Direct surface modification of metallic biomaterials via tyrosine oxidation aiming to accelerate the re-endothelialization of vascular stents.

Rapid in-situ re-endothelialization of coronary stents is one of the most effective approaches to inhibit late thrombosis and restenosis. Strut surfaces allowing excellent adhesion and migration of endothelial cells and endothelial progenitor cells may accelerate in-situ re-endothelialization. Here, a well-known endothelial cell adhesive peptide, Arg-Glu-Asp-Val (REDV), was directly immobilized onto metallic surfaces by means of single-step tyrosine oxidation with copper chloride (II) and hydrogen peroxide, which we recently reported as a new biomaterial modification technique. REDV immobilization on a 316L stainless steel plate improved endothelial cell adhesion and effectively suppressed platelet adhesion in vitro. In addition, a Co-Cr stent immobilized with Ac-Tyr-Gly-Gly-Gly-Arg-Glu-Asp-Val (Y-REDV) was implanted into a rabbit abdominal aorta. On 7 days postimplantation, 80% of the strut surface of the Y-REDV-immobilized stent was covered by a thin neointimal layer and was similar in appearance to native endothelium. Restenosis and late thrombosis were not observed in the Y-REDV-immobilized stent for 42 days. These findings suggest that direct immobilization of Y-REDV peptide onto metallic biomaterials by tyrosine oxidation is effective for promoting in-situ re-endothelialization in vascular stents. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 491-499, 2018.

Micro-CT evaluation of high pressure-decellularized cardiovascular tissues transplanted in rat subcutaneous accelerated-calcification model.

We have succeeded in reducing the calcification of acellular aortas or valves in porcine allogeneic system by removing the DNA and phospholipids, but its further reduction is desirable. Here, the calcification of the acellular tissue was evaluated in rat subcutaneous transplantation model which is known as calcification model. Acellular samples prepared by high-hydrostatic pressure (HHP) protocols with different washing media were implanted and the calcification was monitored under micro-computed tomography for 1 and 3 months. The amount of the calcium deposition was quantitatively evaluated by atomic absorption spectroscopy. A cell culture medium showed very good cell removal ability but led to severe calcification at 1 month, and surprisingly the calcium deposition increased as the washing period increased. This calcification was suppressed by removing the DNA fraction with high DNase concentration. On the other hand, the calcification was greatly reduced when washed with saline even at low DNase concentration after 2 weeks washing. These results suggest that the ion species in the washing medium and the residual DNase cooperatively affect the tendency of in vivo calcification, which led us to the possibility of reduced calcification of acellular cardiac tissues.

Water absorbing and quick degradable PLLA/PEG multiblock copolymers reduce the encapsulation and inflammatory cytokine production.

Biomaterials that contact with soft tissues such as postoperative adhesion prevention membrane or tissue-regenerative scaffolds should possess specific features such as hydrophilicity, mild to no immunogenicity, and quick degradability. The inflammation reaction to multiblock copolymers of poly(L-lactic acid) (PLLA) and poly(ethylene glycol), named as Multi, which we developed as a good adhesion prevention materials with a very high degradation rate were investigated and compared with usual PLLA, non-degradable polyethylene (PE), and acellular collagenous tissue (COL). Tissue encapsulation, inflammatory cell recruitment, and expression of four cytokines (IL-1ß, IL-6, IL-10, and TGFß) affecting the promotion or inhibition of inflammation and wound healing were evaluated. The thick encapsulation for PE might have related to high expression of TGFß, and it was largely reduced in the cases of PLLA and Multi. The cytokine expression pattern in PE was dominantly alternatively activated macrophage (M2) type, while expression patterns to Multi were classically activated macrophage (M1)-type dominant, as with the COL specimen. Thus, multi is a tissue compatible material in spite of the large degradability. By introducing low molecular weight PEG into PLLA as multiblock-type sequence, we successfully prepared biocompatible PLLA derivatives with high molecular weight, large degradation rate, and mild tissue responses.

Expression of the clustered NeuAcα2-3Galß O-glycan determines the cell differentiation state of the cells.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2-3Galß O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

Effects of valproic acid on gene expression during human embryonic stem cell differentiation into neurons.

The widely used antiepileptic drug valproic acid (VPA) is known to exhibit teratogenicity in the form of a failure of the neural tube in humans. Embryonic stem cells (ESCs) are reported to be a promising cell source for evaluating chemical teratogenicity, because they are capable of reproducing embryonic developmental model and enable reduction in the number of experimental animals used. We previously investigated 22 genes for which expressions are altered by teratogens, specifically focusing on neural differentiation of mouse ESCs. In the present study, expressions of the investigated genes were evaluated by quantitative real-time PCR and compared during differentiation of human ESCs into neurons with or without VPA. Under the conditions, almost all gene expressions significantly changed in VPA-containing culture. Specifically, in neural development-related genes such as DCX, ARX, MAP2, and NNAT, more than 2-fold expression was observed. The findings suggest that the genes focused on in this study may help to elucidate the teratogenic effects of VPA and might be a useful tool to analyze embryotoxic potential of chemicals in humans.

Comprehensive genetic analysis of early host body reactions to the bioactive and bio-inert porous scaffolds.

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1ß and IL-10 are important cytokines in tissue responses to biomaterials because IL-1ß promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1ß was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations.

Three-dimensional perfusion cultures of mouse and pig fetal liver cells in a packed-bed reactor: effect of medium flow rate on cell numbers and hepatic functions.

To develop a tissue-engineered bioartificial liver (BAL), perfusion cultures of mouse and pig fetal liver cells (FLCs) immobilized within a three-dimensional (3D) porous scaffold were performed utilizing a packed-bed reactor system. These FLCs were cultured under different medium flow rate conditions, and the effects of the flow rates on cell growth and the hepatic functions of the FLCs were investigated. In the cultures of mouse FLCs, the medium flow suppressed cell growth and the albumin secretion activity of the FLCs, and considerably lower albumin secretion than that in the 3D stationary control cultures was obtained in the perfusion cultures. In the case of pig FLCs, cell growth was also inhibited by the medium flow, however, the cells exhibited higher tolerance to the medium flow compared with mouse FLCs. The albumin secretion activity of pig FLCs was well maintained under an extremely low flow rate condition (4.8 mm/min in the reactor), and activity higher than the 3D stationary cultures was detected at a later stage (after 20 days in the perfusion cultures). These results revealed that FLCs are quite sensitive to medium flow and an extremely low shear stress is required for the perfusion cultures of FLCs.

Cryopreservation of fibroblasts immobilized within a porous scaffold: effects of preculture and collagen coating of scaffold on performance of three-dimensional cryopreservation.

As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs.

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 11 days 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.

Efficient proliferation and maturation of fetal liver cells in three-dimensional culture by stimulation of oncostatin M, epidermal growth factor, and dimethyl sulfoxide.

For the purpose of applying fetal liver cells (FLCs) as a cell source to tissue-engineered bioartificial livers, three-dimensional (3-D) cultures of FLCs using a porous polymer scaffold, as well as monolayer cultures as a control, were simultaneously performed. To achieve efficient growth and differentiation, the FLCs were cultured in the growth medium for the first 3 weeks and then cultured in the differentiation medium for 3 more weeks. In these cultures, stimulating factors (oncostatin M (OSM), epidermal growth factor (EGF), hepatocyte growth factor (HGF), or dimethyl sulfoxide (DMSO)) were added to the media, and their effects were examined. When the growth medium containing OSM and EGF was used, EGF stimulated the growth of FLCs synergistically with OSM. For the differentiation of FLCs into mature hepatocytes, DMSO added to the differentiation medium remarkably enhanced albumin secretion in the 3-D and monolayer cultures, although HGF was effective only in the monolayer culture. Microscopic observation proved that FLCs exhibited hepatocyte-like morphology only in the media containing DMSO. In conclusion, successive supply of the growth medium containing EGF and OSM and the differentiation medium containing DMSO efficiently induced the growth of the 3-D cultured FLCs and their differentiation into mature hepatocytes.

Effects of oncostatin M on secretion of vascular endothelial growth factor and reconstruction of liver-like structure by fetal liver cells in monolayer and three-dimensional cultures.

Vascular endothelial growth factor (VEGF) is crucial for the development and regeneration of the liver. However, there have been no reports about VEGF secretion by cultured fetal liver cells (FLCs). In the present study, the effects of oncostatin M (OSM), which strongly stimulates the growth and albumin secretion of FLCs, on VEGF secretion and morphological changes of long-term cultured FLCs were investigated under three-dimensional (3-D) and monolayer conditions. The cultured FLCs proliferated well and showed stable secretion of VEGF for up to 1 month under both monolayer and 3-D culture conditions. The addition of OSM to cultured cells strongly enhanced VEGF secretion. Compared with 3-D cultures, VEGF secretion per cell was higher in monolayer cultures. After 1 month in culture, the FLCs in 3-D cultures formed large aggregates like liver tissue, and FLCs also formed colonies and duct-like structures after several months of culture even under monolayer conditions. In conclusion, OSM stimulated the secretion of VEGF by cultured FLCs, which seemed to contribute to the development of a liver-like structure.

Three-dimensional culture of porcine fetal liver cells for a bioartificial liver.

A three-dimensional (3-D) culture experiment of porcine fetal liver cells (FLCs) was performed using a porous resin substrate, for the purpose of developing a bioartificial liver. A long-term 3-D culture and monolayer culture as the control were performed for more than 1 month. To promote cell growth and maturation, human oncostatin M (OSM), the human leukemia inhibitory factor (LIF), or cortisol was added to the cultures, and the effect of each agent on cell proliferation and liver-specific cellular functions was investigated. The cell numbers in both the monolayer and 3-D cultures increased gradually with time, irrespective of the supplementation of the stimulating agents. In the monolayer culture, the albumin secretion of FLCs decreased rapidly, and scarce activity was detected from 2 weeks onward under all culture conditions tested. In the 3-D cultures, neither human OSM nor human LIF had any definite effect on the albumin secretion of FLCs. However, in the cultures with cortisol, albumin secretion was maintained for a considerably long period. These findings suggest that a bioartificial liver can be developed by culturing porcine FLCs with cortisol as the stimulant.

Oncostatin M stimulates proliferation and functions of mouse fetal liver cells in three-dimensional cultures.

In order to develop a tissue engineered bioartificial liver (BAL), long-term three-dimensional (3-D) culture of fetal liver cells (FLCs) utilizing porous polymer as a scaffold was performed for up to 1 month. The effects of the basal medium and supplementation with oncostatin M (OSM) on the proliferation and differentiation of mouse FLCs were examined in both 3-D culture and conventional monolayer dish culture. Compared with monolayer culture, cell numbers and hepatic function of FLCs were better maintained by 3-D culture. When two kinds of basal media were tested in this study, Williams' medium E (WE) was superior to minimum essential medium alpha (alphaMEM) in expressing hepatic function of FLCs in both 3-D and monolayer cultures, although higher cell densities were obtained with alphaMEM. OSM potently stimulated both cell growth and metabolic activity, especially in 3-D culture. When WE supplemented with OSM was used for 3-D culture, albumin secretion by FLCs increased dramatically after day 5, and a high level of secretion was maintained until the end of culture. During a period of over 1 month, no decrease of albumin secretion was observed. In conclusion, this 3-D culture method was expected to be one of the realistic attempts to develop a tissue engineered BAL.