIONS: We establish a cartilage explant model of OA that recapitulates several aspects of aged human cartilage. We find that resveratrol and curcumin are effective anti-AGE therapeutics with the potential to decelerate age-related and diabetes-induced OA. This in vitro nonenzymatic glycation-induced model provides a tool for screening OA drugs, to simultaneously evaluate AGE-induced biological and mechanical changes.

Recent advances in targeted drug delivery for treatment of osteoarthritis.

PURPOSE OF REVIEW: Osteoarthritis is associated with severe joint pain, inflammation, and cartilage degeneration. Drugs injected directly into intra-articular joint space clear out rapidly providing only short-term benefit. Their transport into cartilage to reach cellular targets is hindered by the tissue's dense, negatively charged extracellular matrix. This has limited, despite strong preclinical data, the clinical translation of osteoarthritis drugs. Recent work has focused on developing intra-joint and intra-cartilage targeting drug delivery systems (DDS) to enable long-term therapeutic response, which is presented here. RECENT FINDINGS: Synovial joint targeting hybrid systems utilizing combinations of hydrogels, liposomes, and particle-based carriers are in consideration for pain-inflammation relief. Cartilage penetrating DDS target intra-cartilage constituents like aggrecans, collagen II, and chondrocytes such that drugs can reach their cellular and intra-cellular targets, which can enable clinical translation of disease-modifying osteoarthritis drugs including gene therapy. SUMMARY: Recent years have witnessed significant increase in both fundamental and clinical studies evaluating DDS for osteoarthritis. Steroid encapsulating polymeric microparticles for longer lasting pain relief were recently approved for clinical use. Electrically charged biomaterials for intra-cartilage targeting have shown promising disease-modifying response in preclinical models. Clinical trials evaluating safety of viral vectors are ongoing whose success can pave the way for gene therapy as osteoarthritis treatment.

Hyaluronic Acid-Based Shape-Memory Cryogel Scaffolds for Focal Cartilage Defect Repair.

Traumatic joint injuries can result in significant cartilage defects, which can greatly increase the risk of osteoarthritis development. Due to the limited self-healing capacity of avascular cartilage, tissue engineering approaches are required for filling defects and promoting cartilage regeneration. Current approaches utilize invasive surgical procedures for extraction and implantation of autologous chondrocytes; therefore, injectable biomaterials have gained interest to minimize the risk of infection as well as patient pain and discomfort. In this study, we engineered biomimetic, hyaluronic acid (HA)-based cryogel scaffolds that possess shape-memory properties as they contract and regain their shape after syringe injection to noninvasively fill cartilage defects. The cryogels, fabricated with HA and glycidyl methacrylate at -20°C, resulted in an elastic, macroporous, and highly interconnected network that provided a conducive microenvironment for chondrocytes to remain viable and metabolically active after injection through a syringe needle. Chondrocytes seeded within cryogels and cultured for 15 days exhibited enhanced cell proliferation, metabolism, and production of cartilage extracellular matrix glycosaminoglycans compared with HA-based hydrogels. Furthermore, immunohistochemical staining revealed production of collagen type II from chondrocyte-seeded cryogels, indicating the maintenance of cell phenotype. These results demonstrate the potential of chondrocyte-seeded, HA-based, injectable cryogel scaffolds to promote regeneration of cartilage tissue for nonsurgically invasive defect repair. Impact statement Hyaluronic acid-based shape-memory cryogels provide a conducive microenvironment for chondrocyte adhesion, proliferation, and matrix biosynthesis for use in repair of cartilage defects. Due to their sponge-like elastic properties, cryogels can fully recover their original shape back after injection while not impacting metabolism or viability of encapsulated cells. Clinically, they provide an opportunity for filling focal cartilage defects by using a single, minimally invasive injection of a cell encapsulating biocompatible three-dimensional scaffold that can return to its original structure to fit the defect geometry and enable matrix regeneration.

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers.

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

Multi-arm Avidin nano-construct for intra-cartilage delivery of small molecule drugs.

Targeted drug delivery to joint tissues like cartilage remains a challenge that has prevented clinical translation of promising osteoarthritis (OA) drugs. Local intra-articular (IA) injections of drugs suffer from rapid clearance from the joint space and slow diffusive transport through the dense, avascular cartilage matrix comprised of negatively charged glycosaminoglycans (GAGs). Here we apply drug carriers that leverage electrostatic interactions with the tissue's high negative fixed charge density (FCD) for delivering small molecule drugs to cartilage cell and matrix sites. We demonstrate that a multi-arm cationic nano-construct of Avidin (mAv) with 28 sites for covalent drug conjugation can rapidly penetrate through the full thickness of cartilage in high concentration and have long intra-cartilage residence time in both healthy and arthritic cartilage via weak-reversible binding with negatively charged aggrecans. mAv's intra-cartilage mean uptake was found to be 112× and 33× the equilibration bath concentration in healthy and arthritic (50% GAG depleted) cartilage, respectively. mAv was conjugated with Dexamethasone (mAv-Dex), a broad-spectrum glucocorticoid, using a combination of hydrolysable ester linkers derived from succinic anhydride (SA), 3,3-dimethylglutaric anhydride (GA) and phthalic anhydride (PA) in 2:1:1 M ratio that enabled 50% drug release within 38.5 h followed by sustained release in therapeutic doses over 2 weeks. A single 10 µM low dose of controlled release mAv-Dex (2:1:1) effectively suppressed IL-1α-induced GAG loss, cell death and inflammatory response significantly better than unmodified Dex over 2 weeks in cartilage explant culture models of OA. With this multi-arm design, <1 µM Avidin was needed - a concentration which has been shown to be safe, preventing further GAG loss and cytotoxicity. A charge-based cartilage homing drug delivery platform like this can elicit disease modifying effects as well as facilitate long-term symptomatic pain and inflammation relief by enhancing tissue specificity and prolonging intra-cartilage residence time of OA drugs. This nano-construct thus has high translational potential for enabling intra-cartilage delivery of a broad array of small molecule OA drugs and their combinations to chondrocytes, enabling OA treatment with a single injection of low drug doses and eliminating toxicity issues associated with multiple high dose injections.

Interleukin-1 receptor antagonist (IL-1Ra) is more effective in suppressing cytokine-induced catabolism in cartilage-synovium co-culture than in cartilage monoculture.

BACKGROUND: Most in vitro studies of potential osteoarthritis (OA) therapies have used cartilage monocultures, even though synovium is a key player in mediating joint inflammation and, thereby, cartilage degeneration. In the case of interleukin-1 (IL-1) inhibition using its receptor antagonist (IL-1Ra), like chondrocytes, synoviocytes also express IL-1 receptors that influence intra-articular IL-1 signaling and IL-1Ra efficacy. The short residence time of IL-1Ra after intra-articular injection requires the application of frequent dosing, which is clinically impractical and comes with increased risk of infection; these limitations motivate the development of effective drug delivery strategies that can maintain sustained intra-articular IL-1Ra concentrations with only a single injection. The goals of this study were to assess how the presence of synovium in IL-1-challenged cartilage-synovium co-culture impacts the time-dependent biological response of single and sustained doses of IL-1Ra, and to understand the mechanisms underlying any co-culture effects. METHODS: Bovine cartilage explants with or without synovium were treated with IL-1α followed by single or multiple doses of IL-1Ra. Effects of IL-1Ra in rescuing IL-1α-induced catabolism in cartilage monoculture and cartilage-synovium co-culture were assessed by measuring loss of glycosaminoglycans (GAGs) and collagen using DMMB (dimethyl-methylene blue) and hydroxyproline assays, respectively, nitric oxide (NO) release using Griess assay, cell viability by fluorescence staining, metabolic activity using Alamar blue, and proteoglycan biosynthesis by radiolabel incorporation. Day 2 conditioned media from mono and co-cultures were analyzed by mass spectrometry and cytokine array to identify proteins unique to co-culture that contribute to biological crosstalk. RESULTS: A single dose of IL-1Ra was ineffective, and a sustained dose was necessary to significantly suppress IL-1α-induced catabolism as observed by enhanced suppression of GAG and collagen loss, NO synthesis, rescue of chondrocyte metabolism, viability, and GAG biosynthesis rates. The synovium exhibited a protective role as the effects of single-dose IL-1Ra were significantly enhanced in cartilage-synovium co-culture and were accompanied by release of anti-catabolic factors IL-4, carbonic anhydrase-3, and matrilin-3. A total of 26 unique proteins were identified in conditioned media from co-cultures, while expression levels of many additional proteins important to cartilage homeostasis were altered in co-culture compared to monocultures; principal component analysis revealed distinct clustering between co-culture and cartilage and synovium monocultures, thereby confirming significant crosstalk. CONCLUSIONS: IL-1Ra suppresses cytokine-induced catabolism in cartilage more effectively in the presence of synovium, which was associated with endogenous production of anti-catabolic factors. Biological crosstalk between cartilage and synovium is significant; thus, their co-cultures should better model the intra-articular actions of potential OA therapeutics. Additionally, chondroprotective effects of IL-1Ra require sustained drug levels, underscoring the need for developing drug delivery strategies to enhance its joint residence time following a single intra-articular injection.

Cartilage penetrating cationic peptide carriers for applications in drug delivery to avascular negatively charged tissues.

Drug delivery to avascular, negatively charged tissues like cartilage remains a challenge. The constant turnover of synovial fluid results in short residence time of administered drugs in the joint space and the dense negatively charged matrix of cartilage hinders their diffusive transport. Drugs are, therefore, unable to reach their cell and matrix targets in sufficient doses, and fail to elicit relevant biological response, which has led to unsuccessful clinical trials. The high negative fixed charge density (FCD) of cartilage, however, can be used to convert cartilage from a barrier to drug entry into a depot by making drugs positively charged. Here we design cartilage penetrating and binding cationic peptide carriers (CPCs) with varying net charge, spatial distribution and hydrophobicity to deliver large-sized therapeutics and investigate their electro-diffusive transport in healthy and arthritic cartilage. We showed that CPC uptake increased with increasing net charge up to +14 but dropped as charge increased further due to stronger binding interactions that hindered CPC penetrability and uptake showing that weak-reversible binding is key to enable their penetration through full tissue thickness. Even after 90% GAG depletion, while CPC +14 uptake reduced by over 50% but still had a significantly high value of 148× showing that intra-tissue long-range charge-based binding is further stabilized by short-range H-bond and hydrophobic interactions. The work presents an approach for rational design of cationic carriers based on tissue FCD and properties of macromolecules to be delivered. These design rules can be extended to drug delivery for other avascular, negatively charged tissues. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) remains an untreatable disease partly due to short joint residence time of drugs and a lack of delivery methods that can effectively target the dense, avascular, highly negatively charged cartilage tissue. In this study, we designed cartilage penetrating and binding cationic peptide carriers (CPCs) that, due to their optimal charge provide adequate electrical driving force to rapidly transport OA drugs into cartilage and reach their cell and matrix targets in therapeutic doses before drugs exit the joint space. This way cartilage is converted from being a barrier to drug entry into a drug depot that can provide sustained drug release for several weeks. This study also investigates synergistic effects of short-range H-bond and hydrophobic interactions in combination with long-range electrostatic interactions on intra-cartilage solute transport. The work provides rules for rational design of cartilage penetrating charge-based carriers depending on the net charge of tissue (normal versus arthritic), macromolecule to be delivered and whether the application is in drug delivery or tissue imaging.