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.

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.

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.

Physiologic Doses of Transforming Growth Factor-ß Improve the Composition of Engineered Articular Cartilage.

Conventionally, for cartilage tissue engineering applications, transforming growth factor beta (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 (∅2 × 2 mm or ∅3 × 2 mm) 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 (∅2 × 2 mm or ∅3 × 2 mm) and conventional-size constructs (∅4-∅6 mm × 2 mm) were subjected to a range of physiologic (0.1, 0.3, 1 ng/mL) and supraphysiologic (3, 10 ng/mL) TGF-ß doses. At day 56, the physiologic 0.3 ng/mL dose yielded reduced-size constructs with native cartilage-matched Young's modulus (EY) (630 ± 58 kPa) and sulfated glycosaminoglycan (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 10 ng/mL TGF-ß dose. Furthermore, reduced-size constructs exposed to the 0.3 ng/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 10 ng/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.

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.

Label-free 3D molecular imaging of living tissues using Raman spectral projection tomography.

The ability to image tissues in three dimensions (3D) with label-free molecular contrast at the mesoscale would be a valuable capability in biology and biomedicine. Here, we introduce Raman spectral projection tomography (RSPT) for volumetric molecular imaging with optical sub-millimeter spatial resolution. We have developed a RSPT imaging instrument capable of providing 3D molecular contrast in transparent and semi-transparent samples. We also created a computational pipeline for multivariate reconstruction to extract label-free spatial molecular information from Raman projection data. Using these tools, we demonstrate imaging and visualization of phantoms of various complex shapes with label-free molecular contrast. Finally, we apply RSPT as a tool for imaging of molecular gradients and extracellular matrix heterogeneities in fixed and living tissue-engineered constructs and explanted native cartilage tissues. We show that there exists a favorable balance wherein employing Raman spectroscopy, with its advantages in live cell imaging and label-free molecular contrast, outweighs the reduction in imaging resolution and blurring caused by diffuse photon propagation. Thus, RSPT imaging opens new possibilities for label-free molecular monitoring of tissues.

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.

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.

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.

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.

Finite element implementation of mechanochemical phenomena in neutral deformable porous media under finite deformation.

Biological soft tissues and cells may be subjected to mechanical as well as chemical (osmotic) loading under their natural physiological environment or various experimental conditions. The interaction of mechanical and chemical effects may be very significant under some of these conditions, yet the highly nonlinear nature of the set of governing equations describing these mechanisms poses a challenge for the modeling of such phenomena. This study formulated and implemented a finite element algorithm for analyzing mechanochemical events in neutral deformable porous media under finite deformation. The algorithm employed the framework of mixture theory to model the porous permeable solid matrix and interstitial fluid, where the fluid consists of a mixture of solvent and solute. A special emphasis was placed on solute-solid matrix interactions, such as solute exclusion from a fraction of the matrix pore space (solubility) and frictional momentum exchange that produces solute hindrance and pumping under certain dynamic loading conditions. The finite element formulation implemented full coupling of mechanical and chemical effects, providing a framework where material properties and response functions may depend on solid matrix strain as well as solute concentration. The implementation was validated using selected canonical problems for which analytical or alternative numerical solutions exist. This finite element code includes a number of unique features that enhance the modeling of mechanochemical phenomena in biological tissues. The code is available in the public domain, open source finite element program FEBio (http:∕∕mrl.sci.utah.edu∕software).

Influence of the partitioning of osmolytes by the cytoplasm on the passive response of cells to osmotic loading.

Due to the dense organization of organelles, cytoskeletal elements, and protein complexes that make up the intracellular environment, it is likely that membrane-permeant solutes may be excluded from a fraction of the interstitial space of the cytoplasm via steric restrictions, electrostatic interactions, and other long-range intermolecular forces. This study investigates the hypothesis that the intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery in response to hyperosmotic loading, based on prior theoretical and biomimetic experimental studies. Osmotic loading experiments are performed on immature bovine chondrocytes using culture conditions where regulatory volume responses are shown to be insignificant. Osmotic loading with membrane-permeant glycerol (92 Da) and urea (60 Da) are observed to produce partial volume recoveries consistent with the proposed hypothesis, whereas loading with 1,2-propanediol (76 Da) produces complete volume recovery. Combining these experimental results with the previous theoretical framework produces a measure for the intracellular partition coefficient of each of these solutes. At 1000 mOsm, 1,2-propanediol is the only osmolyte to yield a partition coefficient not statistically different from unity, kappa(p)(i) = 1.00 +/- 0.02. For glycerol, the partition coefficient increases with osmolarity from kappa(p)(i) = 0.48 +/- 0.19 at 200 mOsm to kappa(p)(i) = 0.80 +/- 0.07 at 1000 mOsm; urea exhibits no such dependence, with an average value of kappa(p)(i) = 0.87 +/- 0.07 for all osmolarities from 200 to 1000 mOsm. The finding that intracellular partitioning of membrane-permeant solutes manifests itself as a partial volume recovery under osmotic loading offers a simple method for characterizing the partition coefficient. These measurements suggest that significant partitioning may occur even for small membrane-permeant osmolytes. Furthermore, a positive correlation is observed, suggesting that a solute's cytoplasmic partition coefficient increases with increasing hydrophobicity.

Effect of dynamic loading on the transport of solutes into agarose hydrogels.

In functional tissue engineering, the application of dynamic loading has been shown to improve the mechanical properties of chondrocyte-seeded agarose hydrogels relative to unloaded free swelling controls. The goal of this study is to determine the effect of dynamic loading on the transport of nutrients in tissue-engineered constructs. To eliminate confounding effects, such as nutrient consumption in cell-laden disks, this study examines the response of solute transport due to loading using a model system of acellular agarose disks and dextran in phosphate-buffered saline (3 and 70 kDa). An examination of the passive diffusion response of dextran in agarose confirms the applicability of Fick's law of diffusion in describing the behavior of dextran. Under static loading, the application of compressive strain decreased the total interstitial volume available for the 70 kDa dextran, compared to free swelling. Dynamic loading significantly enhanced the rate of solute uptake into agarose disks, relative to static loading. Moreover, the steady-state concentration under dynamic loading was found to be significantly greater than under static loading, for larger-molecular-mass dextran (70 kDa). This experimental finding confirms recent theoretical predictions that mechanical pumping of a porous tissue may actively transport solutes into the disk against their concentration gradient. The results of this study support the hypothesis that the application of dynamic loading in the presence of growth factors of large molecular weight may result in both a mechanically and chemically stimulating environment for tissue growth.

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.

Dynamic loading of deformable porous media can induce active solute transport.

Active solute transport mediated by molecular motors across porous membranes is a well-recognized mechanism for transport across the cell membrane. In contrast, active transport mediated by mechanical loading of porous media is a non-intuitive mechanism that has only been predicted recently from theory, but not yet observed experimentally. This study uses agarose hydrogel and dextran molecules as a model experimental system to explore this mechanism. Results show that dynamic loading can enhance the uptake of dextran by a factor greater than 15 over passive diffusion, for certain combinations of gel concentration and dextran molecular weight. Upon cessation of loading, the concentration reverts back to that achieved under passive diffusion. Thus, active solute transport in porous media can indeed be mediated by cyclical mechanical loading.

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.

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.

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.

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.