Investigating the molecular control of deer antler extract on articular cartilage.

BACKGROUND: Deer antler is considered as a precious traditional Chinese medicinal material and has been widely used to reinforce kidney's yang, nourish essence, and strengthen bone function. The most prominent bioactive components in deer antler are water-soluble proteins that play potential roles in bone formation and repair. The aim of this study was to explore the molecular control and therapeutic targets of deer antler extract (DAE) on articular cartilage. METHODS: DAE was prepared as previously described. All rats were randomly divided into Blank group and DAE group (10 rats per group) after 7-day adaptive feeding. The rats in DAE group were orally administrated with DAE at a dose of 0.2 g/kg per day for 3 weeks, and the rats in Blank group were fed with drinking water. Total RNA was isolated from the articular cartilage of knee joints. RNA sequencing (RNA-seq) experiment combined with quantitative real-time polymerase chain reaction (qRT-PCR) verification assay was carried out to explore the molecular control and therapeutic targets of DAE on articular cartilage. RESULTS: We demonstrated that DAE significantly increased the expression levels of functional genes involved in cartilage formation, growth, and repair and decreased the expression levels of susceptibility genes involved in the pathophysiology of osteoarthritis. CONCLUSIONS: DAE might serve as a candidate supplement for maintaining cartilage homeostasis and preventing cartilage degeneration and inflammation. These effects were possibly achieved by accelerating the expression of functional genes involved in chondrocyte commitment, survival, proliferation, and differentiation and suppressing the expression of susceptibility genes involved in the pathophysiology of osteoarthritis. Thus, our findings will contribute towards deepening the knowledge about the molecular control and therapeutic targets of DAE on the treatment of cartilage-related diseases.

A Novel Multiplex Based Platform for Osteoarthritis Drug Candidate Evaluation.

Osteoarthritis (OA) is characterized by irreversible cartilage degradation with very limited therapeutic interventions. Drug candidates targeted at prototypic players had limited success until now and systems based approaches might be necessary. Consequently, drug evaluation platforms should consider the biological complexity looking beyond well-known contributors of OA. In this study an ex vivo model of cartilage degradation, combined with measuring releases of 27 proteins, was utilized to study 9 drug candidates. After an initial single drug evaluation step the 3 most promising compounds were selected and employed in an exhaustive combinatorial experiment. The resulting most and least promising treatment candidates were selected and validated in an independent study. This included estimation of mechanical properties via finite element modelling (FEM) and quantification of cartilage degradation as glycosaminoglycan (GAG) release. The most promising candidate showed increase of Young's modulus, decrease of hydraulic permeability and decrease of GAG release. The least promising candidate exhibited the opposite behaviour. The study shows the potential of a novel drug evaluation platform in identifying treatments that might reduce cartilage degradation. It also demonstrates the promise of exhaustive combination experiments and a connection between chondrocyte responses at the molecular level with changes of biomechanical properties at the tissue level.

Pulsed Electromagnetic Field Versus Whole Body Vibration on Cartilage and Subchondral Trabecular Bone in Mice With Knee Osteoarthritis.

Pulsed electromagnetic field (PEMF) and whole body vibration (WBV) interventions are expected to be important strategies for management of osteoarthritis (OA). The aim of the study was to investigate the comparative effectiveness of PEMF versus WBV on cartilage and subchondral trabecular bone in mice with knee OA (KOA) induced by surgical destabilization of the medial meniscus (DMM). Forty 12-week-old male C57/BL mice were randomly divided into four groups (n = 10): Control, OA, PEMF, and WBV. OA was induced (OA, PEMF, and WBV groups) by surgical DMM of right knee joint. Mice in PEMF group received 1 h/day PEMF exposure with 75 Hz, 1.6 mT for 4 weeks, and the WBV group was exposed to WBV for 20 min/day with 5 Hz, 4 mm, 0.3 g peak acceleration for 4 weeks. Micro-computed tomography (micro-CT), histology, and immunohistochemistry analyses were performed to evaluate the changes in cartilage and microstructure of trabecular bone. The bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) increased, and bone surface/bone volume (BS/BV) decreased by micro-CT analysis in PEMF and WBV groups. The Osteoarthritis Research Society International (OARSI) scores in PEMF and WBV groups were significantly lower than in the OA group. Immunohistochemical results showed that PEMF and WBV promoted expressions of Aggrecan, and inhibited expressions of IL-1ß, ADAMTS4, and MMP13. Superior results are seen in PEMF group compared with WBV group. Both PEMF and WBV were effective, could delay cartilage degeneration and preserve subchondral trabecular bone microarchitecture, and PEMF was found to be superior to WBV. Bioelectromagnetics. 2020;41:298-307 © 2020 Bioelectromagnetics Society.

Glucose enhances aggrecan expression in chondrocytes via the PKCα/p38-miR141-3p signaling pathway.

Aggrecan is a high molecular weight proteoglycan that plays a critical role in cartilage structure and the function of joints, providing intervertebral disc and cartilage with the ability to resist compressive loads. Aggrecan degradation in articular cartilage is a significant event in early-stage osteoarthritis (OA). Currently, no effective treatment exists for OA other than pain relief. Dextrose (D-glucose) prolotherapy has shown promising activity in the treatment of different musculoskeletal disorders, including OA. However, little is known about the molecular mechanism of the glucose effect in OA and on the regulation of chondrogenesis. We show for the first time that glucose upregulates aggrecan expression and subsequent chondrogenesis in ATDC5 cells. Moreover, we found that glucose-induced aggrecan expression is mediated through the protein kinase Cα (PKCα)- and p38-dependent pathway. As demonstrated by microRNA (miR) and luciferase analyses, the glucose-induced PKCα/p38 signaling axis is responsible for downregulating miR141-3p which targets to the 3'untranslated region of aggrecan. In summary, we show that glucose enhances chondrogenesis by upregulating aggrecan expression via the PKCα-p38-miR141-3p signaling pathway. This result provides new insights into the mechanism of glucose on chondrogenesis.

Interleukin-1ß and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel.

The acute-phase proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1ß and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1ß and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1ß and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1ß and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1ß and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca2+ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1ß and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.

Nanostructure in the trabecular bone of postmenopausal women: Mechanical and chemical analysis.

The possibility of diagnosis and prediction of multiple disorders in trabecular bone through nano-biomechanics and chemical analysis are summarized. Improvements to the understating of the compositional contributors of bone mineral and organic components to mechanical competence are crucial. Viscoelastic properties and Raman characterization have been used to evaluate possible alterations of the trabecular bone associated with aging, disease, or injury. In this study, the trabecular bone of postmenopausal women has been analyzed throughout. (a) Nanomechanical characterization, by using nano-DMA: complex modulus, tan δ, loss modulus (E'), and storage modulus (E'); and (b) Raman analysis: relative presence of minerals, carbonate-to-phosphate ratio (both from the mineral components), the crosslinking and nature/secondary structure of collagen (both from the organic components). Complementary nano-morphological studies were done assessing roughness (SRa) and collagen fibrils width, on this trabecular bone. A general idea of the behavior of the viscoelastic performance can be obtained by the Tan δ (E″/E'), that achieved 0.98GPa of damping. 249nm and 0.898µm of SRa roughness and fibrils width were obtained, respectively. The relative presence of minerals, the carbonate-to-phosphate ratio, the crosslinking and the nature/secondary structure of collagen, between 700 and 1700cm-1, were also obtained, in order to propose a study protocol for trabecular bone characterization.

Developing a Customized Perfusion Bioreactor Prototype with Controlled Positional Variability in Oxygen Partial Pressure for Bone and Cartilage Tissue Engineering.

Skeletal development is a multistep process that involves the complex interplay of multiple cell types at different stages of development. Besides biochemical and physical cues, oxygen tension also plays a pivotal role in influencing cell fate during skeletal development. At physiological conditions, bone cells generally reside in a relatively oxygenated environment whereas chondrocytes reside in a hypoxic environment. However, it is technically challenging to achieve such defined, yet diverse oxygen distribution on traditional in vitro cultivation platforms. Instead, engineered osteochondral constructs are commonly cultivated in a homogeneous, stable environment. In this study, we describe a customized perfusion bioreactor having stable positional variability in oxygen tension at defined regions. Further, engineered collagen constructs were coaxed into adopting the shape and dimensions of defined cultivation platforms that were precasted in 1.5% agarose bedding. After cultivating murine embryonic stem cells that were embedded in collagen constructs for 50 days, mineralized constructs of specific dimensions and a stable structural integrity were achieved. The end-products, specifically constructs cultivated without chondroitin sulfate A (CSA), showed a significant increase in mechanical stiffness compared with their initial gel-like constructs. More importantly, the localization of osteochondral cell types was specific and corresponded to the oxygen tension gradient generated in the bioreactor. In addition, CSA in complementary with low oxygen tension was also found to be a potent inducer of chondrogenesis in this system. In summary, we have demonstrated a customized perfusion bioreactor prototype that is capable of generating a more dynamic, yet specific cultivation environment that could support propagation of multiple osteochondral lineages within a single engineered construct in vitro. Our system opens up new possibilities for in vitro research on human skeletal development.

Acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model.

OBJECTIVE: The overall aim of this study was to evaluate how supplementation of chondrocyte media with recombinant acid ceramidase (rhAC) influenced cartilage repair in a rat osteochondral defect model. METHODS: Primary chondrocytes were grown as monolayers in polystyrene culture dishes with and without rhAC (added once at the time of cell plating) for 7 days, and then seeded onto Bio-Gide® collagen scaffolds and grown for an additional 3 days. The scaffolds were then introduced into osteochondral defects created in Sprague-Dawley rat trochlea by a microdrilling procedure. Analysis was performed 6 weeks post-surgery macroscopically, by micro-CT, histologically, and by immunohistochemistry. RESULTS: Treatment with rhAC led to increased cell numbers and glycosaminoglycan (GAG) production (∼2 and 3-fold, respectively) following 7 days of expansion in vitro. Gene expression of collagen 2, aggrecan and Sox-9 also was significantly elevated. After seeding onto Bio-Gide®, more rhAC treated cells were evident within 4 h. At 6 weeks post-surgery, defects containing rhAC-treated cells exhibited more soft tissue formation at the articular surface, as evidenced by microCT, as well as histological evidence of enhanced cartilage repair. Notably, collagen 2 immunostaining revealed greater surface expression in animals receiving rhAC treated cells as well. Collagen 10 staining was not enhanced. CONCLUSION: The results further demonstrate the positive effects of rhAC treatment on chondrocyte growth and phenotype in vitro, and reveal for the first time the in vivo effects of the treated cells on cartilage repair.

Glucan HBP-A increase type II collagen expression of chondrocytes in vitro and tissue engineered cartilage in vivo.

OBJECTIVE: Although chondroprotective activities have been documented for polysaccharides, the potential target of different polysaccharide may differ. The study was aimed to explore the effect of glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo, especially on the expression of type II collagen. METHODS: Chondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type II collagen. Chondrocyte viability was assessed after being treated with HBP-A in different concentrations. Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope (SEM). The constructs were treated with HBP-A and then injected to nude mice subcutaneously. Six weeks after transplantation, the specimens were observed through transmission electron microscopy (TEM). The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTs-5), aggrecan and type II collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction (PCR). The expression of type II collagen and matrix metalloproteinases-3 (MMP-3) in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: MMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration. In morphological study, there were significant appearance of collagen in those constructs treated by HBP-A. Accordingly, in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs, the expression of type II collagen was increased significantly in HBP-A group when compared with control group (P<0.001). CONCLUSIONS: The study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3, ADAMTs-5, and increasing of type II collagen expression.

Supplementation with platelet-rich plasma improves the in vitro formation of tissue-engineered cartilage with enhanced mechanical properties.

PURPOSE: This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and biomechanical properties of tissue-engineered cartilage. METHODS: Chondrocytes isolated from bovine metacarpal-phalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]). Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from a mature cow. After 2 weeks, samples (n = 8) were analyzed histologically, biochemically, and biomechanically. Data were analyzed using the Wilcoxon test (significance, P < .05). RESULTS: Chondrocytes cultured in 20% PRP formed thicker cartilage tissue (1.6 ± 0.2 mm) than did cells grown in 20% FBS (0.7 ± 0.008 mm; P = .002) and 20% PPP (0.8 ± 0.2 mm; P = .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1 ± 3.6 kPa versus 15.6 ± 1.5 kPa (P = .0002) for 20% PPP and 20.4 ± 3.5 kPa (P = .007) for 20% FBS. Glycosaminoglycan (GAG) content was increased in tissues formed in 20% PRP (176 ± 18.8 µg GAG/mg) compared with those grown in 20% FBS (112 ± 10.6 µg GAG/mg; P = .01) or 20% PPP (131.5 ± 14.8 µg GAG/mg; P = .11). Hydroxyproline content was similar whether the media was supplemented with 20% PRP (8.7 ± 0.9 µg/mg), 20% FBS (7.6 ± 0.9 µg/mg; P = .37), or 20% PPP (6.4 ± 1 µg/mg; P = .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9 ± 3.5 µg/mg), 20% FBS (9.3 ± 2.5 µg/mg; P = .99), or 20% PPP (7.2 ± 1.3 µg/mg; P = .78). Immunostained samples showed prevalence of type II collagen in tissues formed in the presence of 20% PRP. CONCLUSIONS: The presence of PRP in the culture media enhances the in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype. CLINICAL RELEVANCE: Understanding the in vitro effects of PRP on tissue-engineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.

A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage.

The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross-links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross-links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self-assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2-factor, full-factorial study design. Following a 6-wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross-links in all copper sulfate-containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10-fold increase in PYR content over controls. This increase in PYR cross-links manifested in a 3.3-fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross-linking enhancement.

Hyperbaric oxygen treatment prevents nitric oxide-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70.

Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia-induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on (1) interleukin-1ß (IL-1ß)-induced NO production and apoptosis of rabbit chondrocytes and (2) healing of articular cartilage defects. For the in vitro study, RT-PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL-1ß-treated chondrocytes. To clarify that the HSP70 was necessary for anti-iNOS and anti-apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO-induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70.

Scientific evidence and rationale for the development of curcumin and resveratrol as nutraceutricals for joint health.

Interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) are key cytokines that drive the production of inflammatory mediators and matrix-degrading enzymes in osteoarthritis (OA). These proinflammatory cytokines bind to their respective cell surface receptors and activate inflammatory signaling pathways culminating with the activation of nuclear factor κB (NF-κB), a transcription factor that can be triggered by a host of stress-related stimuli including, excessive mechanical stress and ECM degradation products. Once activated, NF-κB regulates the expression of many cytokines, chemokines, adhesion molecules, inflammatory mediators, and several matrix-degrading enzymes. Therefore, proinflammatory cytokines, their cell surface receptors, NF-κB and downstream signaling pathways are therapeutic targets in OA. This paper critically reviews the recent literature and outlines the potential prophylactic properties of plant-derived phytochemicals such as curcumin and resveratrol for targeting NF-κB signaling and inflammation in OA to determine whether these phytochemicals can be used as functional foods.

Osteoarthritis and rheumatoid arthritis: conservative therapeutic management.

Hand therapists need to understand the basic science behind the therapy they carry out and the current evidence to make the best treatment decisions. The purpose of this article was to review current conservative therapeutic management of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) of the hand. Treatment interventions such as orthotics, exercise, joint protection, modalities, and adaptive equipment are discussed from a basic science and evidence-based practice perspective.

[Novel small compound in combination with cell-sheet technology for articular cartilage regeneration].

Aiming at regeneration of articular cartilage, we have established stable lines of mouse chondrogenic ATDC5 cells expressing green fluorescent protein under the control of type II collagen promoter fused with four repeats of a SOX9 enhancer (COL2A1-GFP) , as a monitoring system for chondrogenic differentiation. A screening of natural and synthetic compound libraries using the system identified some novel compounds. Combined with cell-sheet technology, a novel small compound was applied to the treatment of full-thickness knee cartilage defects in murine and canine models.

Harnessing the purinergic receptor pathway to develop functional engineered cartilage constructs.

OBJECTIVE: Mechanical stimulation is a widely used method to enhance the formation and properties of tissue-engineered cartilage. While this approach can be highly successful, it may be more efficient and effective to harness the known underlying mechanotransduction pathways responsible. With this aim, the purpose of this study was to assess the effect of directly stimulating the purinergic receptor pathway through exogenous adenosine 5'-triphosphate (ATP) in absence of externally applied forces. METHODS: Isolated bovine articular chondrocytes were seeded in high density, 3D culture and supplemented with varying doses of ATP for up to 4 weeks. The effects on biosynthesis, extracellular matrix accumulation and mechanical properties were then evaluated. Experiments were also conducted to assess whether exogenous ATP elicited any undesirable effects, such as: inflammatory mediator release, matrix turn-over and mineralization. RESULTS: Supplementation with ATP had a profound effect on the growth and maturation of the developed tissue. Exogenous ATP (62.5-250 microM) increased biosynthesis by 80-120%, and when stimulated for a period of 4 weeks resulted in increased matrix accumulation (80% increase in collagen and 60% increase in proteoglycans) and improved mechanical properties (6.5-fold increase in indentation modulus). While exogenous ATP did not stimulate the release of inflammatory mediators or induce mineralization, high doses of ATP (250 microM) elicited a 2-fold increase in matrix metalloproteinase-13 expression suggesting the emergence of a catabolic response. CONCLUSIONS: Harnessing the ATP-purinergic receptor pathway is a highly effective approach to improve tissue formation and impart functional mechanical properties. However, the dose of ATP needs to be controlled as not to elicit a catabolic response.

Chondroitin sulphate: an effective joint lubricant?

OBJECTIVE: The effect of chondroitin sulphate (CS) treatment on the friction and deformation characteristics of native and glycosaminoglycan (GAG) deficient articular cartilage was investigated. METHODS: Friction tests were conducted at 0.4 MPa load, in Static and Dynamic models, to determine the startup coefficient of friction (COF) and dynamic COF, respectively. Native cartilage: For each cartilage pin and plate couple, the COF was determined under three consecutive tests - (1) baseline COF in PBS (2) COF in CS lubricant and (3) COF again in PBS, after 24h CS treatment. GAG deficient cartilage: For each cartilage pin and plate couple, the baseline COF was determined in PBS initially and again following enzymatic treatment to deplete GAGs. The specimens were then soaked in CS solution for 24h and the COF determined again in PBS. In a similar manner, friction tests were replaced with indentation tests to study the deformation of the tissue. RESULTS: CS at 50mg/ml significantly lowered the startup COF of native cartilage both as a lubricant and a treatment solution. In the dynamic model, where the fluid load support is sustained at a high level, CS failed to have any effect on the COF of native cartilage. GAG depletion raised the friction and deformation levels of cartilage, and subsequent CS treatment failed to lower them to their native levels. CONCLUSION: CS proved to be an effective lubricant for cartilage under mixed-mode lubrication conditions. However, supplemental CS that diffused into the specimens had no influence on the fluid load support of cartilage.

Influence of chondroitin sulfate on the biochemical, mechanical and frictional properties of cartilage explants in long-term culture.

A recent study [Basalo et al., 2007. Chondroitin sulfate reduces the friction coefficient of articular cartilage. J. Biomech. 40(8), 1847-1854] has shown that the friction coefficient of bovine articular cartilage is reduced significantly by the supplementation of chondroitin sulfate (CS) at a concentration of 100mg/ml. This result suggests that intra-articular injection of CS may be used as a prophylactic treatment against the progression of osteoarthritis. The objective of this study was to test the hypothesis that long-term culture of cartilage explants in CS produces no adverse mechanical, biochemical, or cytotoxic effects, while reducing the friction coefficient relative to the control group. Long-term cultures of live bovine articular cartilage explants were performed with incubation in media containing CS of three different concentrations (0, 10 and 100mg/ml). Frictional tests (cartilage-on-glass) were performed under constant stress (0.5MPa) for 3600s and the time-dependent friction coefficient was measured. Samples incubated in a 100mg/ml of CS solution exhibited a significantly lower equilibrium friction coefficient than the control (0.05+/-0.01 vs. 0.18+/-0.02 on Day 0, 0.04+/-0.01 vs. 0.14+/-0.04 on Day 7 and 0.04+/-0.01 vs. 0.15+/-0.06 on Day 14). Samples incubated in 10mg/ml of CS did not exhibit any significant decrease in the friction coefficient. Cell viability and DNA content were maintained in all groups. However, after 28 days of culture, the Young's modulus and glycosaminoglycan content of explants incubated in 100mg/ml of CS decreased to 5% and 40% of their initial levels, respectively. Based on this adverse outcome the hypothesis of this study is rejected, dampening our enthusiasm for the use of intra-articular CS injections as a prophylactic treatment in osteoarthritis.

PGE2 signal via EP2 receptors evoked by a selective agonist enhances regeneration of injured articular cartilage.

OBJECTIVE: The effect of the prostaglandin E2 (PGE2) signal through prostaglandin E receptor 2 (EP2) receptors on the repair of injured articular cartilage was investigated using a selective agonist for EP2. METHODS: Chondral and osteochondral defects were prepared on the rabbit femoral concave in both knee joints, and gelatin containing polylactic-co-glycolic acid microspheres conjugated with or without the EP2 agonist was placed nearby. Animals were sacrificed at 4 or 12 weeks post-operation, and regenerated cartilage tissues and subchondral structure remodeling were evaluated by histological scoring. The quality of regenerated tissues was also evaluated by the immunohistochemical staining of EP2, type II collagen, and proliferating cell nuclear antigen (PCNA). As an evaluation of side effects, the inflammatory reaction of the synovial membrane was analyzed based on histology and the mRNA expression of matrix metalloproteinase3 (MMP3), tissue inhibitor of metalloproteinase 3 (TIMP3), and interleukin-1 beta (IL-1 beta). Also, the activity of MMP3 and the amount of tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein in joint fluid were measured. RESULTS: In both models, the EP2 agonist enhanced the regeneration of the type II collagen-positive tissues containing EP2- and PCNA-positive chondrocytes, and the histological scale of regenerated tissue and subchondral bone was better than that of on the control side, particularly at 12 weeks post-operation. No inflammatory reaction in the synovial membrane was observed, and no induction of pro-inflammatory cytokines was found in joint fluid. CONCLUSION: Selective stimulation of the PGE2 signal through EP2 receptors by a specific agonist promoted regeneration of cartilage tissues with a physiological osteochondral boundary, suggesting the potential usefulness of this small molecule for the treatment of injured articular cartilages.

Chondrocyte death in mechanically injured articular cartilage--the influence of extracellular calcium.

Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium-free media (approximately 0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium-rich media (2-20 mM, ANOVA at 2.5 h, p = 0.002). In calcium-rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium-free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium-free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium-free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.