Atsushi Mahara

In vivo tissue response and degradation behavior of PLLA and stereocomplexed PLA nanofibers.

Biocompatibility of PLLA and stereocomplexed PLA nanofibers was evaluated by subcutaneous implantation in rats for 4-12 weeks. Characterization of the nanofibers was performed by GPC, SEM, wide-angle X-ray diffraction, and optical microscopy of hematoxylin-eosin stained ultrathin sections of explanted nanofibers. Stereocomplexed PLA nanofiber showed slower degradation than PLLA nanofiber and thus retained their shape after prolonged implantation. Furthermore, stereocomplexed PLA nanofiber caused milder inflammatory reaction than PLLA nanofiber. These results offer the potential use of PLLA and stereocomplexed PLA nanofibers as a biomaterial for short-term and long-term tissue regeneration, respectively. Stereocomplexed PLA nanofiber after in vitro degradation showed smaller degree of swelling than PLLA nanofiber. Taking the results of in vivo degradation together with in vitro degradation into consideration, bioabsorption mechanism of the in vivo degradation of the nanofibers is proposed.

Liver-targeted siRNA delivery by polyethylenimine (PEI)-pullulan carrier

Recently, small interfering RNA (siRNA)-based therapeutics have been used to treat diseases. Efficient and stable siRNA delivery into disease cells is important in the use of this agent for treatment. In the present study, pullulan was introduced into polyethylenimine (PEI) for liver targeting. PEI/siRNA or pullulan-containing PEI/siRNA complexes were delivered into mice through the tail vein either by a hydrodynamics- or non-hydrodynamics-based injection. The incidence of mortality was found to increase with an increase in the nitrogen/phosphorus (N/P) ratio of PEI/siRNA complexes. Moreover, the hydrodynamics-based injection increased mice mortality. Introduction of pullulan into PEI dramatically reduced mouse death after systemic injection. After systemic injection, the PEI/fluorescein-labeled siRNA complex increased the level of fluorescence in the lung and the PEI-pullulan/siRNA complex led to an increased fluorescence level in the liver. These results suggest that the PEI-pullulan polymer may be a useful, low toxic means for efficient delivery of siRNA into the liver.

Tissue-engineered acellular small diameter long-bypass grafts with neointima-inducing activity

Researchers have attempted to develop efficient antithrombogenic surfaces, and yet small-caliber artificial vascular grafts are still unavailable. Here, we demonstrate the excellent patency of tissue-engineered small-caliber long-bypass grafts measuring 20-30 cm in length and having a 2-mm inner diameter. The inner surface of an acellular ostrich carotid artery was modified with a novel heterobifunctional peptide composed of a collagen-binding region and the integrin α4β1 ligand, REDV. Six grafts were transplanted in the femoral-femoral artery crossover bypass method. Animals were observed for 20 days and received no anticoagulant medication. No thrombogenesis was observed on the luminal surface and five cases were patent. In contrast, all unmodified grafts became occluded, and severe thrombosis was observed. The vascular grafts reported here are the first successful demonstrations of short-term patency at clinically applicable sizes.

Continuous separation of cells of high osteoblastic differentiation potential from mesenchymal stem cells on an antibody-immobilized column.

Here, we report that two distinctive cell populations with osteoblastic differentiation ability were found in adherent cell populations from bone marrow. Mesenchymal stem cells (MSCs) were conventionally isolated by using adherent property of bone marrow cells onto a plastic culture dish. MSCs enriched on the basis of their adherent property were considered phenotypically and functionally heterogeneous. We developed a ligand-immobilized surface for separating subpopulation of adherent cells derived from bone marrow by the cell rolling process. We successfully isolate two cell populations with high differentiation ability for osteoblasts in adherent bone marrow cells by using the anti-CD34 antibody-immobilized column. The antibody was covalently conjugated with polyacrylic acid and introduced onto the inner surface of a silicone tube. When cell suspension of MSCs was injected into the antibody-immobilized column, different cell populations were isolated. After the cultivation of isolated cells in the osteoblastic differentiation medium for 1 week, few sub-populations were strongly induced to form osteoblastic cells. This study revealed that the ligand-immobilized surface can be used to continually separate cell populations under a labeling-free condition.

Beating behavior of primary neonatal cardiomyocytes and cardiac-differentiated P19.CL6 cells on different extracellular matrix components

Stem cell-based therapy in cardiac tissue engineering is an emerging field that shows great potential for treating heart diseases. However, even preliminary issues, such as the ideal niche for cardiomyocytes, have not been clarified yet. In the present study, the effects of extracellular matrix (ECM) components on the beating duration of neonatal rat cardiomyocytes (RCMs) and on the cardiac differentiation of P19.CL6 carcinoma stem cells were studied. RCMs were cultured on gelatin-, fibronectin-, and collagen type I-coated dishes and on noncoated polystyrene dishes, and their beating rate, beating duration, and cardiac gene expression were evaluated. The beating period and the expression of troponin T type-2 (TNNT2) and troponin C type-1 (TNNC1) of cardiomyocytes cultured on gelatin-coated dishes were longer and higher than for those on dishes with other coatings. For the cardiac differentiation of P19.CL6 cells, troponin T type-2 expression on gelatin- and fibronectin-coated dishes was five times that on collagen type I-coated dishes or polystyrene dishes 11days after induction. These results indicate that a gelatin-coated surface has a high ability not only to maintain the cardiac phenotype but also to enhance cardiac differentiation.

Design and characterization of a polymeric MRI contrast agent based on PVA for in vivo living‐cell tracking

A novel water-soluble MRI contrast agent for in vivo living cell tracking was developed. Unlike the conventional in vivo cell tracking system based on superparamagnetic iron oxide beads, the newly developed contrast agent is eliminated from the body when the contrast agent exits the cells upon cell death, which makes living cell tracking possible. The contrast agent is composed of gadolinium chelates (Gd-DOTA) and a water-soluble carrier, poly(vinyl alcohol) (PVA), which is known to interact with cells and tissues very weakly. Since the Gd-PVA was not taken up by cells spontaneously, the electroporation method was used for cell labeling. The delivered Gd-PVA was localized only in the cytosolic compartment of growing cells with low cytotoxicity and did not leak out of the living cells for long periods of time. This stability may be due to the weak cell-membrane affinity of Gd-PVA, and did not affect cell proliferation at all. After cell labeling, signal enhancement of cells was observed in vitro and in vivo. These results indicate that Gd-PVA can visualize only the living cells in vivo for a long period of time, even in areas deep within large animal bodies.

Biodistribution of vaccines comprised of hydrophobically-modified poly(γ-glutamic acid) nanoparticles and antigen proteins using fluorescence imaging

Fluorophores-modified nanoparticles comprised of poly(γ-glutamic acid)-phenylalanine (γ-PGA-Phe-633) and ovalbumin (OVA-750) termed NPs-633/OVA-750 were prepared to assess their biodistribution using an in vivo fluorescence imager. Dynamic light scattering measurements indicated that NPs-633/OVA-750 were about 200nm in diameter. The release of encapsulated OVA from NPs-633 in PBS was negligible (∼10%) for a week. When subcutaneously injected, the localization period of OVA-750-encapsulated into NPs-633 at the site of injection (SOI) was much longer than that of free OVA-750, but was shorter as compared to a mixture with aluminum hydroxide. The NPs-633 disappeared at the SOI and major organs within 1month after administration. Moreover, intravenously and intraperitoneally administered NPs-633 were mainly observed at the liver, and there was more rapid clearance from all organs as compared with non-biodegradable NPs. These fast clearance and degradation characteristics of γ-PGA-Phe NPs will be important not only for avoiding undesired adverse effects, but also for inducing a strong vaccine effect.

Recombinant Resilin-Based Bioelastomers for Regenerative Medicine Applications.

The outstanding elasticity, excellent resilience at high-frequency, and hydrophilic capacity of natural resilin have motivated investigations of recombinant resilin-based biomaterials as a new class of bio-elastomers in the engineering of mechanically active tissues. Accordingly, here the comprehensive characterization of modular resilin-like polypeptide (RLP) hydrogels is presented and their suitability as a novel biomaterial for in vivo applications is introduced. Oscillatory rheology confirmed that a full suite of the RLPs can be rapidly cross-linked upon addition of the tris(hydroxymethyl phosphine) cross-linker, achieving similar in situ shear storage moduli (20 k ± 3.5 Pa) across various material compositions. Uniaxial stress relaxation tensile testing of hydrated RLP hydrogels under cyclic loading and unloading showed negligible stress reduction and hysteresis, superior reversible extensibility, and high resilience with Youngs moduli of 30 ± 7.4 kPa. RLP hydrogels containing MMP-sensitive domains are susceptible to enzymatic degradation by matrix metalloproteinase-1 (MMP-1). Cell culture studies revealed that RLP-based hydrogels supported the attachment and spreading (2D) of human mesenchymal stem cells and did not activate cultured macrophages. Subcutaneous transplantation of RLP hydrogels in a rat model, which to our knowledge is the first such reported in vivo analysis of RLP-based hydrogels, illustrated that these materials do not elicit a significant inflammatory response, suggesting their potential as materials for tissue engineering applications with targets of mechanically demanding tissues such as vocal fold and cardiovascular tissues.

The Rapid Inactivation of Porcine Skin by Applying High Hydrostatic Pressure without Damaging the Extracellular Matrix

We previously reported that high hydrostatic pressure (HHP) of 200 MPa for 10 minutes could induce cell killing. In this study, we explored whether HHP at 200 MPa or HHP at lower pressure, in combination with hyposmotic distilled water (DW), could inactivate the skin, as well as cultured cells. We investigated the inactivation of porcine skin samples 4 mm in diameter. They were immersed in either a normal saline solution (NSS) or DW, and then were pressurized at 100 and 200 MPa for 5, 10, 30, or 60 min. Next, we explored the inactivation of specimens punched out from the pressurized skin 10 × 2 cm in size. The viability was evaluated using a WST-8 assay and an outgrowth culture. The histology of specimens was analyzed histologically. The mitochondrial activity was inactivated after the pressurization at 200 MPa in both experiments, and no outgrowth was observed after the pressurization at 200 MPa. The arrangement and proportion of the dermal collagen fibers or the elastin fibers were not adversely affected after the pressurization at 200 MPa for up to 60 minutes. This study showed that a HHP at 200 MPa for 10 min could inactivate the skin without damaging the dermal matrix.

Complete Cell Killing by Applying High Hydrostatic Pressure for Acellular Vascular Graft Preparation

Pressure treatment has been developed in tissue engineering application. Although the tissue scaffold prepared by a ultrahydrostatic pressure treatment has been reported, an excessive pressure has a potential to disrupt a structure of extracellular matrix through protein denaturation. It is important to understand the suitable low-pressure condition and mechanisms for cell killing. In this study, cellular morphology, mitochondria activity, and membrane permeability of mammalian cells with various pressure treatments were investigated with in vitro models. When the cells were treated with a pressure of 100 MPa for 10 min, cell morphology and adherence were the same as an untreated cells. Dehydrogenase activity in mitochondria was almost the same as untreated cells. On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed. However, green fluorescence was observed in the live/dead staining images, and the cells were completely stained as red after above 500 MPa. That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa. These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity.