Advances in viscosupplementation and tribosupplementation for early-stage osteoarthritis therapy.

Joint kinematic instability, arising from congenital or acquired musculoskeletal pathoanatomy or from imbalances in anabolism and catabolism induced by pathophysiological factors, leads to deterioration of the composition, structure and function of cartilage and, ultimately, progression to osteoarthritis (OA). Alongside articular cartilage degeneration, synovial fluid lubricity decreases in OA owing to a reduction in the concentration and molecular weight of hyaluronic acid and surface-active mucinous glycoproteins that form a lubricating film over the articulating joint surfaces. Minimizing friction between articulating joint surfaces by lubrication is fundamental for decreasing hyaline cartilage wear and for maintaining the function of synovial joints. Augmentation with highly viscous supplements (that is, viscosupplementation) offers one approach to re-establishing the rheological and tribological properties of synovial fluid in OA. However, this approach has varied clinical outcomes owing to limited intra-articular residence time and ineffective mechanisms of chondroprotection. This Review discusses normal hyaline cartilage function and lubrication and examines the advantages and disadvantages of various strategies for restoring normal joint lubrication. These strategies include contemporary viscosupplements that contain antioxidants, anti-inflammatory drugs or platelet-rich plasma and new synthetic synovial fluid additives and cartilage matrix enhancers. Advanced biomimetic tribosupplements offer promise for mitigating cartilage wear, restoring joint function and, ultimately, improving patient care.

Physiologic Doses of TGF-ß Improve the Composition of Engineered Articular Cartilage.

Conventionally, for cartilage tissue engineering applications, TGF-ß is administered at doses that are several orders of magnitude higher than those present during native cartilage development. While these doses accelerate extracellular matrix (ECM) biosynthesis, they may also contribute to features detrimental to hyaline cartilage function, including tissue swelling, type-I collagen (COL-I) deposition, cellular hypertrophy, and cellular hyperplasia. In contrast, during native cartilage development, chondrocytes are exposed to moderate TGF-ß levels, which serve to promote strong biosynthetic enhancements while mitigating risks of pathology associated with TGF-ß excesses. Here, we examine the hypothesis that physiologic doses of TGF-ß can yield neocartilage with a more hyaline-cartilage-like composition and structure relative to conventionally-administered supraphysiologic doses. This hypothesis was examined on a model system of reduced-size constructs (Ø2×2mm or Ø3×2mm) comprised of bovine chondrocytes encapsulated in agarose, which exhibit mitigated TGF-ß spatial gradients allowing for an evaluation of the intrinsic effect of TGF-ß doses on tissue development. Reduced-size (Ø2×2mm or Ø3×2mm) and conventional-size constructs (Ø4-Ø6mm×2mm) were subjected to a range of physiologic (0.1, 0.3, 1ng/mL) and supraphysiologic (3, 10ng/mL) TGF-ß doses. At day 56, the physiologic 0.3ng/mL dose yielded reduced-size constructs with native-cartilage-matched Young's modulus (EY) (630±58kPa) and sulfated GAG (sGAG) content (5.9±0.6%) while significantly increasing the sGAG-to-collagen ratio, leading to significantly reduced tissue swelling relative to constructs exposed to the supraphysiologic 10ng/mL TGF-ß dose. Further, reduced-size constructs exposed to the 0.3ng/mL dose exhibited a significant reduction in fibrocartilage-associated COL-I and a 77% reduction in the fraction of chondrocytes present in a clustered morphology, relative to the supraphysiologic 10ng/mL dose (p<0.001). EY was significantly lower for conventional-size constructs exposed to physiologic doses due to TGF-ß transport limitations in these larger tissues (p<0.001). Overall, physiologic TGF-ß appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating features detrimental to hyaline cartilage function. While reduced-size constructs are not suitable for the repair of clinical-size cartilage lesions, insights from this work can inform TGF-ß dosing requirements for emerging scaffold-release or nutrient-channel delivery platforms capable of achieving uniform delivery of physiologic TGF-ß doses to larger constructs required for clinical cartilage repair.

A missed opportunity: A scoping review of the effect of sex and age on osteoarthritis using large animal models.

OBJECTIVE: The objective was to critically analyze the published literature accounting for sex differences and skeletal age (open vs. closed physis) in preclinical animal models of OA, including the disaggregation of data by sex and skeletal maturity when data is generated from combined sex and/or multi-aged cohorts without proper confounding. METHOD: A scoping literature review of PubMed, Web of Science, EMBASE, and SCOPUS was performed for studies evaluating the effect of sex and age in experimental studies and clinical trials utilizing preclinical large animal models of OA. RESULTS: A total of 9727 papers were identified in large animal (dog, pig, sheep, goat, horse) models for preclinical OA research, of which 238 ex vivo and/or in vivo studies disclosed model type, animal species, sex, and skeletal age sufficient to analyze their effect on outcomes. Dogs, followed by pigs, sheep, and horses, were the most commonly used models. A paucity of preclinical studies evaluated the effect of sex and age in large animal models of naturally occurring or experimentally induced OA: 26 total studies reported some kind of analysis of the effects of sex or age, with 4 studies discussing the effects of sex only, 11 studies discussing the effects of age only, and 11 studies analyzing both the effects of age and sex. CONCLUSION: Fundamental to translational research, OARSI is uniquely positioned to develop recommendations for conducting preclinical studies using large animal models of OA that consider biological mechanisms linked to sex chromosomes, skeletal age, castration, and gonadal hormones affecting OA pathophysiology and treatment response.

Physiologic Doses of TGF-ß Improve the Composition of Engineered Articular Cartilage.

For cartilage regeneration applications, transforming growth factor beta (TGF-ß) is conventionally administered at highly supraphysiologic doses (10-10,000 ng/mL) in an attempt to cue cells to fabricate neocartilage that matches the composition, structure, and functional properties of native hyaline cartilage. While supraphysiologic doses enhance ECM biosynthesis, they are also associated with inducing detrimental tissue features, such as fibrocartilage matrix deposition, pathologic-like chondrocyte clustering, and tissue swelling. Here we investigate the hypothesis that moderated TGF-ß doses (0.1-1 ng/mL), akin to those present during physiological cartilage development, can improve neocartilage composition. Variable doses of media-supplemented TGF-ß were administered to a model system of reduced-size cylindrical constructs (Ø2-Ø3 mm), which mitigate the TGF-ß spatial gradients observed in conventional-size constructs (Ø4-Ø6 mm), allowing for a novel assessment of the intrinsic effect of TGF-ß doses on macroscale neocartilage properties and composition. The administration of physiologic TGF-ß to reduced-size constructs yields neocartilage with native-matched sGAG content and mechanical properties while providing a more hyaline cartilage-like composition, marked by: 1) reduced fibrocartilage-associated type I collagen, 2) 77% reduction in the fraction of cells present in a clustered morphology, and 3) 45% reduction in the degree of tissue swelling. Physiologic TGF-ß appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating hyaline cartilage compositional deficits. These results can guide the development of novel physiologic TGF-ß-delivering scaffolds to improve the regeneration clinical-sized neocartilage tissues.

Computational and experimental characterizations of the spatiotemporal activity and functional role of TGF-ß in the synovial joint.

TGF-ß is a prominent anabolic signaling molecule associated with synovial joint health. Recent work has uncovered mechanochemical mechanisms that activate the latent form of TGF-ß (LTGF-ß) in the synovial joint-synovial fluid (SF) shearing or cartilage compression-pointing to mechanobiological phenomena, whereby enhanced TGF-ß activity occurs during joint stimulation. Here, we implement computational and experimental models to better understand the role of mechanochemical-activated TGF-ß (aTGF-ß) in regulating the functional biosynthetic activities of synovial joint tissues. Reaction-diffusion models describe the pronounced role of extracellular chemical reactions-load-induced activation, reversible ECM-binding, and cell-mediated internalization-in modulating the spatiotemporal distribution of aTGF-ß in joint tissues. Of note, aTGF-ß from SF shearing predominantly acts on cells in peripheral tissue regions (superficial zone [SZ] chondrocytes and synoviocytes) and aTGF-ß from cartilage compression acts on chondrocytes through all cartilage layers. Further, ECM reversible binding sites in cartilage act to modulate the temporal delivery of aTGF-ß to cells, creating a dynamic where short durations of joint activity give rise to extended periods of aTGF-ß exposure at moderated doses. Ex vivo tissue models were subsequently utilized to characterize the influence of physiologic aTGF-ß activity regimens in regulating functional biosynthetic activities. Physiologic exposure regimens of aTGF-ß in SF induce strong 4-fold to 9-fold enhancements in the secretion rate of the synovial biolubricant, PRG4, from SZ cartilage and synovium explants. Further, aTGF-ß inhibition in cartilage over 1-month culture leads to a pronounced loss of GAG content (30-35% decrease) and tissue softening (60-65% EY reduction). Overall, this work advances a novel perspective on the regulation of TGF-ß in the synovial joint and its role in maintaining synovial joint health.

Raman needle arthroscopy for in vivo molecular assessment of cartilage.

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can "optically biopsy" cartilage and quantify key extracellular matrix (ECM) biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include (1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2 O] scores), and (2) multiplexed polarized Raman spectroscopy to quantify superficial zone (SZ) COL anisotropy via a partial least squares-discriminant analysis-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: (1) chemically GAG-depleted bovine cartilage explants (n = 40), (2) mechanically abraded bovine cartilage explants (n = 30), (3) aging human cartilage explants (n = 14), and (4) anatomical-site-varied ovine osteochondral explants (n = 6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p < 0.001). RCAF values were significantly different for explants with abrasion-induced SZ COL loss (p < 0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2 O scores) predicted 94% of the variation in elastic modulus of ovine explants (p < 0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This study advances Raman arthroscopy toward a transformative low-cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

Multiplexed polarized hypodermic Raman needle probe for biostructural analysis of articular cartilage.

In this Letter, we report a multiplexed polarized hypodermic Raman needle probe for the biostructural analysis of articular cartilage. Using a custom-developed needle probe with a sapphire ball lens, we measure polarized Raman spectra of cartilage. By imaging two polarizations simultaneously on the charge-coupled device (CCD) and binning them separately, we capture both biochemical and structural tissue information in real time. Here, we demonstrate that polarized Raman spectroscopy can distinguish between different collagen fibril alignment orientations in a cartilage explant model system, supporting its capacity for diagnosing the hallmark collagen alignment changes occurring in the early stages of osteoarthritis (OA). Accordingly, this work shows that needle-based polarized Raman spectroscopy has great potential for the monitoring and diagnosis of early OA.

Raman Spectroscopy: Guiding Light for the Extracellular Matrix.

The extracellular matrix (ECM) consists of a complex mesh of proteins, glycoproteins, and glycosaminoglycans, and is essential for maintaining the integrity and function of biological tissues. Imaging and biomolecular characterization of the ECM is critical for understanding disease onset and for the development of novel, disease-modifying therapeutics. Recently, there has been a growing interest in the use of Raman spectroscopy to characterize the ECM. Raman spectroscopy is a label-free vibrational technique that offers unique insights into the structure and composition of tissues and cells at the molecular level. This technique can be applied across a broad range of ECM imaging applications, which encompass in vitro, ex vivo, and in vivo analysis. State-of-the-art confocal Raman microscopy imaging now enables label-free assessments of the ECM structure and composition in tissue sections with a remarkably high degree of biomolecular specificity. Further, novel fiber-optic instrumentation has opened up for clinical in vivo ECM diagnostic measurements across a range of tissue systems. A palette of advanced computational methods based on multivariate statistics, spectral unmixing, and machine learning can be applied to Raman data, allowing for the extraction of specific biochemical information of the ECM. Here, we review Raman spectroscopy techniques for ECM characterizations over a variety of exciting applications and tissue systems, including native tissue assessments (bone, cartilage, cardiovascular), regenerative medicine quality assessments, and diagnostics of disease states. We further discuss the challenges in the widespread adoption of Raman spectroscopy in biomedicine. The results of the latest discovery-driven Raman studies are summarized, illustrating the current and potential future applications of Raman spectroscopy in biomedicine.

Physiologic Medium Maintains the Homeostasis of Immature Bovine Articular Cartilage Explants in Long-Term Culture.

The ability to maintain living articular cartilage tissue in long-term culture can serve as a valuable analytical research tool, allowing for direct examination of mechanical or chemical perturbations on tissue behavior. A fundamental challenge for this technique is the recreation of the salient environmental conditions of the synovial joint in culture that are required to maintain native cartilage homeostasis. Interestingly, conventional media formulations used in explanted cartilage tissue culture investigations often consist of levels of metabolic mediators that deviate greatly from their concentrations in synovial fluid (SF). Here, we hypothesize that the utilization of a culture medium consisting of near-physiologic levels of several highly influential metabolic mediators (glucose, amino acids, cortisol, insulin, and ascorbic acid) will maintain the homeostasis of cartilage explants as assessed by their mechanical properties and extracellular matrix (ECM) contents. Results demonstrate that the aforementioned mediators have a strong effect on the mechanical and biochemical stability of skeletally immature bovine cartilage explants. Most notably, (1) in the absence of cortisol, explants exhibit extensive swelling and tissue softening and (2) in the presence of supraphysiologic levels of anabolic mediators (glucose, amino acids, insulin), explants exhibit increased matrix accumulation and tissue stiffening. In contrast, the administration of physiologic levels of these mediators (as present in native SF) greatly improves the stability of live cartilage explants over one month of culture. These results may have broad applicability for articular cartilage and other musculoskeletal tissue research, setting the foundation for important culture formulations required for examinations into tissue behavior.

Online quantitative monitoring of live cell engineered cartilage growth using diffuse fiber-optic Raman spectroscopy.

Tissue engineering (TE) has the potential to improve the outcome for patients with osteoarthritis (OA). The successful clinical translation of this technique as part of a therapy requires the ability to measure extracellular matrix (ECM) production of engineered tissues in vitro, in order to ensure quality control and improve the likelihood of tissue survival upon implantation. Conventional techniques for assessing the ECM content of engineered cartilage, such as biochemical assays and histological staining are inherently destructive. Raman spectroscopy, on the other hand, represents a non-invasive technique for in situ biochemical characterization. Here, we outline current roadblocks in translational Raman spectroscopy in TE and introduce a comprehensive workflow designed to non-destructively monitor and quantify ECM biomolecules in large (>3 mm), live cell TE constructs online. Diffuse near-infrared fiber-optic Raman spectra were measured from live cell cartilaginous TE constructs over a 56-day culturing period. We developed a multivariate curve resolution model that enabled quantitative biochemical analysis of the TE constructs. Raman spectroscopy was able to non-invasively quantify the ECM components and showed an excellent correlation with biochemical assays for measurement of collagen (R2 = 0.84) and glycosaminoglycans (GAGs) (R2 = 0.86). We further demonstrated the robustness of this technique for online prospective analysis of live cell TE constructs. The fiber-optic Raman spectroscopy strategy developed in this work offers the ability to non-destructively monitor construct growth online and can be adapted to a broad range of TE applications in regenerative medicine toward controlled clinical translation.

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-ß treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-ß treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

Raman Spectroscopy Reveals New Insights into the Zonal Organization of Native and Tissue-Engineered Articular Cartilage.

Tissue architecture is intimately linked with its functions, and loss of tissue organization is often associated with pathologies. The intricate depth-dependent extracellular matrix (ECM) arrangement in articular cartilage is critical to its biomechanical functions. In this study, we developed a Raman spectroscopic imaging approach to gain new insight into the depth-dependent arrangement of native and tissue-engineered articular cartilage using bovine tissues and cells. Our results revealed previously unreported tissue complexity into at least six zones above the tidemark based on a principal component analysis and k-means clustering analysis of the distribution and orientation of the main ECM components. Correlation of nanoindentation and Raman spectroscopic data suggested that the biomechanics across the tissue depth are influenced by ECM microstructure rather than composition. Further, Raman spectroscopy together with multivariate analysis revealed changes in the collagen, glycosaminoglycan, and water distributions in tissue-engineered constructs over time. These changes were assessed using simple metrics that promise to instruct efforts toward the regeneration of a broad range of tissues with native zonal complexity and functional performance.

Heterogeneous engineered cartilage growth results from gradients of media-supplemented active TGF-ß and is ameliorated by the alternative supplementation of latent TGF-ß.

Transforming growth factor beta (TGF-ß) has become one of the most widely utilized mediators of engineered cartilage growth. It is typically exogenously supplemented in the culture medium in its active form, with the expectation that it will readily transport into tissue constructs through passive diffusion and influence cellular biosynthesis uniformly. The results of this investigation advance three novel concepts regarding the role of TGF-ß in cartilage tissue engineering that have important implications for tissue development. First, through the experimental and computational analysis of TGF-ß concentration distributions, we demonstrate that, contrary to conventional expectations, media-supplemented exogenous active TGF-ß exhibits a pronounced concentration gradient in tissue constructs, resulting from a combination of high-affinity binding interactions and a high cellular internalization rate. These gradients are sustained throughout the entire culture duration, leading to highly heterogeneous tissue growth; biochemical and histological measurements support that while biochemical content is enhanced up to 4-fold at the construct periphery, enhancements are entirely absent beyond 1 mm from the construct surface. Second, construct-encapsulated chondrocytes continuously secrete large amounts of endogenous TGF-ß in its latent form, a portion of which undergoes cell-mediated activation and enhances biosynthesis uniformly throughout the tissue. Finally, motivated by these prior insights, we demonstrate that the alternative supplementation of additional exogenous latent TGF-ß enhances biosynthesis uniformly throughout tissue constructs, leading to enhanced but homogeneous tissue growth. This novel demonstration suggests that latent TGF-ß supplementation may be utilized as an important tool for the translational engineering of large cartilage constructs that will be required to repair the large osteoarthritic defects observed clinically.

Matrix Production in Large Engineered Cartilage Constructs Is Enhanced by Nutrient Channels and Excess Media Supply.

Cartilage tissue engineering is a promising approach to resurfacing osteoarthritic joints. Existing techniques successfully engineer small-sized constructs with native levels of extracellular matrix (glycosaminoglycans [GAG] or collagen). However, a remaining challenge is the growth of large-sized constructs with properties similar to those of small constructs, due to consumption and transport limitations resulting in inadequate nutrient availability within the interior of large constructs. This study employed system-specific computational models for estimating glucose requirements of large constructs, with or without channels, to enhance nutrient availability. Based on glucose requirements for matrix synthesis in cartilage constructs, computational simulations were performed to identify the media volume (MV) and the number of nutrient channels (CH) needed to maintain adequate glucose levels within tissue constructs over the 3-day period between media replenishments. In Study 1, the influence of MV (5, 10, 15 mL/construct) and number of nutrient channels (CH: 0, 3, 7, 12 per construct) on glucose availability was investigated computationally for ∅10 × 2.34 mm cylindrical constructs. Results showed that the conventionally used MV 5 led to deleterious glucose depletion after only 40 h of culture, and that MV 15 was required to maintain sufficient glucose levels for all channel configurations. Study 2 examined experimentally the validity of these predictions, for tissue constructs cultured for 56 days. Matrix elaboration was highest in MV 15/CH 12 constructs (21.6% ± 2.4%/ww GAG, 5.5% ± 0.7%/ww collagen, normalized to wet weight (ww) on day 0), leading to the greatest amount of swelling (3.0 ± 0.3 times day-0 volume), in contrast to the significantly lower matrix elaboration of conventional culture, MV 5/CH 0 (11.8% ± 1.6%/ww GAG and 2.5% ± 0.6%/ww collagen, 1.6 ± 0.1 times day-0 volume). The computational analyses correctly predicted the need to increase the conventional media levels threefold to support matrix synthesis in large channeled engineered constructs. Results also suggested that more elaborate computational models are needed for accurate predictive tissue engineering simulations, which account for a broader set of nutrients, cell proliferation, matrix synthesis, and swelling of the constructs.

Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs.

A significant challenge in cartilage tissue engineering is to successfully culture functional tissues that are sufficiently large to treat osteoarthritic joints. Transport limitations due to nutrient consumption by peripheral cells produce heterogeneous constructs with matrix-deficient centers. Incorporation of nutrient channels into large constructs is a promising technique for alleviating transport limitations, in conjunction with simple yet effective methods for enhancing media flow through channels. Cultivation of cylindrical channeled constructs flat in culture dishes, with or without orbital shaking, produced asymmetric constructs with poor tissue properties. We therefore explored a method for exposing the entire construct surface to the culture media, while promoting flow through the channels. To this end, chondrocyte-seeded agarose constructs (∅10mm, 2.34mm thick), with zero or three nutrient channels (∅1mm), were suspended on their sides in custom culture racks and subjected to three media stirring modes for 56 days: uniaxial rocking, orbital shaking, or static control. Orbital shaking led to the highest construct EY, sulfated glycosaminoglycan (sGAG), and collagen contents, whereas rocking had detrimental effects on sGAG and collagen versus static control. Nutrient channels increased EY as well as sGAG homogeneity, and the beneficial effects of channels were most marked in orbitally shaken samples. Under these conditions, the constructs developed symmetrically and reached or exceeded native levels of EY (~400kPa) and sGAG (~9%/ww). These results suggest that the cultivation of channeled constructs in culture racks with orbital shaking is a promising method for engineering mechanically competent large cartilage constructs.

Synthesis rates and binding kinetics of matrix products in engineered cartilage constructs using chondrocyte-seeded agarose gels.

Large-sized cartilage constructs suffer from inhomogeneous extracellular matrix deposition due to insufficient nutrient availability. Computational models of nutrient consumption and tissue growth can be utilized as an efficient alternative to experimental trials to optimize the culture of large constructs; models require system-specific growth and consumption parameters. To inform models of the [bovine chondrocyte]-[agarose gel] system, total synthesis rate (matrix accumulation rate+matrix release rate) and matrix retention fractions of glycosaminoglycans (GAG), collagen, and cartilage oligomeric matrix protein (COMP) were measured either in the presence (continuous or transient) or absence of TGF-ß3 supplementation. TGF-ß3's influences on pyridinoline content and mechanical properties were also measured. Reversible binding kinetic parameters were characterized using computational models. Based on our recent nutrient supplementation work, we measured glucose consumption and critical glucose concentration for tissue growth to computationally simulate the culture of a human patella-sized tissue construct, reproducing the experiment of Hung et al. (2003). Transient TGF-ß3 produced the highest GAG synthesis rate, highest GAG retention ratio, and the highest binding affinity; collagen synthesis was elevated in TGF-ß3 supplementation groups over control, with the highest binding affinity observed in the transient supplementation group; both COMP synthesis and retention were lower than those for GAG and collagen. These results informed the modeling of GAG deposition within a large patella construct; this computational example was similar to the previous experimental results without further adjustments to modeling parameters. These results suggest that these nutrient consumption and matrix synthesis models are an attractive alternative for optimizing the culture of large-sized constructs.

Insulin, ascorbate, and glucose have a much greater influence than transferrin and selenous acid on the in vitro growth of engineered cartilage in chondrogenic media.

The primary goal of this study was to characterize the response of chondrocyte-seeded agarose constructs to varying concentrations of several key nutrients in a chondrogenic medium, within the overall context of optimizing the key nutrients and the placement of nutrient channels for successful growth of cartilage tissue constructs large enough to be clinically relevant in the treatment of osteoarthritis (OA). To this end, chondrocyte-agarose constructs (ø4×2.34 mm, 30×10(6) cells/mL) were subjected to varying supplementation levels of insulin (0× to 30× relative to standard supplementation), transferrin (0× to 30×), selenous acid (0× to 10×), ascorbate (0× to 30×), and glucose (0× to 3×). The quality of resulting engineered tissue constructs was evaluated by their compressive modulus (E(-Y)), tensile modulus (E(+Y)), hydraulic permeability (k), and content of sulfated glycosaminoglycans (sGAG) and collagen (COL); DNA content was also quantified. Three control groups from two separate castings of constructs (1× concentrations of all medium constituents) were used. After 42 days of culture, values in each of these controls were, respectively, E(-Y)=518±78, 401±113, 236±67 kPa; E(+Y)=1420±430, 1140±490, 1240±280 kPa; k=2.3±0.8×10(-3), 5.4±7.0×10(-3), 3.3±1.3×10(-3) mm(4)/N·s; sGAG=7.8±0.3, 6.3±0.4, 4.1±0.5%/ww; COL=1.3±0.2, 1.1±0.3, 1.4±0.4%/ww; and DNA=11.5±2.2, 12.1±0.6, 5.2±2.8 µg/disk. The presence of insulin and ascorbate was essential, but their concentrations may drop as low as 0.3× without detrimental effects on any of the measured properties; excessive supplementation of ascorbate (up to 30×) was detrimental to E(-Y), and 30× insulin was detrimental to both E(+Y) and E(-Y). The presence of glucose was similarly essential, and matrix elaboration was significantly dependent on its concentration (p<10(-6)), with loss of functional properties, composition, and cellularity observed at ≤0.3×; excessive glucose supplementation (up to 3×) showed no detrimental effects. In contrast, transferrin and selenous acid had no influence on matrix elaboration. These findings suggest that adequate distributions of insulin, ascorbate, and glucose, but not necessarily of transferrin and selenous acid, must be ensured within large engineered cartilage constructs to produce a viable substitute for joint tissue lost due to OA.

Dynamic mechanical compression of devitalized articular cartilage does not activate latent TGF-ß.

A growing body of research has highlighted the role that mechanical forces play in the activation of latent TGF-ß in biological tissues. In synovial joints, it has recently been demonstrated that the mechanical shearing of synovial fluid, induced during joint motion, rapidly activates a large fraction of its soluble latent TGF-ß content. Based on this observation, the primary hypothesis of the current study is that the mechanical deformation of articular cartilage, induced by dynamic joint motion, can similarly activate the large stores of latent TGF-ß bound to the tissue extracellular matrix (ECM). Here, devitalized deep zone articular cartilage cylindrical explants (n=84) were subjected to continuous dynamic mechanical loading (low strain: ±2% or high strain: ±7.5% at 0.5Hz) for up to 15h or maintained unloaded. TGF-ß activation was measured in these samples over time while accounting for the active TGF-ß that remains bound to the cartilage ECM. Results indicate that TGF-ß1 is present in cartilage at high levels (68.5±20.6ng/mL) and resides predominantly in the latent form (>98% of total). Under dynamic loading, active TGF-ß1 levels did not statistically increase from the initial value nor the corresponding unloaded control values for any test, indicating that physiologic dynamic compression of cartilage is unable to directly activate ECM-bound latent TGF-ß via purely mechanical pathways and leading us to reject the hypothesis of this study. These results suggest that deep zone articular chondrocytes must alternatively obtain access to active TGF-ß through chemical-mediated activation and further suggest that mechanical deformation is unlikely to directly activate the ECM-bound latent TGF-ß of various other tissues, such as muscle, ligament, and tendon.

Accumulation of exogenous activated TGF-ß in the superficial zone of articular cartilage.

It was recently demonstrated that mechanical shearing of synovial fluid (SF), induced during joint motion, rapidly activates latent transforming growth factor ß (TGF-ß). This discovery raised the possibility of a physiological process consisting of latent TGF-ß supply to SF, activation via shearing, and transport of TGF-ß into the cartilage matrix. Therefore, the two primary objectives of this investigation were to characterize the secretion rate of latent TGF-ß into SF, and the transport of active TGF-ß across the articular surface and into the cartilage layer. Experiments on tissue explants demonstrate that high levels of latent TGF-ß1 are secreted from both the synovium and all three articular cartilage zones (superficial, middle, and deep), suggesting that these tissues are capable of continuously replenishing latent TGF-ß to SF. Furthermore, upon exposure of cartilage to active TGF-ß1, the peptide accumulates in the superficial zone (SZ) due to the presence of an overwhelming concentration of nonspecific TGF-ß binding sites in the extracellular matrix. Although this response leads to high levels of active TGF-ß in the SZ, the active peptide is unable to penetrate deeper into the middle and deep zones of cartilage. These results provide strong evidence for a sequential physiologic mechanism through which SZ chondrocytes gain access to active TGF-ß: the synovium and articular cartilage secrete latent TGF-ß into the SF and, upon activation, TGF-ß transports back into the cartilage layer, binding exclusively to the SZ.

Toward engineering a biological joint replacement.

Osteoarthritis is a major cause of disability and pain for patients in the United States. Treatments for this degenerative disease represent a significant challenge considering the poor regenerative capacity of adult articular cartilage. Tissue-engineering techniques have advanced over the last two decades such that cartilage-like tissue can be cultivated in the laboratory for implantation. Even so, major challenges remain for creating fully functional tissue. This review article overviews some of these challenges, including overcoming limitations in nutrient supply to cartilage, improving in vitro collagen production, improving integration of engineered cartilage with native tissue, and exploring the potential for engineering full articular surface replacements.