Koei Kawakami

An injectable, in situ enzymatically gellable, gelatin derivative for drug delivery and tissue engineering

A phenolic hydroxyl group was incorporated into gelatin, using aqueous-phase carbodiimide activation chemistry, to obtain in situ gellable and injectable protein-based materials for drug delivery and tissue engineering applications. By this means, gelatin derivatives that were gellable via a peroxidase-catalyzed reaction were obtained. The enzymatically cross-linked gelatin gels did not melt at 37 degrees C and showed tunable proteolytic degradability. The time necessary for gelation decreased with increasing content of the phenolic hydroxyl (Ph) group, peroxidase concentration and decreasing H(2)O(2) concentration. Resistance to gel compression also depended on the content of Ph groups, with the gel containing the lowest Ph group content showing the greatest resistance to compression. We encapsulated L929 fibroblast cells in gelatin gels under conditions that induced gelation in about 10 s. The encapsulated cells showed about 95% viability. In addition, L929 cells seeded on the gels showed the same growth profiles as those seeded on an unmodified gelatin-coated dish. Subcutaneous rodent injection experiments demonstrated successful in situ formation of gels at the injected site.

Synthesis and transport characterization of alginate/aminopropyl-silicate/alginate microcapsule: application to bioartificial pancreas.

To develop a novel type of immunoisolation membrane for a microcapsule-shaped bioartificial pancreas, we attempted to use a sol-gel synthesized silicate. An aminopropyl-silicate membrane derived from 3-aminopropyltrimethoxysilane and tetramethoxysilane was formed on Ca-alginate gel beads via electrostatic interaction. The positively charged amino groups remaining on the surface of the resultant gel beads were neutralized by immersion in an aqueous Na-alginate solution. From measurements of the partition coefficients and effective diffusivities of different substances to the gel beads, it was found that the aminopropyl-silicate membrane prepared under optimized composition of silicon alkoxide precursors successfully rejected gamma-globulin, giving good permeability to substances having a low molecular weight. Islets could be encapsulated within the newly developed microcapsules while retaining their ability to secrete insulin.

Novel chitosan derivative soluble at neutral pH and in-situ gellable via peroxidase-catalyzed enzymatic reaction

Chitosan with phenolic hydroxyl groups (Chit-Ph) was synthesized by conjugating chitosan with 3-(p-hydroxyphenyl)propionic acid using aqueous-phase carbodiimide activation chemistry. By this conjugation, we could obtain chitosan derivatives soluble at neutral pH and gellable via a peroxidase-catalyzed reaction within seconds: The Chit-Ph with higher content of Ph groups showed higher solubility at neutral pH. The neutral Chit-Ph solutions gelated via an enzymatic reaction by consuming H2O2. The time necessary for gelation decreased with increasing content of Ph groups, temperature, peroxidase concentration, and decreasing H2O2 concentration. The content of Ph groups also influenced the mechanical property of the resultant gels. In addition, L929 cells, which were contacted to the enzymatically crosslinked Chit-Ph gels for 20 h, showed 96.5% viability, and were observed to grow next to the gels. These results demonstrate that Chit-Ph has the potency for use as injectable hydrogels in applications such as tissue engineering and drug delivery.

In vitro and in vivo evaluation of alginate/sol-gel synthesized aminopropyl-silicate/alginate membrane for bioartificial pancreas

Alginate/aminopropyl-silicate/alginate (Alg/AS/Alg) membrane was prepared on Ca-alginate gel beads by a sol-gel process. The membrane has identical to Si-O-Si identical to bonds as well as electrostatic bonds between amino groups of AS and carboxyl groups of alginate. Permeability and stability were investigated for the membrane. Furthermore, rat islets encapsulated in the membrane (499 +/- 32 microns in diameter, 1000 islets/recipient) were transplanted to the peritoneal cavities of the mice with streptozotocin-induced diabetes. Our data show that the membrane had the molecular weight cut-off point of between 70 and 150 kDa, and hardly inhibited the permeation of glucose and insulin. The Alg/AS/Alg microcapsule was more stable than the well-known Alg/poly-L-lysine (PLL)/Alg microcapsule. After 30 days of soaking in stimulated body fluid, the percentages of intact microcapsule were 98.4 +/- 0.5 (mean +/- SEM)% and 88.0 +/- 1.5% (p < 0.001) for the Alg/AS/Alg and Alg/PLL/Alg microcapsules, respectively. The maximum maintenance period of normoglycemia was 105 days without administration of immunosuppressive drugs.

Enzymatically crosslinked carboxymethylcellulose-tyramine conjugate hydrogel: cellular adhesiveness and feasibility for cell sheet technology.

An aqueous solution of carboxylmethylcellulose with phenolic hydroxyl groups (CMC-Ph) is gellable within 1 min via a peroxidase-catalyzed oxidative reaction under mild conditions suitable for mammalian cells. In this research, we evaluated cellular adhesion and proliferation on the resultant hydrogel, and the feasibility of the hydrogel as a substrate for cell sheet technology. Within 4 h of seeding, 76.9% of L929 fibroblast cells adhered to the gel and showed similar morphology of spreading to that on cell culture dish. Subsequently, the adherent cells proliferated on the gel and formed a confluent monolayer after 168 h of culture. From the confluent monolayer we could harvest a cell sheet after about 5 min of digestion of the gel using cellulase dissolved in medium at 5 U ml(-1). The cells in the cell sheet showed well-preserved morphology similar to that shown before they were detached from the gel. In addition, the harvested cell sheet readhered and proliferated after being transferred to another culture dish. These results demonstrate that CMC-Ph gel is a good candidate material for obtaining cell sheets.

Oxidized alginate-cross-linked alginate/gelatin hydrogel fibers for fabricating tubular constructs with layered smooth muscle cells and endothelial cells in collagen gels.

Hydrogel fibers that possessed a cell-adhesive surface and were degradable via enzymatic reactions were developed for fabricating tubular constructs with smooth muscle cell (SMC) and endothelial cell (EC) layers, similar to native blood vessels, in collagen gels. The fibers were prepared by soaking hydrogel fibers prepared from a solution of sodium alginate and gelatin containing bovine ECs (BECs) in medium containing oxidized alginate (AO). BECs soaked in 8.0% (w/v) AO showed no reduction in viability within 3 h of soaking. Furthermore, mouse SMCs (MSMCs) adhered and proliferated on the AO-cross-linked hydrogels. Based on these results, we prepared AO-cross-linked hydrogel fibers containing BECs, covered their surface with MSMCs, and embedded them in collagen gels. We then degraded the fibers using alginate lyase to obtain channels in the collagen gels. Histological analysis of the released ECs using a specific fluorescent dye revealed the formation of tubular structures with layered BECs and MSMCs.

Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil.

BackgroundThe enzymatic production of biodiesel through alcoholysis of triglycerides has become more attractive because it shows potential in overcoming the drawbacks of chemical processes. In this study, we investigate the production of biodiesel from crude, non-edible Jatropha oil and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substituted hydrophobic silica monolith. We also evaluate the performance of a lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel.ResultsThe Jatropha oil used contained 18% free fatty acids, which is problematic in a base-catalyzed process. In the lipase-catalyzed reaction, the presence of free fatty acids made the reaction mixture homogeneous and allowed bioconversion to proceed to 90% biodiesel yield after a 12 hour reaction time. The optimal molar ratio of methanol to oil was 3.3 to 3.5 parts methanol to one part oil, with water content of 0.6% (w/w). Further experiments revealed that B. cepacia lipase immobilized in hydrophobic silicates was sufficiently tolerant to methanol, and glycerol adsorbed on the support disturbed the reaction to some extent in the present reaction system. The continuous production of biodiesel was performed at steady state using a lipase-immobilized silica monolith bioreactor loaded with 1.67 g of lipase. The yield of 95% was reached at a flow rate of 0.6 mL/h, although the performance of the continuous bioreactor was somewhat below that predicted from the batch reactor. The bioreactor was operated successfully for almost 50 days with 80% retention of the initial yield.ConclusionsThe presence of free fatty acids originally contained in Jatropha oil improved the reaction efficiency of the biodiesel production. A combination of B. cepacia lipase and its immobilization support, n-butyl-substituted silica monolith, was effective in the production of biodiesel. This procedure is easily applicable to the design of a continuous flow-through bioreactor system.

Enzymatically fabricated and degradable microcapsules for production of multicellular spheroids with well-defined diameters of less than 150 μm

Microcapsules with a single, spherical hollow core less than 150 microm in diameter were developed to obtain multicellular spheroids with well-defined sizes of less than 150 microm in diameter. An aqueous solution of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) containing human hepatoma cell line (HepG2) cells and horse radish peroxidase (HRP) was injected into a coflowing stream of liquid paraffin, containing H(2)O(2), resulting in cell-enclosing CMC-Ph microparticles, 135 microm in diameter, via a peroxidase-catalyzed crosslinking reaction. The CMC-Ph microparticles were then coated with a phenolic hydroxyl derivative of alginate (Alg-Ph) gel membrane several dozen micrometers in thickness, crosslinked via the same enzymatic reaction process, followed by further crosslinking between the carboxyl groups of alginate by Sr(2+). A hollow core structure was achieved by immersing the resultant microcapsules in a medium containing cellulase, which degrades the enclosed CMC-Ph microparticles. The HepG2 cells in the microcapsules then grew and completely filled the hollow core. Multicellular spheroids the same size as the CMC-Ph microparticles, with living cells at their outer surface, were collected within 1 min by soaking them in a medium containing alginate lyase to degrade the Alg-Ph gel microcapsule membrane.

Control of molecular weight cut-off for immunoisolation by multilayering glycol chitosan-alginate polyion complex on alginate-based microcapsules

Glycol chitosan is a positively charged polysaccharide which is water-soluble at pH 7.4, and is able to form a polyion complex (PIC) with anionic polymers, such as alginate. The authors attempt to develop a novel type of alginate-based microcapsule using this glycol chitosan for a islets-encapsulated bioartificial pancreas. The number of layers composed of glycol chitosan-alginate (GC-Alg) PIC were optimized, in order to cut off immunoglobulin transport and to protect encapsulated islets from the host immune reaction, and the transport characteristics were evaluated of glucose, bovine serum albumin (BSA) and gammaglobulin. To add mechanical stability to the microcapsule, calcium ions, which crosslinked the alginate polymers close to the interface between core Caalginate and multilayered membrane, were partially substituted with barium ions after the formation of multilayered Ca-alginate gel beads. The partition coefficients of BSA and gamma-globulin were decreased with the increasing number of layers. The immunoisolation was achieved againstgamma-globulin with four layers of the GC-Alg PIC membrane, while BSA could permate the membrane. The four-layered Ba-alginate gel bead had a good permeability for glucose, giving a diffusion coefficient corresponding to 80% of that in pure water. Insulin secretion from the islets in the four-layered Ba-alginate microcapsule was satisfactorily observed with the fractional stimulation ratio of 2.17. This result indicates that the encapsulated islets maintained their viability even after encapsulation. It was, thus, shown that the Ba-alginate microcapsule with four layers of the GC-Alg PIC membrane is promising as the microencapsulation material for a bioartificial pancreas.Glycol chitosan is a positively charged polysaccharide which is water-soluble at pH 7.4, and is able to form a polyion complex (PIC) with anionic polymers, such as alginate. The authors attempt to develop a novel type of alginate-based microcapsule using this glycol chitosan for a islets-encapsulated bioartificial pancreas. The number of layers composed of glycol chitosan-alginate (GC-Alg) PIC were optimized, in order to cut off immunoglobulin transport and to protect encapsulated islets from the host immune reaction, and the transport characteristics were evaluated of glucose, bovine serum albumin (BSA) and gamma-globulin. To add mechanical stability to the microcapsule, calcium ions, which crosslinked the alginate polymers close to the interface between core Ca-alginate and multilayered membrane, were partially substituted with barium ions after the formation of multilayered Ca-alginate gel beads. The partition coefficients of BSA and gamma-globulin were decreased with the increasing number of layers. The immunoisolation was achieved against gamma-globulin with four layers of the GC-Alg PIC membrane, while BSA could permeate the membrane. The four-layered Ba-alginate gel bead had a good permeability for glucose, giving a diffusion coefficient corresponding to 80% of that in pure water. Insulin secretion from the islets in the four-layered Ba-alginate microcapsule was satisfactorily observed with the fractional stimulation ratio of 2.17. This result indicates that the encapsulated islets maintained their viability even after encapsulation. It was, thus, shown that the Ba-alginate microcapsule with four layers of the GC-Alg PIC membrane is promising as the microencapsulation material for a bioartificial pancreas.

Synthesis of a chitosan derivative soluble at neutral pH and gellable by freeze–thawing, and its application in wound care

Conventional chitosan hydrogels exhibit an acidic nature and contain unfavorable additives because (i) chitosan is soluble only in acidic solutions and (ii) toxic chemicals or proteins of non-human origin that serve as antigens are necessary for preparing chitosan hydrogels. These characteristics of the chitosan hydrogels limit their possibilities as wound dressings. In this study, a chitosan-gluconic acid conjugate is developed, soluble in an aqueous solution at neutral pH and gellable by freeze-thawing (cryogelation) without using additives. The viability of L929 fibroblasts cultured in the presence of the chitosan derivative for 24 h was >96%. The degradation rate of the corresponding chitosan cryogels by lysozyme was tunable via the derivative concentration in the gels. The gels had low cellular adhesiveness. The gels promoted the accumulation of inflammatory cells such as polymorphonuclear leukocytes, which have the potential to release chemical mediators effective for wound healing, in full-thickness skin wounds in rats and accelerated the healing of the wounds. These results demonstrate that cryogels are promising for wound care.