Recombinant human midkine stimulates proliferation of articular chondrocytes

Objectives: Midkine, a heparin‐binding growth factor, promotes population growth, survival and migration of several cell types, but its effect on articular chondrocytes remains unknown. The aim of this study was to investigate its role on proliferation of articular chondrocytes in vitro and in vivo. Materials and methods: Bromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out. Results: rhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture‐expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra‐articularly in mice and rats, respectively. Conclusion: Our results demonstrate that rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo. The results of this study warrant further examination of rhMK for treatment of animal models of articular cartilage defects.     

Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction

With the aim of identifying novel genes regulating cartilage development and degeneration, we screened a cartilage-specific expressed sequence tag database. Esophageal cancer related gene 4 (ECRG4) was selected, based on the criteria of ‘chondrocyte-specific’ and ‘unknown function.’ ECRG4 expression was particularly abundant in chondrocytes and cartilage, compared to various other mouse tissues. ECRG4 is a secreted protein that undergoes cleavage after secretion. The protein is specifically expressed in chondrocytes in a manner dependent on differentiation status. The expression is very low in mesenchymal cells, and dramatically increased during chondrogenic differentiation. The ECRG4 level in differentiated chondrocytes is decreased during hypertrophic maturation, both in vitro and in vivo, and additionally in dedifferentiating chondrocytes induced by interleukin-1β or serial subculture, chondrocytes of human osteoarthritic cartilage and experimental mouse osteoarthritic cartilage. However, ectopic expression or exogenous ECRG4 treatment in a primary culture cell system does not affect chondrogenesis of mesenchymal cells, hypertrophic maturation of chondrocytes or dedifferentiation of differentiated chondrocytes. Additionally, cartilage development and organization of extracellular matrix are not affected in transgenic mice overexpressing ECRG4 in cartilage tissue. However, ectopic expression of ECRG4 reduced proliferation of primary culture chondrocytes. While the underlying mechanisms of ECRG4 expression and specific roles remain to be elucidated in more detail, our results support its function as a marker of differentiated articular chondrocytes and cartilage destruction.     

Characterization of caveolins from human knee joint cartilage: expression of caveolin-1, -2, and -3 in chondrocytes and association with integrin β1

Interactions between the extracellular matrix (ECM) and chondrocytes are of great importance for structure and function of cartilage. The present study was undertaken to answer the question whether caveolins take part in integrin-mediated cell–ECM interactions in the human cartilage. In samples of human knee joint cartilage, we detected the caveolin subtypes -1, -2, and -3 by immunohistochemical methods. Double-label experiments revealed a colocalization of caveolin with β1-integrin. Results of immunoprecipitation and immunoblotting assays show that β1-integrins associate with all three caveolin subtypes in human chondrocytes and indicate that they are part of the same complexes. Furthermore, immunoelectron microscopy shows the localization of β1-integrin in caveolae-like structures of the cell membrane. The data stimulate further investigations on the role of the caveolin–integrin complex for integrin-mediated signaling pathways in chondrocytes. Accepted: 17 December 1999     

Notch signaling is involved in human articular chondrocytes de-differentiation during osteoarthritis

Abstract

Context: During osteoarthritis (OA), chondrocytes undergo de-differentiation, resulting in the acquisition of a fibroblast-like morphology, decreased expression of collagen type II (colII) and aggrecan, and increased expression of collagen type I (colI), metalloproteinase 13 (MMP13) and nitric oxide synthase (eNOS). Notch signaling plays a crucial role during embryogenesis. Several studies showed that Notch is expressed in adulthood. Objective: The aim of our study was to confirm the involvement of Notch signaling in human OA at in vitro and ex vivo levels. Materials and methods: Normal human articular chondrocytes were cultured during four passages either treated or not with a Notch inhibitor: DAPT. Human OA cartilage was cultured with DAPT for five days. Chondrocytes secreted markers and some Notch pathway components were analyzed using Western blotting and qPCR. Results: Passaging chondrocytes induced a decrease in the cartilage markers: colII and aggrecan. DAPT-treated chondrocytes and OA cartilage showed a significant increase in healthy cartilage markers. De-differentiation markers, colI, MMP13 and eNOS, were significantly reduced in DAPT-treated chondrocytes and OA cartilage. Notch1 expression was proportional to colI, MMP13 and eNOS expression and inversely proportional to colII and aggrecan expression in nontreated cultured chondrocytes. Notch ligand: Jagged1 increased in chondrocytes culture. DAPT treatment resulted in reduced Jagged1 expression. Notch target gene HES1 increased during chondrocyte culture and was reduced when treated with DAPT. Conclusion: Targeting Notch signaling during OA might lead to the restitution of the typical chondrocyte phenotype and even to chondrocyte redifferentiation during the pathology.     

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants <Emphasis Type="Italic">in vitro</Emphasis>

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)₂ from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)₂ were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.     

Expression of the urokinase-type plasminogen activator receptor in human articular chondrocytes: association with caveolin and β1-integrin

The urokinase-type plasminogen activator (uPA) in concert with other proteolytic enzymes plays a critical role in cartilage degradation during osteoarthritis. Urokinase receptor (uPAR), a glycosyl-phosphatidylinositol-linked glycoprotein present on the cell surface of various cell types such as cancer cells, fibroblasts, synoviocytes, and chondrocytes, is a key regulator of the plasmin-mediated pericellular proteolysis. Recently, in arthritic synovial tissue increased uPAR expression has been detected. By immunohistochemical analysis we observed, in addition, enhanced expression of uPAR in chondrocytes of arthritic samples of human cartilage compared to non-arthritic controls. Using in vitro cultured human chondrocytes, we analyzed whether uPAR is associated with structural proteins, which are known to be involved in cell signaling and activation. uPAR in phorbol-12-myristate-13-acetate-stimulated chondrocytes colocalized with caveolin as well as beta 1-integrin, as demonstrated by double immunostaining with specific antibodies. Furthermore, uPAR was present in caveolae-like structures of chondrocytes as detected by immunoelectron microscopy. Finally, both caveolin and beta 1-integrin were coprecipitated with uPAR-specific antibodies from cell extracts suggesting that these proteins may form functional complexes in human chondrocytes. The localization of uPAR in caveolae and its close association with caveolin and beta 1-integrin points to a significance of uPAR-mediated signaling pathways in human chondrocytes.     

Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients

Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. Since substantial refinements in the isolation, expansion and transplantation of chondrocytes have been made in recent years, the treatment of early stage osteoarthritic lesions using ACT might now be feasible. In this study, we determined the gene expression patterns of osteoarthritic (OA) chondrocytes ex vivo after primary culture and subculture and compared these with healthy chondrocytes ex vivo and with articular chondrocytes expanded for treatment of patients by ACT. Gene expression profiles were determined using quantitative RT-PCR for type I, II and X collagen, aggrecan, IL-1β and activin-like kinase-1. Furthermore, we tested the capability of osteoarthritic chondrocytes to generate hyaline-like cartilage by implanting chondrocyte-seeded collagen scaffolds into immunodeficient (SCID) mice. OA chondrocytes ex vivo showed highly elevated levels of IL-1β mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1β levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage transplants in SCID mice. We conclude that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes are also capable of producing a cartilage-like tissue in the in vivo SCID mouse model. Thus, such chondrocytes seem to fulfil the prerequisites for use in ACT treatment.     

An ex vivo model using human osteoarthritic cartilage demonstrates the release of bioactive insulin‐like growth factor‐1 from a collagen–glycosaminoglycan scaffold

Biomimetic scaffolds hold great promise for therapeutic repair of cartilage, but although most scaffolds are tested with cells in vitro, there are very few ex vivo models (EVMs) where adult cartilage and scaffolds are co‐cultured to optimize their interaction prior to in vivo studies. This study describes a simple, non‐compressive method that is applicable to mammalian or human cartilage and provides a reasonable throughput of samples. Rings of full‐depth articular cartilage slices were derived from human donors undergoing knee replacement for osteoarthritis and a 3 mm core of a collagen/glycosaminoglycan biomimetic scaffold (Tigenix, UK) inserted to create the EVM. Adult osteoarthritis chondrocytes were seeded into the scaffold and cultures maintained for up to 30 days. Ex vivo models were stable throughout experiments, and cells remained viable. Chondrocytes seeded into the EVM attached throughout the scaffold and in contact with the cartilage explants. Cell migration and deposition of extracellular matrix proteins in the scaffold was enhanced by growth factors particularly if the scaffold was preloaded with growth factors. This study demonstrates that the EVM represents a suitable model that has potential for testing a range of therapeutic parameters such as numbers/types of cell, growth factors or therapeutic drugs before progressing to costly pre‐clinical trials. © 2015 The Authors. Cell Biochemistry and Function Published by John Wiley & Sons Ltd.     

β1-Integrins in the cartilage matrix

Integrins are cell-surface receptors that mediate cell attachment to extracellular matrix components. The pericellular matrix in cartilage not only is a mechanical framework, but is also important for chondrocyte differentiation and stabilization of the phenotype. The interaction between chondrocytes and pericellular matrix is mediated, in part, by integrin receptors. We have previously demonstrated the presence of beta1-integrins in the cartilage matrix of organoid culture of limb buds from 12-day-old mouse embryos by immunohistological methods. In order to corroborate these findings, we have further investigated the distribution of integrins in the cartilage matrix by immunoelectron microscopy and by immunoprecipitation methods. Cartilage tissue of limb buds of 17-day-old mouse embryos was treated with collagenase and the cell-free and cellular protein-free supernatant was removed and used for immunoprecipitation experiments. Immunoprecipitation with antibodies against beta1-, alpha1-, alpha3-, and alpha5beta1-integrins and collagen type II, followed by immunoblotting with the same antibodies, demonstrated the presence of these integrins and collagen type II in the supernatant. The integrins found in the cartilage matrix could have been either secreted or shed by the cells. The question as to whether they have a function in the cartilage matrix, such as interlinking, in the matrix organization or in the stabilization of matrix components remains to be elucidated.     

Androgen receptors and gender-specific distribution of alkaline phosphatase in human thyroid cartilage

The degree of mineralization in human thyroid cartilage is gender specific. Until now, laryngeal tissue was tested for sexual hormone receptors by the use of radiolabelled hormones only without exact localization of the receptors. In this study immediately frozen cartilage specimens from seven male and one female patient who underwent laryngectomy were used for immunolocalization of sexual hormone receptors. Additionally, serum sexual hormone levels were measured by means of radioimmunoassay. Alkaline phosphatase was localized enzymohistochemically in another cohort of six male and four female cartilage specimens from laryngectomies and autopsies. Chondrocytes in thyroid cartilage from both sexes reacted with antibodies to the androgen receptor. The low serum testosterone levels, which varied between 1.5 and 3.9 ng/ml, did not correlate with insufficient mineralization of thyroid cartilage in men (r=0.363, P=0.432). Chondrocytes did not react with antibodies to the estrogen receptor α and the progesterone receptor in both sexes. Expression of alkaline phosphatase started about the middle of the second decade. Some chondrocytes near the mineralization front were positive for androgen receptor and alkaline phosphatase, other chondrocytes were negative for both. Our results suggest the involvement of androgen receptor positive chondrocytes in thyroid cartilage mineralization, probably by a testosterone-linked stimulation of alkaline phosphatase.     

Immunotoxins

Immunotoxins (ITs) are chimeric molecules constructed by covalently conjugating monoclonal antibodies (MoAbs) to plant or bacterial toxins (e.g. ricin or pseudomonas exotoxin). The antibody moiety allows specific targeting of ITs to tumor-associated antigens, while the toxin moiety is responsible for cell killing by irreversible inactivation of protein synthesis. Since ITs must reach the cytosol to kill cells, the rates of endocytosis, the pathways of intracellular routing, and the rates of translocation to the cytoplasm are important determinants of the efficacy of an IT. Promisingin vitro andin vivo IT results have been reported by many groups, and phase I clinical trials in cancer patients are currently underway.This research was supported in part by a First Independent Research and Training award from the National Institutes of Health (R29 CA 46134-03).     

THE HISTOGENETIC AND BIOSYNTHETIC CAPABILITIES OF URODELE CHONDROCYTES AFTER SHORT-TERM AND PROLONGED CULTURE IN VITRO*

Urodele larval chondrocytes can be successfully maintained in long-term in vitro cultures with successive serial subculturing. In the monolayered cultures the chondrocytes actively synthesize DNA and undergo mitotic divisions, but no synthesis of mucopolysaccharide matrix takes place. The majority of the cells maintain their normal chromosome complement. Chondrocytes from short-term primary cultures, when implanted into the tail fins of host larvae, organize morphologically into typical cartilage tissue with cellular lacunae and a mucopolysaccharide matrix. When chondrocytes, which have been in monolayered cultures for long periods, are implanted as pellets into the in vivo conditions of the tail fin, they form a discrete trabecularized mass. The cells now display the capability of mucopolysaccharide matrix synthesis, but have apparently lost their histogenetic capability of forming typical cartilage tissue.     

Anemonin attenuates osteoarthritis progression through inhibiting the activation of IL‐1β/NF‐κB pathway

The osteoarthritis (OA) progression is now considered to be related to inflammation. Anemonin (ANE) is a small natural molecule extracted from various kinds of Chinese traditional herbs and has been shown to inhibiting inflammation response. In this study, we examined whether ANE could attenuate the progression of OA via suppression of IL‐1β/NF‐κB pathway activation. Destabilization of the medial meniscus (DMM) was performed in 10‐week‐old male C57BL/6J mice. ANE was then intra‐articularly injected into joint capsule for 8 and 12 weeks. Human articular chondrocytes and cartilage explants challenged with interleukin‐1β (IL‐1β) were treated with ANE. We found that ANE delayed articular cartilage degeneration in vitro and in vivo. In particular, proteoglycan loss and chondrocyte hypertrophy were significantly decreased in ANE ‐treated mice compared with vehicle‐treated mice. ANE decreased the expressions of matrix metalloproteinase‐13 (MMP13), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), collagen X (Col X) while increasing Aggrecan level in murine with DMM surgery. ANE treatment also attenuated proteoglycan loss in human cartilage explants treated with IL‐1β ex vivo. ANE is a potent protective molecule for OA; it delays OA progression by suppressing ECM loss and chondrocyte hypertrophy partially by suppressing IL‐1β/NF‐κB pathway activation.     

Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an <Emphasis Type="Italic">in vitro</Emphasis>study

Background  

Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes.     

Cartilage–Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth

Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage–related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in transgenic mice accelerated the endochondral ossification processes, resulting in increased length of their long bones. Our results also indicate the possible involvement of locally enhanced IGF-I or IGF-II in this extended bone growth.     

Characterization of Human Matrilin-3 (MATN3)

We have isolated a cDNA clone for human matrilin-3 from a cartilage-specific cDNA library. The polypeptide predicted from the nucleotide sequence of this clone shared 83% identity with matrilin-3 from mouse and 61% with that from chicken. It was composed of 486 amino acid residues that were arranged in seven domains: a signal peptide, a von Willebrand factor A domain, four EGF repeats, and an α-helical region. The gene for human matrilin-3 (MATN3) was assigned to chromosome region 2p24–p23. The corresponding mRNA of 2.8 kb was expressed in every type of cartilage investigated thus far. It was also producedin vitroby primary chondrocytes isolated from articular cartilage. However, dedifferentiated chondrocytes of the third passage did not express it at all. Matrilin-3 might therefore serve as a marker for the differentiation state of chondrocytes.     

Ribosome display: next-generation display technologies for production of antibodies in vitro

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.     

Articular Cartilage Chondrocytes are more Advantageous for Generating Hyaline-like Cartilage than Mesenchymal Cells Isolated from Microfracture Repairs

Articular cartilage lacks self-repair capacity. Currently, two methods employing autologous cells are used to stimulate repair of articular cartilage. Micro-fracture induced repair induces autologous mesenchymal cell migration from bone marrow. Autologous chondrocytes' transplantation involves in vitro expansion of chondrocytes, and later implantation. In 15 patients de-differentiated chondrocytes obtained by cartilage biopsy were compared to cells derived from repair tissue induced by micro-fracture. These patients all underwent micro-fracture during the cartilage biopsy procedure. Autologous chondrocytes' transplantation was performed at least two months later then the biopsy. Tissue bits from articular cartilage and micro-fracture repair tissue were incubated in-vitro and explant cell cultures established. The cell cultures were assessed by immunohistochemistry and induced to differentiate. Differentiation into bone tissue was stimulated by addition of basic fibroblast growth factor, ascorbate and dexamethasone. High density (micro-mass) culture was used to stimulate chondrogenesis. Both cell cultures consist of mesenchymal progenitors as indicated by fibroblast growth factor receptor 3 expression and anti-CD-34+ antibodies. However, the micro-fracture generated repair tissue consists of osteocalcin-expressing cells destined to become bone. Collagen type II expression does not occur in these cells compared to autologous chondrocytes. Inducible nitric oxide synthase expression by microfracture cells is likely to damage surrounding articular cartilage in vivo. In conclusion, cells recruited by micro-fracture are inferior for cartilage regeneration purposes to those from cartilage biopsies.