P188 reduces cell death and IGF-I reduces GAG release following single-impact loading of articular cartilage

Prior joint injury predisposes an individual to developing post-traumatic osteoarthritis, for which there is presently no disease modifying treatment. In this condition, articular cartilage degenerates due to cell death and matrix breakdown, resulting in tissue with diminished biomechanical function. P188, a nonionic surfactant, and the growth factor IGF-I have been shown to decrease cell death. Additionally, IGF-I is known to have beneficial effects on cartilage matrix. The objective of this study was to determine the efficacy of P188, IGF-I, and their combination following articular cartilage impact injury with two energy levels, 1.1 J ("low") and 2.8 J ("high"), at 24 h and 1 week. Bovine articular cartilage with attached underlying bone was impacted at the low or high level. Impact sites were explanted and examined immediately, or cultured for 24 h or 1 week in serum-free media supplemented with P188 (8 mgml), IGF-I (100 ngml), or their combination. Gross morphology, cell viability, GAG release to the media, and tissue mechanical properties were assessed. Immediately postimpact, high level impacted tissue had significantly increased gross morphology scores, indicating tissue damage, which were maintained over 1 week. Gross scores following low impact were initially similar to nonimpacted controls, but, at 24 h and 1 week, low impact gross scores significantly increased compared to nonimpacted controls. Additionally, at 24 h, high impact resulted in increased cell death, and both low and high impacts had increased GAG release compared to nonimpacted controls. Furthermore, high impact caused decreased tissue stiffness at 24 h that appeared to worsen over 1 week, evident by the percent decrease from nonimpacted controls increasing from 16% to 26%. No treatment type studied mitigated this loss. The combination did not perform better than either individual treatment; however, following low impact at 1 week, P188 reduced cell death by 75% compared to no treatment and IGF-I decreased GAG release from the tissue by 49%. In conclusion, high impact resulted in immediate tissue changes that worsened over 1 week. Though not causing immediate changes, low impact also resulted in tissue degeneration evident by 24 h. No treatment studied was effective at 24 h, but by 1 week P188 and IGF-I ameliorated established detrimental changes occurring in articular cartilage postimpact. However, further work is needed to optimize treatment strategies to prevent and/or reverse cell death and matrix destruction in a way that maintains tissue mechanical properties, and hence its functionality.     

Temporal assessment of ribose treatment on self-assembled articular cartilage constructs

Articular cartilage cannot repair itself in response to degradation from injury or osteoarthritis. As such, there is a substantial clinical need for replacements of damaged cartilage. Tissue engineering aims to fulfill this need by developing replacement tissues in vitro. A major goal of cartilage tissue engineering is to produce tissues with robust biochemical and biomechanical properties. One technique that has been proposed to improve these properties in engineered tissue is the use of non-enzymatic glycation to induce collagen crosslinking, an attractive solution that may avoid the risks of cytotoxicity posed by conventional crosslinking agents such as glutaraldehyde. The objectives of this study were (1) to determine whether continuous application of ribose would enhance biochemical and biomechanical properties of self-assembled articular cartilage constructs, and (2) to identify an optimal time window for continuous ribose treatment. Self-assembled constructs were grown for 4 weeks using a previously established method and were subjected to continuous 7-day treatment with 30 mM ribose during culture weeks 1, 2, 3, or 4, or for the entire 4-week culture. Control constructs were grown in parallel, and all groups were evaluated for gross morphology, histology, cellularity, collagen and sulfated glycosaminoglycan (GAG) content, and compressive and tensile mechanical properties. Compared to control constructs, it was found that treatment with ribose during week 2 and for the entire duration of culture resulted in significant 62% and 40% increases in compressive stiffness, respectively; significant 66% and 44% increases in tensile stiffness; and significant 50% and 126% increases in tensile strength. Similar statistically significant trends were observed for collagen and GAG. In contrast, constructs treated with ribose during week 1 had poorer biochemical and biomechanical properties, although they were significantly larger and more cellular than all other groups. We conclude that non-enzymatic glycation with ribose is an effective method for improving tissue engineered cartilage and that specific temporal intervention windows exist to achieve optimal functional properties.     

Differential regulation of immature articular cartilage compressive moduli and Poisson’s ratios by in vitro stimulation with IGF-1 and TGF-β1

Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-β1). This study tested the hypothesis that IGF-1 and TGF-β1 regulate immature cartilage compressive moduli and Poisson’s ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-β1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson’s ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-β1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-β1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson’s ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-β1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering.     

In Vivo Evaluation of a Novel Oriented Scaffold-BMSC Construct for Enhancing Full-Thickness Articular Cartilage Repair in a Rabbit Model

Tissue engineering (TE) has been proven usefulness in cartilage defect repair. For effective cartilage repair, the structural orientation of the cartilage scaffold should mimic that of native articular cartilage, as this orientation is closely linked to cartilage mechanical functions. Using thermal-induced phase separation (TIPS) technology, we have fabricated an oriented cartilage extracellular matrix (ECM)-derived scaffold with a Young''s modulus value 3 times higher than that of a random scaffold. In this study, we test the effectiveness of bone mesenchymal stem cell (BMSC)-scaffold constructs (cell-oriented and random) in repairing full-thickness articular cartilage defects in rabbits. While histological and immunohistochemical analyses revealed efficient cartilage regeneration and cartilaginous matrix secretion at 6 and 12 weeks after transplantation in both groups, the biochemical properties (levels of DNA, GAG, and collagen) and biomechanical values in the oriented scaffold group were higher than that in random group at early time points after implantation. While these differences were not evident at 24 weeks, the biochemical and biomechanical properties of the regenerated cartilage in the oriented scaffold-BMSC construct group were similar to that of native cartilage. These results demonstrate that an oriented scaffold, in combination with differentiated BMSCs can successfully repair full-thickness articular cartilage defects in rabbits, and produce cartilage enhanced biomechanical properties.     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

Matrix development in self-assembly of articular cartilage

Background

Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue.

Methodology/Principal Findings

This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks.

Conclusions/Significance

The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs.     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

Purification, cloning, and functional expression of phenylalanine aminomutase: the first committed step in Taxol side-chain biosynthesis

The biomechanical functions of articular cartilage are governed largely by the composition and density of its specialized extracellular matrix. Relationships between matrix density and functional indices such as mechanical properties or interstitial solute diffusivities have been previously explored. However, direct correlations between mechanical properties and solute transport parameters have received less attention, despite potential application of this information for cartilage functional assessment both in vivo and in vitro. The objective of this study was therefore to examine relationships among solute diffusivities, mechanical properties, and matrix density of compressed articular cartilage. Matrix density varied due to natural variation among explants and due to applied static compression. Matrix density of statically compressed cartilage explants was characterized by glycoaminoglycan (GAG) weight fraction and fluid volume fraction, while diffusion coefficients of a wide range of solutes were measured to characterize the transport environment. Explant mechanical properties were characterized by a non-linear Young's modulus (axial stress-strain ratio) and a non-linear Poisson's ratio (radial-to-axial strain ratio). Solute diffusivities were consistently correlated with Young's modulus, as well as with explant GAG weight and fluid volume fractions. Therefore, in vitro mechanical tests may provide a means of assessing transport environments in cartilage-like materials, while in vivo measurements of solute transport (for example with magnetic resonance imaging) may be a useful complement in identifying localized differences in matrix density and mechanical properties.     

Altered responsiveness to TGF-β results in reduced Papss2 expression and alterations in the biomechanical properties of mouse articular cartilage

Introduction

Previous studies have indicated that transforming growth factor β (TGF-β) signaling has a critical role in cartilage homeostasis and repair, yet the mechanisms of TGF-β''s chondroprotective effects are not known. Our objective in this study was to identify downstream targets of TGF-β that could act to maintain biochemical and biomechanical properties of cartilage.

Methods

Tibial joints from 20-week-old mice that express a dominant-negative mutation of the TGF-β type II receptor (DNIIR) were graded histologically for osteoarthritic changes and tested by indentation to evaluate their mechanical properties. To identify gene targets of TGF-β, microarray analysis was performed using bovine articular chondrocytes grown in micromass culture that were either treated with TGF-β or left untreated. Phosphoadenosine phosphosynthetase 2 (PAPSS2) was identified as a TGF-β-responsive gene. Papss2 expression is crucial for proper sulfation of cartilage matrix, and its deficiency causes skeletal defects in mice and humans that overlap with those seen in mice with mutations in TGF-β-signaling genes. Regulation of Papss2 was verified by real time RT-PCR and Western blot analyses. Alterations in sulfation of glycosaminoglycans were analyzed by critical electrolyte concentration and Alcian blue staining and immunofluorescence for chondroitin-4-sulfate, unsulfated chondroitin and the aggrecan core protein.

Results

DNIIR mutants showed reduced mechanical properties and osteoarthritis-like changes when compared to wild-type control mice. Microarray analysis identified a group of genes encoding matrix-modifying enzymes that were regulated by TGF-β. Papss2 was upregulated in bovine articular chondrocytes after treatment with TGF-β and downregulated in cartilage from DNIIR mice. Articular cartilage in DNIIR mice demonstrated reduced Alcian blue staining at critical electrolyte concentrations and reduced chondroitin-4-sulfate staining. Staining for unsulfated chondroitin sulfate was increased, whereas staining for the aggrecan core protein was comparable in DNIIR and wild-type mice.

Conclusion

TGF-β maintains biomechanical properties and regulates expression of Papss2 and sulfation of glycosaminoglycans in mouse articular cartilage.     

Age-related changes in the response of human articular cartilage to IL-1alpha and transforming growth factor-beta (TGF-beta): chondrocytes exhibit a diminished sensitivity to TGF-beta

Cartilage glycosaminoglycan (GAG) synthesis and composition, upon which its structural integrity depends, varies with age, is modified by anabolic and catabolic stimuli, and is regulated by UDP-glucuronate availability. However, how such stimuli, prototypically represented by transforming growth factor-beta1 (TGF-beta1) and IL-1alpha, modify GAG synthesis during aging of normal human articular cartilage is not known. Using explants, we show that chondroitin sulfate (CS):total GAG ratios decrease, whereas C6S:C4S ratios increase with cartilage maturation, and that chondrocytes in the cartilage mid-zone, but not the superficial or deep zones, exhibit uridine 5'-diphosphoglucose dehydrogenase (UDPGD) activity, which is also increased in mature cartilage. We also show that IL-1alpha treatment reduces both total GAG and CS synthesis, decreases C6S:C4S ratios (less C6S), but fails to modify chondrocyte UDPGD activity at all ages. On the other hand, TGF-beta1 increases total GAG synthesis in immature, but not mature, cartilage (stimulates CS but not non-CS), age-independently decreases C6S:C4S (more C4S), and increases chondrocyte UDPGD activity in a manner inversely correlated with age. Our findings show that TGF-beta1, but not IL-1alpha, modifies matrix synthesis such that its composition more closely resembles "less mature" articular cartilage. These effects of TGF-beta1, which appear to be restricted to periods of skeletal immaturity, are closely associated although not necessarily mechanistically linked with increases in chondrocyte UDPGD activity. The antianabolic effects of IL-1alpha are, on the other hand, likely to be independent of any direct modification in UDPGD activity and manifest equally in human cartilage of all ages.     

Mechanical and biochemical characterization of cartilage explants in serum-free culture

Allografts of articular cartilage are both used clinically for tissue-transplantation procedures and experimentally as model systems to study the physiological behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Chemical mediators in poorly defined culture media can stimulate cells to quickly degrade their surrounding extracellular matrix. This is particularly true of juvenile cartilage which is generally more responsive to chemical stimuli than mature tissue. By carefully modulating the culture media, however, it may be possible to preserve allograft tissue over the long-term while maintaining its original mechanical and biochemical properties. In this study juvenile bovine cartilage explants (both chondral and osteochondral) were cultured in both chemically defined medium and serum-supplemented medium for up to 6 weeks. The mechanical properties and biochemical content of explants cultured in chemically defined medium were enhanced after 2 weeks in culture and thereafter remained stable with no loss of cell viability. In contrast, the mechanical properties of explants in serum-supplemented medium were degraded by ( approximately 70%) along with a concurrent loss of biochemical content (30-40% GAG). These results suggest that long-term maintenance of allografts can be extended significantly by the use of a chemically defined medium.     

Doxycycline effects on mechanical and morphometrical properties of early- and late-stage osteoarthritic bone following anterior cruciate ligament injury.

As posttraumatic osteoarthritis (OA) progresses, the mechanical and morphometrical properties of the subchondral bone change and may be linked to damage of the articular cartilage. Potentially to slow that progression, doxycycline was administered orally twice daily (4 mg.kg(-1).day(-1)) in skeletally mature canines after anterior cruciate ligament transection (ACLX). To test if doxycycline significantly altered the structure and function of OA bone, we tested cancellous bone mechanical properties, measured bone mineral content, and analyzed bone structure by microcomputed tomography. Our investigation focused on subchondral trabecular bone changes in the medial femoral condyle at 36 and 72 wk after ACLX. Significant mechanical changes discovered at 36 wk post-ACLX were less obvious at 72 wk in both treated and ACLX groups. Doxycycline treatment conserved bone strain energy density at 72 wk. Doxycycline had little effect on the degradation of superficial osseous tissue at 36 wk post-ACLX; by 72 wk, doxycycline in an ACLX model limited subchondral bone loss within the first 3 mm of periarticular bone with established OA. Significant bone loss occurred in the deeper trabecular bone for all groups. Substantial architectural adaptation within deeper trabecular bone accompanied changes in mechanics in early and established OA.     

Loading-induced changes in synovial fluid affect cartilage metabolism

The object of this study was to determine whether changes in the synovial fluid (SF) induced by in vivo loading can alter the metabolic activity of chondrocytes in vitro, and, if so, whether insulin-like growth factor-I (IGF-I) is responsible for this effect. Therefore, SF was collected from ponies after a period of box rest and after they had been exercised for a week. Normal, unloaded articular cartilage explants were cultured in 20% solutions of these SFs for 4 days and chondrocyte bioactivity was determined by glycosaminoglycan (GAG) turnover (i.e., the incorporation of 35SO4 into GAG and the release of GAG into the medium). Furthermore, the extent to which the bioactivity is IGF-I-dependent was determined in a cartilage explant culture in 20% SF, in the presence and absence of anti-IGF-I antibodies. In explants cultured in post-exercise SF, GAG synthesis was enhanced and GAG release was diminished when compared to cultures in pre-exercise SF. SF analysis showed that IGF-I and IGFBP-3 levels were increased in post-exercise SF. There was a positive correlation between IGF-I levels and proteoglycan synthesis, but no correlation between IGF-I levels and proteoglycan release. Addition of anti-IGF-I antibodies significantly inhibited stimulation of proteoglycan synthesis in explants cultured in SF with 40%. However, there was no difference in inhibition of proteoglycan synthesis between pre- and post-exercise SF which indicated that the relative contribution of IGF-I in the stimulating effect of SF did not change. Proteoglycan release was not influenced by the presence of anti-IGF-I antibodies. It is concluded that chondrocyte metabolic activity is at least partially regulated by changes in the SF induced by in vivo loading. Exercise altered the SF in a way that it had a favourable effect on cartilage PG content by enhancing the PG synthesis and reducing the PG breakdown. IGF-I is an important contributor to the overall stimulating effect of SF on cartilage metabolism. It is, however, unlikely that IGF-I is the only mediator in the exercise-induced increase in this stimulating effect.     

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints

The effect of dynamic mechanical shear and compression on the synthesis of human tissue‐engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA–alginate scaffolds were precultured in shaking T‐flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak‐to‐peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T‐flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3‐ and 10‐fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4‐fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue‐engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Biotechnol. Bioeng. 2012; 109:1060–1073. © 2011 Wiley Periodicals, Inc.     

Immunochemical and mechanical characterization of cartilage subtypes in rabbit.

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.     

Biomarkers affected by impact velocity and maximum strain of cartilage during injury

Osteoarthritis is one of the most common, debilitating, musculoskeletal diseases; 12% associated with traumatic injury resulting in post-traumatic osteoarthritis (PTOA). Our objective was to develop a single impact model with cartilage “injury level” defined in terms of controlled combinations of strain rate to a maximum strain (both independent of cartilage load resistance) to study their sensitivity to articular cartilage cell viability and potential PTOA biomarkers. A servo-hydraulic test machine was used to measure canine humeral head cartilage explant thickness under repeatable pressure, then subject it (except sham and controls) to a single impact having controlled constant velocity V=1 or 100 mm/s (strain rate 1.82 or 182/s) to maximum strain ε=10%, 30%, or 50%. Thereafter, explants were cultured in media for twelve days, with media changed at day 1, 2, 3, 6, 9, 12. Explant thickness was measured at day 0 (pre-injury), 6 and 12 (post-injury). Cell viability, and tissue collagen and glycosaminoglycan (GAG) were analyzed immediately post-injury and day 12. Culture media were tested for biomarkers: GAG, collagen II, chondroitin sulfate-846, nitric oxide, and prostaglandin E₂ (PGE₂). Detrimental effects on cell viability, and release of GAG and PGE₂ to the media were primarily strain-dependent, (PGE₂ being more prolonged and sensitive at lower strains). The cartilage injury model appears to be useful (possibly superior) for investigating the relationship between impact severity of injury and the onset of PTOA, specifically for discovery of biomarkers to evaluate the risk of developing clinical PTOA, and to compare effective treatments for arthritis prevention.     

A modular approach to creating large engineered cartilage surfaces

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries.     

Differential recovery of glycosaminoglycan after IL-1-induced degradation of bovine articular cartilage depends on degree of degradation

In the present study we examined cartilage matrix repair following IL-1-induced matrix depletion. Previous data indicated that, in some cases, chondrocytes can synthesize macromolecules to establish a functional extracellular matrix in response to a matrix-damaging insult or when placed in a three-dimensional environment with inadequate matrix. However, the conditions under which such 'repair' can occur are not entirely clear. Prior studies have shown that chondrocytes in trypsin-depleted young bovine articular cartilage can replenish tissue glycosaminoglycan (GAG) and that the rate of replenishment is relatively uniform throughout the tissue, suggesting that all chondrocytes have similar capacity for repair. In the present study we used the characteristic heterogeneous distribution of matrix depletion in response to IL-1 exposure in order to investigate whether the severity of depletion influenced the rate of GAG replenishment. We used the delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) method to monitor the spatial and temporal evolution of tissue GAG concentration ([GAG]). For both mild (n=4) and moderate (n=10) IL-1-induced GAG depletion, we observed partial recovery of GAG (80% and 50% of baseline values, respectively) over a 3-week recovery period. During the first 2 weeks of recovery, [GAG] increased homogeneously at 10–15 mg/ml per week. However, during the third week the regions most severely depleted following IL-1 exposure showed negligible [GAG] accumulation, whereas those regions affected the least by IL-1 demonstrated the greatest accumulation. This finding could suggest that the most severely degraded regions do not recover fully, possibly because of more severe collagen damage; this possibility requires further examination.     

A mechanical apparatus with microprocessor controlled stress profile for cyclic compression of cultured articular cartilage explants

An apparatus was designed for mechanical compression of cultured articular cartilage explants with acylindrical plain-ended loading head (diameter 2-5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa-12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4 s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.     

Nitric oxide mediates interleukin-1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage

Interleek-1beta (IL-1) is a key mediator of cartilage matrix degradation in osteoarthritis and rheumatoid arthritis. It was found that the IL-1-induced suppression of glycosaminoglycan (GAG) synthesis in rat articular cartilage occurred simultaneously with the accumulation of nitrite (a metabolite of nitric oxide (NO) in aqueous milieu) in the culture medium. NO-synthase inhibitors, L-NMMA and L-NIO, inhibited both these IL-1 effects. Dexamethasone suppressed GAG synthesis additively to IL-1, but did not alter nitrite accumulation. Three NO-donors (GEA 3175, SNAP and SIN-1) also had an inhibitory effect on cartilage GAG synthesis. Therefore, it is concluded that IL-1 induced suppression of GAG synthesis in rat articular cartilage is mediated by the production of NO.