Parathyroid Hormone Rapidly Stimulates Hyaluronan Synthesis by Periosteal Osteoblasts in the Tibial Diaphysis of the Growing Rat

Abstract

Short term treatment (3-24 h) with parathyroid hormone (PTH) stimulated the synthesis and accumulation of hyaluronan (HyA) in explant cultures of tibial diaphyses from young rats. PTH increased the overall HyA content of periosteum 5-fold, with the basal cambium layer exhibiting the greatest enhancement (∼8-fold). PTH increased the HyA content of cortical bone by 2-fold while not affecting the HyA content of bone marrow. PTH treatment greatly enhanced HyA staining throughout all layers of the periosteum, although its most dramatic effect occurred in the basal cambium layer. Here, unlike in the control tissue sections, nearly all cambium-lining osteoblasts stained intensely positive for HyA. PTH treatment enhanced the HyA staining of osteocytes in cortical bone tissue sections to the extent that the lacunocanalicular system became visualized. Three significant findings were revealed in this study. First, mature periosteal osteoblasts, under natural conditions, do not contain much HyA in their surrounding extracellular matrix but dramatically enhance their matrix HyA content when treated with PTH. Second, pre-osteocytes and osteocytes contain more HyA in their natural matrix than mature lining osteoblasts, and they appear to have functional PTH receptors because they responded to PTH treatment with an enhancement of HyA content. Finally, it was observed that the lining cells along the endosteal surface of the diaphysis did not stain strongly positive for HyA either naturally or when exposed to PTH treatment. This indicates that periosteal and endosteal osteoblastic cell populations exhibit metabolic differences in their extracellular matrix responses to PTH. Short term treatment (3-24 h) with parathyroid hormone (PTH) stimulated the synthesis and accumulation of hyaluronan (HyA) in explant cultures of tibial diaphyses from young rats. PTH increased the overall HyA content of periosteum 5-fold, with the basal cambium layer exhibiting the greatest enhancement (∼8-fold). PTH increased the HyA content of cortical bone by 2-fold while not affecting the HyA content of bone marrow. PTH treatment greatly enhanced HyA staining throughout all layers of the periosteum, although its most dramatic effect occurred in the basal cambium layer. Here, unlike in the control tissue sections, nearly all cambium-lining osteoblasts stained intensely positive for HyA. PTH treatment enhanced the HyA staining of osteocytes in cortical bone tissue sections to the extent that the lacunocanalicular system became visualized. Three significant findings were revealed in this study. First, mature periosteal osteoblasts, under natural conditions, do not contain much HyA in their surrounding extracellular matrix but dramatically enhance their matrix HyA content when treated with PTH. Second, pre-osteocytes and osteocytes contain more HyA in their natural matrix than mature lining osteoblasts, and they appear to have functional PTH receptors because they responded to PTH treatment with an enhancement of HyA content. Finally, it was observed that the lining cells along the endosteal surface of the diaphysis did not stain strongly positive for HyA either naturally or when exposed to PTH treatment. This indicates that periosteal and endosteal osteoblastic cell populations exhibit metabolic differences in their extracellular matrix responses to PTH. Parathyroid hormone (PTH) 1The abbreviations used are: PTHparathyroid hormoneHyAhyaluronanbHABCbiotinylated hyaluronan-binding complexPsstripped periosteumPcperiosteal surface. is a key hormone regulating bone mineral homeostasis (1Martin T.J. Raisz L.B. Rodan G. Martin T.J. Raisz L.G. Clinical Endocrinology of Calcium Metabolism. Marcel Dekker, New York1987: 1-52Google Scholar). It contains 84 amino acids, the first 34 of which have most of its biological activity (2Nutt R.F. Caulffield M.P. Levy J.J. Gibbons S.W. Rosenblatt M. McKee R.L. Endocrinology. 1990; 127: 491-493Crossref PubMed Scopus (62) Google Scholar). Under normal physiological conditions PTH stimulates overall remodeling reactions in vivo by stimulating both bone formation and resorption while enhancing formation more (3Frost H.M. Medicina. 1997; 57: 119-126PubMed Google Scholar). This concept explains the net bone gains observed in patients with severe osteoporosis treated with intermittent daily injections of PTH (4Reeve J. Meunier P. Parsons J.A. Bernat M. Bijvet O.L.M. Courpon P. Edouard C. Klenerman L. Neer R.M. Renier J.C. Slovik D. Vismans F.J.F.E. Potts J.T.J. Br. Med. J. 1980; 280 (1377): 1340-1344Crossref PubMed Scopus (528) Google Scholar, 5Slovik D.M. Neer R.M. Potts J.T.J. J. Clin. Invest. 1981; 68: 1261-1271Crossref PubMed Scopus (104) Google Scholar, 6Lindsay R. Nieves J. Formica C. Henneman E. Woelfert L. Shen V. Dempster D. Cosman F. Lancet. 1997; 350: 550-555Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 7Lane N.E. Sanchez S. Modin G.W. Genant H.K. Ini E. Arnaud C.D. J. Clin. Invest. 1998; 102: 1627-1633Crossref PubMed Scopus (499) Google Scholar). There is ample evidence that intermittent PTH treatments in vivo increase both the mass and mechanical strength of bone tissue, although there is still no consensus as to how in vivo PTH therapy works at the cellular and molecular level (8Dempster D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1995; 4: 247-250Google Scholar, 9Cosman F. Lindsay R. Calcif. Tissue Int. 1998; 62: 475-480Crossref PubMed Scopus (87) Google Scholar, 10Hock J.M. J. Clin. Invest. 1999; 104: 371-373Crossref PubMed Scopus (24) Google Scholar). parathyroid hormone hyaluronan biotinylated hyaluronan-binding complex stripped periosteum periosteal surface. Hyaluronan (HyA) 2We have chosen to abbreviate hyaluronan as HyA to distinguish this abbreviation from one typically used for hydroxyapatite (HA), thereby avoiding any potential confusion. is a large polysaccharide consisting of several thousand repeats of a single disaccharide unit (1,4-glucuronic acid-β-1,3-N-acetylglucosamine-β) and can reach a relative molecular weight of several million daltons (11Laurent T.C. Kennedy J.F. Phillips G.O. Williams P.A. Hascall V.C. Hyaluronan: Chemical, Biochemical and Biological Aspects. Vol. 1. Woodhead Publishing Limited, Cambridge, UK2002: 3-16Google Scholar). It is an important component of several connective tissue matrices promoting tissue hydration and imparting high viscosity in certain tissue fluids (12Laurent T.C. Balaza E.A. Chemistry and Molecular Biology of the Intercellular Matrix. Academic Press, New York1970: 703-732Google Scholar), acting as a macromolecular scaffold for three-dimensional matrix assembly (13Maleski M.P. Knudson C.B. Connect. Tissue Res. 1996; 34: 75-86Crossref PubMed Scopus (41) Google Scholar), modulating cell-cell or cell-matrix interactions (14Knudson C.B. Knudson W. FASEB J. 1993; 13: 1233-1241Crossref Scopus (610) Google Scholar), affecting the differentiation of some progenitor cells (15Kujawa M.J. Pechak D.G. Fitzman M.Y. Caplan A.I. Dev. Biol. 1986; 113: 10-16Crossref PubMed Scopus (92) Google Scholar, 16Kujawa M.J. Carrino D.A. Caplan A.I. Dev. Biol. 1986; 114: 519-528Crossref PubMed Scopus (73) Google Scholar), and regulating tissue inflammation via immune cell reactions (17de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Am. J. Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). PTH stimulates the production of HyA in cultures of calvarial explants (18Severson A.R. Rothberg P.F. Pratt R.M. Goggins J.F. Endocrinology. 1973; 92: 1282-1285Crossref PubMed Scopus (21) Google Scholar, 19Luben R.A. Goggins J.F. Raisz L.G. Endocrinology. 1974; 94: 737-745Crossref PubMed Scopus (36) Google Scholar, 20Luben R.A. Cohn D.V. Endocrinology. 1976; 98: 413-419Crossref PubMed Scopus (48) Google Scholar, 21Severson A.R. Exp. Cell Biol. 1983; 51: 70-76PubMed Google Scholar) and in cultures of primary cells isolated from bone tissues (22Luben R.A. Wong G.L. Cohn D.V. Endocrinology. 1976; 99: 526-534Crossref PubMed Scopus (344) Google Scholar, 23Wong G.L. Luben R.A. Cohn D.V. Science. 1977; 197: 663-665Crossref PubMed Scopus (177) Google Scholar, 24Wong G.L. Calcif. Tissue Int. 1982; 34: 67-75Crossref PubMed Scopus (22) Google Scholar). HyA has been detected in bone tissues by several investigators, and these studies suggest that the resident osteogenic cells produce HyA (25Noonan K.J. Stevens J.W. Tammi R. Tammi M. Hernandez J.A. Midura R.J. J. Orthop. Res. 1996; 14: 573-581Crossref PubMed Scopus (50) Google Scholar, 26Genever P.G. Dickson I.R. Bone. 1996; 18: 429-435Crossref PubMed Scopus (11) Google Scholar). It has been reported that HyA is produced by isolated osteoclastic (22Luben R.A. Wong G.L. Cohn D.V. Endocrinology. 1976; 99: 526-534Crossref PubMed Scopus (344) Google Scholar, 23Wong G.L. Luben R.A. Cohn D.V. Science. 1977; 197: 663-665Crossref PubMed Scopus (177) Google Scholar, 24Wong G.L. Calcif. Tissue Int. 1982; 34: 67-75Crossref PubMed Scopus (22) Google Scholar) and osteoblastic (27Fedarko N.S. Termine J.D. Young M.F. Robey P.G. J. Biol. Chem. 1990; 265: 12200-12209Abstract Full Text PDF PubMed Google Scholar, 28Ecarot-Charrier B. Broekhuyse H. J. Biol. Chem. 1987; 262: 5345-5351Abstract Full Text PDF PubMed Google Scholar) cells in vitro, as well as by osteoblast-like cell lines (29Midura R.J. Evanko S.P. Hascall V.C. J. Biol. Chem. 1994; 269: 13200-13206Abstract Full Text PDF PubMed Google Scholar, 30Melick R.A. Mercuri S. Bingham P.J. Calcif. Tissue Res. 1977; 22: 508-510Crossref PubMed Scopus (2) Google Scholar). Thus, it is still not known which types of cells in bone are the predominant ones to synthesize HyA in response to PTH treatment (31Wong G.L. J. Biol. Chem. 1984; 259: 4019-4022Abstract Full Text PDF PubMed Google Scholar). The functions of HyA in bone are not known presently. Furthermore, it is not clear why PTH treatment greatly enhances the HyA content of bone tissue. The present study sought to answer three basic questions: 1) Does PTH rapidly stimulate HyA production in bone? 2) Are the primary target cells involved in this response the mature osteoblasts? 3) Is PTH-stimulated HyA deposition in bone tissue regions more associated with active bone formation? An affirmative answer to the first question would suggest it is a primary response to the hormone, whereas affirmative results to the latter two would suggest a in active bone these explants of tibial diaphyses from rapidly were vivo in the or of PTH and for from to This has several to the bone tissue and cells that to PTH treatment with an increased HyA First, the tibial diaphyses from rapidly exhibit by bone deposition at the periosteal surface and any bone resorption at the endosteal surface G.W. J. Am. J. PubMed Scopus Google Scholar). Thus, the of active bone formation and the of the osteoblasts are known in this bone explant Second, have the to the tissue the tibial diaphysis and cortical Finally, have a to HyA content and with a biotinylated complex J.A. J. PubMed Scopus (177) Google Scholar, R. J.A. Tammi M. J. Invest. Full Text PDF PubMed Google Scholar, C. Tammi M. Tammi R. 98: PubMed Scopus Google Scholar) to HyA in tissue of the used in this study were of the was from and were from and were from was from was from Tissue were from were from was from were from was from The complex and were from were from of was from Tissue and of the used in this study were and by the and were by with from the diaphysis of was from the and the of the bone were to of the the explants were for or with or in and at the tibial diaphyses were to bone periosteum, and cortical tissue was isolated from explants by of the a periosteal tissue as well as more than of the basal cambium cell were stripped the bone by this and at this bone was at both bone This was to of to the periosteal bone surface The cambium cells and matrix the periosteal surface of the bone were isolated in with for at bone is as the bone tissue the HyA synthesis some explant cultures of tibial diaphysis were with of and for and in the of HyA in Tissue periosteum, and cortical bone were exposed to a with for at by with the tissue from these this the from tissue matrices J.D. H.K. S.W. Martin 1981; Full Text PDF PubMed Scopus Google Scholar). bone tissue was with the to the and the matrix molecular mass the were with and for at The disaccharide were isolated from the and by with a molecular mass or as M. Tammi M. Hascall V.C. Midura R.J. PubMed Scopus Google Scholar, R.J. M. Hascall V.C. 1994; 4: PubMed Scopus Google Scholar). HyA were detected and by M. Tammi M. Hascall V.C. Midura R.J. PubMed Scopus Google Scholar) or R.J. M. Hascall V.C. 1994; 4: PubMed Scopus Google Scholar). HyA were R.J. M. Hascall V.C. 1994; 4: PubMed Scopus Google Scholar) and from by HyA in Tissue were in and the of in for at The were in and in in the for at The were in and in with a to of were and were which contains the regions of and was from and HyA was as R. J.A. Tammi M. J. Invest. Full Text PDF PubMed Google Scholar, C. Tammi M. Tammi R. 98: PubMed Scopus Google Scholar). The of HyA was by of the with HyA disaccharide repeats of of and it of in the control for the of some tissue sections with to the sections were with in for to tissue with and in in for to sections were with in at with and treated with complex for at The sections were with and the was a the sections were with and with Tissue bone of were from the of young and and by and to bone periosteum, and cortical bone tissue The bone tissue both and cells and their associated extracellular as well as most of the endosteal lining cells their in stripped periosteum all of the tissue layers and nearly of the basal cambium cell layer periosteal lining with its associated extracellular matrix of the all cambium osteoblasts and matrix from the periosteal surface and The cortical bone tissue all of the although there were some osteoblasts lining and an by HyA of of HyA was from isolated tibial diaphyses at this with HyA detected in from all tissue The HyA content of tissue was of than its content. a of the HyA content the tibial diaphysis with and from and more HyA was in the tissue of cortical bone than that detected in the tissue of cortical bone and of isolated tibial as under and by as under and by to by the HyA content by the content from the and by the the from the to by to which with the as under and by as under and by by the HyA content by the content from the and by the the from the to by to which with the in a diaphyses were as explants in with or for because these conditions a nearly response for HyA content not the tibial diaphyses were their tissue and the HyA content in was PTH treatment increased the of HyA in all tissue tissue The periosteal layers were the most with and in HyA content detected in and The relative in HyA content for both the and cortical bone tissue were exhibiting a 2-fold with a was used to the of HyA synthesis by tibial explants in response to PTH treatment. cultures were in with or for because this is the still exhibiting a of HyA not PTH the periosteum the greatest of HyA of the in and in the of the periosteum to the it was the of in the of the of the in the mineral and bone of HyA synthesis as with The of to in the from these tissues was that the activity of the in tissue was M. Hascall V.C. PubMed Scopus (48) Google Scholar). all of the bone tissue in the of the as the of PTH treatment The periosteum the greatest increase in content in response to PTH and in and both the and of the cortical bone tissue in HyA synthesis as a of PTH treatment. all the tissue bone did not to PTH treatment with an increase in HyA The of to in the from all tissues was in as with their control This would suggest that PTH treatment the activity of the in tissue but that the extent of this was for all bone tissues not It that PTH treatment enhances HyA synthesis that for in bone explant with findings for osteoblast-like cells (29Midura R.J. Evanko S.P. Hascall V.C. J. Biol. Chem. 1994; 269: 13200-13206Abstract Full Text PDF PubMed Google Scholar). HyA that the periosteum most of the HyA in the tibial diaphysis and that it the greatest response to PTH treatment with to an increase in HyA all of these bone tissue contain several cell the periosteum and Thus, is to cells in bone tissue that produce HyA and that synthesize increased of HyA in response to PTH treatment. An HyA staining was chosen the that any cell of HyA would exhibit some positive staining for this in its surrounding extracellular matrix and that cells exhibiting enhanced HyA synthesis from PTH treatment would an enhanced staining for HyA in their surrounding extracellular sections of isolated tibial diaphysis stained with a in the or of HyA revealed the of this for HyA in tissue this of HyA staining were detected in the extracellular matrices of all bone tissue The cell layers of the periosteum stained strongly positive for the of matrix HyA the basal or cambium layer of the periosteum, of the not lining the periosteal bone surface stained strongly positive for the of matrix HyA. most of the mature osteoblasts lining the periosteal bone surface did not stain positive for the of matrix HyA This periosteal cortical bone with of the and mature staining positive for the of HyA The layers of both the periosteum and bone stained intensely for HyA There was HyA staining along the endosteal and bone there was HyA staining the tissue, although it to and relative to the or the periosteum or cells were observed in the that stained positive for HyA of tibial explants vivo for and stained with a a increase in HyA staining of periosteum as with sections of tissue This is by the of in the used vivo a of the HyA staining in the extracellular matrix of the periosteum of tibial diaphyses revealed relative The to this was that the mature osteoblasts lining the periosteal bone surface of explants to some staining for the of HyA is that the tissue the endosteal bone surface of explants has some matrix an increase in HyA staining as with that of isolated bone tissue and of tibial explants vivo for in the of or PTH and stained with a in HyA staining of periosteum as with sections of control tissue The most dramatic in HyA staining stimulated by PTH occurred the and mature osteoblasts the basal or cambium layer there was still evidence of HyA staining in the cell layers lining the endosteal bone surface of bone explants when PTH was used to that hormone would the bone tissue the not evidence that PTH the tissue in these explant cultures was by a matrix along the endosteal bone surface as with that in The of HyA staining in isolated bone tissue and and in control explant cultures This staining was enhanced by PTH treatment to the that were observed of both control and cortical bone the that the of osteocytes staining positive for HyA in their extracellular matrix was the in both the control and cortical bone was that the HyA staining surrounding osteocytes was greatly enhanced by to the hormone and studies have reported that PTH treatment stimulates the synthesis of HyA in cultures of calvarial explants (18Severson A.R. Rothberg P.F. Pratt R.M. Goggins J.F. Endocrinology. 1973; 92: 1282-1285Crossref PubMed Scopus (21) Google Scholar, 19Luben R.A. Goggins J.F. Raisz L.G. Endocrinology. 1974; 94: 737-745Crossref PubMed Scopus (36) Google Scholar, 20Luben R.A. Cohn D.V. Endocrinology. 1976; 98: 413-419Crossref PubMed Scopus (48) Google Scholar, 21Severson A.R. Exp. Cell Biol. 1983; 51: 70-76PubMed Google Scholar), in isolated primary bone cells (22Luben R.A. Wong G.L. Cohn D.V. Endocrinology. 1976; 99: 526-534Crossref PubMed Scopus (344) Google Scholar, 23Wong G.L. Luben R.A. Cohn D.V. Science. 1977; 197: 663-665Crossref PubMed Scopus (177) Google Scholar, 24Wong G.L. Calcif. Tissue Int. 1982; 34: 67-75Crossref PubMed Scopus (22) Google Scholar), and in an osteoblast-like cell (29Midura R.J. Evanko S.P. Hascall V.C. J. Biol. Chem. 1994; 269: 13200-13206Abstract Full Text PDF PubMed Google Scholar). several to this biological response of bone tissue to PTH The present study to answer three of these First, is the by PTH of HyA synthesis a primary response of the hormone bone cells or a Second, are the primary target cells involved in this response the mature osteoblasts? Finally, PTH-stimulated HyA deposition in extracellular matrix regions of bone tissue associated with active bone formation? HyA synthesis is a primary response to PTH was in this study by how rapidly PTH stimulate HyA production in bone tissue. that an of HyA synthesis stimulated by PTH was the first of which the a to the hormone in a increase in HyA accumulation both the periosteum and cortical bone not This is more with a that the of HyA synthesis is a primary effect of PTH and not a effect by J.J. C. Endocrinology. 1987; PubMed Scopus Google Scholar). to the predominant cell types that HyA in bone in vivo revealed that the cell layers of the periosteum the greatest of naturally HyA. of tissue that most of this HyA is in the layers of the The or layer of the periosteum most of its HyA staining in the cell layers and not the mature osteoblasts lining the periosteal bone surface. pre-osteocytes and mature osteocytes HyA staining along their HyA surrounding osteocytes as a matrix that in the system or the osteocytes from the mineral of a in HyA staining that with of osteoblastic differentiation that this matrix a in bone The functional of a of HyA surrounding mature periosteal lining osteoblasts a of these cells naturally exhibiting of HyA it more to a mature not to HyA in its natural extracellular This latter from a of receptors to HyA at the surface (25Noonan K.J. Stevens J.W. Tammi R. Tammi M. Hernandez J.A. Midura R.J. J. Orthop. Res. 1996; 14: 573-581Crossref PubMed Scopus (50) Google Scholar, D.M. R. J. Res. 1994; PubMed Scopus Google Scholar) or an increased of HyA in the extracellular matrix by the of an by the of results that the cell most to a of HyA synthesis by PTH treatment was the mature osteoblasts lining the periosteal bone by in cortical bone a increase in HyA synthesis PTH treatment. This indicates that osteocytes in young bone have functional PTH osteocytes in bone have functional PTH they target cell for intermittent PTH therapy as a treatment for osteoporosis (4Reeve J. Meunier P. Parsons J.A. Bernat M. Bijvet O.L.M. Courpon P. Edouard C. Klenerman L. Neer R.M. Renier J.C. Slovik D. Vismans F.J.F.E. Potts J.T.J. Br. Med. J. 1980; 280 (1377): 1340-1344Crossref PubMed Scopus (528) Google Scholar, 5Slovik D.M. Neer R.M. Potts J.T.J. J. Clin. Invest. 1981; 68: 1261-1271Crossref PubMed Scopus (104) Google Scholar, 6Lindsay R. Nieves J. Formica C. Henneman E. Woelfert L. Shen V. Dempster D. Cosman F. Lancet. 1997; 350: 550-555Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 7Lane N.E. Sanchez S. Modin G.W. Genant H.K. Ini E. Arnaud C.D. J. Clin. Invest. 1998; 102: 1627-1633Crossref PubMed Scopus (499) Google Scholar, D.W. Cosman F. Parisien M. Shen V. Lindsay R. Endocr. Rev. 1995; 4: 247-250Google Scholar, 9Cosman F. Lindsay R. Calcif. Tissue Int. 1998; 62: 475-480Crossref PubMed Scopus (87) Google Scholar, 10Hock J.M. J. Clin. Invest. 1999; 104: 371-373Crossref PubMed Scopus (24) Google Scholar). to their periosteal osteoblastic cells lining the endosteal surface of bone did not produce much HyA did they exhibit a increased production of HyA when treated with PTH. The for this is not known at although have that endosteal lining cells in the diaphysis PTH receptors P. R. H. Endocrinology. PubMed Scopus Google Scholar). that the PTH the of the tissue by matrix along the endosteal bone surface that an of hormone the and the that bone deposition in the young diaphysis at the periosteal surface and not the endosteal surface G.W. J. Am. J. PubMed Scopus Google Scholar), a positive the production and accumulation of HyA with of bone formation and not with of bone This for the tibial tissues from rats. the of young the of bone deposition from periosteal to endosteal G.W. J. Am. J. PubMed Scopus Google Scholar), and the of HyA accumulation from a periosteal to an endosteal (25Noonan K.J. Stevens J.W. Tammi R. Tammi M. Hernandez J.A. Midura R.J. J. Orthop. Res. 1996; 14: 573-581Crossref PubMed Scopus (50) Google Scholar). functional for HyA associated with of bone is not known at HyA in the of formation and Scopus Google Scholar, J. Dev. Biol. PubMed Scopus Google Scholar). HyA dramatically the extracellular matrix as they of (25Noonan K.J. Stevens J.W. Tammi R. Tammi M. Hernandez J.A. Midura R.J. J. Orthop. Res. 1996; 14: 573-581Crossref PubMed Scopus (50) Google Scholar, P. C.B. J. Cell 1994; Google Scholar). of a in HyA and staining with of osteoblastic differentiation in the periosteum is with these functional for HyA active periosteal bone formation cell-matrix and cell-cell reactions L. J. Invest. 1993; Full Text PDF PubMed Google Scholar), and hydration of the extracellular which would and (11Laurent T.C. Kennedy J.F. Phillips G.O. Williams P.A. Hascall V.C. Hyaluronan: Chemical, Biochemical and Biological Aspects. Vol. 1. Woodhead Publishing Limited, Cambridge, UK2002: 3-16Google Scholar, T.C. Balaza E.A. Chemistry and Molecular Biology of the Intercellular Matrix. Academic Press, New York1970: 703-732Google Scholar), affecting the of F. S. Science. PubMed Scopus Google Scholar), or modulating to and cellular D.V. Rev. Biol. 1994; PubMed Scopus Google Scholar). the of the of this study.