Hyaluronan Based Biomaterials

To obtain materials that are mechanically and chemically robust, a variety of crosslinking strategies based on the chemistry described above have been explored to produce insoluble or gel-like hyaluronan materials. We separate hyaluronan-based biomaterials into two categories based on the crosslinking mechanism: physical and chemical. In physical crosslinking, hydrophobic and ionic interactions convert hyaluronan or its derivatives into hydrogels. Chemically crosslinked materials have new covalent inter- and intramolecular bonds that create an infinite molecular network.

A. Esterified Hyaluronan (HYAFF®)

The HYAFF® esterfied hyaluronans are versatile and well-studied biomaterials. Esterfication results in the loss of hydrophilicity and hydrogen bonding by masking the free carboxylic group, and the simultaneous increase of hydrophobilicity by the introduction of alkyl or benzyl groups. As a result, most HYAFF® materials are insoluble in water. Instead, HYAFF® materials can be dissolved in organic solvents and extruded to produce membranes and fibers, lyophilized to obtain sponges, or spray-dried to produce microspheres.

The degree of esterification and the type of alcohol used influence the size of hydrophobic clusters. These clusters create a polymer chain network that is more rigid and stable, and less susceptible to enzymatic degradation. Hyaluronan esters undergo spontaneous de-esterification in an aqueous environment, and the hydrolytic degradation of ester bonds of HYAFF® 7 and HYAFF® 11 was mostly complete within 2-3 months in artificial plasma (62,63). The materials degrade in vivo within 110 days, synchronous with neotissue formation (64). The biocompatibility of HYAFF® materials has been extensively documented in vitro and in vivo and recently reviewed (8).

Drug release from HYAFF®-based devices was examined for entrapped or covalently attached molecules. For example, release of the steroids hydrocortisone and a-methylpredisolone from microspheres fabricated from different HYAFF® materials was examined with the drug either dispersed within or bound to the polymer. However, the release of physically entrapped small molecular weight drug with considerable aqueous solubility from HYAFF®-based devices was too rapid; hydrocortisone diffused out of microspheres in 10 min. The covalent attachment of steroid drugs to HYAFF® dramatically retarded drug release. The release showed zero-order kinetics with a half-time of 100 h, consistent with ester bond hydrolysis (65,66). Preclinical in vivo evaluations in a rabbit model demonstrated that an a-methylpredisolone carried by a HYAFF® 11 formulation could maintain its anti-inflammatory and chondroprotective properties (65-67) and increase the residence time of the drug in the tear fluid (68,69).

HYAFF® 11 matrix has more acceptable controlled-release properties for macromolecules. Polypeptides and proteins such as recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) diffuse out slowly from these matrices (70). Furthermore, the mucoadhesive properties of HYAFF® materials permit drug delivery via nasal, oral, and vaginal mucosal routes. For example, HYAFF® materials have been used for intranasal delivery of insulin in sheep (71), vaginal delivery of calcitonin in rats (72,73), and vaginal delivery of flu vaccine in rodents (74).

Hyaluronan has many roles in biology, particularly during embryonic development (75-77). Therefore, hyaluronan derivatives may create a suitable environment not only for the growth of cells derived from organ biopsy samples, and may stimulate stem or precursor cells to divide and differentiate into specific cell types in this embryo-like environment (78,79).

Hyaluronan and its various chemical derivatives have been used already in wound healing and related tissue repair application. These unique properties of hyaluronan are attributed to its biophysical properties and also its biological properties as an ECM molecule as well as a biological signaling molecule (80,81). HYAFF® scaffolds have been employed to produce hyaluronan-based tissue engineered skin grafts. Epithelialization is achieved by employing autologous keratinocyte grafts delivered on Laserskin®, a HYAFF® micro-perforated transparent membrane designed to facilitate graft handling procedures and to enable grafting in preconfluence. The dermal component of the skin has been addressed by using a 3D HYAFF® fiber mesh scaffold, named Hyalograft 3D®, designed to support fibroblast cultures. Under these conditions, fibroblasts proliferate within the interstices of the scaffolds, depositing extracellular components such as type I, III, and IV collagen, and adhesion molecules such as laminin and fibronectin (82-89). To expedite vascularization and thereby facilitate transport of oxygen and nutrients and the removal of toxins, reconstruction of a dermal-like tissue provided with a capillary-like structure would have important advantages. To this end, HUVECs, either alone or with fibroblasts at 1:1 ratio, were seeded into HYAFF® 11 non-woven mesh. HYAFF® 11 non-woven mesh was found to be a suitable substrate for HUVEC adhesion, proliferation, and reorganization into microcapillary network (90).

Primary hepatocytes were seeded onto HYAFF® 7 and HYAFF® 11 films and non-woven fabrics in order to develop a potential bioartificial liver support device. After in vitro culture for 1 week, the cells in HYAFF® 7 non-woven fabrics exhibited an ammonia elimination approximately equal to that of cells on collagen films. The urea synthesis was more than threefold greater than that of adherent cells on collagen films, a widely used substrata for monolayer hepatocyte culture. Thus, HYAFF® 7 non-woven fabrics are promising substrata for hepatocyte culture in bioartificial liver devices (91).

Tissue reconstruction after surgical intervention or trauma tissue reconstruction represents an unresolved clinical problem. A biohybrid construct, consisting of hyaluronic acid-based scaffolds (HYAFF® 11) and human adipocyte precursor cells, was developed and the implantation of such bioconstructs into nude mice resulted in local differentiation of pre-adipocytes, suggesting the potential of the bioconstructs as autologous soft tissue-filler for clinical application in humans (92,93).

The use of HYAFF® as a scaffold for articular cartilage and bone tissue engineering has also been explored (64,94-96). For example, avian chondrocytes were shown to adhere and proliferate onto non-woven meshes of the hyaluronan-based scaffolds (HYAFF® 7 and HYAFF® 11). The adherent cells also synthesized a glycosaminoglycan- and collagen type II-rich ECM (64). HYAFF® 11 also supported the growth of human chondrocytes, and the cells maintained their original phenotype. Human chondrocytes seeded on HYAFFw 11 expressed and produced collagen type II and aggrecan and down-regulated the production of collagen type I, indicating the therapeutic potential of HYAFFw 11 as a delivery vehicle in a tissue-engineering approaches to the repair of articular cartilage defects (97). HYAFFw 11 was an effective human chondrocyte delivery matrix for the treatment of articular cartilage defects (98). Hyaluronan also showed a molecular weight-specific and dose-specific stimulation of osteoblasts, suggesting a mechanism to enhance the osteogenic and osteoinductive properties of bone graft materials (95).

Solchaga and co-workers (99) hypothesized that synthetic matrices based on hyaluronan derivatives could mimic the embryonic hyaluronan-rich environment supportive of progenitor cell development. When used as a delivery vehicle for cell-based therapies in adult organisms, the hyaluronan matrix should promote a recapitulation of these embryonic events and thereby facilitate tissue repair. Scaffolds made from HYAFF 11 and an autocrosslinked derivative of hyaluronan (ACP™) were loaded with rabbit mesenchymal progenitor cells and compared with a control of porous, fibronectin-coated calcium phosphate ceramic cubes. HYAFF® 11 scaffolds retained 90% more cells than did the ACP™ scaffolds and the fibronectin-coated ceramics. When implanted sub-cutaneously in nude mice, HYAFFw 11 scaffolds retained their integrity after 3-6 weeks, whereas the ACP® scaffolds rapidly degraded within 7-10 days. Bone, cartilage, and fibrous tissue formation was evident in the pores of the implanted HYAFFw 11 and ceramic scaffolds, with the former showing a 30% increase in the relative amount of bone and cartilage per unit area (96,99). The HYAFFw 11 scaffold was also a good substrata for adhesion and proliferation of bone marrow-derived mesenchymal progenitor cells (100). When a bioconstruct of HYAFFw 11 scaffold seeded with autologous bone marrow-derived mesenchymal progenitor cells was implanted in a full-thickness osteochondral lesion in rabbit, the histological evidence revealed that lesions filled with the biomaterial, either seeded or unseeded with cells, healed faster and more completely than controls.

In bone repair, HYAFFw 11 scaffolds promoted mineralization of rat bone marrow stromal cells and basic fibroblast growth factor (bFGF) further enhanced the mineralization in vitro (101). Implantation of HYAFF® 11 scaffold seeded with bone marrow stromal cells, which had been previously grown in vitro in medium supplemented with bFGF, was used to treat large segmental radius defects in rat. The presence of bFGF significantly accelerated bone mineralization (102).

In addition to the HYAFF® materials, long alkyl chains (dodecyl and octadecyl) have also been covalently linked to hyaluronan via ester functions with low degree of substitution. In dilute aqueous solutions, these amphiphilic derivatives exhibited the rheological properties typical of hydrophobically associated polymers. These properties included the tremendous enhancement of zero shear rate Newtonian viscosity, steep shear-thinning behavior, and formation of physically crosslinked gel-like networks. Such physically associated polymeric materials could be used for synovial fluid viscosupplementation as well as in cartilage replacement (103).

B. Ionic Crosslinking HA-Based Biomaterials

GYNECARE INTERGEL® (LifeCore) is a viscous formulation of hyaluronan formed by chelation with ferric hydroxide. This reddish gel can be used during gynecological surgery to separate and protect tissues as they heal, and was approved by the FDA in November 2001 (104,105). Adhesions—bands of scar tissue that form during healing—are a common complication of surgery on the female reproductive organs (ovaries, uterus, or fallopian tubes). Adhesions inappropriately connect organs or tissues that should normally be separate, leading to pelvic pain, bowel obstruction, and infertility. In clinical use, the inner tissues were coated with GYNECARE INTERGEL® after surgical procedures were finished, but before the abdominal cavity was closed. The gel was meant to lubricate the tissue surfaces and prevent contact and adhesion formation. After about a week, the tissues absorbed the gel and the metabolites were excreted. However, in 2003 this product caused inflammatory responses during clinical use and was withdrawn from the market.

A thermoreversible hyaluronan gel was fabricated for drug release by simply mixing hyaluronan with doxycycline and a divalent metal cation in an aqueous solution (106). Two kinds of electrostatic interactions were invoked for the crosslinking and drug retention. The desired drug release profiles were achieved by controlling the absolute concentration of hyaluronan and doxycycline and the molecular weight of hyaluronan.

Polycations form robust polyelectrolyte complexes with polyanions. Such complexes have been suggested as scaffolds for tissue regeneration. Chitosan, the only natural positively charged polysaccharide with good biocompatibility and biodegradability, has been widely studied for biomedical applications (107). A chitosan-hyaluronan complex was obtained by simply adding chitosan hydrochloride solution into hyaluronan solution The resulting gel was well tolerated by chondrocytes and keratinocytes in vitro and in vivo in rats (108,109). However, the results showed that chitosan alone gave better results than the complex. This was attributed to the dissociation of the complex at physiological pH. In another study, chitosan-hyaluronan microspheres were prepared containing gentamycin as model drug, and the combination of hyaluronan with chitosan appeared to combine the mucoadhesive potential of hyaluronan with the penetration enhancing effect of chitosan (110).

Under physiological conditions, PLL has a higher charge density than chitosan, and thus would form stronger electrostatic interactions with hyaluronan. A biocompatible film based on PLL and hyaluronan was developed by deposition of alternating layers of PLL and hyaluronan. The molecular basis for this buildup was investigated by Picart and co-workers (111-113) using fluorescently labeled hyaluronan and PLL. These multilayered polyelectrolyte films may provide a novel approach to surface modification for cell adhesion and growth.

Oxidized polypyrrole (PP) is a polycationic electronic conductor, and recent results have shown that PP is able to support the growth and differentiation of multiple cell types in vitro, including neurons and endothelial cells. PP can also facilitate the in vivo regeneration of damaged peripheral nerves in rats (114,115). A PP-hyaluronan film was fabricated to combine the biological activity of hyaluronan and the electrical conductivity of PP. These films were promising candidates for tissue engineering and wound-healing applications that could benefit from both electrical stimulation and enhanced vascularization (116).

Alginate sponges loaded with hyaluronan provided an adapted environment for proteoglycan and collagen synthesis by chondrocytes (117). However, the supplemented hyaluronan rapidly diffused out of the beads, thus limiting the in vitro development of a cartilage repair material (118). Altenatively, the deacetylation of hyaluronan as described above creates new amino functionalities, thereby increasing the electrostatic interaction between alginate and hyaluronan. Additional biomaterials can be fabricated based on alginate and deacetylated hyaluronan (60).

C. Covalent Crosslinked Hyaluronan-Based Biomaterials

1. Hylans

Originally developed in the early 1980s by Balazs and commercialized by Biomatrix, Inc., Hylan® is produced by chemically crosslinking the hyaluronan containing residual proteins with formaldehyde (soluble gel) or divinyl sulfone (119). These materials are now marketed by Genzyme Corporation. Soluble Hylan A® is a very high molecular weight form (8-23 MDa) of hyaluronan (5-6 MDa) that exhibits enhanced rheological properties compared to native hyaluronan, and is used in ophthalmic viscosurgery (Hylashieldw). Hylan Bw was the first crosslinked, water-insoluble hyaluronan derivative with appropriate physical and biological properties for clinical use. In an alkaline environment, divinyl sulfone reacts with the hydroxyl groups of hyaluronan, creating bis-ethyl sulfone crosslinks. Hylan Bw gel has greater elasticity and viscosity than soluble Hylan Aw and retains the biocompatibility of the native hyaluronan (non-immunogenic, non-inflammatory and non-toxic).

Hylan B® gel for viscoaugmentation was developed as a "bulking agent" that is physically and biologically compatible with various soft tissues, including subdermal and sphincter muscle tissue. Hylan Bw gel can be injected through a 30 gauge needle as small, elastically deformable gel particles. This gel is available in some countries for soft tissue augmentation to correct dermal wrinkles and depressed scars (Hylaform®) (120,121), and the approval by the FDA for use in the US is pending. Preclinical studies also showed that Hylan B® gel could be used to augment the sphincter muscle for the treatment of urinary stress incontinence. Additionally, the injection of viscoelastic hylan B® gel gave a durable augmentation of the soft tissue in the vocal fold in a rabbit model for the treatment of glottic insufficiency in laryngeal disorders (122).

Hylans have also been used for the treatment of degenerative joint disease and rheumatoid arthritis. The gel protects cartilage and prevents further chondrocyte injury. However, the effect was found to be reversible and viscosity dependent (123). Synvisc® (Hylan G-F 20®) is an FDA-approved local therapy that provides lubrication for the knee joint and acts as a "shock absorber." Successful with treatment Synvisc® can help reduce osteoarthritis knee pain, which can lead to increased mobility (124,125).

Hylan gel (Sepragel®) has been offered as an anti-adhesion in surgery (126), but has not been used in tissue regeneration due to its poor capacity to support cell growth. Cells from eight established cell lines originating from fibroblasts, epithelial or endothelial cells, chondrocytes, tumor cells, and stem cells were seeded on the hylan B® gel surface. Though all but the endothelial-origin cells attached to the gel, only the L929 fibroblasts and stem cells multiplied (127). To enhance cell attachment, coating with PLL (128), or the addition of matrix factors such as collagen type I, laminin, or fibronectin (129), was required.

2. Diepoxide-Crosslinked Hyaluronan

The methodology of using diepoxides to prepare hyaluronan gel was reported by Laurent (48) in the 1960s. Similar to the Hylans, this gel is mainly used in dermal augmentation in Europe under the tradename Restlane® (Q-Med Inc., Sweden). FDA approval for use in the United States is pending. Additionally, several reports have appeared for the use of this gel for drug delivery (130-132) and wound healing therapies after modification by the cell attachment domain sequence Arg-Gly-Asp (RGD) (133,134).

3. Auto-Crosslinked Hyaluronan

The internally crosslinked ACP™ was developed by Fidia Advanced Polymers, Inc. based on the intra-molecular esterfication of the carboxyl groups and hydroxyl groups of hyaluronan. This biomaterial, due to its highly viscous gel formulation, allows application even on vertical areas between adjacent organs. It has been shown to have a longer residence time than native hyaluronan, being slowly reabsorbed from peritioneal surfaces over 6-7 days. Thus, the potential of ACP™ gel as an anti-adhesive barrier after abdominal and gynecological surgery has been investigated (135,136).

In addition, porous ACP™ could behave as a scaffold for tissue regeneration. ACP was investigated as a cell delivery vehicle for the repair of bone and cartilage, but relatively poor cell growth was observed (99,137). The addition of BMP-2, as a bone enhancing osteogenic substrate, to ACP™ clearly increased both the volume and density in critically sized bony defects in a dog model (138). The cell affinity of ACP™ will need to be improved for the further use of ACP™ for tissue regeneration. Recently, studies have indicated that fibronectin coating can enhance the tissue regeneration properties of ACP™ (139).

4. Photo-Crosslinking Hyaluronan

Methacrylate and methacryamide derivatized hyaluronans were synthesized, and then used for hydrogel fabrication under UV or visible light with an initiator (18, 47,51-53). The potential uses for these in situ photopolymerized hydrogels include prevention of adhesions and tissue regeneration. The poor cell attachment of these in situ photopolymerized hydrogels can be significantly enhanced by RGD modification (18).

5. Carbodiimide-Mediated Crosslinking

Hyaluronan was claimed to be directly crosslinked by a water-soluble carbodiimide through intra-esterfication of the carboxyl groups and hydroxyl groups, but supporting physicochemical data was lacking (140). Recently, flexible, mucoadhesive, biocompatible controlled release films of hyaluronan "crosslinked" with 2 mM EDCI and containing 10% glycerol and 5% paclitaxel were synthesized (141) and examined for prevention of post-surgical adhesion formation. However, the materials degraded quickly, most likely due to the very low incidence of crosslinking relative to N-acylurea formation as described above.

The O-acyl to N-acylurea rearrangement could be used to advantage in a novel crosslinking approach. Thus, using biscarbodiimide coupling agents in the absence of added nucleophiles, hyaluronan was crosslinked by the joining two carboxyl groups in an inter- or intramolecular fashion. In most cases, a water-miscible organic cosolvent was used due to the poor aqueous solubility of biscarbodiimide agents (4). The water-insoluble gels, films and sponges of hyaluronic acid with biscarbodiimide were produced, and may be used as surgical aids to prevent adhesions (INCERT®, Anika Therapeutics Inc.).

As described above, the use of bishydrazide, trishydrazide, or polyvalent hydrazide compounds as nucleophiles in combination with EDCI has become the most versatile methodology for crosslinking hyaluronan. Hydrogels with physico-chemical properties ranging from soft-pourable gels to more mechanically rigid and brittle gels were created by varying reaction conditions and the molar ratios of the reagents involved (5,25). Applications of this chemistry were licensed in the United States by Clear Solutions Biotech, Inc. (Stony Brook, NY).

An alternative strategy to produce hyaluronan gels involves the synthesis of hyaluronan derivatives with unique properties followed by crosslinking with commercially available homobifunctional reagents. Through carbodiimide-mediated chemistry as described above, hyaluronan derivatives with primary amino or hydrazide functionalities were synthesized. The derivatives were then further crosslinked by glutaraldehyde, polyethylene glycol bis(succinimidyl propionate), and polyethylene glycol dialdehyde to produce hydrogels for drug delivery or tissue engineering (5,20-22,142,143). While biocompatible materials could be made, the crosslinking conditions per se were cytotoxic, and precluded the development of cell-seeded materials for in situ crosslinking approaches.

To work towards a cytocompatible crosslinking strategy, new chemistry was required. Thus, at the University of Utah, thiolated hyaluronan derivatives were synthesized using EDCI-hydrazide chemistry (33). Murine L-929 fibroblasts in cell culture medium were added to a solution of thiolated hyaluronan, and air oxidation of the thiols produced disulfide-crosslinked hyaluronan hydrogels. The encapsulated cells remained viable and proliferated slowly in vitro. However, the poor cell attachment and slow crosslinking kinetics limited the further use of this hydrogel for tissue regeneration (33). We next investigated the addition of a covalently linked ECM protein to create a covalently crosslinked biomimic of the ECM. Using thiol-modified gelatin, a solubilized collagen derivative, the cell attachment of this hyaluronan hydrogel was significantly improved. Thus, co-crosslinking of a 50:50 mixture of thiol-modified HA and thiol-modified gelatin produced a hydrogel that could be lyophilized to give a macroporous sponge that constituted a synthetic, covalent ECM mimetic. Both hydrogel films and sponges were successfully employed for cell culture in vitro and for the growth of new fibrous tissue in vivo (34,35). However, while these materials could be pre-seeded with cells prior to air-induced crosslinking, surgical implantation of the seeded scaffold was still required.

To proceed towards an injectable, cytocompatible gel for in vivo cell delivery, we sought to accelerate the gelation step. Conjugate addition of a thiol to a thiol acceptor was selected as the desired chemistry, and several different homobifunctional thiol acceptors based on the PEG chain were evaluated. PEG diacrylate (PEGDA) emerged as the optimal choice for crosslinking thiol-modified hyaluronan, both for cytocompatibility and speed of crosslinking—less than 10 min was required for gelation. This approach satisfied the key requirements that we had envisioned for injectable in vivo tissue engineering applications (36). Preliminary results showed that T31 human tracheal scar fibroblasts maintained the same phenotype and proliferated in this hydrogel 10-fold during 4 weeks culture in vitro. In addition, these human fibroblasts also proliferated and secreted ECM in vivo when implanted subcutaneously into nude mice. However, crosslinked hyaluronan alone was too hydrophilic and polyanionic and inhibited cell attachment. This limitation was overcome by covalent attachment of RGD-containing peptides to the hydrogel (Fig. 3). The attachment, spreading, and proliferation of human and murine fibroblasts were significantly enhanced on the surface of PEGDA-crosslinked thiol-modified hydrogels. Moreover, essentially perfect fibrous tissues were formed in vivo by injecting the viscous, gelling mixture of NIH 3T3 fibroblasts, thiol-modified HA, and PEGDA subcutaneously into the flanks of nude mice (37).

CRGDS

CRGDS

Hylan Crosslink

HA-DTPH-RGD-PEGDA

Figure 3 CRGDS peptide modified HA-DTPH hydrogel crosslinked by PEGDA.

HA-DTPH-RGD-PEGDA

Figure 3 CRGDS peptide modified HA-DTPH hydrogel crosslinked by PEGDA.

6. Glutaraldehyde-Crosslinked Hyaluronan

Hyaluronan was crosslinked in glutaraldehyde aqueous solution and then the glutaraldehyde-hyaluronan adduct was resurfaced using a PLL coating to improve the biocompatibility and fibroblast attachment (144). The chemistry of the glutaraldhyde-hyaluronan adduct is that of a hemiacetal and is inherently hydrolytically unstable. Partial stabilization can occur with a polycationic polyamine such as PLL, but the linkages are still hydrolytically labile. In any case, residual aldehyde functionality from monovalent coupling rather than bivalent linkages can be quite cytotoxic and cause an inflammatory response. Recently, the use of glutaraldehyde in vapor phase as a crosslinking agent was reported to improve gel biocompatibility (145).

7. Multiple-Component Crosslinked Hyaluronan

Hyaluronan derivatives can be prepared via three- or four-component reactions known as the Passerini reaction and Ugi reaction. While this chemistry is extremely powerful and very versatile, only very limited information concerning the modification of hyaluronan by this chemistry is available (146). The removal of by-products, characterization of the linkages, and immobilization of partial reaction products remain concerns to be addressed before the potential of this chemistry for the fabrication of hyaluronan biomaterials can be realized.

8. Hyaluronan-Grafted Copolymers

Three new kinds of hyaluronan-grafted copolymer were described above, and each has been investigated for a specific application. Hyaluronan-grafted PEG was explored as a drug delivery vehicle (26). The temperature-sensitive hyaluronan-gra/t-poly(N-isopropylacrylamide) (61) was developed for injectable use, and the PLL-gra/t-hyaluronan has been studied for gene delivery (58).

9. Composites

Hyaluronan has been blended with other materials in order to produce interpenetrating molecular networks with good physicochemical, mechanical, and biocompatible properties. Several of these composites are described below.

a. Hyaluronan-Liposome Composites (147-149).

Unmodified hyaluronan has been incorporated into liposomes, and the solution of hyaluronan and liposomes was developed as a topically administered pharmaceutical agent. This composite was found to effectively treat skin disorders while minimizing systemic circulation. Bioadhesive liposomes, in which hyaluronan is the surface-anchored bioadhesive ligand on a liposome surface, were prepared by the pre-activation of hyaluronan with a carbodiimide. This activated form was then added to a suspension of multi-lamellar liposomes consisting of phosphatidylcholine, phosphatidylethanolamine, and cholesterol. In principle, a hyaluronan-coated liposome functionally resembles the PEG-coated "stealth" liposomes. Liposomes that avoid detection have been investigated for their ability to act as site-adherent and sustained-release carriers of epidermal growth factor for the topical therapy of wounds and burns.

b. Hyaluronan-Polysaccharide Composites.

Composites of chitosan-hyaluronan and alginate-hyaluronan composites based on physical interactions were prepared for use in drug delivery and tissue regeneration (60,108-110,117,118). This was described in detail above.

High-viscosity solutions of hyaluronan have been used in combination with basic fibroblast growth factor (FGF-2) as an injectable formulation for the treatment of bone fractures (150). The combination of hyaluronan and heparin has the potential to provide an even more efficient system for the sustained delivery of heparin-binding growth factors such as FGF-2. Therefore, polymeric gels of hyaluronan and heparin were synthesized, and the stability and activity of the released FGF-2 increased significantly (57).

Hyaluronan and carboxymethylcellulose (CMC), followed by carbodiimide-mediated "crosslinking" with lysine produces a bioresorbable membrane (Seprafilmw, Genzyme Corporation). The crosslinking chemistry most likely results in a material with N-acylurea linkages that confer electrostatic interactions as well as some covalent linkages. This membrane was approved by the FDA in 1996 for use in patients undergoing abdominal or pelvic laparotomy to reduce the incidence, extent and severity of postoperative adhesion (151). However, Seprafilm® is reported to suffer from handling difficulties—most likely due to the low percentage of covalent crosslinks—hampering its acceptance by surgeons for many applications. The same technology has been expanded to reduce the development of adhesions to synthetic non-absorbable mesh, such as hernia repair products. Seprameshw is a composite of polypropylene mesh and a form of the Seprafilm® membrane. This composite reduces the development of adhesions to the surface of the hernia repair (152).

c. Hyaluronan-Collagen Composites.

Hyaluronan plays a prominent role in preventing scar formation during fetal wound healing. The in vivo evidence showed that application of hyaluronan reduced scar formation (153) and prevented joint contractures (154). The combination of hyaluronan and collagen may provide a suitable scaffold as a non-contractible biomaterial for skin regeneration. To prolong the persistence of hyaluronan in collagen fibrillar matrix (CFM), and simulate the lasting effects of hyaluronan in fetal wound healing, hyaluronan was crosslinked to collagen via periodate modification or CNBr-activation. The inhibition of CFM contraction by fibroblast was minimized for at least 7 days (155). Similar results were also observed in crosslinked CNBr-activated hyaluronan-collagen matrices (55). Biochemical and cytological analysis of these matrices suggested that hyaluronan strengthened the collagen fibrils and blocked direct communication between fibroblasts and the collagen fibrils.

Recently, a porous scaffold containing collagen and hyaluronan was fabricated by freezing-drying of the coagulates and subsequently crosslinking with EDCI to improve the mechanical stability of the composite matrix (156). The in vivo tests reported suggest that this matrix has promise for dermal tissue restoration (157).

Hyaluronan has been employed extensively in the treatment of osteoarthritis patients (158). However, controversial studies have been documented on the effects of hyaluronan on glycosaminoglycan synthesis by chondrocytes. Depending on molecular weight and biological source, hyaluronan has been reported to elicit inhibitory (159-161) as well as no effects (162,163) on cultured chondrocytes in vitro. However, in a 3D culture (collagen gel), hyaluronan enhanced chondrocyte proliferation as well as synthesis of both glycosaminoglycans and type II collagen, suggesting a benefit for repair of osteochondral defects (164).

A new osteoconductive collagen-hyaluronate matrix was synthesized for bone regeneration by crosslinking collagen fibers with peroxide-modified hyaluronate bearing active formyl groups (56). The resulting matrix was a 3D scaffold consisting of interconnected pores with an average size of 40 mm and a high pore volume/surface area ratio. The fraction of covalently bound hyaluronate in the matrix ranged from 5 to 25 wt%. When implanted in cranial defects in rats, the matrix demonstrated good biocompatibility and exhibited greater osteo-conductive potential than matrices composed of either crosslinked collagen or crosslinked hyaluronate alone. The incorporation of hydroxyapatite may further improve the osteoconductivity in hyaluronan-collagen composites (165).

Composite materials consisting of hyaluronan and type I collagen also have been prepared by complexing both components into a coagulate followed by crosslinking with either glyoxal or starch dialdehyde (166). However, polyelec-trolyte complex of hyaluronan and soluble type II collagen are readily formed and will precipitate from aqueous solutions. A new methodology was thus developed to make hyaluronan-type II collagen scaffold. In this method, 0.4 M NaCl was added to diminish the polymer-polymer electrostatic interactions. Next, EDCI was added to create the amide crosslinks between hyaluronan and collagen (167). This lyophilized and porous scaffold may be beneficial for cartilage regeneration, because hyaluronan and type II are the main components of cartilage.

Gelatin, the denatured form of collagen, is an excellent substrate for cell attachment, proliferation and differentiation. For instance, the physical incorporation of gelatin into esterified hyaluronan facilitated the osteochondral differentiation of culture-expanded, bone marrow-derived mesenchymal progenitor cell (168). The disadvantages of using gelatin as a scaffold material for tissue repair are its low biomechanical stiffness and rapid biodegradation. The conjugation of hyaluronan to gelatin matrices to prepare a covalent mimic of the ECM retarded degradation both in vitro and in vivo, and improved the biomechanical strength at the same time, which makes it useful as a cell delivery vehicle for tissue regeneration (34,35). Dipping an uncrosslinked hyaluronan-gelatin sponge into 90% (w/v) acetone-water containing EDCI resulted in a porous matrix, which could be used for either wound dressing or as a scaffold for tissue engineering (169). The incorporation of an antibiotic (silver sulfadiazine) in the sponge enhanced wound healing in a rat model (169).

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