Abstract Hydrogels are used as cell culture scaffolds for tissue engineering and regeneration. Th... more Abstract Hydrogels are used as cell culture scaffolds for tissue engineering and regeneration. These hydrogel designs are inevitably complicated because favorable scaffolds should have stiffness to sustain alignment of the cells and mimic the structure of the extracellular matrix (ECM) in the targeted tissue. However, the incorporation of biodegradability, which is an essential property for practical applications, into complex hydrogels is not easily attained. Herein, we established a new concept for constructing biodegradable hydrogels with an interpenetrating polymer network (IPN) structure, composed of a covalent cross-linked network and peptide self-assembling networks, to solve this dilemma of selecting between the complicated structure and facile biodegradability. Assuming that the diffusion of the self-assembled peptides out of the IPN hydrogel would be facilitated by the disappearance of the covalent cross-linked networks, we designed an IPN hydrogel with chitosan cross-linked with poly(ethylene glycol)-block-poly( dl -lactide)-block-poly(ethylene glycol) as the covalent cross-linked networks with hydrolysis properties and RADA16 peptides as the self-assembling networks. This IPN hydrogel showed overall degradation, based on hydrolysis of the poly( dl -lactide) domain, and was more effective as a scaffold for culturing chondrocytes to form articular cartilage tissues compared with the IPN hydrogel without the poly( dl -lactide) domain, likely owing to the promotion of ECM deposition. These results verified our strategy of constructing a hydrogel with a complicated, but biodegradable, structure.
Surface engineering techniques for cellular micropatterning are emerging as important tools to cl... more Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behavior. Cells usually integrate with and respond to the microscale environment, and they are affected by the chemical and mechanical properties of the surrounding fluid and extracellular matrix, soluble protein factors, small signal molecules, and contacts with neighboring cells. Furthermore, recent progress in cellular micropatterning has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. In this regard, the ability to control with high precision the shape and spreading of attached cells and cell-cell contacts through the form and dimension of cell-adhesive patches is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlling microenvironments through engineered surfaces may not only be a valuable approach toward fundamental cell-biological studies, but could also be of great importance to the design of cell culture substrates for tissue engineering. To develop this kind of cellular microarray composed of a cell-resistant surface and cell attachment region, micropatterning a protein-repellent surface is important because cellular adhesion and proliferation are regulated by protein adsorption. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional surfaces, with the goal of providing an introductory overview of what is described in the literature. In particular, the importance of nonfouling surface chemistries is discussed.
The realization that soluble factors secreted by heterotypic cells play an importanta role in par... more The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co‐culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co‐culture models that are used to study paracrine signaling between heterotypic cells have been identified as long‐term viability and retention of cell‐specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co‐culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two‐dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co‐cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell‐to‐cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.
This paper deals with novel approaches established by our group for the construction of a functio... more This paper deals with novel approaches established by our group for the construction of a functionalized poly(ethylene glycol) (PEG) layer, PEG-brushed layer possessing a reactive group at the free end of tethered PEG chain, on substrates. An AB-type block copolymer composed of /spl alpha/-acetal-poly(ethylene glycol) (PEG) as the hydrophilic segment and polylactide (PLA) as the hydrophobic segment was synthesized, and utilized to construct the functionalized PEG layer on the biodegradable polylactide surface by simple coating. In this way, a PEG-brushed layer with a terminal aldehyde group was readily prepared which may have both non-fouling and ligand-binding properties. Non-fouling property of PEG strands eliminates nonspecific and uncontrolled interactions of the surface with biological components, including cells and proteins, while presentation of tethered ligands attached to the chain end of PEG brush allows cell behavior at the surface to be modulated in a specific manner via receptor-mediated signaling. Based on the characterization of these PEGylated surfaces from a physicochemical (contact angle, atomic force microscopy, electron spin resonance) as well as biological (protein adsorption/cell adhesion) point of view, our strategy to construct a functionalized PEG layer was confirmed. Active functional groups were present at the tethered PEG-chain end, these materials will provide new insights into controlling cell behavior at surfaces for tissue engineering and biomedical applications.
Abstract Hydrogels are used as cell culture scaffolds for tissue engineering and regeneration. Th... more Abstract Hydrogels are used as cell culture scaffolds for tissue engineering and regeneration. These hydrogel designs are inevitably complicated because favorable scaffolds should have stiffness to sustain alignment of the cells and mimic the structure of the extracellular matrix (ECM) in the targeted tissue. However, the incorporation of biodegradability, which is an essential property for practical applications, into complex hydrogels is not easily attained. Herein, we established a new concept for constructing biodegradable hydrogels with an interpenetrating polymer network (IPN) structure, composed of a covalent cross-linked network and peptide self-assembling networks, to solve this dilemma of selecting between the complicated structure and facile biodegradability. Assuming that the diffusion of the self-assembled peptides out of the IPN hydrogel would be facilitated by the disappearance of the covalent cross-linked networks, we designed an IPN hydrogel with chitosan cross-linked with poly(ethylene glycol)-block-poly( dl -lactide)-block-poly(ethylene glycol) as the covalent cross-linked networks with hydrolysis properties and RADA16 peptides as the self-assembling networks. This IPN hydrogel showed overall degradation, based on hydrolysis of the poly( dl -lactide) domain, and was more effective as a scaffold for culturing chondrocytes to form articular cartilage tissues compared with the IPN hydrogel without the poly( dl -lactide) domain, likely owing to the promotion of ECM deposition. These results verified our strategy of constructing a hydrogel with a complicated, but biodegradable, structure.
Surface engineering techniques for cellular micropatterning are emerging as important tools to cl... more Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behavior. Cells usually integrate with and respond to the microscale environment, and they are affected by the chemical and mechanical properties of the surrounding fluid and extracellular matrix, soluble protein factors, small signal molecules, and contacts with neighboring cells. Furthermore, recent progress in cellular micropatterning has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. In this regard, the ability to control with high precision the shape and spreading of attached cells and cell-cell contacts through the form and dimension of cell-adhesive patches is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlling microenvironments through engineered surfaces may not only be a valuable approach toward fundamental cell-biological studies, but could also be of great importance to the design of cell culture substrates for tissue engineering. To develop this kind of cellular microarray composed of a cell-resistant surface and cell attachment region, micropatterning a protein-repellent surface is important because cellular adhesion and proliferation are regulated by protein adsorption. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional surfaces, with the goal of providing an introductory overview of what is described in the literature. In particular, the importance of nonfouling surface chemistries is discussed.
The realization that soluble factors secreted by heterotypic cells play an importanta role in par... more The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co‐culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co‐culture models that are used to study paracrine signaling between heterotypic cells have been identified as long‐term viability and retention of cell‐specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co‐culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two‐dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co‐cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell‐to‐cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.
This paper deals with novel approaches established by our group for the construction of a functio... more This paper deals with novel approaches established by our group for the construction of a functionalized poly(ethylene glycol) (PEG) layer, PEG-brushed layer possessing a reactive group at the free end of tethered PEG chain, on substrates. An AB-type block copolymer composed of /spl alpha/-acetal-poly(ethylene glycol) (PEG) as the hydrophilic segment and polylactide (PLA) as the hydrophobic segment was synthesized, and utilized to construct the functionalized PEG layer on the biodegradable polylactide surface by simple coating. In this way, a PEG-brushed layer with a terminal aldehyde group was readily prepared which may have both non-fouling and ligand-binding properties. Non-fouling property of PEG strands eliminates nonspecific and uncontrolled interactions of the surface with biological components, including cells and proteins, while presentation of tethered ligands attached to the chain end of PEG brush allows cell behavior at the surface to be modulated in a specific manner via receptor-mediated signaling. Based on the characterization of these PEGylated surfaces from a physicochemical (contact angle, atomic force microscopy, electron spin resonance) as well as biological (protein adsorption/cell adhesion) point of view, our strategy to construct a functionalized PEG layer was confirmed. Active functional groups were present at the tethered PEG-chain end, these materials will provide new insights into controlling cell behavior at surfaces for tissue engineering and biomedical applications.
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