Oxidative stress inhibits insulin-like growth factor-I induction of chondrocyte proteoglycan synthesis through differential regulation of phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK signaling pathways.

The ability of insulin-like growth factor I (IGF-I) to stimulate cartilage matrix synthesis is reduced in aged and osteoarthritic cartilage. Aging and osteoarthritis are associated with an increase in reactive oxygen species, which we hypothesized would interfere with normal IGF-I signaling. We compared IGF-I signaling in normal and osteoarthritic human articular chondrocytes and investigated the effects of oxidative stress induced by tert-butylhydroperoxide (tBHP). In normal human chondrocytes, IGF-I initiated a strong and sustained phosphorylation of IRS-1 (Tyr-612) and Akt (Ser-473) and transient ERK phosphorylation. In contrast, in osteoarthritic chondrocytes, which possessed elevated basal IRS-1 (Ser-312) and ERK phosphorylation, IGF-I failed to stimulate IRS-1 (Tyr-612) or Akt phosphorylation. In normal human chondrocytes, tBHP triggered strong IRS-1 (Ser-312 and Ser-616) and ERK phosphorylation and inhibited IGF-I-induced IRS-1 (Tyr-612) and Akt phosphorylation. Lentivirus-mediated overexpression of constitutively active (CA) Akt significantly enhanced proteoglycan synthesis, whereas both dominant negative Akt and CA MEK inhibited proteoglycan synthesis. CA Akt also promoted type II collagen and Sox9 expression, whereas tBHP treatment and CA MEK inhibited aggrecan, collagen II, and Sox9 mRNA expression. In osteoarthritic chondrocytes, the antioxidants Mn(III) tetrakis(4-benzoic acid)porphyrin and N-acetylcysteine increased the ratio of Akt to ERK phosphorylation and promoted IGF-I-mediated proteoglycan synthesis. Chemical inhibition of ERK significantly enhanced IGF-I phosphorylation of Akt and alleviated tBHP inhibition of Akt phosphorylation. These results demonstrate opposing roles for phosphatidylinositol 3-kinase-Akt and MEK-ERK in cartilage matrix synthesis and suggest that elevated levels of reactive oxygen species cause chondrocyte IGF-I resistance by altering the balance of Akt to ERK activity.

Ankle synovium-derived mesenchymal stem cells for the treatment of osteochondral lesion of the talus: a novel cell harvesting technique and clinical applications.

OBJECTIVE: To describe a novel technique for harvesting mesenchymal stem cells (MSCs) from ankle synovium and demonstrate their multipotency for chondrogenesis and osteogenesis in the treatment of a large osteochondral lesion of the talus (OLT). PATIENTS AND METHODS: MSCs were harvested under local anesthesia from a biopsy of the ankle synovium of a patient with OLT and cultured for 4 weeks. In accordance with the International Society for Cellular Therapy (ISCT) criteria, synovial-derived MSCs were analyzed for cell surface markers using flow cytometry and grown in differentiation induction medium to demonstrate multilineage differentiation potentials in vitro. The patient received an injection of synovial-derived MSCs in a collagen matrix with a fibrin sealant to augment surgery for the lesion. RESULTS: Harvesting of ankle synovium yielded a culture of 30 million spindle-shaped stem cells in 4 weeks. Cell surface marker expression of the MSCs met the ISCT standards. After 21 days, cultured cells could be differentiated into adipocytes, osteocytes and chondrocytes. The patient recovered uneventfully from surgery and showed satisfactory improvements in pain and ankle motion. CONCLUSIONS: Ankle synovium is a potential source of MSCs for the treatment of OLT. We developed a novel harvesting technique that was simple, convenient and had no complications.

Intra-individual comparison of human ankle and knee chondrocytes in vitro: relevance for talar cartilage repair.

OBJECTIVE: As compared to knee chondrocytes (KC), talar chondrocytes (TC) have superior synthetic activity and increased resistance to catabolic stimuli. We investigated whether these properties are maintained after TC are isolated and expanded in vitro. METHODS: Human TC and KC from 10 cadavers were expanded in monolayer and then cultured in pellets for 3 and 14 days or in hyaluronan meshes (Hyaff-11) for 14 and 28 days. Resulting tissues were assessed biochemically, histologically, biomechanically and by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The proteoglycan and collagen synthesis rates in the pellets were also measured following exposure to Interleukin-1 beta (IL-1 beta). RESULTS: After 14 days of pellet culture, TC and KC expressed similar levels of type I collagen (CI) and type II collagen (CII) mRNA and the resulting tissues contained comparable amounts of glycosaminoglycans (GAG) and displayed similar staining intensities for CII. Also proteoglycan and collagen synthesis were similar in TC and KC pellets, and dropped to a comparable extent in response to IL-1 beta. Following 14 days of culture in Hyaff-11, TC and KC generated tissues with similar amounts of GAG and CI and CII. After 28 days, KC deposited significantly larger fractions of GAG and CII than TC, although the trend was not reflected in the measured biomechanical properties. CONCLUSION: After isolation from their original matrices and culture expansion, TC and KC displayed similar biosynthetic activities, even in the presence of catabolic stimuli. These in vitro data suggest a possible equivalence of TC and KC as autologous cell sources for the repair of talar cartilage lesions.

Mechanical responses and integrin associated protein expression by human ankle chondrocytes.

Metabolic, biochemical and biomechanical differences between ankle and knee joint cartilage and chondrocytes including resistance to the effects of catabolic cytokines and fibronectin fragments may be relevant to differences in prevalence of OA in these joints. Although there is increasing information available on how chondrocytes from knee and hip joint cartilage recognise and respond to mechanical stimuli, knowledge of mechanotransduction in ankle joint chondrocytes is limited. This study was undertaken to (i) establish whether the response of normal ankle joint derived chondrocytes to mechanical stimulation in vitro was similar to that of normal and osteoarthritic knee joint derived chondrocytes and (ii) to investigate whether these chondrocytes showed differences in expression of integrin associated regulatory and signalling molecules. Unlike normal knee joint chondrocytes, ankle joint chondrocytes did not show an increase in relative levels of aggrecan mRNA when mechanically stimulated. No obvious change in protein tyrosine phosphorylation was seen in ankle chondrocytes subsequent to mechanical stimulation but these cells expressed elevated levels of tyrosine phosphorylated proteins at rest when compared to normal knee joint chondrocytes. Ankle joint chondrocytes showed an increase in protein kinase B phosphorylation following 1 min 0.33 Hz stimulation which was inhibited by the presence of antibodies to alpha5beta1 integrin. Ankle joint chondrocytes appeared to show significant differences in levels of the integrin-associated proteins CD98, CD147 and galectin 3, PKCgamma and differences in responses to glutamate were seen. Chondrocytes from ankle and knee joint cartilage respond differently to 0.33 Hz mechanical stimulation. This may be related to modified integrin-dependent mechanotransduction as a result of changes in expression of integrin regulatory molecules such as CD98 or differential expression and function of downstream components of the mechanotransduction pathway such as PKC or NMDA receptors.

Tissue engineered ceramic artificial joint--ex vivo osteogenic differentiation of patient mesenchymal cells on total ankle joints for treatment of osteoarthritis.

Total joint arthroplasty is the common treatment of severe cases of osteoarthritis. However, complications involving failure of the bone-prosthesis interface are significant, especially in ankle arthroplasty. To prevent this complication, we attempted a tissue engineering approach using the mesenchymal cells of the patient. We collected a small amount of fresh bone marrow cells from the patient's iliac crest and expanded the number of mesenchymal cells. We then applied the mesenchymal cells to a ceramic ankle prosthesis and cultured them to form an osteoblasts/bone matrix on the prosthesis. We used tissue engineered prostheses on three patients suffering from ankle arthritis and followed their progress for at least 2 years. Follow-up X-ray examinations revealed early radiodense appearance (bone formation) around the cell-seeded areas of the prostheses about 2 months after the operation after which a stable host bone-prosthesis interface was established. All patients showed high clinical scores after the operation and did not exhibit inflammatory reactions. These preliminary results indicate that the tissue engineering approach using autologous cultured marrow mesenchymal cells might prevent aseptic loosening of the total ankle arthroplasty.

Cultured human ankle and knee cartilage differ in susceptibility to damage mediated by fibronectin fragments.

According to numerous cadaveric, radiographic, and clinical studies, ankle and knee joints differ in susceptibility to osteoarthritis. To test for biochemical differences in susceptibility to damage, a chondrocytic chondrolysis system has been utilized. In this system, fibronectin fragments are added to cultured cartilage explants, resulting in enhanced release of catabolic cytokines, induction of matrix metalloproteinases, temporary suppression of proteoglycan synthesis, and consequently, severe loss of cartilage proteoglycan. We found that the addition of an amino-terminal thrombin-generated 29-kDa fibronectin fragment to cultured knee cartilage from 14 donors (average age: 53 years) usually caused a 30-50% decrease in proteoglycan content by day 7. However, of the ankle cartilage specimens examined from 21 donors (average age: 50 years), only three showed damage by day 7, one by day 14, and six by day 21, and 11 were not damaged until day 28. For eight of the donors (average age: 44 years), both knee and ankle cartilages were obtained: this allowed comparison between tissues from the same donor. The analysis showed that the ankle cartilage was much more refractory to damage than was the knee cartilage from the same donor. These data clearly show differences between ankle and knee cartilage in susceptibility to the fibronectin fragments and suggest the feasibility of use of these fragments for discerning differences in homeostasis of the ankle and knee cartilage.

Spatial and temporal heterogeneity of superficial muscle strain during in situ fixed-end contractions.

Numerical models of contracting muscle offer a powerful tool to study local mechanical load. For validation of these models, the spatial and temporal distribution of strain was quantified in fixed-end contracting rat tibialis anterior muscle in situ at optimal muscle length (L(o)) and at 120 degrees plantar flexion as well as at 125 and 33Hz stimulation frequency. We studied the hypothesis that after termination of stimulation in situ muscle segments near the motor endplates elongate while segments away from the endplates shorten. We show that both spatial and temporal inhomogeneities in muscle deformation occurred during contraction. Muscle plateau shortening strain equalled 4.1%. Maximal plateau shortening of a muscle segment was much larger (9.6%) and occurred distally (at 0.26 of the scaled length of the muscle). Manipulating torque levels by decreasing the stimulation frequency at the same muscle length induced a decrease in torque ( approximately 20%) with a smaller effect on the level and no effect on the pattern of muscle deformation. During relaxation, distal segments actively shortened at the expense of proximal muscle segments, which elongated. The segments undergoing lengthening were nearer to motor endplates than segments undergoing shortening. In conclusion, the present study provides experimental data on magnitude of contraction-induced deformation needed for validation of numerical models. Local muscle deformation is heterogeneous both temporally and spatially and may be related to proximity to the motor endplates.

Anatomical and histologic investigation of the syndesmotic area in the ankle joint.

The syndesmotic area in the ankle joint is analyzed by reviewing the literature and by examining its gross anatomy and histology. Seventy-five ankles were dissected and examined, with an emphasis on the syndesmotic area. The gross anatomical results of this study differ somewhat with those of other authors. The average height, attachments, consistency, color, and shape are reported. In addition to gross examination, four ankles were examined histologically, and the findings are discussed. The results of this study define in detail the anatomy of the syndesmotic area.

Current concepts: tissue engineering and regenerative medicine applications in the ankle joint.

Tissue engineering and regenerative medicine (TERM) has caused a revolution in present and future trends of medicine and surgery. In different tissues, advanced TERM approaches bring new therapeutic possibilities in general population as well as in young patients and high-level athletes, improving restoration of biological functions and rehabilitation. The mainstream components required to obtain a functional regeneration of tissues may include biodegradable scaffolds, drugs or growth factors and different cell types (either autologous or heterologous) that can be cultured in bioreactor systems (in vitro) prior to implantation into the patient. Particularly in the ankle, which is subject to many different injuries (e.g. acute, chronic, traumatic and degenerative), there is still no definitive and feasible answer to 'conventional' methods. This review aims to provide current concepts of TERM applications to ankle injuries under preclinical and/or clinical research applied to skin, tendon, bone and cartilage problems. A particular attention has been given to biomaterial design and scaffold processing with potential use in osteochondral ankle lesions.

Citrullination of fibronectin modulates synovial fibroblast behavior.

INTRODUCTION: Rheumatoid arthritis is an autoimmune arthritis characterized by joint destruction. Anti-citrullinated protein antibodies are pathologic in rheumatoid arthritis, but the role of the citrullinated proteins themselves is much less clear. Citrullination is the conversion of the arginine residues of a protein to citrulline. In the inflamed rheumatoid joint there is increased protein citrullination. Several proteins are citrullinated in rheumatoid arthritis, including collagen type II, fibrinogen, and fibronectin. Fibronectin is thought to mediate the adhesion of joint-invading synovial fibroblasts to the rheumatoid cartilage in addition to regulating other synovial fibroblast functions. However, the effect of citrullinated fibronectin on synovial fibroblasts is unknown. METHODS: To investigate the effect of citrullinated fibronectin on synovial fibroblast behavior, we cultured normal murine, arthritic murine, and human rheumatoid synovial fibroblasts. We then compared several synovial fibroblast functions in the presence of fibronectin versus citrullinated fibronectin. We assessed adhesion with time-lapse microscopy, migration with transwell assays, focal adhesion kinase and paxillin phosphorylation by western blot, and focal matrix degradation by fluorescent gelatin degradation. RESULTS: Normal synovial fibroblasts have impaired adhesion, spreading, migration, and integrin-mediated phosphorylation of focal adhesion kinase and paxillin on citrullinated fibronectin. Murine arthritic and human rheumatoid synovial fibroblasts also have impaired adhesion and spreading on citrullinated fibronectin, but focal matrix degradation is unaffected by citrullinated fibronectin. CONCLUSION: Citrullination of fibronectin alters synovial fibroblast behavior and may affect how these cells adhere to and invade the joint and travel through the bloodstream. This work suggests an important role for the interaction of synovial fibroblasts with citrullinated matrix in the pathophysiology of rheumatoid arthritis.

A review of the differences between normal and osteoarthritis articular cartilage in human knee and ankle joints.

BACKGROUND: Osteoarthritis (OA) is the most common joint disease yet its pathophysiology is still poorly understood. It is more prevalent in some lower limb joints than others; in particular the knee is more commonly affected than the ankle. Research into articular cartilage and OA has primarily focussed on using animal models. However, it is apparent that articular cartilage differs between species, so more research is concentrating on human cartilage. OBJECTIVE: This paper reviews recent studies that have been undertaken to elucidate the reasons for this, and to discover if the findings would alter the conception that articular cartilage is not capable of repair. METHOD: Primary research papers into human knee and ankle cartilage published since 1997 have been reviewed. RESULTS: Differences in the structure, metabolism, physical properties and response to trauma have been found, implying that ankle cartilage may be more resistant to damage. CONCLUSIONS: More research is needed before definitive conclusions can be reached, but the findings so far suggest that OA should not be accepted as the inevitable outcome of joint injury and individuals and practitioners, such as podiatrists, may be able to use simple measures to prevent or delay its onset.