The response of primary articular chondrocytes to micrometric surface topography and sulphated hyaluronic acid-based matrices

Understanding the response of chondrocytes to topographical cues and chemical patterns could provide invaluable information to advance the repair of chondral lesions. We studied the response of primary chondrocytes to nano- and micro-grooved surfaces, and sulphated hyaluronic acid (HyalS). Cells were grown on grooves ranging from 80 nm to 9 microm in depth, and from 2 microm to 20 microm in width. Observations showed that the cells did not spread appreciably on any groove size, or alter morphology or F-actin organization, although cells showed accelerated movement on 750 nm deep grooves in comparison to flat surfaces. On chemical patterns, the cells migrated onto, and preferentially attached to, HyalS and showed a greater degree of spreading and F-actin re-arrangement. This study shows that 750 nm deep grooves and sulphated hyaluronic acid elicit responses from primary chondrocytes, and this could have implications for the future direction of cartilage reconstruction and orthopaedic treatments in general.     

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.     

Nitric oxide and energy production in articular chondrocytes

Addition of human, recombinant interleukin-1β (hrIL-1β) to cultures of lapine articular chondrocytes provoked a delayed increase in the production of both nitric oxide (NO) and lactate. These two phenomena followed a similar time course and shared a parallel dose-response sensitivity to hrIL-1β. A causal relationship is suggested by the ability of N-monomethyl-L-arginine (NMA), an inhibitor of NO synthase, to blunt the glycolytic response to hrIL-1β. Furthermore, addition of S-nitroso-N-acetylpenicillamine (SNAP), which spontaneously generates NO in culture, increased lactate production to the same degree as IL-1. However, 8-Br-cGMP and isobutylmethylxanthine (IBMX) had no effect either in the presence or absence of IL-1. Even under standard, aerobic, cell culture conditions, chondrocytes consumed little oxygen, either in the presence or absence of IL-1 or NMA. Furthermore, cyanide at concentrations up to 100 μM had no effect upon NO synthesis or lactate production. Thus, the increases in glycolysis under study were not secondary to reduced mitochondrial activity. Although cells treated with IL-1 had increased rates of glycolysis, their concentrations of ATP fell below those of untreated chondrocytes in a time-dependent, but NMA-independent, manner. Transforming growth factor-β (TGF-β) and synovial cytokines (CAF) also increased lactate production. However, TGF-β failed to induce NO, and its effect on glycolysis was independent of NMA. Furthermore, cells treated with TGF-β were not depleted in ATP. These data are consistent with hypotheses that rates of proteoglycan synthesis are, in part, regulated by the intracellular concentration of ATP or by changes in pericellular pH. These two possibilities are not mutually exclusive. © 1994 wiley-Liss, Inc.     

Extracellular matrix metabolism by chondrocytes. 4. Role of glutamine in glycosaminoglycan synthesis in vitro by chondrocytes

The rate of synthesis of glycosaminoglycans by cartilage was shown to be dependent on an exogenous source of L-glutamine. In the absence of L-glutamine the tissue and cellular levels of this amino acid were rapidly depleted. The levels of nucleotide sugars and their precursors were measured after separation on Dowex 1 (formate form) in cartilage incubated with and without L-glutamine. It was found that the levels of N-acetylhexoamine 6-phosphate and UDP-N-acetylhexosamine were decreased by 27 and 40% respectively. This demonstrates that L-glutamine is required as the amido group donor in the synthesis of glucosamine 6-phosphate and that the decrease in glycosaminoglycan synthesis is due to the limitation in synthesis of UDP-N-acetylhexoamine.     

Cell hypertrophy and type X collagen synthesis in cultured articular chondrocytes

Articular cartilage is a permanent tissue whose cells do not normally take part in the endochondral ossification process. To determine whether articular chondrocytes possess the potential to express traits associated with this process such as cell hypertrophy and type X collagen, chondrocytes were isolated from adult chicken tibial articular cartilage and maintained in long-term suspension cultures. As a positive control in these experiments, we used parallel cultures of chondrocytes from the caudal portion of chick embryo sternum. Both articular and sternal chondrocytes readily proliferated and progressively increased in size with time in culture. Many had undergone hypertrophy by 4-5 weeks. Analysis of medium-released collagenous proteins revealed that both articular and sternal chondrocytes initiated type X collagen synthesis between 3 and 4 weeks of culture; synthesis of this macromolecule increased with further growth. Immunofluorescence analysis of 5-week-old cultures showed that about 15% of articular chondrocytes and 30% of sternal chondrocytes produced type X collagen; strikingly, there appeared to be no obvious relationship between type X collagen production and cell size. The results of this study show that articular chondrocytes from adult chicken tibia possess the ability to express traits associated with endochondral ossification when exposed to a permissive environment. They suggest also that the process of cell hypertrophy and initiation of type X collagen synthesis are independently regulated both in articular and sternal chondrocytes.     

New collagen markers of 'derepression' synthesized by rabbit articular chondrocytes in culture.

Chondrocytes isolated from rabbit articular cartilage undergo a progressive ‘derepression’ in culture and synthesize Type I, Type III and an unidentified collagen designated Peak ‘X’. In contrast, cartilage slices synthesize predominantly Type II collagen with increasing amounts of Peak ‘X’ during prolonged culture.     

Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes,subcultured chondrocytes,and fibroblasts

The objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor-β (TGF-β) is the most potent mitogen among a variety of factors tested. All three isoforms of TGF-β caused similar dose-dependent increases in chondrocyte proliferation. Other members of the TGF-β family, including bone morphogenetic protein 2B (BMP2B), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet-derived growth factor-AA (PDGF-AA), basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1) also stimulated proliferation but were less effective than TGF-β. In contrast to findings with other cell types, the effects of TGF-β on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines Interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) gave no stimulation, but IL-1 inhibited chondrocyte proliferation induced by TGF-β or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL-1 caused greater increases in proliferation than TGF-β. In summary, these results describe growth factor responses that are characteristic for chondrocytes and provide a basis for the analysis of changes in chondrocyte growth proliferation that occur in aging and tissue injury. © 1994 Wiley-Liss, Inc.     

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E₂ and F₂α, 6 keto PGF₁α and thromboxane B₂ by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10⁻⁷M – 10⁻³M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E₂ and F₂, and to a lesser extent, 6 keto PGF₁α, but T×B₂ production was only slightly inhibited by the drug in the absenced of arachidonic acid and markedly increased in its presence. Colchicine (10⁻⁷M – 10⁻³M) had the opposite effect, causing an inhibition of T×B₂ and stimulating PGE₂ and 6 keto PGF₁α production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A₂ and cyclo-oxygenase, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using ³⁵SO₄ labeling methodology. The results showed that the concentrations tested (10⁻⁵M to 10⁻⁷M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited ³⁵SO₄ incorporation into newly synthesized proteoglycan molecules both in the presence (10⁻⁶M) and absence of exogenous arachidonic acid. In the same concentration range choroquine had no effect.These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.     

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E2 and F2 alpha, 6 keto PGF1 alpha and thromboxane B2 by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10(-7)M - 10(-13)M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E2 and F2 alpha, and to a lesser extent, 6 keto PGF 1 alpha, but TxB2 production was only slightly inhibited by the drug in the absence of arachidonic acid and markedly increased in its presence. Colchicine (10(-7)M-10(-3)M) had the opposite effect, causing an inhibition of TxB2 and stimulating PGE2 and 6 keto PGF1 alpha production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A2 and cyclo-oxygenases, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using 35SO4 labeling methodology. The results showed that at the concentrations tested (10(-5)M to 10(-7)M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited 35SO4 incorporation into newly synthesized proteoglycan molecules both in the presence (10(-6)M) and absence of exogenous arachidonic acid. In the same concentration range chloroquine had no effect. These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.     

Effects of phospholipids on sphingomyelin hydrolysis induced by intestinal alkaline sphingomyelinase: an in vitro study

Digestion of dietary sphingomyelin (SM) is catalyzed by intestinal alkaline sphingomyelinase (SMase) and may have important implications in colonic tumorigenesis. Previous studies demonstrated that the digestion and absorption of dietary SM was slow and incomplete and that the colon was exposed to SM and its hydrolytic products including ceramide. In the present work, we studied the influences of glycerophospholipids and hydrolytic products of phosphatidylcholine (PC; i.e., lyso-PC, fatty acid, diacylglycerol, and phosphorylcholine) on SM hydrolysis induced by purified rat intestinal alkaline SMase in the presence of 10 mM taurocholate. It was found that various phospholipids including PC, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidic acid (PA) inhibit alkaline SMase activity in a dose-dependent manner, with the degree of inhibition being in the order PA > PS > PI > PC > PE. Similar inhibition was also seen in a buffer of pH 7.4, which is close to the physiologic pH in the middle of the small intestine. When the effects of hydrolytic products of PC were studied, lyso-PC, oleic acid, and 1,2-dioleoyl glycerol also inhibited alkaline SMase activity, whereas phosphorylcholine enhanced SMase activity. However, in the absence of bile salt, acid phospholipids including PA, PS, and PI mildly stimulated alkaline SMase activity whereas PC and PE had no effect. It is concluded that in the presence of bile salts, glycerophospholipids and their hydrolytic products inhibit intestinal alkaline SMase activity. This may contribute to the slow rate of SM digestion in the upper small intestine.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

Subchondral bone influences chondrogenic differentiation and collagen production of human bone marrow-derived mesenchymal stem cells and articular chondrocytes

Introduction

Osteoarthritis (OA) is characterized by an imbalance in cartilage and underlying subchondral bone homeostasis. We hypothesized that signals from the subchondral bone may modulate production of matrix components, alter chondrogenic differentiation potential of cocultured bone marrow-derived mesenchymal stem cells (BMSC) and induce a phenotypic shift in differentiated OA chondrocytes.

Methods

We established a novel coculture model between BMSC, mixed cultures (BMSC and chondrocytes) and chondrocytes embedded in fibrin gel with OA and normal subchondral bone explants (OAB and NB). Tissues and cells were either derived from OA or trauma patients. In addition, we used adipose-derived stem cells (ASC) from liposuction. With gene expression analysis, biochemical assays, immunofluorescence and biomechanical tests we characterized the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiating BMSC cocultured with OAB or NB in comparison with monocultures (cultures without bone explants).

Results

Overall, gene expression of collagens of OAB and NB cocultured cells was reduced compared to monocultures. Concomitantly, we observed significantly lower collagen I, II and III and glycosaminoglycan (GAG) production in OAB cocultured cell lysates. In parallel, we detected increased concentrations of soluble GAGs and basic fibroblast growth factor (bFGF), interleukin (IL)-6 and IL-8 in supernatants of OAB and NB cocultures mainly at early time points. IL-1ß concentration was increased in supernatants of OAB cocultures, but not in NB cocultures. Cell-free NB or OAB explants released different amounts of IL-1ß, bFGF and soluble GAG into cell culture supernatants. In comparison to cocultures, monocultures exhibited higher Young’s modulus and equilibrium modulus. Stimulation of monocultures with IL-1ß led to a downregulation of aggrecan (ACAN) gene expression and in general to induced matrix metalloprotease (MMP)2, MMP3 and MMP-13 gene expression while IL-6 and IL-8 stimulation partly reduced ACAN, MMP3 and MMP-13 gene expression.

Conclusions

Our results suggest an alteration of molecular composition and mechanical properties of the newly formed ECM in subchondral bone cocultures. We suggest that soluble factors, that is interleukins and bFGF, released in cocultures exert inhibitory effects on collagen and temporary effects on proteoglycan production, which finally results in a reduction of mechanical strength of newly formed fibrillar networks.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0453-9) contains supplementary material, which is available to authorized users.     

YKL-40 (cartilage gp-39) induces proliferative events in cultured chondrocytes and synoviocytes and increases glycosaminoglycan synthesis in chondrocytes

YKL-40 (cartilage gp-39), is a mammalian glycoprotein related in sequence to chitinases. Its function is unknown, but it is thought to be involved in tissue remodeling. Immunocytochemical staining of YKL-40 in guinea pig chondrocytes (GPC), rabbit chondrocytes (RC), and rabbit synoviocytes (RS) was higher in dividing cells than in confluent cells, suggesting a participation of YKL-40 in cell cycle events. As assessed by the MTT assay, YKL-40 at 1.9-7.6 nM had dose-dependent mitogenic activity toward the three cell types. At 7.6 nM, YKL-40 increased the number of cells of 42% in GPC, 75% in RC, and 86% in RS after 72 h. YKL-40 also stimulated total proteoglycan synthesis by chondrocytes in a dose-dependent manner as assessed by Na[35SO4] incorporation and cetylpyridinium chloride precipitation. At 9.4 nM, YKL-40 increased proteoglycan synthesis of 42% in GPC and 58% in RC after 24 h. The growth factor properties of YKL-40 may explain the increased tissue remodeling associated with high levels of YKL-40 in joint diseases, and possibly, in malignant pathologies such as breast cancer or colorectal cancer.     

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.     

Thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes

Thrombospondin, a multifunctional adhesive glycoprotein originally identified in platelets, was isolated and identified from an extract of ovine articular cartilage. Immunoreactive material from a cartilage extract comigrated on gel electrophoresis with purified human platelet thrombospondin. When articular chondrocytes were cultured in the presence of 35S-methionine, metabolically labeled thrombospondin was immunoprecipitated from the culture medium and cell layer extract. These results demonstrate that thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

Influence of perfusion on metabolism and matrix production by bovine articular chondrocytes in hydrogel scaffolds

Articular cartilage has a limited capacity for self-repair after damage. Engineered cartilage is a promising treatment to replace or repair damaged tissue. The growth of engineered cartilage is sensitive to the extracellular culture environment. Chondrocytes were seeded into alginate beads and agarose scaffolds at 4 millions/mL, and the response to static and perfusion culture was examined over period of up to 12 days. For both types of scaffolds, the chondrocytes kept their differentiated morphology over 12 days in all culture conditions. In alginate beads, more glycosaminoglycans (GAGs) were produced in perfusion culture than in static conditions. GAG distribution in alginate constructs was more uniform in perfusion culture than in static culture. However, in agarose constructs there was no significant difference in GAG production between static culture and perfusion culture. Under perfusion culture, the retention rate of GAG in alginate was higher than in agarsoe. It is suggested that the positive effect of perfusion culture only can be achieved by an appropriate choice of other factors such as scaffold materials.     

The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell culture

Summary This report describes a method for preparing primary cell cultures of differentiated rabbit sternal and human vertebral cartilage cells. These cell cultures were shown to synthesize primarily α1 chains, which is taken to mean that at least 82% of the collagen produced is cartilage specific collagen (type II). This work was supported in part by grant HD-05505 from NIH.