An immunohistochemical study of matrix components in primary and secondary cartilages of embryonic chick skull.

OBJECTIVES: This study aimed to compare the extracellular matrix of primary cartilage with the secondary cartilage of chicks using immunohistochemical analyses in order to understand the features of chick secondary chondrogenesis. METHODS: Immunohistochemical analysis was performed on the extracellular matrix of quadrate (primary), squamosal, surangular, and anterior pterygoid secondary cartilages using various antibodies targeting the extracellular matrix of cartilage and bone. RESULTS: The localization of collagen types I, II, and X, versican, aggrecan, hyaluronan, link protein, and tenascin-C was identified in the quadrate cartilage, with variations within and between the regions. Newly formed squamosal and surangular secondary cartilages showed simultaneous immunoreactivity for all molecules investigated. However, collagen type X immunoreactivity was not observed, and there was weak immunoreactivity for versican and aggrecan in the anterior pterygoid secondary cartilage. CONCLUSIONS: The immunohistochemical localization of extracellular matrix in the quadrate (primary) cartilage was comparable to that of long bone (primary) cartilage in mammals. The fibrocartilaginous nature and rapid differentiation into hypertrophic chondrocytes, which are known structural features of secondary cartilage, were confirmed in the extracellular matrix of squamosal and surangular secondary cartilages. Furthermore, these tissues appear to undergo developmental processes similar to those in mammals. However, the anterior pterygoid secondary cartilage exhibited unique features that differed from primary and other secondary cartilages, suggesting it is formed through a distinct developmental process.

Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes - Then and Now.

Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilage during movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as during the development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthritic diseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.

Nanomechanics of Aggrecan: A New Perspective on Cartilage Biomechanics, Disease and Regeneration.

Articular cartilage is a hydrated macromolecular composite mainly composed of type II collagen fibrils and the large proteoglycan, aggrecan. Aggrecan is a key determinant of the load bearing and energy dissipation functions of cartilage. Previously, studies of cartilage biomechanics have been primarily focusing on the macroscopic, tissue-level properties, which failed to elucidate the molecular-level activities that govern cartilage development, function, and disease. This chapter provides a brief summary of Dr. Alan J. Grodzinsky's seminal contribution to the understanding of aggrecan molecular mechanics at the nanoscopic level. By developing and applying a series of atomic force microscopy (AFM)-based nanomechanical tools, Grodzinsky and colleagues revealed the unique structural and mechanical characteristics of aggrecan at unprecedented resolutions. In this body of work, the "bottle-brush"-like ultrastructure of aggrecan was directly visualized for the first time. Meanwhile, molecular mechanics of aggrecan was studied using a physiological-like 2D biomimetic assembly of aggrecan on multiple fronts, including compression, dynamic loading, shear, and adhesion. These studies not only generated new insights into the development, aging, and disease of cartilage, but established a foundation for designing and evaluating novel cartilage regeneration strategies. For example, building on the scientific foundation and methodology infrastructure established by Dr. Grodzinsky, recent studies have elucidated the roles of other proteoglycans in mediating cartilage integrity, such as decorin and perlecan, and evaluated the therapeutic potential of biomimetic proteoglycans in improving cartilage regeneration.

Comprehensive analysis of chondroitin sulfate and aggrecan in the head cartilage of bony fishes: Identification of proteoglycans in the head cartilage of sturgeon.

Cartilage in the head of sturgeon or salmon has been gaining attention as a rich source of functional chondroitin sulfate (CS) or proteoglycans. Although the cartilage was found in the heads of other bony fishes, the structure of CS and its core protein, especially aggrecan, was not fully investigated. In this study, comprehensive analysis of CS and aggrecan in the head cartilage of 10 bony fishes including sturgeon and salmon was performed. The 4-O-sulfation to 6-O-sulfation ratio (4S/6S ratio; S: sulfate residue) of CS in Perciformes was ≧1.0, while the 4S/6S ratios of CS from sturgeons and salmon were less than 0.5. Dot blotting and proteomic analysis revealed that aggrecan was a major core protein in head cartilage of all bony fishes. These results suggest that the head cartilage of bony fishes is a promising source for the preparation of CS or proteoglycans as a health food ingredient.

Expanding the Spectrum of EWSR1-NFATC2-rearranged Benign Tumors: A Common Genomic Abnormality in Vascular Malformation/Hemangioma and Simple Bone Cyst.

A simple bone cyst (SBC) is a cystic bone lesion predominantly affecting young males. The cyst is lined by a fibrous membrane and filled with serosanguinous fluid. EWSR1/FUS-NFATC2 rearrangements were recently identified in SBC. We here report exactly the same rearrangement in 3 lesions diagnosed as vascular malformations of 2 elderly patients. In total, through Archer FusionPlex, fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction the EWSR1-NFATC2 rearrangement was identified in 6 of 9 SBC, 3 of 12 benign vascular tumors, and none of 5 aneurysmal bone cyst lacking USP6 fusion. Using fluorescence in situ hybridization, it was apparent that amplification of the fusion, as seen in EWSR1-NFATC2 round cell sarcomas, was absent, and that in the vascular tumors the fusion was present both in the lining cells as well as in the surrounding spindle cells. Of note, not all of the spaces in the vascular malformations were lined by endothelial cells. Aggrecan was positive in all cases but was not specific. NKX2-2 and NKX3-1 staining were negative in all cases. Thus, even though the overlap between the 2 entities is limited to the presence of few thick-walled cysts lacking endothelial lining in the benign vascular malformations, the spectrum of benign tumors containing NFATC2 fusions should be expanded and contains not only SBC in the young, but also vascular malformation/hemangioma in elderly patients.

Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Mechanism of TGF-ß3 promoting chondrogenesis in human fat stem cells.

Clinically deficient cartilage is difficult to regenerate, and the availability of chondrocytes is very limited. However, human adipose-derived stem cells (ADSCs) can be obtained easily and in sufficient quantities. Therefore, we will find a way of replacing chondrocytes with fat stem cells to solve the problem of seed cell origin. Previous studies have revealed that transforming growth factor-ß (TGF-ß) can promote chondrocyte differentiation and maturation. In this study, we found that TGF-ß3 in the transforming growth factor family can effectively promote the transformation process from fat stem cells to chondrocytes, thus promoting chondrogenesis. At the same time, we also further reviewed and considered the mechanism of this process. Through flow cytometry, immunohistochemical, fluorescent microscopy, qRCR, Wb etc., we found that TGF-ß3 mainly plays a role through wnt5a/ß-catenin, promoting human fat stem cell growing into the cartilage. This discovery is expected to provide new ideas in the field of cartilage regeneration.

The study of targeted blocking SDF-1/CXCR4 signaling pathway with three antagonists on MMPs, type II collagen, and aggrecan levels in articular cartilage of guinea pigs.

OBJECTIVE: To explore the possibility and mechanism of targeted blocking SDF-1/CXCR4 signaling pathway using three antagonists TN14003, T140, and AMD3100 in vivo, and to investigate the function of three antagonists in delay degeneration process of articular cartilage. METHODS: Ninety-six male Duncan-Hartley guinea pigs (6 months old) were divided into groups A, B, C, and D randomly. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group A, and TN14003 with concentration of 180 µg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group B, and T140 with concentration of 180 µg/ml was pumped every day. Alzet trace pump was implanted in the back subcutaneous tissue of pigs in group C, and AMD3100 with concentration of 180 µg/ml was pumped every day. Hartley guinea pigs in group D remained untreated as the blank control group. At 2, 4, 6, 8, 10, and 12 weeks of treatment, 5 to 8 animals in each group were randomly chosen for blood collection via cardiac puncture. SDF-1 content using enzyme-linked immunosorbent assay (ELISA). At 12 weeks, all guinea pigs were sacrificed by injecting pentobarbital sodium (30 mg/kg) into the peritoneal cavity. Cartilages from the tibial plateau in each group were harvested for PCR testing and western blot analysis. SPSS19.0 was used for data analysis. RESULTS: Result of ELISA: the serum levels of SDF-1 of groups A, B, and C decreased gradually with time. Significant drop of SDF-1 level was seen in group A while increased SDF-1 was shown in group D. At the same time, the serum levels of SDF-1 of the group A were significantly lower than that of group B; those of group B were significantly lower than that of group C, which was significantly lower than that of group D, and their difference is statistically significant (P < 0.05). Real time quantitative PCR result: The mRNA levels of MMPs in group A were significantly lower than group B, and those of group B were significantly lower than group C, which was significantly lower than group D, and there was statistically significant (P < 0.05). The mRNA levels of type II collagen, aggrecan in group A were significantly more than group B; those of group B were significantly more than group C, which was significantly more than group D, and the difference was statistically significant (P < 0.05). H&E staining result: cartilage of group C was more significantly degenerative than other groups. CONCLUSIONS: The three antagonists can target SDF-1/CXCR4 signaling pathway in vivo, reduce the expression and secretion of MMP-3, MMP-9, and MMP-13 in cartilage tissue, and reduce the degradation of collagen II and aggregating proteoglycan, thus delaying the degeneration of articular cartilage, of which TN14003 has the strongest regulatory effect. Targeted blockade of SDF-1/CXCR4 signaling pathway by TN14003 in vivo delays articular cartilage degeneration more effectively than T140 and AMD3100.

Abnormal expression of chondroitin sulfate sulfotransferases in the articular cartilage of pediatric patients with Kashin-Beck disease.

The objective of this study is to investigate the expression of enzymes involved in the sulfation of articular cartilage from proximal metacarpophalangeal (PMC) joint cartilage and distal metacarpophalangeal (DMC) joint cartilage in children with Kashin-Beck disease (KBD). The finger cartilage samples of PMC and DMC were collected from KBD and normal children aged 5-14 years old. Hematoxylin and eosin staining as well as immunohistochemical staining were used to observe the morphology and quantitate the expression of carbohydrate sulfotransferase 3 (CHST-3), carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), uronyl 2-O-sulfotransferase (UST), and aggrecan. In the results, the numbers of chondrocyte decreased in all three zones of PMC and DMC in the KBD group. Less positive staining cells for CHST-3, CHST-12, CHST-13, UST, and aggrecan were observed in almost all three zones of PMC and DMC in KBD. The positive staining cell rates of CHST-12 were higher in superficial and middle zones of PMC and DMC in KBD, and a significantly higher rate of CHST-13 was observed only in superficial zone of PMC in KBD. In conclusion, the abnormal expression of chondroitin sulfate sulfotransferases in chondrocytes of KBD children may provide an explanation for the cartilage damage, and provide therapeutic targets for the treatment.

Composition of Cell Clusters in Torn Menisci and Their Extracellular Matrix Components.

Cell clusters, or groups of cells sharing a single chondron-like structure, are frequently found in degenerated areas of the osteoarthritic (OA) meniscus. However, little is known about these meniscal clusters in humans. The aim of our study was to determine the composition of the extracellular matrix deposition around cell clusters in human OA menisci. Twenty-six menisci were obtained through total knee arthroplasty from patients with OA knee joints. The specimens were subjected to safranin O staining and immunostaining for Sry-type HMG box 9 (SOX9), type II collagen, and aggrecan. Their signal density after staining was assessed using ImageJ software. Five regions of interest were analyzed within each tissue sample. The SOX9, type II collagen, and aggrecan densities were considerably higher in cluster areas than in intact superficial layers of the meniscus. In addition, a substantial difference was detected between cluster areas and degenerative areas without cell clusters. We demonstrated that cell clusters localized near fissures and clefts showed remarkable uniformity in menisci exposed to a broad range of injuries. In addition, the chondrogenic proteins SOX9, type II collagen, and aggrecan were highly expressed in these tissues.

Aggrecan Directs Extracellular Matrix-Mediated Neuronal Plasticity.

In the adult brain, the extracellular matrix (ECM) influences recovery after injury, susceptibility to mental disorders, and is in general a strong regulator of neuronal plasticity. The proteoglycan aggrecan is a core component of the condensed ECM structures termed perineuronal nets (PNNs), and the specific role of PNNs on neural plasticity remains elusive. Here, we genetically targeted the Acan gene encoding for aggrecan using a novel animal model. This allowed for conditional and targeted loss of aggrecan in vivo, which ablated the PNN structure and caused a shift in the population of parvalbumin-expressing inhibitory interneurons toward a high plasticity state. Selective deletion of the Acan gene in the visual cortex of male adult mice reinstated juvenile ocular dominance plasticity, which was mechanistically identical to critical period plasticity. Brain-wide targeting improved object recognition memory.SIGNIFICANCE STATEMENT The study provides the first direct evidence of aggrecan as the main functional constituent and orchestrator of perineuronal nets (PNNs), and that loss of PNNs by aggrecan removal induces a permanent state of critical period-like plasticity. Loss of aggrecan ablates the PNN structure, resulting in invoked juvenile plasticity in the visual cortex and enhanced object recognition memory.

Neuroanatomical characterization of perineuronal net components in the human cochlear nucleus and superior olivary complex.

The human auditory brainstem, especially the cochlear nucleus (CN) and the superior olivary complex (SOC) are characterized by a high density of neurons associated with perineuronal nets (PNs). PNs build a specific form of extracellular matrix surrounding the neuronal somata, proximal dendrites and axon initial segments. They restrict synaptic plasticity and control high-frequency synaptic activity, a prominent characteristic of neurons of the auditory brainstem. The distribution of PNs within the auditory brainstem has been investigated in a number of mammalian species. However, much less is known regarding PNs in the human auditory brainstem. The present study aimed at the immunohistochemical identification of PNs in the cochlear nucleus (CN) and superior olivary complex (SOC) in the human brainstem. We focused on the complex nature and molecular variability of PNs in the CN and SOC by using specific antibodies against the main PN components (aggrecan, brevican, neurocan and hyaluronan and proteoglycan link protein 1). Virtually all subnuclei within the ventral CN and SOC were found to be associated with PNs. Direct comparison between gerbil and human yielded similar fine structure of PNs and confirmed the typical tight interdigitation of PNs with synaptic terminals in both species. Noticeably, an elaborate combination of immunohistochemical labelings clearly supports the still debated existence of the medial nucleus of trapezoid body (MNTB) in the human brain. In conclusion, the present study demonstrates that PNs form a prominent extracellular structure on CN and SOC neurons in the human brain, potentially stabilizing synaptic contacts, which is in agreement with many other mammalian species.

The adolescent idiopathic scoliotic IVD displays advanced aggrecanolysis and a glycosaminoglycan composition similar to that of aged human and ovine IVDs.

PURPOSE: The present study was designed to ascertain how altered biomechanics in adolescent idiopathic scoliotic (AIS) intervertebral discs (IVDs) affected tissue compositions and aggrecan processing compared to age matched and aged human IVDs. Newborn, 2- and 10-year-old ovine IVDs were also examined. METHODS: Aggrecan populations were separated by Sepharose CL2B chromatography, composite agarose polyacrylamide gel electrophoresis (CAPAGE) and identified by immunoblotting. The KS and CS content of IVD tissue extracts from AIS IVDs were compared with age-matched normal adolescent IVDs and with old human IVDs. Extracts from newborn, 2- and 10-year-old ovine IVDs were also examined in a similar manner. RESULTS: Adolescent idiopathic scoliotic IVD Aggrecan populations shared similar levels of polydispersity and aggregatability with hyaluronan as old IVD proteoglycans. CAPAGE demonstrated three aggrecan populations in AIS, aged human and ovine IVDs increased polydispersity and mobility in CAPAGE. AIS IVDs had GAG compositions similar to aged human and ovine IVDs. Sulphated KS (5-D-4) and chondroitin-6-sulphate, 3-B-3(+) were markers of tissue maturation, and chondroitin-4-sulphate, 2-B-6(+) was prominent in immature IVDs but its levels were lower in mature IVDs. DISCUSSION: Sulphated KS and 3-B-3(+) CS were prominently associated with IVD maturation and AIS IVDs, while the 2-B-6(+) CS isomer was associated with immature IVD tissues. The polydispersity of aggrecan in AIS IVDs, which was similar to in old human and ovine IVDs, reflected altered processing in the AIS IVDs in response to the biomechanical microenvironments the disc cells were exposed to in AIS IVDs. These slides can be retrieved under Electronic Supplementary Material.

Nano-hydroxyapatite/collagen film as a favorable substrate to maintain the phenotype and promote the growth of chondrocytes cultured in vitro.

Autologous chondrocyte implantation (ACI) has emerged as a novel approach to cartilage repair through the use of harvested chondrocytes. However, the expansion of the chondrocytes from the donor tissue in vitro is restricted by the limited cell numbers and the dedifferentiation of the chondrocytes. The present study investigated the effect of collagen-based films, including collagen, hydroxyapatite (HA)/collagen (HC) and in situ synthesis of nano­HC (nHC), on monolayer cultures of chondrocytes. As a substrate for the chondrocytes monolayer culture in vitro, nHC was able to restrain the dedifferentiation of chondrocytes and facilitate cell expansion, which was detected by methyl thiazolyl tetrazolium assay, scanning electron microscopy, calcein­acetoxymethyl/propidium iodide staining, hematoxylin and eosin staining, Safranin O staining, immunohistochemical staining and reverse transcription­quantitative polymerase chain reaction. Furthermore, the nHC films significantly facilitated cell growth and enhanced the expression of cartilage­specific extracellular matrix (ECM) components, including aggrecan and type II collagen. In addition, nHC films markedly downregulated the expression of collagen type I, an indicator of dedifferentiation. The results indicated that nHC, a collagen­based substrate optimized by nanoparticles, was able to better support cell growth and preserve cell phenotype compared with collagen alone or HC. The nHC film, which favors cell growth and prevents the dedifferentiation of chondrocytes, may therefore serve as a useful cartilage­like ECM for chondrocytes. In conclusion, nHC film is a promising substrate for the culture of chondrocytes in cell-based therapy.

Prediction of progression of damage to articular cartilage 2 years after anterior cruciate ligament reconstruction: use of aggrecan and type II collagen biomarkers in a retrospective observational study.

BACKGROUND: We aimed to determine whether synovial fluid (SF) biomarkers can predict the progression of articular cartilage damage as determined by arthroscopic evaluation during and after anterior cruciate ligament (ACL) reconstruction. METHODS: Arthroscopic assessment of articular cartilage damage was performed twice in 62 patients, first during ACL reconstruction and then approximately 2 years later during implant removal for ligament fixation. SF levels of the collagenase-generated cleavage neoepitope of type II collagen (C2C) and proteoglycan glycosaminoglycans keratan sulfate (KS), chondroitin-4-sulfate (Δdi-C4S), and chondroitin-6-sulfate (Δdi-C6S) were measured at ACL reconstruction. Associations between baseline biomarker levels and subsequent progression of cartilage damage were determined using receiver operating characteristic analysis and multivariable logistic regression analysis. RESULTS: No radiographic changes were observed in any of the patients. Progression of high-grade cartilage damage, observed arthroscopically, was negatively correlated with levels of Δdi-C6S and KS, as well as the ratio of Δdi-C6S to Δdi-C4S (C6S/C4S). Logistic regression analysis revealed significant associations of Δdi-C6S (cut-off: 55.7 nmol/ml, odds ratio (OR) 0.231, 95% confidence interval (CI) 0.061-0.879), KS (cut-off: 10.6 µg/ml, OR 0.114, 95% CI 0.024-0.529), and C6S/C4S ratio (cut-off: 4.6, OR 0.060, 95% CI 0.005-0.737) with the progression of high-grade cartilage damage after adjusting for age, the duration from injury to first surgery, sex, and the number of high-grade lesions (grades III and IV) at baseline. CONCLUSIONS: The progression of high-grade cartilage damage was significantly associated with baseline levels of proteoglycan glycosaminoglycan biomarkers; namely, Δdi-C6S, KS, and C6S/C4S ratio.

Impact of a daily exercise dose on knee joint cartilage - a systematic review and meta-analysis of randomized controlled trials in healthy animals.

OBJECTIVE: To investigate the impact of a daily exercise dose on cartilage composition and thickness, by conducting a systematic review of randomized controlled trials (RCTs) involving healthy animals. METHODS: A narrative synthesis of the effect of a daily exercise dose on knee cartilage aggrecan, collagen and thickness was performed. A subset of studies reporting sufficient data was combined in meta-analysis using a random-effects model. Meta-regression analyses were performed to investigate the impact of covariates. RESULTS: Twenty-nine RCTs, involving 64 comparisons, were included. In the low dose exercise group, 21/25 comparisons reported decreased or no effect on cartilage aggrecan, collagen and thickness. In the moderate dose exercise group, all 12 comparisons reported either no or increased effect. In the high dose exercise group, 19/27 comparisons reported decreased effect. A meta-analysis of 14 studies investigating cartilage thickness showed no effect in the low dose exercise group (SMD -0.02; 95% CI -0.42 to 0.38; I2 = 0.0%), large but non-significant cartilage thickening in the moderate dose exercise group (SMD 0.95; 95% CI -0.33 to 2.23; I2 = 72.1%) and non-significant cartilage thinning in the high dose exercise group (SMD -0.19; 95% CI -0.49 to 0.12; I2 = 0.0%). Results were independent of analyzed covariates. The overall quality of the studies was poor because of inadequate reporting of data and high risk of bias. CONCLUSIONS: Our results suggest that the relationship between daily exercise dose and cartilage composition, but not necessarily cartilage thickness, may be non-linear. While we found inconclusive evidence for a low daily dose of exercise, a high daily dose of exercise may have negative effects and a moderate daily dose of exercise may have positive effects on cartilage matrix composition in healthy animals.

[Indirect coculture of human infrapatellar fat pad-derived stem cells with osteoarthritic chondrocytes induces their chondrogenesis].

Objective: To promote phenotype recovery of osteoarthritic articular chondrocytes( OACs) and induce chondrogenic differentiation of infrapatellar fat pad-derived stem cells( IPFPSCs) by indirectly coculturing these two types of cells. Methods: The OACs and IPFPSCs were isolated and cultured in vitro. This experiment included single IPFPSCs group,single OACs group,and coculture group. After cells were cultured in vitro with chondrogenic medium for 21 days,the chondrocyte phenotypes were determined by HE staining( cell morphology),Alcian blue staining( glycosaminoglycan content) and immunofluorescence cytochemistry( collagen 1,collagen 2,collagen 3,aggrecan,SOX9). Results: In coculture group,the OACs aggregated into microspheres,and the IPFPSCs were oval in shape. In single culture groups,the OACs were less aggregated and the spheres were smaller; and the IPFPSCs were spindle in shape. HE staining showed that,in the coculture group,the nuclei of OACs spheres were dark,and the IPFPSCs were rich in cytoplasm; while in single culture groups,the nuclei of OAC spheres were less dark,and the IPFPSCs were less stained compared with the coculture group. Alcian blue staining indicated that glycosaminoglycan content was higher in the coculture group than in single culture groups. Immunofluorescent staining showed that the intensity of chondrogenic markers( collagen 2,aggrecan,and SOX9)was stronger,while the intensity of collagen 1 and collagen 10 was weaker in the coculture group as compared with single culture groups. Conclusion: The indirect coculture of IPFPSCs with OACs can contribute to the phenotype recovery of OACs and induce the chondrogenic differentiation of IPFPSCs.

Salubrinal Suppresses IL-17-Induced Upregulation of MMP-13 and Extracellular Matrix Degradation Through the NF-kB Pathway in Human Nucleus Pulposus Cells.

Matrix metalloproteinase 13 (MMP-13) plays an important role in the process of pro-inflammatory cytokine-induced intervertebral disc degeneration (IDD). This study examined the effect of IL-17 on the regulation of MMP-13 and the extracellular matrix (ECM) in the intervertebral disc (IVD). We then examined whether salubrinal, a known inhibitor of eIF2α dephosphorylation, inhibited the IL-17-induced changes mentioned above. Furthermore, we demonstrated a potential therapeutic role for salubrinal in alleviating the chronic inflammatory-dependent degenerative state commonly observed in IDD. After inflammatory distress with IL-17, RT-PCR and western blot were employed to investigate the expression of MMP-13, collagen type II (COL2A1), collagen type I (COL1A1), and aggrecan (ACAN) in nucleus pulpous (NP) tissue. Activation of the NF-kB pathway was measured by western blot and immunocytochemistry following IL-17 treatment. We also examine the level of eIF2α phosphorylation after IL-17 treatment with or without salubrinal. Then, we investigated interactions of the NF-kB pathway to eIF2α phosphorylation. Moreover, we employed salubrinal and a specific inhibitor of NF-kB (BAY11-7082) to evaluate their effects on IL-17-driven regulation of MMP-13 and the ECM, as well as on the activation of NF-kB. The results showed that IL-17 increased the production of MMP-13 and decreased expression of COL2A1 and ACAN via the NF-kB pathway. Either IL-17 or salubrinal increased the level of eIF2α phosphorylation, but the effects of BAY11-7082 on the level of p-eIF2α were not detectable. BAY11-7082 and salubrinal significantly suppressed IL-17-driven intervertebral disc degeneration. Furthermore, salubrinal produced stronger effects than BAY11-7082. These results imply the potential involvement of IL-17 in IDD through activation of NF-kB signaling, which successively upregulated the expression of MMP-13 and led to the degradation of the ECM. Furthermore, salubrinal can inhibit this process through inhibition of NF-kB activation that is not directly linked to eIF2α phosphorylation, suggesting a potential therapeutic role in IDD.

Aggrecan heterogeneity in articular cartilage from patients with osteoarthritis.

BACKGROUND: Aggrecan degradation is the hallmark of cartilage degeneration in osteoarthritis (OA), though it is unclear whether a common proteolytic process occurs in all individuals. METHODS: Aggrecan degradation in articular cartilage from the knees of 33 individuals with OA, who were undergoing joint replacement surgery, was studied by immunoblotting of tissue extracts. RESULTS: Matrix metalloproteinases (MMPs) and aggrecanases are the major proteases involved in aggrecan degradation within the cartilage, though the proportion of aggrecan cleavage attributable to MMPs or aggrecanases was variable between individuals. However, aggrecanases were more associated with the increase in aggrecan loss associated with OA than MMPs. While the extent of aggrecan cleavage was highly variable between individuals, it was greatest in areas of cartilage adjacent to sites of cartilage erosion compared to sites more remote within the same joint. Analysis of link protein shows that in some individuals additional proteolytic mechanisms must also be involved to some extent. CONCLUSIONS: The present studies indicate that there is no one protease, or a fixed combination of proteases, responsible for cartilage degradation in OA. Thus, rather than targeting the individual proteases for OA therapy, directing research to techniques that control global protease generation may be more productive.

Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.