Designing tailored biomaterial surfaces to direct keratinocyte morphology, attachment, and differentiation.

Precisely engineering the surface chemistry of biomaterials to modulate the adsorption and functionality of biochemical signaling molecules that direct cellular functions is critical in the development of tissue engineered scaffolds. Specifically, this study describes the use of functionalized self-assembled monolayers (SAMs) as a model system to assess the effects of biomaterial surface properties on controlling fibronectin (FN) conformation and concentration as well as keratinocyte function. By systematically analyzing FN adsorption at low and saturated surface densities, we distinguished between SAM-dependent effects of FN concentration and conformation on presenting cellular binding domains that direct cellular functions. Quantitative image analyses of immunostained samples showed that modulating the availability of the FN synergy site directly correlated with changes in keratinocyte attachment, spreading, and differentiation, through integrin-mediated signaling mechanisms. The results of this study will be used to elucidate design features that can be incorporated into dermal equivalents and percutaneous implants to enhance the rate of re-epithelialization and tissue regeneration. Furthermore, these findings indicate that SAM-based model systems are a valuable tool for designing and investigating the development of scaffolds that regulate the conformation of extracellular matrix cues and cellular functions that accelerate the rate of tissue regeneration.

Plasmin triggers rapid contraction and degradation of fibroblast-populated collagen lattices.

We examined the role of the serine proteinase plasmin in regulating fibroblast-mediated tissue remodeling during wound healing. As an in vitro model system, collagen lattices were seeded with human dermal fibroblasts, and various concentrations of plasmin were added to the medium of the contracting lattices. Within 16 h, fibroblast-populated collagen lattices treated with plasmin rapidly contracted from approximately 20 mm to less than 2 mm in diameter. Measurements of collagen lattices with radiolabeled collagen indicated that, when these lattices included either fibroblasts or conditioned medium derived from fibroblast-populated collagen lattices, exogenous plasmin induced collagen degradation and rapid lattice contraction. Western blot analyses of conditioned medium demonstrated that fibroblasts in collagen lattices secreted the latent matrix metalloproteinase, MMP-1, which was subsequently cleaved by plasmin. Additionally, rapidlattice contraction and collagen degradation were blocked when collagen lattices were treated simultaneously with plasmin and aprotinin or a tissue inhibitor of metalloproteinases, TIMP-1. These results provide strong evidence that plasmin regulates rapid contraction of collagen lattices by activating fibroblast-secreted MMP-1 that triggers collagen degradation. The findings from this study suggest that fibroblast-populated collagen lattices can be used as an in vitro model system to investigate the mechanisms by which plasmin and cell-secreted plasminogen activators control MMP-1 mediated extracellular lattice degradation and remodeling during wound healing.

Microfabrication of an analog of the basal lamina: biocompatible membranes with complex topographies.

A microfabrication approach was used to produce novel analogs of the basal lamina with complex topographic features. A test pattern of ridges and channels with length scales (40 to 310 micrometer) similar to the invaginations found in a native basal lamina was laser machined into the surface of a polyimide master chip. Negative replicates of the chip were produced using polydimethylsiloxane silicone elastomer and these replicates were used as templates for the production of thin ( approximately 21 micrometer) membranes of collagen or gelatin. The resulting membranes had a complex topography of ridges and channels that recapitulated the features of the master chip. To demonstrate their utility, these complex membranes were laminated to type I collagen sponges and their surfaces were seeded with cultured human epidermal keratinocytes to form a skin equivalent. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microfabricated membrane. The topography of the membrane influenced the differentiation of the keratinocytes because stratification was enhanced in the deeper channels. Membrane topography also controlled the gross surface features of the skin equivalent; infolds of the epidermis increased as channel depth increased. These novel microfabricated analogs of the basal lamina will help to elucidate the influence of topography on epithelial cell proliferation and differentiation and should have applications in the tissue engineering of skin equivalents as well as other basal lamina-containing tissues.

Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties.

Collagen is the primary structural element in extracellular matrices. In the form of fibers it acts to transmit forces, dissipate energy, and prevent premature mechanical failure in normal tissues. Deformation of collagen fibers involves molecular stretching and slippage, fibrillar slippage, and, ultimately, defibrillation. Our laboratory has developed a process for self-assembly of macroscopic collagen fibers that have structures and mechanical properties similar to rat tail tendon fibers. The purpose of this study is to determine the effects of subfibrillar orientation and decorin incorporation on the mechanical properties of collagen fibers. Self-assembled collagen fibers were stretched 0-50% before cross-linking and then characterized by microscopy and mechanical testing. Results of these studies indicate that fibrillar orientation, packing, and ultimate tensile strength can be increased by stretching. In addition, it is shown that decorin incorporation increases ultimate tensile strength of uncross-linked fibers. Based on the observed results it is hypothesized that decorin facilitates fibrillar slippage during deformation and thereby improves the tensile properties of collagen fibers.

Preparation and use of fibrin glue in surgery.

Fibrin glue (FG) is used to control bleeding, to adhere tissues together, and to seal tissue defects. FG is prepared from platelet-rich plasma or by mixing concentrated fibrinogen solutions with thrombin. Concentrated fibrinogen solutions are produced by cryoprecipitation or by chemical precipitation of plasma. The literature on FG preparation is reviewed in order to compare the advantages and disadvantages of the different products reported and to summarize the clinical applications. It is concluded that additional studies are needed to fully evaluate the advantages and disadvantages of fibrinogen concentrated using cryoprecipitation and chemical precipitation and that specific advantages exist for use of both pooled homologous and autologous blood.

Preparation of fibrin glue: a study of chemical and physical methods.

Concentrated fibrinogen was prepared from whole blood by cryoprecipitation or chemical precipitation and combined with thrombin to make fibrin glue (FG). Surgical applications of FG include control of bleeding, adhesion of tissues, and sealing of tissue defects. The purpose of this study was to compare cryoprecipitation (cryo) of fibrinogen to precipitation using ethanol, ammonium sulfate (AS), and poly(ethylene glycol) (PEG). Our results suggest that AS precipitation is as effective as cryo in yielding fibrin glues with high bond strengths and is more effective than ethanol and PEG precipitation. In addition, the volume of FG per milliliter of plasma is greater after AS precipitation than after a single freeze-thaw cycle. It is concluded that AS is an efficient means for preparing FG from autologous blood.

Collagen fibres with improved strength for the repair of soft tissue injuries.

Our laboratory has developed a process for self-assembly of high strength collagen fibres in vitro which exhibit the characteristic D period. These fibres can be cross-linked by severe dehydration (dehydrothermal cross-linking) at elevated temperature and formulated into devices used to repair soft tissues. This study was conducted to evaluate the effects of dehydrothermal cross-linking time and temperature on the tensile mechanical properties of collagen fibres. The results discussed indicate that the tensile strength of reconstituted collagen fibres is optimized by cross-linking for 5 d at 110 degrees C. Tensile strength and modulus values of 91.8 and 896 MPa are reported for fibres cross-linked in this manner. High tensile strength and modulus values are especially important in developing biodegradable materials that promote healing of orthopaedic structures.