Polysaccharide-based films for the prevention of unwanted postoperative adhesions at biological interfaces.

Postoperative adhesions protect, repair, and supply nutrients to injured tissues; however, such adhesions often remain permanent and complicate otherwise successful surgeries by tethering tissues together that are normally separated. An ideal adhesion barrier should not only effectively prevent unwanted adhesions but should be easy to use, however, those that are currently available have inconsistent efficacy and are difficult to handle or to apply. A robust hydrogel film composed of alginate and a photo-crosslinkable hyaluronic acid (HA) derivative (glycidyl methacrylate functionalized hyaluronic acid (GMHA)) represents a solution to this problem. A sacrificial porogen (urea) was used in the film manufacture process to impart macropores that yield films that are more malleable and tougher than equivalent films produced without the sacrificial porogen. The robust mechanical behavior of these templated alginate/GMHA films directly facilitated handling characteristics of the barrier film. In a rat peritoneal abrasion model for adhesion formation, the polysaccharide films successfully prevented adhesions with statistical equivalence to the leading anti-adhesion technology on the market, Seprafilm®. STATEMENT OF SIGNIFICANCE: Postoperative adhesions often remain permanent and complicate otherwise successful surgeries by tethering tissues together that are normally separated and pose potentially significant challenges to patients. Therefore, the generation of adhesion barriers that are easy to deploy during surgery and effectively prevent unwanted adhesions is a big challenge. In this study robust hydrogel films composed of alginate and a photo-crosslinkable hyaluronic acid (HA) derivative (glycidyl methacrylate functionalized HA, GMHA) were fabricated and investigated for their potential to act as a solution to this problem using a rat peritoneal abrasion model for adhesion formation. We observed the polysaccharide films successfully prevented adhesions with statistical equivalence to the leading anti-adhesion technology on the market, Seprafilm®, suggesting that such films represent a promising strategy for the prevention of postoperative adhesions.

Fibrillar films obtained from sodium soap fibers and polyelectrolyte multilayers.

An objective of tissue engineering is to create synthetic polymer scaffolds with a fibrillar microstructure similar to the extracellular matrix. Here, we present a novel method for creating polymer fibers using the layer-by-layer method and sacrificial templates composed of sodium soap fibers. Soap fibers were prepared from neutralized fatty acids using a sodium chloride crystal dissolution method. Polyelectrolyte multilayers (PEMs) of polystyrene sulfonate and polyallylamine hydrochloride were deposited onto the soap fibers, crosslinked with glutaraldehyde, and then the soap fibers were leached with warm water and ethanol. The morphology of the resulting PEM structures was a dense network of fibers surrounded by a nonfibrillar matrix. Microscopy revealed that the PEM fibers were solid structures, presumably composed of polyelectrolytes complexed with residual fatty acids. These fibrillar PEM films were found to support the attachment of human dermal fibroblasts.

Crystal templating dendritic pore networks and fibrillar microstructure into hydrogels.

Native tissues contain space-filling dendritic pore networks, such as vasculature, for the efficient distribution of oxygen and nutrients; however, it is not yet possible to create tissue-engineered scaffolds with dendritic porosity. Fibers are also important structural features of native tissues because they provide sites for cell anchorage, promote cell guidance and contribute to mechanical stability. Here, we have developed a "crystal templating" technique, which is simple and inexpensive, for fabricating polymer scaffolds with space-filling dendritic pore networks and fibrillar microtopography. To do this, we grow dendritic urea crystals in solution cast films of hyaluronic acid (HA), photocrosslink the HA around the crystal network to lock in the dendritic configuration, and dissolve the crystals to obtain empty pores. During in situ crystal growth the HA biopolymer is phase separated from the long narrow urea crystals and shaped into a fibrillar microstructure. The porous fibrillar HA scaffolds created by crystal templating may be applicable as regenerative patches for skin and other tissues.

Simple benchtop patterning of hydrogel grids for living cell microarrays.

A living cell microarray consists of an orderly arrangement of cells attached to a solid support such as a glass microscope slide. The chief difficulty of obtaining such arrays is the fabrication of substrates patterned with micro-wells, adhesive spots, or other features to guide orderly cell attachment. Here we report a novel method using woven Nylon mesh to micropattern three-dimensional alginate hydrogel grids on glass slides. The Nylon mesh is both inexpensive and off-the-shelf. By using Nylon mesh we have eliminated any need for lithography, clean room equipment, and microarray printers to generate microarray patterns; thus, this technique can be easily adopted by biological research labs that may lack microfabrication expertise and facilities. We have demonstrated that glass slides micropatterned with hydrogel grids guide the orderly attachment of single fibroblast cells and Schwann cell clusters in microarrays. The fibroblast arrays consisted of 70 microm square compartments at a density of 21,000 compartments per cm(2). The Schwann cell arrays consisted of 100 microm square compartments at a density of 6000 per cm(2). This patterning technique addresses the need for a simple, inexpensive, benchtop method for micro-patterning glass slides and obtaining living cell microarrays.

Photopatterned anisotropic swelling of dual-crosslinked hyaluronic acid hydrogels.

The purpose of a tissue engineered (TE) scaffold is to provide a support structure that can aid the regeneration of damaged tissue. Unlike native tissues, currently existing TE scaffolds are structurally simple, with homogeneous bulk properties that are unable to induce cells to regenerate architecturally complex healthy tissue. Thus, there is a need for methods that can create structural complexity within TE scaffolds to guide tissue regeneration. In this paper we have engineered novel dual-crosslinked hyaluronic acid hydrogel scaffolds with photopatterned anisotropic swelling. Anisotropic swelling can produce zonal distributions of crosslink density, water content and viscoelasticity on the macro- and micro-scales within the hydrogel scaffold. We have found that anisotropically swelling hydrogels can be obtained by a combination of chemical crosslinks and patterned photocrosslinks within a single dual-crosslinked hydrogel. According to our method an unswollen chemically crosslinked hydrogel substrate was spatially patterned with photocrosslinks that restricted swelling at selected sites. The resulting dual-crosslinked hydrogel swelled anisotropically because of differential crosslink densities between the photopatterned and non-photopatterned regions. Anisotropic swelling permitted the hydrogel to contort and evolve a shape different from that of the unswollen hydrogel. A biodegradable hydrogel with this unique swelling behavior yields a new, unexplored type of shape-changing TE scaffold.

Drug-binding hydrogels of hyaluronic acid functionalized with beta-cyclodextrin.

Hyaluronic acid (HA) hydrogels are attractive materials for biomedical applications because they are porous, water-swelling, biocompatible, biodegradable, and resistant to non-specific cell adhesion. A limitation of HA hydrogels is that incorporation of bioactive drugs can be restricted by low solubility of drug within the hydrogel environment. Our goal was to synthesize HA hydrogels that bind drug through hydrophobic interactions as a method for increasing drug loading. We functionalized photocrosslinked HA hydrogels with a methacryloyl derivative of beta-cyclodextrin (betaCD). betaCD is a molecular "basket" with a hydrophilic exterior and a hydrophobic cavity. Inclusion complexes are formed when betaCD hosts all or part of a hydrophobic drug within the cavity. HA hydrogels functionalized with methacryloyl-betaCD monomer gained the property of inclusion complexation which greatly enhanced the uptake of a model hydrophobic drug, hydrocortisone. Pre-incubation of the hydrogels with adamantane carboxylic acid (ACA) inhibited hydrocortisone uptake by competition for betaCD cavities. In addition, control hydrogels of HA functionalized with alphaCD monomer were not efficient at hydrocortisone uptake because the alphaCD cavity is too small for efficient complexation. These experiments confirmed that the betaCD monomer enhances drug loading by the mechanism of inclusion complexation. Drug-binding HA-betaCD hydrogels may be further engineered to create HA-based biomaterials with a built in drug delivery capability.

Optimized acellular nerve graft is immunologically tolerated and supports regeneration.

To replace the autologous graft as a clinical treatment of peripheral nerve injuries we developed an optimized acellular (OA) nerve graft that retains the extracellular structure of peripheral nerve tissue via an improved chemical decellularization treatment. The process removes cellular membranes from tissue, thus eliminating the antigens responsible for allograft rejection. In the present study, the immunogenicity and regenerative capacity of the OA grafts were tested. Histological examination of the levels of CD(8+) cells and macrophages that infiltrated the OA grafts suggested that the decellularization process averted cell-mediated rejection of the grafts. In a subsequent experiment, regeneration in OA grafts was compared with that in isografts (comparable to the clinical autograft) and two published acellular graft models. After 84 days, the axon density at the midpoints of OA grafts was statistically indistinguishable from that in isografts, 910% higher than in the thermally decellularized model described by Gulati (J. Neurosurg. 68, 117, 1988), and 401% higher than in the chemically decellularized model described by Sondell et al. (Brain Res. 795, 44, 1998). In summary, the results imply that OA grafts are immunologically tolerated and that the removal of cellular material and preservation of the matrix are beneficial for promoting regeneration through an acellular nerve graft.