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Objective: To query the transcript-level changes in the medial and lateral tibial plateau cartilage in tandem with obesity in patients with end-stage osteoarthritis (OA)., Design: Cartilage was obtained from 23 patients (20 obese [body mass index > 30 kg/m 2 ], 3 overweight [body mass index < 30 kg/m 2 ]) at the time of total knee replacement. Cartilage integrity was assessed using Outerbridge scale, while radiographic changes were scored on preoperative X-rays using Kellgren-Lawrence (K-L) classification. RNA was probed for differentially expressed transcripts between medial and lateral compartments using Affymetrix Gene 2.0 ST Array and validated via real-time polymerase chain reaction. Gene ontology and pathway analyses were also queried., Results: Scoring of cartilage integrity by the Outerbridge scale indicated that the medial and lateral compartments were similar, while scoring by the K-L classification indicated that the medial compartment was more severely damaged than the lateral compartment. We observed a distinct transcript profile with >50% of transcripts unique between medial and lateral compartments. MMP13 and COL2A1 were more highly expressed in medial versus lateral compartment. Polymerase chain reaction confirmed expression of 4 differentially expressed transcripts. Numerous transcripts, biological processes, and pathways were significantly different between overweight and obese patients with a differential response of obesity on medial and lateral compartments., Conclusions: Our findings support molecular differences between medial and lateral compartments reflective of the greater severity of OA in the medial compartment. The K-L system better reflected the molecular results than did the Outerbridge. Moreover, the molecular effect of obesity was different between the medial and lateral compartments of the same knee plausibly reflecting the molecular effects of differential biomechanical loading.

Objective: The etiology of osteoarthritis (OA) is unknown, however, there appears to be a significant contribution from genetics. We have identified recombinant inbred strains of mice derived from LG/J (large) and SM/J (small) strains that vary significantly in their ability to repair articular cartilage and susceptibility to post-traumatic OA due to their genetic composition. Here, we report cartilage repair phenotypes in the same strains of mice in which OA susceptibility was analyzed previously, and determine the genetic correlations between phenotypes., Design: We used 12 recombinant inbred strains, including the parental strains, to test three phenotypes: ear-wound healing (n = 263), knee articular cartilage repair (n = 131), and post-traumatic OA (n = 53) induced by the surgical destabilization of the medial meniscus (DMM). Genetic correlations between various traits were calculated as Pearson's correlation coefficients of strain means., Results: We found a significant positive correlation between ear-wound healing and articular cartilage regeneration (r = 0.71; P = 0.005). We observed a strong inverse correlation between articular cartilage regeneration and susceptibility to OA based on maximum (r = -0.54; P = 0.036) and summed Osteoarthritis Research Society International (OARSI) scores (r = -0.56; P = 0.028). Synovitis was not significantly correlated with articular cartilage regeneration but was significantly positively correlated with maximum (r = 0.63; P = 0.014) and summed (r = 0.70; P = 0.005) OARSI scores. Ectopic calcification was significantly positively correlated with articular cartilage regeneration (r = 0.59; P = 0.021)., Conclusions: Using recombinant inbred strains, our study allows, for the first time, the measurement of genetic correlations of regeneration phenotypes with degeneration phenotypes, characteristic of OA (cartilage degeneration, synovitis). We demonstrate that OA is positively correlated with synovitis and inversely correlated with the ability to repair cartilage. These results suggest an addition to the risk paradigm for OA from a focus on degeneration to regeneration., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Delivery of therapeutic small interfering RNAs (siRNAs) in an effective dose to articular cartilage is very challenging as the cartilage dense extracellular matrix renders the chondrocytes inaccessible, even to intra-articular injections. Herein, we used a self-assembling peptidic nanoparticle (NP) platform featuring a cell penetrating peptide complexed to NF-κB p65 siRNA. We show that it efficiently and deeply penetrated human cartilage to deliver its siRNA cargo up to a depth of at least 700 μm. To simulate osteoarthritis in vitro, human articular cartilage explants were placed in culture and treated with IL-1β, a cytokine with known cartilage catabolic and pro-inflammatory effects. Exposure of peptide-siRNA NP to cartilage explants markedly suppressed p65 activation, an effect that persisted up to 3 weeks after an initial 48 h exposure to NP and in the presence of continuous IL-1β stimulation. Suppression of IL-1β-induced p65 activity attenuated chondrocyte apoptosis and maintained cartilage homeostasis. These findings confirm our previous in vivo studies in a murine model of post-traumatic osteoarthritis and suggest that the ability of peptide-siRNA NP to specifically modulate NF-κB pathway, a central regulator of the inflammatory responses in chondrocytes, may potentially mitigate the progression of cartilage degeneration.

Objective: Anterior cruciate ligament (ACL) injury initiates a cascade of events often leading to osteoarthritis (OA). ACL reconstruction does not alter the course of OA, suggesting that heightened OA risk is likely due to factors in addition to the joint instability. We showed that torn ACL remnants express periostin (POSTN) in the acute phase of injury. Considering that ACL injury predisposes to OA and that POSTN is associated with cartilage metabolism, we hypothesize that ACL injury affects chondrocytes via POSTN., Design: Cartilage was obtained from osteoarthritic patients and ACL remnants were collected from patients undergoing ACL reconstruction. Crosstalk between ACL remnants and chondrocytes was studied in a transwell co-culture system. Expression of POSTN and other anabolic and catabolic genes was assessed via real-time polymerase chain reaction (PCR). Immunostaining for periostin was performed in human and mouse cartilage. The impact of exogenous periostin and siRNA-mediated ablation of periostin on matrix metabolism and cell migration was examined. Furthermore, the effect of anabolic (transforming growth factor beta 1 [TGF-β1]) and catabolic (interleukin 1 beta [IL-1β]) factors on POSTN expression was investigated., Results: ACL remnants induced expression of POSTN, MMP13 and ADAMTS4. Periostin levels were significantly higher in osteoarthritic compared to normal cartilage. Exogenous periostin induced MMP13 expression and cell migration, and repressed COL1A1 expression while POSTN knockdown inhibited expression of both anabolic and catabolic genes and impeded cell migration. TGF-β1 and IL-1β treatment did not alter POSTN expression but influenced chondrocyte metabolism as determined by quantification of anabolic and catabolic genes via real-time PCR., Conclusions: ACL remnants can exert paracrine effects on cartilage, altering cellular homeostasis. Over time, this metabolic imbalance could contribute to OA development., (Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Purpose/Aim: Meniscus tears are a common injury to the knee associated with the development of osteoarthritis. Gene expression in the injured meniscus may be associated with early degeneration in the articular cartilage. The purpose of this study was to test the hypothesis that gene expression in meniscus tears is associated with early degenerative changes in the articular cartilage at the time of partial meniscectomy., Materials and Methods: Torn meniscus was removed at the time of partial meniscectomy in 68 patients without radiographic osteoarthritis. Meniscal mRNA expression was measured by quantitative PCR for multiple molecular markers of osteoarthritis and cartilage homeostasis. The presence of early degenerative changes in the knee was recorded by X-ray (N = 63), magnetic resonance imaging (MRI, N = 48), and arthroscopy (N = 63). Gene expression was tested for correlation with the presence/absence of degenerative changes after adjusting for age, sex, and body mass index., Results: Overall gene expression varied significantly with degenerative changes based on X-ray (P = 0.047) and MRI (P = 0.018). The linear combination of gene variation was also significant. However, only adiponectin (ADIPOQ) (P = 0.015) was expressed at a significantly lower level in patients with chondrosis on MRI, while the expression of ADIPOQ (P = 0.035) and resistin (RETN) (P = 0.017) was higher in patients with early degenerative changes on X-ray. None of the genes varied significantly with presence/absence of chondrosis as measured by arthroscopy., Conclusions: There is an overall association of gene expression in meniscal tears to early degenerative changes in the knee, but only a limited number of specific genes demonstrate this relationship. The roles of adiponectin and resistin in knee injury and osteoarthritis deserve further study.

Objectives: (i) To provide baseline knowledge of gene expression in macroscopically normal articular cartilage, (ii) to test the hypothesis that age, body-mass-index (BMI), and sex are associated with cartilage RNA transcriptome, and (iii) to predict individuals at potential risk for developing "pre-osteoarthritis" (OA) based on screening of genetic risk-alleles associated with OA and gene transcripts differentially expressed between normal and OA cartilage., Design: Healthy-appearing cartilage was obtained from the medial femoral notch of 12 knees with a meniscus tear undergoing arthroscopic partial meniscectomy. Cartilage had no radiographic, magnetic-resonance-imaging or arthroscopic evidence for degeneration. RNA was subjected to Affymetrix microarrays followed by validation of selected transcripts by microfluidic digital polymerase-chain-reaction. The underlying biological processes were explored computationally. Transcriptome-wide gene expression was probed for association with known OA genetic risk-alleles assembled from published literature and for comparison with gene transcripts differentially expressed between healthy and OA cartilage from other studies., Results: We generated a list of 27,641 gene transcripts in healthy cartilage. Several gene transcripts representing numerous biological processes were correlated with age and BMI and differentially expressed by sex. Based on disease-specific Ingenuity Pathways Analysis, gene transcripts associated with aging were enriched for bone/cartilage disease while the gene expression profile associated with BMI was enriched for growth-plate calcification and OA. When segregated by genetic risk-alleles, two clusters of study patients emerged, one cluster containing transcripts predicted by risk studies. When segregated by OA-associated gene transcripts, three clusters of study patients emerged, one of which is remarkably similar to gene expression pattern in OA., Conclusions: Our study provides a list of gene transcripts in healthy-appearing cartilage. Preliminary analysis into groupings based on OA risk-alleles and OA-associated gene transcripts reveals a subset of patients expressing OA transcripts. Prospective studies in larger cohorts are needed to assess whether these patterns are predictive for OA.

Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: C-C chemokine receptor type 5 (CCR5) has been implicated in rheumatoid arthritis and several inflammatory diseases, where its blockade resulted in reduced joint destruction. However, its role in modulating cartilage and bone changes in post-traumatic osteoarthritis (OA) has not yet been investigated. In this study, we investigated changes in articular cartilage, synovium and bone in a post-traumatic OA model using CCR5-deficient (CCR5(-/-)) mice., Method: Destabilization of the medial meniscus (DMM) was performed on the right knee of 10-week old CCR5(-/-) and C57BL/6J wild-type (WT) mice to induce post-traumatic OA. The contralateral left knee served as sham-operated control. Knee joints were analyzed at 4-, 8- and 12-weeks after surgery to evaluate cartilage degeneration and synovitis by histology, and bone changes via micro-CT., Results: Our findings showed that CCR5(-/-) mice exhibited significantly less cartilage degeneration than WT mice at 8- and 12-weeks post-surgery. CCR5(-/-) mice showed some altered bone parameters 18- and 22-weeks of age, but body size and weight were not affected. The effect of CCR5-ablation was insignificant at all time points post-surgery for synovitis and for bone parameters such as bone volume/total volume, connectivity density index (CDI), structure model index (SMI), subchondral bone plate thickness, and trabecular bone number, thickness and spacing., Conclusion: These findings suggest that CCR5(-/-) mice developed less cartilage degeneration, which may indicate a potential protective role of CCR5-ablation in cartilage homeostasis. There were no differences in bone or synovial response to surgery suggesting that CCR5 functions primarily in cartilage during the development of post-traumatic OA., (Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Mammals rarely regenerate their lost or injured tissues into adulthood. MRL/MpJ mouse strain initially identified to heal full-thickness ear wounds now represents a classical example of mammalian wound regeneration since it can heal a spectrum of injuries such as skin and cardiac wounds, nerve injuries and knee articular cartilage lesions. In addition to MRL/MpJ, a few other mouse strains such as LG/J (a parent of MRL/MpJ) and LGXSM-6 (arising from an intercross between LG/J and SM/J mouse strains) have now been recognized to possess regenerative/healing abilities for articular cartilage and ear wound injuries that are similar, if not superior, to MRL/MpJ mice. While some mechanisms underlying regenerative potential have been begun to emerge, a complete set of biological processes and pathways still needs to be elucidated. Using a panel of healer and non-healer mouse strains, our recent work has provided some insights into the genes that could potentially be associated with healing potential. Future mechanistic studies can help seek the Holy Grail of regenerative medicine. This review highlights the regenerative capacity of selected mouse strains for articular cartilage, in particular, and lessons from other body tissues, in general., (Copyright © 2014 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Objective: Aging and obesity contribute to the initiation and progression of osteoarthritis with little information on their relation to gene expression in joint tissues, particularly the meniscus. Here, we test the hypothesis that patient age and body mass index (BMI) correlate with the expression of osteoarthritis- and obesity-related gene signatures in the meniscus., Design: Meniscus was obtained from patients (N=68) undergoing arthroscopic partial meniscectomy. The mRNA expression of 24 osteoarthritis-related and 4 obesity-related genes in meniscus was assessed by quantitative real-time PCR. The relationship between gene expression and patient age and BMI was analyzed using Spearman's rank-order correlation. Hierarchical cluster dendrogram and heat map were generated to study inter-gene associations., Results: Age was negatively correlated (P<0.05) with the expression of MMP-1 (r=-0.447), NFκB2 (r=-0.361), NFκBIA (r=-0.312), IκBA (r=-0.308), IL-8 (r=-0.305), ADAMTS-4 (r=-0.294), APLN (apelin) (r=-0.250) and IL-6 (r=-0.244). Similarly, BMI was negatively correlated with the expression of APLN (r=-0.328), ACAN (r=-0.268) and MMP-1 (r=-0.261). After adjusting for the correlation between age and BMI (r=0.310; P=0.008), the only independent effect of BMI on gene expression was for APLN (r=-0.272). However, age had an independent effect on the expression on ADAMTS-4 (r=-0.253), MMP-1 (r=-0.399), IL-8 (r=-0.327), COL1A1 (r=-0.287), NFκBIA (r=-0.278), NFκB2 (r=-0.312) and IκBA (r=-0.299). The gene correlation analysis identified four clusters of potentially relevant genes: transcription factors, matrix-degrading enzymes, cytokines and chemokines, and obesity genes., Conclusion: Age and BMI were negatively correlated with several osteoarthritis- and obesity-related genes. Although the bulk of these changes appeared to be driven by age, expression of APLN was related to BMI. Inter-gene correlation analysis implicated a common role for strongly correlated genes. Although age-related variations in gene expression appear to be more relevant than obesity-related differences for the role of the meniscus in osteoarthritis development, further investigation into the role of APLN in meniscus and joint health is warranted.

Background: Femoroacetabular impingement is a frequent cause of hip pain and may lead to secondary osteoarthritis, yet little is known about the molecular events linking mechanical hip impingement and articular cartilage degeneration. The first goal of this study was to quantify the expression of inflammatory cytokine and chemokine, matrix-degrading, and extracellular matrix genes in articular cartilage harvested from control hips and hips with femoroacetabular impingement and end-stage osteoarthritis. The second goal was to analyze the relative expression of these genes in articular cartilage harvested at various stages of osteoarthritis., Methods: Cartilage samples were obtained from thirty-two hips undergoing hip preservation surgery for femoroacetabular impingement or hip arthroplasty. Three control cartilage samples were also analyzed. Specimens were graded intraoperatively with regard to the severity of cartilage damage, the radiographic osteoarthritis grade was recorded, and quantitative RT-PCR (real-time polymerase chain reaction) was performed to determine relative gene expression., Results: Except for interleukin-1β (IL-1β) and CXCL2, the mRNA (messenger RNA) expression of all other chemokine (IL-8, CXCL1, CXCL3, CXCL6, CCL3, and CCL3L1), matrix-degrading (matrix metalloproteinase [MMP]-13 and ADAMTS-4), and structural matrix (COL2A1 [collagen, type II, alpha] and ACAN [aggregan]) genes was higher overall in cartilage from hips with femoroacetabular impingement compared with hips with osteoarthritis and normal controls. The differences reached significance (p ≤ 0.05) for seven of these ten quantified genes, with CXCL3, CXCL6, and COL2A1 being elevated in the femoroacetabular impingement group compared with only the control group and IL-8, CCL3L1, ADAMTS-4, and ACAN being elevated compared with both the osteoarthritis and control groups. When samples were grouped according to the stage of the degenerative cascade, mRNA expression was relatively higher in one of the two middle stages of femoroacetabular impingement (chondromalacia or cleavage/thinning), with the difference reaching significance for IL-8, CXCL2, CXCL3, CCL3L1, and ACAN. ACAN expression was diminished in hips with osteoarthritis compared with femoroacetabular impingement but elevated compared with the control articular cartilage., Conclusions: Articular cartilage from the impingement zone of hips with femoroacetabular impingement (and particularly those hips in the cleavage/thinning stage) expressed higher levels of certain inflammatory, anabolic, and catabolic genes, representing a heightened metabolic state., Clinical Relevance: The articular cartilage from the impingement zone of hips with femoroacetabular impingement was metabolically hyperactive, supporting the concept that such impingement is a structural precursor to hip osteoarthritis.

Objective: Emerging evidence suggests that genetic components contribute significantly to cartilage degeneration in osteoarthritis pathophysiology, but little information is available on the genetics of cartilage regeneration. Therefore, this study was undertaken to investigate cartilage regeneration in genetic murine models using common inbred strains and a set of recombinant inbred (RI) lines generated from LG/J (healer of ear wounds) and SM/J (nonhealer) inbred mouse strains., Methods: An acute full-thickness cartilage injury was introduced in the trochlear groove of 8-week-old mice (n=265) through microsurgery. Mouse knee joints were sagittally sectioned and stained with toluidine blue to evaluate regeneration. For the ear wound phenotype, a bilateral 2-mm through-and-through puncture was created in 6-week-old mice (n=229), and healing outcomes were measured after 30 days. Broad-sense heritability and genetic correlations were calculated for both phenotypes., Results: Time-course analysis of the RI mouse lines showed no significant regeneration until 16 weeks after surgery; at that time, the strains could be segregated into 3 categories: good, intermediate, and poor healers. Analysis of heritability (H2) showed that both cartilage regeneration (H2=26%; P=0.006) and ear wound closure (H2=53%; P<0.00001) were significantly heritable. The genetic correlations between the two healing phenotypes for common inbred mouse strains (r=0.92) and RI mouse lines (r=0.86) were found to be extremely high., Conclusion: Our findings indicate that articular cartilage regeneration in mice is heritable, the differences between the mouse lines are due to genetic differences, and a strong genetic correlation between the two phenotypes exists, indicating that they plausibly share a common genetic basis. We therefore surmise that LG/J by SM/J intercross mice can be used to dissect the genetic basis of variation in cartilage regeneration., (Copyright © 2012 by the American College of Rheumatology.)

Introduction: Little evidence is available on the natural course of osteoarthritis (OA) development and the genes that protect and predispose individuals to it. This study was designed to compare strain-dependent development of OA and its association with tissue regeneration in mice. Two recombinant inbred lines LGXSM-6 and LGXSM-33 generated from LG/J and SM/J intercross were used. Previous studies indicated that LGXSM-6 can regenerate both articular cartilage and ear hole punch while LGXSM-33 cannot., Methods: Transection of the medial meniscotibial ligament was performed on 10-week-old male mice to induce OA. Cartilage damage was analyzed by histology and bone morphology was evaluated using micro-computed tomography (CT). Ear punches were performed and evaluated by measurement of residual hole diameter., Results: Cartilage analysis showed that LGXSM-33 developed a significantly higher grade of OA than LGXSM-6. Bone analysis showed that LGXSM-33 had substantial subchondral bone and trabecular bone thickening 8 weeks post-surgery, while LGXSM-6 showed bone loss over time. We also confirmed that LGXSM-6 can heal ear tissues significantly better than LGXSM-33., Conclusions: OA was found to be negatively correlated with the degree of tissue regeneration. LGXSM-33, a poor healer of ear tissues (and articular cartilage), developed more OA compared to LGXSM-6, which had better regenerative ability for ear tissues and articular cartilage. The phenotypic differences observed here are due to genetic differences further suggesting that similar sets of physiological processes and gene variants may mediate variation in OA development and tissue regeneration., (Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To elucidate the effects of resistin on human articular chondrocytes and to generate a picture of their regulation at the transcriptional and posttranscriptional levels., Methods: Human articular chondrocytes were cultured with resistin. Changes in gene expression were analyzed at various doses and times. Cells were also treated with the transcription inhibitor actinomycin D after resistin treatment or with the NF-kappaB inhibitor IKK-NBD before resistin treatment. Gene expression was tested by quantitative real-time polymerase chain reaction. Computational analysis for transcription factor binding motifs was performed on the promoter regions of differentially expressed genes. TC-28 chondrocytes were transfected with CCL3 and CCL4 promoter constructs, pNF-kappaB reporter, and NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) expression vectors with or without resistin., Results: Resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. Levels of messenger RNA (mRNA) for matrix metalloproteinase 1 (MMP-1), MMP-13, and ADAMTS-4 also increased, while type II collagen alpha1 (COL2A1) and aggrecan were down-regulated. The cytokine and chemokine genes could be categorized into 3 groups according to the pattern of mRNA expression over a 24-hour time course. One pattern suggested rapid regulation by mRNA stability. The second and third patterns were consistent with transcriptional regulation. Computational analysis suggested the transcription factors NF-kappaB and C/EBPbeta were involved in the resistin-induced up-regulation. This prediction was confirmed by the cotransfection of NF-kappaB and C/EBPbeta and the IKK-NBD inhibition., Conclusion: Resistin has diverse effects on gene expression in human chondrocytes, affecting chemokines, cytokines, and matrix genes. Messenger RNA stabilization and transcriptional up-regulation are involved in resistin-induced gene expression in human chondrocytes.

This study has assessed the relative proportions of type I and II collagens and IIA procollagen in full depth biopsies of repair tissue in a large sample of patients treated with autologous chondrocyte implantation (ACI). Sixty five full depth biopsies were obtained from knees of 58 patients 8-60 months after treatment by ACI alone (n=55) or in combination with mosaicplasty (n=10). In addition articular cartilage was examined from eight individuals (aged 10-50) as controls. Morphology and semi-quantitative immunohistochemistry for collagen types I and II and procollagen IIA in the repair tissue were studied. Repair cartilage thickness was 2.89+/-1.5 mm and there was good basal integration between the repair cartilage, calcified cartilage and subchondral bone. Sixty five percent of the biopsies were predominantly fibrocartilage (mostly type I collagen and IIA procollagen), 15% were hyaline cartilage (mostly type II collagen), 17% were of mixed morphology and 3% were fibrous tissue (mostly type I collagen). Type II collagen and IIA procollagen were usually found in the lower regions near the bone and most type II collagen was present 30-60 months after treatment. The presence of type IIA procollagen in the repair tissue supports our hypothesis that this is indicative of a developing cartilage, with the ratio of type II collagen:procollagen IIA increasing from <2% in the first two years post-treatment to 30% three to five years after treatment. This suggests that cartilage repair tissue produced following ACI treatment, is likely to take some years to mature.

Articular cartilage deterioration commonly occurs following traumatic joint injury. Patients with posttraumatic osteoarthritis (PTA) experience pain and stiffness in the involved joint causing limited mobility and function. The mechanism by which PTA occurs has not been fully delineated. The goal of this study was to determine if a single high-energy impact load could cause the development of PTA in 3-month-old NZ White rabbits. Each rabbit underwent the application of a single, rapid, high-energy impact load to the posterior aspect of their right medial femoral condyle using a previously validated mechanism. At regular intervals (0, 1, 6 months) the injured cartilage was harvested and analyzed for the presence of PTA. Each specimen was assessed histologically for cell and tissue morphology and chondrocyte metabolism, including BMP-2 production and synthesis of extracellular matrix (type II procollagen mRNA). Cartilage from the contralateral sham limb, as well as uninjured cartilage from the experimental limb served as internal controls for each animal. Significant changes were found in the morphology of the cartilage including proteoglycan loss along with decreased BMP-2 and type II procollagen mRNA staining. These findings confirm that a single high-energy impact load can cause the development of PTA by disrupting the extracellular matrix and by causing a decrease in chondrocyte metabolism., ((c) 2008 Orthopaedic Research Society.)

Objective: To provide a more complete picture of the effect of interleukin-1 beta (IL-1beta) on adult human articular chondrocyte gene expression, in contrast to the candidate gene approach., Design: Chondrocytes from human knee cartilage were cultured in medium containing IL-1beta. Changes in gene expression were analyzed by microarray and reverse transcriptase-polymerase chain reaction analysis. The ability of transforming growth factor beta-1 (TGF-beta1), fibroblast growth factor (FGF)-18, and bone morphogenetic protein 2 (BMP-2) to alter the effects of IL-1beta was analyzed. Computational analysis of the promoter regions of differentially expressed genes for transcription factor binding motifs was performed., Results: IL-1beta-treated human chondrocytes showed significant increases in the expression of granulocyte colony stimulating factor-3, endothelial leukocyte adhesion molecule 1 and leukemia inhibitory factor as well as for a large group of chemokines that include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL8, CCL20, CCL3L1, CX3CL1 and the cytokine IL-6. As expected, the mRNA for matrix metalloproteinase (MMP)-13 and BMP-2 also increased while mRNA for the matrix genes COL2A1 and aggrecan was down-regulated. A subset of chemokines increased rapidly at very low levels of IL-1beta. The phenotype induced by IL-1beta was partially reversed by TGF-beta1, but not by BMP-2. In the presence of IL-1beta, FGF-18 increased expression of ADAMTS-4, aggrecan, BMP-2, COL2A1, CCL3, CCL4, CCL20, CXCL1, CXCL3, CXCL6, IL-1beta, IL-6, and IL-8 and decreased ADAMTS-5, MMP-13, CCL2, and CCL8. Computational analysis revealed a high likelihood that the most up-regulated chemokines are regulated by the transcription factors myocyte enhancer binding factor-3 (MEF-3), CCAAT/enhancer binding protein (C/EBP) and nuclear factor-kappa B (NF-kappaB)., Conclusion: IL-1beta has a diverse effect on gene expression profile in human chondrocytes affecting matrix genes as well as chemokines and cytokines. TGF-beta1 has the ability to antagonize some of the phenotype induced by IL-1beta.

Objective: Oxidative stress occurs when the metabolic balance of a cell is disrupted through exposure to excess pro-oxidant. Whilst it is known that unregulated production or exposure to exogenous sources of pro-oxidants induces chondrocyte cell death and degrades matrix components in vitro, relatively little is known of the effects of pro-oxidants on articular cartilage in situ. The objective of this study was to determine if a single exposure to the pro-oxidant hydrogen peroxide (H(2)O(2)) induces a degenerative phenotype., Methods: Articular cartilage explants were obtained from skeletally mature bovine steers and exposed to a single dose of hydrogen peroxide (0.1-1.0 mM) and cultured for up to 21 days. Cell death, and sulfated glycosaminoglycan loss into the medium and gene expression were quantitatively determined. Adoption of an abnormal chondrocyte phenotype was analyzed through the expression of 3B3(-), nitrotyrosine and procollagen type IIA epitopes in cartilage explants., Results: Cell death occurred primarily at the surface zone of cartilage in a dose-dependent manner in H(2)O(2) treated explants, and supplementation of standard serum-free medium with insulin-selenium-transferrin significantly reduced cell death (>fourfold). Nitric oxide synthase-2 gene expression and proteoglycan loss increased in oxidant treated explants in a concentration-dependent manner. Antibody labeling to 3B3(-), procollagen type IIA and nitrotyrosine was present in all treated explants but absent in untreated explants., Conclusions: This study demonstrates that a single exposure to high levels of pro-oxidant causes the expression of genes and antibody epitopes that are associated with early degenerative changes observed in experimental osteoarthritis.

While a great deal of information is available on the cellular and molecular biology of cartilage degradation, less is known about anabolism in normal cartilage and degenerating cartilage. A consistent amount of evidence is now available on the neo-synthesis of matrix molecules and enzymes in OA: the entire cell metabolism appears to be increased leading to the hypothesis that chondrocytes in OA are actually "activated". This chapter will focus on anabolic events that are now known to occur in articular cartilage. We will begin to view articular cartilage as a complex three-dimensional tissue in which local events may be different. We will also be interested in viewing the development of degenerative arthritis as a continuum from functionally normal tissue to degeneration.

We have measured the concentration of cartilage-derived retinoic-acid-sensitive protein (CD-RAP) in synovial fluid (SF) from the knees of 49 patients with osteoarthritis (OA) and 79 with rheumatoid arthritis (RA) in order to investigate the correlation between the type of joint disease and level of CD-RAP. The mean concentration of CD-RAP in synovial fluid was significantly higher in OA than in RA. The level of CD-RAP in the group of patients with mild OA was significantly higher than in the moderate or severe groups and that in the group with mild RA was also significantly higher than in the other RA groups and decreased with progression of the disease. Immunohistochemical studies showed expression of CD-RAP in the cytoplasm of chondrocytes in newly-formed fibrocartilage. Since CD-RAP is mainly produced in young and proliferating chondrocytes, our results suggest that the level of CD-RAP in synovial fluid reflects remodelling of articular cartilage and may be used as a marker to estimate objectively the restorative reaction of chondrocytes.

The control of extracellular matrix (ECM) production is important for the development, maintenance, and repair of cartilage tissues. Matrix molecule synthesis is generally regulated by the rate of gene transcription determined by DNA transcription factors. We have shown that transcription factors Sox9, AP-2, and [delta]EF1 are able to alter the rate of CD-RAP transcription in vitro: Sox9 upregulates, AP-2 exhibits biphasic effects, and [delta]EF1 represses expression of the CD-RAP gene. To correlate these in vitro activities in vivo, transcription factors were co-immunolocalized with ECM proteins in three different cartilage tissues in which the rates of biosynthesis are quite different: articular, meniscal, and growth plate. Immunoreactivities of type II collagen and CD-RAP were higher in growth plate than in either the articular or meniscal cartilages and correlated positively with Sox9 protein. Sox9 staining decreased with hypertrophy and was low in articular and meniscal cartilages. In contrast, AP-2 and [delta]EF1 were low in proliferating chondrocytes but high in lower growth plate, articular, and meniscal cartilages. This increase was also accompanied by intense nuclear staining. These immunohistochemical results are the first to localize both [delta]EF1 and AP-2 to adult articular, meniscal, and growth plate cartilages and provide in vivo correlation of previous molecular biological studies.

Cartilage is comprised of a large amount of functional extracellular matrix that is made and maintained by a small number of chondrocytes, the sole resident cell type. Normal cartilage exists in a relatively steady state: that is, the anabolic processes (those that result in the synthesis of cartilage matrix components) are in equilibrium with the catabolic processes (those that result in the normal turnover of matrix molecules). If the functional extracellular matrix is disturbed by physical or molecular means, the cells respond in an attempt to repair the matrix. This stimulated activity does not result in repair due to the extent and complexity of the extracellular matrix. Eventually, the newly synthesized and activated catabolic enzymes degrade the matrix components. This review presents the cellular and molecular mechanisms that account for this activity and provides some possible solutions.

This study evaluated the constitutive insulin-like growth factor-I (IGF-I) gene expression pattern in spontaneously healing cartilage defects over the course of 16 weeks, and correlated the tissue morphology and matrix gene expression with IGF-I mRNA levels. Full-thickness 15 mm cartilage defects were debrided in the femoral trochlea of both femoropatellar joints of 8 horses and the healing defects examined 2, 4, 8, or 16 weeks after surgery. Samples were harvested for histologic assessment of tissue healing using H&E staining, toluidine blue histochemical reaction for proteoglycan deposition, and in situ hybridization and immunohistochemistry procedures to demonstrate collagen type II mRNA and protein expression. Total RNA was isolated for Northern analysis to measure cartilage matrix molecule expression, and for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) to determine IGF-I gene expression patterns in healing cartilage defects. Full-thickness cartilage defects in horses were slow to heal compared to smaller lesions in similar locations in other animals. However, a progressive decline in tissue cellularity and vascularity, and increased tissue organization were observed on H&E stained specimens over the 16-week experiment. Evidence of early chondrogenic repair was detected through collagen type II in situ hybridization and immunohistochemistry. However, levels of collagen type II and aggrecan mRNA in lesions were not abundant on Northern analysis indicating incomplete chondrogenesis. IGF-I message expression followed a cyclic pattern with low levels at 2 weeks, followed by an increase at 4 and 8 weeks, and a subsequent decline at 16 weeks. There was no direct correlation between the stage of healing and cartilage matrix message expression, and the abundance of IGF-I mRNA in the healing lesions. In conclusion, this study demonstrated that the spontaneous healing of articular defects was accompanied by a temporal fluctuation in IGF-I gene expression which was discoordinate to the steady rise in expression of cartilage matrix molecules such as procollagen type II.

Study Design: The expression of cartilage-derived retinoic acid-sensitive protein (CD-RAP) was measured in cerebrospinal fluid from patients with spinal diseases., Objectives: To quantify the levels of CD-RAP in human cerebrospinal fluid and to clarify its character., Summary of Background Data: Cartilage-derived retinoic acid-sensitive protein is a newly discovered, secreted molecule that is expressed during the chondrogenesis phase of endochondral bone formation and in articular cartilage. In recent studies CD-RAP has been detected in the serum of patients with melanoma and breast cancer, and it has been used to monitor tumor activity. However, the function of CD-RAP is unknown, and the expression of CD-RAP in human cerebrospinal fluid has never been reported., Methods: The concentration of CD-RAP in human cerebrospinal fluid was measured by enzyme-linked immunosorbent assay with antihuman CD-RAP antibodies. Cerebrospinal fluid samples were collected from two groups of patients. Group 1, the control group, consisted of 40 patients: 22 with trauma and 18 with gynecologic diseases. Group 2 consisted of 172 patients with spinal diseases: 5 with meningioma, 5 with neurinoma, 5 with arachnoid cyst, 30 with cervical spondylotic myelopathy, 35 with lumbar disc herniation, 56 with lumbar canal stenosis, and 36 with scoliosis., Results: The concentration of CD-RAP in the control group was 16.5 +/- 8.3 ng/mL. The concentrations of CD-RAP in Group 2 were: 35.3 +/- 14.7 ng/mL in meningioma, 23.5 +/- 7.41 ng/mL in neurinoma, 26.0 +/- 22.2 ng/mL in arachnoid cyst, 41.7 +/- 22.3 ng/mL in cervical myelopathy, 27.8 +/- 14.7 ng/mL in lumbar disc herniation, 36.5 +/- 18.4 ng/mL in lumbar canal stenosis, and 13.4 +/- 7.48 ng/mL in scoliosis. The concentrations of CD-RAP in cervical myelopathy, lumbar canal stenosis, and lumbar disc herniation were significantly higher than in the control group (P < 0.001)., Conclusions: The CD-RAP concentration was low in the control group, whereas it was significantly higher in spinal diseases that cause spinal stenosis. CD-RAP is expressed in cerebrospinal fluid as a result of damage to or stressing of neural structures and could be a marker for spinal diseases.

The reaction patterns of chondrocytes in osteoarthritis can be summarized in five categories: (1) proliferation and cell death (apoptosis); changes in (2) synthetic activity and (3) degradation; (4) phenotypic modulation of the articular chondrocytes; and (5) formation of osteophytes. In osteoarthritis, the primary responses are reinitiation of synthesis of cartilage macromolecules, the initiation of synthesis of types IIA and III procollagens as markers of a more primitive phenotype, and synthesis of active proteolytic enzymes. Reversion to a fibroblast-like phenotype, known as "dedifferentiation", does not appear to be an important component. Proliferation plays a role in forming characteristic chondrocyte clusters near the surface, while apoptosis probably occurs primarily in the calcified cartilage.

Objective: To test for the reexpression of the chondroprogenitor splice variant of the gene COL2A1, type IIA procollagen (containing a cysteine-rich NH2 propeptide), in adult articular chondrocytes in osteoarthritic (OA) joint disease., Methods: In situ hybridization and immunohistochemical localization were performed on normal and OA articular cartilage specimens. The presence of type IIA procollagen messenger RNA (mRNA) expression was confirmed by Northern blot analysis., Results: In normal articular cartilage, no expression of mRNA or presence of type IIA procollagen was found. In OA articular cartilage, focally intense staining for type IIA protein was detected. Consistent with this, chondrocytes, particularly in the middle zones of articular cartilage, expressed type IIA procollagen mRNA. OA repair cartilage typically showed a broad zone of cells expressing type IIA mRNA and protein., Conclusion: Type IIA procollagen is reexpressed by adult articular chondrocytes in OA cartilage degeneration, indicating the potential reversion of the cells to a chondroprogenitor cellular phenotype. The absence of type IIA mRNA and protein in normal adult articular cartilage and its onset in the diseased state suggests type IIA procollagen as a marker of OA.

Objective: To define the relative contributions of procollagen IIA and IIB messenger RNA (mRNA) in the 8-fold elevation of total (IIA + IIB) type II procollagen mRNA in cartilage from joints with early experimental osteoarthritis (OA)., Methods: Unilateral cruciate ligament transection was performed on skeletally mature animals. Total RNA was extracted from articular cartilage from control joints and from joints with experimental OA. Northern blot hybridization with oligoprobes was used to distinguish between types IIA and IIB mRNA., Results: Levels of IIA mRNA were similar in control and experimental cartilage. Type IIB mRNA accounted for the increase in total type II mRNA expression in OA joints., Conclusion: Although OA chondrocytes adapt to making immature forms of aggrecan proteoglycan, they do not revert to the prechondrocyte stage characterized by IIA mRNA expression.

Retinoic acid (RA) is known to play a role in various aspects of skeletal development in vivo, including morphogenesis, growth plate maturation, and apoptosis. In cell culture, RA treatment of chondrocytes suppresses the differentiated phenotype characterized by production of type II collagen and aggrecan. In an effort to discover molecules involved in regulation of the chondrocyte phenotype or related to developmental processes such as chondrogenesis, mRNAs from bovine chondrocytes cultured with and without RA were amplified by reverse transcription-polymerase chain reaction (PCR) and compared by differential display. PCR products whose expression was inhibited by RA treatment were cloned. One cDNA encodes a molecule we call cartilage-derived retinoic acid-sensitive protein (CD-RAP), and its properties are described here. The full-length bovine CD-RAP mRNA was cloned after amplification by the rapid amplification of cDNA ends procedure, and a part of the rat CD-RAP mRNA was amplified by reverse transcription-PCR using sequence-specific primers. The bovine CD-RAP mRNA contains an open reading frame of 130 amino acids. CD-RAP mRNA expression, as determined by Northern blot analysis and in situ hybridization, was present only in cartilage primordia and cartilage. The inhibition of CD-RAP mRNA expression by RA in vitro was time- and dose-dependent and was tested over concentrations from 10(-8) to 10(-6) M. Southern blot analysis of genomic DNA indicated that CD-RAP was encoded by a single copy gene and that no other genes were closely related. What appears to be the human homologue of CD-RAP was recently isolated and cloned from a melanoma cell line and shown to function as a growth inhibitory protein (Blesch, A., Boberhoff, A.-K., Apfel, R., Behl, C., Hessdoerfer, B., Schmitt, A., Jachimcza, P., Lottspeich, F., Buettner, R., and Bogdahn, U. (1994) Cancer Res. 54, 5695-5701). Neither CD-RAP nor this protein showed any homology to known proteins. We speculate that, in vivo, CD-RAP functions during cartilage development and maintenance.

This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum. mRNAs for aggrecan, types I and II collagen, and beta-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by 35SO4 uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.

Objective: To quantify the gene expression of aggrecan core protein and type II collagen in an experimental animal model of osteoarthritis (OA)., Methods: Total RNA was extracted from the articular cartilage of unoperated knee joints and from OA joints produced by anterior cruciate ligament transection. The relative amounts of type II collagen and aggrecan core protein messenger RNA (mRNA) were evaluated by Northern blot analysis., Results: Total RNA was elevated 2.5 times, aggrecan mRNA was elevated 2 times, and type II collagen mRNA was elevated 8 times, in OA knees compared to unoperated controls., Conclusion: Chondrocytes are activated metabolically in response to joint injury. Discoordinate gene expression of aggrecan and type II collagen is characteristic of early experimental OA, and we speculate that it may contribute to its pathogenesis.

To define the influence of time in culture on gene expression for extracellular matrix proteins we have examined the progression of changes in gene expression for chondrocyte extracellular matrix proteins from the time the chondrocytes are initially isolated from their native cartilaginous matrix through 13 days of high-density culture. We have also determined the effect of matrix depletion and shape change by enzymatically resuspending cells after 6 days in culture and sampling the replated cells at intervals up to 13 days. Northern blots of chondrocyte RNA were hybridized with probes for collagen alpha 1(II), alpha 1(I), aggrecan, link protein, and decorin mRNA. The steady-state level of alpha 1(II) collagen mRNA dropped to 45% of the initial value within 24 h, with a further decrease to 21% by Day 3. A similar decline occurred, but less rapidly in ascorbate supplemented cultures with values of 78%, at 24 h and 62% at Day 3. Very low levels of alpha 1(I) collagen mRNA were detectible in cells maintained for 2 weeks without ascorbate supplementation. Type I collagen alpha 1(I) mRNA was not detected in freshly isolated chondrocytes or at the earliest times in culture but was increasingly abundant from Days 5-13 in the presence of ascorbate. Ascorbic acid supplementation altered the pattern of aggrecan expression over time. Without ascorbate there was an increase in steady-state aggrecan mRNA with time in culture, but in the presence of ascorbate, aggrecan mRNA levels peaked at early culture times and progressively diminished. Decorin steady-state mRNA levels in cultures not supplemented with ascorbic acid steadily increased over time in culture following a lag of several days. In cultures treated with ascorbate, however, there was a progressive increase in decorin steady-state mRNA levels from the first day in culture. Resuspending chondrocytes by digestion of the cell layer with pronase and collagenase at Day 6, which resulted in a transient shape change as well as matrix depletion, resulted in a greater than 2-fold increase in alpha 1(II) mRNA at Day 7 in ascorbate supplemented cultures. Only with ascorbate was there a small increase in decorin mRNA at Day 7, after resuspension. Aggrecan mRNA, however, showed a 3-fold increase without ascorbate and a 10-fold increase with ascorbate within 24 h of resuspension. Similarly, link protein steady-state mRNA showed an 8-fold increase without ascorbate and a 9-fold increase with ascorbate within 24 h after resuspension.

We have developed a method to isolate RNA in high yield from adult articular cartilage. Homogenization of the articular cartilage with a freezer mill, extraction with 4 M guanidinium isothiocyanate/acid-phenol, and ultracentrifugation in cesium trifluoroacetate was found to be an effective and practical method for isolating a high yield of intact RNA from adult canine articular cartilage. The total RNA was suitable for Northern blot analysis. The mRNA that could then be isolated by oligo-dT affinity chromatography was found to be a suitable substrate for in vitro translation, for making a cDNA library, and for PCR amplification.

Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two procollagens was examined in order to establish a potential functional significance for the two type II procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II procollagens. Secondly, to test the hypothesis that differential expression of type II procollagens may be a marker for a distinct population of cells, specific procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of type II collagen, primarily type IIA, are also expressed in embryonic spinal ganglion. While small amounts of type II collagen have been previously detected in noncartilagenous tissues, the detection of this new form of the collagen in relatively high abundance in embryonic nerve tissue is unique. Taken together, these findings imply a potential functional difference between type IIA and type IIB procollagens and indicate that the removal of exon 2 from the pre-mRNA, and consequently the NH2-propeptide from the collagen molecule, may be an important step in chondrogenesis. In addition, type II procollagen, specifically type IIA, may function in noncartilage tissues, particularly during development.

We have studied the biosynthesis of cartilage dermatan sulfate proteoglycan II (DS-PGII) (decorin) using in vitro translation of mRNA to determine the size of the primary gene product and by radiolabeling the protein in the presence of tunicamycin to inhibit the addition of Asn-linked oligosaccharides. Pulse-chase experiments were performed to examine post-translational processing and secretion. Inhibitors of oligosaccharide processing were used to determine whether DS-PGII molecules containing partially processed oligosaccharides could become proteoglycans and be secreted. Cell-free translation of sucrose gradient-fractionated RNA and subsequent immunoprecipitation of the core protein confirmed that the functional translated mRNA is in the size range of the two mRNA species observed by hybridization of chondrocyte RNA with a bone PGII cloned probe and that the translation product is a single protein with an apparent molecular mass of 42 kDa. Digestion of the intact proteoglycan (average molecular mass = 103 kDa) with chondroitinase ABC or AC results in an approximately 48-49-kDa product. Chondrocytes treated with tunicamycin to inhibit Asn-linked oligosaccharide addition synthesize and secrete a glycosaminoglycan (GAG)-substituted proteoglycan (average molecular mass = 86 kDa), yielding a 42-kDa core protein after chondroitinase ABC digestion, showing that Asn-linked oligosaccharides are not required for the addition of GAG chains or secretion. Following a short pulse (10 min) of [3H]leucine, three glycosylated forms of the DS-PGII core protein were observed, one of which is likely to be the precursor form of PGII predicted by the implied protein sequence of both bovine and human cDNA clones. Following the apparent cleavage of the propeptide, GAG-substituted intracellular core protein is detectable. Susceptibility to endoglycosidase H indicates that approximately one-third of the secreted core protein contains exclusively complex-type Asn-linked oligosaccharides and approximately two-thirds contain high mannose as well as complex-type oligosaccharides. Secreted DS-PGII appears to be fully substituted with three Asn-linked oligosaccharide chains. Inhibitors of oligosaccharide processing, however, permitted secretion of GAG-substituted DS-PGII that was fully (three chains) or incompletely (one or two chains) substituted with partially processed Asn-linked carbohydrate chains. By comparison of chondrocyte DS-PGII with fibroblast DS-PGII, we conclude that the addition and processing of Asn-linked carbohydrate chains are directed by the amino acid sequence of the core protein. The results reported here also suggest that the addition of xylose, the initial step in GAG chain synthesis, occurs early in biosynthesis and is determined by the primary amino acid sequence of the core protein.(ABSTRACT TRUNCATED AT 400 WORDS)

We have examined posttranslational modifications which are responsible for converting an apparently single precursor (Hering, T. M., and Sandell, L. J. (1988) J. Biol. Chem. 263, 1030-1036) to the two major forms of link protein in bovine articular cartilage. Resistance to endoglycosidases H and F suggests that Asn-linked oligosaccharides of link protein secreted by bovine chondrocytes in culture are of the complex or hybrid type. There is no evidence for O-linked oligosaccharides. There is no apparent precursor-product relationship between link protein (LP)1 and LP2, since after a short pulse with [3H]leucine two forms are present, consistent with the existence of two glycosylation sites. An immunoprecipitate of LP1 from pulse-labeled chondrocytes was observed to show a decrease in electrophoretic mobility and increased microheterogeneity during transit through the Golgi, whereas LP2 did not change. During processing both LP1 and LP2 become endoglycosidase H resistant. LP1, but not LP2, can be biosynthetically labeled with [35S]sulfate. Incorporation of [35S]sulfate is inhibited by tunicamycin, indicating that the sulfate is associated with Asn-linked carbohydrate. Sulfation may be important for normal processing, secretion, or degradation of link protein and with sialylation may confer considerable charge heterogeneity upon LP1. We conclude that there are considerable biochemical differences between glycoproteins LP1 and LP2 which may provide a basis for functional differences.

Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of procollagens, the steady state levels of types I and II procollagen mRNAs, and the expression of major histocompatibility complex class II (Ia-like) antigens on the cell surface. Adult articular chondrocytes synthesized mainly type II collagen during weeks 1-3 of primary culture, whereas types I and III collagens were also produced after longer incubation and predominated after the first subculture. Juvenile costal chondrocytes synthesized no detectable alpha 2(I) collagen chains until after week 1 of primary culture; type II collagen was the predominant species even after weeks of culture. The relative amounts of types I and II collagens synthesized were reflected in the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. In articular chondrocytes, the levels of alpha 1(I) procollagen mRNA were disproportionately low (alpha 1(I)/alpha 2(I) less than 1.0) compared with costal chondrocytes (alpha 1 (I)/alpha 2(I) approximately 2). Recombinant IFN-gamma (0.1-100 units/ml) inhibited synthesis of type II as well as types I and III collagens associated with suppression of the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. IFN-gamma suppressed the levels of alpha 1(I) and alpha 1(II) procollagen mRNAs to a greater extent than alpha 2(I) procollagen mRNA in articular but not in costal chondrocytes. Human leukocyte interferon (IFN-alpha) at 1000 units/ml suppressed collagen synthesis and procollagen mRNA levels to a similar extent as IFN-gamma at 1.0 unit/ml. In addition, IFN-gamma but not IFN-alpha induced the expression of HLA-DR antigens on intact cells. The lymphokine IFN-gamma could, therefore, have a role in suppressing cartilage matrix synthesis in vivo under conditions in which the chondrocytes are in proximity to T lymphocytes and their products.