Journal References

Journal Articles

Classification by general topic

Section 1: 3-D cell culture
Section 2: Angiogenesis
Section 3: Electrospinning
Section 4: ECM incorporation
Section 5: Centrifugal casting of tubes
Section 6: Collagen stenting

Section 7: Growth factor release
Section 8: Hydrogel compliance
Section 9: Protein/peptide incorporation
Section 10: Tissue engineering
Section 11: Tumor xenograft
Section 12: ADH-based materials
Section 13: Stem Cells

Book Chapters

Section 1: 3-D cell culture

M.A. Serban, Y. Liu, and G.D. Prestwich, “Effects of Synthetic Extracellular Matrices on Primary Human Fibroblast Behavior,” Acta Biomaterialia, 4, 67-75 (2008).

In vitro cell culture is a vital research tool for cell biology, pharmacology, toxicology, protein production, systems biology and drug discovery. Traditional culturing methods on plastic surfaces do not accurately represent the in vivo environment, and a paradigm shift from two-dimensional to three-dimensional (3-D) experimental techniques is underway. To enable this change, a variety of natural, synthetic and semi-synthetic extracellular matrix (ECM) equivalents have been developed to provide an appropriate cellular microenvironment. We describe herein an investigation of the properties of four commercially available ECM equivalents on the growth and proliferation of primary human tracheal scar fibroblast behavior, both in 3-D and pseudo-3-D conditions. We also compare subcutaneous tissue growth of 3-D encapsulated fibroblasts in vivo in two of these materials, Matrigeltrade mark and Extraceltrade mark. The latter shows increased cell proliferation and remodeling of the ECM equivalent. The results provide researchers with a rational basis for selection of a given ECM equivalent based on its biological performance in vitro and in vivo, as well as the practicality of the experimental protocols. Biomaterials that use a customizable glycosaminoglycan-based hydrogel appear to offer the most convenient and flexible system for conducting in vitro research that accurately translates to in vivo physiology needed for tissue engineering.

G.D. Prestwich, “Evaluating Drug Toxicity and Efficacy in Three Dimensions: Using Synthetic Extracellular Matrices in Drug Discovery,” Acc. Chem. Res., in press (2008).

Synthetic extracellular matrices (sECMs) developed for engineering of vascularized tissues in vivo can also be used for in vitro three-dimensional (3D) cell culture. The sECMs, prepared from cross-linked derivatives of glycosaminoglycans and gelatin, can be customized for specific cell types. Primary hepatocytes retain biochemical function and live longer in 3D in sECMs, providing a new tool for determining hepatotoxicity. "Tumor engineering", a novel xenograft model for testing anticancer drug candidates, affords orthotopic, vascularized tumors. Future clinically relevant applications may include high-content chemogenomic screens and organotypic 3D cultures for drug discovery and drug evaluation.

M.A. Serban, G. D. Prestwich, “Making Modular Extracellular Matrices: Solutions for the Puzzle,” Methods in Extracellular Matrix Research, in press (2008)

No abstract available.

G.D. Prestwich, Y. Liu, M. Serban, B. Yu, X.Z. Shu, and A. Scott, “3-D Culture in Synthetic Extracellular Matrices: New Tissue Models for Drug Toxicology and Cancer Drug Discovery,” invited, Adv. Enz. Res. in press (2007).

No abstract available.

E.M. Horn, M. Beaumont, X.Z. Shu, A. Harvey, G.D. Prestwich, K.M. Horn, A.R. Gibson, M.C. Preul, A. Panitch, “Influence of cross-linked hyaluronic acid hydrogels on neurite outgrowth and recovery from spinal cord injury,” J of Neurosurg Spine, 6(2), 133-40 (2007).

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.

K. Ghosh, X.-D. Ren, X.Z. Shu, G.D. Prestwich, and R.A.F. Clark, “Fibronectin Functional Domains Coupled to Hyaluronan Stimulate Adult Human Dermal Fibroblast Responses Critical for Wound Healing,” Tissue Eng., 12, 601-613 (2006).

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.

Y. Liu, X.Z. Shu, and G.D. Prestwich, “Biocompatibility and Stability of Disulfide-Crosslinked Hyaluronan Films,” Biomaterials, 26, 4737-4746 (2005).

Hyaluronan (HA) can be chemically modified to engineer robust materials with pre-selected mechanical properties and resorption rates that can be dictated by the intended clinical use. Disulfide-crosslinked HA films were prepared by air oxidation of thiol-modified HA, followed by treatment with 0.3% hydrogen peroxide. The degradation of the disulfide-crosslinked films in vitro was very slow (<10% in 7 days) in buffer alone and shorter (t1/2=3-5 days) in the presence of hyaluronidase (HAse). The cytocompatibility of the disulfide-crosslinked HA films was determined using two separate conditions: (i) in vitro culture of mouse fibroblasts in indirect contract with the films, and (ii) in vitro culture of fibroblasts directly on films coated with poly d-lysine. Excellent cytocompatibility was observed in murine fibroblasts that were cultured in indirect contact with thiolated HA films. Although cells were unable to attach and spread on thiolated HA films, pre-coating the thiolated HA films with poly D-lysine resulted in attachment and spreading equivalent to that observed on polystyrene. Rates of resorption in vivo were obtained by subcutaneous implantation of disulfide-crosslinked HA films into the backs of Wistar rats. Biocompatibility in vivo was determined in both subcutaneous flank and peritoneal cavity implantation of the films in Wistar rats. The disulfide-crosslinked HA films were less than 30% resorbed after 42 days in vivo, and histochemical and cytochemical analysis indicated that the films were well-tolerated with mild inflammatory response at both sites of implantation.

Y. Liu, X.Z. Shu, S.D. Gray, and G.D. Prestwich, “Disulfide-Crosslinked Hyaluronan-Gelatin Sponge: Growth of Fibrous Tissue in vivo,” J. Biomed. Mater. Res., 68A, 142-149 (2004).

The modification of hyaluronan (HA) and gelatin using dithiobis(propanoic dihydrazide) (DTP) has provided two thiolated macromolecular components of the extracellular matrix (ECM), specifically HA-DTPH and gelatin-DTPH. Blends of these thiolated ECM components were crosslinked in air to form hydrogels that were interpenetrating disulfide-crosslinked networks. Lyophilization of the hydrogels afforded sponge-like macroporous scaffolds suitable for cell attachment and proliferation. Increasing percentages of gelatin-DTPH (0, 25, 50, and 75%) were blended with HA-DTPH, and the resulting sponges were evaluated in vitro and in vivo as scaffolds for tissue engineering by seeding with human tracheal scar (HTS) fibroblasts. While cells failed to attach and grow in HA-only sponges, the gelatin-modified HA sponges promoted cell adhesion, proliferation, and spreading in vitro. Optimal attachment and growth was observed with 50% gelatin-HA sponges. Cell attachment to the gelatin-HA sponge could be blocked by preincubation of cells with a soluble fibronectin peptide Gly-Arg-Gly-Asp (GRGD). Finally, HTS fibroblast-seeded gelatin-HA sponges were implanted into the flanks of nude mice and evaluated at 2 and 8 weeks postimplantation. The sponges were fully biocompatible and new fibrous tissue formed, gradually replacing the sponge-like scaffold. The gelatin-HA sponges act as synthetic, macroporous, covalent mimics of the ECM and constitute novel scaffolds for cell growth and tissue augmentation. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 142-149, 2004

X.Z. Shu, Y. Liu, F. Palumbo, and G.D. Prestwich, “Disulfide-Crosslinked Hyaluronan-Gelatin Hydrogel Films: A Covalent Mimic of the Extracellular Matrix for In Vitro Cell Growth,” Biomaterials, 24, 3825-3834 (2003).

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.

X. Z. Shu, Y. Liu, Y. Luo, M.C. Roberts, and G.D. Prestwich, “Disulfide Crosslinked Hyaluronan Hydrogels,” Biomacromolecules, 3, 1304-1311 (2002).

A new disulfide cross-linking strategy was developed to prepare hyaluronic acid (HA) hydrogel from thiol-modified HA. First, dithiobis(propanoic dihydrazide) (DTP) and dithiobis(butyric dihydrazide) (DTB) were synthesized and then coupled to HA with carbodiimide chemistry. Next, disulfide bonds of the initially formed gel were reduced using dithiothreitol (DTT) to give, after exhaustive dialysis, the corresponding thiol-modified macromolecular derivatives HA-DTPH and HA-DTBH. The degree of substitution of HA-DTPH and HA-DTBH could be controlled from 20% to 70% of available glucuronate carboxylic acid groups. The pK(a) values of the HA-thiol derivatives were determined spectrophotometrically to be pK(a) = 8.87 (HA-DTPH) and pK(a) = 9.01 (HA-DTBH). The thiol groups could be oxidized in air to reform disulfide linkages, which resulted in HA-DTPH and HA-DTBH hydrogel films. Further oxidation of these hydrogels with dilute H(2)O(2) created additional cross-links and afforded poorly swellable films. The disulfide cross-linking was reversible, and films could be again reduced to sols with DTT. Release of blue dextran from cross-linked films was used as a model for drug release. The rapid gelation of the HA-DTPH solution under physiological conditions was also achieved, which demonstrated the capacity for in situ cell encapsulation. Thus, L-929 murine fibroblasts were encapsulated in HA-DTPH hydrogel; these cells remained viable and proliferated during 3 days of culture in vitro.

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Section 2: Angiogenesis

L.W. Hosack, M. A. Firpo, J.A. Scott, G.D. Prestwich, R.A. Peattie, " Microvascular maturity elicited in tissue treated with cytokine-loaded hyaluronan-based hydrogels," Biomaterials, 29, 2336-2347 (2008).

Hydrogels composed of crosslinked, chemically modified hyaluronic acid (HA), gelatin (Gtn) and heparin (Hp) were preloaded with vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) or platelet derived growth factor (PDGF) either individually or in combination with VEGF and implanted into the Balb/c mouse ear pinna. At 7 and 14 days post-surgery, elicited vascular maturity levels were quantified using immunohistochemical (IHC) staining techniques and reported as a vascular maturity index (VMI). At both time points, it was discovered that the dual cytokine combinations elicited greater maturity levels than that of cytokine administered individually. For example, VEGF and KGF-containing HA:Hp implants at day 7 yielded VMI values of -0.1375 and -0.092, respectively, whereas their combination resulted in a VMI of 0.176 ( p < 0.007). At day 7, only one of the seven HA:Hp experimental cases yielded a positive VMI (VEGF þ KGF), whereas four of the seven HA:Hp cases yielded positive VMI values at day 14, indicating a sustained maturity response. The same general trends were found to exist in tissue treated with HA:Hp:Gtn experimental implants. Differences in elicited maturity also existed between tissue treated with HA:Hp and HA-containing hydrogels (VMI ¼ 0.176 for HA:Hp-VEGF þ KGF vs. -0.064 for HAVEGF þ KGF, p < 0.012), and these differences are thought to result from differences in characteristic cytokine release rates. This result also suggests that the presentation of multiple growth factors (GFs) on immobilized Hp may actively contribute to cytokine related signal transduction, a characteristic that may be exploited in the future to improve the efficacy of cytokine-loaded implants towards tissue regeneration therapeutic strategies.

C.M. Riley, P.W. Fuegy, M.A. Firpo, X.Z. Shu, G.D. Prestwich, R.A. Peattie, “Stimulation of In Vivo Angiogenesis Using Dual Growth Factor-loaded Crosslinking Glycosaminoglycan Hydrogels,“ Biomaterials, 27, 5935-5943 (2006).

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.

R.A. Peattie, E. Rieke, E. Hewett, R.J. Fisher, X.Z. Shu, and G.D. Prestwich, “Dual growth factor-induced angiogenesis in vivo using hyaluronan hydrogel implants,” Biomaterials, 27, 1868-1875 (2006).

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.

D. B. Pike, S. Cai, K.R. Pomraning; M.A. Firpo, R.J. Fisher, X.Z. Shu, G.D. Prestwich, R.A. Peattie, “Heparin-regulated Release of Growth Factors In Vitro and Angiogenic Response In Vivo to Implanted Hyaluronan Hydrogels Containing VEGF and bFGF”, Biomaterials, 27, 5242-5251 (2006).

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.

S. Cai, Y. Liu, X.Z. Shu, and G.D. Prestwich, “Injectable Glycosaminoglycan Hydrogels for Controlled Release of Basic Fibroblast Growth Factor,” Biomaterials, 26, 6054-6067 (2005).

Synthetic hydrogel mimics of the extracellular matrix (ECM) were created by crosslinking a thiol-modified analog of heparin with thiol-modified hyaluronan (HA) or chondroitin sulfate (CS) with poly(ethylene glycol) diacrylate (PEGDA). The covalently bound heparin provided a crosslinkable analog of a heparan sulfate proteoglycan, thus providing a multivalent biomaterial capable of controlled release of basic fibroblast growth factor (bFGF). Hydrogels contained >97% water and formed rapidly in <10min. With as little as 1% (w/w) covalently bound heparin (relative to total glycosaminoglycan content), the rate of release of bFGF in vitro was substantially reduced. Total bFGF released increased with lower percentages of heparin; essentially quantitative release of bFGF was observed from heparin-free hydrogels. Moreover, the hydrogel-released bFGF retained 55% of its biological activity for up to 28 days as determined by a cell proliferation assay. Finally, when these hydrogels were implanted into subcutaneous pockets in Balb/c mice, neovascularization increased dramatically with HA and CS hydrogels that contained both bFGF and crosslinked heparin. In contrast, hydrogels lacking bFGF or crosslinked heparin showed little increase in neovascularization. Thus, covalently linked, heparin-containing glycosaminoglycan hydrogels that can be injected and crosslinked in situ constitute highly promising new materials for controlled release of heparin-binding growth factors in vivo.

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Section 3: Electrospinning

Y. Ji, K. Ghosh, X.Z. Shu, B. Li., J.C. Sokolov, G.D. Prestwich, R.A.F. Clark, M.H. Rafailovich, “Electrospun Three-dimensional Hyaluronic Acid Nanofibrous Scaffolds,” Biomaterials, 27, 3782-92 (2006).

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.

Section 4: ECM incorporation

T. Mehra, K. Ghosh, X.Z. Shu, G.D. Prestwich and R.A.F. Clark, “Molecular Stenting with a Crosslinked Hyaluronan Derivative Inhibits Collagen Gel Contraction,” Journal of Investigative Dermatology, 126(10), 2202-9 (2006).

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.

Section 5: Centrifugal casting of tubes

V. Mironov, V. Kasyanov, X.Z. Shu, C. Eisenberg, L. Eisenberg, S. Gonda, T. Trusk, R.R. Markwald, and G.D. Prestwich, “Fabrication of Tubular Tissue Constructs by Centrifugal Casting of Cells Suspended in an In Situ Crosslinkable Hyaluronan-Gelatin Hydrogel,” Biomaterials, 26, 7628-7635 (2005).

Achieving the optimal cell density and desired cell distribution in scaffolds is a major goal of cell seeding technologies in tissue engineering. In order to reach this goal, a novel centrifugal casting technology was developed using in situ crosslinkable hyaluronan-based (HA) synthetic extracellular matrix (sECM). Living cells were suspended in a viscous solution of thiol-modified HA and thiol-modified gelatin, a polyethyleneglycol diacrylate crosslinker was added, and a hydrogel was formed during rotation. The tubular tissue constructs consisting of a densely packed cell layer were fabricated with the rotation device operating at 2000 rpm for 10 min. The majority of cells suspended in the HA mixture before rotation were located inside the layer after centrifugal casting. Cells survived the effect of the centrifugal forces experienced under the rotational regime employed. The volume cell density (65.6%) approached the maximal possible volume density based on theoretical sphere packing models. Thus, centrifugal casting allows the fabrication of tubular constructs with the desired redistribution, composition and thickness of cell layers that makes the maximum efficient use of available cells. Centrifugal casting in this sECM would enable rapid fabrication of tissue-engineered vascular grafts, as well as other tubular and planar tissue-engineered constructs.

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Section 6: Collagen stenting

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Section 7: Growth factor release

Y. Liu, S. Cai, X.Z. Shu, J. Shelby, G.D. Prestwich, “Release of basic fibroblast growth factor from a crosslinked glycosaminoglycan hydrogel promotes wound healing," Wound Repair Regen, 15(2), 245-51 (2007).

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.

X.Z. Shu, S. Ahmad, Y. Liu, G.D. Prestwich, “Synthesis and evaluation of injectable, in situ crosslinkable synthetic extracellular matrices for tissue engineering,” J Biomed Mater Res A, 79A(4), 901-912 (2006).

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.

S. Cai, Y. Liu, X.Z. Shu, and G.D. Prestwich, “Injectable Glycosaminoglycan Hydrogels for Controlled Release of Basic Fibroblast Growth Factor,” Biomaterials, 26, 6054-6067 (2005).

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Section 8: Hydrogel compliance

K. Ghosh, Z. Pan, E. Guan, S. Ge, Y. Liu, T. Nakamura, X. Ren, M. Rafailovich, R. Clark, “Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties,” Biomaterials 28, 671–679 (2007).

To successfully induce tissue repair or regeneration in vivo, bioengineered constructs must possess both optimal bioactivity and mechanical strength. This is because cell interaction with the extracellular matrix (ECM) produces two different but concurrent signaling mechanisms: ligation-induced signaling, which depends on ECM biological stimuli, and traction-induced signaling, which depends on ECM mechanical stimuli. In this report, we provide a fundamental understanding of how alterations in mechanical stimuli alone, produced by varying the viscoelastic properties of our bioengineered construct, modulate phenotypic behavior at the whole-cell level. Using a physiologically relevant ECM mimic composed of hyaluronan and fibronectin, we found that adult human dermal fibroblasts modify their mechanical response in order to match substrate stiffness. More specifically, the cells on stiffer substrates had higher modulus and a more stretched and organized actin cytoskeleton (and vice versa), which translated into larger traction forces exerted on the substrate. This modulation of cellular mechanics had contrasting effects on migration and proliferation, where cells migrated faster on softer substrates while proliferating preferentially on the stiffer ones. These findings implicate substrate rigidity as a critical design parameter in the development of bioengineered constructs aimed at eliciting maximal cell and tissue function

K. Ghosh, X.Z. Shu, R. Mou, J. Lombardi, G.D. Prestwich, M.H. Rafailovich, and R.A.F. Clark, “Rheological Characterization of in Situ Cross-Linkable Hyaluronan Hydrogels,” Biomacromolecules, 6, 2857-2865 (2005).

This report investigates the rheological properties of cross-linked, thiol-functionalized HA (HA-DTPH) hydrogels prepared by varying the concentration and molecular weight (MW) of the cross-linker, poly(ethylene glycol) diacrylate (PEGDA). Hydrogels were subsequently cured for either short-term (hours) or long-term (days) and subjected to oscillatory shear rheometry (OSR). OSR allows the evaluation and comparison of the shear storage moduli (G'), an index of the total number of effective cross-links formed in the hydrogels. While the oscillatory time sweep monitored the evolution of G' during in situ gelation, the stress and frequency sweeps measured the G' of preformed and subsequently cured hydrogels. From stress sweeps, we found that, for the hydrogels, G' scaled linearly with PEGDA concentration and was independent of its MW. Upon comparison with the classical Flory's theory of elasticity, stress sweep tests on short-term cured hydrogels revealed the simultaneous, but gradual, formation of spontaneous disulfide cross-links in the hydrogels. Results from time and frequency sweeps suggested that the formation of a stable, three-dimensional network depended strictly on PEGDA concentration. Results from the equilibrium swelling of hydrogels concurred with those obtained from oscillatory stress sweeps. Such a detailed rheological characterization of our HA-DTPH-PEGDA hydrogels will aid in the design of biomaterials targeted for biomedical or pharmaceutical purposes, especially in applications involving functional tissue engineering.

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Section 9: Protein/peptide incorporation

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.

Y. Liu, H. Li, X.Z. Shu, S.D. Gray, G.D. Prestwich, “Crosslinked Hyaluronan Hydrogels Containing Mitomycin C Reduce Post-operative Abdominal Adhesions,” Fertil. & Steril., 83, 1275-1283 (2005).

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.

H. Li, Y. Liu, X.Z. Shu, S.D. Gray, and G.D. Prestwich, “Synthesis and Biological Evaluation of a Cross-Linked Hyaluronan -Mitomycin C Hydrogel,” Biomacromolecules, 5, 895-902 (2004).

A cross-linked hyaluronan (HA) hydrogel that contained a covalently bound derivative of the anti-proliferative drug mitomycin C (MMC) was synthesized and evaluated in vitro and in vivo. The HA-MMC hydrogel was prepared by coupling MMC-aziridinyl-N-acrylate with thiol-modified HA followed by cross-linking with poly(ethylene glycol) diacrylate (PEGDA). MMC was released from 0.5% and 2.0% MMC films by hydrolysis in proportion to the MMC loading. When incubated in vitro with human T31 tracheal scar fibroblasts, 0.5% MMC films inhibited proliferation, whereas 2.0% MMC films were cytotoxic. When implanted in vivo into a rat peritoneal cavity, neither 0.5% nor 2.0% HA-MMC films elicited a severe peritoneal fluid leukocyte response. Importantly, MMC reduced the thickness of fibrous tissue formed surrounding the implanted films. Thus, cross-linked HA-MMC films have strong potential as anti-fibrotic barriers for the prevention of post-surgical adhesions.

X.Z. Shu, K. Ghosh, Y. Liu, F.S. Palumbo, Y. Luo, R.A. Clark, and G.D. Prestwich, “Attachment and Spreading of Fibroblasts on an RGD Peptide-Modified Injectable Hyaluronan Hydrogel,” J. Biomed. Mat. Res., 68A, 365-375 (2004).

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 365-375, 2004

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Section 10: Tissue engineering

Section 10.1: General materials

G.D. Prestwich, “Simplifying the Extracellular Matrix for 3-D Cell Culture and Tissue Engineering: A Pragmatic Approach,” J. Cell. Biochem., 101, 1370-1383 (2007).

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.

G.D. Prestwich and J.W. Kuo, “Chemically-Modified HA for Therapy and Regenerative Medicine,” invited article, Curr. Pharm. Biotech., in press (2007).

No abstract available.

X.Z. Shu, S. Ahmad, Y. Liu, and G.D. Prestwich, “Synthesis and Evaluation of Injectable, In Situ Crosslinkable Synthetic Extracellular Matrices (sECMs) for Tissue Engineering,” J. Biomed. Mater. Res. A, 79A(4), 901-912 (2006).

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.

Section 10.2: Adhesion prevention

R.C. Connors, J.J. Muir, Y. Liu, G.R. Reiss, P.C. Kouretas, M.G Whitten, T.K. Sorenson, G.D. Prestwich, and D.A. Bull, “Postoperative Pericardial Adhesion Prevention Using Carbylan‑SX in a Rabbit Model,” J. Surg. Res., 140, 237-242 (2007).

INTRODUCTION: The presence of dense adhesions within the pericardial space complicates reoperative cardiac surgery. Prior attempts to reduce adhesion formation after primary cardiac surgery using medications or biomaterials have had variable success. Carbylan-SX (Carbylan Biosurgery Inc., Palo Alto, CA) is a hyaluronan-based biomaterial, which has been shown to be effective at reducing adhesions in a nonthoracic rat model. This study evaluates whether Carbylan-SX can effectively reduce postoperative adhesions within the pericardial cavity. METHODS: Thirty-eight New Zealand white rabbits underwent a left lateral thoracotomy. A pericardiotomy was made and epicardial adhesions were induced on the anterior surface of the heart using a Dremel device (Racine, WI). The rabbits were divided into four groups: controls with abrasions only receiving no treatment (n=10), Carbylan-SX films (n=10), Carbylan-SX aerosolized hydrogel (n=10), and Seprafilm (n=8). The pericardial sac and chest were subsequently closed. Rabbits were sacrificed at a mean of 15 days. For histological analysis, each heart was divided into 12 separate 1 mm sections. Computer imaging software was used to measure the adhesion thickness and the mean of 12 random measurements for each animal was recorded and statistical analysis performed. RESULTS: Histological analysis revealed all treatment groups to be significantly better than the control (2159 mum thickness, P<0.0001) at preventing adhesions. The Carbylan-SX film and Carbylan-SX aerosolized hydrogel both proved to be better at preventing adhesions than Seprafilm (Genzyme Corp., Cambridge, MA) with an average adhesion thickness of 454 and 577 microm, respectively, compared with 1319 microm for Seprafilm (P<0.0001 and P<0.0005, respectively). The Carbylan-SX film and Carbylan-SX aerosolized hydrogel were equally effective at preventing adhesion formation. CONCLUSION: Carbylan-SX film and Carbylan-SX aerosolized crosslinkable hydrogel are equally effective methods of reducing postoperative pericardial adhesions within the pericardial cavity. Both the Carbylan-SX film and aerosolized hydrogel showed a significantly greater reduction in adhesions than Seprafilm. Clinical application of Carbylan-SX could have significant therapeutic implications in the future.

Y. Liu, X.Z. Shu, and G.D. Prestwich, “Reduced Post-operative Intra-abdominal Adhesions Using Carbylan™-SX, a Semi-synthetic Glycosaminoglycan Hydrogel,” Fertil. & Steril., 87(4), 940-948 (2007).

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.

Y. Liu, H. Li, X.Z. Shu, S.D. Gray, G.D. Prestwich, “Crosslinked Hyaluronan Hydrogels Containing Mitomycin C Reduce Post-operative Abdominal Adhesions,” Fertil. & Steril., 83, 1275-1283 (2005).

Section 10.3: Bladder repair

A.L. Brown, E.M. Srokowski, X.Z. Shu, G.D. Prestwich, and K.A. Woodhouse, “Development of a Model Bladder Extracellular Matrix Combining Disulfide Cross-linked Hyaluronan with Decellularized Bladder Tissue,” Macromolecular Biosci., 6(8), 648-657 (2006).

In this work we investigate the feasibility of modifying porcine-derived BAM to include HA with a view to developing a model, artificial extracellular matrix for the study of bladder cell-matrix interactions. HA-DPTH was incorporated into BAM disks and then cross-linked oxidatively to a disulfide containing hydrogel. Disks were seeded with bladder smooth muscle cells (BSMC) and UEC under three culture configurations and incubated for 3, 7, and 14 d. At each time point, matrix contraction was measured, and media supernatants assayed for cell-secreted gelatinase activity. To evaluate cell adherence and organization, triple immunofluorescent labeling of cell nuclei, actin cytoskeleton, and focal contacts was performed. HA-modified BAM exhibited a significant increase in matrix contraction and induced a higher level of cell-secreted gelatinase activity compared to unmodified BAM. Immunofluorescent labeling demonstrated that BSMCs remained adherent to both scaffold types over time. The distribution and organization of the cytoskeleton and focal contacts did not appear to be altered by the presence of HA. Interestingly, cellular infiltration into modified BAM was evident by 7 d and continued beyond 14 d, while BSMCs seeded onto unmodified BAM remained localized to the surface out to 14 d, with minimal infiltration evident only at day 28. These differences in cell infiltration support the gelatinase activity results. Increases in cell migration and matrix proteolysis in the presence of HA may be contributing factors toward BAM remodeling leading to increased matrix contraction with time. The model ECM developed in this work will be utilized for future studies aimed at elucidating the mechanisms controlling key remodeling events associated with bladder repair.Matrix contraction of cell-seeded BAM scaffolds.

A.L. Brown, M.J. Ringuette, G.D. Prestwich, D.J. Bagli, and K.A. Woodhouse, “Effects of Hyaluronan and SPARC on Fibroproliferative Events Assessed in an In Vitro Bladder Acellular Matrix Model,” Biomaterials, 27, 3825-3835 (2006).

Bladder acellular matrix (BAM) is a promising candidate for urinary biomaterials development. In the current work we have modified the BAM construct to include two biologically active components; hyaluronan (HA) and a peptide (SP4.2) derived from secreted protein, acidic, rich in cysteine (SPARC), a matricellular glycoprotein. In order to assess the potential of an HA/SP4.2 modified BAM to influence cellular functions associated with bladder healing, experiments were conducted to evaluate the individual and combined effects of these molecules on in vitro fibroproliferative endpoints within a co-culture model. Thiol-modified HA (246 kDa, 15 mg/ml)+/-SP4.2 (200 microm) was incorporated and cross-linked into BAM disks through disulfide bond formation. The following scaffolds compositions were then evaluated in a bladder smooth muscle cell (SMC)-urothelial (UEC) cell co-culture model: BAM unmodified; BAM+HA, BAM+SP4.2 (media addition); BAM+HA+SP4.2 (media addition); BAM+HA+SP4.2 (matrix incorporated). At 3, 7 and 14 days post-seeding, SMC-mediated matrix contraction and gelatinolytic activity were evaluated. HA-modified BAM exhibited a significantly higher degree of contraction and gelatinase activity compared to unmodified BAM. In contrast, addition of SP4.2 to BAM produced a negligible effect on contraction, while significantly reducing gelatinase activity. Matrices containing both molecules displayed significant increases in contraction, while gelatinase activity was dependent upon the method of peptide delivery. These results demonstrate that both HA and SP4.2 have significant, yet distinct effects on the contractile and proteolytic activity of bladder SMCs and suggest that a modified BAM may be capable of modulating processes associated with post-surgical graft contracture and scar formation.

Section 10.4: Bone and cartilage repair

Y. Liu, X.Z. Shu, G.P. Prestwich, “Osteochondral Defect Repair with Autologous Bone Marrow-Derived Mesenchymal Stem Cells in an Injectable, In Situ Crosslinked Synthetic Extracellular Matrix,” Tissue Eng., 12(12), 3405-3416 (2006).

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.

Y. Liu, S. Ahmad, X.Z. Shu, R.K. Sanders, S.A. Kopesec, and G.D. Prestwich, “Accelerated Repair of Cortical Bone Defects Using a Synthetic Extracellular Matrix to Deliver Human Demineralized Bone Matrix,” J. Orthoped. Res., 24(7), 1454-1462 (2006).

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.

Section 10.5: Sinus repair

M. Proctor, K. Proctor, X.Z. Shu, L.D. McGill, G.D. Prestwich, and R.R. Orlandi, “Composition of Hyaluronan Affects Wound Healing in the Rabbit Maxillary Sinus,” Am. J. Rhinology, 20, 206-211 (2006).

BACKGROUND: Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. HA and its derivatives have been used in the sinuses to reduce scarring and possibly promote wound healing. However, in recent animal studies, HA esters exhibited inflammatory effects. Mitomycin C (MMC) is another potential antiscarring treatment. This study prospectively evaluated the effects of three different HA constructs on wound healing in the rabbit maxillary sinus: (i) a novel cross-linked HA hydrogel, (ii) the cross-linked HA gel containing covalently bound MMC, and (iii) a commercially available woven HA ester (Merogel). METHODS: Ostia were created with a 4-mm otologic drill in the maxillary sinuses of 15 New Zealand white rabbits with one side randomly chosen for treatment. After 14 or 21 days the size of the maxillary ostia were recorded and the tissue was examined under light microscopy. RESULTS: Sinuses treated with the novel HA and HA-MMC hydrogels showed an increased ostial diameter compared with untreated controls. Woven HA ester-treated sinuses showed no improvement, with a trend toward a smaller ostium than controls. Histological examination showed that woven HA ester tended to cause increased fibrosis and granulomatous inflammation, and heterophilia was slightly increased in the HA hydrogel-treated sinuses. Blinded observation noted foamy macrophages surrounding the residual woven HA ester in each specimen while no similar reaction was noted near the residual HA or HA-MMC hydrogels. CONCLUSION: This study suggests that the degree of ostial narrowing, inflammation, and fibrosis depends on the formulation of the HA used. Minimal, if any, additional benefit is seen with addition of MMC to the HA hydrogel in this pilot study.

C. Sondrup, Y. Liu, X.Z. Shu, G.D. Prestwich, and M.E. Smith, “Cross-linked hyaluronan-coated stents in the prevention of airway stenosis,” Otolaryngol. Head & Neck Surg., 135(1), 28-35 (2006).

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.

Section 10.6: Spinal cord injury

E.M. Horn, M. Beaumont, X.Z. Shu, A. Harvey, G.D. Prestwich, K.M. Horn, A.R. Gibson, M.C. Preul, A. Panitch, “Influence of cross-linked hyaluronic acid hydrogels on neurite outgrowth and recovery from spinal cord injury”, Journal of Neurosurg Spine, 6(2), 133-40 (2007).

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.

Section 10.7: Tympanic membrane repair

A.H. Park, C.W. Hughes, A. Jackson, L. Hunter, L. McGill, S.E. Simonsen, S.C. Alder, X.Z. Shu, and G.D. Prestwich, “Crosslinked Hydrogels for Tympanic Membrane Repair,” Otolaryngol. Head Neck Surg., 135(6), 877-883 (2006).

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.

Section 10.8: Vocal fold repair

R.R. Orlandi, X.Z. Shu, L. McGill, E. Petersen, and G.D. Prestwich, “Structural Variations in a Single Hyaluronan Derivative Significantly Alter Wound-Healing Effects in the Rabbit Maxillary Sinus,” Laryngoscope, 117, 1288-1295 (2007). * EN

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.

S. Duflo, S.L. Thibeault, W. Li, X.Z. Shu, and G.D. Prestwich, “Vocal Fold Tissue Repair In Vivo Using a Synthetic Extracellular Matrix,” Tissue Eng., 12(8), 2171-2180. (2006).

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.

J.K. Hansen, S.L. Thibeault, J.F. Walsh, X.Z. Shu, and G.D. Prestwich, “In Vivo Engineering of the Vocal Fold ECM with Injectable HA Hydrogels: Early Effects on Tissue Repair and Biomechanics in a Rabbit Model,” Ann. Otol. Rhinol. Laryngol., 114, 662-670 (2005).

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.

Y. Liu, A. Skardal, X.Z. Shu, and G.D. Prestwich, “Prevention of Peritendinous Adhesions Following Flexor Tendon Injury with CarbylanÔ-SX, a Semisynthetic Glycosaminoglycan Hydrogel,” J. Orthopedic Res., in press (2008).

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(R), (iii) Carbylantrade mark-SX in situ crosslinked hydrogel, and (iv) preformed Carbylantrade mark-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 Carbylantrade mark-SX film was statistically superior to Seprafilm(R) and Carbylantrade mark-SX gel in preventing tendon adhesion formation. In biomechanical tests, the Carbylantrade mark-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). Carbylantrade mark-SX gel was less effective than Carbylantrade mark-SX film but superior to Seprafilm(R) for all evaluations. A crosslinked HA-derived film promoted healing of a flexor tendon injury without the formation of fibrosis at 3 weeks postoperatively.

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Section 11: Tumor xenograft

C.L. Scaife, J.E. Shea, Q. Dai, M.A. Firpo, G.D. Prestwich, S.J. Mulvihill, “Synthetic Extracellular Matrix Enchances Tumor Growth and Metastasis in an Orthotopic Mouse Model of Pancreatic Adenocarcinoma”, J Gastrointest Surg in press (2007)

Individuals with pancreatic cancer have one of the poorest survival rates among the major cancers, suggesting the need to develop new therapeutic approaches. An effective animal model that mimics the progression and metastases of human pancreatic adenocarcinoma does not exist. The goal of this investigation was to develop a model that would compare the growth and metastasis of orthotopically injected pancreatic cancer cells to cells encapsulated within a synthetic extracellular matrix (sECM). The hypotheses tested were that the cells within the sECM would grow more quickly and more frequently develop metastasis to distant organs. MiaPaCa-2 cells expressing red fluorescent protein, either in serum-free media or within a hyaluronan-based hydrogel, were injected into the pancreas of nude mice. Tumors were monitored for 8 weeks via intravital red fluorescent protein imaging. Cells encapsulated within the sECM grew more quickly and produced larger tumors compared with the cells alone. In addition, the cells within the sECM developed metastasis more frequently. Therefore, the encapsulation of human pancreatic cancer cells within an injectable sECM improved the rate of tumor growth and metastasis in an orthotopic mouse model. The advantages of this new approach can be utilized to investigate the mechanisms of tumor progression and test novel therapeutic agents in vivo.

Y. Liu, X. Z. Shu, and G.D. Prestwich, “Tumor Engineering: Orthotopic Cancer Models in Mice Using Cell-Loaded, Injectable, Crosslinked Hyaluronan-Derived Hydrogels” Tissue Engineering, 13(5), 1091-1101 (2007).

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.

Section 12: ADH-based materials

K.R. Kirker, Y. Luo, J.H. Nielson, J. Shelby, and G.D. Prestwich, “Glycosaminoglycan Hydrogel Films as Biointeractive Dressings for Wound Healing,” Biomaterials, 23, 3661-3671 (2002).

Chemically crosslinked glycosaminoglycan (GAG) hydrogel films were evaluated as biointeractive dressings in a porcine model for donor-site autograft wounds. Multiple 5 x 5 x 0.03 cm wounds were created on the dorsum of pigs. Half of the wounds were treated with a GAG film plus an occlusive dressing (Tegaderm), whereas the other half were treated with Tegaderm alone. At 3, 5, or 7 days after surgery, the partially healed wounds were excised and evaluated histologically for three animals at each time point. By day 3, epithelial cells had proliferated and migrated from wound edges and from epithelial islands associated with residual hair follicles to begin to cover the wound bed. A statistically significant increase in coverage was observed for GAG + Tegaderm-dressed wounds than for those with Tegaderm alone at day 3 and day 5 post-surgery. By day 7, all treatment groups were completely healed. Thus, GAG hydrogels accelerated wound healing by enhancing re-epithelialization.

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Section 13: Stem cells

L. Flynn, G.D. Prestwich, J.L Semple, and K.A. Woodhouse, “Adipose Tissue Engineering with Naturally-derived Scaffolds and Adipose-derived Stem Cells,” Biomaterials, 28, 3834‑3842 (2007).

A tissue-engineered adipose substitute would have numerous applications in plastic and reconstructive surgery. This work involves the characterization of the in vitro cellular response of primary human adipose-derived stem cells (ASC) to three dimensional, naturally derived scaffolds. To establish a more thorough understanding of the influence of the scaffold environment on ASC, we have designed several different soft tissue scaffolds composed of decellularized human placenta and crosslinked hyaluronan (XLHA). The cellular organization within the scaffolds was characterized using confocal microscopy. Adipogenic differentiation was induced and the ASC response was characterized in terms of glycerol-3-phosphate dehydrogenase (GPDH) activity and intracellular lipid accumulation. The results indicate that the scaffold environment impacts the ASC response and that the adipogenic differentiation of the ASC was augmented in the non-adhesive XLHA gels.

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Book Chapters

G.D. Prestwich, X.Z. Shu, Y. Liu, S. Cai, J.F. Walsh, C.W. Hughes, K.R. Kirker, R.R. Orlandi, A.H. Park, S.L. Thibeault, and M.E. Smith, “Injectable Synthetic Extracellular Matrices for Tissue Engineering and Repair,” in Tissue Engineering (J. Fisher, ed.), Springer, New York, pp. 125-134 (2006).

G.D. Prestwich, X.Z. Shu, Y. Liu, K.R. Kirker, H. Li, J. Shelby, S.E. Morris, and S.D. Gray, “In Situ Crosslinkable Synthetic Extracellular Matrices For Tissue Repair and Prevention of Surgical Adhesions,” in Hyaluronan: Structure, Metabolism, Biological Activities, Therapeutic Applications, Volume I (E.A. Balazs and V.C. Hascall, eds.), Matrix Biology Institute, Edgewater, New Jersey, pp. 409-414 (2005).

J. Tanyi, D. Croetzer, J. Wolf, S. Yu, Y. Hasegawa, J. Lahad, K.W. Cheng, M. Umezu-Goto, G.D. Prestwich, A.J. Morris, R.A. Newman, E.A. Felix, R. Lapis, and G.B. Mills, “Functional Lipidomics: Lysophosphatidic Acid as a Target for Molecular Diagnosis and Therapy of Ovarian Cancer,” in Functional Lipidomics (L. Feng and G.D. Prestwich, eds.), CRC Press/Taylor & Francis, New York, pp.101-123 (2006).

K.R. Kirker, Y. Luo, S.E. Morris, J. Shelby, and G.D. Prestwich, “Glycosaminoglycan Hydrogels for Wound Healing,” in Hyaluronan: Structure, Metabolism, Biological Activities, Therapeutic Application, Volume I (E.A. Balazs and V.C. Hascall, eds.), Matrix Biology Institute, Edgewater, New Jersey, pp. 397-400 (2005).

L. Feng, C.G. Ferguson, P.O. Neilsen, L. Chakravarty, P. Rzepecki, and G.D. Prestwich, Methods of Probing Phosphoinositides-Protein Interactions,” in Functional Lipidomics (L. Feng and G.D. Prestwich, eds.), CRC Press/Taylor & Francis, New York, pp. 189-210 (2006).

Q. Chen, S. Cai, K.G. Shadrach, G.D. Prestwich, and Joe G. Hollyfield, “Spacrcan Binding to Hyaluronan: Molecular and Biochemical Studies,” in Hyaluronan: Structure, Metabolism, Biological Activities, Therapeutic Applications, Volume II (E.A. Balazs and V.C. Hascall, eds.), Matrix Biology Institute, Edgewater, New Jersey, pp. 739-746 (2005).

R.R. Orlandi, H. Li, X.Z. Shu, Y. Liu, and G.D. Prestwich, “Preventing Ostia Closure During Sinus Surgery Using In Situ Crosslinked Hyaluronan Hydrogels,” in Hyaluronan: Structure, Metabolism, Biological Activities, Therapeutic Applications, Volume I (E.A. Balazs and V.C. Hascall, eds.), Matrix Biology Institute, Edgewater, New Jersey, pp. 405-408 (2005).

X.Z. Shu, Y. Liu, and G.D. Prestwich, “Injectable, In Situ-Crosslinkable Biomimetic Hydrogels for Tissue Engineering,” in Hyaluronan: Structure, Metabolism, Biological Activities, Therapeutic Applications, Volume I (E.A. Balazs and V.C. Hascall, eds.), Matrix Biology Institute, Edgewater, New Jersey, pp. 415-419 (2005).

Y. Liu, H. Li, X.Z. Shu, S.D. Gray, and G.D. Prestwich, “Reduction of Post-Operative Adhesions by In Situ Crosslinked Hyaluronan Hydrogels,” in Hyaluronan: Structure, Metabolism, Biological Activities, Therapeutic Applications, Volume I (E.A. Balazs and V.C. Hascall, eds.), Matrix Biology Institute, Edgewater, New Jersey, pp. 381-384 (2005).

Y. Liu, X.Z. Shu, S.D. Gray, and G.D. Prestwich, “Tissue Growth in a Disulfide-Crosslinked Hyaluronan-Gelatin Sponge,” in Hyaluronan: Structure, Metabolism, Biological Activities, Therapeutic Applications, Volume I (E.A. Balazs and V.C. Hascall, eds.), Matrix Biology Institute, Edgewater, New Jersey, pp. 377-380 (2005).

X.Z. Shu and G.D. Prestwich, “Therapeutic Biomaterials from Chemically Modified Hyaluronan, in Chemistry and Biology of Hyaluronan (H.G. Garg and C.A. Hales, eds.), Elsevier Press, Amsterdam, pp. 475-504 (2004).

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"Glycosan Systems: We see the future in 3-D™ - Hydrogels for 3-D culture, tumor xenografts, angiogenesis, growth factor release, and tissue engineering.

Journal References

Journal Articles

Classification by general topic

Section 1: 3-D cell culture

Section 2: Angiogenesis

Section 3: Electrospinning

Section 4: ECM incorporation

Section 5: Centrifugal casting of tubes

Section 6: Collagen stenting

Section 7: Growth factor release

Section 8: Hydrogel compliance

Section 9: Protein/peptide incorporation

Section 10: Tissue engineering

Section 11: Tumor xenograft

Section 12: ADH-based materials

Section 13: Stem cells

Book Chapters