Proc. Natt Acad. Sci. USAVol. 79, pp. 307-309, January 1982Biochemistry

Ultrastructure of a hyaluronic acid matrix(glycosaminoglycan/synovial fluid/connective tissue)

NORTIN M. HADLER*, ROBERT R. DOURMASHKINt, MILAN V. NERMUTt, AND LYNN D. WILLIAMSt*Departments of Medicine and Bacteriology (Immunology), University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514; tSection of ElectronMicroscopy, Clinical Research Centre, Harrow, England; and *Laboratory of Biological Ultrastructure, National Institute for Medical Research, Mill Hill, England

Communicated by Charles N. Reilley, September 21, 1981

ABSTRACT Freeze-etch replicas of a hylauronic acid matrixwere visualized by electron microscopy. In water a coarse branch-ing fibrillar network of hyaluronic acid aggregates was seen. Thehigh solvent permeability of this matrix suggests that the spacesobserved are relatively devoid ofunaggregated polymer. Additionof calcium disordered the matrix, resulting in a more dispersedfelt of polymer.

Hyaluronic acid is an unbranched polysaccharide consisting ofregularly alternating 3-D-glucuronic acid and 2-acetamido-2-deoxy-(3D-glucose. Although widely distributed throughoutthe extracellular space, it is the principal glycosaminoglycan insuch body fluids as synovial fluid, the vitreous humor, andWharton's jelly of the umbilical cord. In synovial fluid, for ex-ample, it has an average molecular mass of 6 x 106 daltons. Insolution the hyaluronate chain is folded into a solvated sphericalmolecule or domain with a diameter often exceeding 200 nm(1). At the known range of concentrations in the fluids men-tioned it is clear that these domains greatly overlap, resultingin the concentration-dependent, non-Newtonian viscoelasticproperties of the fluids (2). It was postulated initially that theconformation of the hyaluronic acid in this continuous three-dimensional chain network (matrix) was that of a random coil(3). However, assessment of the hyaluronic acid matrix by usingmeasurements ofcircular dichroism, optical rotatory dispersion(4), and nuclear magnetic resonance (5) suggests a considerabledegree of orderedness in solution.

In the current studies we directly visualized the orderednessof the hyaluronic acid matrix by electron microscopy of freeze-etch replicas.

METHODSThe hyaluronic acid used was from human umbilical cord. Thismaterial (Sigma, grade 1, no. H-1751, lot 68C-0379) had po-tassium and sodium contents of 5.32% and 0.04%, respectively.The 2.5% solutions we studied were prepared by placing thehyaluronate in sterile tubes with the solvent layered on top. Thetubes were allowed to stand for 24 hr at 37°C; this was sufficienttime for the hyaluronic acid to dissolve and to allow all bubblesto rise to the top. The tubes were maintained at 40C until used.The solvent system always employed sterile distilled water.

Freeze-fracturing was carried out by two methods describedby Nermut and Williams (6). In one method, a drop of hyalu-ronate was placed on a piece of freshly cleaved mica (9 x 4 mm).After a few minutes another piece of cleaved mica (same size)was attached crosswise without force and the sandwich wassnap-frozen in Freon 22; this was followed by freezing in liquidN2. The sandwich was then fractured by using a special double-replica device. In the second method, a frozen droplet was frac-

tured with a knife. Both methods produced similar results. Thefracture surface was etched for 30-60 sec, in a BAF 300 freeze-etch unit (Balzers) at - 100'C, using an electron beam gun forPt/C shadowing. The thickness of the Pt/C layer, controlledwith a quartz crystal monitor (usually about 200 Hz), was30-35K Replicas were cleaned with sodium hypochlorite (1 hr)and washed with distilled water. The grids were examined ina Phillips 300 electron microscope.

RESULTS

With water (Fig. 1 Top) or saline buffered with 0.01 M sodiumphosphate at pH 7.4 (Fig. 2) as the solvent, the matrix was acoarse network of branching fibrils. Every structure visualizedwas an aggregate ofcolinear hyaluronic acid molecules. The fol-lowing arguments support that assertion: (i) Because the hyalu-ronic acid molecule itself is linear and unbranched, the ob-served branching must represent the crossover of members ofone bundle to run parallel with those of another bundle of hy-aluronic acid molecules. (ii) Single molecules ofhyaluronic acidhave not been visualized with heavy metal shadowing in theelectron microscope unless spread in the presence of a posi-tively charged protein such as cytochrome c (7). (iii) Hydratedfilms and putty ofvarious hyaluronate salts have been subjectedto x-ray crystallographic structural analysis (8, 9). An upper limitestimate for the diameter of a single molecule of hyaluronateapproaches 0.4 nm. The lower limit estimate for the thinneststructure observed (Fig. 2) approaches 2.0 nm when the thick-ness of the Pt/C layer is taken into account. The packing of thehyaluronate chains within the fibrils cannot be visualized.

Because a single hyaluronate molecule cannot be visualized,is it possible that the spaces in the matrix (Figs. 1 Top and 2)are a fine feltwork of disaggregated molecules? In our labora-tory, studies of the permeability of the hyaluronate matrix (10)bear on this issue. [The apparatus we employ is similar to onedescribed (11).] Solvent permeation reflects the continuity andextent of channels prerequisite to bulk flow. The effect of cal-cium ion concentration on the permeability of a 1% hyaluronatematrix is considerable. Matrices with calcium concentrations>5 mM are one-fifth as permeable as those with no added cal-cium. Therefore, in the absence of calcium one would expectcontinuous channels; these are probably represented by thespaces in the matrix in Figs. 1 Top and 2. With the addition ofcalcium, the hyaluronate is disordered (5). Concomitantly (Fig.1 Middle and Bottom), the spaces in the matrix must becomea feltwork of disaggregated hyaluronate molecules that can nolonger be resolved.

DISCUSSIONGlycosaminoglycans, generally of the heparan sulfate and hy-aluronate classes, are synthesized by many, if not all, mam-malian cells grown in vitro (12, 13). In vivo, as well, these mac-

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308 Biochemistry: Hadler et aL

FIG. 1. Negative image electron micrographs of matrices of 2.5%hyaluronate freeze-etched in parallel. (x63,000.) The solvents arewater (Top), 5.0 mM calcium chloride (Middle), and 10.0 mM calciumchloride (Bottom). (Top) Matrix appears as a coarse network of branch-ing fibrillar strands. With the addition of calcium the matrix becomesmore heterogeneous, with fewer areas exhibiting this degree of or-deredness. Rather, structure as in Middle and Bottom predominates.(Middle) Network appears to have a finer mesh than in Top. (Bottom)Only a few strands are resolved. At 25 mM calcium chloride or greater,essentially no orderedness is demonstrable.

FIG. 2. Negative image electron micrograph of freeze-etched ma-trix of 2.5% hyaluronate in saline buffered at pH 7.4 with 0.01 M so-dium phosphate. (x300,000.) The detail of the coarse branching fi-brillar network is apparent. This orderedness cannot be distinguishedfrom that in water as a solvent (Fig. 1 Top). The thinnest fibril detectedmeasures 4.0 nm in diameter. Of that thickness, 30-50% is the Pt/C coating. The lower limit estimate of fibril diameter is 2.0 nm. There-fore, all discernible structures are aggregates of hyaluronate molecules.

romolecules are demonstrable in intimate association with theplasma membrane (14). In this site, glycosaminoglycans are in-volved in adherence to surfaces (15) and cell-cell contact (16,17). Furthermore, the glycosaminoglycans of the extracellularmatrix critically influence various differentiated cell functionsinvolved in chondrogenesis (18), embryogenesis (19), oncogen-esis (20), and other phenomena.

These and many other biological properties of the extracel-lular space (21) depend on the glycosaminoglycan class, on thepresence of serum constituents in the matrix (22, 23), and onthe structure of the matrix (5, 24). Freeze-etch microscopy-similar to the method used to study a thick hyaluronate ma-trix-might yield insights into regional variations in the struc-ture ofthe extracellular matrix in various tissues. Furthermore,extrapolating from the influence of calcium concentration onhyaluronate, one would anticipate that local physiological fac-tors would perturb matrix structure and vice versa.

During the course of these studies N.M. H. was a visiting scientistin the Division of Immunological Medicine, Clinical Research Centre,Harrow, England, as part of his tenure as an Established Investigatorof the American Heart Association. This work was supported in part byResearch Grant AM-19992 from the National Institutes of Health.

Proc. Nad Acad. Sci. USA 79 (1982)

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