Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

Biochemical and Biophysical Research Communications 405 (2011) 575–580

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Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid Michinao Kawano a,b, Wataru Ariyoshi b, Kenjiro Iwanaga a, Toshinori Okinaga b, Manabu Habu a, Izumi Yoshioka c, Kazuhiro Tominaga a,d, Tatsuji Nishihara b,d,⇑ a

Division of Maxillofacial Diagnostic and Surgical Science, Department of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu 803-8580, Japan Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580, Japan Division of Oral and Maxillofacial Surgery, Department of Medicine of Sensory and Motor Organs, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan d Oral Bioresearch Center, Kyushu Dental College, Kitakyushu 803-8580, Japan b c

a r t i c l e

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Article history: Received 14 January 2011 Available online 23 January 2011 Keywords: BMP-2 Hyaluronic acid Osteoblast ALP Smad MAPK

a b s t r a c t Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA. Conclusions: These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved.

1. Introduction Hyaluronic acid (HA) is a long polysaccharide consisting of repeating disaccharide units of N-acetylglucosamine and D-glucuronic acid, and a major component of extracellular matrix (ECM) proteins in mammalian tissues [1]. Together with its structural role in the matrix, HA regulates diverse cellular responses including Abbreviations: BMP-2, bone morphogenetic proteins-2; HA, hyaluronic acid; ALP, alkaline phosphatase; ECM, extracellular matrix; TGF-b, transforming growth factor beta; MAPK, mitogen-activated protein kinase; ERK, extracellular signalregulated kinase; HBSS, Hank’s balanced salt solution; FCS, fetal calf serum; realtime RT-PCR, real-time reverse transcription polymerase chain reaction; PBS, phosphate-buffered saline. ⇑ Corresponding author at: Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental College, Kitakyushu 803-8580, Japan. Fax: +81 93 581 4984. E-mail address: [email protected] (T. Nishihara).

proliferation, differentiation, motility, adhesion, and gene expression [2]. A variety of cell types function to shield against potentially harmful extracellular molecules or pathogenesis [3–9]. In bone, HA and its receptor CD44 interact to restrict osteoblast-mediated osteoclast genesis, which may play a role in inter-osteocyte and osteocyte–osteoclast communication [10,11]. Furthermore, those interactions might also provide stop signals for inducing bone resorption [12] and osteoclastogenesis [13,14]. Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta (TGF-b) superfamily, were originally identified as unique proteins in demineralised bone matrix and also shown to induce ectopic bone formation when implanted into muscular tissues [15]. It is well known that BMPs regulate the differentiation and function of cells involved in bone and cartilage formation and deformation, including osteoblasts, chondrocytes, and osteoclasts [16].

0006-291X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.01.071

M. Kawano et al. / Biochemical and Biophysical Research Communications 405 (2011) 575–580

BMP signaling is initiated by binding to type I and type II receptors, which are specific trans-membrane receptors [17]. Type I receptors are activated by ligand bound-type II receptors and then phosphorylate downstream molecules in the cytoplasm. Furthermore, Smad 1/5/8 transcription factors are phosphorylated by the BMP receptor (BMPR) in the cytoplasm as substrates and accumulate in the nucleus within 1 h after BMP stimulation [18]. Phosphorylated Smads directly regulate the expression of primary target genes by binding to their promoter or enhancer elements, together with Smad 4 and other transcription factors [19]. Recently, we found that heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMPR [20], while HA was reported to facilitate TGF-b1-mediated fibroblast proliferation [21] and chondrocyte response to BMP-7 [22]. Although HA is a member of the glycosaminoglycan family, such as heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, and chondroitin6-sulfate, the mechanism by which HA regulates osteogenic activities has not been fully elucidated. In the present study, we examined the effects of HA on osteoblast differentiation induced by BMP-2 in vitro and found that it enhanced osteogenesis. 2. Materials and methods

phate-buffered saline (PBS; pH 7.2) and lysed in cell lysis buffer (75 mM Tris–HCl containing 2% SDS and 10% glycerol, pH 6.8). Samples were then subjected to 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). Non-specific binding sites were blocked by immersing the membranes in 10% skim milk in PBS for 60 min at room temperature, then the membrane was washed six times with PBS, followed by incubation with the diluted primary antibody at 4 °C. Anti-Smad 1/5/8, anti-phospho-Smad 1/5/8, anti-p38 MAPK, antiphospho-p38 MAPK, anti-ERK, anti-phospho-ERK, and anti-b-actin were used as the primary antibodies, while horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were used as secondary antibodies (Santa Cruz Biotechnology Inc.) in this experiment. After washing the membranes, chemiluminescence was produced using ECL reagent (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and detected with Hyperfilm-ECL (GE Healthcare Bio-Sciences Corp.). Gel images were subjected to densitometric analysis using Image Lab™ software (Bio-Rad Laboratories Inc., Hercules, CA, USA).

A

High molecular weight HA (2000 kDa) and recombinant human BMP-2 were kindly supplied by Seikagaku Corp. (Tokyo, Japan) and Astellas Pharmaceutical Inc. (Tokyo, Japan), respectively. Anti-phospho-Smad 1/5/8 polyclonal, anti-p38 mitogen-activated protein kinase (MAPK) polyclonal, anti-phospho-p38 MAPK polyclonal, antiextracellular signal-regulated kinase (ERK) monoclonal, and antiphospho-ERK polyclonal antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-Smad 1/5/8 polyclonal antibody was obtained from Abcam plc (Cambridge, MA, USA). Anti-b-actin monoclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

* 5 (normalized to control)

2.1. Reagents

Fold change of ALP activity

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2.2. Cell culture The human osteoblastic cell line MG63 cells were maintained in

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a-minimum essential medium (a-MEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Equitech-Bio Inc., Kerrville, TX, USA), 100 U/ml penicillin G, and 100 lg/ml streptomycin at 37 °C in an atmosphere of 5% CO2. 2.3. Alkaline phosphatase (ALP) activity

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MG63 cells were seeded at a density of 1  10 /well in a 24-well plate, then ALP activity was evaluated, as described below. Cells were stimulated with BMP-2 (50 ng/ml) and HA (100 lg/ml) for 96 h, washed twice with Hank’s balanced salt solution (HBSS), and solubilized with HBSS containing 0.2% Nonidet P-40. ALP activity of the lysate was determined using p-nitrophenylphosphate (pNPP; Wako, Osaka, Japan) using the Lowry method. After a 30-min incubation at 37 °C, absorbance of pNPP at 405 nm was measured using a Multiskan JX microplate reader (Thermo Fisher Scientific, Rockford, IL, USA). Protein contents were determined using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). The specific activity of ALP was calculated as lM/lg protein. 2.4. Western blot analysis MG63 cells (4  105 cells/well) were cultured with BMP-2 (50 ng/ml) in the presence of HA (100 lg/ml) in 6-well plates for various time periods. Adherent cells were washed twice with phos-

time (min)

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BMP-2 ( 50 ng/ml ) Fig. 1. (A) HA enhances ALP activity induced by BMP-2 in MG63 cells. MG63 cells (4  105 cells/well) were stimulated with BMP-2 (50 ng/ml) in the presence or absence of HA (100 lg/ml) for 4 days. The specific activity of ALP (lM/lg protein) was determined as described in Section 2. Values are expressed as fold increases relative to the untreated controls. Data are expressed as the mean ± SD of triplicate cultures. The experiment was performed three times, with similar results obtained in each. ⁄p < 0.05, as measured using an unpaired Student’s t-test. (B) HA enhances BMP-2-induced Smad 1/5/8 phosphorylation. MG63 cells (4  105 cells/well) were stimulated with BMP-2 (50 ng/ml) in the presence or absence of HA (100 lg/ml) for the indicated time periods, then whole lysates were analyzed by SDS–PAGE and Western blotting analyses. Gel images were subjected to densitometric analysis using Image Lab™ software. The relative band intensity (RBI) of the control was set to 1.

M. Kawano et al. / Biochemical and Biophysical Research Communications 405 (2011) 575–580

2.5. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) Following incubation with BMP-2 (50 ng/ml) and HA (100 lg/ ml), total RNA was extracted using an RNeasy Mini Kit (QIAGEN Inc. Valencia, CA, USA), according to the manufacturer’s instructions. Obtained RNA was transcribed with SuperScript Reverse Transcriptase II (Invitrogen, Carlsbad, CA, USA) and amplified using a Mastercycler gradient (Eppendorf AG, Hamburg, Germany). PCR products Ò were detected using FAST SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) using the following primer sequences: GAPDH forward; 50 -ATGGAAATCCCATCACCATCTT-30 and reverse; 50 -CGCCCCACTTGATTTTGG-30 , noggin forward; 50 -GCGCTGCGGC TGGAT-30 and reverse; 50 -AGCACTTGCACTCGGAAATGA-30 , and follistatin forward; 50 -CGAGGAGGACGTGAATGACA-30 and reverse; 50 -GCGCCCCCGTTGAAA-30 . Thermal cycling and fluorescence detection were performed using a StepOne™ Real-Time PCR System (Applied Biosystems). Real-time RT-PCR efficiency (E) was calculated according to the equation provided by Rasmussen et al. [23] as E = 10 [1/slope] for b-actin and various target genes. The slope was determined from the cDNA substrate (x-axis) versus the cycle number at the crossing point (CP) (y-axis). The CP value was the PCR cycle number that represented the CP in SYBRÒ Green fluorescence intensity above the automatic noise-based threshold. The fold increase in copy numbers of mRNA was determined as the relative ratio of the target gene to GAPDH, using the following mathematical model introduced by Pfaffl [24].

Fold increase ¼

ðETARGET ÞCP TARGET

ðMEAN controlMEAN subjectÞ

CP GAPDH ðMEAN controlMEAN subjectÞ

ðEGAPDH Þ

2.6. Immunofluorescence microscopy Cells were cultured in wells of Lab-TekÒ II chamber slides (Nalge Nunc International, Rochester, NY, USA) at a density of 2  103/

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well with or without BMP-2 (100 ng/ml) and HA (50 lg/ml) for 6– 24 h. The cells were fixed with 4% paraformaldehyde, quenched with 0.2 M glycine in PBS, and then permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. After washing in PBS, specific binding sites were blocked with 10% bovine serum albumin in PBS and the cells were incubated for 1 h at 4 °C with rabbit polyclonal anti-phospho-Smad 1/5/8. Next, the cells were washed and incubated with Cy3-conjugated AffiniPure goat antirabbit IgG antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 1 h at room temperature, followed by addition of the nuclear staining agent 40 ,6-diamidino-2-phenylindole (DAPI). The cells were visualized using a Fluorescence Microscope BZ-9000 (KEYENCE Corp., Osaka, Japan). Images were captured digitally in real time and processed using BZ-II imaging software (KEYENCE Corp.). 2.7. Statistical analysis Statistical differences were determined using an unpaired Student’s t-test with Bonferroni’s correction for multiple comparisons. All data are expressed as the mean ± standard deviation (SD). 3. Results 3.1. Effect of HA on osteoblast differentiation induced by BMP-2 To determine the effect of HA on osteoblast differentiation induced by BMP-2, we assessed ALP activity as a typical marker of osteoblast differentiation. BMP-2 (50 ng/ml) slightly enhanced ALP activity in MG63 cells. However, ALP activity was much higher when the cells were incubated with BMP-2 (50 ng/ml) and HA (100 lg/ml) (Fig. 1A), whereas HA alone had no effect on ALP activity in MG63 cells. Next, we examined the levels of Smad 1/5/8 phosphorylation by Western blot analysis. HA alone caused no

Fig. 2. HA enhances BMP-2-induced nuclear localization of phospho-Smad 1/5/8 protein. MG63 cells (2  103 cells/well) were stimulated with BMP-2 (50 ng/ml) in the presence or absence of HA (100 lg/ml) for 60 min, then the cells were fixed, permeabilized, and incubated with anti-phospho-Smad 1/5/8 antibody. Intracellularly localized phospho-Smad 1/5/8 protein was visualized using Cy3-conjugated secondary antibody. The nuclei were identified by blue fluorescence due to 40 -6-diamidino-2-phynlindole staining. (A) Non-stimulated cells. (B) HA-stimulated cells. (C) BMP-2-stimulated cells. (D) BMP-2- and HA-stimulated cells.

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mRNA expressions of noggin and follistatin, which are soluble BMP antagonists. After 90 min of culture, stimulation with BMP-2 enhanced their expression levels by 2.2- and 3.0-fold, respectively (Fig. 3A and B). In contrast, those mRNA expressions were remarkably suppressed when the cells were cultured with both BMP-2 and HA.

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3.4. Effect of HA on down-regulation of BMP-2-induced MAPK phosphorylation

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4. Discussion

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To elucidate the roles of the ERK and p38 MAPK pathways in the regulation of BMP-2 responses, the expressions of phospho-ERK and phospho-p38 MAPK were detected by Western blot analysis. BMP-2 alone up-regulated ERK phosphorylation after 30 min, which then gradually decreased (Fig. 4). When MG63 cells were cultured with HA (100 lg/ml) for 30 min, the phosphorylation level of ERK decreased from 6.4- to 3.5-fold. In contrast, the phosphorylation of p38 MAPK was not changed when the cells were cocultured with both BMP-2 and HA (100 lg/ml).

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BMP-2 ( 50 ng / ml ) Fig. 3. HA suppresses BMP-2-induced noggin and follistatin expression. MG63 cells (4  105 cells/well) were incubated with BMP-2 (50 ng/ml) in the presence or absence of HA (100 lg/ml) for the indicated time periods, then total RNA was isolated and reverse transcribed into cDNA using PCR amplification with SYBR green. PCR amplification was performed using primers specific for (A) noggin, (B) follistatin, and GAPDH. Fold changes in noggin and follistatin mRNA copy number values represent the average ± SD of data derived from triplicate cultures. ⁄⁄p < 0.01, as measured using an unpaired Student’s t-test. ⁄p < 0.05 as measured using an unpaired Student’s t-test.

changes in Smad 1/5/8 phosphorylation (data not shown). On the other hand, the level of Smad 1/5/8 phosphorylation induced by BMP-2 was up-regulated following addition of HA in a time-dependent manner for up to 90 min (Fig. 1B). 3.2. Effect of HA on nuclear translocation of phospho-Smad 1/5/8 in osteoblastic cells Nuclear translocation of specific receptor-regulated Smad 1/5/8 is an early cellular response to stimulation by BMP-2. Nuclear translocation of Smad 1/5/8 was detected in MG63 cells treated with BMP-2 (50 ng/ml). Furthermore, treatment with BMP-2 (50 ng/ml) and HA (100 lg/ml) enhanced the nuclear translocation of Smad 1/5/8 (Fig. 2). 3.3. Effect of HA on noggin and follistatin mRNA expression induced by BMP-2 To clarify the mechanisms involved in suppression of BMP-2-induced osteogenesis by HA, we investigated the effect of HA on the

The actions of BMPs are dependent on concentration and their combination with a variety of other factors or ECM components, as well as cell type and cellular age [25]. It has also been shown that BMP-2/4 interactions with ECM are essential for the differentiation of osteoblastic cells [26]. HA, a ubiquitous component of most ECM components, is important for maintaining matrix stability and tissue hydration, while it also plays a major role in regulating cell functions through interactions with cell surface receptors and binding proteins [27–34]. However, it remains unclear how HA regulates bone metabolism induced by BMP-2. To the best of our knowledge, this is the first report of the extending effects of HA on osteoblast differentiation induced by BMP-2 (Fig. 1A). Members of the Smad protein family are known to play pivotal roles in signaling by the intracellular TGF-b family, such as TGF-b, BMP-2, and activins. Among them, Smad 1, Smad 2, Smad 3, and Smad 5 become phosphorylated by specific activated type I serine/threonine kinase receptors and thus act in a pathway-restricted fashion [35]. It is well known that Smads play critical roles in stimulation of osteoblastic cells by BMP-2 and HA was found to enhance their signaling pathway in the present study (Figs. 1B and 2). This finding is consistent with previous reports that demonstrated that addition of HA to matrix-depleted chondrocytes restored Smad 1 and Smad 4 nuclear translocation in response to BMP [22]. We also examined the mechanism by which HA enhances BMP-2-mediated bioactivity. It is well known that BMP signaling is determined by the binding of BMPs and their receptors. Soluble BMP antagonists such as noggin and follistatin are known to prevent BMP signaling by binding directly to BMPs, therefore precluding their binding to specific cell surface receptors [16,36]. Noggin expression in osteoblasts is limited, however, its mRNA and protein levels are induced after exposure of cells to BMP-2, -4, or -6, which may be a protective mechanism to prevent excessive exposure of skeletal cells to BMPs [37,38]. Noggin blocks the effects of BMPs in undifferentiated and differentiated cells of osteoblastic lineage, while its addition to cultured osteoblasts blocks the stimulatory effects of BMPs on collagen, non-collagen protein synthesis, and alkaline phosphatase activity [37]. Follistatin was initially identified as an activin binding protein that precluded activin signaling [39]. Unlike noggin, its expression is down-regulated by BMPs and induced by TGF-b, indicating that different signaling pathways can regulate specific BMP antagonists,

M. Kawano et al. / Biochemical and Biophysical Research Communications 405 (2011) 575–580

BMP-2 ( 50 ng/ml ) phospho ERK 1.0 6.4 3.5

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Fig. 4. HA suppresses BMP-2-induced ERK phosphorylation. MG63 cells (4  105 cells/well) were stimulated with BMP-2 (50 ng/ml) in the presence or absence of HA (100 lg/ml) for the indicated time periods, then whole lysates were analyzed by SDS–PAGE and Western blotting analyses. Gel images were subjected to densitometric analysis using Image Lab™ software. The relative band intensity (RBI) of the control values was set to 1.

such as follistatin [40,41]. As shown in Fig. 4, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA. These results suggest that HA up-regulates BMP-2-induced osteogenic activity in combination with BMP-2 antagonists. It is well known that BMP receptors determine the intensity of BMP signals via Smad 1 C-terminal phosphorylations, and that the duration of the activated pSmad signal is regulated by sequential Smad linker region phosphorylation at conserved MAPK and GSK sites [42]. In addition, Elshaier et al. reported that longer cultures with BMP inhibited MAPK signaling and promoted BMP-mediated receptor-regulated Smad (R-Smad) phosphorylation [43]. Those findings led us to speculate that induction of BMP-2 signaling by HA in human osteoblastic cells may occur due to MAPK signaling. In our experiments, we found that HA strongly suppressed ERK phosphorylation in MG63 cells stimulated with BMP-2 (Fig. 4), which indicates that enhancement of osteoblast differentiation by HA is responsible for the regulation of ERK. In conclusion, we found that HA enhanced BMP-2 osteogenic bioactivity in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. Further study is required to clarify the roles of these inhibitors on the regulation of BMP-2 by HA in vivo. References [1] C.B. Knudson, W. Knudson, Hyaluronan-binding proteins in development, tissue homeostasis, and disease, FASEB J. 7 (1993) 1233–1241. [2] J.Y. Lee, A.P. Spicer, Hyaluronan: a multifunctional, megaDalton, stealth molecule, Curr. Opin. Cell Biol. 12 (2000) 581–586. [3] B.J. Clarris, J.R. Fraser, On the pericellular zone of some mammalian cells in vitro, Exp. Cell Res. 49 (1968) 181–193. [4] R.L. Goldberg, B.P. Toole, Pericellular coat of chick embryo chondrocytes: structural role of hyaluronate, J. Cell Biol. 99 (1984) 2114–2122. [5] P. Heldin, H. Pertoft, Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells, Exp. Cell Res. 208 (1993) 422–429. [6] C.B. Knudson, B.P. Toole, Changes in the pericellular matrix during differentiation of limb bud mesoderm, Dev. Biol. 112 (1985) 308–318. [7] W. Knudson, C.B. Knudson, Assembly of a chondrocyte-like pericellular matrix on non-chondrogenic cells. Role of the cell surface hyaluronan receptors in the assembly of a pericellular matrix, J. Cell Sci. 99 (Pt. 2) (1991) 227–235. [8] P.G. McGuire, J.J. Castellot Jr., R.W. Orkin, Size-dependent hyaluronate degradation by cultured cells, J. Cell. Physiol. 133 (1987) 267–276.

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