Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

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.     

In vitro comparison of six different matrix systems for the cultivation of human chondrocytes

Summary In recent years, a great variety of different matrix systems for the cultivation of chondrocytes have been developed. Although some of these scaffolds show promising experimental results in vitro, the potential clinical value remains unclear. In this comparative study, we propagated human articular chondrocytes precultivated in monolayer culture on six different scaffolds (collagen gels, membranes and sponges) under standardized in vitro conditions. Mechanical properties of the matrix systems were not improved significantly by cultivation of human chondrocytes under the given in vitro conditions. The gel systems (CaReS, Ars Artho, Germany and Atelocollagen, Koken, Japan) showed a homogeneous cell distribution; chondrocytes propagated on Chondro-Gide (Geistlich Biomaterials, Switzerland) and Integra membranes (Integra, USA) were building multilayers. Only few cells penetrated the two Atelocollagen honeycomb sponges (Koken, Japan). During cultivation, chondrocytes propagated on all systems showed a partial morphological redifferentiation, which was best with regard to the gel systems. In general, only small amounts of collagen type-II protein could be detected in the pericellular region and chondrocytes failed to build a territorial matrix. During the first two weeks of cultivation, the two gel systems showed a significantly higher collagen type-II gene expression and a lower collagen type-I gene expression than the other investigated matrix systems. Although collagen gels seem to be superior when dealing with deep cartilage defects, membrane systems might rather be useful in improving conventional autologous chondrocyte transplantation or in combination with gel systems.     

Glucosamine prevents <Emphasis Type="Italic">in vitro</Emphasis> collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation

Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.     

DIFFERENTIATION OF MESENCHYMAL LIMB BUD CELLS TO CHONDROCYTES IN ALGINATE BEADS

Manyin vitromodels of embryonic material used for the cultivation of chondrocytes yield mixed cultures consisting of chondrocytes and fibroblast-like cells. For the optimization of cartilage cell cultures, alginate, a semisolid medium, was employed to obtain pure chondrocyte cultures. Isolated mesenchymal cells from 12-day-old mouse limb buds were grown in alginate for up to 4 weeks. A sub-population of the cells differentiated to chondrocytes and exhibited a stable phenotype until the end of the culture period. After 3 to 4 days a cartilage-specific matrix started to develop. Fibroblast-like cells from this mixed culture did not survive; they became necrotic. When alginate was later on dissolved by chelating agents, only chondrocytes were isolated. During dissolution of alginate and centrifugation, chondrocytes did not lose their contact with their new matrix present on their surfaces. Cultivation of these chondrocytes or chondrones in mass culture yields a pure chondrocyte population. Immunoelectron microscopic investigations revealed collagen type II, fibronectin, decorin and chondroitin sulfate-proteoglycans in the chondrocyte capsules and in mass culture.     

Src kinase inhibition promotes the chondrocyte phenotype

Regulated differentiation of chondrocytes is essential for both normal skeletal development and maintenance of articular cartilage. The intracellular pathways that control these events are incompletely understood, and our ability to modulate the chondrocyte phenotype in vivo or in vitro is therefore limited. Here we examine the role played by one prominent group of intracellular signalling proteins, the Src family kinases, in regulating the chondrocyte phenotype. We show that the Src family kinase Lyn exhibits a dynamic expression pattern in the chondrogenic cell line ATDC5 and in a mixed population of embryonic mouse chondrocytes in high-density monolayer culture. Inhibition of Src kinase activity using the pharmacological compound PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine) strongly reduced the number of primary mouse chondrocytes. In parallel, PP2 treatment increased the expression of both early markers (such as Sox9, collagen type II, aggrecan and xylosyltransferases) and late markers (collagen type X, Indian hedgehog and p57) markers of chondrocyte differentiation. Interestingly, PP2 repressed the expression of the Src family members Lyn, Frk and Hck. It also reversed morphological de-differentiation of chondrocytes in monolayer culture and induced rounding of chondrocytes, and reduced stress fibre formation and focal adhesion kinase phosphorylation. We conclude that the Src kinase inhibitor PP2 promotes chondrogenic gene expression and morphology in monolayer culture. Strategies to block Src activity might therefore be useful both in tissue engineering of cartilage and in the maintenance of the chondrocyte phenotype in diseases such as osteoarthritis.     

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 <Emphasis Type="Italic">in vitro</Emphasis>

Introduction  

Chondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.     

Co-expression of collagen types II and X mRNAs in newly formed hypertrophic chondrocytes of the embryonic chick vertebral body demonstrated by double-fluorescence in situ hybridization

Summary Collagen types II and X mRNAs have been demonstrated simultaneously in newly formed hypertrophic chondrocytes of embryonic chick vertebral cartilage using a double-fluorescence in situ hybridization technique. Digoxigenin- and biotin-labelled type-specific collagen II and X cDNA probes were used. In the embryonic chick vertebra at stage 45, two different fluorescence signals (Fluorescein isothiocyanate and Rhodamine) - one for collagen type II mRNA, the other for type X mRNA - showed differential distribution of the two collagen mRNAs in the proliferating and hypertrophic chondrocyte zones. Several layers of newly formed hypertrophic chondrocytes expressing both collagen types II and X genes were identified in the same section as two different fluorescent colour signals. Low levels of fluorescent signals for collagen type II mRNA were also detected in the hypertrophic chondrocyte zone. Cytological identification of maturing chondrocyte phenotypes, expressing collagen mRNAs, is easier in sections processed by non-radioactive in situ hybridization than in those subjected to radioactive in situ hybridization using ³H-labelled cDNA probes.This study demonstrates that double-fluorescence in situ hybridization is a useful tool for simultaneously detecting the expression of two collagen genes in the same chondrocyte population.     

P38 mitogen‐activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture

Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell‐based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes ‘dedifferentiation’ into fibroblastic cells. Mitogen‐activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up‐regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up‐regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow‐on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell‐based therapies.     

Proliferation and differentiation potential of chondrocytes from osteoarthritic patients

Autologous chondrocyte transplantation (ACT) has been shown, in long-term follow-up studies, to be a promising treatment for the repair of isolated cartilage lesions. The method is based on an implantation of in vitro expanded chondrocytes originating from a small cartilage biopsy harvested from a non-weight-bearing area within the joint. In patients with osteoarthritis (OA), there is a need for the resurfacing of large areas, which could potentially be made by using a scaffold in combination with culture-expanded cells. As a first step towards a cell-based therapy for OA, we therefore investigated the expansion and redifferentiation potential in vitro of chondrocytes isolated from patients undergoing total knee replacement. The results demonstrate that OA chondrocytes have a good proliferation potential and are able to redifferentiate in a three-dimensional pellet model. During the redifferentiation, the OA cells expressed increasing amounts of DNA and proteoglycans, and at day 14 the cells from all donors contained type II collagen-rich matrix. The accumulation of proteoglycans was in comparable amounts to those from ACT donors, whereas total collagen was significantly lower in all of the redifferentiated OA chondrocytes. When the OA chondrocytes were loaded into a scaffold based on hyaluronic acid, they bound to the scaffold and produced cartilage-specific matrix proteins. Thus, autologous chondrocytes are a potential source for the biological treatment of OA patients but the limited collagen synthesis of the OA chondrocytes needs to be further explained.     

Effects of Phosphorylatable Short Peptide-Conjugated Chitosan-Mediated IL-1Ra and igf-1 Gene Transfer on Articular Cartilage Defects in Rabbits

Previously, we reported an improvement in the transfection efficiency of the plasmid DNA-chitosan (pDNA/CS) complex by the utilization of phosphorylatable short peptide-conjugated chitosan (^pSP-CS). In this study, we investigated the effects of ^pSP-CS-mediated gene transfection of interleukin-1 receptor antagonist protein (IL-1Ra) combined with insulin-like growth factor-1 (IGF-1) in rabbit chondrocytes and in a rabbit model of cartilage defects. pBudCE4.1-IL-1Ra+igf-1, pBudCE4.1-IL-1Ra and pBudCE4.1-igf-1 were constructed and combined with ^pSP-CS to form pDNA/^pSP-CS complexes. These complexes were transfected into rabbit primary chondrocytes or injected into the joint cavity. Seven weeks after treatment, all rabbits were sacrificed and analyzed. High levels of IL-1Ra and igf-1 expression were detected both in the cell culture supernatant and in the synovial fluid. In vitro, the transgenic complexes caused significant proliferation of chondrocytes, promotion of glycosaminoglycan (GAG) and collagen II synthesis, and inhibition of chondrocyte apoptosis and nitric oxide (NO) synthesis. In vivo, the exogenous genes resulted in increased collagen II synthesis and reduced NO and GAG concentrations in the synovial fluid; histological studies revealed that pDNA/^pSP-CS treatment resulted in varying degrees of hyaline-like cartilage repair and Mankin score decrease. The co-expression of both genes produced greater effects than each single gene alone both in vitro and in vivo. The results suggest that ^pSP-CS is a good candidate for use in gene therapy for the treatment of cartilage defects and that igf-1 and IL-1Ra co-expression produces promising biologic effects on cartilage defects.     

The roles of matrix molecules in mediating chondrocyte aggregation, attachment, and spreading

The most abundant macromolecules in cartilage are hyaluronan, collagen, aggrecan, and link protein, which are believed to play roles in maintaining a unique three-dimensional network for a functional joint. This study was designed to investigate the roles of the major extracellular molecules in mediating chondrocyte-matrix interactions. We employed specific approaches to remove components individually or in combination: hyaluronan was digested with hyaluronidase; type II collagen was digested with collagenase; aggrecan expression was inhibited with antisense and beta-xyloside approaches; and link protein expression was inhibited with antisense oligonucleotides. Digestion of hyaluronan induced chondrocyte attachment to tissue culture plates, collagen-coated plates, and fibroblast-like chondrocyte cultures, and induced chondrocyte aggregation. Treated chondrocytes exhibited a fibroblast-like morphology, and the effects of hyaluronidase were dose-dependent. Conversely, the effect of collagenase on chondrocyte adhesion and aggregation was far less pronounced. Treatment with Arg-Gly-Asp peptide inhibited chondrocyte-collagen interaction. Chondrocyte attachment was enhanced by antisense oligonucleotides complementary to aggrecan and link protein and by beta-xyloside treatment. Nevertheless, hyaluronan seems to predominate over the other molecules in mediating chondrocyte-matrix interactions.     

INTEGRIN EXPRESSION AND COLLAGEN TYPE II IMPLICATED IN MAINTENANCE OF CHONDROCYTE SHAPE IN MONOLAYER CULTURE: AN IMMUNOMORPHOLOGICAL STUDY

Chondrocytes grown in monolayer culture at low density, with serum added, either dedifferentiate after several days whereby their cell shape changes or they are overgrown by fibroblast-like cells. The aim of this study was to optimize the cultivation of chondrocytes in monolayer culture and to slow down their transformation or their overgrowth by fibroblast-like cells. For this purpose freshly isolated chondrocytes of cartilage anlagen from 17-day-old mouse embryos were grown on plastic or collagen type II-coated substrates. With this model: (a) chondrocytes grown on plastic substrates had almost completely changed to fibroblast-like cells after 5 days in culture. (b) When grown on collagen type II, the chondrocytes maintained their round phenotype for more than 2 weeks in culture. (c) Immunomorphological investigations showed that chondrocytes produce collagen type II and fibronectin and express specific surface receptors (integrins of the β1-group) on the membrane from day 1 until the end of the culture period when grown on collagen type II. (d) Treatment with β1-integrin antibodies clearly reduces chondrocyte adhesion on collagen type II by about 70%. Hence, these data indicate that the most probable influence of collagen type II on cellular behaviour depends on the integrins participating in a chondrocyte—collagen type II interaction, and this model represents a pure chondrocyte culture which allows cell growth for an extended period.     

Hyaluronan receptor-directed assembly of chondrocyte pericellular matrix

Initial assembly of extracellular matrix occurs within a zone immediately adjacent to the chondrocyte cell surface termed the cell- associated or pericellular matrix. Assembly within the pericellular matrix compartment requires specific cell-matrix interactions to occur, that are mediated via membrane receptors. The focus of this study is to elucidate the mechanisms of assembly and retention of the cartilage pericellular matrix proteoglycan aggregates important for matrix organization. Assembly of newly synthesized chondrocyte pericellular matrices was inhibited by the addition to hyaluronan hexasaccharides, competitive inhibitors of the binding of hyaluronan to its cell surface receptor. Fully assembled chondrocyte pericellular matrices were displaced using hyaluronan hexasaccharides as well. When exogenous hyaluronan was added to matrix-free chondrocytes in combination with aggrecan, a pericellular matrix equivalent in size to an endogenous matrix formed within 30 min of incubation. Addition of hyaluronan and aggrecan to glutaraldehyde-fixed chondrocytes resulted in matrix assembly comparable to live chondrocytes. These matrices could be inhibited from assembling by the addition of excess hyaluronan hexasaccharides or displaced once assembled by subsequent incubation with hyaluronan hexasaccharides. The results indicate that the aggrecanrich chondrocyte pericellular matrix is not only on a scaffolding of hyaluronan, but actually anchored to the cell surface via the interaction between hyaluronan and hyaluronan receptors.     

Monoclonal antibodies against human chondrocytes

Summary Cell-specific antigens are mainly found in cells or membrane surfaces rather than in the surrounding matrix. However, until now it was not possible to produce antibodies specific for cellular structures of chondrocytes. In 1989, Lance (Immunol. Lett. 21:63–73; 1989) first established specific monoclonal antibodies for human articular chondrocytes tested only by immunofluorescence. Studies describing the specificity of these five antibodies (HUMC 1–5) and their relevance for immunohistological analysis of cartilage tissue were not available until now. Therefore, the aim of the following study was to investigate the distribution of HUMC 1, 2, 3, 4, and 5 in mesenchymal cellsin vivo andin vitro immunohistochemically. Further investigations concentrate on the localization of chondrocyte specific antigens using immunoelectron microscopy. Immunohistological studies showed positive immunostainings with all five antibodies in human chondrocytesin vivo andin vitro. A cross-reaction with human fibroblasts and osteoblasts for the antibodies HUMC 2 and HUMC 5 was observed. furthermore, a parallel loss of immunoreactivity for HUMC 1, HUMC 3, and HUMC 4 was observed in cultured chondrocytes indicating that the specific antigens vanish during differentiation observedin vitro. Subsequent immunoblot analysis employing collagens as antigens did not show any reactivity. Using immunoelectron microscopy, gold particle labeling was observed in intracytoplasmatic vesicles of isolated chondrocytes. Our results indicate that HUMC 1, HUMC 3, and HUMC 4 are specific for cartilage cells and might be suitable for immunohistological analysis of different cartilage tissues and pathologically altered chondrocytes.     

Effects of Strontium on Collagen Content and Expression of Related Genes in Rat Chondrocytes Cultured In Vitro

Strontium stimulates cartilage matrix formation in vitro. However, the mechanisms governing these effects have not yet been extensively reported. In this study, chondrocytes were isolated from rat articular cartilage by enzymatic digestion and cultured for 24–72 h with 1–5 mM strontium. We investigated the effects of different concentrations of strontium on collagen content, type II collagen, insulin-like growth factor (IGF-1) and matrix metalloproteinase (MMP)-13 expression in rat cultured articular chondrocytes in vitro. The collagen content of the chondrocytes, determined as hydroxyproline, was measured by a colorimetry method. Type II collagen, IGF-1, and MMP-13 mRNA abundance and protein expression levels were determined by real-time polymerase chain reaction (real-time PCR) and western blot, respectively. The results showed that collagen content from the chondrocytes extracellular matrix increased with increasing strontium concentration. Moreover, 3 and 5 mM strontium strongly stimulated protein expression and mRNA levels of type II collagen and IGF-1. Conversely, MMP-13 expression in chondrocytes decreased dose-dependently with increasing strontium concentration. These results should provide insight into the ability of strontium to promote chondrocyte extracellular matrix synthesis. Strontium could promote collagen synthesis and suppress collagen degradation via the repression of MMP-13 expression.     

Imaging cell interactions with native and crosslinked polyelectrolyte multilayers

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films. Both authors kcontributed equally.