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     

FT-IR Microspectroscopy of Rat Ear Cartilage

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140–820 cm^-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of –SO₃^- groups at 1064 cm^-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of –SO₃^- groups (1236–1225 cm^-1) overlapped with that of amide III bands, it is not recommended for evaluation of the –SO₃^- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027–1016 cm^-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.     

Caprine articular,meniscus and intervertebral disc cartilage: An integral analysis of collagen network and chondrocytes

Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM. Although it is known that the various (fibro)cartilaginous tissues (articular cartilage, annulus fibrosus, nucleus pulposus, and meniscus) differ from one each other with respect to their molecular make-up, remarkable little quantitative information is available with respect to its biochemical constituents, such as collagen content, or the various posttranslational modifications of collagen. Furthermore, we have noticed that tissue-engineering strategies to replace cartilaginous tissues pay in general little attention to the biochemical differences of the tissues or the phenotypical differences of the (fibro)chondrocytes under consideration. The goal of this paper is therefore to provide quantitative biochemical data from these tissues as a reference for further studies. We have chosen the goat as the source of these tissues, as this animal is widely accepted as an animal model in orthopaedic studies, e.g. in the field of cartilage degeneration and tissue engineering. Furthermore, we provide data on mRNA levels (from genes encoding proteins/enzymes involved in the synthesis and degradation of the ECM) from (fibro)chondrocytes that are freshly isolated from these tissues and from the same (fibro)chondrocytes that are cultured for 18 days in alginate beads. Expression levels of genes involved in the cross-linking of collagen were different between cells isolated from various cartilaginous tissues. This opens the possibility to include more markers than the commonly used chondrogenic markers type II collagen and aggrecan for cartilage tissue-engineering applications.     

Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis

Bone marrow mesenchymal stem cells (MSCs) are candidate cells for cartilage tissue engineering. This is due to their ability to undergo chondrogenic differentiation after extensive expansion in vitro and stimulation with various biomaterials in three-dimensional (3-D) systems. Collagen type II is one of the major components of the hyaline cartilage and plays a key role in maintaining chondrocyte function. This study aimed at analyzing the MSC chondrogenic response during culture in different types of extracellular matrix (ECM) with a focus on the influence of collagen type II on MSC chondrogenesis. Bovine MSCs were cultured in monolayer as well as in alginate and collagen type I and II hydrogels, in both serum free medium and medium supplemented with transforming growth factor (TGF) beta1. Chondrogenic differentiation was detected after 3 days of culture in 3-D hydrogels, by examining the presence of glycosaminoglycan and newly synthesized collagen type II in the ECM. Differentiation was most prominent in cells cultured in collagen type II hydrogel, and it increased in a time-dependent manner. The expression levels of the of chondrocyte specific genes: sox9, collagen type II, aggrecan, and COMP were measured by quantitative "Real Time" RT-PCR, and genes distribution in the hydrogel beads were localized by in situ hybridization. All genes were upregulated by the presence of collagen, particularly type II, in the ECM. Additionally, the chondrogenic influence of TGF beta1 on MSCs cultured in collagen-incorporated ECM was analyzed. TGF beta1 and dexamethasone treatment in the presence of collagen type II provided more favorable conditions for expression of the chondrogenic phenotype. In this study, we demonstrated that collagen type II alone has the potential to induce and maintain MSC chondrogenesis, and prior interaction with TGF beta1 to enhance the differentiation.     

Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen

The functional integrity of articular cartilage is dependent on the maintenance of the extracellular matrix (ECM), a process which is controlled by chondrocytes. The regulation of ECM biosynthesis is complex and a variety of substances have been found to influence chondrocyte metabolism. In the present study we have investigated the effect of degraded collagen on the formation of type II collagen by mature bovine chondrocytes in a cell culture model. The culture medium was supplemented with collagen hydrolysate (CH) and biosynthesis of type II collagen by chondrocytes was compared to control cells treated with native type I and type II collagen and a collagen-free protein hydrolysate. The quantification of type II collagen by means of an ELISA technique was confirmed by immunocytochemical detection as well as by the incorporation of (14)C-proline in the ECM after a 48 h incubation. Chondrocytes in the control group were maintained in the basal medium for 11 days. The presence of extracellular CH led to a dose-dependent increase in type II collagen secretion. However, native collagens as well as a collagen-free hydrolysate of wheat proteins failed to stimulate the production of type II collagen in chondrocytes. These results clearly indicate a stimulatory effect of degraded collagen on the type II collagen biosynthesis of chondrocytes and suggest a possible feedback mechanism for the regulation of collagen turnover in cartilage tissue.     

Accumulation of advanced glycation end products decreases collagen turnover by bovine chondrocytes

The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that chondrocytes maintain a functional collagen network. We investigated the effects of advanced glycation end products (AGEs) on the turnover of collagen by articular cartilage chondrocytes. Increased AGE levels (by culturing in the presence of ribose) resulted in decreased collagen synthesis (P < 0.05) and decreased MMP-mediated collagen degradation (P < 0.02). The latter could be attributed to increased resistance of the collagen network to MMPs (P < 0.05) as well as the decreased production of MMPs by chondrocytes (P < 0.02). Turnover of a protein is determined by its synthesis and degradation rates and therefore these data indicate that collagen turnover is decreased at enhanced AGE levels. Since AGE levels in human cartilage increase approximately 50 fold between age 20 and 80, cartilage collagen turnover likely decreases with increasing age. Impaired collagen turnover adversely affects the capacity of chondrocytes to remodel and/or repair its extracellular matrix. Consequently, age-related accumulation of AGE (via decreased collagen turnover) may contribute to the development of cartilage damage in osteoarthritis.     

Analysis of cartilage maturation using micromass cultures of primary chondrocytes

A micromass culture (MM-C) system of primary immature chondrocytes for functional analysis of soluble factors involved in the maturation step of cartilage was previously developed. Ectopically expressed BMP-2 was shown to induce the expression of the Ihh and Noggin genes. Here it is demonstrated that, upon longer culture, secreted bone morphogenetic protein-2 (BMP-2) further promotes the maturation step as judged by the induction of type X collagen and BMP-6 expression, which are known to be detectable in the later phase of cartilage maturation. Induction of all of these genes by secreted BMP-2 was not inhibited by ectopic expression of parathyroid hormone-related peptide (PTHrP) induced by retrovirus vector infection, although the same virus vector showed strong inhibitory effects on the expression of type X collagen gene or alkaline phosphatase activity in mature chondrocytes. These results suggest that the maturation-promoting activity exhibited by BMP-2 is dominant over the suppressive effect of PTHrP in immature chondrocytes. When the BMP-6 gene was introduced into the same virus vector as that used for BMP-2, it induced the same sets of genes (Ihh, Noggin, type X collagen and endogenous BMP-6) as BMP-2 did. These results also suggest that BMP-6 would autonomously maintain and/or promote a later stage of chondrocytic maturation.     

Human polymer-based cartilage grafts for the regeneration of articular cartilage defects

The use of autologous chondrocyte implantation (ACI) and its further development combining autologous chondrocytes with bioresorbable matrices may represent a promising new technology for cartilage regeneration in orthopaedic research. Aim of our study was to evaluate the applicability of a resorbable three-dimensional polymer of pure polyglycolic acid (PGA) for the use in human cartilage tissue engineering under autologous conditions. Adult human chondrocytes were expanded in vitro using human serum and were rearranged three-dimensionally in human fibrin and PGA. The capacity of dedifferentiated chondrocytes to re-differentiate was evaluated after two weeks of tissue culture in vitro and after subcutaneous transplantation into nude mice by propidium iodide/fluorescein diacetate (PI/FDA) staining, scanning electron microscopy (SEM), gene expression analysis of typical chondrocyte marker genes and histological staining of proteoglycans and type II collagen. PI/FDA staining and SEM documented that vital human chondrocytes are evenly distributed within the polymer-based cartilage tissue engineering graft. The induction of the typical chondrocyte marker genes including cartilage oligomeric matrix protein (COMP) and cartilage link protein after two weeks of tissue culture indicates the initiation of chondrocyte re-differentiation by three-dimensional assembly in fibrin and PGA. Histological analysis of human cartilage tissue engineering grafts after 6 weeks of subcutaneous transplantation demonstrates the development of the graft towards hyaline cartilage with formation of a cartilaginous matrix comprising type II collagen and proteoglycan. These results suggest that human polymer-based cartilage tissue engineering grafts made of human chondrocytes, human fibrin and PGA are clinically suited for the regeneration of articular cartilage defects.     

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.     

Integrin–ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. We recently identified Diedel, a secreted protein/cytokine, as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. Here, we show that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, we uncovered a resilience mechanism of epithelial tissues, mediated by Integrin–ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, we found that resilient epithelial cells, enriched for Diedel–Integrin–ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin–ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin–ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

A key feature of the resilience mechanisms that underlie the control of epithelial tissue damage is the balance between cell death and survival. This study shows that the anti-oxidant enzyme catalase can relocate to membranes in order to promote the resilience of the Drosophila midgut epithelium, synergizing with integrin-ECM interactions to prevent the spread of cell death.     

Effect of different ascorbate supplementations on in vitro cartilage formation in porcine high-density pellet cultures

Articular cartilage has only very limited potential for self-repair and regeneration. For this reason, various tissue engineering approaches have been developed to generate cartilage tissue in vitro. Usually, most strategies require ascorbate supplementation to promote matrix formation by isolated chondrocytes. In this study, we evaluate and compare the effect of different ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate, sodium l-ascorbate, and l-ascorbate-2-phosphate were administered in 100 μM, 200 μM, and 400 μM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation were evaluated histologically and could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied by real-time RT-PCR and compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. Again, all supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes. Besides that, we could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations.     

Hypoxia-inducible Factor-1 (HIF-1) but Not HIF-2 Is Essential for Hypoxic Induction of Collagen Prolyl 4-Hydroxylases in Primary Newborn Mouse Epiphyseal Growth Plate Chondrocytes

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α₂β₂ tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I–III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.     

Peripheral blood derived mononuclear cells enhance osteoarthritic human chondrocyte migration

IntroductionA major problem in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into defects. Cartilage is essentially avascular and therefore its healing is not considered to involve mononuclear cells. Peripheral blood derived mononuclear cells (PBMC) offer a readily available autologous cell source for clinical use and therefore this study was designed to evaluate the effects of PBMCs on chondrocytes and cartilage.MethodsHuman primary chondrocytes and cartilage tissue explants were taken from patients undergoing total knee replacement (n = 17). Peripheral blood samples were obtained from healthy volunteers (n = 12) and mononuclear cells were isolated by density-gradient centrifugation. Cell migration and chemokinetic potential were measured using a scratch assay, xCELLigence and CyQuant assay. PCR array and quantitative PCR was used to evaluate mRNA expression of 87 cell motility and/or chondrogenic genes.ResultsThe chondrocyte migration rate was 2.6 times higher at 3 hour time point (p < 0.0001) and total number of migrating chondrocytes was 9.7 times higher (p < 0.0001) after three day indirect PBMC stimulus and 8.2 times higher (p < 0.0001) after three day direct co-culture with PBMCs. A cartilage explant model confirmed that PBMCs also exert a chemokinetic role on ex vivo tissue. PBMC stimulation was found to significantly upregulate the mRNA levels of 2 chondrogenic genes; collagen type II (COL2A1 600–fold, p < 0.0001) and SRY box 9 (SOX9 30–fold, p < 0.0001) and the mRNA levels of 7 genes central in cell motility and migration were differentially regulated by 24h PBMC stimulation.ConclusionThe results support the concept that PBMC treatment enhances chondrocyte migration without suppressing the chondrogenic phenotype possibly via mechanistic pathways involving MMP9 and IGF1. In the future, peripheral blood mononuclear cells could be used as an autologous point-ofcare treatment to attract native chondrocytes from the diseased tissue to aid in cartilage repair.