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.

Autoclaving-Triggered Hydrogelation of Chitosan-Gluconic acid Conjugate Aqueous Solution for Wound Healing.

Moist wound healing is known to heal wounds faster than dry wound healing. Hydrogel wound dressings are suitable for moist wound healing because of their hyperhydrous structure. Chitosan, a natural polymer, promotes wound healing by stimulating inflammatory cells and releasing bioactive compounds. Therefore, chitosan hydrogel has great potential as a wound dressing. In our previous study, physically crosslinked chitosan hydrogels were successfully prepared solely by freeze-thawing of chitosan-gluconic acid conjugate (CG) aqueous solution without using any toxic additives. Furthermore, the CG hydrogels could be sterilized by autoclaving (steam sterilization). In this study, we showed that autoclaving (121 °C, 20 min) of a CG aqueous solution simultaneously achieved gelation of the solution and sterilization of the hydrogel. Hydrogelation of CG aqueous solution by autoclaving is also physically crosslinking without any toxic additives. Further, we showed that the CG hydrogels retained favorable biological properties of the CG hydrogels prepared by freeze-thawing and subsequent autoclaving. These results indicated that CG hydrogels prepared by autoclaving were promising as wound dressings.

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.

Novel hydrophobically modified agarose cryogels fabricated using dimethyl sulfoxide.

Drug delivery systems (DDS) are devices able to adsorb therapeutic drugs in vitro before being either injected or surgically implanted into the body before releasing the drugs in vivo. Hydrogels are interesting for DDS researchers as they mimic soft tissue and can absorb large quantities of liquid. This research reported the successful fabrication of hydrophobically modified agarose (HMA) as well as the creation of a novel approach to the formation of hydrophobically modified agarose cryogels. By activating the hydroxyl groups in agarose, hydrophobic modification could occur through the bonding of the activated hydroxyl groups and the amines in fatty aldehydes. It was found that HMA was insoluble in water, and as such a new method of cryogel creation was produced using dimethyl sulfoxide. Further testing of HMA cryogels showed that cell adhesiveness and cytotoxicity were low. Adsorption tests showed that HMA cryogels had the ability to adsorb larger amounts of hydrophobic dye than unmodified agarose cryogels and that the release of the hydrophobic dye from HMA cryogels could be controlled. These results showed that the HMA cryogels made using this novel approach have the potential to be used as drug delivery systems.

Hydrophobically Modified Gelatin Particles for Production of Liquid Marbles.

The unique properties and morphology of liquid marbles (LMs) make them potentially useful for various applications. Non-edible hydrophobic organic polymer particles are widely used to prepare LMs. It is necessary to increase the variety of LM particles to extend their use into food and pharmaceuticals. Herein, we focus on hydrophobically modified gelatin (HMG) as a base material for the particles. The surface tension of HMG decreased as the length of alkyl chains incorporated into the gelatin and the degree of substitution (DS) of the alkyl chains increased. HMG with a surface tension of less than 37.5 mN/m (determined using equations based on the Young-Dupré equation and Kaelble-Uy theory) successfully formed LMs of water. The minimum surface tension of a liquid in which it was possible to form LMs using HMG particles was approximately 53 mN/m. We also showed that the liquid-over-solid spreading coefficient SL/S is a potential new factor for predicting if particles can form LMs. The HMG particles and the new system for predicting LM formation could expand the use of LMs in food and pharmaceuticals.

Optimized hydrophobically modified chitosan cryogels for strength and drug delivery systems.

This research reports the success of the fabrication of hydrophobically modified chitosan cryogel. Chitosan was modified with alkyl groups through reductive animation. By varying the alkyl chain length and the substitution degree, the resulting cryogel could be optimized for strength, and the adsorption and release of hydrophobic dye, a model for hydrophobic medicines. By optimizing these attributes, the hydrogel was found to have the potential as a biomedical material.

In situ gellable oxidized citrus pectin for localized delivery of anticancer drugs and prevention of homotypic cancer cell aggregation.

The aim of this study was to develop in situ gellable hydrogels composed of periodate oxidized citrus pectin (OP) for localized anticancer drug delivery and evaluate the potential of OP to inhibit cancer metastasis. Doxorubicin (Dox) was coupled to OP by imine bonds. Adipic dihydrazide (ADH) was used for cross-linking of the Dox-OP conjugates. The Dox-OP conjugate solution gelled within 2 min after addition of ADH. The release rate of Dox from the hydrogels was controllable by an additive amount of ADH. The released Dox retained anticancer activity. OP was shown to have a potency to prevent homotypic cancer cell aggregation compared to unmodified citrus pectin, strongly suggesting that OP released from hydrogels in vivo will inhibit cancer metastasis. These results indicate that OP hydrogels have the potential to prevent progression of primary cancer by the released Dox and generation of metastatic cancer by the released OP.

Hydrophobically-modified gelatin hydrogel as a carrier for charged hydrophilic drugs and hydrophobic drugs.

Gelatin molecules have been chemically crosslinked using potentially cytotoxic reagents to prepare stable hydrogels. Hydrophobic interaction is a means of forming physical crosslinks that is a good candidate for enhancing the stability of gelatin hydrogels without using cytotoxic chemicals. In this study, we proposed a new method to fabricate hydrogels from hydrophobically-modified gelatin (HMG) with high content of hydrophobic segments. HMG was first dissolved in dimethyl sulfoxide and poured into a vial with the desired shape. After the solution was freeze-dried, the solid construct was hydrated. The HMG hydrogel containing basic fibroblast growth factor promoted angiogenesis in vivo, indicating that the positively charged hydrophilic growth factor formed an electrostatic complex with negatively charged HMG hydrogel and was gradually released in vivo with the degradation of the hydrogel. In addition, we showed that the hydrophobic segments of HMG enhanced the adsorption of fluorescein sodium, a model for hydrophobic therapeutic agents, to the hydrogel through hydrophobic interaction. Furthermore, in vitro experiments indicated that the hydrophobic agents would be released from the hydrogel in a controlled manner in vivo. These results show that the HMG hydrogel has significant potential as a carrier for both charged hydrophilic drugs and hydrophobic drugs.

Core-Shell Beads Made by Composite Liquid Marble Technology as A Versatile Microreactor for Polymerase Chain Reaction.

Over the last three decades, the protocols and procedures of the DNA amplification technique, polymerase chain reaction (PCR), have been optimized and well developed. However, there have been no significant innovations in processes for sample dispersion for PCR that have reduced the amount of single-use or unrecyclable plastic waste produced. To address the issue of plastic waste, this paper reports the synthesis and successful use of a core-shell bead microreactor using photopolymerization of a composite liquid marble as a dispersion process. This platform uses the core-shell bead as a simple and effective sample dispersion medium that significantly reduces plastic waste generated compared to conventional PCR processes. Other improvements over conventional PCR processes of the novel dispersion platform include increasing the throughput capability, enhancing the performance and portability of the thermal cycler, and allowing for the contamination-free storage of samples after thermal cycling.

Millimeter-sized capsules prepared using liquid marbles: Encapsulation of ingredients with high efficiency and preparation of spherical core-shell capsules with highly uniform shell thickness using centrifugal force.

HYPOTHESIS: In our previous study, we prepared millimeter-sized spherical hard capsules by solidifying droplets of liquid monomer or polymer solution placed on superamphiphobic surface. Application of liquid marbles in place of the naked droplets for capsule preparation has a great potential to increase encapsulation efficiency of high volatile ingredients. Further, interfacial thermodynamic prediction of internal configuration of capsules from spreading coefficients may be effective to prepare core/shell capsule. EXPERIMENTS: Droplets of liquid monomer containing a volatile ingredient were rolled on superamphiphobic powders to prepare liquid marbles and solidified by photopolymerization. For preparation of core/shell capsules, the liquid marbles injected with an immiscible water droplet were also solidified. FINDINGS: A volatile ingredient could be encapsulated with higher efficiency than our previous method. Interfacial thermodynamic prediction of internal configuration of capsules from spreading coefficients indicated successful formation of core/shell capsules. However, photopolymerization of the liquid marbles in a static condition resulted in formation of not only core/shell capsules but also acorn-type capsules. Furthermore, the core/shell capsules were distorted and the shell thickness was not uniform. Rolling of the liquid marbles, which generated centrifugal force inside of the liquid marbles, was effective to prepare spherical capsules with highly uniform shell thickness.

Use of ANGPTL2 mRNA levels in formalin-fixed paraffin-embedded tissues as a biomarker to diagnose gastric cancer and to evaluate the extent of vascular invasion.

With the recent advances in medical technologies, gastric cancer can often be removed with minimally invasive surgical techniques when identified early. Surgery must remove all gastric cancer, since residual cancerous tissue may lead to recurrence. Resected cancerous tissues are pathologically evaluated to determine whether all cancerous areas have been removed, but such assessments are rarely straightforward, and cancer markers could inform such pathological evaluations of cancer. An ideal marker would be identifiable in formalin-fixed paraffin-embedded (FFPE) tumor tissue. The first objective of the present study was to compare levels of angiopoietin-like protein 2 (ANGPTL2) in cancerous and noncancerous areas of FFPE tissues to determine whether ANGPTL2 is a marker relevant to the pathological diagnosis of cancer. The second objective was to evaluate whether ANGPTL2 mRNA is useful as a marker of the extent of vascular invasion of gastric cancer. Out of the 15 patients studied, 12 had a higher ANGPTL2 mRNA levels in cancerous areas compared with noncancerous areas. This finding indicated that ANGPTL2 mRNA is useful as a biomarker for identifying cancerous areas in FFPE tissues, at least for male patients. Spearman's rank correlation analysis showed a significant correlation between the ANGPTL2 mRNA level and the degree of vascular invasion of cancer (r=0.66; P=0.01). In receiver operating characteristic curve analysis of the association between the ANGPTL2 mRNA level and the degree of vascular invasion, the area under the curve was 0.92 (95% confidence interval, 0.78-1.00; P=0.01), indicating a significant association. The present study demonstrates that ANGPTL2 mRNA in FFPE tissues is a potential biomarker that informs the pathological diagnosis of gastric cancer and that ANGPTL2 mRNA may be predictive of vascular invasion, which is an indicator of metastasis in gastric cancer.

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.

Efficient mixing of microliter droplets as micro-bioreactors using paramagnetic microparticles manipulated by external magnetic field.

Magnetic manipulation of paramagnetic particles had great potential for efficient bioprocessing. In this study, we stirred microliter-volume water droplets formed on a superhydrophobic surface as micro-bioreactors by using paramagnetic magnetite microparticles manipulated by an external magnetic field. We showed that magnetite microparticles in the droplets spontaneously formed rod-like aggregates, which were like commercial stir bars, in an external magnetic field and spun with rotating of magnetic field. Increasing the rotating rate of the magnetic field and increasing the concentration of the microparticles caused the microparticles to fixate at the air/water interface of the droplets and their rotation at the interface with rotating of magnetic field. The active mixing enhanced the enzyme reaction and microorganism proliferation in the droplets. These results demonstrated that manipulating the magnetite microparticles by an external magnetic field efficiently mixed the small droplets as micro-bioreactors.

Autoclavable physically-crosslinked chitosan cryogel as a wound dressing.

Moist wounds were known to heal more rapidly than dry wounds. Hydrogel wound dressings were suitable for the moist wound healing because of their hyperhydrous structure. Chitosan was a strong candidate as a base material for hydrogel wound dressings because the polymer had excellent biological properties that promoted wound healing. We previously developed physically-crosslinked chitosan cryogels, which were prepared solely by freeze-thawing of a chitosan-gluconic acid conjugate (CG) aqueous solution, for wound treatment. The CG cryogels were disinfected by immersing in 70% ethanol before applying to wounds in our previous study. In the present study, we examined the influence of autoclave sterilization (121°C, 20 min) on the characteristics of CG cryogel because complete sterilization was one of the fundamental requirements for medical devices. We found that optimum value of gluconic acid content of CG, defined as the number of the incorporated gluconic acid units per 100 glucosamine units of chitosan, was 11 for autoclaving. An increased crosslinking level of CG cryogel on autoclaving enhanced resistance of the gels to enzymatic degradation. Furthermore, the autoclaved CG cryogels retained favorable biological properties of the pre-autoclaved CG cryogels in that they showed the same hemostatic activity and efficacy in repairing full-thickness skin wounds as the pre-autoclaved CG cryogels. These results showed the great potential of autoclavable CG cryogels as a practical wound dressing.

Angiopoietin-like Protein 2 is a Useful Biomarker for Pancreatic Cancer that is Associated with Type 2 Diabetes Mellitus and Inflammation.

Pancreatic cancer is one of the tumors with the worst prognosis, with the 5-year survival rate reported to be 6%. The number of patients suffering from pancreatic cancer in recent years has continued to increase dramatically. Carbohydrate antigen 19-9 is an established biomarker of pancreatic cancer, but it does not have sufficient ability to detect pancreatic cancer at an early stage. We focused on angiopoietin-like protein 2 (ANGPTL2), which has been reported to be related to chronic inflammation and Type 2 diabetes mellitus. In this study, whether ANGPTL2 can detect early pancreatic cancer was evaluated. It was found that the concentration of serum ANGPTL2 was significantly higher in pancreatic cancer patients and tumor stage 0-I patients than in healthy individuals (5.84 ± 1.82 ng/mL vs 3.61 ± 0.64 ng/mL; P < 0.001) (5.68 ± 0.79 ng/mL vs 3.61 ± 0.64 ng/mL; P = 0.010). In addition, the diagnostic capability of serum ANGPTL2 levels for pancreatic cancer was evaluated using receiver operating characteristic (ROC) curve analysis. The area under the ROC curve (AUC) for ANGPTL2 was 0.906 (95% confidence interval (CI): 0.815-0.997; P < 0.001). To identify the risk factors for pancreatic cancer, multivariate regression models were used. Ten factors were included, and increasing age (odds ratio (OR), 1.318, 95% CI, 1.058-1.642; P = 0.014) and high ANGPTL2 levels (OR, 22.219, 95% CI, 1.962-251.659, P = 0.012) were found to be independent risk factors for pancreatic cancer, with ANGPTL2 having the strongest relationship. In addition, serum ANGPTL2 levels were strongly correlated with inflammatory markers, with blood sugar levels showing the strongest correlation with serum ANGPTL2 levels. In conclusion, this study suggested that an elevated serum ANGPTL2 level has the potential to be a biomarker capable of early detection of pancreatic cancer, and it was correlated with inflammation of the pancreas and the risk of developing diabetes mellitus.

Expression of a liver-specific function by a hepatoblastoma cell line cocultured with three-dimensional endothelialized tubes in collagen gels.

A human hepatoblastoma cell line, Hep G2, showed albumin production activity, a hepatic function, when cocultured with three-dimensional endothelialized tubes in collagen gels. The albumin production rate of the collagen-based liver-like constructs increased with increasing length of the endothelialized tube in the construct.

Novel technique for fabricating double-layered tubular constructs consisting of two vascular cell types in collagen gels used as templates for three-dimensional tissues.

Double-layered tubular constructs consisting of two vascular cell types in collagen gels were fabricated using a previously developed technique [Takei et al., Biotechnol. Bioeng., 95, 1-7 (2006)]. Histological examination suggested that shear stress (4.0 dyn/cm2) on the luminal surface of the constructs induced morphological changes in their walls.

In vitro formation of vascular-like networks using hydrogels.

Tissue engineering has great potential to create tissue and organ constructs of clinically relevant sizes. The main obstacle to creating volumetric tissue constructs was the lack of a technique for fabricating dense and perfusable vascular networks in vitro within the constructs. In significant efforts to develop such a technique, hydrogels have been used as materials for templates and support architectures of vascular-like networks because of their excellent properties, such as high biocompatibility, flexibility, and the rapid diffusion of oxygen and nutrients compared with solid materials. Herein, we reviewed current hydrogel-based strategies to fabricate vascular-like networks in vitro. The first strategy was based on the ability of vascular endothelial cells to form capillary-like tubes. The second was an engineering-based strategy that can be categorized into templating, modular assembly, microfabrication, rapid prototyping technique, and their hybrid model. Finally, we discussed future directions in tissue engineering for creating transplantable and volumetric constructs.

Vascular-like network prepared using hollow hydrogel microfibers.

One major challenge in the field of tissue engineering was the creation of volumetric tissues and organs in vitro. To achieve this goal, the development of a three-dimensional vascular-like network that extended throughout the tissue-engineered construct was essential to supply sufficient oxygen and nutrients to all of the cells in the constructs. For sufficient oxygenation and nutrition of the tissue-engineered constructs, the distance between each microvessel-like channel in the network should ideally be within 100-200 µm. In addition, the medium or blood should be perfused through the microchannels as soon as possible after the seeding of cells into the templates (scaffolds) of the constructs. In the present study, we proposed a novel technique for fabricating an engineered vascular-like network that satisfied these two requirements. The network comprised assembled hollow alginate hydrogel microfibers with mammalian cells enclosed in the gel portions. We controlled the distance between each flow microchannel (hollow core portions and interspace of the microfibers) to be within 150 µm by using microfibers with a gel thickness of approximately 50 µm. Furthermore, we confirmed that medium could be perfused into the flow channels quickly (within 10 min) after immobilization of the cells in the assembly. A human hepatoblastoma cell line (HepG2) proliferated in the gel portions of the microfibers and maintained their specific function during perfusion culture for 7 days. These results showed that the novel vascular-like networks fabricated here had the potential to allow the creation of volumetric tissues in vitro.

Necrotic regions are absent in fiber-shaped cell aggregates, approximately 100 µm in diameter.

Microscopic, fiber-shaped cell aggregates, have been used as building blocks for fabricating macroscopic three-dimensional tissue architectures, in the field of tissue engineering. In this study, we examined the occurrence of necrotic regions in the most widely used, fiber-shaped cell aggregates, approximately 100 µm in diameter. Alginate hydrogel hollow microfibers were used as templates for the cell aggregates. We demonstrated negligible necrotic region formation occurred in the cell aggregates formed in the hollow microfibers. Furthermore, we improved on previously-reported methods for preparing the hollow microfibers to avoid common microfiber tangling during the fiber preparation process.