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Current auricular cartilage replacements for pediatric microtia fail to address the need for long-term integration and neocartilage formation. While collagen hydrogels have been successful in fostering neocartilage formation, the toughness and extensibility of these materials do not match that of native tissue. This study used the N-terminal functionalization of collagen with alginate oligomers to improve toughness and extensibility through metal–ion complexation. Alginate conjugation was confirmed via FTIR spectroscopy. The retention of native collagen fibrillar structure, thermal gelation, and helical conformation in functionalized gels was confirmed via scanning electron microscopy, oscillatory shear rheology, and circular dichroism spectroscopy, respectively. Alginate–calcium complexation enabled a more than two-fold increase in modulus and work density in functionalized collagen with the addition of 50 mM CaCl2, whereas unmodified collagen decreased in both modulus and work density with increasing calcium concentration. Additionally, the extensibility of alginate-functionalized collagen was increased at 25 and 50 mM CaCl2. Following 2-week culture with auricular chondrocytes, alginate-functionalization had no effect on the cytocompatibility of collagen gels, with no effects on cell density, and increased glycosaminoglycan deposition. Custom MATLAB video analysis was then used to quantify fracture toughness, which was more than 5-fold higher following culture in functionalized collagen and almost three-fold higher in unmodified collagen. [ABSTRACT FROM AUTHOR]

Proteoglycans, especially small leucine rich proteoglycans (SLRPs), play major roles in facilitating the development and regulation of collagen fibers and other extracellular matrix components. However, their roles in fibrocartilage have not been widely reviewed. Here, we discuss both SLRP and large aggregating proteoglycan's roles in collagen fibrillogenesis and extracellular matrix assembly in fibrocartilage tissues such as the meniscus, annulus fibrosus (AF), and TMJ disc. We also discuss their expression levels throughout development, aging and degeneration, as well as repair. A review of literature discussing proteoglycans and collagen fibrillogenesis in fibrocartilage was conducted and data from these manuscripts were analyzed and grouped to discuss trends throughout the tissue's architectural zones and developmental stage. The spatial collagen architecture of these fibrocartilaginous tissues is reflected in the distribution of proteoglycans expressed, suggesting that each proteoglycan plays an important role in the type of architecture presented and associated mechanical function. The unique structure-function relationship of fibrocartilage makes the varied architectures throughout the tissues imperative for their success and understanding the functions of these proteoglycans in developing and maintaining the fiber structure could inform future work in fibrocartilage replacement using tissue engineered constructs. [ABSTRACT FROM AUTHOR]

Fibrin sealants are commonly used in cartilage repair surgeries to adhere cells or grafts into a cartilage defect. Both autologous and commercial allogeneic fibrin sealants are used in cartilage repair surgeries, yet there are no studies characterizing and comparing the mechanical properties of fibrin sealants from all-autologous sources. The objectives of this study were to investigate (i) the effect of fibrinogen and thrombin sources on failure mechanics of sealants, and (ii) how sealants affect the adhesion of particulated cartilage graft material (BioCartilage) to surrounding cartilage under physiological loading. Allogeneic thrombin and fibrinogen were purchased (Tisseel), and autologous sources were prepared from platelet-rich plasma (PRP) and platelet-poor plasma (PPP) generated from human blood. To compare failure characteristics, sealants were sandwiched between cartilage explants and pulled to failure. The effect of sealant on the adhesion of BioCartilage graft to cartilage was determined by quantifying microscale strains at the graft-cartilage interface using an in vitro cartilage defect model subjected to shear loading at physiological strains well below failure thresholds. Fibrinogen sources were not equivalent; PRP fibrinogen created sealants that were more brittle, failed at lower strains, and resulted in sustained higher strains through the graft-cartilage interface depth compared to PPP and allogeneic sources. PPP clotted slower compared to PRP, suggesting PPP may percolate deeper into the repair to provide more stability through the tissue depth. There was no difference in bulk failure properties or microscale strains at the graft-cartilage interface between the purely autologous sealant (autologous thrombin + PPP fibrinogen) and the commercial allogeneic sealant. Clinical Significance: All-autologous fibrin sealants fabricated with PPP have comparable adhesion strength as commercial allogeneic sealants in vitro, whereas PRP creates an inferior all-autologous sealant that sustains higher strains through the graft-cartilage interface depth. [ABSTRACT FROM AUTHOR]

Molecular transport of osteoarthritis (OA) therapeutics within articular cartilage is influenced by many factors, such as solute charge, that have yet to be fully understood. This study characterizes how solute charge influences local diffusion and convective transport of antibodies within the heterogeneous cartilage matrix. Three fluorescently tagged solutes of varying isoelectric point (pI) (4.7-5.9) were tested in either cyclic or passive cartilage loading conditions. In each case, local diffusivities were calculated based on local fluorescence in the cartilage sample, as observed by confocal microscopy. In agreement with past research, local solute diffusivities within the heterogeneous cartilage matrix were highest around 200-275 μm from the articular surface, but 3-4 times lower at the articular surface and in the deeper zones of the tissue. Transport of all 150 kDa solutes was significantly increased by the application of mechanical loading at 1 Hz, but local transport enhancement was not significantly affected by changes in solute isoelectric point. More positively charged solutes (higher pI) had significantly higher local diffusivities 200-275 μm from the tissue surface, but no other differences were observed. This implies that there are certain regions of cartilage that are more sensitive to changes in solute charge than others, which could be useful for future development of OA therapeutics. [ABSTRACT FROM AUTHOR]

ABSTRACT: Hyaluronic acid (HA) is widely injected as a viscosupplement in the treatment of osteoarthritis. Despite its extensive use, it is not currently known if cartilage degradation alters how HA‐based solutions lubricate the articular surface. In this study we utilized a model of cartilage degradation by IL‐1β along with a recently developed framework to study role of cartilage degradation on lubrication by clinically‐approved HA‐based lubricants with high viscosities. Cartilage explants were cultured up to 8 days with 10 ng/ml IL‐1β. After culture, samples were examined histologically, immunohistochemically, biochemically, mechanically, topographically, and tribologically. The tribological testing analyzed both boundary and mixed lubrication modes to assess individual effects of viscosity and boundary lubricating ability. Friction testing was carried out using PBS and two clinically approved HA‐based viscosupplements in a cartilage‐glass configuration. After culture with IL‐1β, boundary mode friction was elevated after both 4 and 8 days. Additionally, friction in mixed mode lubrication, where HA is most effective as a lubricant, was significantly elevated after 8 days of culture. As cartilage became rougher, softer, and more permeable after culture, the boundary mode plateau was extended, and as a result, significantly increased lubricant viscosities or sliding speeds were necessary to achieve effective mixed lubrication. Overall, this study revealed that lubrication of cartilage by HA is degradation‐dependent and coincides with changes in mechanics and roughness. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1456–1464, 2018. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) have been investigated with promising results for meniscus healing and tissue engineering. While MSCs are known to contribute to extracellular matrix (ECM) production, less is known about how MSCs produce and align large organized fibers for application to tissue engineering the meniscus. The goal of this study was to investigate the capability of MSCs to produce and organize ECM molecules compared to meniscal fibrochondrocytes (FCCs). Bovine FCCs and MSCs were encapsulated in an anatomically accurate collagen meniscus using monoculture and co-culture of each cell type. Each meniscus was mechanically anchored at the horns to mimic the physiological fixation by the meniscal entheses. Mechanical fixation generates a static mechanical boundary condition previously shown to induce formation of oriented fiber by FCCs. Samples were cultured for 4 weeks and then evaluated for biochemical composition and fiber development. MSCs increased the glycosaminoglycan (GAG) and collagen production in both co-culture and monoculture groups compared to FCC monoculture. Collagen organization was greatest in the FCC monoculture group. While MSCs had increased matrix production, they lacked the fiber organization capabilities of FCCs. This study suggests that GAG production and fiber formation are linked. Co-culture can be used as a means of balancing the synthetic properties of MSCs and the matrix remodeling capabilities of FCCs for tissue engineering applications. [ABSTRACT FROM AUTHOR]

Sensation of mechanical stimuli by chondrocytes is critical to cartilage homeostasis and osteoarthritis development. The earliest responses in chondrocyte mechanotransduction pathways involve calcium influx and changes in mitochondrial function, which occur in seconds to minutes. Deeper understanding of these events can elucidate new therapeutic targets for early intervention to prevent osteoarthritis. [ABSTRACT FROM AUTHOR]

Though remarkably robust, articular cartilage becomes susceptible to damage at high loading rates, particularly under shear. While several studies have measured the local static and steady-state shear properties of cartilage, it is the local viscoelastic properties that determine the tissue's ability to withstand physiological loading regimens. However, measuring local viscoelastic properties requires overcoming technical challenges that include resolving strain fields in both space and time and accurately calculating their phase offsets. This study combined recently developed high-speed confocal imaging techniques with three approaches for analyzing time- and location-dependent mechanical data to measure the depth-dependent dynamic modulus and phase angles of articular cartilage. For sinusoidal shear at frequencies f= 0.01 to 1 Hz with no strain offset, the dynamic shear modulus \G*\ and phase angleδ reached their minimum and maximum values (respectively) approximately 100 nm below the articular surface, resulting in a profound focusing of energy dissipation in this narrow band of tissue that increased with frequency. This region, known as the transitional zone, was previously thought to simply connect surface and deeper tissue regions. Within 250 [μn of the articular surface, \G*\ increased from 0.32 ± 0.08 to 0.42 ± 0.08 MPa across the five frequencies tested, while δ decreased from 12 deg ± 1 deg to 9.1 deg±0.5 deg. Deeper into the tissue, \G*\ increased from 1.5 ± 0.4 MPa to 2.1 ± 0.6MPa and <5 decreased from 13 deg ± 1 deg to 5.5 deg ± 0.2 deg. Viscoelastic properties were also strain-dependent, with localized energy dissipation suppressed at higher shear strain offsets. These results suggest a critical role for the transitional zone in dissipating energy, representing a possible shift in our understanding of cartilage mechanical function. Further, they give insight into how focal degeneration and mechanical trauma could lead to sustained damage in this tissue. [ABSTRACT FROM AUTHOR]

A new cell-printed scaffold consisting of poly( ϵ-caprolactone) (PCL) and cell-embedded alginate struts is designed. The PCL and alginate struts are stacked in an interdigitated pattern in successive layers to acquire a three-dimensional (3D) shape. The hybrid scaffold exhibits a two-phase structure consisting of cell (MC3T3-E1)-laden alginate struts able to support biological activity and PCL struts able to provide controllable mechanical support of the cell-laden alginate struts. The hybrid scaffolds exhibit an impressive increase in tensile modulus and maximum strength compared to pure alginate scaffolds. Laden cells are homogeneously distributed throughout the alginate struts and the entire scaffold, resulting in cell viability of approximately 84%. [ABSTRACT FROM AUTHOR]

Intra‐articular injections of hyaluronic acid have been a mainstay of osteoarthritis treatment for decades. However, controversy surrounds the mechanism of action and efficacy of this therapy. As such, there has been recent interest in developing synthetic lubricants that lubricate cartilage. Recently, a synthetic 4 wt% polyacrylamide (pAAm) hydrogel was shown to effectively decrease lameness in horses. However, its mechanism of action and ability to lubricate cartilage is unknown. The goal of this study was to characterize the lubricating ability of this hydrogel and determine its efficacy for healthy and degraded cartilage. The study utilized previously established IL‐1β‐induced biochemical degradation and mechanical impact injury models to degrade cartilage. The lubricating ability of the hydrogel was then characterized using a custom‐built tribometer using a glass counterface and friction was evaluated using the Stribeck framework for articular cartilage. pAAm hydrogel was shown to significantly lower the friction coefficient of cartilage explants from both degradation models (30%–40% reduction in friction relative to controls). A striking finding from this study was the aggregation of the pAAm hydrogel at the articulating surface. The surface aggregation was observed in the histological sections of explants from all treatment groups after tribological evaluation. Using the Stribeck framework, the hydrogel was mapped to higher Sommerfeld numbers and was characterized as a viscous lubricant predominantly in the minimum friction mode. In summary, this study revealed that pAAm hydrogel lubricates native and degraded cartilage explants effectively and may have an affinity for the articulating surface of the cartilage. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the hypothesis that dynamic compression loading enhances tissue formation and increases mechanical properties of anatomically shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2%w/v alginate, crosslinked with CaSO4, injected into μCT based molds, and post crosslinked with CaCl2. Samples were loaded via a custom bioreactor with loading platens specifically designed to load anatomically shaped constructs in unconfined compression. Based on the results of finite element simulations, constructs were loaded under sinusoidal displacement to yield physiological strain levels. Constructs were loaded 3 times a week for 1h followed by 1h of rest and loaded again for 1h. Constructs were dynamically loaded for up to 6 weeks. After 2 weeks of culture, loaded samples had 2–3.2 fold increases in the extracellular matrix (ECM) content and 1.8–2.5 fold increases in the compressive modulus compared with static controls. After 6 weeks of loading, glycosaminoglycan (GAG) content and compressive modulus both decreased compared with 2 week cultures by 2.3–2.7 and 1.5–1.7 fold, respectively, whereas collagen content increased by 1.8–2.2 fold. Prolonged loading of engineered constructs could have altered alginate scaffold degradation rate and/or initiated a catabolic cellular response, indicated by significantly decreased ECM retention at 6 weeks compared with 2 weeks. However, the data indicates that dynamic loading had a strikingly positive effect on ECM accumulation and mechanical properties in short term culture. [ABSTRACT FROM AUTHOR]

Abstract: Lubrication of articular cartilage occurs in distinct modes with various structural and biomolecular mechanisms contributing to the low-friction properties of natural joints. In order to elucidate relative contributions of these factors in normal and diseased tissues, determination and control of lubrication mode must occur. The objectives of these studies were (1) to develop an in vitro cartilage on glass test system to measure friction coefficient, μ; (2) to implement and extend a framework for the determination of cartilage lubrication modes; and (3) to determine the effects of synovial fluid on μ and lubrication mode transitions. Patellofemoral groove cartilage was linearly oscillated against glass under varying magnitudes of compressive strain utilizing phosphate buffered saline (PBS) and equine and bovine synovial fluid as lubricants. The time-dependent frictional properties were measured to determine the lubricant type and strain magnitude dependence for the initial friction coefficient (μ 0=μ(t→0)) and equilibrium friction coefficient (μ eq=μ(t→∞)). Parameters including tissue-glass co-planarity, normal strain, and surface speed were altered to determine the effect of the parameters on lubrication mode via a ‘Stribeck surface’. Using this testing apparatus, cartilage exhibited biphasic lubrication with significant influence of strain magnitude on μ 0 and minimal influence on μ eq, consistent with hydrostatic pressurization as reported by others. Lubrication analysis using ‘Stribeck surfaces’ demonstrated clear regions of boundary and mixed modes, but hydrodynamic or full film lubrication was not observed even at the highest speed (50mm/s) and lowest strain (5%). [Copyright &y& Elsevier]

Abstract: Cell–matrix interactions transmit a wealth of information about the extracellular environment. In return, a variety of responses from the cell are initiated by changes in the matrix. One such response involves the positive regulation of matrix metalloproteinases (MMPs) by α2β1 integrin attaching to a specific extracellular matrix component, collagen. This study explores the relationship between mechanical and biochemical functions of α2β1 integrins as it pertains to regulating matrix remodeling. To understand this relationship, the individual influences of MMP activity and α2β1 integrin function on collagen gel contraction were studied. We have observed little evidence of mutual participation in matrix remodeling by the α2β1 integrin and MMP activity in cell models where α2 is minimally expressed. In cells expressing high levels of α2, we see an increase in gel contraction that is enhanced by MMP activity. Measuring tension as it builds within the gel reveals that α2β1 integrin presence correlates with force output but is insensitive to MMP activity. These data strongly suggest that α2β1 regulates collagen gel remodeling through multiple simultaneous mechanisms including force generation and modulation of MMP activity. [Copyright &y& Elsevier]

The proliferation and hypertrophy of growth plate chondrocytes set the pace and pattern for growth of endochondral bones. Complex signaling pathways regulating chondrocyte differentiation during development and growth have been discovered in recent years, but as yet little is known about how chondrocytes are able to orient themselves to align properly with respect to the direction of bone growth. Histomorphometric methods developed for analysis of growth plates rely to a significant extent on assessments of the relative heights of the zones of proliferating and hypertrophic chondrocytes. In a growing number of osteopetrotic mutations, however, it is becoming apparent that growth plates lack clearly demarcated zones of chondrocyte differentiation, and they show other notable histological abnormalities that cannot be measured with standard approaches. This is particularly true of mutations in which osteoclasts are altogether absent. We therefore developed a new approach that measures the salient features of this type of chondrodysplasia and have applied it to the toothless ( tl ) rat. The tl rat has a frameshift mutation in the Csf-1 gene that renders it null, resulting in severe osteopetrosis. An accompanying pathology is a severe, progressive growth plate chondrodysplasia. We measured cell orientation, cell area, and local columnar organization as functions of distance from the upper margin of the growth plate, in addition to growth plate thickness and cell density. All these parameters were markedly abnormal in the tl rats, thus implicating Csf-1 not only in its well-established role in regulating osteoclastic bone resorption, but also in endochondral ossification. This approach should prove useful in distinguishing among growth plate chondrodysplasias, most especially in the growing number of osteopetrotic mutations having growth plates that lack the normal zonal organization and in which the chondrocytes are mis-oriented. In turn, detailed assessments of chondrocyte misorientation may give insights into how they normally are able to arrange themselves with such precision. [ABSTRACT FROM AUTHOR]

Loading of articular cartilage during weight bearing is essential for the maintenance of cartilage function. Although certain cyclic loading protocols stimulate extracellular matrix synthesis, constant or static compression decreases proteoglycan and collagen synthesis in cartilage explants. The goal of this study was to determine whether the compression-induced decrease in proteoglycan synthesis involves an interleukin-1 (IL-1) signaling pathway. Cartilage explants were compressed 50% in the presence of IL-1 receptor antagonist (IL-1ra), and the incorporation of [35S]sulfate into macromolecules was measured. IL-1ra increased sulfate incorporation in compressed cartilage but not in cartilage maintained at the in situ thickness (0% compression). IL-1α and IL-1β mRNAs were detected in cartilage compressed 50% for at least 3 h, while nitric oxide synthase II mRNA was only detected in cartilage compressed 50% for 6 h. The data support a role for the IL-1 receptor in the pathway linking static compression to reduced proteoglycan synthesis. [Copyright &y& Elsevier]

Mathematical models to describe extracellular matrix (ECM) deposition and scaffold degradation in cell–polymer constructs for the design of engineered cartilage were developed and validated. The ECM deposition model characterized a product-inhibition mechanism in the concentration of cartilage molecules, collagen and glycosaminoglycans (GAG). The scaffold degradation model used first-order kinetics to describe hydrolysis (not limited by diffusion) of biodegradable polyesters, polyglycolic acid and polylactic acid. Each model was fit to published accumulation and degradation data. As experimental validation, cell–polymer constructs (n=24) and unseeded scaffolds (n=24) were cultured in vitro. Biochemical assays for ECM content and measurements of scaffold mass were performed at 1, 2, 4, 6, 8, or 10 weeks (n=8 per time point). The models demonstrated a strong fit with published data and experimental results (R2=0.75 to 0.99) and predicted the temporal total construct mass with reasonable accuracy (30% RMS error). This approach can elucidate mechanisms governing accumulation/degradation and may be coupled with structure–function relationships to describe time-dependent changes in construct elastic properties. [Copyright &y& Elsevier]

The aim of this study was to determine whether the biomechanical and biochemical properties of tissue-engineered human septal cartilage vary with donor age and in vitro culture time. Chondrocytes were isolated from human septal cartilage of patients from 15 to 60 year old and maintained in primary monolayer culture for 14 days. Cells were seeded onto 0.5% PLA coated PGA disks and kept in stationary three-dimensional culture for either 1 day or 3 weeks. Specimens were then implanted subcutaneously into athymic nude mice and harvested after either 4 or 8 weeks. Upon harvest, the equilibrium confined compression modulus was measured as to quantify mechanical properties, and the glycosaminoglycan, hydroxyproline, and DNA contents were determined as measures of tissue proteoglycans, collagen, and cell density. This study demonstrated that native nasal cartilage showed distinct changes in these parameters with age, but cartilage engineered using the cells of these specimens showed no significant dependence on the age of the donor. There was little difference in quality of cartilage between samples cultured for 3 weeks in vitro and those implanted directly after seeding. Together, the results of this study suggest that the process of extracellular matrix assembly by chondrocytes on three-dimensional scaffolds may be independent of in vivo conditions experienced by the tissue prior to harvest. [Copyright &y& Elsevier]

Inflammation of the synovium, known as synovitis, plays an important role in the pathogenesis of osteoarthritis (OA). Synovitis involves the release of a wide variety of pro‐inflammatory mediators in synovial fluid (SF) that damage the articular cartilage extracellular matrix and induce death and apoptosis in chondrocytes. The composition of synovial fluid is dramatically altered by inflammation in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. However, the relationship between key biochemical markers of joint inflammation and mechanical function of SF is not well understood. Here, we demonstrate the application of a novel analytical framework to measure the effective viscosity for SF lubrication of cartilage, which is distinct from conventional rheological viscosity. Notably, in a well‐established equine model of synovitis, this effective lubricating viscosity decreased by up to 10,000‐fold for synovitis SF compared to a ~4 fold change in conventional viscosity measurements. Further, the effective lubricating viscosity was strongly inversely correlated (r = −0.6 to −0.8) to multiple established biochemical markers of SF inflammation, including white blood cell count, prostaglandin E2 (PGE2), and chemokine ligand (CCLs) concentrations, while conventional measurements of viscosity were poorly correlated to these markers. These findings demonstrate the importance of experimental and analytical approaches to characterize functional lubricating properties of synovial fluid and their relationships to soluble biomarkers to better understand the progression of OA. [ABSTRACT FROM AUTHOR]

In various biological systems, analyzing how cell behaviors are coordinated over time would enable a deeper understanding of tissue-scale response to physiologic or superphysiologic stimuli. Such data is necessary for establishing both normal tissue function and the sequence of events after injury that lead to chronic disease. However, collecting and analyzing these large datasets presents a challenge—such systems are time-consuming to process, and the overwhelming scale of data makes it difficult to parse overall behaviors. This problem calls for an analysis technique that can quickly provide an overview of the groups present in the entire system and also produce meaningful categorization of cell behaviors. Here, we demonstrate the application of an unsupervised method—the Variational Autoencoder (VAE)—to learn the features of cells in cartilage tissue after impact-induced injury and identify meaningful clusters of chondrocyte behavior. This technique quickly generated new insights into the spatial distribution of specific cell behavior phenotypes and connected specific peracute calcium signaling timeseries with long term cellular outcomes, demonstrating the value of the VAE technique. [ABSTRACT FROM AUTHOR]

Articular cartilage is one of the most unique materials found in nature. This tissue's ability to provide low friction and low wear over decades of constant use is not surpassed, as of yet, by any synthetic materials. Lubrication of the body's joints is essential to mammalian locomotion, but breakdown and degeneration of cartilage is the leading cause of severe disability in the industrialized world. In this paper, we review how theories of cartilage lubrication have evolved over the past decades and connect how theories of cartilage lubrication have been translated to lubrication-based therapies. Here, we call upon these historical perspectives and highlight the open questions in cartilage lubrication research. Additionally, these open questions within the field's understanding of natural lubrication mechanisms reveal strategic directions for lubrication therapy. [ABSTRACT FROM AUTHOR]

Posttraumatic osteoarthritis (PTOA) involves the mechanical and biological deterioration of articular cartilage that occurs following joint injury. PTOA is a growing problem in health care due to the lack of effective therapies combined with an aging population with high activity levels. Recently, acute mitochondrial dysfunction and altered cellular respiration have been associated with cartilage degeneration after injury. This finding is particularly important because recently developed mitoprotective drugs, including SS peptides, can preserve mitochondrial structure and function after acute injury in other tissues. It is not known, however, if cartilage injury induces rapid structural changes in mitochondria, to what degree mitochondrial dysfunction in cartilage depends on the mechanics of injury or the time frame over which such dysfunction develops. Similarly, it is unknown if SS‐peptide treatment can preserve mitochondrial structure and function after cartilage injury. Here, we combined fast camera elastography, longitudinal fluorescence assays, and computer vision techniques to track the fates of thousands of individual cells. Our results show that impact induces mechanically dependent mitochondrial depolarization within a few minutes after injury. Electron microscopy revealed that impact causes rapid structural changes in mitochondria that are related to reduced mitochondrial function, namely, fission and loss of cristae structure. We found that SS‐peptide treatment prior to impact protects the mitochondrial structure and preserves mitochondrial function at levels comparable with that of unimpacted control samples. Overall, this study reveals the vital role of mitochondria in mediating cartilage's peracute (within minutes) response to traumatic injury and demonstrates mitoprotection as a promising therapeutic strategy for injury‐induced cartilage damage. [ABSTRACT FROM AUTHOR]

Abstract Developing effective therapeutics for osteoarthritis (OA) necessitates that such molecules can reach and target chondrocytes within articular cartilage. However, predicting how well very large therapeutic molecules diffuse through cartilage is often difficult, and the relationship between local transport mechanics for these molecules and tissue heterogeneities in the tissue is still unclear. In this study, a 150 kDa antibody diffused through the articular surface of healthy and enzymatically degraded cartilage, which enabled the calculation of local diffusion mechanics in tissue with large compositional variations. Local cartilage composition and structure was quantified with Fourier transform infrared (FTIR) spectroscopy and second harmonic generation (SHG) imaging techniques. Overall, both local concentrations of aggrecan and collagen were correlated to local diffusivities for both healthy and surface-degraded samples (0.3 > R2 < 0.9). However, samples that underwent surface degradation by collagenase exhibited stronger correlations (R2 > 0.75) compared to healthy samples (R2 < 0.46), suggesting that the highly aligned collagen at the surface of cartilage acts as a barrier to macromolecular transport. [ABSTRACT FROM AUTHOR]

Developing therapeutic molecules that target chondrocytes and locally produced inflammatory factors within arthritic cartilage is an active area of investigation. The extensive studies that have been conducted over the past 50 years have enabled the accurate prediction and reliable optimization of the transport of a wide variety of molecules into cartilage. In this Review, the factors that can be used to tune the transport kinetics of therapeutics are summarized. Overall, the most crucial factor when designing new therapeutic molecules is solute size. The diffusivity and partition coefficient of a solute both decrease with increasing solute size as indicated by molecular mass or by hydrodynamic radius. Surprisingly, despite having an effective pore size of ~6 nm, molecules of ~16 nm radius can diffuse through the cartilage matrix. Alteration of the shape or charge of a solute and the application of physiological loading to cartilage can be used to predictably improve solute transport kinetics, and this knowledge can be used to improve the development of therapeutic agents for osteoarthritis that target the cartilage. [ABSTRACT FROM AUTHOR]

Cracks in articular cartilage are a common sign of joint damage, but failure properties of cartilage are poorly understood, especially for damage initiation. Cartilage failure may be further complicated by rate-dependent and depth-dependent properties, including the compliant surface layer. Existing blunt impact methods do not resolve local cartilage inhomogeneities and traditional fracture mechanics tests induce crack blunting and may violate underlying assumptions of linear elasticity. To address this knowledge gap, we developed and applied a method to indent cartilage explants with a sharp blade and initiate damage across a range of loading rates (strain rates 0.5%/s–500%/s), while recording local sample deformation and strain energy fields using confocal elastography. To investigate the importance of cartilage’s compliant surface, we repeated the experiment for samples with the surface removed. Bulk data suggest a critical force at which the tissue cuts, but local strains reveals that the deformation the sample can sustain before reaching this force is significantly higher in the surface layer. Bulk and local results also showed significant rate dependence, such that samples were easier to cut at faster speeds. This result highlights the importance of rate for understanding cracks in cartilage and parallels recent studies of rate-dependent failure in hydrogels. Notably, local sample deformation fields were well fit by classical Hookean elasticity. Overall, this study illustrates how local and global measurements surrounding the initiation of damage in articular cartilage can be combined to reveal the importance of cartilage’s zonal structure in protecting against failure across physiologically relevant loading rates. [ABSTRACT FROM AUTHOR]

ABSTRACT: Mitochondrial (MT) dysfunction is known to occur in chondrocytes isolated from end‐stage osteoarthritis (OA) patients, but the role of MT dysfunction in the initiation and early pathogenesis of post‐traumatic OA (PTOA) remains unclear. The objective of this study was to investigate chondrocyte MT function immediately following mechanical injury in cartilage, and to determine if the response to injury differed between a weight bearing region (medial femoral condyle; MFC) and a non‐weight bearing region (distal patellofemoral groove; PFG) of the same joint. Cartilage was harvested from the MFC and PFG of 10 neonatal bovids, and subjected to injurious compression at varying magnitudes (5‐17 MPa, 5‐34 GPa/s) using a rapid single‐impact model. Chondrocyte MT respiratory function, MT membrane polarity, chondrocyte viability, and cell membrane damage were assessed in situ. Cartilage impact resulted in MT depolarization and impaired MT respiratory function within 2 h of injury. Cartilage from a non‐weight bearing region of the joint (PFG) was more sensitive to impact‐induced MT dysfunction and chondrocyte death than cartilage from a weight‐bearing surface (MFC). Our findings suggest that MT dysfunction is an acute response of chondrocytes to cartilage injury, and that MT may play a key mechanobiological role in the initiation and early pathogenesis of PTOA. Clinical significance: Direct therapeutic targeting of MT function in the early post‐injury time frame may provide a strategy to block perpetuation of tissue damage and prevent the development of PTOA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:739–750, 2018. [ABSTRACT FROM AUTHOR]

ABSTRACT The diagnosis of ankle osteoarthritis (OA) is increasing as a result of advancements in non-invasive imaging modalities such as magnetic resonance imaging, improved arthroscopic surgical technology and heightened awareness among clinicians. Unlike OA of the knee, primary or age-related ankle OA is rare, with the majority of ankle OA classified as post-traumatic (PTOA). Ankle trauma, more specifically ankle sprain, is the single most common athletic injury, and no effective therapies are available to prevent or slow progression of PTOA. Despite the high incidence of ankle trauma and OA, ankle-related OA research is sparse, with the majority of clinical and basic studies pertaining to the knee joint. Fundamental differences exist between joints including their structure and molecular composition, response to trauma, susceptibility to OA, clinical manifestations of disease, and response to treatment. Considerable evidence suggests that research findings from knee should not be extrapolated to the ankle, however few ankle-specific preclinical models of PTOA are currently available. The objective of this article is to review the current state of ankle OA investigation, highlighting important differences between the ankle and knee that may limit the extent to which research findings from knee models are applicable to the ankle joint. Considerations for the development of new ankle-specific, clinically relevant animal models are discussed. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:440-453, 2017. [ABSTRACT FROM AUTHOR]

he goal of this study was to characterize antibody penetration through cartilage tissue under mechanical loading. Mechanical stimulation aids in the penetration of some proteins, but this effect has not characterized molecules such as antibodies (>100kDa), which may hold some clinical value for treating osteoarthritis (OA). For each experiment, fresh articular cartilage plugs were obtained and exposed to fluorescently labeled antibodies while under cyclic mechanical load in unconfined compression for several hours. Penetration of these antibodies was quantified using confocal microscopy, and finite element (FE) simulations were conducted to predict fluid flow patterns within loaded samples. Transport enhancement followed a linear trend with strain amplitude (0.25-5%) and a nonlinear trend with frequency (0.25-2.60Hz), with maximum enhancement found to be at 5% cyclic strain and 1Hz, respectively. Regions of highest enhancement of transport within the tissue were associated with the regions of highest interstitial fluid velocity, as predicted from finite-element simulations. Overall, cyclic compression-enhanced antibody transport by twofold to threefold. To our knowledge, this is the first study to test how mechanical stimulation affects the diffusion of antibodies in cartilage and suggest further study into other important factors regarding macromolecular transport. [ABSTRACT FROM AUTHOR]

Articular cartilage has limited healing capacity and no drugs are available that can prevent or slow the development of osteoarthritis (OA) after joint injury. Mesenchymal stromal cell (MSC)-based regenerative therapies for OA are increasingly common, but questions regarding their mechanisms of action remain. Our group recently reported that although cartilage is avascular and relatively metabolically quiescent, injury induces chondrocyte mitochondrial dysfunction, driving cartilage degradation and OA. MSCs are known to rescue injured cells and improve healing by donating healthy mitochondria in highly metabolic tissues, but mitochondrial transfer has not been investigated in cartilage. Here, we demonstrate that MSCs transfer mitochondria to stressed chondrocytes in cell culture and in injured cartilage tissue. Conditions known to induce chondrocyte mitochondrial dysfunction, including stimulation with rotenone/antimycin and hyperoxia, increased transfer. MSC-chondrocyte mitochondrial transfer was blocked by non-specific and specific (connexin-43) gap-junction inhibition. When exposed to mechanically injured cartilage, MSCs localized to areas of matrix damage and extended cellular processes deep into microcracks, delivering mitochondria to chondrocytes. This work provides insights into the chemical, environmental, and mechanical conditions that can elicit MSC-chondrocyte mitochondrial transfer in vitro and in situ, and our findings suggest a new potential role for MSC-based therapeutics after cartilage injury. [ABSTRACT FROM AUTHOR]

Cellular response to stimulation governs tissue scale processes ranging from growth and development to maintaining tissue health and initiating disease. To determine how cells coordinate their response to such stimuli, it is necessary to simultaneously track and measure the spatiotemporal distribution of their behaviors throughout the tissue. Here, we report on a novel SpatioTemporal Response Analysis IN Situ (STRAINS) tool that uses fluorescent micrographs, cell tracking, and machine learning to measure such behavioral distributions. STRAINS is broadly applicable to any tissue where fluorescence can be used to indicate changes in cell behavior. For illustration, we use STRAINS to simultaneously analyze the mechanotransduction response of 5000 chondrocytes—over 20 million data points—in cartilage during the 50 ms to 4 hours after the tissue was subjected to local mechanical injury, known to initiate osteoarthritis. We find that chondrocytes exhibit a range of mechanobiological responses indicating activation of distinct biochemical pathways with clear spatial patterns related to the induced local strains during impact. These results illustrate the power of this approach. [ABSTRACT FROM AUTHOR]

While mechanical stimulation of cells seeded within scaffolds is widely thought to be beneficial, the amount of benefit observed is highly variable between experimental systems. Although studies have investigated specific experimental loading protocols thought to be advantageous for cartilage growth, less is known about the physical stimuli (e.g., pressures, velocities, and local strains) cells experience during these experiments. This study used results of a literature survey, which looked for patterns in the efficacy of mechanical stimulation of chondrocyte seeded scaffolds, to inform the modeling of spatial patterns of physical stimuli present in mechanically stimulated constructs. The literature survey revealed a large variation in conditions used in mechanical loading studies, with a peak to peak strain of 10% (i.e., the maximum amount of deformation experienced by the scaffold) at 1 Hz on agarose scaffolds being the most frequently studied parameters and scaffold. This loading frequency was then used as the basis for simulation in the finite element analyses. 2D axisymmetric finite element models of 2×4 mm2 scaffolds with 360 modulus/permeability combinations were constructed using COMSOL MULTIPHYSICS software. A time dependent coupled pore pressure/effective stress analysis was used to model fluid/solid interactions in the scaffolds upon loading. Loading was simulated using an impermeable frictionless loader on the top boundary with fluid and solid displacement confined to the radial axis. As expected, all scaffold materials exhibited classic poro-elastic behavior having pressurized cores with low fluid flow and edges with high radial fluid velocities. Under the simulation parameters of this study, PEG scaffolds had the highest pressure and radial fluid velocity but also the lowest shear stress and radial strain. Chitosan and KLD-12 simulated scaffold materials had the lowest radial strains and fluid velocities, with collagen scaffolds having the lowest pressures. Parametric analysis showed maximum peak pressures within the scaffold to be more dependent on scaffold modulus than on permeability and velocities to depend on both scaffold properties similarly. The dependence of radial strain on permeability or modulus was more complex; maximum strains occurred at lower permeabilities and moduli, and the lowest strain occurred at the stiffest most permeable scaffold. Shear stresses within all scaffolds were negligible. These results give insight into the large variations in metabolic response seen in studies involving mechanical stimulation of cell-seeded constructs, where the same loading conditions produce very different results due to the differences in material properties. [ABSTRACT FROM AUTHOR]

While mechanical stimulation of cells seeded within scaffolds is widely thought to be beneficial, the amount of benefit observed is highly variable between experimental systems. Although studies have investigated specific experimental loading protocols thought to be advantageous for cartilage growth, less is known about the physical stimuli (e.g., pressures, velocities, and local strains) cells experience during these experiments. This study used results of a literature survey, which looked for patterns in the efficacy of mechanical stimulation of chondrocyte seeded scaffolds, to inform the modeling of spatial patterns of physical stimuli present in mechanically stimulated constructs. The literature survey revealed a large variation in conditions used in mechanical loading studies, with a peak to peak strain of 10% (i.e., the maximum amount of deformation experienced by the scaffold) at 1 Hz on agarose scaffolds being the most frequently studied parameters and scaffold. This loading frequency was then used as the basis for simulation in the finite element analyses. 2D axisymmetric finite element models of 2×4 mm2 scaffolds with 360 modulus/permeability combinations were constructed using COMSOL MULTIPHYSICS software. A time dependent coupled pore pressure/effective stress analysis was used to model fluid/solid interactions in the scaffolds upon loading. Loading was simulated using an impermeable frictionless loader on the top boundary with fluid and solid displacement confined to the radial axis. As expected, all scaffold materials exhibited classic poro-elastic behavior having pressurized cores with low fluid flow and edges with high radial fluid velocities. Under the simulation parameters of this study, PEG scaffolds had the highest pressure and radial fluid velocity but also the lowest shear stress and radial strain. Chitosan and KLD-12 simulated scaffold materials had the lowest radial strains and fluid velocities, with collagen scaffolds having the lowest pressures. Parametric analysis showed maximum peak pressures within the scaffold to be more dependent on scaffold modulus than on permeability and velocities to depend on both scaffold properties similarly. The dependence of radial strain on permeability or modulus was more complex; maximum strains occurred at lower permeabilities and moduli, and the lowest strain occurred at the stiffest most permeable scaffold. Shear stresses within all scaffolds were negligible. These results give insight into the large variations in metabolic response seen in studies involving mechanical stimulation of cell-seeded constructs, where the same loading conditions produce very different results due to the differences in material properties. [ABSTRACT FROM AUTHOR]

Reports on the use of tissue-engineered distal phalanx to replace the bone in a patient who had partial avulsion of the thumb. Details of the injury; Procurement and in vitro culture of autologous cells; Implantation of the cell-scaffold complex using an implant of natural coral in the thumb; Documentation of the development of bony tissue; Results.

Background: Bone marrow mesenchymal stem cells (MSCs) have shown positive therapeutic effects for meniscus regeneration and repair. Preliminary in vitro work has indicated positive results for MSC applications for meniscus tissue engineering; however, more information is needed on how to direct MSC behavior. The objective of this study was to examine the effect of MSC co-culture with primary meniscal fibrochondrocytes (FCCs) in a threedimensional collagen scaffold in fibrochondrogenic media. Co-culture of MSCs and FCCs was hypothesized to facilitate the transition of MSCs to a FCC cell phenotype as measured by matrix secretion and morphology. Methods: MSCs and FCCs were isolated from bovine bone marrow and meniscus, respectively. Cells were seeded in a 20 mg/mL high-density type I collagen gel at MSC:FCC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs were cultured for up to 2 weeks and then analyzed for cell morphology, glycosaminoglycan content, collagen content, and production of collagen type I, II, and X. Results: Cells were homogeneously mixed throughout the scaffold and cells had limited direct cell-cell contact. After 2 weeks in culture, MSCs transitioned from a spindle-like morphology toward a rounded phenotype, while FCCs remained rounded throughout culture. Although MSC shape changed with culture, the overall size was significantly larger than FCCs throughout culture. While 75:25 and 100:0 (MSC mono-culture) culture groups produced significantly more glycosaminoglycan (GAG)/DNA than FCCs in mono-culture, GAG retention was highest in 50:50 co-cultures. Similarly, the aggregate modulus was highest in 100:0 and 50:50 co-cultures. All samples contained both collagen types I and II after 2 weeks, and collagen type X expression was evident only in MSC mono-culture gels. Conclusions: MSCs shift to a FCC morphology in both mono- and co-culture. Co-culture reduced hypertrophy by MSCs, indicated by collagen type X. This study shows that MSC phenotype can be influenced by indirect homogeneous cell culture in a three-dimensional gel, demonstrating the applicability of MSCs in meniscus tissue engineering applications. [ABSTRACT FROM AUTHOR]

Articular cartilage is a heterogeneous soft tissue that dissipates and distributes loads in mammalian joints. Though robust, cartilage is susceptible to damage from loading at high rates or magnitudes. Such injurious loads have been implicated in degenerative changes, including chronic osteoarthritis (OA), which remains a leading cause of disability in developed nations. Despite decades of research, mechanisms of OA initiation after trauma remain poorly understood. Indeed, although bulk cartilage mechanics are measurable during impact, current techniques cannot access microscale mechanics at those rapid time scales. We aimed to address this knowledge gap by imaging the microscale mechanics and corresponding acute biological changes of cartilage in response to rapid loading. In this study, we utilized fast-camera and confocal microscopy to achieve roughly 85 µm spatial resolution of both the cartilage deformation during a rapid (~3 ms), localized impact and the chondrocyte death following impact. Our results showed that, at these high rates, strain and chondrocyte death were highly correlated ( p <0.001) with a threshold of 8% microscale strain norm before any cell death occurred. Additionally, chondrocyte death had developed by two hours after impact, suggesting a time frame for clinical therapeutics. Moreover, when the superficial layer was removed, strain – and subsequently chondrocyte death – penetrated deeper into the samples ( p <0.001), suggesting a protective role for the superficial layer of articular cartilage. Combined, these results provide insight regarding the detailed biomechanics that drive early chondrocyte damage after trauma and emphasize the importance of understanding cartilage and its mechanics on the microscale. [ABSTRACT FROM AUTHOR]

Collagen is the most abundant component of mammalian extracellular matrices. As such, the development of materials that mimic the biological and mechanical properties of collagenous tissues is an enduring goal of the biomaterials community. Despite the development of molded and 3D printed collagen hydrogel platforms, their use as biomaterials and tissue engineering scaffolds is hindered by either low stiffness and toughness or processing complexity. Here, we demonstrate the development of stiff and tough biohybrid composites by combining collagen with a zwitterionic hydrogel through simple mixing. This combination led to the self-assembly of a nanostructured fibrillar network of collagen that was ionically linked to the surrounding zwitterionic hydrogel matrix, leading to a composite microstructure reminiscent of soft biological tissues. The addition of 5–15 mg mL−1 collagen and the formation of nanostructured fibrils increased the elastic modulus of the composite system by 40% compared to the base zwitterionic matrix. Most notably, the addition of collagen increased the fracture energy nearly 11-fold (Γ= 180 J m−2) and clearly delayed crack initiation and propagation. These composites exhibit elastic modulus (E= 0.180 MJ) and toughness (W*= 0.617 MJ m−3) approaching that of biological tissues such as articular cartilage. Maintenance of the fibrillar structure of collagen also greatly enhanced cytocompatibility, improving cell adhesion more than 100-fold with >90% cell viability. [ABSTRACT FROM AUTHOR]

This study investigated the effect of mechanical anchoring on the development of fiber organization and anisotropy in anatomically shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were mixed with collagen and injected into molds designed to produce meniscus implants with 12 mm extensions at each horn. After a day of static culture, 10 and 20 mg/ml collagen menisci were either clamped or unclamped and cultured for up to 8 weeks. Clamped menisci were anchored in culture trays throughout culture to mimic the native meniscus horn attachment sites, restrict contraction circumferentially, and encourage circumferential alignment. Clamped menisci retained their size and shape, and by 8 weeks developed circumferential and radial fiber organization that resembled native meniscus. Clamping also increased collagen accumulation and improved mechanical properties compared to unclamped menisci. Enhanced organization in clamped menisci was further reflected in the development of anisotropic tensile properties, with 2-3 fold higher circumferential moduli compared to radial moduli, a similar ratio to native meniscus. Ten and 20 mg/ml clamped menisci had similar levels of organization, with 20 mg/ml menisci producing larger diameter fibers and significantly better mechanical properties. Collectively, these data demonstrate the benefit of using bio-inspired mechanical boundary conditions to drive the formation of a highly organized collagen fiber network. [ABSTRACT FROM AUTHOR]

In this study we analyzed the effects of IGF-I on the boundary lubricating ability of engineered meniscal tissue using a high density collagen gel seeded with meniscal fibrochondrocytes. Biochemical, histological, immunohistochemical, and tribological analyses were carried out to determine a construct's ability to functionally localize lubricin. Our study revealed that supplementation with IGF-I enhanced both the proliferation of cells within the construct as well as enhanced the anabolic activity of the seeded cells. Growth factor supplementation also facilitated the localization of ECM constituents (i.e. fibronectin and type II collagen) near the tissue surface that are important for the localization of lubricin, a boundary lubricant. Consequently, we found localized lubricin in the constructs supplemented with IGF-I. Tribologically, we demonstrated that lubricin serves as a boundary lubricant adsorbed to native meniscal surfaces. Lubricin removal from the native meniscus surface increased boundary friction coefficient by 40%. For the engineered constructs, the lubricin localization facilitated by growth factor supplementation also reduced friction coefficient by a similar margin, but similar results were not evident in control constructs. This study demonstrates that the use of growth factors in meniscal tissue engineering can enhance tribological properties by facilitating the localization of boundary lubricants at the surface of engineered tissue. [ABSTRACT FROM AUTHOR]

Low back pain is the leading cause of work absences and years lived with disability, and it is often associated with degenerative disc disease. In recent years, biological treatment approaches such as the use of growth factors, cell injections, annulus fibrosus (AF) repair, nucleus pulposus replacement, and tissue-engineered discs have been explored as means for preventing or reversing degenerative disc disease. Both animal and clinical studies have shown promising results for cell-based therapy on the grounds of its regenerative potential. Clinical data also indicate that stem cell injection is safe when appropriately performed, albeit its long-term safety and efficacy are yet to be explored. Numerous challenges also remain to be overcome, such as isolating, differentiating, and preconditioning the disc cells, as well as managing the nutrient-deficient and oxygen-deficient micromilieu of the intervertebral disc (IVD). AF repair methods including devices used in clinical trials have shown success in decreasing reherniation rates and improving overall clinical outcomes. In addition, recent studies that combined AF repair and nucleus pulposus replacement have shown improved biomechanical stability in IVDs after the combined treatment. Tissue-engineered IVDs for total disc replacement are still being developed, and future studies are necessary to overcome the challenges in their delivery, efficacy, and safety. [ABSTRACT FROM AUTHOR]

In this study, we propose a new cell encapsulation methodconsistingof a dispensing method and an aerosol-spraying method. The aerosolspray using a cross-linking agent, calcium chloride (CaCl2), was used to control the surface gelation of dispensed alginatestruts during dispensing. To show the feasibility of the method, weused preosteoblast (MC3T3-E1) cells. By changing the relationshipbetween the various dispensing/aerosol-spraying conditions and cellviability, we could determine the optimal cell-dispensing process:a nozzle size (240 μm) and an aerosol spray flow rate (0.93± 0.12 mL min–1), 10 mm s–1nozzle moving speed, a 10 wt % concentration of CaCl2in the aerosol solution, and 2 wt % concentration of CaCl2in the second cross-linking process. Based on these optimized processconditions, we successfully fabricated a three-dimensional, pore-structured,cell-laden alginate scaffold of 20 × 20 × 4.6 mm3and 84% cell viability. During long cell culture periods (16, 25,33, and 45 days), the preosteoblasts in the alginate scaffold survivedand proliferated well. [ABSTRACT FROM AUTHOR]

Abstract: The interaction between chondrocytes and their surrounding extracellular matrix plays an important role in regulating cartilage metabolism in response to environmental cues. This study characterized the role of cell adhesion on the calcium signaling response of chondrocytes to fluid flow. Bovine chondrocytes were suspended in alginate hydrogels functionalized with RGD at concentrations of 0–400μM. The hydrogels were perfused and the calcium signaling response of the cells was measured over a range of fluid velocities from 0 to 68μm/s. Attachment to RGD-alginate doubled the sensitivity of chondrocytes to flows in the range of 8–13μm/s, but at higher fluid velocities, the contribution of cell adhesion to the observed calcium signaling response was no longer apparent. The enhanced sensitivity to flow was dependent on the density of RGD-ligand present in the scaffolds. The RGD-enhanced sensitivity to flow was completely inhibited by the addition of soluble RGD which acted as a competitive inhibitor. The results of this study indicate a role for matrix adhesion in regulating chondrocyte response to fluid flow through a calcium dependent mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Background context: Needle puncture of the intervertebral disc (IVD) is required for delivery of therapeutic agents to the nucleus pulposus and for some diagnostic procedures. Needle puncture has also been implicated as an initiator of disc degeneration. It is hypothesized that needle puncture may initiate IVD degeneration by altering microscale mechanical behavior in the annulus fibrosus (AF). Purpose: Quantify the changes in AF microscale strain behavior resulting from puncture with a hypodermic needle. Study design: Cadaveric IVD tissue explant study. Methods: Annulus fibrosus explants from bovine caudal IVDs that had been punctured radially with hypodermic needles were loaded in dynamic sinusoidal shear while being imaged with a confocal microscope. Digital image analysis was used to quantify local tissue strain and damage propagation with repeated shearing. Results: Needle puncture changed the distribution of microscale shear strains in the AF under load from homogenous (equal to far field) to a distinct pattern of high (4× far field) and low (0.25× far field) strain areas. Repeated loading did not cause further growth of the disruption beyond the second cycle. Conclusions: Needle puncture results in a drastic alteration of microscale strain behavior in the AF under load. This alteration may directly initiate disc degeneration by being detrimental to tissue-cell mechanotransduction. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the hypothesis that controlled media mixing will enhance tissue formation and increase mechanical properties of anatomically-shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2% w/v alginate, cross-linked with 0.02 g/mL CaSO4, and injected into molds of menisci. Engineered menisci were incubated for up to 6 weeks. A mixing media bioreactor was designed to ensure proper mixing of culture medium while protecting constructs from the spinning impeller. Impeller speeds were calibrated to produce Reynolds number (Re) of 0.5, 2.9, 5.8, 10.2, and 21.8. Constructs were divided a tested in confined compression and in tension to determine the equilibrium and tensile moduli, respectively. Media stimulation resulted in a 2–5 fold increase in mechanical properties and a 2–3 fold increase in matrix accumulation in constructs over 6 weeks in culture. Benefits from mixing stimulation for collagen accumulation and compressive modulus appeared to peak near Re 2.9, and decreased with increased mixing intensity. This study suggests that fluid mixing can be optimized to enhance mechanical properties of anatomically-shaped engineered constructs. [Copyright &y& Elsevier]

Abstract: Shear strain has been implicated as an initiator of intervertebral disc anulus failure, however a clear, multi-scale picture of how shear strain affects the tissue microstructure has been lacking. The purposes of this study were to measure microscale deformations in anulus tissue under dynamic shear in two orie ntations, and to determine the role of elastin in regulating these deformations. Bovine AF tissue was simultaneously shear loaded and imaged using confocal microscopy following either a buffer or elastase treatment. Digital image analysis was used to track through time local shear strains in specimens sheared transversely, and stretch and rotation of collagen fiber bundles in specimens sheared circumferentially. The results of this study suggest that sliding does not occur between AF plies under shear, and that interlamellar connections are governed by collagen and fibrilin rather than elastin. The transverse shear modulus was found to be approximately 1.6 times as high in plies the direction of the collagen fibers as in plies across them. Under physiological levels of in-plane shear, fiber bundles stretched and re-oriented linearly. Elastin was found to primarily stiffen plies transversely. We conclude that alterations in the elastic fiber network, as found with IVD herniation and degeneration, can therefore be expected to significantly influence the AF response to shear making it more susceptible to micro failure under bending or torsion loading. [Copyright &y& Elsevier]

Collagen glycated with ribose (250 mM) in solution (pre-glycation) and as a gel (post-glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre-glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks ( p < 0.05), although at both time points there were no statistical differences in cell number between pre-glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre-glycated constructs ( p < 0.05). Total collagen content in these pre-glycated gels was also significantly higher than unglycated gels at 4 weeks ( p < 0.05). With post-glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls ( p < 0.05). Post-glycation also inhibited GAG retention by the constructs ( p < 0.05). Given these results, pre-glycation may be an improved processing method for collagen gels for tissue engineering techniques. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1434-1439, 2008 [ABSTRACT FROM AUTHOR]

Abstract: Determining the depth dependence of the shear properties of articular cartilage is essential for understanding the structure–function relation in this tissue. Here, we measured spatial variations in the shear modulus G of bovine articular cartilage using a novel technique that combines shear testing, confocal imaging and force measurement. We found that G varied by up to two orders of magnitude across a single sample, exhibited a global minimum 50–250μm below the articular surface in a region just below the superficial zone and was roughly constant at depths >1000μm (the “plateau region”). For plateau strains γ plateau≈0.75% and overall compressive strains ε≈5%, G min and G plateau were ≈70 and ≈650kPa, respectively. In addition, we found that the shear modulus profile depended strongly on the applied shear and axial strains. The greatest change in G occurred at the global minimum where the tissue was highly nonlinear, stiffening under increased shear strain, and weakening under increased compressive strain. Our results can be explained through a simple thought model describing the observed nonlinear behavior in terms of localized buckling of collagen fibers and suggest that compression may decrease the vulnerability of articular cartilage to shear-induced damage by lowering the effective strain on individual collagen fibrils. [Copyright &y& Elsevier]

In the preparation of bioengineered reparative strategies for damaged or diseased tissues, the processes of biomaterial degradation and neotissue synthesis combine to affect the developing mechanical state of multiphase, composite engineered tissues. Here, cell-polymer constructs for engineered cartilage have been fabricated by seeding chondrocytes within three-dimensional scaffolds of biodegradable polymers. During culture, synthetic scaffolds degraded passively as the cells assembled an extracellular matrix (ECM) composed primarily of glycosaminoglycan and collagen. Biochemical and biomechanical assessment of the composite (cells, ECM, and polymer scaffold) were modeled at a unit-cell level to mathematically solve stress-strain relationships and thus construct elastic properties (n=4 samples per seven time points). This approach employed a composite spheres, micromechanical analysis to determine bulk moduli of: (1) the cellular-ECM inclusion within the supporting scaffold structure; and (2) the cellular inclusion within its ECM. Results indicate a dependence of constituent volume fractions with culture time (p<0.05). Overall mean bulk moduli were variably influenced by culture, as noted for the cell-ECM inclusion (Kc-m=29.7 kPa, p=0.1439), the cellular inclusion (Kc=5.5 kPa, p=0.0067), and its surrounding ECM (Km=373.9 kPa, p=0.0748), as well as the overall engineered construct (K=165.0 kPa, p=0.6899). This analytical technique provides a framework to describe the time-dependent contribution of cells, accumulating ECM, and a degrading scaffold affecting bioengineered construct mechanical properties. [ABSTRACT FROM AUTHOR]

Posttraumatic osteoarthritis (PTOA) is typically initiated by momentary supraphysiologic shear and compressive forces delivered to articular cartilage during acute joint injury and develops through subsequent degradation of cartilage matrix components and tissue remodeling. PTOA affects 12% of the population who experience osteoarthritis and is attributed to over $3 billion dollars annually in healthcare costs. It is currently unknown whether articulation of the joint post‐injury helps tissue healing or exacerbates cellular dysfunction and eventual death. We hypothesize that post‐injury cartilage articulation will lead to increased cartilage damage. Our objective was to test this hypothesis by mimicking the mechanical environment of the joint during and post‐injury and determining if subsequent joint articulation exacerbates damage produced by initial injury. We use a model of PTOA that combines impact injury and repetitive sliding with confocal microscopy to quantify and track chondrocyte viability, apoptosis, and mitochondrial depolarization in a depth‐dependent manner. Cartilage explants were harvested from neonatal bovine knee joints and subjected to either rapid impact injury (17.34 ± 0.99 MPa, 21.6 ± 2.45 GPa/s), sliding (60 min at 1 mm/s, under 15% axial compression), or rapid impact injury followed by sliding. Explants were then bisected and fluorescently stained for cell viability, caspase activity (apoptosis), and mitochondria polarization. Results show that compared to either impact or sliding alone, explants that were both impacted and slid experienced higher magnitudes of damage spanning greater tissue depths. [ABSTRACT FROM AUTHOR]

The extracellular matrix not only provides a structural scaffold for cells to inhabit but also forms a conduit by which mechanical information may be transmitted. Fibroblasts undergo a variety of changes when activated, including upregulating matrix metalloproteinase (MMP) activity and establishing a smooth muscle-like contractile apparatus. The relationship between MMP activity and matrix contraction has yet to be established. Here we report that inhibition of MMP activity correlates with a significant reduction in collagen gel contraction, however, force development does not change respective to MMP activity. These results suggest cellular controls of contractile forces are independent of MMP activity. Our results also raise the possibility that the material properties of the matrix dynamically change during remodeling. [Copyright &y& Elsevier]

Tissue-engineered osteochondral implants manufactured from condensed mesenchymal stem cell bodies have shown promise for treating focal cartilage defects. Notably, such manufacturing techniques have shown to successfully recapture the bulk mechanical properties of native cartilage. However, the relationships among the architectural features, local composition, and micromechanical environment within tissue-engineered cartilage from cell-based aggregates remain unclear. Understanding such relationships is crucial for identifying critical parameters that can predict in vivo performance. Therefore, this study investigated the relationship among architectural features, composition, and micromechanical behavior of tissue-engineered osteochondral implants. We utilized fast-confocal microscopy combined with a strain mapping technique to analyze the micromechanical behavior under quasi-static loading, as well as Fourier Transform Infrared Spectroscopy to analyze the local compositions. More specifically, we investigated the architectural features and compositional distributions generated from tissue maturation, along with macro- and micro-level strain distributions. Our results showed that under compression, cell-based aggregates underwent deformation followed by body movement, generating high local strain around the boundaries, where local aggrecan concentration was low and local collagen concentration was high. By analyzing the micromechanics and composition at the single aggregate length scale, we identified a strong threshold relationship between local strain and compositions. Namely at the aggrecan concentration below 0.015 arbitrary unit (A.U.) and the collagen concentration above 0.15 A.U., the constructs experienced greater than threefold increase in compressive strain. Overall, this study suggests that local compositional features are the primary driver of the local mechanical environment in tissue-engineered cartilage constructs, providing insight into potential quality control parameters for manufacturing tissue-engineered constructs. [ABSTRACT FROM AUTHOR]

Inferior synovial lubrication is a hallmark of osteoarthritis (OA), and synovial fluid (SF) lubrication and composition are variable among OA patients. Hyaluronic acid (HA) viscosupplementation is a widely used therapy for improving SF viscoelasticity and lubrication, but it is unclear how the effectiveness of HA viscosupplements varies with arthritic endotype. The objective of this study was to investigate the effects of the HA viscosupplement, Hymovis®, on the lubricating properties of diseased SF from patients with noninflammatory OA and inflammatory arthritis (IA). The composition (cytokine, HA, and lubricin concentrations) of the SF was measured as well as the mechanical properties (rheology, tribology) of the SF alone and in a 1:1 mixture with the HA viscosupplement. Using rotational rheometry, no difference in SF viscosity was detected between disease types, and the addition of HA significantly increased all fluids' viscosities. In noninflammatory OA SF, friction coefficients followed a typical Stribeck pattern, and their magnitude was decreased by the addition of HA. While some of the IA SF also showed typical Stribeck behavior, a subset showed more erratic behavior with highly variable and larger friction coefficients. Interestingly, this aberrant behavior was not eliminated by the addition of HA, and it was associated with low concentrations of lubricin. Aberrant SF exhibited significantly lower effective viscosities compared to noninflammatory OA and IA SF with typical tribological behavior. Collectively, these results suggest that different endotypes of arthritis exist with respect to lubrication, which may impact the effectiveness of HA viscosupplements in reducing friction. [ABSTRACT FROM AUTHOR]