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.

Effects of dexamethasone and dynamic loading on cartilage of human osteochondral explants challenged with inflammatory cytokines.

Post-traumatic osteoarthritis (PTOA), characterized by articular cartilage degradation initiated in an inflammatory environment after traumatic joint injury, can lead to alterations in cartilage biomechanical properties. Low dose dexamethasone (Dex) shows chondroprotection in cartilage challenged with inflammatory cytokines, but little is known about the structural biomechanical response of human cartilage to Dex in such a diseased state. This study examined changes in the biomechanical properties and biochemical composition of the cartilage within human osteochondral explants in response to treatment with exogenous cytokines, Dex, and a regimen of cyclic loading at the start and end of culture. Osteochondral explants were harvested from five pairs of human ankle talocrural joints (Collins grade 0-1) and cultured for 10 days with/without exogenous cytokines (100 ng/mL TNFα, 50 ng/mL IL-6, 250 ng/mL sIL-6R) ± Dex (100 nM). Biomechanical testing on day-0 and day-10 enabled estimation of the unconfined compression equilibrium modulus (Ey), dynamic stiffness (Ed) and hydraulic permeability (kp) of cartilage excised from bone, accompanied by biochemical assessment of media and cartilage tissue. Dex preserved chondrocyte cell viability and decreased sulfated glycosaminoglycan (sGAG) loss and nitric oxide release, but did not alter Ey, Ed and kp (before or after loading) on day-10. In the cytokine/cytokine+Dex treated groups, sGAG content exhibited a weaker correlation with Ey and Ed than at baseline, suggesting an important role for structural rather than biochemical changes in producing biomechanical alterations in response to cytokines and Dex. These findings aid in forming a more complete profile of potential clinical effects of Dex for use in OA/PTOA treatment regimens.

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.

Microfracture Augmentation With Trypsin Pretreatment and Growth Factor-Functionalized Self-assembling Peptide Hydrogel Scaffold in an Equine Model.

BACKGROUND: Microfracture augmentation can be a cost-effective single-step alternative to current cartilage repair techniques. Trypsin pretreatment combined with a growth factor-functionalized self-assembling KLD hydrogel ("functionalized hydrogel") has been shown to improve overall cartilage repair and integration to surrounding tissue in small animal models of osteochondral defects. HYPOTHESIS: Microfracture combined with trypsin treatment and a functionalized hydrogel will improve reparative tissue quality and integration as compared with microfracture alone in an equine model. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral cartilage defects (15-mm diameter) were created on the medial trochlear ridge of the femoropatellar joints in 8 adult horses (16 defects total). One defect was randomly selected to receive the treatment, and the contralateral defect served as the control (microfracture only). Treatment consisted of 2-minute trypsin pretreatment of the surrounding cartilage, subchondral bone microfracture, and functionalized hydrogel premixed with growth factors (platelet-derived growth factor and heparin-binding insulin-like growth factor 1). After surgery, all horses were subjected to standardized controlled exercise on a high-speed treadmill. Clinical evaluation was conducted monthly, and radiographic examinations were performed at 2, 16, 24, 32, 40, and 52 weeks after defect creation. After 12 months, all animals were euthanized. Magnetic resonance imaging, arthroscopy, gross pathologic evaluation of the joint, histology, immunohistochemistry, and biomechanical analyses were performed. Generalized linear mixed models (with horse as random effect) were utilized to assess outcome parameters. When P values were <.05, pairwise comparisons were made using least squares means. RESULTS: Improved functional outcome parameters were observed for the treatment group, even though mildly increased joint effusion and subchondral bone sclerosis were noted on imaging. Microscopically, treatment resulted in improvement of several histologic parameters and overall quality of repaired tissue. Proteoglycan content based on safranin O-fast green staining was also significantly higher in the treated defects. CONCLUSION: Trypsin treatment combined with functionalized hydrogel resulted in improved microfracture augmentation. CLINICAL RELEVANCE: Therapeutic strategies for microfracture augmentation, such as those presented in this study, can be cost-effective ways to improve cartilage healing outcomes, especially in more active patients.

Enzyme Pretreatment plus Locally Delivered HB-IGF-1 Stimulate Integrative Cartilage Repair In Vitro.

IMPACT STATEMENT: A critical attribute for the long-term success of cartilage defect repair is the strong integration between the repair tissue and the surrounding native tissue. Current approaches utilized by physicians fail to achieve this attribute, leading to eventual relapse of the defect. This article demonstrates the concept of a simple, clinically viable approach for enhancing tissue integration via the combination of a safe, transient enzymatic treatment with a locally delivered, retained growth factor through an in vitro hydrogel/cartilage explant model.

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.

Near infrared spectroscopic assessment of developing engineered tissues: correlations with compositional and mechanical properties.

Articular cartilage degeneration causes pain and reduces the mobility of millions of people annually. Regeneration of cartilage is challenging, due in part to its avascular nature, and thus tissue engineering approaches for cartilage repair have been studied extensively. Current techniques to assess the composition and integrity of engineered tissues, including histology, biochemical evaluation, and mechanical testing, are destructive, which limits real-time monitoring of engineered cartilage tissue development in vitro and in vivo. Near infrared spectroscopy (NIRS) has been proposed as a non-destructive technique to characterize cartilage. In the current study, we describe a non-destructive NIRS approach for assessment of engineered cartilage during development, and demonstrate correlation of these data to gold standard mid infrared spectroscopic measurements, and to mechanical properties of constructs. Cartilage constructs were generated using bovine chondrocyte culture on polyglycolic acid (PGA) scaffolds for six weeks. BMP-4 growth factor and ultrasound mechanical stimulation were used to provide a greater dynamic range of tissue properties and outcome variables. NIR spectra were collected daily using an infrared fiber optic probe in diffuse reflectance mode. Constructs were harvested after three and six weeks of culture and evaluated by the correlative modalities of mid infrared (MIR) spectroscopy, histology, and mechanical testing (equilibrium and dynamic stiffness). We found that specific NIR spectral absorbances correlated with MIR measurements of chemical composition, including relative amount of PGA (R = 0.86, p = 0.02), collagen (R = 0.88, p = 0.03), and proteoglycan (R = 0.83, p = 0.01). In addition, NIR-derived water content correlated with MIR-derived proteoglycan content (R = 0.76, p = 0.04). Both equilibrium and dynamic mechanical properties generally improved with cartilage growth from three to six weeks. In addition, significant correlations between NIRS-derived parameters and mechanical properties were found for constructs that were not treated with ultrasound (PGA (R = 0.71, p = 0.01), water (R = 0.74, p = 0.02), collagen (R = 0.69, p = 0.04), and proteoglycan (R = 0.62, p = 0.05)). These results lay the groundwork for extension to arthroscopic engineered cartilage assessment in clinical studies.

High-bandwidth AFM-based rheology is a sensitive indicator of early cartilage aggrecan degradation relevant to mouse models of osteoarthritis.

Murine models of osteoarthritis (OA) and post-traumatic OA have been widely used to study the development and progression of these diseases using genetically engineered mouse strains along with surgical or biochemical interventions. However, due to the small size and thickness of murine cartilage, the relationship between mechanical properties, molecular structure and cartilage composition has not been well studied. We adapted a recently developed AFM-based nano-rheology system to probe the dynamic nanomechanical properties of murine cartilage over a wide frequency range of 1 Hz to 10 kHz, and studied the role of glycosaminoglycan (GAG) on the dynamic modulus and poroelastic properties of murine femoral cartilage. We showed that poroelastic properties, highlighting fluid-solid interactions, are more sensitive indicators of loss of mechanical function compared to equilibrium properties in which fluid flow is negligible. These fluid-flow-dependent properties include the hydraulic permeability (an indicator of the resistance of matrix to fluid flow) and the high frequency modulus, obtained at high rates of loading relevant to jumping and impact injury in vivo. Utilizing a fibril-reinforced finite element model, we estimated the poroelastic properties of mouse cartilage over a wide range of loading rates for the first time, and show that the hydraulic permeability increased by a factor ~16 from knormal=7.80×10(-16)±1.3×10(-16) m(4)/N s to kGAG-depleted=1.26×10(-14)±6.73×10(-15) m(4)/N s after GAG depletion. The high-frequency modulus, which is related to fluid pressurization and the fibrillar network, decreased significantly after GAG depletion. In contrast, the equilibrium modulus, which is fluid-flow independent, did not show a statistically significant alteration following GAG depletion.

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.

Stress-vs-time signals allow the prediction of structurally catastrophic events during fracturing of immature cartilage and predetermine the biomechanical, biochemical, and structural impairment.

OBJECTIVE: Trauma-associated cartilage fractures occur in children and adolescents with clinically significant incidence. Several studies investigated biomechanical injury by compressive forces but the injury-related stress has not been investigated extensively. In this study, we hypothesized that the biomechanical stress occurring during compressive injury predetermines the biomechanical, biochemical, and structural consequences. We specifically investigated whether the stress-vs-time signal correlated with the injurious damage and may allow prediction of cartilage matrix fracturing. METHODS: Superficial and deeper zones disks (SZDs, DZDs; immature bovine cartilage) were biomechanically characterized, injured (50% compression, 100%/s strain-rate), and re-characterized. Correlations of the quantified functional, biochemical and histological damage with biomechanical parameters were zonally investigated. RESULTS: Injured SZDs exhibited decreased dynamic stiffness (by 93.04±1.72%), unresolvable equilibrium moduli, structural damage (2.0±0.5 on a 5-point-damage-scale), and 1.78-fold increased sGAG loss. DZDs remained intact. Measured stress-vs-time-curves during injury displayed 4 distinct shapes, which correlated with histological damage (p<0.001), loss of dynamic stiffness and sGAG (p<0.05). Damage prediction in a blinded experiment using stress-vs-time grades was 100%-correct and sensitive to differentiate single/complex matrix disruptions. Correlations of the dissipated energy and maximum stress rise with the extent of biomechanical and biochemical damage reached significance when SZDs and DZDs were analyzed as zonal composites but not separately. CONCLUSIONS: The biomechanical stress that occurs during compressive injury predetermines the biomechanical, biochemical, and structural consequences and, thus, the structural and functional damage during cartilage fracturing. A novel biomechanical method based on the interpretation of compressive yielding allows the accurate prediction of the extent of structural damage.

High-bandwidth AFM-based rheology reveals that cartilage is most sensitive to high loading rates at early stages of impairment.

Utilizing a newly developed atomic-force-microscopy-based wide-frequency rheology system, we measured the dynamic nanomechanical behavior of normal and glycosaminoglycan (GAG)-depleted cartilage, the latter representing matrix degradation that occurs at the earliest stages of osteoarthritis. We observed unique variations in the frequency-dependent stiffness and hydraulic permeability of cartilage in the 1 Hz-to-10 kHz range, a frequency range that is relevant to joint motions from normal ambulation to high-frequency impact loading. Measurement in this frequency range is well beyond the capabilities of typical commercial atomic force microscopes. We showed that the dynamic modulus of cartilage undergoes a dramatic alteration after GAG loss, even with the collagen network still intact: whereas the magnitude of the dynamic modulus decreased two- to threefold at higher frequencies, the peak frequency of the phase angle of the modulus (representing fluid-solid frictional dissipation) increased 15-fold from 55 Hz in normal cartilage to 800 Hz after GAG depletion. These results, based on a fibril-reinforced poroelastic finite-element model, demonstrated that GAG loss caused a dramatic increase in cartilage hydraulic permeability (up to 25-fold), suggesting that early osteoarthritic cartilage is more vulnerable to higher loading rates than to the conventionally studied "loading magnitude". Thus, over the wide frequency range of joint motion during daily activities, hydraulic permeability appears the most sensitive marker of early tissue degradation.

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.

Vulnerability of the superficial zone of immature articular cartilage to compressive injury.

OBJECTIVE: The zonal composition and functioning of adult articular cartilage causes depth-dependent responses to compressive injury. In immature cartilage, shear and compressive moduli as well as collagen and sulfated glycosaminoglycan (sGAG) content also vary with depth. However, there is little understanding of the depth-dependent damage caused by injury. Since injury to immature knee joints most often causes articular cartilage lesions, this study was undertaken to characterize the zonal dependence of biomechanical, biochemical, and matrix-associated changes caused by compressive injury. METHODS: Disks from the superficial and deeper zones of bovine calves were biomechanically characterized. Injury to the disks was achieved by applying a final strain of 50% compression at 100%/second, followed by biomechanical recharacterization. Tissue compaction upon injury as well as sGAG density, sGAG loss, and biosynthesis were measured. Collagen fiber orientation and matrix damage were assessed using histology, diffraction-enhanced x-ray imaging, and texture analysis. RESULTS: Injured superficial zone disks showed surface disruption, tissue compaction by 20.3 ± 4.3% (mean ± SEM), and immediate biomechanical impairment that was revealed by a mean ± SEM decrease in dynamic stiffness to 7.1 ± 3.3% of the value before injury and equilibrium moduli that were below the level of detection. Tissue areas that appeared intact on histology showed clear textural alterations. Injured deeper zone disks showed collagen crimping but remained undamaged and biomechanically intact. Superficial zone disks did not lose sGAG immediately after injury, but lost 17.8 ± 1.4% of sGAG after 48 hours; deeper zone disks lost only 2.8 ± 0.3% of sGAG content. Biomechanical impairment was associated primarily with structural damage. CONCLUSION: The soft superficial zone of immature cartilage is vulnerable to compressive injury, causing superficial matrix disruption, extensive compaction, and textural alteration, which results in immediate loss of biomechanical function. In conjunction with delayed superficial sGAG loss, these changes may predispose the articular surface to further softening and tissue damage, thus increasing the risk of development of secondary osteoarthritis.

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.