Aged tendons lack adaptive response to acute compressive injury.

Rotator cuff tendinopathy has a multifactorial etiology, with both aging and external compression found to influence disease progression. However, tendon's response to these factors is still poorly understood and in vivo animal models make it difficult to decouple these effects. Therefore, we developed an explant culture model that allows us to directly apply compression to tendons and then observe their biological responses. Using this model, we applied a single acute compressive injury to C57BL/6J flexor digitorum longus tendon explants and observed changes in viability, metabolic activity, matrix composition, matrix biosynthesis, matrix structure, gene expression, and mechanical properties. We hypothesized that a single acute compressive load would result in an injury response in tendon and that this effect would be amplified in aged tendons. We found that young tendons had increased matrix turnover with a decrease in small leucine-rich proteoglycans, increase in compression-resistant proteoglycan aggrecan, increase in collagen synthesis, and an upregulation of collagen-degrading MMP-9. Aged tendons lacked any of these adaptive responses and instead had decreased metabolic activity and collagen synthesis. This implies that aged tendons lack the adaptation mechanisms required to return to homeostasis, and therefore are at greater risk for compression-induced injury. Overall, we present a novel compressive injury model that demonstrates lasting age-dependent changes and has the potential to examine the long-term response of tendon to a variety of compressive loading conditions.

Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system.

BACKGROUND: Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease. METHODS: Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin's grade 0-2, 23-83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile. RESULTS: Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified. CONCLUSION: CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.

Cyclic loading regime considered beneficial does not protect injured and interleukin-1-inflamed cartilage from post-traumatic osteoarthritis.

Injurious overloading and inflammation perturbate homeostasis of articular cartilage, leading to abnormal tissue-level loading during post-traumatic osteoarthritis. Our objective was to gain time- and cartilage depth-dependent insights into the early-stage disease progression with an in vitro model incorporating for the first time the coaction of (1) mechanical injury, (2) pro-inflammatory interleukin-1 challenge, and (3) cyclic loading mimicking walking and considered beneficial for cartilage health. Cartilage plugs (n = 406) were harvested from the patellofemoral grooves of young calves (N = 6) and subjected to injurious compression (50% strain, rate 100%/s; INJ), interleukin-1α-challenge (1 ng/ml; IL), and cyclic loading (intermittent 1 h loading periods, 15% strain, 1 Hz; CL). Plugs were assigned to six groups (control, INJ, IL, INJ-IL, IL-CL, INJ-IL-CL). Bulk and localized glycosaminoglycan (GAG) content (DMMB assay, digital densitometry), aggrecan biosynthesis (35S-sulfate incorporation), and chondrocyte viability (fluorescence microscopy) were assessed on days 3-12. The INJ, IL, and INJ-IL groups exhibited rapid early (days 2-4) GAG loss in contrast to CL groups. On day 3, deep cartilage of INJ-IL-CL group had higher GAG content than INJ group (p < 0.05). On day 12, INJ-IL-CL group showed more accumulated GAG loss (normalized with control) than INJ-IL group (average fold changes 1.97 [95% CI: 1.23-2.70]; 1.66 [1.42-1.89]; p = 0.007). Aggrecan biosynthesis increased in CL groups on day 12 compared to day 0. Despite promoting aggrecan biosynthesis, this cyclic loading protocol seems to be beneficial early-on to deep cartilage, but later becoming incapable of restricting further degradation triggered by marked but non-destructive injury and cytokine transport.

Green fluorescent proteins engineered for cartilage-targeted drug delivery: Insights for transport into highly charged avascular tissues.

Osteoarthritis (OA), the most common form of arthritis, is a multi-factorial disease that primarily affects cartilage as well as other joint tissues such as subchondral bone. The lack of effective drug delivery, due to the avascular nature of cartilage and the rapid clearance of intra-articularly delivered drugs via the synovium, remains a major challenge in the development of disease modifying drugs for OA. Cationic delivery carriers can significantly enhance the uptake, penetration and retention of drugs in cartilage by interacting with negatively charged matrix proteoglycans. In this study, we used "supercharged" green fluorescent proteins (GFPs), engineered to have a wide range of net positive charge and surface charge distributions, to characterize the effects of carrier charge on transport into cartilage in isolation of other factors such as carrier size and shape. We quantified the uptake, extent of cartilage penetration and cellular uptake of the GFP variants into living human knee cartilage and bovine cartilage explants. Based on these results, we identified optimal net charges of GFP carriers for potential drug targets located within cartilage extracellular matrix as well as the resident live chondrocytes. These cationic GFPs did not have adverse effects on cartilage in terms of measured cell viability and metabolism, cartilage cell biosynthesis and matrix degradation at doses needed for drug delivery. In addition to quantifying the kinetics of GFP uptake, we developed a predictive mathematical model for transport of the GFP variants that exhibited the highest uptake and penetration into cartilage. This model was further used to predict the transport behavior of GFPs during scale-up to in vivo applications such as intra-articular injection into human knees. The insights gained from this study set the stage for development of cartilage-targeted delivery systems to prevent cartilage degeneration, improve tissue regeneration and reduce inflammation that may cause degradation of other joint tissues affected by OA.

Dynamic nanomechanics of individual bone marrow stromal cells and cell-matrix composites during chondrogenic differentiation.

Dynamic nanomechanical properties of bovine bone marrow stromal cells (BMSCs) and their newly synthesized cartilage-like matrices were studied at nanometer scale deformation amplitudes. The increase in their dynamic modulus, |E(*)| (e.g., 2.4±0.4 kPa at 1 Hz to 9.7±0.2 kPa at 316 Hz at day 21, mean±SEM), and phase angle, δ, (e.g., 15±2° at 1 Hz to 74±1° at 316 Hz at day 21) with increasing frequency were attributed to the fluid flow induced poroelasticity, governed by both the newly synthesized matrix and the intracellular structures. The absence of culture duration dependence suggested that chondrogenesis of BMSCs had not yet resulted in the formation of a well-organized matrix with a hierarchical structure similar to cartilage. BMSC-matrix composites demonstrated different poro-viscoelastic frequency-dependent mechanical behavior and energy dissipation compared to chondrocyte-matrix composites due to differences in matrix molecular constituents, structure and cell properties. This study provides important insights into the design of optimal protocols for tissue-engineered cartilage products using chondrocytes and BMSCs.

Effects of the combination of microfracture and self-assembling Peptide filling on the repair of a clinically relevant trochlear defect in an equine model.

BACKGROUND: The goal of this study was to test the ability of an injectable self-assembling peptide (KLD) hydrogel, with or without microfracture, to augment articular cartilage defect repair in an equine cartilage defect model involving strenuous exercise. METHODS: Defects 15 mm in diameter were created on the medial trochlear ridge and debrided down to the subchondral bone. Four treatment groups (n = 8 each) were tested: no treatment (empty defect), only defect filling with KLD, only microfracture, and microfracture followed by filling with KLD. Horses were given strenuous exercise throughout the one-year study. Evaluations included lameness, arthroscopy, radiography, and gross, histologic, immunohistochemical, biochemical, and biomechanical analyses. RESULTS: Overall, KLD-only treatment of defects provided improvement in clinical symptoms and improved filling compared with no treatment, and KLD-only treatment protected against radiographic changes compared with microfracture treatment. Defect treatment with only microfracture also resulted in improved clinical symptoms compared with no treatment, and microfracture treatment resulted in repair tissue containing greater amounts of aggrecan and type-II collagen compared with KLD-only treatment. Microfracture treatment also protected against synovial fibrosis compared with no treatment and KLD-only treatment. Treatment with the self-assembling KLD peptide in combination with microfracture resulted in no additional improvements over microfracture-only treatment. In general, the nature of the predominant tissue in the defects was a mix of noncartilaginous and fibrocartilage tissue, with no significant differences among the treatments. CONCLUSIONS: Treatment of defects with only KLD or with only microfracture resulted in an improvement in clinical symptoms compared with no treatment; the improvement likely resulted from different causes depending on the treatment. Whereas microfracture improved the quality of repair tissue, KLD improved the amount of filling and protected against radiographic changes. CLINICAL RELEVANCE: Treatment of defects with only microfracture and with KLD only resulted in clinical improvements compared with untreated defects, despite differing with respect to the structural improvements that they induced.

Prediction of cartilage compressive modulus using multiexponential analysis of T(2) relaxation data and support vector regression.

Evaluation of mechanical characteristics of cartilage by magnetic resonance imaging would provide a noninvasive measure of tissue quality both for tissue engineering and when monitoring clinical response to therapeutic interventions for cartilage degradation. We use results from multiexponential transverse relaxation analysis to predict equilibrium and dynamic stiffness of control and degraded bovine nasal cartilage, a biochemical model for articular cartilage. Sulfated glycosaminoglycan concentration/wet weight (ww) and equilibrium and dynamic stiffness decreased with degradation from 103.6 ± 37.0 µg/mg ww, 1.71 ± 1.10 MPa and 15.3 ± 6.7 MPa in controls to 8.25 ± 2.4 µg/mg ww, 0.015 ± 0.006 MPa and 0.89 ± 0.25MPa, respectively, in severely degraded explants. Magnetic resonance measurements were performed on cartilage explants at 4 °C in a 9.4 T wide-bore NMR spectrometer using a Carr-Purcell-Meiboom-Gill sequence. Multiexponential T2 analysis revealed four water compartments with T2 values of approximately 0.14, 3, 40 and 150 ms, with corresponding weight fractions of approximately 3, 2, 4 and 91%. Correlations between weight fractions and stiffness based on conventional univariate and multiple linear regressions exhibited a maximum r(2) of 0.65, while those based on support vector regression (SVR) had a maximum r(2) value of 0.90. These results indicate that (i) compartment weight fractions derived from multiexponential analysis reflect cartilage stiffness and (ii) SVR-based multivariate regression exhibits greatly improved accuracy in predicting mechanical properties as compared with conventional regression.

Avidin as a model for charge driven transport into cartilage and drug delivery for treating early stage post-traumatic osteoarthritis.

Local drug delivery into cartilage remains a challenge due to its dense extracellular matrix of negatively charged proteoglycans enmeshed within a collagen fibril network. The high negative fixed charge density of cartilage offers the unique opportunity to utilize electrostatic interactions to augment transport, binding and retention of drug carriers. With the goal of developing particle-based drug delivery mechanisms for treating post-traumatic osteoarthritis, our objectives were, first, to determine the size range of a variety of solutes that could penetrate and diffuse through normal cartilage and enzymatically treated cartilage to mimic early stages of OA, and second, to investigate the effects of electrostatic interactions on particle partitioning, uptake and binding within cartilage using the highly positively charged protein, Avidin, as a model. Results showed that solutes having a hydrodynamic diameter ≤10 nm can penetrate into the full thickness of cartilage explants while larger sized solutes were trapped in the tissue's superficial zone. Avidin had a 400-fold higher uptake than its neutral same-sized counterpart, NeutrAvidin, and >90% of the absorbed Avidin remained within cartilage explants for at least 15 days. We report reversible, weak binding (K(D) ~ 150 µM) of Avidin to intratissue sites in cartilage. The large effective binding site density (N(T) ~ 2920 µM) within cartilage matrix facilitates Avidin's retention, making its structure suitable for particle based drug delivery into cartilage.

Transport and binding of tumor necrosis factor-α in articular cartilage depend on its quaternary structure.

The effect of tumor necrosis factor-α (TNFα) on cartilage matrix degradation is mediated by its transport and binding within the extracellular matrix (ECM) of the tissue, which mediates availability to cell receptors. Since the bioactive form of TNFα is a homotrimer of monomeric subunits, conversion between trimeric and monomeric forms during intratissue transport may affect binding to ECM and, thereby, bioactivity within cartilage. We studied the transport and binding of TNFα in cartilage, considering the quaternary structure of this cytokine. Competitive binding assays showed significant binding of TNFα in cartilage tissue, leading to an enhanced uptake. However, studies in which TNFα was cross-linked to remain in the trimeric form revealed that the binding of trimeric TNFα was negligible. Thus, binding of TNFα to ECM was associated with the monomeric form. Binding of TNFα was not disrupted by pre-treating cartilage tissue with trypsin, which removes proteoglycans and glycoproteins but leaves the collagen network intact. Therefore, proteoglycan loss during osteoarthritis should only alter the passive diffusion of TNFα but not its binding interaction with the remaining matrix. Our results suggest that matrix binding and trimer-monomer conversion of TNFα both play crucial roles in regulating the accessibility of bioactive TNFα within cartilage.

Transport of anti-IL-6 antigen binding fragments into cartilage and the effects of injury.

The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules.

Time-dependent nanomechanics of cartilage.

In this study, atomic force microscopy-based dynamic oscillatory and force-relaxation indentation was employed to quantify the time-dependent nanomechanics of native (untreated) and proteoglycan (PG)-depleted cartilage disks, including indentation modulus E(ind), force-relaxation time constant τ, magnitude of dynamic complex modulus |E(∗)|, phase angle δ between force and indentation depth, storage modulus E', and loss modulus E″. At ∼2 nm dynamic deformation amplitude, |E(∗)| increased significantly with frequency from 0.22 ± 0.02 MPa (1 Hz) to 0.77 ± 0.10 MPa (316 Hz), accompanied by an increase in δ (energy dissipation). At this length scale, the energy dissipation mechanisms were deconvoluted: the dynamic frequency dependence was primarily governed by the fluid-flow-induced poroelasticity, whereas the long-time force relaxation reflected flow-independent viscoelasticity. After PG depletion, the change in the frequency response of |E(∗)| and δ was consistent with an increase in cartilage local hydraulic permeability. Although untreated disks showed only slight dynamic amplitude-dependent behavior, PG-depleted disks showed great amplitude-enhanced energy dissipation, possibly due to additional viscoelastic mechanisms. Hence, in addition to functioning as a primary determinant of cartilage compressive stiffness and hydraulic permeability, the presence of aggrecan minimized the amplitude dependence of |E(∗)| at nanometer-scale deformation.

Transport and equilibrium uptake of a peptide inhibitor of PACE4 into articular cartilage is dominated by electrostatic interactions.

The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be approximately 1 x 10(-6) cm(2)/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery.

Dynamic mechanical properties of the tissue-engineered matrix associated with individual chondrocytes.

The success of cell-based tissue engineering approaches in restoring biological function will be facilitated by a comprehensive fundamental knowledge of the temporal evolution of the structure and properties of the newly synthesized matrix. Here, we quantify the dynamic oscillatory mechanical behavior of the engineered matrix associated with individual chondrocytes cultured in vitro for up to 28 days in alginate scaffolds. The magnitude of the complex modulus (|E*|) and phase shift (delta) were measured in culture medium using Atomic Force Microscopy (AFM)-based nanoindentation in response to an imposed oscillatory deformation (amplitude approximately 5nm) as a function of frequency (f=1-316Hz), probe tip geometry (2.5microm radius sphere and 50nm radius square pyramid), and in the absence and presence of growth factors (GF, insulin growth factor-1, IGF-1, and osteogenic protein-1, OP-1). |E*| for all conditions increased nonlinearly with frequency dependence approximately f(1/2) and ranged between approximately 1 and 25kPa. This result, along with theoretical calculations of the characteristic poroelastic relaxation frequency, f(p), (approximately 50-90Hz) suggested that this time-dependent behavior was governed primarily by fluid flow-dependent poroelasticity, rather than flow-independent viscoelastic processes associated with the solid matrix. |E*(f)| increased, (f) decreased, and the hydraulic permeability, k, decreased with time in culture and with growth factor treatment. This trend of a more elastic-like response was thought to be associated with increased macromolecular biosynthesis, density, and a more mature matrix structure/organization.

Influence of tissue maturation and antioxidants on the apoptotic response of articular cartilage after injurious compression.

OBJECTIVE: To study the influence of tissue maturation and antioxidants on apoptosis in bovine articular cartilage induced by injurious compression. METHODS: Bovine articular cartilage disks were obtained from the femoropatellar groove of animals ages 0.5-23 months and placed in culture. Cartilage disks were preincubated overnight with the cell-permeable superoxide dismutase (SOD) mimetic Mn(III) porphyrin (0-12.5 microM) or alpha-tocopherol (0-50 microM) and then injured by a single unconfined compression to a final strain of 50% at a velocity of 1 mm/second. After 4 days of additional incubation, the disks were fixed and embedded for light and electron microscopy. Apoptotic cells were quantified morphologically by the appearance of nuclear blebbing on light microscopy. Biosynthetic activity was demonstrated by incorporation of radiolabeled proline. The antioxidative action of the SOD mimetic was confirmed by histologic examination of cartilage after incubation with nitroblue tetrazolium. RESULTS: Injurious compression induced significantly more apoptosis in cartilage disks from newborn calves (22% of cells) than in cartilage from more mature cows (2-6%). In cartilage from 22-month-old animals, the SOD mimetic reduced the percentage of apoptotic cells induced by injury in a dose-dependent manner (complete inhibition with 2.5 microM), while alpha-tocopherol had no effect. Neither antioxidant altered protein biosynthesis or cellular ultrastructure. CONCLUSION: Our data suggest that the apoptotic response of articular cartilage to mechanical injury is affected by maturation and is mediated in part by reactive oxygen species. The antioxidative status of the tissue might be important for the prevention of mechanically induced cell death in articular cartilage.

Transport and binding of insulin-like growth factor I through articular cartilage.

This study focused on the role of insulin-like growth factor (IGF) binding proteins (IGFBPs) in cartilage on the transport and binding of IGF-I within the tissue. We have developed experimental and theoretical modeling techniques to quantify and contrast the roles of diffusion, binding, fluid convection, and electrical migration on the transport of IGF-I within cartilage tissue. Bovine articular cartilage disks were equilibrated in buffer containing 125I-IGF-I and graded levels of unlabeled IGF-I. Equilibrium binding, as measured by the uptake ratio of 125I-IGF-I in the tissue (free plus bound) to the concentration of labeled species in the buffer, was found to be consistent with a first-order reversible binding model involving one dominant family of binding sites within the matrix. Western ligand blots revealed a major IGF binding doublet around 23 kDa, which has been previously shown to coincide with IGFBP-6. Diffusive transport of 125I-IGF-I through cartilage was measured and found to be consistent with a diffusion-limited reaction theoretical model incorporating first-order reversible binding. Addition of excess amounts of unlabeled IGF-I during steady state transport of 125I-IGF-I resulted in release of bound 125I-IGF-I from the tissue, as predicted by the diffusion-reaction model. In contrast, addition of the low-affinity Des(1-3)IGF-I analog did not result in release of bound 125I-IGF-I. Application of electric current was used to augment transport of IGF-I through cartilage via electroosmosis and electrophoresis. Taken together, our results suggest that a single dominant substrate family, the high-affinity IGFBPs, is responsible for much of the observed binding of IGF-I within cartilage. The data suggest that intratissue fluid flow, such as that induced by mechanical loading of cartilage in vivo may be expected to enhance IGF transport by an order of magnitude and that this increment may help to counterbalance the restrictions encountered by the immobilization of IGFs by the binding proteins.