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`Hyaluronan: Structure and Physical Properties
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`Dec. 15, 1997
`
`Hyaluronan: Structure and
`Physical Properties （1997 Vol.1, A2）
`
`Vincent C. Hascall / Torvard C. Laurent
`
`1. Introduction
`2. Chemical Structure
`3. Polymer Structure
`4. Solution Structure
`5. Hyaluronan in Tissues
`6. Metabolism of Hyaluronan
`7. Viscoelastic Properties
`8. Medical Application
`9. Concluding Remarks
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`Hyaluronan: Structure and Physical Properties
`
`Vincent C. Hascall
`Department of Biomedical Engineering (Wb3), Lerner Research Institute, Cleveland
`Clinic Foundation, Cleveland, Ohio 44195
`
`Described in Biosketch of Editors
`
`Torvard C. Laurent
`Dr. Torvard Laurent received his Doctor of Medicine from the Karolinska Institute,
`Stockholm in 1958. After a 3 year fellowship from the Retina Foundation in Boston, Dr.
`Laurent established a research program at the University of Uppsala, where he attained
`a Professorship in 1966. He is currently Professor emeritus at this institution and
`Science Secretary for the Wenner-Gren Foundations, Stockholm. Dr. Laurent has
`conducted pioneering, internationally recognized research throughout his career on the
`chemistry of connective tissues, most notably on the physical and physiological
`properties, and medical applications of hyaluronan. His numerous honors include:
`Deputy Chairman, Swedish Medical Research Council, 1973-77; Chairman, Swedish
`Biochemical Society, 1973-76; President, Royal Swedish Academy of Sciences, 1991-
`94; and Chairman, Council of the Nobel Foundation, 1994-present. His busy schedule
`includes responsibility for organizing international conferences for the Wenner-Gren
`Foundations, where he recently organized and edited one on the 'Structure, Biology and
`Medical Applications of Hyaluronan'.
`
`1. Introduction
`
`1In 1934, Karl Meyer and his assistant, John Palmer, described a procedure
`for isolating a novel glycosaminoglycan from the vitreous of bovine eyes [1].
`They showed that this substance contained an uronic acid and an
`'we propose, for
`aminosugar, but no sulfoesters.
`In
`their words:
`convenience, the name "hyaluronic acid", from hyaloid (vitreous) +
`uronic acid.' This marked the birth announcement for one of nature's most
`versatile and fascinating macromolecules. Today, this macromolecule is most
`frequently referred to as 'Hyaluronan', reflecting the fact that it exists in vivo
`as a polyanion and not in the protonated acid form.
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`Hyaluronan: Structure and Physical Properties
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`2. Chemical Structure
`
`It would take an additional 20 years before Meyer's laboratory finally
`completed the work that determined the precise chemical structure of the
`basic disaccharide motif that forms hyaluronan [2]. During these years they
`showed that the uronic acid and aminosugar in the disaccharide are D-
`glucuronic acid and D-N-acetylglucosamine, and that they are linked together
`through alternating beta-1,4 and beta-1,3 glycosidic bonds, Fig. 1. Both
`sugars are spatially related to glucose which in the beta configuration allows
`all of its bulky groups (the hydroxyls, the carboxylate moiety and the
`anomeric carbon on the adjacent sugar) to be in sterically favorable
`equatorial positions while all of the small hydrogen atoms occupy the less
`sterically favorable axial positions. Thus, the structure of the disaccharide
`shown in Fig. 1 is energetically very stable.
`
`Fig. 1
`the repeat disaccharide of
`Relationship between beta-D-glucose (A) and
`hyaluronan, D-glucuronic acid-beta-1, 3-N-acetylglucosamine-beta-1, 4 (B)
`H ; axial hydrogens that contribute to the hydrophobic face
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`Hyaluronan: Structure and Physical Properties
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`3. Polymer Structure
`
`Hyaluronan synthase enzymes synthesize large, linear polymers of the
`repeating disaccharide structure of hyaluronan by alternate addition of
`glucuronic acid and N-acetylglucosamine to the growing chain using their
`activated nucleotide sugars
`(UDP
`- glucuronic acid and UDP-N-
`acetlyglucosamine) as substrates. The number of repeat disaccharides, n, in
`1
`a completed hyaluronan molecule can reach 10,000 or more, a molecular
`mass of ~4 million daltons (each disaccharide is ~400 daltons). The average
`length of a disaccharide is ~1 nm. Thus, a hyaluronan molecule of 10,000
`repeats could extend 10 ｵ m if stretched from end to end, a length
`approximately equal to the diameter of a human erythrocyte. Fig. 2 shows an
`electron micrograph of a few intertwined hyaluronan molecules that have
`been deposited on a flat surface and rotary shadowed with heavy metal for
`contrast.
`
`1
`
`These enzymes and the mechanism of hyaluronan synthesis will be the
`subject of later articles in this series.
`
`Fig. 2
`The electron micrograph was kindly provided by Dr. Richard Mayne and Dr.
`Randolph Brewton, University of Alabama at Birmingham.
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`Hyaluronan: Structure and Physical Properties
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`4. Solution Structure
`
`In a physiological solution, the backbone of a hyaluronan molecule is
`stiffened by a combination of the chemical structure of the disaccharide,
`internal hydrogen bonds, and interactions with solvent. The axial hydrogen
`atoms (indicated in Fig. 1B) form a non-polar, relatively hydrophobic face
`while the equatorial side chains form a more polar, hydrophilic face, thereby
`2
`creating a twisting ribbon structure. Consequently, a hyaluronan molecule
`assumes an expanded random coil structure in physiological solutions which
`occupies a very large domain, Fig. 3. The actual mass of hyaluronan within
`this domain is very low, ~0.1% (wt/vol) or less when the macromolecule is
`present at a very dilute concentration in saline. This means that the domains
`of individual molecules would overlap each other at concentrations of 1 mg
`hyaluronan per ml or higher.
`
`2
`See article 2 by John Scott.
`
`Fig. 3 Model of hyaluronan ribbon in a 3-dimensional domain.
`The light blue box represents the domain of the molecule in solution. The
`alternating blue and red strand represents the ribbon structure with blue
`(hydrophilic) and red (hydrophobic) faces.
`The slice is represented in Fig. 4.
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`Hyaluronan: Structure and Physical Properties
`important
`The domain structure of hyaluronan has
`interesting and
`consequences. Small molecules such as water, electrolytes and nutrients can
`freely diffuse through the solvent within the domain. However, large
`molecules such as proteins will be partially excluded from the domain
`because of their hydrodynamic sizes in solution. As shown in Fig. 4, the
`hyaluronan network in the domain allows less and less space for other
`molecules the larger they are. This leads both to slower diffusion of
`macromolecules through the network and to their lower concentration in the
`network compared to the surrounding hyaluronan free compartments.
`Interestingly, the hyaluronan chains are constantly moving in the solution, and
`the effective 'pores' in the network continuously change in size. Statistically,
`all sizes of pores can exist, but with different probabilities. This means that in
`principle, all molecules can pass through a hyaluronan network, but with
`different degrees of retardation depending on their hydrodynamic volumes.
`
`Fig. 4 Vertical slice from Fig. 3 illustrating average pore size and partial
`exclusion of large molecules.
`The red and blue are tracings of regions in Fig. 3 representing portions of the
`hyaluronan backbone in the representative slice. The fuzzy halo (shading) around
`the hyaluronan fragments would be the volume of the slice inaccessible to a
`diffusing molecule. The 3 circles of different size represent areas available to
`diffusing molecules. The smallest would have access to most of the volume not
`occupied by hyaluronan while the largest would have access to only the place it is
`located and would clearly have a harder time moving through the hyaluronan
`domain (Fig. 3) than the smaller ones.
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`Hyaluronan: Structure and Physical Properties
`The pK of the carboxyl groups on the glucuronic acid residues is 3-4,
`depending on ion conditions. At pH 7, then, these groups are predominantly
`ionized, and the hyaluronan molecule is a polyanion that has associated,
`exchangeable cation counterions to maintain charge neutrality. Directional
`flow of electrolyte through such a polyanionic domain can lead to sufficient
`charge separation to create a streaming potential.
`
`5. Hyaluronan in Tissues
`
`Hyaluronan is present in all vertebrates, perhaps arising in animals with
`notochords. It is also present in the capsule of some strains of Streptococci
`that quite likely pirated the enzymatic machinery for its synthesis from
`vertebrate hosts. Hyaluronan is a major constituent of the extracellular
`3
`matrices in which most tissues differentiate.
`It is also an essential
`component of many extracellular matrices in mature tissues. In some cases,
`hyaluronan is a major constituent; as, for example, in the vitreous of the
`human eye (0.1-0.4 mg/g wet weight), or in synovial joint fluid (3-4 mg/ml), or
`in the matrix produced by the cumulus cells around the oocyte prior to
`4
`ovulation (~0.5 mg/ml), or in the pathological matrix that occludes the artery
`5
`in coronary restenosis.
`
`In others, while representing less of the mass of the tissue, hyaluronan
`serves as an essential structural element in the matrix. For example,
`hyaluronan is present at ~1 mg/g wet weight in hyaline cartilages, enough to
`fill the tissue volume in the absence of other constituents. However,
`aggrecan, the large chondroitin sulfate proteoglycan, is present at a much
`higher concentration (25-50 mg/g wet weight), and hyaluronan retains
`aggrecan molecules in the matrix through specific protein-hyaluronan
`6
`interactions which mask the hyaluronan backbone. Hyaluronan is less
`concentrated in the matrix of other connective tissues, such as those
`surrounding smooth muscle cells in the aorta and fibroblasts in the dermis of
`skin. Like cartilage however, hyaluronan forms a scaffold for binding large
`chondroitin sulfate proteoglycans in the matrices of these tissues.
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`Hyaluronan: Structure and Physical Properties
`
`The largest amount of hyaluronan (7-8 g per average adult human, ~50% of
`the total in the body) resides in skin tissue, where it is present in both the
`dermis (~0.5 mg/g wet tissue) and the epidermis (~0.1 mg/g wet tissue).
`Interestingly, while dermis consists primarily of extracellular matrix with a
`sparse population of cells, the epidermis is the reverse; the keratinocytes fill
`all but a few percent of the tissue. Thus, the actual concentrations of
`hyaluronan in the matrix around the cells in the epidermis (estimated to be 2-
`4 mg/ml) is an order of magnitude higher than in the dermis (estimated to be
`7
`~0.5 mg/ml). The matrix around keratinocytes, then, may have a hyaluronan
`concentration as high as that in umbilical cord (~4 mg/ml) considered to be
`the mammalian tissue with one of the highest concentrations. Interestingly,
`rooster comb, a specialized piece of skin, has even higher amounts of
`hyaluronan (up to 7.5 mg/ml).
`
`3
`The role of hyaluronan in tissue morphogenesis will be the subject of a later
`article in this series.
`4
`See article 3 by Antonietta Salustri and Csaba Fulop.
`5
`The role of hyaluronan in formation of the arterial matrix in restenosis will be
`the subject of a later article in this series.
`6
`The role of hyaluronan for cartilage structure and function will be the subject
`of a later article in this series.
`7
`The role of hyaluronan in the epidermis will be the subject of a later article in
`this series.
`
`6. Metabolism of Hyaluronan
`
`The metabolism of hyaluronan is very dynamic. Some cells, such as
`chondrocytes in cartilages, actively synthesize and catabolize hyaluronan
`throughout the lifetime of the tissue. Synthesis is usually balanced by
`catabolism, thereby maintaining a constant concentration in the tissue.
`Metabolic studies have shown that the half life of a hyaluronan molecule in
`cartilage is normally 2-3 weeks. Keratinocytes in epidermis are another
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`Hyaluronan: Structure and Physical Properties
`example of cells that actively synthesize and catabolize hyaluronan. In this
`case, the half life of a hyaluronan molecule is surprisingly short, less than a
`day.
`
`they catabolize
`Sometimes cells either predominantly synthesize or
`hyaluronan. For example the cells in the dermis actively synthesize more
`hyaluronan than they catabolize. A large proportion of the hyaluronan
`molecules escape from this tissue only to be rapidly captured by receptors on
`reticulo-endothelial cells in lymph nodes and in the liver which internalize
`them for subsequent catabolism in lysosomes. The half life of a hyaluronan
`molecule in the blood is very short, only a few minutes. Tissues in joints, such
`as the lining cells of the joint capsule of the knee, synthesize hyaluronan and
`release it into the synovial fluid, where it becomes a major component that
`contributes to the viscoelastic properties of the fluid. Also, the synovial fluid
`drains through the lymphatics before entry into the bloodstream. Reticulo-
`endothelial cells lining the lymphatics actively remove almost 90 percent of
`the hyaluronan before the remainder reaches the vascular system. It has
`been estimated that almost one-third of the total hyaluronan in the human
`body is metabolically removed and replaced during an average day.
`
`7. Viscoelastic Properties
`
`The concentration of hyaluronan in tissues is often higher than would be
`expected
`if
`individual molecules maintained
`their expanded domain
`structures. In many cases the hyaluronan is organized into the extracellular
`matrix by specific interactions with other matrix macromolecules. However,
`high molecular weight hyaluronan at high concentration in solution (for
`example, 5 million daltons at concentrations above 0.1 mg/ml) can also form
`entangled molecular networks through steric interactions and self association
`between and within individual molecules. The latter can occur when a stretch
`of the hydrophobic face of the ribbon structure of the backbone interacts
`reversibly with the hydrophobic face on a comparable stretch of hyaluronan
`on another molecule or in a different region of the same molecule. Such
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`Hyaluronan: Structure and Physical Properties
`networks exhibit different properties
`than would
`isolated hyaluronan
`molecules. They can resist rapid, short-duration fluid flow through the
`network, thereby exhibiting elastic properties which can distribute load or
`shear forces within the network, Fig. 5. On the other hand, slow fluid flow of
`longer duration can partially separate and align the molecules, allowing their
`movement and exhibiting viscous properties. Procedures for introducing
`covalent cross-links in hyaluronan matrices have been developed to create
`stable networks and semi-solid materials exhibiting pronounced viscoelastic
`8
`properties.
`
`Fig. 5 Model demonstrating the viscous and elastic properties of
`hyaluronan solutions.
`
`8
`Cross-linked hyaluronan matrices and their applications will be the topic of a
`future article in this series.
`
`8. Medical Application
`
`Appropriately, the first medical application of hyaluronan for humans was as a
`vitreous supplement/replacement during eye surgery in the late 1950s. The
`hyaluronan used was isolated initially from human umbilical cord, and shortly
`thereafter from rooster combs in a highly purified and high molecular weight
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`Hyaluronan: Structure and Physical Properties
`form. This latter preparation, now sold under the trade name of Healon
`(Pharmacia), is currently widely used for ophthalmic viscosurgery and in other
`forms of surgery, as is Opegan (Seikagaku), a hyaluronan product also
`prepared from rooster comb. Another hyaluronan product, Artz (Seikagaku),
`was developed for use as a supplement in the synovium of osteoarthritic
`joints, and a covalently cross-linked form of hyaluronan, Synvisc (Biomatrix),
`with more pronounced viscoelastic properties, is also being used for the
`same purpose.
`
`9. Concluding Remarks
`
`We hope that this inaugural article in "The Science of Hyaluronan Today", will
`provide an overview and background information that you will find useful for
`understanding and appreciating the multi-faceted world of hyaluronan
`research that will unfold in the subsequent articles of this series.
`
`1. Meyer K, Palmer JW: The polysaccharide of the vitreous humor. J. Biol. Chem. 107,
`629-634, 1934.
`2. Weissman B, Meyer K: The structure of hyalobiuronic acid and of hyaluronic acid
`from umbilical cord. J. Am. Chem. Soc. 76, 1753-1757, 1954.
`
`A recent general overview of hyaluronan
`
`3. Laurent TC, ed: The Chemistry, Biology and Medical Applications of Hyaluronan and
`its Derivatives. Wenner-Gren International Series, Vol 72, Portland Press, London,
`1998.
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