High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-κB ligand through Rho kinase.

OBJECTIVE: To determine the effects of high molecular weight hyaluronic acid (HMW-HA) on osteoclast differentiation by monocytes co-cultured with stromal cells. METHODS: Mouse bone marrow stromal cell line ST2 cells were incubated with HMW-HA or 4-methylunbeliferone (4-MU) for various times. In some experiments, cells were pre-treated with the anti-CD44 monoclonal antibody (CD44 mAb) or Rho kinase pathway inhibitors (simvastatin or Y27632), then treated with HMW-HA. The expression of receptor activator of NF-κB ligand (RANKL) was determined using real-time reverse transcription polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence microscopy, while the amount of active RhoA was measured by a pull-down assay. To further clarify the role of HMW-HA in osteoclastogenesis, mouse monocyte RAW 264.7 cells were co-cultured with ST2 cells pre-stimulated with 1,25(OH)2D3. Osteoclast-like cells were detected by staining with tartrate-resistant acid phosphatase (TRAP). RESULTS: HMW-HA decreased RANKL mRNA and protein expressions, whereas inhibition of hyaluronic acid (HA) synthesis by 4-MU enhanced RANKL expression. Blockage of HA-CD44 binding by CD44 mAb suppressed HMW-HA-mediated inhibition of RANKL. Pull-down assay findings also revealed that HMW-HA transiently activated RhoA in ST2 cells and pre-treatment with CD44 mAb inhibited the activation of RhoA protein mediated by HMW-HA. Moreover pre-treatment with Rho kinase pathway inhibitors also blocked the inhibition of RANKL by HMW-HA. Co-culture system results showed that HMW-HA down-regulated differentiation into osteoclast-like cells by RAW 264.7 cells induced by 1,25(OH)2D3-stimulated ST2 cells. CONCLUSIONS: These results indicated that HA-CD44 interactions down-regulate RANKL expression and osteoclastogenesis via activation of the Rho kinase pathway.

Hyaluronan oligosaccharide treatment of chondrocytes stimulates expression of both HAS-2 and MMP-3, but by different signaling pathways.

OBJECTIVE: Small hyaluronan (HA) oligosaccharides displace HA from the cell surface and induce cell signaling events. In articular chondrocytes this cell signaling is mediated by the HA receptor CD44 and includes stimulation of genes involved in matrix degradation such as matrix metalloproteinases (MMPs) as well as matrix repair genes including collagen type II, aggrecan and HA synthase-2 (HAS-2). The objective of this study was to determine whether stimulation of HAS-2 and MMP-3 by HA oligosaccharides is due to the activation of a single, cascading pathway or multiple signaling pathways. METHOD: Bovine articular chondrocytes were pre-treated with a variety of inhibitors of major signaling pathways prior to the addition of HA oligosaccharides. Changes in HA were monitored by real time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HAS-2 mRNA, HA ELISA and HA accumulation at the cell surface. A 1900 base pair sequence containing the proximal promoter of HAS-2 was inserted into a luciferase reporter construct, transfected into human immortalized chondrocytes and assayed in a similar fashion. RESULTS: While our previous studies demonstrated that HA oligosaccharides stimulate MMP-13 activity via activation of p38 MAP kinase and NF-kappaB, inhibitors of these pathways did not affect the stimulation of HAS-2 mRNA expression. However, inhibiting the phosphatidylinositol-3-kinase pathway blocked HA oligosaccharide-mediated stimulation of HAS-2 yet had no effect on MMP-3. Wortmannin and LY294002 also blocked HA oligosaccharide-induced serine and threonine Akt phosphorylation. Treatment of transfected immortalized chondrocytes with HA oligosaccharides resulted in stimulation of HAS-2 mRNA, activation of Akt and enhanced luciferase activity-activity that was blocked by inhibitors of Akt phosphorylation. CONCLUSIONS: Changes in chondrocyte-matrix interactions by HA oligosaccharides induce altered matrix metabolism by the activation of least two distinct signaling pathways.

Hyaluronan promotes the chondrocyte response to BMP-7.

OBJECTIVE: Chondrocytes exhibit specific responses to bone morphogenetic proteins (BMPs) and transforming growth factor-betas (TGF-betas). The bioactivity of these growth factors is regulated by numerous mediators. In our previous study, Smad1 was found to interact with the cytoplasmic domain of the hyaluronan receptor CD44. The purpose of this study was to determine the ability of hyaluronan in the pericellular matrix to modulate the chondrocyte responses to BMP-7 or TGF-beta1. EXPERIMENTAL DESIGN: Nuclear translocation of Smad1, Smad2 and Smad4 was studied in bovine articular chondrocytes in response to BMP-7 and TGF-beta1. The effects of matrix disruption by hyaluronidase treatment and the initiation of matrix repair by the addition of hyaluronan on the nuclear translocation of Smad proteins, Smad1 phosphorylation and luciferase expression by a CD44 reporter construct in response to BMP-7 were also studied. RESULTS: The disruption of the hyaluronan-dependent pericellular matrix of chondrocytes resulted in diminished nuclear translocation of endogenous Smad1 and Smad4 in response to BMP-7; however, the nuclear translocation of Smad2 and Smad4 in these matrix-depleted chondrocytes in response to TGF-beta1 was not diminished. Incubation of the matrix-depleted chondrocytes with exogenous hyaluronan restored Smad1 and Smad4 nuclear translocation and increased pCD44(499)-Luc luciferase expression in response to BMP-7. Both exogenous hyaluronan and matrix re-growth enhanced by hyaluronan synthase-2 (HAS2) transfection restored Smad1 phosphorylation. CONCLUSIONS: Disruption of hyaluronan-CD44 interactions has little effect on the TGF-beta responses; however, re-establishing CD44-hyaluronan ligation promotes a robust cellular response to BMP-7 by articular chondrocytes. Thus, changes in cell-hyaluronan interactions may serve as a mechanism to modulate cellular responsiveness to BMP-7.

Expression and cellular localization of human hyaluronidase-2 in articular chondrocytes and cultured cell lines.

OBJECTIVE: There is debate whether hyaluronan (HA) can be enzymatically degraded within the extracellular matrix of cartilage and other tissues or whether its catabolism occurs strictly within the lysosomal compartment of chondrocytes and other cell types. Previous studies have suggested that one of the lysosomal hyaluronidases (hyaluronidase-2) can be expressed as a functionally-active glycosyl phosphatidylinositol-linked protein at the surface of mammalian cells. If this form of hyaluronidase expression occurs in chondrocytes, this could represent a possible mechanism for extracellular HA cleavage. Thus, which hyaluronidases are expressed and where was the objective of this study. METHODS: mRNA for hyaluronidases was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and enzymatic activity by HA zymograms. Recombinant forms of hyaluronidase-2 were generated and expressed in model cell lines. A peptide-specific polyclonal antiserum was prepared to localize endogenous human hyaluronidase-2 in human articular chondrocytes. RESULTS: Hyaluronidase-2 is the principal mRNA transcript expressed by primary human articular chondrocytes as well as various model cell lines. Recombinant hyaluronidase-2, containing N-terminal or C-terminal epitope tags, was strictly localized intracellularly and not released by treatment with a phosphatidylinositol-specific phospholipase. Endogenous hyaluronidase-2 expressed by human chondrocytes as well as HeLa cells could only be detected following detergent permeabilization of the plasma membranes. CONCLUSIONS: These data suggest that on chondrocytes and other cell types examined, hyaluronidase-2 is not present or functional at the external plasma membrane. Thus, local turnover of HA is dependent on receptor-mediated endocytosis and delivery to low pH intracellular organelles for its complete degradation.

Expression of superficial zone protein in mandibular condyle cartilage.

OBJECTIVE: Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle. METHODS: The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta) on SZP expression were examined in condyle explants and fibroblasts derived from the fibrous zone of condyle cartilage. RESULTS: SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-beta in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1beta. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-beta. CONCLUSIONS: These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself, and exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies.

Induction of MMP-3 by hyaluronan oligosaccharides in temporomandibular joint chondrocytes.

Low-molecular-weight hyaluronan (LMW-HA) is often increased in osteoarthritic joints; however, its biological function in cartilage has not been clarified. We hypothesize that LMW-HA causes the catabolic activation of chondrocytes through its interaction with CD44. Cartilage explants and chondrocytes, derived from bovine temporomandibular joints (TMJ), were examined for matrix loss and the expression of matrix metalloproteinase-3 (MMP-3) following treatment with hyaluronan oligosaccharides (HA(oligos)). Hyaluronan and CD44 were uniformly distributed throughout the fibrous and cartilaginous zones of the TMJ condyle. Treatment of cartilage explants with HA(oligos) resulted in cartilage matrix loss with increased secreted caseinolytic activity. HA(oligos) treatment of TMJ chondrocytes resulted in enhanced MMP-3 expression, whereas wash-out of the HA(oligos) in the middle of the experimental period reduced this induction. These results suggest that HA(oligos) activate chondrocytes, resulting in a substantial enhancement of proteinase expression, and the removal of HA(oligos) by wash-out reverses this catabolic activation.

Extracellular matrix recovery by human articular chondrocytes after treatment with hyaluronan hexasaccharides or Streptomyces hyaluronidase.

Abstract The treatment of human articular chondrocytes with Streptomyces hyaluronidase (St-HA'ase) or hyaluronan hexasaccharides (HA6) provides two approaches to the selective depletion of specific components of the extracellular matrix, and an opportunity to follow the reparative responses initiated by these changes. In this study, changes in the relative expression of messenger RNA for hyaluronan synthase-2, CD44, and aggrecan were determined by competitive, quantitative reverse transcriptase-polymerase chain reaction. Changes in the size of the cell-associated matrix surrounding live chondrocytes were analyzed by the particle exclusion assay, and hyaluronan accumulation was characterized using a biotin-labeled hyaluronan-specific binding protein. Both Streptomyces hyaluronidase as well as hyaluronan hexasaccharide treatment of chondrocytes resulted in an approximately 2-fold increase in hyaluronan synthase-2 mRNA copy numbers, together with a 1.8-fold increase in the mRNA copy number for the proteoglycan aggrecan. However, although matrix biosynthesis was enhanced, the chondrocytes failed to retain these components. Both treatments resulted in a diminished accumulation of extracellular hyaluronan as well as a loss of the chondrocyte proteoglycan-rich cell-associated matrix. Thus, this model is similar to the early stages of osteoarthritis. Upon removal of the Streptomyces hyaluronidase or hyaluronan hexasaccharides, the normal, healthy, adult human chondrocytes used in this study regained their capacity to retain extracellular hyaluronan and to reassemble and retain a cell-associated matrix. This stimulation of hyaluronan synthase-2 (HAS-2) and aggrecan mRNA expression, and the subsequent capacity to retain the newly synthesized extracellular matrix, illustrate the events which are necessary for adult human articular chondrocytes to undergo effective repair.

Antisense inhibition of CD44 tailless splice variant in human articular chondrocytes promotes hyaluronan internalization.

OBJECTIVE: To determine whether alternatively spliced variants of CD44, in particular a short, intracellular tail CD44 isoform, are used by articular chondrocytes to modulate the functions of this matrix receptor. METHODS: Normal human articular chondrocytes were cultured with or without interleukin-1alpha (IL-1alpha), and the relative expression of CD44 exon 19 and CD44 exon 20, hyaluronan synthase 2, aggrecan, and GAPDH messenger RNA (mRNA) was determined using reverse transcriptase-polymerase chain reaction. Next, CD44 exon 19 mRNA was selectively inhibited by the use of antisense oligonucleotides. The effects of exon 19 loss were analyzed by matrix assembly and hyaluronan internalization assays. RESULTS: Human articular chondrocytes express varying levels of exon 19 (short tail)- and exon 20 (long tail)-containing CD44 mRNA. Both CD44 mRNA are up-regulated by IL-1alpha. Selective inhibition of CD44 exon 19 results in enhanced hyaluronan internalization and smaller cell-associated matrices. CONCLUSION: The expression of a natural CD44 decoy-like receptor by articular chondrocytes modulates the function of this matrix receptor.

Cartilage proteoglycans.

The predominant proteoglycan present in cartilage is the large chondroitin sulfate proteoglycan 'aggrecan'. Following its secretion, aggrecan self-assembles into a supramolecular structure with as many as 50 monomers bound to a filament of hyaluronan. Aggrecan serves a direct, primary role providing the osmotic resistance necessary for cartilage to resist compressive loads. Other proteoglycans expressed during chondrogenesis and in cartilage include the cell surface syndecans and glypican, the small leucine-rich proteoglycans decorin, biglycan, fibromodulin, lumican and epiphycan and the basement membrane proteoglycan, perlecan. The emerging functions of these proteoglycans in cartilage will enhance our understanding of chondrogenesis and cartilage degeneration.

Mechanisms of chondrocyte adhesion to cartilage: role of beta1-integrins, CD44, and annexin V.

The initial adhesion of transplanted chondrocytes to surrounding host cartilage may be important in the repair of articular defects. Adhesion may position cells to secrete molecules that fill the defect and integrate repair tissue with host tissue. While chondrocytes are known to become increasingly adherent to cartilage with time, the molecular basis for this is unknown. The objective of this study was to investigate the role of beta1-integrin, CD44, and annexin V receptors in chondrocyte adhesion to cartilage. Chondrocytes were cultured in high density monolayer, released with trypsin, and allowed to recover in suspension for 2 h at 37 degrees C. Under these conditions, flow cytometry analysis showed that chondrocytes expressed beta1-integrins, CD44, and annexin V. In a rapid screening assay to assess chondrocyte adhesion to cartilage, cell detachment decreased from 79% at 10 min following transplantation to 10% at 320 min. Treatment of cells with a monoclonal antibody to block beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls. Similarly, results from a parallel-plate shear flow adhesion assay showed that blocking beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls at each level of applied shear (0-70 Pa). In both assays, treatment of cells with reagents that block CD44 (hyaluronan oligosaccharides or monoclonal Ab IM7) or annexin V (polyclonal Ab #8958) had no detectable effect on adhesion. With cartilage treated with chondroitinase ABC, blocking beta1-integrins also increased chondrocyte detachment, while blocking CD44 and annexin V also had no detectable effect. Under the conditions studied here, beta1-integrins appear to mediate chondrocyte adhesion to a cut cartilage surface. Delineation of the mechanisms of adhesion may have clinical implications by allowing cell manipulations or matrix treatments to enhance chondrocyte adhesion and retention at a defect site.

Hyaluronan oligosaccharides perturb cartilage matrix homeostasis and induce chondrocytic chondrolysis.

OBJECTIVE: To determine the role of CD44-mediated matrix assembly in maintaining cartilage homeostasis. METHODS: Articular cartilage tissue slices as well as isolated chondrocytes were treated with hyaluronan (HA) hexasaccharides. Tissues and cells were processed for histology, immunohistochemistry, colorimetric assay, reverse transcriptase-polymerase chain reaction, and zymography. RESULTS: HA oligosaccharides induced a dose-dependent state of chondrocytic chondrolysis, including near-total loss of stainable proteoglycan-rich matrix, activation of gelatinolytic activity, and exhibition of the NITEGE epitope. However, HA oligosaccharides also induced an increase of proteoglycan synthesis, including elevation of aggrecan and HA synthase 2 messenger RNA. CONCLUSION: Uncoupling chondrocytes from the matrix results in deleterious changes in matrix structure and modifications in chondrocyte metabolism. The close interaction of chondrocytes with their matrix and their ability to "sense" changes in receptor occupancy are needed for maintenance of cartilage homeostasis.

Osteogenic protein-1 promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures.

OBJECTIVE: We have used recombinant osteogenic protein-1 to investigate our hypothesis that proper repair and maintenance of cartilage requires not only enhanced biosynthesis and replenishment of the extracellular matrix but also the enhancement of components necessary for matrix retention. DESIGN: The effects of osteogenic protein-1 were examined on bovine articular cartilage slices as well as isolated chondrocytes grown in alginate beads. Cartilage slices were examined for accumulation of proteoglycan by incorporation of 35S-sulfate and staining using Safranin O or, a biotinylated probe specific for hyaluronan. Bovine chondrocytes were characterized by use of a particle exclusion assay, in-situ hybridization, quantitative-competitive RT-PCR and a hyaluronan-binding assay. RESULTS: Osteogenic protein-1 treatment substantially enhanced the accumulation of hyaluronan and proteoglycan within cartilage tissue slices. As with the tissue, osteogenic protein-1 enhanced the size of cell-associated matrices assembled and retained by chondrocytes in vitro. This enhanced matrix assembly was paralleled by an increased expression of mRNA for aggrecan, hyaluronan synthase-2 and CD44. Of the two hyaluronan synthase genes expressed by chondrocytes, only hyaluronan synthase-2 was upregulated by osteogenic protein-1. Coupled with the increase in the CD44 mRNA was an increase in functional hyaluronan binding activity present at the chondrocyte cell surface. CONCLUSIONS: These results demonstrate that osteogenic protein-1 stimulates not only the synthesis of the major cartilage extracellular matrix component aggrecan, but also two associated molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.

Osteogenic protein 1 stimulates cells-associated matrix assembly by normal human articular chondrocytes: up-regulation of hyaluronan synthase, CD44, and aggrecan.

OBJECTIVE: To determine the effects of osteogenic protein 1 (OP-1) on hyaluronan (HA), CD44, and aggrecan biosynthesis as well as the contribution of these molecules in promoting matrix assembly by human articular chondrocytes. METHODS: Normal human chondrocytes were cultured with or without OP-1 treatment. Changes in the relative expression of messenger RNA (mRNA) for HA synthases 2 and 3 (HAS-2 and HAS-3), CD44, and aggrecan were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Accumulation of HA was characterized by indirect staining, CD44 by flow cytometry, and aggrecan biosynthesis by 35SO4 incorporation. RESULTS: OP-1 stimulated the expression of HAS-2, CD44, and aggrecan mRNA in a time-dependent manner, resulting in increased expression of HA, CD44, and aggrecan. Prominent increases in HA-rich cell-associated matrices were also observed. CONCLUSION: OP-1 stimulates not only the synthesis of matrix macromolecules such as aggrecan, but also the synthesis of other molecules required for matrix retention, namely, HA and CD44.

Internalization of the hyaluronan receptor CD44 by chondrocytes.

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.

Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly.

In order to define the role of cell-associated hyaluronan in cartilage matrix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antisense to the mRNA of human HA synthase-2 (HAS-2). As a prerequisite for these studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, or HAS-3, is primarily responsible for HA synthesis in human articular chondrocytes. The copy number of each HAS mRNA expressed in cultured human articular chondrocytes was determined using quantitative (competitive) reverse transcription-polymerase chain reaction (RT-PCR). Only HAS-2 and HAS-3 mRNA expression was detected. The level of HAS-2 mRNA expression was 40-fold higher than that of HAS-3. Cultures of human articular chondrocytes and cartilage tissue slices were then transfected with HAS-2-specific antisense oligonucleotides. This treatment resulted in time-dependent inhibition of HAS-2 mRNA expression, as measured by quantitative RT-PCR, and a significant loss of cell-associated HA staining. Sense and reverse HAS-2 oligonucleotides showed no effect. The consequences of reduced HA levels (due to HAS-2 antisense inhibition) were a decrease in the diameter of the cell-associated matrix and a decreased capacity to retain newly synthesized proteoglycan. These results suggest that HA synthesized by HAS-2 plays a crucial role in matrix assembly and retention by human articular chondrocytes.

Antisense inhibition of chondrocyte CD44 expression leading to cartilage chondrolysis.

OBJECTIVE: To better define critical functions of the hyaluronan receptor CD44 in cartilage. METHODS: Articular chondrocytes and cartilage tissue slices were treated with CD44 sequence-specific antisense phosphorothioate oligonucleotides. CD44 expression was probed by immunofluorescence microscopy, enzyme-linked immunosorbent assay, and Western blotting. RESULTS: Antisense oligonucleotides demonstrated a dose- and time-dependent inhibition of CD44 protein expression; negative controls showed no effect. Similar to osteoarthritic cartilage, antisense-treated cartilage slices displayed a near-total loss of stainable proteoglycan-rich matrix. CONCLUSION: CD44 expression is needed for maintenance of cartilage homeostasis.

CD44-anchored hyaluronan-rich pericellular matrices: an ultrastructural and biochemical analysis.

The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.

Hyaluronan-mediated aggregation of limb bud mesenchyme and mesenchymal condensation during chondrogenesis.

Cell condensations are the initial structures in the formation of proper cartilage and skeletal patterning in the developing vertebrate limb. Chondrogenic differentiation is dependent upon the cell-cell and/or cell-matrix interactions which take place during the condensation process. Coincident with the onset of condensation is the expression by limb mesenchyme of specific cell surface binding sites for the extracellular matrix macromolecule hyaluronan. The association of hyaluronan with the cell surface can influence the behavior of cells, especially cell aggregation. In this study the possible involvement of hyaluronan as an extracellular linker molecule in the cell-cell adhesion event during mesenchymal condensation was investigated in the avian limb model. Hyaluronan hexasaccharides were used to prevent the multivalent interactions that occur between native hyaluronan macromolecules and the cell surface. Our studies show that hyaluronan is required for early adhesive cell-cell interactions of limb bud mesenchyme and that perturbations of hyaluronan-cell interactions with hyaluronan hexasaccharides result in a delay in the formation of condensations as well as a delay in chondrogenic differentiation of mesenchymal cells in micromass cultures.