Injectable hydrogels can be useful tools for facilitating wound healing since they conform to the irregular shapes of wounds, serving as a temporary matrix during the healing process. However, the lack of inherent pore structures of most injectable hydrogels prohibits desired interactions with the cells of the surrounding tissues limiting their clinical efficacy. Here, we introduce a simple, cost-effective and highly biofunctional injectable macroporous hydrogel made of gelatin microgels crosslinked by microbial transglutaminase (mTG). Pores are created by the interstitial space among the microgels. A water-in-oil emulsion technique was used to create gelatin microgels of an average size of 250μm in diameter. When crosslinked with mTG, the microgels adhered to each other to form a bulk hydrogel with inherent pores large enough for cell migration. The viscoelastic properties of the porous hydrogel were similar to those of nonporous gelatin hydrogel made by adding mTG to a homogeneous gelatin solution. The porous hydrogel supported higher cellular proliferation of human dermal fibroblasts (hDFs) than the nonporous hydrogel over two weeks, and allowed the migration of hDFs into the pores. Conversely, the hDFs were unable to permeate the surface of the nonporous hydrogel. To demonstrate its potential use in wound healing, the gelatin microgels were injected with mTG into a cut out section of an excised porcine cornea. Due to the action of mTG, the porous hydrogel stably adhered to the cornea tissue for two weeks. Confocal images showed that a large number of cells from the cornea tissue migrated into the interstitial space of the porous hydrogel. The porous hydrogel was also used for the controlled release of platelet-derived growth factor (PDGF), increasing the proliferation of hDFs compared to the nonporous hydrogel. This gelatin microgel-based porous hydrogel will be a useful tool for wound healing and tissue engineering.

Experimental and theoretical studies on a backflush hollow-fiber enzymatic reactor (HFER) were conducted in this work for a lactose/ lactase system. An A. niger lactase was chosen, from the four lactases tested, for reversible immobilization in the sponge layers of the fibers. An enzyme loading procedure was developed that allowed reliable and reproducible operation of the hollow-fiber reactor and produced industrially significant conversions without apparent change in the activity or stability of the lactase used. This reversible immobilization scheme also permitted easy replacement of the enzyme used. The performance of the backflush HFER was investigated and a large number of data concerning its operation were obtained and interpreted. Momentum and mass transports in such a HFER were analyzed, and mathematical models that took the experimental findings into consideration were also developed and solved analytically and/or numerically. Predictions from the computer model developed in this work were found to be in excellent agreement with the experimental data collected, suggesting the possibility of a priori design of a process-scale backflush HFER. With minor modifications, the models developed are expected to be applicable to hollow-fiber reactors with a wide selection of immobilized cells, organelles, and other enzymes. C. K. S. Jones, R. Y. K. Yang, E. T. WhiteDepartment of Chemical Engineering University of Queensland St. Lucia, QLD 4067 Australia Introduction A large number of enzyme immobilization schemes involve chemical coupling of enzymes to a solid support. In addition to chemical binding, physical techniques such as adsorption, gel entrapment, and encapsulation have been used. Enzymes immobilized by encapsulation usually can expect to experience an environment similar to that of free enzymes in an aqueous solution. In particular, if the enzyme is encapsulated after the formation of an encapsulating medium, it should retain its intrinsic kinetic properties.Examples of the last method of immobilization are the hollow-fiber enzymatic reactor (HFER) investigated by Breslau and Kilcullen (1975), Robertson et al. (1976), Rony (1971), and Waterland et al. (1974. All these reactors allow the enzymes to be truly immobilized under mild conditions without causing alteration of their inherent kinetic behaviors. However, Corrspondencc concerning this paper should be addressed to R. Y. K. Yang. whose current address is Department of Chemical Engineering, West Virginia University. Morgantown. WV 26506-6101. AIChE Journal February 1988with the notable exception of the first work, all these reactor schemes relied solely on diffusion as the means of contacting substrates and enzymes. Breslau and Kilcullen have suggested a number of HFER's in which the bulk flow is an important transport mechanism. Of great interest is the backflush mode of operating a HFER, in which substrates flow from the shell of a hollow-fiber cartridge to the lumina of the fibers, and enzymes are loaded into the sponge layers of the anisotropic ...

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The use of hollow fibers for enzyme immobilization offers considerable advantages over other types of immobilization techniques. It allows enzymes to be immobilized under mild conditions without causing significant alteration of their kinetic properties and permits easy removal and replacement of the immobilized enzymes. For some enzyme-substrate combinations, with proper choice of hollow fibers and operation strategies, the method also allows simultaneous execution of reaction and separation within the same reactors.These considerations have prompted considerable research efforts over the past decade to investigate the use of commercially available hollow-fiber cartridges as reactors for carrying out enzymatic reactions (For example: Breslau and Kilcullen,* Chambers et al.,' Davis,4 Georgakis et al.,' Henley et u I . ,~ Jones and Yang,n Kohlwey and Cheryan,' Korus and Olson,'" Robertson et aI.,l4 Rony," Waterland et al.".") However, with the exception of the works by Breslau and Kilcullen involving liquid substrate and by Henley et al. and Jones and Yang involving solid substrates, all the investigated reactor schemes have used only diffusion as the major means of contacting substrates and enzymes. The backflush scheme proposed by Breslau and Kilcullen. in which bulk flow is expected to play a role in the enzymatic conversion of liquid substrates, has been investigated further in this work. Major findings are summarized here; details are described elsewhere.Lactase (P-galactosidase) was chosen as the enzyme to be immobilized in the sponge layer of the hollow fibers. Four commercially available lactases derived from different microbial sources (Escherichia coli, Kluyveromyces lactis, K. fragilis and Aspergillus niger) were evaluated with respect to their suitability for this type of immobilization. The activity and the stability of the free and the immobilized enzymes, as well as their compatibility with Amicon hollow fibers of different molecular weight cut-offs, were the major factors of consideration. "Lactase N", derived from A . niger (G. B. Fermentation), was chosen as the best candidate for the study.This lactase showed a broad range of pH tolerance with an optimum pH of 4.4 at 37°C. The temperature at which maximum activity was displayed at a pH of 4.4 was 58OC. Further kinetic studies using both the initial rate method (Allison and Purichl) and the progress curve method (Orsi and Tipton"), together with nonlinear regression analysis, confirmed that the rate expression associated with this enzyme was that of competitive product inhibition (by galactose). The values for the kinetic parameters associated with the rate expression were determined and, together with the rate expression, are shown in TABLE I . The relatively high value (approximately 30 to 80)