The subacromial bursa modulates tendon healing after rotator cuff injury in rats.

Rotator cuff injuries result in more than 500,000 surgeries annually in the United States, many of which fail. These surgeries typically involve repair of the injured tendon and removal of the subacromial bursa, a synovial-like tissue that sits between the rotator cuff and the acromion. The subacromial bursa has been implicated in rotator cuff pathogenesis and healing. Using proteomic profiling of bursa samples from nine patients with rotator cuff injury, we show that the bursa responds to injury in the underlying tendon. In a rat model of supraspinatus tenotomy, we evaluated the bursa's effect on the injured supraspinatus tendon, the uninjured infraspinatus tendon, and the underlying humeral head. The bursa protected the intact infraspinatus tendon adjacent to the injured supraspinatus tendon by maintaining its mechanical properties and protected the underlying humeral head by maintaining bone morphometry. The bursa promoted an inflammatory response in injured rat tendon, initiating expression of genes associated with wound healing, including Cox2 and Il6. These results were confirmed in rat bursa organ cultures. To evaluate the potential of the bursa as a therapeutic target, polymer microspheres loaded with dexamethasone were delivered to the intact bursae of rats after tenotomy. Dexamethasone released from the bursa reduced Il1b expression in injured rat supraspinatus tendon, suggesting that the bursa could be used for drug delivery to reduce inflammation in the healing tendon. Our findings indicate that the subacromial bursa contributes to healing in underlying tissues of the shoulder joint, suggesting that its removal during rotator cuff surgery should be reconsidered.

In vitro inflammatory multi-cellular model of osteoarthritis.

Objective: Osteoarthritis (OA) is a chronic joint disease, with limited treatment options, characterized by inflammation and matrix degradation, and resulting in severe pain or disability. Progressive inflammatory interaction among key cell types, including chondrocytes and macrophages, leads to a cascade of intra- and inter-cellular events which culminate in OA induction. In order to investigate these interactions, we developed a multi-cellular in vitro OA model, to characterize OA progression, and identify and evaluate potential OA therapeutics in response to mediators representing graded levels of inflammatory severity. Methods: We compared macrophages, chondrocytes and their co-culture responses to "low" Interleukin-1 (IL-1) or "high" IL-1/tumor necrosis factor (IL-1/TNF) levels of inflammation. We also investigated response changes following the administration of dexamethasone (DEX) or mesenchymal stromal cell (MSC) treatment via a combination of gene expression and secretory changes, reflecting not only inflammation, but also chondrocyte function. Results: Inflamed chondrocytes presented an osteoarthritic-like phenotype characterized by high gene expression of pro-inflammatory cytokines and chemokines, up-regulation of ECM degrading proteases, and down-regulation of chondrogenic genes. Our results indicate that while MSC treatment attenuates macrophage inflammation directly, it does not reduce chondrocyte inflammatory responses, unless macrophages are present as well. DEX however, can directly attenuate chondrocyte inflammation. Conclusions: Our results highlight the importance of considering multi-cellular interactions when studying complex systems such as the articular joint. In addition, our approach, using a panel of both inflammatory and chondrocyte functional genes, provides a more comprehensive approach to investigate disease biomarkers, and responses to treatment.

Sex differences in the biomechanical and biochemical responses of caudal rat intervertebral discs to injury.

Background: Intervertebral disc degeneration (IDD) is a major cause of low back pain (LBP) worldwide. Sexual dimorphism, or sex-based differences, appear to exist in the severity of LBP. However, it is unknown if there are sex-based differences in the inflammatory, biomechanical, biochemical, and histological responses of intervertebral discs (IVDs). Methods: Caudal (Coccygeal/Co) bone-disc-bone motion segments were isolated from multiple spinal levels (Co8 to Co14) of male and female Sprague-Dawley rats. Changes in motion segment biomechanics and extracellular matrix (ECM) biochemistry (glycosaminoglycan [GAG], collagen [COL], water, and DNA content) were evaluated at baseline and in response to chemical insult (lipopolysaccharide [LPS]) or puncture injury ex vivo. We also investigated the contributions of Toll-like receptor (TLR4) signaling on responses to LPS or puncture injury ex vivo, using a small molecule TLR4 inhibitor, TAK-242. Results: Findings indicate that IVD motion segments from female donors had greater nitric oxide (NO) release in LPS groups compared to male donors. HMGB1 release was increased in punctured discs, but not LPS injured discs, with no sex effect. Although both male and female discs exhibited reductions in dynamic moduli in response to LPS and puncture injuries, dynamic moduli from female donors were higher than male donors across all groups. In uninjured (baseline) samples, a significant sex effect was observed in nucleus pulposus (NP) DNA and water content. Female annulus fibrosus (AF) also had higher DNA, GAG, and COL content (normalized by dry weight), but lower water content than male AF. Additional injury- and sex-dependent effects were observed in AF GAG/DNA and COL/DNA content. Finally, TAK-242 improved the dynamic modulus of female but not male punctured discs. Conclusions: Our findings demonstrate that there are differences in rat IVD motion segments based on sex, and that the response to injury in inflammatory, biomechanical, biochemical, and histological outcomes also exhibit sex differences. TLR4 inhibition protected against loss of mechanical integrity of puncture-injured IVD motion segments, with differences responses based on donor sex.

Hedgehog Activation for Enhanced Rotator Cuff Tendon-to-Bone Healing.

BACKGROUND: Rotator cuff repair is a common orthopaedic procedure, yet the rate of failure to heal after surgery is high. Repair site rupture is due to poor tendon-to-bone healing and lack of regeneration of the native fibrocartilaginous enthesis. During development, the enthesis is formed and mineralized by a pool of progenitors activated by hedgehog signaling. Furthermore, hedgehog signaling drives regenerative enthesis healing in young animals, in contrast to older animals, in which enthesis injuries heal via fibrovascular scar and without participation of hedgehog signaling. HYPOTHESIS: Hedgehog activation improves tendon-to-bone healing in an animal model of rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 78 adult Sprague-Dawley rats were used. Supraspinatus tendon injury and repair were completed bilaterally, with microsphere-encapsulated hedgehog agonist administered to right shoulders and control microspheres administered to left shoulders. Animals were sacrificed after 3, 14, 28, or 56 days. Gene expression and histological, biomechanical, and bone morphometric analyses were conducted. RESULTS: At 3 days, hedgehog signaling pathway genes Gli1 (1.70; P = .029) and Smo (2.06; P = .0173), as well as Runx2 (1.69; P = .0386), a transcription factor of osteogenesis, were upregulated in treated relative to control repairs. At 14 days, transcription factors of tenogenesis, Scx (4.00; P = .041), and chondrogenesis, Sox9 (2.95; P = .010), and mineralized fibrocartilage genes Col2 (3.18; P = .031) and Colx (1.85; P = .006), were upregulated in treated relative to control repairs. Treatment promoted fibrocartilage formation at the healing interface by 28 days, with improvements in tendon-bone maturity, organization, and continuity. Treatment led to improved biomechanical properties. The material property strength (2.43 vs 1.89 N/m2; P = .046) and the structural property work to failure (29.01 vs 18.09 mJ; P = .030) were increased in treated relative to control repairs at 28 days and 56 days, respectively. Treatment had a marginal effect on bone morphometry underlying the repair. Trabecular thickness (0.08 vs 0.07 mm; P = .035) was increased at 28 days. CONCLUSION: Hedgehog agonist treatment activated hedgehog signaling at the tendon-to-bone repair site and prompted increased mineralized fibrocartilage production. This extracellular matrix production and mineralization resulted in improved biomechanical properties, demonstrating the therapeutic potential of hedgehog agonism for improving tendon-to-bone healing after rotator cuff repair. CLINICAL RELEVANCE: This study demonstrates the therapeutic potential of hedgehog agonist treatment for improving tendon-to-bone healing after rotator cuff injury and repair.

The subacromial bursa is a key regulator of the rotator cuff and a new therapeutic target for improving repair.

Rotator cuff injuries result in over 500,000 surgeries performed annually, an alarmingly high number of which fail. These procedures typically involve repair of the injured tendon and removal of the subacromial bursa. However, recent identification of a resident population of mesenchymal stem cells and inflammatory responsiveness of the bursa to tendinopathy indicate an unexplored biological role of the bursa in the context of rotator cuff disease. Therefore, we aimed to understand the clinical relevance of bursa-tendon crosstalk, characterize the biologic role of the bursa within the shoulder, and test the therapeutic potential for targeting the bursa. Proteomic profiling of patient bursa and tendon samples demonstrated that the bursa is activated by tendon injury. Using a rat to model rotator cuff injury and repair, tenotomy-activated bursa protected the intact tendon adjacent to the injured tendon and maintained the morphology of the underlying bone. The bursa also promoted an early inflammatory response in the injured tendon, initiating key players in wound healing. In vivo results were supported by targeted organ culture studies of the bursa. To examine the potential to therapeutically target the bursa, dexamethasone was delivered to the bursa, prompting a shift in cellular signaling towards resolution of inflammation in the healing tendon. In conclusion, contrary to current clinical practice, the bursa should be retained to the greatest extent possible and provides a new therapeutically target for improving tendon healing outcomes. One Sentence Summary: The subacromial bursa is activated by rotator cuff injury and regulates the paracrine environment of the shoulder to maintain the properties of the underlying tendon and bone.

Red blood cell exposure increases chondrocyte susceptibility to oxidative stress following hemarthrosis.

OBJECTIVE: The detrimental effects of blood exposure on articular tissues are well characterized, but the individual contributions of specific whole blood components are yet to be fully elucidated. Better understanding of mechanisms that drive cell and tissue damage in hemophilic arthropathy will inform novel therapeutic strategies. The studies here aimed to identify the specific contributions of intact and lysed red blood cells (RBCs) on cartilage and the therapeutic potential of Ferrostatin-1 in the context of lipid changes, oxidative stress, and ferroptosis. METHODS: Changes to biochemical and mechanical properties following intact RBC treatment were assessed in human chondrocyte-based tissue-engineered cartilage constructs and validated against human cartilage explants. Chondrocyte monolayers were assayed for changes to intracellular lipid profiles and the presence of oxidative and ferroptotic mechanisms. RESULTS: Markers of tissue breakdown were observed in cartilage constructs without parallel losses in DNA (control: 786.3 (102.2) ng/mg; RBCINT: 751 (126.4) ng/mg; P = 0.6279), implicating nonlethal chondrocyte responses to intact RBCs. Dose-dependent loss of viability in response to intact and lysed RBCs was observed in chondrocyte monolayers, with greater toxicity observed with lysates. Intact RBCs induced changes to chondrocyte lipid profiles, upregulating highly oxidizable fatty acids (e.g., FA 18:2) and matrix disrupting ceramides. RBC lysates induced cell death via oxidative mechanisms that resemble ferroptosis. CONCLUSIONS: Intact RBCs induce intracellular phenotypic changes to chondrocytes that increase vulnerability to tissue damage while lysed RBCs have a more direct influence on chondrocyte death by mechanisms that are representative of ferroptosis.

Modeling Inelastic Responses Using Constrained Reactive Mixtures.

This study reviews the progression of our research, from modeling growth theories for cartilage tissue engineering, to the formulation of constrained reactive mixture theories to model inelastic responses in any solid material, such as theories for damage mechanics, viscoelasticity, plasticity, and elasto-plastic damage. In this framework, multiple solid generations α can co-exist at any given time in the mixture. The oldest generation is denoted by α=s and is called the master generation, whose reference configuration Xs is observable. The solid generations α are all constrained to share the same velocity vs, but may have distinct reference configurations Xα. An important element of this formulation is that the time-invariant mapping Fαs=∂Xα/∂Xs between these reference configurations is a function of state, whose mathematical formulation is postulated by constitutive assumption. Thus, reference configurations Xα are not observable (α≠s). This formulation employs only observable state variables, such as the deformation gradient Fs of the master generation and the referential mass concentrations ρrα of each generation, in contrast to classical formulations of inelastic responses which rely on internal state variable theory, requiring evolution equations for those hidden variables. In constrained reactive mixtures, the evolution of the mass concentrations is governed by the axiom of mass balance, using constitutive models for the mass supply densities ρˆrα. Classical and constrained reactive mixture approaches share considerable mathematical analogies, as they both introduce a multiplicative decomposition of the deformation gradient, also requiring evolution equations to track some of the state variables. However, they also differ at a fundamental level, since one adopts only observable state variables while the other introduces hidden state variables. In summary, this review presents an alternative foundational approach to the modeling of inelastic responses in solids, grounded in the classical framework of mixture theory.

Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-ß signaling.

In tissue development and homeostasis, transforming growth factor (TGF)-ß signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-ß signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-ß signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-ß gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-ß signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.

Blocking toll-like receptor 4 mitigates static loading induced pro-inflammatory expression in intervertebral disc motion segments.

While the anabolic effects of mechanical loading on the intervertebral disc (IVD) have been extensively studied, inflammatory responses to loading have not been as well characterized. Recent studies have highlighted a significant role of innate immune activation, particularly that of toll-like receptors (TLRs), in IVD degeneration. Biological responses of intervertebral disc cells to loading depend on many factors that include magnitude and frequency. The goals of this study were to characterize the inflammatory signaling changes in response to static and dynamic loading of IVD and investigate the contributions of TLR4 signaling in response to mechanical loading. Rat bone-disc-bone motion segments were loaded for 3 hr under a static load (20 % strain, 0 Hz) with or without an additional low-dynamic (4 % dynamic strain, 0.5 Hz) or high-dynamic (8 % dynamic strain, 3 Hz) strain, and results were compared to unloaded controls. Some samples were also loaded with or without TAK-242, an inhibitor of TLR4 signaling. The magnitude of NO release into the loading media (LM) was correlated with the applied frequency and strain magnitudes across different loading groups. Injurious loading profiles, such as static and high-dynamic, significantly increased Tlr4 and Hmgb1 expression while this result was not observed in the more physiologically relevant low-dynamic loading group. TAK-242 co-treatment decreased pro-inflammatory expression in static but not dynamic loaded groups, suggesting that TLR4 plays a direct role in mediating inflammatory responses of IVD to static compression. Overall, the microenvironment induced by dynamic loading diminished the protective effects of the TAK-242, suggesting that TLR4 plays a direct role in mediating inflammatory responses of IVD to static loading injury.

Toward defining the role of the synovium in mitigating normal articular cartilage wear and tear.

Cartilage repair has been studied extensively in the context of injury and disease, but the joint's management of regular sub-injurious damage to cartilage, or 'wear and tear,' which occurs due to normal activity, is poorly understood. We hypothesize that this cartilage maintenance is mediated in part by cells derived from the synovium that migrate to the worn articular surface. Here, we demonstrate in vitro that the early steps required for such a process can occur. First, we show that under physiologic mechanical loads, chondrocyte death occurs in the cartilage superficial zone along with changes to the cartilage surface topography. Second, we show that synoviocytes are released from the synovial lining under physiologic loads and attach to worn cartilage. Third, we show that synoviocytes parachuted onto a simulated or native cartilage surface will modify their behavior. Specifically, we show that synoviocyte interactions with chondrocytes lead to changes in synoviocyte mechanosensitivity, and we demonstrate that cartilage-attached synoviocytes can express COL2A1, a hallmark of the chondrogenic phenotype. Our findings suggest that synoviocyte-mediated repair of cartilage 'wear and tear' as a component of joint homeostasis is feasible and is deserving of future study.

Superficial zone chondrocytes can get compacted under physiological loading: A multiscale finite element analysis.

Recent in vivo and in vitro studies have demonstrated that superficial zone (SZ) chondrocytes within articular layers of diarthrodial joints die under normal physiologic loading conditions. In order to further explore the implications of this observation in future investigations, we first needed to understand the mechanical environment of SZ chondrocytes that might cause them to die under physiological sliding contact conditions. In this study we performed a multiscale finite element analysis of articular contact to track the temporal evolution of a SZ chondrocyte's interstitial fluid pressure, hydraulic permeability, and volume under physiologic loading conditions. The effect of the pericellular matrix modulus and permeability was parametrically investigated. Results showed that SZ chondrocytes can lose ninety percent of their intracellular fluid after several hours of intermittent or continuous contact loading, resulting in a reduction of intracellular hydraulic permeability by more than three orders of magnitude. These findings are consistent with loss of cell viability due to the impediment of cellular metabolic pathways induced by the loss of fluid. They suggest that there is a simple mechanical explanation for the vulnerability of SZ chondrocytes to sustained physiological loading conditions. Future studies will focus on validating these specific findings experimentally. STATEMENT OF SIGNIFICANCE: As with any mechanical system, normal 'wear and tear' of cartilage tissue lining joints is expected. Yet incidences of osteoarthritis are uncommon in individuals younger than 45. This counter-intuitive observation suggests there must be an intrinsic repair mechanism compensating for this wear and tear over many decades of life. Recent experimental studies have shown superficial zone chondrocytes die under physiologic loading conditions, suggesting that this repair mechanism may involve cell replenishment. To better understand the mechanical environment of these cells, we performed a multiscale computational analysis of articular contact under loading. Results indicated that normal activities like walking or standing can induce significant loss of intracellular fluid volume, potentially hindering metabolic activity and fluid transport properties, and causing cell death.

Bendable osteochondral allografts for patellar resurfacing: A finite element analysis of congruence.

Osteochondral allograft (OCA) transplantation provides a safe and effective treatment option for large cartilage defects, but its use is limited partly due to the difficulty of matching articular surface curvature between donor and recipient. We hypothesize that bendable OCAs may provide better curvature matching for patella transplants in the patellofemoral joint (PFJ). This finite element study investigates PFJ congruence for unbent and bendable OCAs, at various flexion angles. Finite element models were created for 12 femur-patella OCA pairings. Two grooves were cut in each OCA bony substrate, allowing the articular layer to bend. PFJs with either unbent (OCA) or permanently bent (BOCA) allografts were articulated from 40 to 70 degrees flexion and contact area was calculated. OCAs and BOCAs were then shifted 6 mm distally toward the tibia (S-OCA, S-BOCA) to investigate the influence of proximal-distal alignment on congruence. On average, no significant difference in contact area was found between native PFJs and either OCAs or BOCAs (p > 0.25), indicating that both types of allografts restored native congruence. This result provides biomechanical support in favor of an emerging surgical procedure. S-BOCAs resulted in a significant increase in contact area relative to the remaining groups (p < 0.02). The fact that BOCAs produced equally good results implies that bendable allografts may prove useful in future surgical procedures, with the possibility of transplanting them with a small distal shift. Surgeons who are reluctant to use OCAs for resurfacing patellae based on curvature matching capabilities may be more amenable to adopting BOCAs.

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation.

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

Biophysical Modulation of Mesenchymal Stem Cell Differentiation in the Context of Skeletal Repair.

A prominent feature of the skeleton is its ability to remodel in response to biophysical stimuli and to repair under varied biophysical conditions. This allows the skeleton considerable adaptation to meet its physiological roles of stability and movement. Skeletal cells and their mesenchymal precursors exist in a native environment rich with biophysical signals, and they sense and respond to those signals to meet organismal demands of the skeleton. While mechanical strain is the most recognized of the skeletal biophysical stimuli, signaling phenomena also include fluid flow, hydrostatic pressure, shear stress, and ion-movement-related electrokinetic phenomena including, prominently, streaming potentials. Because of the complex interactions of these electromechanical signals, it is difficult to isolate the significance of each. The application of external electrical and electromagnetic fields allows an exploration of the effects of these stimuli on cell differentiation and extra-cellular matrix formation in the absence of mechanical strain. This review takes a distinctly translational approach to mechanistic and preclinical studies of differentiation and skeletal lineage commitment of mesenchymal cells under biophysical stimulation. In vitro studies facilitate the examination of isolated cellular responses while in vivo studies permit the observation of cell differentiation and extracellular matrix synthesis.

Toward Development of a Diabetic Synovium Culture Model.

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and inflammation of synovium, the specialized connective tissue that envelops the diarthrodial joint. Type 2 diabetes mellitus (DM) is often found in OA patients, with nearly double the incidence of arthritis reported in patients with diabetes (52%) than those without it (27%). The correlation between OA and DM has been attributed to similar risk factors, namely increasing age and joint loading due to obesity. However, a potential causative link is not well understood due to comorbidities involved with treating diabetic patients, such as high infection rates and poor healing response caused by hyperglycemia and insulin resistance. The purpose of this study was to investigate the effect of hyperglycemic and insulin culture conditions on synovium properties. It was hypothesized that modeling hyperglycemia-induced insulin resistance in synovium would provide novel insights of OA pathogenesis in DM patients. To simulate DM in the synovial joint, healthy synovium was preconditioned in either euglycemic (EG) or hyperglycemic (HG) glucose concentrations with insulin in order to induce the biological response of the diseased phenotype. Synovium biochemical composition was evaluated to determine ECM remodeling under hyperglycemic culture conditions. Concurrent changes in AKT phosphorylation, a signaling pathway implicated in insulin resistance, were measured along with gene expression data for insulin receptors, glucose transporters, and specific glycolysis markers involved in glucose regulation. Since fluid shear stress arising during joint articulation is a relevant upstream stimulus for fibroblast-like synoviocytes (FLS), the predominant cell type in synovium, FLS mechanotransduction was evaluated via intracellular calcium ([Ca2+]i). Incidence and length of primary cilia, a critical effector of cell mechanosensing, were measured as potential mechanisms to support differences in [Ca2+]i responses. Hyperglycemic culture conditions decreased collagen and GAG content compared to EG groups, while insulin recovered ECM constituents. FLS mechanosensitivity was significantly greater in EG and insulin conditions compared to HG and non-insulin treated groups. Hyperglycemic treatment led to decreased incidence and length of primary cilia and decreased AKT phosphorylation, providing possible links to the mechanosensing response and suggesting a potential correlation between glycemic culture conditions, diabetic insulin resistance, and OA development.

Pulsed Electromagnetic Field Therapy and Direct Current Electric Field Modulation Promote the Migration of Fibroblast-like Synoviocytes to Accelerate Cartilage Repair In Vitro.

Articular cartilage injuries are a common source of joint pain and dysfunction. As articular cartilage is avascular, it exhibits a poor intrinsic healing capacity for self-repair. Clinically, osteochondral grafts are used to surgically restore the articular surface following injury. A significant challenge remains with the repair properties at the graft-host tissue interface as proper integration is critical toward restoring normal load distribution across the joint. A key to addressing poor tissue integration may involve optimizing mobilization of fibroblast-like synoviocytes (FLS) that exhibit chondrogenic potential and are derived from the adjacent synovium, the specialized connective tissue membrane that envelops the diarthrodial joint. Synovium-derived cells have been directly implicated in the native repair response of articular cartilage. Electrotherapeutics hold potential as low-cost, low-risk, non-invasive adjunctive therapies for promoting cartilage healing via cell-mediated repair. Pulsed electromagnetic fields (PEMFs) and applied direct current (DC) electric fields (EFs) via galvanotaxis are two potential therapeutic strategies to promote cartilage repair by stimulating the migration of FLS within a wound or defect site. PEMF chambers were calibrated to recapitulate clinical standards (1.5 ± 0.2 mT, 75 Hz, 1.3 ms duration). PEMF stimulation promoted bovine FLS migration using a 2D in vitro scratch assay to assess the rate of wound closure following cruciform injury. Galvanotaxis DC EF stimulation assisted FLS migration within a collagen hydrogel matrix in order to promote cartilage repair. A novel tissue-scale bioreactor capable of applying DC EFs in sterile culture conditions to 3D constructs was designed in order to track the increased recruitment of synovial repair cells via galvanotaxis from intact bovine synovium explants to the site of a cartilage wound injury. PEMF stimulation further modulated FLS migration into the bovine cartilage defect region. Biochemical composition, histological analysis, and gene expression revealed elevated GAG and collagen levels following PEMF treatment, indicative of its pro-anabolic effect. Together, PEMF and galvanotaxis DC EF modulation are electrotherapeutic strategies with complementary repair properties. Both procedures may enable direct migration or selective homing of target cells to defect sites, thus augmenting natural repair processes for improving cartilage repair and healing.

Attachment of cartilage wear particles to the synovium negatively impacts friction properties.

Cartilage wear particles are released into the synovial fluid by mechanical and chemical degradation of the articular surfaces during osteoarthritis and attach to the synovial membrane. Accumulation of wear particles could alter key tissue-level mechanical properties of the synovium, hindering its characteristically low-friction interactions with underlying articular surfaces in the synovial joint. The present study employs a custom loading device to further the characterization of native synovium friction properties, while investigating the hypothesis that attachment of cartilage wear particles increases friction coefficient. Juvenile bovine synovium demonstrated characteristically low friction coefficients in sliding contact with glass, in agreement with historical measurements. Friction coefficient increased with higher normal load in saline, while lubrication with native synovial fluid maintained low friction coefficients at higher loads. Cartilage wear particles generated from juvenile bovine cartilage attached directly to synovium explants in static culture, with incorporation onto the tissue denoted by cell migration onto the particle surface. In dilute synovial fluid mimicking the decreased lubricating properties during osteoarthritis, wear particle attachment significantly increased friction coefficient against glass, and native cartilage and synovium. In addition to providing a novel characterization of synovial joint tribology this work highlights a potential mechanism for cartilage wear particles to perpetuate the degradative environment of osteoarthritis by modulating tissue-level properties of the synovium that could impact macroscopic wear as well as mechanical stimuli transmitted to resident cells.

Sustained Delivery of SB-431542, a Type I Transforming Growth Factor Beta-1 Receptor Inhibitor, to Prevent Arthrofibrosis.

Fibrosis of the knee is a common disorder resulting from an aberrant wound healing response and is characterized by extracellular matrix deposition, joint contraction, and scar tissue formation. The principal regulator of the fibrotic cascade is transforming growth factor beta-1 (TGF-ß1), a factor that induces rapid proliferation and differentiation of resident fibroblasts. In this study, we demonstrate successful inhibition of TGF-ß1-driven myofibroblastic differentiation in human fibroblast-like synoviocytes using a small molecule TGF-ß1 receptor inhibitor, SB-431542. We also demonstrate successful encapsulation of SB-431542 in poly(D,L-lactide-co-glycolide) (PLGA) as a potential prophylactic treatment for arthrofibrosis and characterize drug release and bioactivity in a three-dimensional collagen gel contraction assay. We assessed the effects of TGF-ß1 and SB-431542 on cell proliferation and viability in monolayer cultures. Opposing dose-dependent trends were observed in cell proliferation, which increased in TGF-ß1-treated cultures and decreased in SB-431542-treated cultures relative to control (p < 0.05). SB-431542 was not cytotoxic at the concentrations studied (0-50 µM) and inhibited TGF-ß1-induced collagen gel contraction in a dose-dependent manner. Specifically, TGF-ß1-treated gels contracted to 18% ± 1% of their initial surface area, while gels treated with TGF-ß1 and ≥10 µM SB-431542 showed no evidence of contraction (p < 0.0001). Upon removal of the compound, all gels contracted to control levels after 44 h in culture, necessitating sustained delivery for prolonged inhibition. To this end, SB-431542 was encapsulated in PLGA microspheres (SBMS) that had an average diameter of 87.5 ± 24 µm and a loading capacity of 4.3 µg SB-431542 per milligram of SBMS. Functional assessment of SBMS revealed sustained inhibition of TGF-ß1-induced gel contraction as well as hallmark features of myofibroblastic differentiation, including α-smooth muscle actin expression and connective tissue growth factor production. These results suggest that SB-431542 may be used to counter TGF-ß1-driven events in the fibrotic cascade in the knee cartilage. Impact statement Arthrofibrosis is the most prevalent comorbidity resulting from orthopedic procedures such as total knee arthroplasty that is characterized by excess deposition and accumulation of extracellular matrix. Despite its prevalence, treatments are generally palliative, and there is no effective prophylactic therapy. We report that the small molecule transforming growth factor beta-1 (TGF-ß1) receptor inhibitor, SB-431542, can inhibit the TGF-ß1-driven myofibroblastic differentiation of fibroblast-like synoviocytes. To provide sustained inhibition, we explored the use of SB-laden microspheres as a prophylactic therapy in a three-dimensional contraction model of fibrosis and propose that such therapies will have the potential to improve the standard of care for arthrofibrosis.

Direct Osmotic Pressure Measurements in Articular Cartilage Demonstrate Nonideal and Concentration-Dependent Phenomena.

The osmotic pressure in articular cartilage serves an important mechanical function in healthy tissue. Its magnitude is thought to play a role in advancing osteoarthritis. The aims of this study were to: (1) isolate and quantify the magnitude of cartilage swelling pressure in situ; and (2) identify the effect of salt concentration on material parameters. Confined compression stress-relaxation testing was performed on 18 immature bovine and six mature human cartilage samples in solutions of varying osmolarities. Direct measurements of osmotic pressure revealed nonideal and concentration-dependent osmotic behavior, with magnitudes approximately 1/3 those predicted by ideal Donnan law. A modified Donnan constitutive behavior was able to capture the aggregate behavior of all samples with a single adjustable parameter. Results of curve-fitting transient stress-relaxation data with triphasic theory in febio demonstrated concentration-dependent material properties. The aggregate modulus HA increased threefold as the external concentration decreased from hypertonic 2 M to hypotonic 0.001 M NaCl (bovine: HA=0.420±0.109 MPa to 1.266±0.438 MPa; human: HA=0.499±0.208 MPa to 1.597±0.455 MPa), within a triphasic theory inclusive of osmotic effects. This study provides a novel and simple analytical model for cartilage osmotic pressure which may be used in computational simulations, validated with direct in situ measurements. A key finding is the simultaneous existence of Donnan osmotic and Poisson-Boltzmann electrostatic interactions within cartilage.

Immature bovine cartilage wear by fatigue failure and delamination.

This study investigated wear damage of immature bovine articular cartilage using reciprocal sliding of tibial cartilage strips against glass or cartilage. Experiments were conducted in physiological buffered saline (PBS) or mature bovine synovial fluid (SF). A total of 63 samples were tested, of which 47 exhibited wear damage due to delamination of the cartilage surface initiated in the middle zone, with no evidence of abrasive wear. There was no difference between the friction coefficient of damaged and undamaged samples, showing that delamination wear occurs even when friction remains low under a migrating contact area configuration. No difference was observed in the onset of damage or in the friction coefficient between samples tested in PBS or SF. The onset of damage occurred earlier when testing cartilage against glass versus cartilage against cartilage, supporting the hypothesis that delamination occurs due to fatigue failure of the collagen in the middle zone, since stiffer glass produces higher strains and tensile stresses under comparable loads. The findings of this study are novel because they establish that delamination of the articular surface, starting in the middle zone, may represent a primary mechanism of failure. Based on preliminary data, it is reasonable to hypothesize that delamination wear via subsurface fatigue failure is similarly the primary mechanism of human cartilage wear under normal loading conditions, albeit requiring far more cycles of loading than in immature bovine cartilage.