Effect of chitosan-gluconic acid conjugate/poly(vinyl alcohol) cryogels as wound dressing on partial-thickness wounds in diabetic rats.

We previously developed chitosan cryogels from chitosan-gluconic acid conjugate without using toxic additives for wound care. In this study, we improved physiological characteristics of the previous cryogels by incorporating poly(vinyl alcohol) that also form cryogels. Mechanical strength of the cryogels was more than two times higher than that of the previous cryogels. Furthermore, the incorporation of poly(vinyl alcohol) enhanced water retention and resistance to degradation of the gels by lysozyme. The cryogels retained the favorable biological properties of the previous cryogels that they accelerate infiltration of inflammatory cells into wound sites. Time period for repairing 50 % of initial area of partial-thickness skin wound treated with the cryogels (4.0 ± 1.1 days) was shorter than those with gauze (6.5 ± 0.3 days) or a commercial hydrogel dressing (5.7 ± 0.3 days). Finally, we confirmed that incorporation of basic fibroblast growth factor into the cryogels was effective to further accelerate wound healing (2.7 ± 1.0 days). These results demonstrate that the cryogels in this study are promising for wound care.

Rapidly serum-degradable hydrogel templating fabrication of spherical tissues and curved tubular structures.

Development of the techniques for fabricating three-dimensional tissues still poses significant challenges for tissue engineering. We used hydrogels obtained from phenol-substituted amylopectin (AP-Ph) as templates for preparing multicellular spherical tissues (MSTs) and endothelialized curved tubular structures in type I collagen gel. AP-Ph hydrogel microparticles of diameter 200 µm and fibers of diameter 500 µm disappeared within hours of soaking in a serum-containing medium. HeLa cells and human endothelial cells were enclosed in the microparticles and hydrogel fibers, respectively, and then embedded in Ca-alginate microcapsules or the collagen gel. The enclosed cells were released in cavities formed by hydrogel degradation in the serum-containing medium. The released HeLa cells in the spherical cavities grew and formed MSTs, eventually filling the cavities. The spherical tissues were easily harvested by liquefying the Ca-alginate hydrogel microcapsule membrane by chelation using sodium citrate. The released endothelial cells grew on the tubular cavity surfaces and formed tubular structures. An endothelial cell network was formed by cell migration into the collagen gel. These results demonstrate the potential of serum-degradable AP-Ph hydrogels in constructing three-dimensional tissues.

Fabrication of capillary-like network in a matrix of water-soluble polymer using poly(methyl methacrylate) microfibers.

Poly(methyl methacrylate) (PMMA) microfibers were used as a template for development of a capillary-like network in agarose hydrogel. Microfibers with diameter 10-20 µm, which is comparable to the diameter of native capillary vessels, were fabricated using a wet spinning technique. The microfibers were embedded in agarose gel and dissolved by immersing the gel in dichloromethane. The resultant microchannels in the gel had the same diameter as the microfibers, and allowed an aqueous solution to be perfused through the gel. The methodology is promising for fabricating a capillary-like network in tissue engineering scaffolds of various water-soluble polymers.

Hematin is an alternative catalyst to horseradish peroxidase for in situ hydrogelation of polymers with phenolic hydroxyl groups in vivo.

Hematin, an iron-containing porphyrin used in the management of porphyria attacks, was evaluated as an alternative catalyst to horseradish peroxidase (HRP) for in situ gelation of polymers with phenolic hydroxyl (Ph) moieties in vivo. An aqueous solution of gelatin derivative with Ph moieties was gellable in the presence of both hematin and H2O2. A total of 98.6% adhesion of L929 fibroblast cells 4 h after seeding and their similar morphology to those on substrate coated with unmodified gelatin indicated no obvious substrate cytotoxicity. High cytocompatibility of the gelation process under conditions inducing gelation within 20 s was demonstrated by 95.0% viability of enclosed cells in vitro. Furthermore, no adverse effects of hematin were found compared with HRP by histological observation of cutaneous tissue surrounding in situ formed gels. The versatility of hematin for gelation of a variety of polymers possessing Ph groups was demonstrated by the gelation of a carboxymethyl cellulose derivative.

Immobilization of Pseudomonas cepacia lipase onto electrospun polyacrylonitrile fibers through physical adsorption and application to transesterification in nonaqueous solvent.

The lipase of Pseudomonas cepacia was immobilized onto electrospun polyacrylonitrile (PAN) fibers and used for the conversion of (S)-glycidol with vinyl n-butyrate to glycidyl n-butyrate in isooctane. The rate of reaction with the adsorbed lipase was 23-fold higher than the initial material. After 10 recyclings, the initial reaction rate was 80% of the original rate. This system of enzyme immobilization is therefore suitable for carrying out transesterification reactions in nonaqueous solvents.

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.

Synthesis of enzymatically-gellable carboxymethylcellulose for biomedical applications.

We synthesized a carboxymethylcellulose with phenol moieties by covalently incorporating tyramine into carboxymethylcellulose using aqueous-phase carbodiimide activation chemistry. The resulting hydrogel was obtained from an aqueous solution of the conjugate via the horseradish peroxidase-catalyzed oxidation reaction of phenols by consuming H(2)O(2), where the gelation speed depended on the concentrations of enzyme and H(2)O(2). The viability of the mammalian cells enclosed within the hydrogel prepared from 1.5% (w/v) conjugate solution containing 5 units/ml horseradish peroxidase and 1 mM H(2)O(2), was 80% after 24 h. These results demonstrate that this carboxymethylcellulose with phenol moieties has potential for biomedical applications including tissue-engineering.

Synthesis of an agarose-gelatin conjugate for use as a tissue engineering scaffold.

We synthesized a conjugate in which gelatin was covalently crosslinked to agarose using 1,1-carbonyldiimidazole (CDI) in dimethyl sulfoxide in order to obtain gels with cellular adhesiveness that showed a sol-to-gel transition, but no gel-to-sol transition, around body temperature. The gelatin content of the conjugate increased by 2.7-fold when the concentration of CDI was increased from 1.3 to 32.7 mM. Aqueous solutions of the conjugate gelled upon cooling from 40 degrees C to 20 degrees C, but did not remelt at 37 degrees C. The percentage of adhered cells after 4 h of culture on a gel created from a conjugate containing about 25 wt% gelatin was similar to that for cells grown on tissue culture dishes. The adhered cells proliferated on the conjugate gel during culture for a further 5 d. In addition, the conjugate used in this study did not result in mechanical instability of the resultant gel compared to that of an unmodified agarose gel. These results demonstrate that this agarose-gelatin conjugate is a good candidate material for tissue engineering.

Biocompatibility of subsieve-size capsules versus conventional-size microcapsules.

Biocompatibility of cell-enclosing capsules, defined as suppression of pericapsular cellular reactions, is one of the factors governing the success of enclosed cell transplantation in in vivo cell therapy. Agarose capsules of subsieve size, less than 100 microm in diameter, and conventional size, approximately 300-1,000 microm in diameter, were implanted into the peritoneal cavity and epididymal fat pads of mice and rats, respectively, to determine the effect of a reduction in diameter to subsieve size. The degree of cellular reaction to the subsieve-size capsules was much lower than that of the conventional-size microcapsules, independent of implantation site. The frequency of overgrown subsieve-size capsules retrieved from the peritoneal cavities was less than 5% in contrast to approximately 20% for capsules 387 microm in diameter. In addition, no increase in floating cells, which are generated through capsule stimulation, was observed in the peritoneal cavity only with subsieve-size capsules. From these results, we concluded that subsieve-size capsules are more biocompatible than microcapsules of conventional size.

Development of mammalian cell-enclosing subsieve-size agarose capsules (<100 microm) for cell therapy.

Agarose capsules were prepared using a droplet breakup method in a coflowing stream. Subsieve-size capsules 76+/-9 microm in diameter were obtained by extruding 4 wt% agarose solution from a needle (300 microm inner diameter) at a velocity of 1.2 cm/s into an ambient liquid paraffin flow of 20.8 cm/s. Increasing the flow rate of the liquid paraffin and decreasing that of the agarose solution resulted in a decreased resultant capsule diameter. Reduction in diameter from several hundred micrometers to subsieve-size (<100 microm) enhanced molecular exchange and mechanical stability. Measurements based on the percentage of intact mitochondria in the cells demonstrated that the viability of the enclosed cells was independent of capsule diameter. No significant difference was observed between the viabilities of cells enclosed in capsules with diameters of 79+/-8 and 351+/-41 microm (p=0.43). Compared with cells seeded in a tissue culture dish, the cells enclosed in the subsieve-size capsules showed 89.2% viability.

Higher viscous solution induces smaller droplets for cell-enclosing capsules in a co-flowing stream.

Mechanical strength of cell-enclosing capsules governs the success of the transplantation of enclosed cells in vivo for cell therapy. Mechanical strength closely correlates with the concentration and molecular weight of the polymers present in the aqueous solution that end up in the capsules, and the viscosity of the aqueous polymer solution also depends on these two factors. Three aqueous solutions differing in viscosity (1.0, 36, and 194 mPa s) were extruded from a needle (300 microm inner diameter) at a velocity of 1.2 cm/s into an ambient co-flowing liquid paraffin laminar stream. Smaller droplets were obtained from a higher viscous solution. At a liquid paraffin velocity of 23.5 cm/s, the diameter of droplets obtained from the highest viscous solution (194 mPa s)) was 44 +/- 4 microm, and it represented 40% and 20% of that from droplets in solutions of 36 and 1.0 mPa s viscosity, respectively. The cells enclosed in these droplets maintained more than 95% viability during the droplet breakup process independent of the viscosity of the aqueous solution (p > 0.50). In addition, retrieved cells from the droplets showed the same proliferation profiles as the cells that were not subjected to the droplet breakup process, on tissue culture dishes (p > 0.13).

Promotion of monolayer formation and high expression of ammonia metabolism of primary rat hepatocytes on arginine-glycine-aspartic acid-containing peptide-coated polystyrene dish.

Cell adhesive peptide Arg-Gly-Asp (RGD) was immobilized using ProNectin F (PnF) on a nontreated polystyrene petri dish at a PnF density of 20 ng cm(-2), which is sufficient for primary rat hepatocyte immobilization. The density of PnF on the dish affects cell morphology and expression of the differentiated functions within the range of 2-2500 ng cm(-2). An optimal monolayer state with defined cell boundaries and hepatocyte nuclei was formed on a 2500 ng cm(-2) PnF-coated petri dish, and the ammonia metabolic function was expressed at as high a level as in the hepatocyte/spheroid. We conclude that 2500 ng cm(-2) PnF enhances the morphological stability and expression of liver-specific functions of the hepatocyte.

Transition of mechanical property of porous alginate scaffold with cells during culture period.

The rupture forces of porous alginate scaffolds seeded with hepatocytes or fibroblast-like cells increased during 3 d of culture and then decreased. The phenomenon was independent of the number of viable cells within the scaffolds, but dependent on protein adsorption to the alginate as well as a reduction in the degree of crosslinks of the calcium-alginate gel.

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.

Alginate/aminopropyl-silicate/alginate membrane immunoisolatability and insulin secretion of encapsulated islets.

We utilized the sol-gel reaction to prepare an immunoisolatable membrane for a microcapsule-shaped bioartificial pancreas. The membrane, derived from two precursors, 3-(aminopropyl)trimethoxysilane (APTrMOS) and tetramethoxysilane (TMOS), was formed onto calcium-alginate gel beads via electrostatic interaction. The molecular weight cutoff point of less than 150 000 required for immunoisolation was achieved at molar ratios ([APTrMOS]/[TMOS]) ranging from 0.60 to 2.40 with the amount of APTrMOS fixed at 3.40 mmol/(10 mL of calcium-alginate). When encapsulated in a membrane prepared at the molar ratio of 0.60, the islets contracted in volume and showed no response to stimulation by a high glucose concentration. However, islets in a membrane prepared at the molar ratio of 2.40 showed no contraction and responded to the glucose stimulation at almost the same level as free islets. These results demonstrated that the molar ratio of the precursors was a dominant factor affecting membrane permeability and the insulin secretion activity of the encapsulated islets.

Adipose tissue engineering using adipose-derived stem cells enclosed within an injectable carboxymethylcellulose-based hydrogel.

In situ gelation of an aqueous solution of carboxymethylcellulose derivative bearing phenolic hydroxyl groups (CMC-Ph) that contained suspended adipose-derived stem cells (ASCs) was studied in vitro and in vivo for evaluating feasibility in adipose tissue-engineering strategies. The rat ASCs that were enclosed in the CMC-Ph gels through a horseradish peroxidase-catalysed reaction showed 92.8% viability, good proliferation and adipogenic differentiation in vitro. Ten weeks after the subcutaneous injection of ASCs-suspending CMC-Ph for in situ gelation, clearly visible new vascularized adipose tissue formed at the injection site. The number of blood vessels and the area occupied by adipose tissues were five and eight times larger, respectively, than those found in the implanted acellular gel. The adipogenesis and neovascularization were further enhanced by incorporation of fibroblast growth factor into the CMC-Ph gel containing ASCs.

Bone regeneration of tibial defects in rats with enzymatic hydrogelation of gelatin derivative and recombinant human platelet-derived growth factor-BB complex.

PURPOSE: To evaluate the capabilities of gelatin derivatives that incorporate phenolic hydroxyl groups (gelatin-Ph) as a delivery carrier with recombinant human platelet-derived growth factor-BB (rhPDGF-BB) for bone regeneration. MATERIALS AND METHODS: The growth factor release profile from gelatin-Ph gel or atelocollagen was analyzed to evaluate the capability of gelatin-Ph as a carrier. The biocompatibility of gelatin-Ph (ie, the survival rates of the cells during gelation, and the morphology and proliferation of the cells) were investigated with rat bone marrow cells (RBMCs) in vitro. To evaluate the effect of the gelatin-Ph-rhPDGF-BB complex on bone formation, the complex was applied to bone defects in rat tibiae and undecalcified specimens were fabricated for histologic analysis. RESULTS: Scarce amounts of rhPDGF-BB from gelatin-Ph gel were detected during 30 days, but the addition of protease induced the release of rhPDGF-BB on day 31. No differences were observed in the survival rates of RBMCs during gelation and in the morphology and proliferation of RBMCs on the gel sheet between groups. Histological analyses demonstrated that the complex enhanced bone formation and mineralization at 2 and 4 weeks. CONCLUSION: These results suggest that gelatin-Ph gel can be an ideal delivery carrier, and the localized delivery of rhPDGF-BB in gelatin-Ph can contribute to bone formation.

Production of butyl-biodiesel using lipase physically-adsorbed onto electrospun polyacrylonitrile fibers.

Butyl-biodiesel production using electrospun polyacrylonitrile fibers with Pseudomonas cepacia lipase immobilized through physical adsorption was studied. About 80% conversion to butyl-biodiesel was achieved after 24h by suspending the catalyst at 2.4 mg/mL in a mixture of rapeseed oil and n-butanol at a molar ratio of 1:3, containing water at 8000 ppm at 40 degrees C. A further 24h of operation resulted in 94% conversion. The initial reaction rate detected for this process was 65-fold faster than those detected for Novozym 435 on a total catalyst mass basis. The immobilized lipase continued to work as a catalyst for 27 d, within a 15% reduction in conversion yield at the outlet of the reactor compared with the average value detected during the first 3d of operation in a continuous butyl-biodiesel production system.

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.

Surface immobilization of poly(ethyleneimine) and plasmid DNA on electrospun poly(L-lactic acid) fibrous mats using a layer-by-layer approach for gene delivery.

The method of coating electrospun ultrafine poly(L-lactic acid) fibers with DNA, by building up polyelectrolyte layer(s) of poly(ethyleneimine) (PEI) and plasmid DNA using an electrostatic layer-by-layer deposition method, for gene delivery is presented. The pGL3 encoding luciferase was applied as plasmid DNA. The quantity of pGL3 immobilized on individual fibers increased with increasing pGL3 concentration in the immersion solution (0.017-0.870 mg/mL) and increasing bilayer number of PEI/pGL3 (single-triple). With the exception of one specimen prepared under the condition 0.870 mg/mL pGL3 solution and double PEI/pGL3 layers, the transfection efficiency of COS-7 cells, defined by the ratio of fluorescence intensity (resulting from the presence of luciferase) with respect to the quantity of cellular protein on the fibrous mat increased with increasing quantity of pGL3 on the fibers. In addition to the ease of controlling the quality of polyelectrolyte bilayer(s) by simply changing the concentrations of substances and number of immersing cycles, the features of the electrospun fibrous mat such as a very large surface-to-volume ratio and flexibility, could potentially be employed as a strategy for gene therapy combined with tissue engineering technology.