Transglutaminase crosslinked gelatin as a tissue engineering scaffold.

Gelatin is one of the most commonly used biomaterials for creating cellular scaffolds due to its innocuous nature. In order to create stable gelatin hydrogels at physiological temperatures (37 degrees C), chemical crosslinking agents such as glutaraldehyde are typically used. To circumvent potential problems with residual amounts of these crosslinkers in vivo and create scaffolds that are both physiologically robust and biocompatible, a microbial transglutaminase (mTG) was used in this study to enzymatically crosslink gelatin solutions. HEK293 cells encapsulated in mTG-crosslinked gelatin proliferated at a rate of 0.03 day(-1). When released via proteolytic degradation with trypsin, the cells were able to recolonize tissue culture flasks, suggesting that cells for therapeutic purposes could be delivered in vivo using an mTG-crosslinked gelatin construct. Upon submersion in a saline solution at 37 degrees C, the mTG-crosslinked gelatin exhibited no mass loss, within experimental error, indicating that the material is thermally stable. The proteolytic degradation rate of mTG-crosslinked gelatin at RT was slightly faster than that of thermally-cooled (physically-crosslinked) gelatin. Thermally-cooled gelatin that was subsequently crosslinked with mTG resulted in hydrogels that were more resistant to proteolysis. Degradation rates were found to be tunable with gelatin content, an attribute that may be useful for either long-time cell encapsulation or time-released regenerative cell delivery. Further investigation showed that proteolytic degradation was controlled by surface erosion.

Poly(vinyl alcohol) : a potential matrix for glucose oxidase immobilization?

Considerable effort has been focused on the method of immobilizing glucose oxidase (GOD) for amperometric glucose biosensors since the technique employed may influence the available activity of the enzyme and thus affect the performance of the sensor. Narrow measuring range and low current response are still considered problems in this area. In this work, poly(vinyl alcohol)(PVA) was investigated as a potential matrix for GOD immobilization. GOD was entrapped in cross-linked PVA. The use of a PVA-GOD membrane as the enzymatic component of a glucose biosensor was found to be promising in both the magnitude of its signal and its relative stability over time. The optimum PVA-GOD membrane (cross-linking density of 0.06) was obtained through careful selection of the cross-linking density of the PVA matrix.

Poly(vinyl alcohol) : a potential matrix for glucose oxidase immobilization?

Considerable effort has been focused on the method of immobilizing glucose oxidase (GOD) for amperometric glucose biosensors since the technique employed may influence the available activity of the enzyme and thus affect the performance of the sensor. Narrow measuring range and low current response are still considered problems in this area. In this work, poly(vinyl alcohol)(PVA) was investigated as a potential matrix for GOD immobilization. GOD was entrapped in cross-linked PVA. The use of a PVA-GOD membrane as the enzymatic component of a glucose biosensor was found to be promising in both the magnitude of its signal and its relative stability over time. The optimum PVA-GOD membrane (cross-linking density of 0.06) was obtained through careful selection of the cross-linking density of the PVA matrix.

Histologic evaluation of the inflammatory response around implanted hollow fiber membranes.

Hollow fiber membranes are enjoying widespread use as barrier materials in many implanted applications. In order to predict in vivo device behavior, it is important to understand and quantify the changes to the membrane and to the tissue immediately surrounding it that occur following implantation. We have considered a range of commercially available hollow fiber membranes for their suitability as candidates for subcutaneously implanted applications. Through analysis of excised tissue sections by light microscopy, membranes were screened at 3, 6, and 12 weeks post-implantation for the ability to maintain integrity, foreign-body reaction, and thickness of the external fibrotic capsule layer. The polysulfone microfiltration membranes and cellulose diacetate membranes investigated were found to be unsuitable owing to extensive degradation. All membranes exhibited typical foreign body reaction with fibrotic capsule formation. The thinnest capsules were observed on the regenerated cellulose microdialysis membranes and the polysulfone ultrafiltration membranes. Extensive cellular penetration into the membrane matrix of the latter was observed, but did not appear to affect the foreign body reaction. A heat-sealing method was also considered for thermoplastic membranes and found to effectively prevent cellular penetration into the lumen of the hollow fiber for the duration of the 12-week implantation.

Gel-impregnated pore membranes with mesh-size asymmetry for biohybrid artificial organs.

Membranes based on mechanically supported poly(vinyl alcohol) (PVA) hydrogels with mesh-size asymmetry were developed for potential application in biohybrid artificial organs. The pores of cellulose ester microfiltration membranes were impregnated with a PVA solution, which was lightly crosslinked with glutaraldehyde and then modified under a glutaraldehyde gradient to produce mesh-size asymmetry. Permeation experiments were performed with the resulting homogeneous and asymmetric gel-impregnated pore membranes (GIPMs). Creatinine (MW: 113), goat Fab (MW: 50 kD) and human IgG (MW: 150 kD) were used to simulate the molecular size of nutrients, therapeutic proteins, and immunological molecules, respectively. The transport properties of the GIPMs were compared to those of conventional ultrafiltration (UF) and dialysis membranes. Experimental results indicate that GIPMs with mesh-size asymmetry have thickness-normalized creatinine permeabilities that are slightly higher than those in cellulosic UF membranes but as much as 100% greater than those in polysulfone UF or cellulosic dialysis membranes. IgG permeabilities in the GIPMs are from 5 to 50 times lower than those in the UF membranes. Fab permeabilities are 6 to 40 times higher in the UF membranes than those in the GIPMs, but the required permeability for a therapeutic protein is application specific. GIPMs may also be suitable as an alternative for hemodialysis.

Modeling the response time of an in vivo glucose affinity sensor.

A mathematical model was developed to describe the dose-response relationship of an optical glucose sensor. The basis for glucose detection is the reversible competitive displacement of a ligand from a receptor protein with specific binding sites for certain carbohydrates. Detection of glucose is based on measurements of the change in fluorescent lifetime of the donor-labeled protein, as it binds to the acceptor-labeled ligand. The sensor was modeled as a hollow fiber membrane, permeable to glucose, which encapsulates a solution of the receptor protein and competing ligand. Model equations that describe the diffusion of glucose through the fiber membrane and the subsequent displacement reactions within the fiber lumen were solved numerically to predict the response time of the sensor following a step change in bulk glucose concentration. The incorporation of an external mass transfer boundary layer was found to increase the response time by a factor of 3.7 over the well-stirred case. On the basis of the results of a parametric study, the response time of the sensor was found to be most sensitive to the diffusion coefficient of glucose in the membrane. When compared to experimental response times for an intensity-based affinity sensor using Concanavalin A as the receptor protein and dextran as the competing ligand, the model predictions were found to be significantly shorter than those observed. The effect of the in vivo environment on the performance of the sensor was also investigated through the incorporation of a fibrotic capsule layer. The additional diffusional resistance offered by the capsular tissue resulted in a 5-fold increase in the response time of the sensor.