Takayuki Takei

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.

Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering.

The construction of a functional liver-tissue equivalent using tissue engineering is a very important goal because the liver is a central organ in the body. However, the construction of functional organ-scale liver tissue is impossible because it requires a high-density blood vessel network. In this study, we focused on decellularization technology to solve this problem. Decellularized liver tissue with a fine vascular tree network template was obtained using Triton X-100. The distance between each vascular structure was less than 1 mm. Endothelialization of the blood vessel network with human umbilical vein endothelial cells (HUVECs) was successfully performed without any leakage of HUVECs to the outside of the vessel structure. Furthermore, hepatocytes/spheroids could be located around the blood vessel structure. This study indicates that decellularized liver tissue is a potential scaffold for creating a practical liver tissue using tissue engineering technology.

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.

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.

Fabrication of Artificial Endothelialized Tubes with Predetermined Three‐Dimensional Configuration from Flexible Cell‐Enclosing Alginate Fibers

One possible strategy for creating three‐dimensional (3D) tissue‐engineered organs in vitro is to develop a vasculature for sufficient transport of oxygen and nutrients within these constructs. Here, we describe a novel technique to fabricate endothelialized tubes with predetermined 3D configuration, as a starting point for self‐developing capillary‐like networks in vitro. Calcium‐alginate hydrogel fibers of ca. 250 and 500 μm in diameter, enclosing bovine carotid artery vascular endothelial cells (BECs), were used as templates for endothelialized tubes. Fibers were prepared by extruding a 2% (w/v) sodium alginate solution containing BECs into a 100 mM calcium chloride solution flowing in the same direction. Fibers were embedded in type I collagen gels and enzymatically degraded by alginate lyase, resulting in channels with predetermined 3D configuration filled with a BEC suspension. Cells attached to and covered the surfaces of the channels. Exposing the cells to medium containing basic fibroblast growth factor resulted in their migration into the ambient collagen gel and self‐assembly into capillary‐like structures. These results demonstrate that using artificial endothelialized tubes with predetermined 3D configuration, as a starting point for a self‐developing capillary‐like network, could be potentially useful for constructing 3D tissue‐engineered organs.

Growth factor/heparin-immobilized collagen gel system enhances viability of transplanted hepatocytes and induces angiogenesis

Hepatocyte transplantation is being explored as a treatment strategy for end-stage liver disease; however, the main limitation is the insufficient vascularization of transplanted hepatocytes. To overcome this problem, a suitable 3D microenvironment and the types of transplanted cells must be considered for hepatocyte transplantation. In this study, a growth factor (GF)/heparin-immobilized collagen gel-filled polyurethane foam (PUF) scaffold was developed for angiogenesis induction and hepatocyte transplantation. First, a vascular endothelial growth factor (VEGF)/heparin-immobilized, collagen-gel-filled PUF scaffold was developed to establish a prevascularized cavity in the subcutaneous space in rats. Second, accompanied by 70% partial hepatectomy (PH), hepatocytes were embedded inside heparin-immobilized, collagen-gel-filled PUF scaffolds, and were transplanted into the VEGF-induced prevascularized cavity. The benefits of using this system were confirmed by using three types of hepatocytes, namely single hepatocyte, hepatocyte spheroids, and fetal hepatocytes. The normalized hemoglobin content and live nucleus numbers were determined separately to evaluate the angiogenesis and viability of transplanted hepatocytes. In summary, after PH pretreatment, transplantation of fetal hepatocyte-embedded, heparin-immobilized, collagen-gel-filled PUF scaffold into a VEGF-induced prevascularized cavity appears to be a promising strategy for future liver tissue engineering.

Effect of a hepatocyte growth factor/heparin-immobilized collagen system on albumin synthesis and spheroid formation by hepatocytes

A hepatocyte growth factor (HGF)/heparin-immobilized collagen system was used as a synthetic extracellular matrix for hepatocyte culture. The albumin synthesis, nucleus numbers and morphology of the hepatocytes were determined separately to evaluate the hepatocyte number and hepatocyte-specific function under this system. The benefits of the HGF/heparin-immobilized collagen system for hepatocyte culture were confirmed by three types of culture methods in vitro, namely 2D film cultures, 2D gel cultures and 3D gel cultures. In 2D collagen film cultures, hepatocytes exhibited the highest albumin synthesis (1.42 microg/well/day) in HGF/heparin-immobilized collagen films at 7 days of culture. Heparin inhibited hepatocyte adhesion while HGF promoted hepatocyte migration, and spheroid formation was easily detected in HGF/heparin-immobilized collagen films. In 2D collagen gel cultures, albumin synthesis of around 15 microg/well/day was detected and maintained for more than 18 days on HGF/heparin-immobilized collagen gels. Similar findings were obtained in 3D HGF/heparin-immobilized collagen gel cultures, which exhibited albumin synthesis of up to 30 microg/well/day. The albumin synthesis by hepatocytes was two-fold higher in 3D gel cultures compared with 2D gel cultures, and was maintained for over 2 weeks compared with 2D film cultures using the HGF/heparin-immobilized collagen system. Taken together, the HGF/heparin-immobilized collagen system was effective for albumin synthesis by hepatocytes in both 2D film cultures and 3D gel cultures, and therefore shows good potential for tissue engineering use.

In situ gellable sugar beet pectin via enzyme-catalyzed coupling reaction of feruloyl groups for biomedical applications

In situ gellable hydrogels are more attractive in many biomedical and biopharmaceutical applications than pre-formed hydrogels because they can be implanted simply by injection and allow homogeneous incorporation of bioactive materials. In this study, the potential suitability of in situ gellable sugar beet pectin (SBP) for biomedical and biopharmaceutical applications was investigated. SBP aqueous solution gelled within 1 min after addition of appropriate amounts of horseradish peroxidase (HRP) and H₂O₂ via HRP-catalyzed oxidative coupling reaction of feruloyl groups on SBP molecules. The resultant gels gradually degraded under simulated physiological condition. L929 fibroblast cells encapsulated in the gels were scarcely damaged during the gelation process. A subcutaneously injected mixture of SBP, HRP and H₂O₂ solutions successfully gelled, and the gel did not induce necrosis in the surrounding tissue 1 week after implantation. These results demonstrate that the in situ gellable SBP gels are useful for biomedical and biopharmaceutical applications.

Base structure consisting of an endothelialized vascular-tree network and hepatocytes for whole liver engineering.

Reconstructed liver has been desired as a liver substitute for transplantation. However, reconstruction of a whole liver has not been achieved because construction of a vascular network at an organ scale is very difficult. We focused on decellularized liver (DC-liver) as an artificial scaffold for the construction of a hierarchical vascular network. In this study, we obtained DC-liver and the tubular network structure in which both portal vein and hepatic vein systems remained intact. Furthermore, endothelialization of the tubular structure in DC-liver was achieved, which prevented blood leakage from the tubular structure. In addition, hepatocytes suspended in a collagen sol were injected from the surroundings using a syringe as a suitable procedure for liver cell inoculation. In summary, we developed a base structure consisting of an endothelialized vascular-tree network and hepatocytes for whole liver engineering.

Lactic acid bacteria-enclosing poly(ɛ-caprolactone) microcapsules as soil bioamendment

Free plant growth-promoting bacteria in soil bioamendments (SBA) are easily outnumbered by competitors and predators in agricultural soils. Microencapsulation of the bacteria is an effective technique that provides a suitable microenvironment for their survival. In this study, we attempted to prepare poly(epsilon-caprolactone) (PCL) microcapsules enclosing lactic acid bacteria (LAB), a plant growth-promoting bacteria, using the solvent-evaporation method via water-in-oil-in-water (W/O/W) emulsion. Three preparation parameters in the emulsion system were optimized based on the lactic acid production activity of the encapsulated LAB. A sodium alginate aqueous solution suspending the bacteria, a dichloromethane solution with dissolved PCL, and a poly(vinyl alcohol) aqueous solution were used as the inner aqueous phase (W(i)), the oil phase (O), and the outer aqueous phase (W(o)), respectively. Suitable volume ratio of W(i) to O, concentration of sodium alginate in W(i), and the molecular weight of PCL in O were 0.1, 1.0%, and 40 kDa, respectively. The lactic acid production activity of the microcapsules prepared under the optimized conditions was approximately nine times higher than that of commercial SBA. Application to soil demonstrated that the microcapsules are effective in the removal of the root-knot nematodes.