D

Formation of microfibrils l.5nm](Z3S5ES

Collagen molecule

End to end aggregation

Col kigen fiber

FIG* 1 • Collagen, like other proteins, is distinguished by several levels of structural order. (A) Primary structure—the complete sequence of amino acids along each polypeptide chain. An example is the triple chain sequence of type i calf skin collagen at the N-end of the molecule. Roughly 5% of a complete molecule is shown. No attempt has been made to indicate the coiling of the chains. Amino acid residues participating in the triple helix are numbered, and the residue-to-residue spacing (0.286 nm) is shown as a constant within the triple helical domain, but not outside it. Bold capitals indicate charged residues which occur in groups (underlined) (Reprinted from J. A. Chapman and D. J. S. Hulmes, in Ultrastructure of the Connective Tissue Matrix, A Ruggeri and P. M. Motta, eds., Martinus Nijhoff, 1984, Chap. 1, Fig. 1, p. 2, with permission.)

structural order in proteins, and we specialize it to the case of type 1 collagen (Fig. 1).

The primary structure denotes the complete sequence of amino acids along each of three polypeptide chains as well as the location of interchain cross-links in relation to this sequence. Approximately one-third of the residues are glycine and another quarter or so are proline or hydroxyproline. The structure of the (afunctional interchain cross-link is the relatively complex condensation product of a reaction involving lysine and hydroxylysine residues; this reaction continues as the organism matures, thereby rendering the collagens of older animal« more difficult to extract.

The secondary structure is the local configuration of a polypeptide chain that results from satisfaction of stereochemical angles and hydrogen-bonding potential of peptide residues. In collagen, the abundance of glycine residues (Gly) plays a key configurational role in the triplet Gly-X-Y, where X and Y are frequently proline or hydroxyproline, respectively, the two amino acids that direct the chain configuration locally by the rigidity of their ring structures. On the other hand, the absence of a side chain in glycine permits the close approach of polypeptide chains in the collagen triple helix. The tertiary structure refers to the global configuration of the polypeptide chains; it represents the pattern according to which the secondary structures are packed together within the complete macromole-cule and it constitutes the structural unit that can exist as a physicochemically stable entity in solution, namely, the triple helical collagen molecule.

In type I collagen, two of the three polypeptide chains have identical amino acid composition, consist of 1056 residues, and are termed a l il) chains, while the third has a different composition; it consists of 1038 residues and is termed a2(I). The three polypeptide chains fold to produce a left-handed helix while the three-chain supercoil is actually right-handed with an estimated pitch of about 100 nm (30—40 residues). The helical structure extends over 1014 of the residues in each of the three chains, leaving the remaining residues at the ends (telopeptides) in a nonhelical configuration. The residue spacing is 0.286 nm and the length of the helical portion of the molecule is, therefore, about 1014 x 0.286 or 290 nm.

The fourth-order or quaternary structure denotes the repeating supermolecular unit structure, comprising several molecules packed in a specific lattice, which constitutes the basic element of the solid state (microfibril). Collagen molecules are packed in a quasi-hexagonal lattice at an interchain distance of about 1.3 nm which shrinks considerably when the microfibril is dehydrated. Adjacent molecules in the microfibril are approximately parallel to the fibril axis; they all point in the same direction along the fibril and are staggered regularly, giving rise to the well-known D-period of collagen, about 64 nm, which is visible in the electron microscope. Higher levels of order, eventually leading to gross anatomical features which can be readily seen with the naked eye, have been proposed but there is no agreement on their definition.

physical modifications of the native, structure of collagen

Crystallinity in collagen can, according to Fig. 1, be detected at two discrete levels of structural order: the tertiary (triple helix) (Fig. 1C) and the quaternary (la ttice of triple helices) (Fig. ID). Each of these levels of order corresponds, interestingly enough, to a separate melting transformation. A solution of collagen triple helices is thus converted to the randomly coiled gelatin by heating above the helix-coil transition temperature, which is approximately 37°C for bovine collagen, or by exceeding a critical concentration of certain highly polarizable anions, e.g., bromide or thiocyanate, in the solution of collagen molecules. Infrared spectroscopic procedures, based on helical marker bands in the mid- and far infrared, have been developed to assay the gelatin content of collagen in the solid or semisolid states in which collagen is commonly used as an implant. Since implanted gelatin is much more rapidly degradable than collagen, these assays are essential tools for quality control of collagen-based biomaterials. Frequently such biomaterials have been processed under manufacturing conditions which may threaten the integrity of the triple helix.

Collagen fibers also exhibit a characteristic banding pattern

(B) Secondary structure—the local configuration of a polypeptide chain. The triplet sequence Gly-Pro-Hyp illustrates elements of collagen triple-helix stabilization. The numbers identify peptide backbone atoms. The conformation is determined by trans peptide bonds (3-4, 6-7, and 9-1); fixed rotation angle of bond in proline ring (4-5); limited rotation of proline past the C=0 group (bond 5-6); interchain hydrogen bonds (dots) involving the NH hydrogen at position 1 and the C=0 at position 6 in adjacent chains; and the hydroxy group of hydroxyproline, possibly through water-bridged hydrogen bonds. (Reprinted from K. A. Piez and A. H. Reddi, eds., Extracellular Matrix Biochemistry, Elsevier, 1984, Chap. 1, Fig. 1.6, p. 7, with permission.)

(C) Tertiary structure—the global configuration of polypeptide chains, representing the pattern according to which the secondary structures are packed together within the unit substructure. A schematic view of the type I collagen molecule, a triple helix 300 nm long. (Reprinted from K. A. Piez and A. H. Reddi, eds., Extracellular Matrix Biochemistry, Elsevier, 1984, Chap. 1, Fig. 1.22, p. 29, with permission.) (D) Quaternary structure—the unit supermolecular structure. The most widely accepted unit is one involving five collagen molecules (microfibril). Several microfibrils aggregate end to end and also laterally to form a collagen fiber which exhibits a regular banding pattern in the electron microscope with a period of 65 nm. (Reprinted from M. E. Nimni, ed.., Collagen, Vol. 1, Biochemistry. CRC Press, Boca Raton, 1988, Chap. 1, Fig. 10, p. 14, with permission.)

with a period of 65 nm (quarternary structure). This pattern is lost reversibly when the pH of a suspension of collagen fibers in acetic acid is lowered below 4.25 ± 0.30. Transmission electron microscopy or small-angle X-ray diffraction can be used to determine the fraction of fibrils which possess banding as the pH of the system is altered. During this transformation, which appears to be a first-order thermodynamic transition, the triple helical structure remains unchanged. Changes in pH can, therefore, be used to selectively abolish the quarternary structure while maintaining the tertiary structure intact.

This experimental strategy has made it possible to show that the well-known phenomenon of blood platelet aggregation by collagen fibers (the reason for using collagen sponges as hemostatic devices) is a specific property of the quarternary rather than the tertiary structure. Thus collagen which is thromboresistant in vitro has been prepared by selectively "melting out" the packing order of helices while preserving the triple helices themselves. Figure 2 illustrates the banding pattern of such collagen fibers. Notice that short segments of banded fibrils persist even after very long treatment at low pH, occasionally interrupting long segments of non banded fibrils (Fig. 2, inset).

The porosity of collagenous implants normally makes an indispensable contribution to its performance. A porous structure provides an implant with two critical functions. First, pore channels are ports of entry for cells migrating from adjacent tissues into the bulk of the implant or for the capillary suction of blood from a hemorrhaging blood vessel nearby. Second, pores endow a solid with a frequently enormous specific surface which is made available either for specific interactions with invading cells (e.g., collagen-glycosaminoglycan (CG) copolymers which induce regeneration of skin in burned patients) or for interaction with coagulation factors in blood flowing into the device (e.g., hemostatic sponges).

Pores can be incorporated by first freezing a dilute suspension of collagen fibers and then inducing sublimation of the ice crystals by exposing the suspension to a low-temperature vacuum. The resulting pore structure is a negative replica of the network of ice crystals (primarily dendrites). It follows that control of the conditions for ice nucleation and growth can lead to a large variety of pore structures (Fig. 3).

In practice, the average pore diameter decreases with decreasing temperature of freezing while the orientation of pore channel axes depends on the magnitude of the heat flux vector during freezing. In experimental implants, the mean pore diameter has ranged between about 1 and 800 mm; pore volume fractions have ranged up to 0.995; the specific surface has been varied between about 0.01 and 100 m2/g dry matrix; and the orientation of axes of pore channels has ranged from strongly uniaxial to highly radial. The ability of collagen-glycosamino-glycans to induce regeneration of tissues such as skin and nerve depends critically, among other factors, on the adjustment of the pore structure to desired levels, e.g., about 20—125 ¿im for skin regeneration and less than 10 jum for sciatic nerve regeneration. Determination of pore structure is based on principles of stereology, the discipline which allows the quantitative statistical properties of three-dimensional implant structures to be related to those of two-dimensional projections, e.g., sections used for histological analysis.

chemical modification of collagen

The primary structure of collagen is made up of long sequences of some 20 different amino acids. Since each amino acid has its own chemical identity, there are 20 types of pendant side groups, each with its own chemical reactivity, attached to the polypeptide chain backbone. As examples, there are carboxylic side groups (from glutamic acid and aspartic acid residues), primary amino groups (lysine, hydroxylysine, and arginine residues), and hydroxylic groups (tyrosine and hydroxylysine), The collagen molecule is therefore subject to modification by a large variety of chemical reagents. Such versatility comes with a price: Even though the choice of reagents is large, it is imporant to ascertain that use of a given reagent has led to modification of a given fraction of the residues of a certain amino acid in the molecule. This is equivalent to proof that a reaction has proceeded to a desired "yield." Furthermore, proof that a given reagent has attacked only a specific type of amino acid, rather than all amino acid residue types carrying the same functional group, also requires chemical analysis.

Historically, the chemical modification of collagen has been practiced in the leather industry (since about 50% of the protein content of cowhide is collagen) and in the photographic gelatin industry. Today, the increasing use of collagen in biomaterials applications has provided renewed incentive for novel chemical modification, primarily in two areas. First, implanted collagen is subject to degradative attack by collagenases, and chemical cross-linking is a well-known means of decelerating the degradation rate. Second, collagen extracted from an animal source elicits production of antibodies (immunogenicity). Although it is widely accepted that collagen elicits synthesis of a far smaller concentration of antibodies than other proteins (e.g., albumin), treatment with specific reagents, including enzymatic treatment, is occasionally used to reduce the immunogenicity of collagen.

Collagen-based implants are normally degraded by collagenases, naturally occurring enzymes which attack the triple helical molecule at a specific location. Two characteristic products result, namely, the N-terminal fragment which amounts to about two thirds of the molecule, and the one-quarter C-terminal fragment. Both of these fragments become spontaneously transformed (denatured) to gelatin at physiological temperatures via the helix-coil transition and the gelatinized fragments are then cleaved to oligopeptides by naturally occurring enzymes which degrade several other tissue proteins (non specific proteases).

Collagenases are naturally present in healing wounds and are credited with a major role in the degradation of collagen fibers at the site of trauma. At about the same time that degradation of collagen and of other ECM components proceeds in the wound bed, these components are being synthesized de novo by cells in the wound bed. Eventually, new architectural arrangements, such as scar tissue, are synthesized. While it is not a replica of the intact tissue, scar tissue forms a stable endpoint to the healing process, and forms a tissue barrier between adjacent organs which allows the healed organ to continue functioning at a nearly physiological level. The combined process of collagen degradation and scar synthesis is

FIG* 2* Following exposure to pH below 4.25 ± Q30, the banding pattern of type I bovine hide collagen practically disappears. Short lengths of banded collagen (B) do, however, persist next to very long lengths ot nonbanded collagen (NB) which has tertiary but not quaternary structure. This preparation does not include platelet aggregation provided that the fibers are prevented from recrystallizing to form banded structures when the pH is adjusted to neutral in order to perform the platelet assay. Stained with 0*5 wt.% phosphotungstic acid. Banded collagen period, about 65 nm. x 12,750. Inset: >'63,750. (Reprinted from M. J. Forbes, M. S. dissertation, Massachusetts Institute oi Technology, 1980* coiurtesy of MtT.>

FIG. 3. illustration of tlit; variety of porous structures which can be obtained with collagen-CAG copolymers by ■■l;iis!:iuv, the kinetics ol crysta Urea lion ot ice to the appropriate magnitude and direction, rotes lorm when the ice dendrites .ire eveiitualK' .Mihtiuied. ic.innini; electron microscopy. (Courtesy of MIT.)

FIG. 3. illustration of tlit; variety of porous structures which can be obtained with collagen-CAG copolymers by ■■l;iis!:iuv, the kinetics ol crysta Urea lion ot ice to the appropriate magnitude and direction, rotes lorm when the ice dendrites .ire eveiitualK' .Mihtiuied. ic.innini; electron microscopy. (Courtesy of MIT.)

often referred to as remodeling. One of the frequent challenges in the design of collagen implants is to modify collagen chemically in a way which either accelerates or slows down the rate of its degradation at the implantation site to a desired level.

An effective method for reducing the degradation rate of collagen hy naturally occurring enzymes is chemical cross-linking. A very simple self-cross-1inkmg procedure, dehydrative cross-linking, is based on the fact that removal of water below about 1 wt,% tnsolubiiizes collagen as welt as gelatin by inducing formation of interchain peptide bonds. The nature of the cross-links formed can be inferred from the results of studies using chemically modified gelatins. Gelatin which had been modified either by esrerification of the carboxylic groups of aspartvl—glutamyl residues or by acetylation of the E-amino groups of lysyl residues remained soluble iri aqueous solvents after exposure of the solid protein to high temperature, while unmodified gelatins lost their solubility. Insolubilization of collagen and gelatin following severe dehydration has been, accordingly, interpreted as the resuit of drastic removal of the aqueous product of a condensation reaction which led to the formation of interchain amide links. The proposed mechanism is consistent with results, obtained by titration, showing that the number of free carboxylic groups and free amino groups in collagen arc both significantly decreased followiog high-temperature treatment.

Removal of water to the extent necessary to achieve a density of cross-links in excess of iO"J moles of cross-links/g dry gelatin, which corresponds to an average molecular weight between cross-links, AT., of about 70 kDa. can be achieved within hours by exposure to temperatures in excess of 105' C

under atmospheric pressure. The possibility that the cross-linking achieved under these conditions is caused by a pyrolytic reaction has been ruled out. Furthermore, chromatographic data have shown that the amino acid composition of collagen remains intact after exposure to 105°C for several days. In fact, it has been observed that gelatin can be cross-linked by exposure to temperatures as low as 25°C provided that a sufficiently high vacuum is present to achieve the drastic moisture removal which appears to drive the cross-linking reaction.

Exposure of highly hydrated collagen to temperatures in excess of about 37°C is known to cause reversible melting of the triple helical structure, as described earlier. The melting point of the triple helix increases with the collagen-diluent ratio from 37°C, the helix-coil transition of the infinitely dilute solution, to about 120°C for collagen swollen with as little as 20% wt. diluent and up to about 210°C, the melting point of anhydrous collagen. Accordingly, it is possible to cross-link collagen using the drastic dehydration procedure described earlier without loss of the triple helical structure. It is sufficient to adjust the moisture content of collagen to a low enough level prior to exposure to the high temperature levels required for rapid dehydration.

Dialdehydes have been long known in the leather industry as effective tanning agents and in histological laboratories as useful fixatives. Both of these applications are based on the reaction between the dialdehyde and the e-amino group of lysyl residues in the protein, which induces formation of interchain cross-links. Glutaraldehyde cross-linking is a relatively widely used procedure. The nature of the cross-link formed has been the subject of controversy, primarily because of the complex, apparently polymeric, character of this reagent. Considerable evidence supports the proposed anabilysine structure, which is derived from two lysine side chains and two molecules of glutaraldehyde:

modulus of films that have been treated to induce cross-linking and have subsequently been gelatinized by treatment in 1 M NaCl at 70°C. Under such conditions, only films which have been converted into a three-dimensional network support an equilibrium tensile force; by contrast, uncross-linked specimens dissolve readily in the hot medium.

The immunogenicity of the collagen used in implants is significant and has been studied assiduously using laboratory preparations. However, the clinical significance of such immunogenicity has been shown to be very low, and is often considered to be negligible. This immense simplification of the immunological problem of using collagen as a biomaterial was recognized a long time ago by manufacturers of collagen-based sutures. The apparent reason for the low antigenicity of type I collagen stems from the small species difference among type I collagens (e.g., cow vs. human). Such similarity is, in turn, probably understandable in terms of the inability of the triple helical configuration to incorporate the substantial amino acid substitutions which characterize species differences with other proteins. The relative constancy of the structure of the triple helix among the various species is, in fact, the reason why collagen is sometimes referred to as a "successful" protein in terms of its evolution or, rather, the lack of it.

In order to modify the immunogenicity of collagen, it is useful to consider the location of its antigenic determinants, i.e., the specific chemical groups which are recognized as foreign by the immunological system of the host animal. The configura-tional (or conformational) determinants of collagen depend on the presence of the intact triple helix and, consequently, are abolished when collagen is denatured into gelatin; the latter event (see earlier discussion) occurs spontaneously after the triple helix is cleaved by a collagenase. Gelatinization exposes the sequential determinant of collagen over the short period during which gelatin retains its macromolecule character, before it is cleared away following attack by one of several non-

Evidence for other mechanisms has been presented. Compared with other aldehydes, glutaraldehyde has shown itself to be a particularly effective cross-linking agent, as judged, for example, by its ability to increase the cross-link density. The Mc values provide the experimenter with a series of collagens in which the enzymatic degradation rate can be studied over a wide range, thereby affording implants which effectively disappear from tissue between a few days and several weeks following implantation. Although the mechanism of the reaction between glutaraldehyde and collagen at neutral pH is understood in part, the reaction in acidic media has not been studied extensively. Evidence that covalent cross-linking is involved comes from measurements of the equilibrium tensile specific proteases. Controlling the stability of the triple helix during processing of collagen, therefore, prevents the display of the sequential determinants.

Sequential determinants also exist in the nonhelical end (telopeptide region) of the collagen molecule and this region has been associated with most of the immunogenicity of collagenbased implants. Several enzymatic treatments have been devised to cleave the telopeptide region without destroying the triple helix. Treating collagen with glutaraldehyde not only reduces its degradation rate by collagenase but also appears to reduce its antigenicity as well. The mechanism of this effect is not well understood. Certain applications of collagen-based biomaterials are shown in Table 2.

TABLE 2 Certain Applications of Collagen-Based Bionnaterials

Application

Physical state

Sutures

Hemostatic agents Blood vessels

Heart valves

Tendon, ligaments Burn treatment (dermal regeneration)

Peripheral nerve regeneration

Meniscus regeneration

Intradermal augmentation

Gynecological applications Drug-delivery systems

Extruded tape (Schmitt, 1985)

Powder, sponge, fleece (Stengel et ai, 1974; Chvapil, 1979)

Extruded collagen tube, processed human or animal blood vessel (Nimni, 1988)

Processed porcine heart valve (Nimni, 1988)

Processed tendon (Piez, 1985)

Porous collagen-glycosaminoglycan (GAG) polymer" (Yannas et al, 1981, 1982, and 1989)

Porous collagen-GAG copolymer (Chang and Yannas, 1992)

Porous collagen-GAG copolymers (Stone et al., 1989)

Injectable suspension of collagen particles (Piez, 1985)

Sponges (Chvapil, 1979)

Various forms (Stenzel et ai., 1974, Chvapil, 1979)

"See also Chapter 7.10.

proteoglycans and glycosaminoglycans

Glycosaminoglycans (GAGs) occur naturally as polysaccharide branches of a protein chain, or protein core, to which they are covalently attached via a specific oligosaccharide link. The entire branched macromolecule, which has been described as having a "bottle brush" configuration, is known as a proteoglycan and has a molecular weight of about 103 kDa.

The structure of GAGs can be generically described as that of an alternating copolymer, the repeat unit consisting of a hexosamine (glucosamine or galactosamine) and of another sugar (galactose, glucuronic acid or iduronic acid). Individual GAG chains are known to contain occasional substitutions of one uronic acid for another; however, the nature of the hexosamine component remains invariant along the chain. There are other deviations from the model of a flawless alternating copolymer, such as variations in sulfate content along the chain. It is, nevertheless, useful for the purpose of getting acquainted with the GAGs to show their typical (rather, typified) repeat unit structure, as in Fig. 4. The molecular weights of GAGs are in the range of 5—60 kDa, with the exception of hyaluronic acid, the only GAG which is not sulfated; it exhibits molecular weights in the range of 50—500 kDa. Sugar units along GAG chains are linked by a or /3 glycosidic bonds and are 1, 3, or 1, 4 (Fig. 4). There are several naturally occurring enzymes which degrade specific GAGs, the most well-known being hyaluronidase. These enzymes are primarily responsible for the physiological turnover rate of GAGs, which is in the range of 2-14 days.

The nature of the oligosaccharide link appears to be identical for the GAGs, except for keratan sulfate, and is a galactosyl—galactosyl—xylose, with the latter glycosidically linked to the hydroxyl group of serine in the protein core.

The very high molecular weight of hyaluronic acid is the basis of most uses of this GAG as a biomaterial; almost all applications make use of the exceptionally high viscosity and the facility to form gels which characterize this polysaccharide. Hyaluronic acid gels have found considerable use in ophthalmology because they facilitate cataract surgery as well as retinal reattachment. Other uses of this GAG reported are the treatment of degenerative joint dysfunction in horses and experimental treatment of certain orthopedic dysfunctions in humans. On the other hand, sulfated GAGs are anionically charged and can induce precipitation of collagen at acidic pH levels, a process which yields collagen-GAG coprecipitates that can be subsequently freeze dried and covalently cross-linked to yield biomaterials which have been shown capable of inducing regeneration of skin (dermis), peripheral nerve, and the meniscus of joints (Table 2).

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