The clinical outcomes of new hyaluronan nasal dressing: a prospective, randomized, controlled study.

BACKGROUND: Poor postoperative wound healing after endoscopic sinus surgery (ESS) remains a significant problem. This study evaluates the efficacy and safety of a new absorbable hyaluronan hydrogel. METHODS: A prospective, randomized, controlled trial was conducted. Fifty-five patients with bilateral ESS were recruited and randomized to receive absorbable hyaluronan hydrogel in one side as treated and the opposite side without absorbable hyaluronan hydrogel as control. Clinical outcome measures were assessed at postoperative 1, 2, 4, 8, and 12 weeks. RESULTS: Fifty-four patients completed the study. Overall, absorbable hyaluronan hydrogel significantly promotes the reepithelization process and reduces the presence of obstructing synechia, nonobstructing synechia, edema, crust, and mild mucopurulent drainage (all p ≤ 0.0002). At all postoperative follow-up visits, the promotion in reepithelization is statistically significant at 2, 4, and 8 weeks, and the reductions in the presence of nonobstructing synechia, edema, crust, and mild mucopurulent drainage are all statistically significant except for the presence of crust at 12 weeks and mild mucopurulent drainage at 1 and 12 weeks. Although the presence of obstructing synechia at each follow-up visit between groups does not reach statistical significance, the incidence ranges from 5.56 to 12.96% in the control group and from 0 to 3.70% in the treated group. No adverse event related to treatment was observed. CONCLUSION: In this clinical study, data analyses suggest that this new absorbable hyaluronan hydrogel, as nasal dressing/packing after ESS is safe and promotes the postoperative reepithelization process and reduces the presence of synechia, edema, crust, and mild mucopurulent drainage.

The evaluation of two new hyaluronan hydrogels as nasal dressing in the rabbit maxillary sinus.

BACKGROUND: The postoperative scaring, ostial stenosis, and adhesions after functional endoscopic sinus surgery for chronic rhinosinusitis remains a major problem. This study was designed to evaluate two new hyaluronan (HA) hydrogels for neo-ostium antistenosis and promoting wound healing in a rabbit maxillary sinus model. METHODS: The anterior wall of the maxillary sinus of 48 rabbits was removed to create a 4-mm circumferential wound both on the nasal and on the sinus sides. A rapid-gelling HA hydrogel or preformed HA hydrogel was filled randomly into the right or left sinus, while the opposite sinus served as blank control or was treated with Merogel (Medtronic Xomed Surgical Products, Jacksonville, FL) as control. The neo-ostium diameter and histological scores were evaluated and analyzed postoperatively. RESULTS: The neo-ostium diameter in the rapid-gelling HA hydrogel-treated side was significantly larger than that in the blank control side with a mean difference of 1.46 ± 0.99 mm (p = 0.03), 1.30 ± 0.61 mm (p = 0.0087), and 1.60 ± 0.25 mm (p = 0.00015) at 2, 3, and 4 weeks, respectively; the neo-ostium diameter in the preformed HA hydrogel-treated side at 2 weeks was significantly larger than that in the blank control side or Merogel control side with a mean difference of 1.46 ± 0.76 mm (p = 0.002) or 0.54 ± 0.36 mm (p = 0.007), respectively. The preformed HA hydrogel-treated side showed better histology scores at 2 weeks in heterophils, fibrosis, and osteogenesis than the blank control, and the chronic inflammation (lymphocyte/plasmacyte infiltration) was not prevalent. CONCLUSION: During the postoperative follow-up period both of the two HA hydrogels significantly prevented neo-ostium stenosis and the preformed HA hydrogel promoted wound healing.

Effect of gelatin on heparin regulation of cytokine release from hyaluronan-based hydrogels.

The hypothesis that incorporation of small amounts (0.3% w/w) of modified heparin in thiol-modified hyaluronan or HA and gelatin hydrogels would regulate release of cytokine growth factors (GFs) from those gels has been investigated in vitro. In addition, the physiologic response to gel implantation has been evaluated in vivo. Tests were performed with 6 GFs: basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor, platelet-derived growth factor-AA (PDGF), and transforming growth factor-beta 1. Release profiles for all 6 over several weeks were well fit by first order exponential kinetics (R(2) > 0.9 for all cases). The most remarkable result of the experiment was a dramatic variation in the total mass ultimately released, which varied from as much as 90.2% of the initial load for bFGF to as little as 1.8% for PDGF, a 45-fold difference. Furthermore, gels containing either VEGF of Ang-1 produced twice the vascularization response in vivo as gels not containing a growth factor. Thus, those GFs maintained strong physiologic effectiveness.

Prevention of peritendinous adhesions using a hyaluronan-derived hydrogel film following partial-thickness flexor tendon injury.

Peritendinous adhesions are an important complication of flexor tendon injury. Three hyaluronan (HA)-derived biomaterials were evaluated for the reduction of peritendinous adhesions following partial-thickness tendon injury in rabbits. Rabbits (n = 24) were divided into three groups (n = 8 per group), which were used for gross evaluation, histologic assessment, or biomechanical testing. The fourth and third toes from both hindpaws of each rabbit were randomly assigned to one of four treatments: (i) untreated control, (ii) Seprafilm, (iii) Carbylan-SX in situ crosslinked hydrogel, and (iv) preformed Carbylan-SX film. Rabbits were sacrificed at 3 weeks postsurgery and evaluated anatomically, histologically, and mechanically. All materials used reduced adhesions relative to untreated controls for all three evaluations. Both the gross anatomic and histologic results revealed that Carbylan-SX film was statistically superior to Seprafilm and Carbylan-SX gel in preventing tendon adhesion formation. In biomechanical tests, the Carbylan-SX film-treated hindpaws required the least force to pull the tendon from the sheath. This force was statistically indistinguishable from that required to extrude an unoperated tendon (n = 8). Carbylan-SX gel was less effective than Carbylan-SX film but superior to Seprafilm for all evaluations. A crosslinked HA-derived film promoted healing of a flexor tendon injury without the formation of fibrosis at 3 weeks postoperatively.

Structural variations in a single hyaluronan derivative significantly alter wound-healing effects in the rabbit maxillary sinus.

BACKGROUND: Biomaterials based on hyaluronan (HA) are currently used after sinus surgery but have not been found to decrease scarring or enhance wound healing. Chemical composition of these modified HA molecules may impact their biological and clinical effects. OBJECTIVE: To analyze chemical variations of a single crosslinked HA-based hydrogel, chemically modified thiolated HA (CMHA-SX). METHODS: Four different components of the hydrogel composition were altered, yielding 54 variations. These were subjected to biomechanical testing, and then potential clinically relevant variations were further tested for swelling and degradation characteristics. Using a rabbit maxillary sinus model, the ability of the material variations to stent a neo-ostium was tested. Histologic measures were also assessed. Biomechanical and biological effects were correlated. RESULTS: Minor compositional changes had profound biomechanical and biological effects. Swelling and rate of enzymatic degradation were closely related. CMHA-SX hydrogels that were the most effective stents in maintaining the neo-ostium also generated the lowest level of acute inflammation, as determined by histology. CONCLUSIONS: Chemical composition has a significant impact on the clinical potential of modified HA materials. Histocompatibility appears to most significantly affect ostium preservation. SIGNIFICANCE: Different CMHA-SX hydrogels perform differently in vivo, even when the chemical compositions are quite similar. Objective prospective testing of modified HA materials should precede their clinical use in sinus surgery.

Tumor engineering: orthotopic cancer models in mice using cell-loaded, injectable, cross-linked hyaluronan-derived hydrogels.

Current cancer xenograft models used to evaluate new anticancer therapies are limited to "good take" cell lines, fail to mimic normal human disease, and poorly predict clinical outcomes. We now describe the use of an injectable, in situ cross-linkable synthetic extracellular matrix (sECM) to deliver and grow cancer cells in vivo. The hyaluronan (HA)-derived sECMs were seeded with breast, colon, and ovarian cancer cells prior to gelation, and then injected subcutaneously into mammary fat pads, subserosally in colons, and intracapsularly in ovaries, respectively. Two cell lines were used for each type of cancer, and results were compared with orthotopic injection of cells in serum-free medium. At 4 weeks postinjection, four parameters were measured: (i) incidence and size of cancer at the injection site, (ii) vascularization or necrosis of new cancer tissue, (iii) cancer seeding in adjacent tissues, and (iv) metastasis to lymph nodes and other vital organs. In addition, the activation of the phosphoinositide 3-kinase (PI 3-K) signaling pathway was analyzed immunohistochemically. Overall, orthotopic delivery of cancer cells in sECM hydrogels showed clear advantages: (i) increased incidence of cancer formation and reduced variability in tumor size, (ii) enhanced growth of organ-specific cancers with good tumor-tissue integration, (iii) improved vascularization and reduced necrosis within the tumor, (iv) reduced cancer seeding on adjacent tissues, and (v) better general health of animals. Thus, engineered tumors represent an improved approach to traditional tumor xenografts, and facilitate studies in cancer biology, invasion and metastasis, as well as the investigation of new therapeutic and diagnostic protocols.

Influence of cross-linked hyaluronic acid hydrogels on neurite outgrowth and recovery from spinal cord injury.

OBJECT: Therapies that use bioactive materials as replacement extracellular matrices may hold the potential to mitigate the inhibition of regeneration observed after central nervous system trauma. Hyaluronic acid (HA), a nonsulfated glycosaminoglycan ubiquitous in all tissues, was investigated as a potential neural tissue engineering matrix. METHODS: Chick dorsal root ganglia were cultured in 3D hydrogel matrices composed of cross-linked thiol-modified HA or fibrin. Samples were cultured and images were acquired at 48-, 60-, and 192-hour time points. Images of all samples were analyzed at 48 hours of incubation to quantify the extent of neurite growth. Cultures in crosslinked thiolated HA exhibited more than a 50% increase in neurite length compared with fibrin samples. Furthermore, cross-linked thiolated HA supported neurites for the entire duration of the culture period, whereas fibrin cultures exhibited collapsed and degenerating extensions beyond 60 hours. Two concentrations of the thiolated HA (0.5 and 1%) were then placed at the site of a complete thoracic spinal cord transection in rats. The ability of the polymer to promote regeneration was tested using motor evoked potentials, retrograde axonal labeling, and behavioral assessments. There were no differences in any of the parameters between rats treated with the polymer and controls. CONCLUSIONS: The use of a cross-linked HA scaffold promoted robust neurite outgrowth. Although there was no benefit from the polymer in a rodent spinal cord injury model, the findings in this study represent an early step in the development of semisynthetic extracellular matrice scaffolds for the treatment of neuronal injury.

Release of basic fibroblast growth factor from a crosslinked glycosaminoglycan hydrogel promotes wound healing.

We describe synthetic extracellular matrix (sECM) hydrogel films composed of co-crosslinked thiolated derivatives of chondroitin 6-sulfate (CS) and heparin (HP) for controlled-release delivery of basic fibroblast growth factor (bFGF) to full-thickness wounds in genetically diabetic (db/db) mice. In this model for chronic wound repair, full-thickness wounds were treated with CS, CS-bFGF, or CS-HP-bFGF films. At 2 and 4 weeks postinjury, wound closure and formation of the new epidermis and dermis were determined. Both CS and CS-HP hydrogel films accelerated wound repair, even without bFGF. Addition of bFGF to CS films showed partial dose-dependent acceleration of wound repair. Importantly, addition of bFGF to co-crosslinked CS-HP sECM films showed a dramatic bFGF dose-dependent acceleration of wound healing, as well as improved dermis formation and vascularization. Compared with 27% wound closure in 2 weeks in the controls, 89% wound closure was observed for mice treated with the CS-HP-bFGF films. The synthetic CS-HP sECM films mimic the chemistry and biology of heparan sulfate proteoglycans, and may have clinical potential for topical delivery of growth factors to patients with compromised wound healing.

Reduced postoperative intra-abdominal adhesions using Carbylan-SX, a semisynthetic glycosaminoglycan hydrogel.

OBJECTIVE: To compare the efficacy of crosslinked Carbylan-SX (Carbylan BioSurgery, Inc., Palo Alto, CA) hydrogel films and sprayable gels as physical barriers in reducing postoperative intra-abdominal adhesions in the rat cecum-abdominal wall and rat uterine horn models. DESIGN: Pre-formed crosslinked Carbylan-SX films and sprayable in situ crosslinkable Carbylan-SX gels were evaluated in rat cecum-abdominal wall and rat uterine horn models and compared with commercially available and clinically used Seprafilm. SETTING: University animal research facility. ANIMALS: Female Wistar rats. INTERVENTION(S): Abrasions were made with the foot-pedal-operated Flex-shaft (Dremel, Racine, WI) on both the cecum and abdominal wall (each area 10 mm in diameter) in female rats as one model and on both uterine horns (3 x 10 mm) in female rats as the other model. In each of the two adhesion models, four groups were assigned with eight rats in each group: (1) untreated control, (2) treated with Seprafilm (Genzyme Corporation, Cambridge, MA), (3) treated with preformed Carbylan-SX hydrogel films, and (4) treated with sprayable Carbylan-SX gel. MAIN OUTCOME MEASURE(S): Extent and severity of postoperative adhesions between the cecum and the abdominal wall in rat cecum-abdominal wall model and between the uterine horns in rat uterine horn model. RESULT(S): The Carbylan-SX film and the Carbylan-SX sprayable gel led to fewer adhesions than Seprafilm in both rat adhesion models. Interestingly, a single physical form was not optimal for both models: the Carbylan film was more efficacious in the rat uterine horn model, whereas Carbylan gel gave the best results in the rat cecum-abdominal wall model. CONCLUSION(S): Both Carbylan-SX film and gel were efficacious in reducing postoperative intra-abdominal adhesion formation in rat cecum-abdominal wall and uterine horn models.

Crosslinked hydrogels for tympanic membrane repair.

PROBLEM: To provide a less expensive and more convenient protocol for the treatment of tympanic membrane perforations (TMPs). METHODS: Several materials were prepared and compared for TMP repair including Carbylan-SX, Gelatin-DTPH-PEGDA (GX), Carbylan-S/Gelatin-DTPH (Carbylan-GSX) (injectable and sponge), Gelfoam, Epifilm, and crosslinked thiolated chondroitin sulfate (CS-DTPH-PEGDA [CS-SX]). Hartley pigmented guinea pigs (Elm Hill) underwent bilateral myringotomy with 1 ear left as a control and the other treated with one of the previously mentioned materials. RESULTS: Carbylan-GSX (injectable and sponge), Gelfoam with saline, and CS-SX had the shortest time for TMP closure. Epifilm, Carbylan, and gelatin preparations resulted in closure rates similar to controls. CS-SX showed a marked inflammatory reaction compared with controls and other materials based on neutrophil, lymphocyte, epitheloid counts, and degree of fibrosis. CONCLUSIONS: This study shows the validity of Carbylan-GSX compared with Gelfoam as a material to promote TMP closure in an acute TMP guinea pig model.

Stimulation of in vivo angiogenesis using dual growth factor-loaded crosslinked glycosaminoglycan hydrogels.

Crosslinked, chemically modified hyaluronan (HA) hydrogels pre-loaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), were employed to elicit new microvessel growth in vivo, in both the presence and absence of heparin (Hp) in the gels. HA hydrogel film samples were surgically implanted in the ear pinnae of mice, and the ears were harvested at 7 or 14 days post-implantation. Analysis of neovascularization showed that each of the treatment groups receiving an implant, except for HA/Hp at day 14, demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p<0.015). Treatment groups receiving either Ang-1 alone, or aqueous co-delivery of both Ang-1 and VEGF, were statistically unchanged with time. In contrast, film delivery of both growth factors produced continuing increases in vascularization from day 7 to day 14 in the absence of Hp, but decreases in its presence. However, presentation of both VEGF and Ang-1 in crosslinked HA gels containing Hp generated intact microvessel beds with well-defined borders. The HA hydrogels containing Ang-1+VEGF produced the greatest angiogenic response of any treatment group tested at day 14 (NI=7.44 in the absence of Hp and 4.67 in its presence, where NI is a neovascularization index). Even in the presence of Hp, this had 29% greater vessel density than the next largest treatment group receiving HA/Hp+VEGF (NI=3.61, p=0.04). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and Ang-1 from crosslinked HA films.

Cross-linked hydrogels for middle ear packing.

OBJECTIVE: To develop an ideal supportive packing material for ossiculoplasty, tympanoplasty, or other otologic procedures. MATERIALS AND METHODS: Several materials, namely, Carbylan-SX (P-C; Sentrx Surgical, Inc., Salt Lake City, UT), Gelfoam (P-GF; Pharmacia & Upjohn, Kalamazoo, MI), and Merogel (P-MG; Medtronics, Inc., Minneapolis, MN), were prepared and then placed into a Hartley guinea pig's (Elm Hill, Chelmsford, MA) middle ear cavities through a large myringotomy incision. The contralateral ear underwent a large myringotomy without packing material being placed. Preoperative and posteroperative auditory brainstem response studies were performed using Intelligent Hearing system software. The animals were examined weekly. Two weeks after packing placement, the animals were killed, and the temporal bones were harvested. Whole temporal bone sectioning was performed to analyze the presence of implant, surrounding inflammation, presence of osteoneogenesis and fibrosis, or adhesions. RESULTS: All the materials, except the P-MG, were easy to place into the middle ear cavity. The P-MG contains woven strands that are difficult to trim into the small sizes needed for placement. The P-MG group had a smaller average amount of implant present compared with the other groups at 2 weeks. The degree of osteoneogenesis was similar among the P-GF, P-C, and P-MG groups. The P-MG and P-C groups contained the lowest amount of fibrosis between the implant and surrounding middle ear structures. CONCLUSION: This study demonstrates promising results with P-C as a potential supportive packing material for otologic procedures. P-C compares favorably with P-MG and P-GF in a guinea pig model with respect to ease of placement and amount of fibrosis.

Vocal fold tissue repair in vivo using a synthetic extracellular matrix.

Chemically modified hyaluronic acid (HA)-gelatin hydrogels have been documented to support attachment, growth, and proliferation of fibroblasts in vitro and to facilitate repair and engineering of tissues in vivo. The objective of this study was to determine the optimal composition of a synthetic extracellular matrix (sECM) that would promote wound repair and induce tissue regeneration in a rabbit vocal fold wound healing model. The sECM was formed using a thiol-modified semisynthetic glycosaminoglycan (GAG) derived of HA (Carbylan-SX) mixed with a thiolated gelatin derivative, co-cross-linked with poly(ethylene glycol) diacrylate to form Carbylan-GSX. Forty rabbits underwent vocal fold biopsy bilaterally. Rabbits were treated with Carbylan-SX, which lacks gelatin, or with Carbylan-GSX with different gelatin concentrations (2.5%, 5%, 10%, and 20%) via unilateral injection of the vocal fold at the time of biopsy. Saline was injected in the contralateral vocal fold as a control. Three weeks after biopsy and injection, animals were euthanized and mRNA levels of procollagen type 1, fibronectin, transforming growth factor beta 1 (TGF-beta1), fibromodulin, HA synthase 2, hyaluronidase 2, and tissue biomechanics were evaluated. Hyaluronidase mRNA levels were found to be significantly elevated in for Carbylan-GSX 20% w/w gelatin compared to controls. Both Carbylan-SX and Carbylan-GSX significantly improved tissue elasticity and viscosity. Carbylan-GSX containing 5% w/w gelatin showed the most promise as a scaffold material for vocal fold tissue regeneration.

Synthesis and evaluation of injectable, in situ crosslinkable synthetic extracellular matrices for tissue engineering.

Simple and effective biocompatible materials that mimic the natural extracellular matrix (ECM) were developed for a variety of uses in regenerative medicine. These synthetic ECMs (sECMs) were designed to recapitulate the minimal composition required to obtain functional ECMs. The sECM components are crosslinkable in situ, and may be seeded with cells prior to injection in vivo, without compromising either the cells or the recipient tissues. Several sECM compositions were evaluated to establish which formulation would be most beneficial for cell growth and tissue remodeling. Three natural ECM macromonomeric building blocks were employed: hyaluronan (HA), chondroitin sulfate (CS), and gelatin (Gtn). The carboxyl-rich glycosaminoglycans and Gtn were each chemically modified to give the corresponding thiolated dithiopropionylhydrazide (DTPH) derivatives (CS-DTPH, HA-DTPH, and Gtn-DTPH). Different compositions of CS-Gtn and HA-Gtn hydrogels were fabricated by crosslinking the thiolated biomacromonomers with polyethylene glycol diacrylate. Each sECM had high water content (>96%), biologically suitable mechanical properties, and a useful gelation time ( approximately 2-6 min). The bioerosion rates for the sECMs were determined, and a given composition could be selected to meet the requirements of a given clinical application. Both the HA-Gtn and CS-Gtn sECM hydrogels supported cell growth and proliferation with cultured murine fibroblasts in vitro. Moreover, subcutaneous injection of a suspension of murine fibroblasts in each of the two sECM hydrogels into nude mice in vivo resulted in the formation of viable and uniform soft tissue in vivo.

Cross-linked hyaluronan-coated stents in the prevention of airway stenosis.

OBJECTIVE: This project studies the use of airway stents coated with a cross-linked derivative of hyaluronan (HA) in a rabbit airway model of subglottic stenosis (SGS). STUDY DESIGN AND SETTING: An acute subglottic mucosal injury and airway stent placement design were used in a rabbit model. Thirty-six rabbits were randomized to 6 different study groups. Four groups had the subglottic mucosa denuded at the cricoid, and 2 groups received no injury. Airway stents coated with Carbylan-SX, a cross-linked derivative of HA, and controls were placed for 3 weeks. After sacrifice at 6 weeks, morphometric measurements of subglottic lumen were taken. RESULTS: In posttraumatic models, no significant differences were seen in airway area measures between groups (P = 0.86). In non-injury groups, a significant difference between Carbylan-SX versus non-HA-derivative-coated stents was seen (P = 0.05). CONCLUSION: In this model of acute subglottic mucosal injury, the HA-derivative-coated stent did not improve healing. However, in the absence of mucosal injury, the Carbylan-SX film-coated stent yielded significantly larger airway areas compared with a noncoated stent. SIGNIFICANCE: Stents or endotracheal tubes coated with a cross-linked derivative of HA may prevent stenosis in patients without airway injury but require long-term intubation or laryngotracheal stenting.

Molecular stenting with a crosslinked hyaluronan derivative inhibits collagen gel contraction.

Adult burn wounds, which lack hyaluronan (HA), often undergo excessive tissue remodeling and contraction. In contrast fetal wounds, which contain large amounts of HA, undergo remodeling that culminates in a scarless repair or regeneration. Therefore, adding a HA derivative to burn wounds would better mimic the fetal extracellular matrix and could reduce contraction. To test this hypothesis, we determined the effects of HA and its two derivatives on fibroblast-mediated, collagen gel contraction, an assay widely used to mimic in vivo wound contraction. Interestingly, high molecular weight HA (HMW HA) facilitated collagen gel contraction, whereas a thiol-functionalized derivative HA-DTPH weakly inhibited contraction. In contrast, polyethylene glycol diacrylate (PEGDA)-crosslinked HA-DTPH (HA-DTPH-PEGDA) strongly inhibited contraction in a concentration-dependent manner. Immunofluorescence staining of cellular actin showed that this inhibition was not owing to reduced cell attachment or spreading. Furthermore, the supernatant of contracted collagen-HMW HA gels contained greater amounts of HA than those found in the supernatant of collagen-HA-DTPH-PEGDA gels, suggesting that HMW HA facilitates contraction by effectively diffusing out of the collagen gels. Therefore, the results suggest that the crosslinking of HA-DTPH enhances the structural mechanics of collagen/HA-DTPH composites, which resists the fibroblast contractile forces and may, therefore, be able to reduce excessive wound contraction observed in pathological conditions.

Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF.

Controlled release of human vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) from hydrogels composed of chemically modified hyaluronan (HA) and gelatin (Gtn) was evaluated both in vitro and in vivo. We hypothesized that inclusion of small quantities of heparin (Hp) in these gels would regulate growth factor (GF) release over an extended period, while still maintaining the in vivo bioactivity of released GFs. To test this hypothesis, HA, Gtn, and Hp (15 kDa) were modified with thiol groups, then co-crosslinked with poly (ethylene glycol) diacrylate (PEGDA). Either VEGF or bFGF was incorporated into the gels before crosslinking with PEGDA. Release of these GFs in vitro could be sustained over 42 days by less than 1% Hp content, and was found to decrease monotonically with increasing Hp concentration. As little as 0.03% Hp in the gels reduced the released VEGF fraction from 30% to 21%, while 3% Hp reduced it to 19%. Since the minimum Hp concentration capable of effective controlled GF release in vitro was found to be 0.3% (w/w), this concentration was selected for subsequent in vivo experiments. To evaluate the bioactivity of released GFs in vivo, gel samples were implanted into the ear pinnas of Balb/c mice and the resulting neovascularization response measured. In the presence of Hp, vascularization was sustained over 28 days. GF release was more rapid in vitro from gels containing Gtn than from gels lacking Gtn, though unexpectedly, the in vivo neovascularization response to Gtn-containing gels was decreased. Nevertheless significant numbers of neovessels were generated. The ability to stimulate localized microvessel growth at controlled rates for extended times through the release of GFs from covalently linked, Hp-supplemented hydrogels will ultimately provide a powerful therapeutic tool.

Aminooxy pluronics: synthesis and preparation of glycosaminoglycan adducts.

Novel biomaterials have been prepared in which glycosaminoglycans (GAGs) are chemically modified to create amphiphilic multiblock copolymers that are able to adhere to hydrophobic surfaces and can self-assemble into cross-linker-free hydrogels. First, the triblock poly(ethylene oxide)-polypropylene oxide copolymers (Pluronics) were converted into the previously unknown aminooxy (AO) derivatives. Both mono-AO and bis-AO Pluronics (AOPs) were synthesized and fully characterized in order to prepare tetrablock and pentablock copolymers, respectively. Second, the AOPs were coupled to the uronic acid carboxylates of heparin (HP) and hyaluronic acid (HA) using carbodiimide chemistry in order to give the previously undescribed amidooxy GAG derivatives. The coupling chemistry was confirmed using a newly prepared fluorescent AO reagent. Third, AOP-heparin and AOP-fluorescently labeled heparin were shown to adsorb efficiently to polystyrene surfaces, as determined by IL-8 based ELISA and fluorescence measurements, respectively. Fourth, AOP-linked fluorescently labeled HA was shown to adsorb efficiently to plastic surfaces. Finally, three different AOPs were evaluated for self-assembling hydrogel formation by AOP-HA pentablock polymers. In short, AOP-GAG adducts are semisynthetic amphiphilic biomacromolecules that offer a range of valuable practical opportunities for surface modification, preparation of cross-linker-free hydrogels, and formation of self-assembling mimics of the extracellular matrix.