Yes-associated protein nuclear translocation promotes anabolic activity in human articular chondrocytes.

OBJECTIVE: Yes-associated protein (YAP) has been widely studied as a mechanotransducer in many cell types, but its function in cartilage is controversial. The aim of this study was to identify the effect of YAP phosphorylation and nuclear translocation on the chondrocyte response to stimuli relevant to osteoarthritis (OA). DESIGN: Cultured normal human articular chondrocytes from 81 donors were treated with increased osmolarity media as an in vitro model of mechanical stimulation, fibronectin fragments (FN-f) or IL-1ß as catabolic stimuli, and IGF-1 as an anabolic stimulus. YAP function was assessed with gene knockdown and inhibition by verteporfin. Nuclear translocation of YAP and its transcriptional co-activator TAZ and site-specific YAP phosphorylation were determined by immunoblotting. Immunohistochemistry and immunofluorescence to detect YAP were performed on normal and OA human cartilage with different degrees of damage. RESULTS: Chondrocyte YAP/TAZ nuclear translocation increased under physiological osmolarity (400 mOsm) and IGF-1 stimulation, which was associated with YAP phosphorylation at Ser128. In contrast, catabolic stimulation decreased the levels of nuclear YAP/TAZ through YAP phosphorylation at Ser127. Following YAP inhibition, anabolic gene expression and transcriptional activity decreased. Additionally, YAP knockdown reduced proteoglycan staining and levels of type II collagen. Total YAP immunostaining was greater in OA cartilage, but YAP was sequestered in the cytosol in cartilage areas with more severe damage. CONCLUSIONS: YAP chondrocyte nuclear translocation is regulated by differential phosphorylation in response to anabolic and catabolic stimuli. Decreased nuclear YAP in OA chondrocytes may contribute to reduced anabolic activity and promotion of further cartilage loss.

Transcriptional response of human articular chondrocytes treated with fibronectin fragments: an in vitro model of the osteoarthritis phenotype.

OBJECTIVE: Fibronectin is a matrix protein that is fragmented during cartilage degradation in osteoarthritis (OA). Treatment of chondrocytes with fibronectin fragments (FN-f) has been used to model OA in vitro, but the system has not been fully characterized. This study sought to define the transcriptional response of chondrocytes to FN-f, and directly compare it to responses traditionally observed in OA. DESIGN: Normal human femoral chondrocytes isolated from tissue donors were treated with either FN-f or PBS (control) for 3, 6, or 18 h. RNA-seq libraries were compared between time-matched FN-f and control samples in order to identify changes in gene expression over time. Differentially expressed genes were compared to a published OA gene set and used for pathway, transcription factor motif, and kinome analysis. RESULTS: FN-f treatment resulted in 3,914 differentially expressed genes over the time course. Genes that are up- or downregulated in OA were significantly up- (P < 0.00001) or downregulated (P < 0.0004) in response to FN-f. Early response genes were involved in proinflammatory pathways, whereas many late response genes were involved in ferroptosis. The promoters of upregulated genes were enriched for NF-κB, AP-1, and IRF motifs. Highly upregulated kinases included CAMK1G, IRAK2, and the uncharacterized kinase DYRK3, while growth factor receptors TGFBR2 and FGFR2 were downregulated. CONCLUSIONS: FN-f treatment of normal human articular chondrocytes recapitulated many key aspects of the OA chondrocyte phenotype. This in vitro model is promising for future OA studies, especially considering its compatibility with genomics and genome-editing techniques.

The combination of mitogenic stimulation and DNA damage induces chondrocyte senescence.

OBJECTIVE: Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating senescence, one emerging theme is that damaged cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. DESIGN: This study utilized healthy cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated to initiate DNA damage, and mitogenic stimulation was provided through serum-containing medium and treatment with transforming growth factor ß1 and basic fibroblastic growth factor. Readouts of senescence were a quantitative flow cytometry assay to detect senescence-associated ß galactosidase activity (SA-ß-gal), immunofluorescence for p16 and γH2AX, and qPCR for the expression of inflammatory genes. RESULTS: Human cartilage explants required both irradiation and mitogenic stimulation to induce senescence as compared to baseline control conditions (7.16% vs 2.34% SA-ß-gal high, p = 0.0007). These conditions also resulted in chondrocyte clusters within explants, a persistent DNA damage response, increased p16, and gene expression changes. CONCLUSIONS: Treatment of cartilage explants with mitogenic stimuli in the context of cellular damage reliably induces high levels of SA-ß-gal activity and other senescence markers, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.

Articular chondrocytes isolated from the knee and ankle joints of human tissue donors demonstrate similar redox-regulated MAP kinase and Akt signaling.

OBJECTIVE: To compare key intracellular redox-regulated signaling pathways in chondrocytes derived from knee joint femoral cartilage and ankle joint talar cartilage in order to determine if differences exist that might contribute to the lower prevalence of ankle osteoarthritis (OA). METHODS: Femoral and talar chondrocytes were treated with H2O2 generators (menadione or 2-3-dimethoxy-1,4-napthoquinone (DMNQ), fragments of fibronectin (FN-f)) to stimulate MAP kinase signaling (MAPK), or with IGF-1 to stimulate the Akt signaling pathway. Hyperoxidation of the peroxiredoxins, used as a measure of redox status, and phosphorylation of proteins pertinent to MAPK (p38, ERK, JNK, c-Jun) and Akt (Akt, PRAS40) signaling cascades were detected by immunoblotting. RESULTS: Treatment of femoral and talar chondrocytes with menadione, DMNQ or FN-f led to a time dependent increase in extracellular-regulated kinase (ERK) and p38 phosphorylation. DMNQ and FN-f stimulation enhanced phosphorylation of JNK and its downstream substrate, c-Jun. Menadione treatment did not stimulate JNK activity but hyperoxidized the peroxiredoxins and inhibited IGF-1-induced Akt activation. In all experiments, chondrocytes derived from the femur and talar joints displayed comparable MAP kinase responses after treatment with various catabolic stimuli, as well as similar Akt signaling responses after IGF-1 treatment. CONCLUSIONS: MAP kinase and Akt signaling in response to factors that modulate the intracellular redox status were similar in chondrocytes from knee and ankle joints suggesting that redox signaling differences do not explain differences in OA prevalence. Talar chondrocytes, when isolated from their native matrix, can be used to examine redox-regulated cell signaling events relevant to OA in either joint.

Cell-surface markers identify tissue resident multipotential stem/stromal cell subsets in synovial intimal and sub-intimal compartments with distinct chondrogenic properties.

OBJECTIVE: Synovium contains multipotent progenitor/stromal cells (MPCs) with potential to participate in cartilage repair. Understanding the identity of these MPCs will allow their therapeutic potential to be fully exploited. Hence this study aimed to identify primary synovial MPCs and characterize them in the context of cartilage regeneration. METHODS: Primary MPC/MPC-subset specific markers in synovium were identified by FACS analysis of uncultured cells. MPC-subsets from human synovium obtained from patients undergoing total knee arthroplasty were FACS sorted, cultured, immunophenotyped and chondrogenically differentiated. The anatomical localization of MPCs in synovium was examined using immunohistochemistry. Finally, the presence of these MPC subsets in healthy synovium obtained from human organ donors was examined. RESULTS: A combination of CD45, CD31, CD73 and CD90 can isolate two distinct MPC-subsets in synovium. These MPC-subsets, freshly isolated from synovium, did not express CD45 or CD31, but expressed CD73. Additionally, a sub-population of CD73+ cells also expressed CD90. CD45-CD31-CD73+CD90- cells were significantly more chondrogenic than CD45-CD31-CD73+CD90+ cells in the presence of TGFß1. Interestingly, reduced chondrogenic ability of CD73+CD90+ cells could be reversed by the addition of BMP2, showing discrete chondrogenic factor requirements by distinct cell-subsets. In addition, these MPCs had distinct anatomical localization; CD73 was expressed both in intimal and sub-intimal region while CD90 was enriched in the sub-intimal region. We further demonstrated that these subsets are also present in healthy synovium. CONCLUSIONS: We provide indications that primary MPCs in synovial intima and sub-intima are phenotypically and functionally distinct with different chondrogenic properties.

Wnt5a induces catabolic signaling and matrix metalloproteinase production in human articular chondrocytes.

OBJECTIVE: Aberrant Wnt signaling may contribute to osteoarthritis (OA) but the Wnt family members involved have not been fully identified. The purpose of this study was to investigate the role of Wnt5a as a potential mediator of cartilage destruction in OA. DESIGN: Immunohistochemistry to detect Wnt5a was performed using normal and OA human articular cartilage. Cultured normal human chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus or recombinant Wnt5a protein with or without pretreatment using a panel of signaling inhibitors. Expression of Wnt5a, anabolic genes and catabolic genes were determined by quantitative real-time PCR. Production of Wnt5a protein and matrix metalloproteinases (MMPs) as well as activation of signaling proteins were analyzed by immunoblotting. RESULTS: Wnt5a was present in human articular cartilage with OA changes and its expression and secretion were increased in FN-f stimulated chondrocytes. FN-f stimulated Wnt5a production through the c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) pathways. Wnt5a reduced aggrecan gene expression after 48 h of treatment. Wnt5a seemed to promote MMP1, -3, and -13 expression as well as MMP1 and MMP13 protein production in normal human chondrocytes. Wnt5a inhibitor peptides did not affect FN-f induced MMP production. Wnt5a activated ß-catenin independent signaling including calmodulin-dependent protein kinase II (CaMKII), JNK, p38, ERK1/2, p65 and Akt. Inhibition of JNK, p38, ERK, PI-3 kinase and CaMKII by specific signaling inhibitors suppressed Wnt5a mediated MMP1 and MMP13 production. CONCLUSIONS: Wnt5a is present in human OA cartilage and can promote chondrocyte catabolic activity through non-canonical Wnt signaling, which suggests a potential role in OA.

High fat-diet and saturated fatty acid palmitate inhibits IGF-1 function in chondrocytes.

INTRODUCTION: Insulin-like growth factor-1 (IGF-1) promotes matrix synthesis and cell survival in cartilage. Chondrocytes from aged and osteoarthritic cartilage have a reduced response to IGF-1. The purpose of this study was to determine the effect of free fatty acids (FFA) present in a high-fat diet on IGF-1 function in cartilage and the role of endoplasmic reticulum (ER) stress. METHODS: C57BL/6 male mice were maintained on either a high-fat (60% kcal from fat) or a low-fat (10% kcal from fat) diet for 4 months. Mice were then sacrificed; femoral head cartilage caps were collected and treated with IGF-1 to measure proteoglycan (PG) synthesis. Cultured human chondrocytes were treated with 500 µM FFA palmitate or oleate, followed by stimulation with (100 ng/ml) IGF-1 overnight to measure CHOP (a protein marker for ER stress) and PG synthesis. Human chondrocytes were pre-treated with palmitate or 1 mM 4-phenyl butyric acid (PBA) or 1 µM C-Jun N terminal Kinase (JNK) inhibitor, and IGF-1 function (PG synthesis and signaling) was measured. RESULTS: Cartilage explants from mice on the high fat-diet showed reduced IGF-1 mediated PG synthesis compared to a low-fat group. Treatment of human chondrocytes with palmitate induced expression of CHOP, activated JNK and inhibited IGF-1 function. PBA, a small molecule chemical chaperone that alleviates ER stress rescued IGF-1 function and a JNK inhibitor rescued IGF-1 signaling. CONCLUSIONS: Palmitate-induced ER stress inhibited IGF-1 function in chondrocytes/cartilage via activating the mitogen-activated protein (MAP) kinase JNK. This is the first study to demonstrate that ER stress is metabolic factor that regulates IGF-1 function in chondrocytes.

Reduced response of human meniscal cells to Osteogenic Protein 1 during osteoarthritis and pro-inflammatory stimulation.

OBJECTIVE: Many cell types lose responsiveness to anabolic factors during inflammation and disease. Osteogenic Protein 1 (OP1/BMP7) was evaluated for the ability to enhance extracellular matrix synthesis in healthy and OA meniscus cells. Mechanisms of cell response to OP1 were explored. DESIGN: Meniscus and cartilage tissues from healthy tissue donors and osteoarthritis (OA) patients undergoing total knee arthroplasties were acquired. Primary cell cultures were stimulated with OP1 and/or inflammatory factors (IL1α, IL1ß, or fibronectin fragments (FnF)) and cellular responses were analyzed by RT-qPCR and immunoblots. Frozen section immunohistochemistry (IHC) was conducted to assess OP1 and receptor proteins in normal and OA meniscus. RESULTS: OP1 treatment of normal meniscus cells resulted in significant, dose-dependent increases in ACAN (aggrecan) and COL2A1, and decreased MMP13 gene transcription, while only ACAN was upregulated (P < 0.01) at the highest dose of OP1 in OA meniscus cells. OP1 induced significantly more ACAN gene transcription in normal meniscus than normal articular cartilage (P = 0.05), and no differences between normal and OA cartilage were detected. Receptor expression and kinetics of canonical signaling activation were similar between normal and OA specimens. Normal meniscus cells treated with inflammatory factors were refractory to OP1 stimulation. Smad1 phosphorylation at an inhibitory site was induced (P = 0.01 for both normal and OA meniscus) by inflammatory cytokine treatment. CONCLUSIONS: The meniscus demonstrates resistance to OP1 stimulation in OA and in the presence of inflammatory mediators. MAPK-mediated Smad1 linker phosphorylation is a possible mediator of the loss of anabolic extracellular matrix production in the inflammatory cytokine affected meniscus.

Regenerative approaches for the treatment of early OA.

The diagnosis and the prompt treatment of early osteoarthritis (OA) represent vital steps for delaying the onset and progression of fully blown OA, which is the most common form of arthritis, involving more than 10 % of the world's population older than 60 years of age. Nonsurgical treatments such as physiotherapy, anti-inflammatory medications, and other disease-modifying drugs all have modest and short-lasting effect. In this context, the biological approaches have recently gained more and more attention. Growth factors, blood derivatives, such as platelet concentrates, and mesenchymal adult stem cells, either expanded or freshly isolated, are advocated amongst the most promising tool for the treatment of OA, especially in the early phases. Primarily targeted towards focal cartilage defects, these biological agents have indeed recently showed promising results to relieve pain and reduce inflammation in patients with more advanced OA as well, with the final aim to halt the progression of the disease and the need for joint replacement. However, despite of a number of satisfactory in vitro and pre-clinical studies, the evidences are still limited to support their clinical efficacy in OA setting.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities.

OBJECTIVE: We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. METHODS: HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. RESULTS: HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. CONCLUSION: HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage.

Effects of insulin-like growth factor-1 and dexamethasone on cytokine-challenged cartilage: relevance to post-traumatic osteoarthritis.

OBJECTIVE: Interleukin-1 is one of the inflammatory cytokines elevated after traumatic joint injury that plays a critical role in mediating cartilage tissue degradation, suppressing matrix biosynthesis, and inducing chondrocyte apoptosis, events associated with progression to post-traumatic osteoarthritis (PTOA). We studied the combined use of insulin-like growth factor-1 (IGF-1) and dexamethasone (Dex) to block these multiple degradative effects of cytokine challenge to articular cartilage. METHODS: Young bovine and adult human articular cartilage explants were treated with IL-1α in the presence or absence of IGF-1, Dex, or their combination. Loss of sulfated glycosaminoglycans (sGAG) and collagen were evaluated by the DMMB and hydroxyproline assays, respectively. Matrix biosynthesis was measured via radiolabel incorporation, chondrocyte gene expression by qRT-PCR, and cell viability by fluorescence staining. RESULTS: In young bovine cartilage, the combination of IGF-1 and Dex significantly inhibited the loss of sGAG and collagen, rescued the suppression of matrix biosynthesis, and inhibited the loss of chondrocyte viability caused by IL-1α treatment. In adult human cartilage, only IGF-1 rescued matrix biosynthesis and only Dex inhibited sGAG loss and improved cell viability. Thus, the combination of IGF-1 + Dex together showed combined beneficial effects in human cartilage. CONCLUSIONS: Our findings suggest that the combination of IGF-1 and Dex has greater beneficial effects than either molecule alone in preventing cytokine-mediated cartilage degradation in adult human and young bovine cartilage. Our results support the use of such a combined approach as a potential treatment relevant to early cartilage degradative changes associated with joint injury.

Serum free light chains, interferon-alpha, and interleukins in systemic lupus erythematosus.

OBJECTIVE: Interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-alpha (IFN-α), and free light chains (FLCs: lambda, kappa) have all been noted to be of importance in systemic lupus erythematosus (SLE). Herein, we quantified and explored the relationship between these inflammatory mediators and disease activity in SLE; and stratified by their current anti-dsDNA antibody status. METHODS: Seventy-seven SLE patients underwent assessment of disease activity using the SLE disease activity index (SLEDAI). Serum FLC (lambda, kappa, and total), IL-6, IL-10, and IFN-α were quantified. Demographics of disease characteristics were determined by chart reviews. Statistical analyses included Mann-Whitney test, chi square, and linear regression analyses. RESULTS: Mean (SD) age of the patients was 44.9 ± 12.7 years; SLEDAI (mean ± SD) was 3.4 ± 4.0. Serum lambda FLC levels had a moderate correlation (r = 0.46 with physician global assessment, 0.44 with SLEDAI) and the strongest correlation with disease activity as compared with other inflammatory mediators including current dsDNA antibody status. After adjusting for prednisone use, the correlation of lambda FLC with PGA (r = 0.48) and SLEDAI (r = 0.52) was better than of current dsDNA antibody status with PGA (r = 0.33) and adjusted SLEDAI (r = 0.24), respectively. IL-10 and IFN-α activity did not correlate with disease activity. Serum FLC and IL-6 levels could differentiate between active and inactive SLE patients. Serum lambda FLC and IL-6 levels differed significantly among patients with and without current dsDNA antibodies. Serum lambda FLC levels accounted for 31% of variance in SLEDAI scores. CONCLUSION: Serum FLC and IL-6 are potentially useful biomarkers of disease activity in SLE. Further studies, with larger study sample and longitudinal design, are indicated.

Moderate dynamic compression inhibits pro-catabolic response of cartilage to mechanical injury, tumor necrosis factor-α and interleukin-6, but accentuates degradation above a strain threshold.

OBJECTIVE: Traumatic joint injury can initiate early cartilage degeneration in the presence of elevated inflammatory cytokines (e.g., tumor necrosis factor (TNF)-α and interleukin (IL)-6). The positive/negative effects of post-injury dynamic loading on cartilage degradation and repair in vivo are not well-understood. This study examined the effects of dynamic strain on immature bovine cartilage in vitro challenged with TNF-α + IL-6 and its soluble receptor (sIL-6R) with/without initial mechanical injury. METHODS: Groups of mechanically injured or non-injured explants were cultured in TNF-α + IL-6/sIL-6R for 8 days. Intermittent dynamic compression was applied concurrently at 10%, 20%, or 30% strain amplitude. Outcome measures included sulfated glycosaminoglycan (sGAG) loss (dimethylmethylene blue (DMMB)), aggrecan biosynthesis ((35)S-incorporation), aggrecanase activity (Western blot), chondrocyte viability (fluorescence staining) and apoptosis (nuclear blebbing via light microscopy), and gene expression (qPCR). RESULTS: In bovine explants, cytokine alone and injury-plus-cytokine treatments markedly increased sGAG loss and aggrecanase activity, and induced chondrocyte apoptosis. These effects were abolished by moderate 10% and 20% strains. However, 30% strain amplitude greatly increased apoptosis and had no inhibitory effect on aggrecanase activity. TNF + IL-6/sIL-6R downregulated matrix gene expression and upregulated expression of inflammatory genes, effects that were rescued by moderate dynamic strains but not by 30% strain. CONCLUSIONS: Moderate dynamic compression inhibits the pro-catabolic response of cartilage to mechanical injury and cytokine challenge, but there is a threshold strain amplitude above which loading becomes detrimental to cartilage. Our findings support the concept of appropriate loading for post-injury rehabilitation.

Methylation of the OP-1 promoter: potential role in the age-related decline in OP-1 expression in cartilage.

OBJECTIVE: An age-related decline in chondrocyte production of osteogenic protein-1 (OP-1) (Bone Morphogenetic Protein-7) may contribute to cartilage loss in osteoarthritis. This study was designed to determine if increased methylation of the OP-1 promoter might serve as a mechanism for the age-related decline in OP-1 expression. METHODS: Human articular chondrocytes were isolated from cartilage obtained after death from tissue donors (ages 19-86 years) without a known history of arthritis. DNA was obtained from isolated chondrocytes in primary culture and analyzed for OP-1 promoter methylation by polymerase chain reaction (PCR) after bisulfite treatment. Cultured cells were treated with the DNA methyltransferase inhibitor 5-azacytidine and OP-1 production was measured in the media by enzyme-linked immunosorbent assay (ELISA). RNA was isolated to measure expression of insulin-like growth factor-1 (IGF-1), the IGF-1 receptor (IGF-1R), aggrecan, and OP-1 by real-time PCR. RESULTS: Methylation of the OP-1 promoter was detected in chondrocytes isolated from tissue obtained from older adults and there was a positive correlation between age and OP-1 methylation status (n=22, R(2)=0.277, P=0.014). Inhibition of methylation in cultured cells with 5-azacytidine increased chondrocyte production of OP-1 protein and increased the expression of the IGF-1, the IGF-1R, aggrecan, and OP-1 genes but not GAPDH. CONCLUSION: Age-related methylation of the OP-1 promoter may contribute to a decrease in OP-1 production in cartilage and a decrease in expression of OP-1 responsive genes such as IGF-1, the IGF-1R, and aggrecan.

Anti-apoptotic treatments prevent cartilage degradation after acute trauma to human ankle cartilage.

OBJECTIVES: To investigate the effect of anti-apoptotic agents on cartilage degradation after a single impact to ankle cartilage. DESIGN: Ten human normal tali were impacted with the impulse of 1 Ns generating peak forces in the range of 600 N using a 4 mm diameter indenter. Eight millimeter cartilage plugs containing the 4 mm diameter impacted core and a 4 mm adjacent ring were removed and cultured with or without P188 surfactant (8 mg/ml), caspase-3 (10 uM), or caspase-9 (2 uM) inhibitors for 48 h. Results were assessed in the superficial and middle-deep layers immediately after injury at day 0 and at 2, 7 and 14 days after injury by live/dead cell and Tunel assays and by histology with Safranin O/fast green staining. RESULTS: A single impact to human articular cartilage ex vivo resulted in cell death, cartilage degeneration, and radial progression of apoptosis to the areas immediately adjacent to the impact. The P188 was more effective in preventing cell death than the inhibitors of caspases. It reduced cell death by more than 2-fold (P<0.05) in the core and by about 30% in the ring in comparison with the impacted untreated control at all time points. P188 also prevented radial expansion of apoptosis in the ring region especially in the first 7 days post-impaction (7.5% Tunel-positive cells vs 46% in the untreated control; P<0.01). Inhibitors of caspase-3 or -9 were effective in reducing cell death in the impacted core only at early time points, but were ineffective in doing so in the ring. Mankin score was significantly improved in the P188 and caspase-3 treated groups. CONCLUSIONS: Early intervention with the P188 and caspase-3 inhibitor may have therapeutic potential in the treatment of cartilage defects immediately after injury.

Tissue engineering a human phalanx.

A principal purpose of tissue engineering is the augmentation, repair or replacement of diseased or injured human tissue. This study was undertaken to determine whether human biopsies as a cell source could be utilized for successful engineering of human phalanges consisting of both bone and cartilage. This paper reports the use of cadaveric human chondrocytes and periosteum as a model for the development of phalanx constructs. Two factors, osteogenic protein-1 [OP-1/bone morphogenetic protein-7 (BMP7)], alone or combined with insulin-like growth factor (IGF-1), were examined for their potential enhancement of chondrocytes and their secreted extracellular matrices. Design of the study included culture of chondrocytes and periosteum on biodegradable polyglycolic acid (PGA) and poly-l-lactic acid (PLLA)-poly-ε-caprolactone (PCL) scaffolds and subsequent implantation in athymic nu/nu (nude) mice for 5, 20, 40 and 60 weeks. Engineered constructs retrieved from mice were characterized with regard to genotype and phenotype as a function of developmental (implantation) time. Assessments included gross observation, X-ray radiography or microcomputed tomography, histology and gene expression. The resulting data showed that human cell-scaffold constructs could be successfully developed over 60 weeks, despite variability in donor age. Cartilage formation of the distal phalanx models enhanced with both OP-1 and IGF-1 yielded more cells and extracellular matrix (collagen and proteoglycans) than control chondrocytes without added factors. Summary data demonstrated that human distal phalanx models utilizing cadaveric chondrocytes and periosteum were successfully fabricated and OP-1 and OP-1/IGF-1 accelerated construct development and mineralization. The results suggest that similar engineering and transplantation of human autologous tissues in patients are clinically feasible. Copyright © 2016 John Wiley & Sons, Ltd.

ADAM-10 message is expressed in human articular cartilage.

The depletion of the pericellular and territorial matrices in articular cartilage is considered to be one of the earliest events in pathobiology of osteoarthritis (OA). A newly discovered family of proteins with a disintegrin-like and metalloproteinase-like domain (ADAM) may be involved in matrix degradation as well as in cell-cell and cell-matrix interactions. The purpose of this study was to determine by in situ hybridization whether human articular chondrocytes from newborn, normal adult, and OA cartilages express messenger RNA for ADAM-10, one of the members of this family, and by semiquantitative RT-PCR to compare the levels of this expression. The results confirmed the expression of ADAM-10 by human articular chondrocytes and revealed the highest levels of expression in the continuously remodeling cartilage of newborns and the most fibrillated areas of OA cartilage, especially the regions of cell clusters. Importantly, ADAM-10 mRNA expression was evident in tissues with the greatest loss of Safranin O staining from the territorial and interterritorial matrix of the chondrocytes. Messenger RNA was upregulated in OA tissue compared to the age-matched normal cartilage, as detected by RT-PCR. Upregulated levels of ADAM-10 mRNA expression appear to be related to the degree of cartilage damage and/or degradation, which suggests a potential role for at least one member of this new family in the cartilage matrix destruction accompanying OA.

ADAM-10 protein is present in human articular cartilage primarily in the membrane-bound form and is upregulated in osteoarthritis and in response to IL-1alpha in bovine nasal cartilage.

The objective of our study was to determine the tissue distribution and localization of ADAM-10 protein in human and bovine cartilage and the changes it undergoes with cartilage degeneration seen in osteoarthritis (OA) and under the influence of interleukin-1 (IL-1). Human normal and OA articular cartilage and bovine nasal cartilage cultured in the presence of IL-1alpha were processed for histology and immunohistochemistry. ADAM-10 protein was extracted from human cartilage and analyzed by Western blotting using anti-ADAM-10 antibodies. Fluor S Image analyzer and Quantity One software program were applied to quantify the total amount of ADAM-10. ADAM-10 protein was detected in both human and bovine cartilage. The strongest immunostaining was found in the cytoplasm and/or cell membranes of the superficial and upper middle layer of normal adult human cartilage, in the clusters and fibrillated areas of OA cartilage, and in IL-1alpha-stimulated bovine nasal cartilage. The distribution of ADAM-10 protein in bovine nasal cartilage was dependent on the length of exposure to IL-1alpha and corresponded to the areas of proteoglycan depletion. By Western blotting analysis of human cartilage, ADAM-10 was primarily detected in the membrane-enriched fraction and its levels were increased in degenerated and OA cartilage compared to normal cartilage. The results of this study suggest that ADAM-10 might be an important factor associated with cartilage degenerative processes. (J Histochem Cytochem 49:1165-1176, 2001)

Gene expression by human articular chondrocytes cultured in alginate beads.

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of alpha1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The alpha1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.

Human articular chondrocytes express osteogenic protein-1.

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)