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.

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.

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.

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.

Dual growth factor-induced angiogenesis in vivo using hyaluronan hydrogel implants.

Crosslinked hyaluronan (HA) hydrogels preloaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), were employed to elicit new microvessel growth in vivo. As a major glycosaminoglycan (GAG) component of extracellular matrix (ECM), HA is an excellent biopolymeric building block for new biomimetic, biocompatible therapeutic materials. 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. Histologic analysis showed that each of the groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p<0.001). Treatment groups receiving either co-delivery of both KGF and VEGF, an HA hydrogel lacking a growth factor or HA hydrogels containing a single cytokine were statistically unchanged with time, whereas treatment with KGF alone produced continuing increases in vascularization from day 7 to day 14. Strikingly, presentation of both VEGF and KGF in crosslinked HA generated intact microvessel beds with well-defined borders. In addition, an additive response to co-delivery of both cytokines in the HA hydrogel was observed. The HA hydrogels containing KGF+VEGF produced the greatest angiogenic response of any treatment group tested (NI=5.4 at day 14, where NI is a neovascularization index). This was 33% greater vessel density than in the next largest treatment group, that received HA+KGF (NI=4.0, p<0.002). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and KGF from crosslinked HA films.

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.

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.

Electrospun three-dimensional hyaluronic acid nanofibrous scaffolds.

A three-dimensional (3D) hyaluronic acid (HA) nanofibrous scaffold was successfully fabricated to mimic the architecture of natural extracellular matrix (ECM) based on electrospinning. Thiolated HA derivative, 3,3'-dithiobis(propanoic dihydrazide)-modified HA (HA-DTPH), was synthesized and electrospun to form 3D nanofibrous scaffolds. In order to facilitate the fiber formation during electrospinning, Poly (ethylene oxide) (PEO) was added into the aqueous solution of HA-DTPH at an optimal weight ratio of 1:1. The electrospun HA-DTPH/PEO blend scaffold was subsequently cross-linked through poly (ethylene glycol)-diacrylate (PEGDA) mediated conjugate addition. PEO was then extracted in DI water to obtain an electrospun HA-DTPH nanofibrous scaffold. NIH 3T3 fibroblasts were seeded on fibronectin-adsorbed HA-DTPH nanofibrous scaffolds for 24h in vitro. Fluorescence microscopy and laser scanning confocal microscopy revealed that the 3T3 fibroblasts attached to the scaffold and spread, demonstrating an extended dendritic morphology within the scaffold, which suggests potential applications of HA-DTPH nanofibrous scaffolds in cell encapsulation and tissue regeneration.

Osteochondral defect repair with autologous bone marrow-derived mesenchymal stem cells in an injectable, in situ, cross-linked synthetic extracellular matrix.

A co-cross-linked synthetic extracellular matrix (sECM) composed of chemically modified hyaluronic acid and gelatin was used as a cell delivery vehicle for osteochondral defect repair in a rabbit model. A full-thickness defect was created in the patellar groove of the femoral articular cartilage in each of 2 rabbit joints, and 4 experimental groups were assigned (12 rabbits/group): untreated control, autologous mesenchymal stem cells (MSCs) only, sECM only, and MSCs + sECM. The sECM hydrogels were allowed to cross-link in the defect in situ. Rabbits were sacrificed at 4, 8, and 12 weeks post-surgery, and cartilage repair was evaluated and scored. In the controls, defects were filled with fibrous tissue. In the MSC-only group, hyaline-like cartilage filled the peripheral area of the defect, but the center was filled with fibrous tissue. In the sECM-only group, hyaline cartilage with zonal architecture filled the defect at 12 weeks, but an interface between repaired and adjacent host cartilage was evident. In the MSCs + sECM group, defects were completely filled with elastic, firm, translucent cartilage at 12 weeks and showed superior integration of the repair tissue with the normal cartilage. The sECM delivers and retains MSCs, and the injectable cell-seeded sECM could be delivered arthroscopically in the clinic.

Effect of a synthetic extracellular matrix on vocal fold lamina propria gene expression in early wound healing.

An improved understanding of molecular response in the vocal folds to a synthetic extracellular matrix (sECM) during early wound repair is essential for understanding functional improvement of the tissue and implementation of future tissue-engineering strategies. The present study used real-time reverse transcriptase polymerase chain reaction to measure transcript expression of selected markers (procollagen alpha 2 type I, fibronectin, fibromodulin, hyaluronan synthase 2, and hyaluronidase 2) in 20 rabbits that underwent vocal fold biopsy bilaterally. After the biopsy, Carbylan-GSX 5% was injected immediately into the left vocal fold, and saline was injected into the right vocal fold. Two unwounded normal rabbit larynges were also harvested. Animals were sacrificed at days 1, 3, 5, and 10 post-surgery. At days 1, 3, and 10, no significant differences were found between the Carbylan-GSX-treated and saline-treated groups. At day 5, significant differences in procollagen (p = 0.02), fibronectin (p = 0.02), and transforming growth factor beta1 (p = 0.02) between the Carbylan-GSX-treated and saline-treated groups were measured. The presence of a sECM in the wound bed during the early stages of repair amplified the normal rabbit vocal fold wound-healing response over a short period of time. This amplification provided an optimal environment for tissue regeneration and may lead to the recovery of the functional biomechanical properties of the vocal folds necessary for voice production.

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.

Fibronectin functional domains coupled to hyaluronan stimulate adult human dermal fibroblast responses critical for wound healing.

Fibronectin (FN) facilitates dermal fibroblast migration during normal wound healing. Proteolytic degradation of FN in chronic wounds hampers healing. Previously, three FN functional domains (FNfd) have been shown to be sufficient for optimal adult human dermal fibroblast migration. Here we report the development of an acellular hydrogel matrix comprised of the FNfds coupled to a hyaluronan (HA) backbone to stimulate wound repair. Employing Michael-type addition, the cysteine- tagged FNfds were first coupled to a homobifunctional PEG derivative. Thereafter, these PEG derivative FNfd solutions, containing bifunctional PEG-derivative crosslinker were coupled to thiol-modified HA (HA-DTPH) to obtain a crosslinked hydrogel matrix. When evaluated in vitro, these acellular hydrogels were completely cytocompatible. While spreading and proliferation of adult human dermal fibroblasts plateaued at higher FNfd bulk densities, their rapid and robust migration followed a typical bell-shaped response. When implanted in porcine cutaneous wounds, these acellular matrices, besides being completely biocompatible, induced rapid and en masse recruitment of stromal fibroblasts that was not observed with RGD-tethered or unmodified hydrogels. Such constructs might be of great benefit in clinical settings where rapid formation of new tissue is needed.

Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix.

Injectable hydrogel and porous sponge formulations of Carbylan-GSX, a crosslinked synthetic extracellular matrix (ECM), were used to deliver human demineralized bone matrix (DBM) in a rat femoral defect model. A cortical, full-thickness 5-mm defect was created in two femurs of each rat. Six rats were assigned to each of five experimental groups (thus, 12 defects per group). The defects were either untreated or filled with Carbylan-GSX hydrogel or sponges with or without 20% (w/v) DBM. Radiographs were obtained on day 1 and at weeks 2, 4, 6, and 8 postsurgery of each femur. Animals were sacrificed at week 8 postsurgery and each femur was fixed, embedded, sectioned, and processed for Masson's Trichrome staining. The bone defects were measured from radiographs and the fraction of bone healing was calculated. The average fractions of bone healing for each group were statistically different among all groups, and all treatment groups were significantly better than the control group. The Carbylan-GSX sponge with DBM was superior to the sponge without DBM and to the hydrogel with DBM. Histology showed that defects treated with the Carbylan-GSX sponge plus DBM were completely filled with newly generated bone tissue with a thickness comparable to native bone. Carbylan-GSX sponge was an optimal delivery vehicle for human DBM to accelerate bone healing.

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.

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.

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.

Crosslinked hyaluronan hydrogels containing mitomycin C reduce postoperative abdominal adhesions.

OBJECTIVE: To evaluate the efficacy of crosslinked hyaluronan (HA) hydrogels that contained covalently-bound mitomycin C (MMC) in reducing postoperative adhesions in a rat uterine horn model. DESIGN: Two independent parameters were investigated: [1] the quantity of MMC in preformed crosslinked hydrogel films and [2] the efficacy of intraperitoneal injection of in situ crosslinkable solutions. SETTING: University animal research facility. ANIMAL(S): Female Wistar rats. INTERVENTION(S): Injuries (3 x 10 mm) were made to contacting serosal surfaces of the medial uterine wall musculature in female rats. Two treatment protocols were used. In the first, sterile crosslinked HA films that contained different MMC loadings (0, 0.5%, and 2%) were applied to two injured uterine horns; control animals received no films. In the second protocol, MMC-loaded crosslinked HA gels that contained different MMC loadings (0.31%, 0.625%, and 1.25%) were spread on the site of uterine horn injury (1 mL); then, an additional 4 mL of the same formulation was injected into the peritoneal cavity after abdominal closure. Control animals were injected with 5 mL of buffer only. MAIN OUTCOME MEASURE(S): Extent of postoperative adhesions between uterine horns and with surrounding tissues and organs. RESULT(S): Mitomycin C-loaded crosslinked HA films and in situ crosslinked gels were more effective in reducing postoperative adhesion formation than were buffer controls or crosslinked HA films without MMC. CONCLUSION(S): Mitomycin C-loaded crosslinked HA films and gels reduced formation of postoperative intraperitoneal adhesions.

In vivo engineering of the vocal fold extracellular matrix with injectable hyaluronic acid hydrogels: early effects on tissue repair and biomechanics in a rabbit model.

OBJECTIVES: A prospective, controlled animal study was performed to determine whether the use of injectable, chemically modified hyaluronic acid (HA) derivatives at the time of intentional vocal fold resection might facilitate wound repair and preserve the unique viscoelastic properties of the vocal fold extracellular matrix. METHODS: We performed bilateral vocal fold biopsies on 33 rabbits. Two groups of rabbits were unilaterally treated with 2 different HA derivatives--Carbylan-SX and HA-DTPH-PEGDA--at the time of resection. Saline was injected as a control into the contralateral fold. The animals were painlessly sacrificed 3 weeks after biopsy and injection. The outcomes measured included histologic fibrosis level, tissue HA level, and tissue viscosity and elasticity. RESULTS: The Carbylan-SX-treated vocal folds were found to have significantly less fibrosis than the saline-treated controls. The levels of HA in the treated vocal folds were not significantly different from those in the controls at 3 weeks as measured by enzyme-linked immunosorbent assay. The Carbylan-SX-treated vocal folds had significantly improved biomechanical properties of elasticity and viscosity. The HA-DTPH-PEGDA injections yielded significantly improved viscosity, but not elasticity. CONCLUSIONS: Prophylactic in vivo manipulation of the extracellular matrix with an injectable Carbylan-SX hydrogel appears to induce vocal fold tissue regeneration to yield optimal tissue composition and biomechanical properties favorable for phonation.

Disulfide-crosslinked hyaluronan-gelatin hydrogel films: a covalent mimic of the extracellular matrix for in vitro cell growth.

A new disulfide crosslinking method was developed for the preparation of blended hyaluronan (HA)-gelatin hydrogels to form a synthetic, covalently linked mimic of the extracellular matrix (ECM). The HA and gelatin were chemically modified using 3,3'-dithiobis(propionic hydrazide) (DTP). After reduction with dithiothreitol (DTT), the thiol derivatives of HA (HA-DTPH) and gelatin (gelatin-DTPH) were obtained and characterized. To minimize interference with biological function, the degree of substitution of HA-DTPH and gelatin-DTPH was kept below 50%. Solutions of HA-DTPH and gelatin-DTPH in varying blends (20%, 40%, 60%, 80% gelatin) were prepared in 1% w/v NaCl and crosslinked by disulfide bond formation in air. Hydrogel films were dried and further crosslinked with dilute hydrogen peroxide. Disulfide crosslinked HA-DTPH, gelatin-DTPH, and blends thereof, were degradable enzymatically by collagenase and by hyaluronidase (HAse). The rapid digestion of the crosslinked 100% gelatin-DTPH film by collagenase was significantly retarded by the presence of 20% or 40% HA-DTPH. Addition of at least 40% w/v gelatin into the 100% HA-DTPH films significantly improved the attachment and spreading of Balb/c 3T3 murine fibroblasts seeded on the surface of the hydrogel. These results demonstrate that disulfide-crosslinked HA-gelatin hydrogels, a new type of covalent synthetic ECM, constitute biocompatible and biodegradable substrata for cell culture in vitro.