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Decellularized extracellular matrix (dECM) derived from natural ECM is receiving considerable interest as a promising component of tissue adhesives because of its high biocompatibility and tissue regenerative ability. However, the availability of dECM as a tissue adhesive is limited because of the lack of a gelator that can crosslink low concentrations of dECM to form hydrogels. Here, we report dECM-based tissue adhesives using a genipin gelator. Based on the pH-dependent reactivity of genipin, genipin-terminated 4 arm-poly(ethylene glycol) (GeniPEG) was synthesized. dECM-based hydrogels were formed within a few seconds of mixing GeniPEG and dECM at an optimum pH through crosslinking of dECM and self-crosslinking between GeniPEG molecules. The hydrogels crosslinked with GeniPEG exhibited greater tissue adhesive strength to porcine-derived aorta tissue than those crosslinked with genipin. Moreover, GeniPEG can be applied to various dECMs, including those from the urinary bladder, heart, liver, pancreas, and small intestine. In vivo implantation experiments demonstrated biocompatibility and biodegradability of the dECM-GeniPEG hydrogels. Therefore, this dECM-based hydrogel may extend the possibility and availability of dECM as an organ-specific tissue adhesive and contribute to successful minimally invasive surgery. STATEMENT OF SIGNIFICANCE: There is a strong need to develop highly functional tissue adhesives with high biocompatibility, tissue adhesive strength, and tissue regenerative ability. In this report, dECM-based tissue adhesives were reported using a pH-driven genipin-gelator. Focusing on the pH-dependent reactivity of genipin, genipin-based gelators were synthesized to form dECM-based hydrogels in response to pH changes. The crosslinking reaction proceeded within a few seconds to form hydrogels. The hydrogels obtained had greater tissue adhesion to aorta tissue than that of the free genipin crosslinker. This gelator can be applied to various types of dECMs. This dECM-based hydrogel had high biocompatibility and tissue adhesive properties and is useful for sealing wounds and preventing postoperative complications.

Despite many efforts focusing on regenerative medicine, there are few clinically-available cell-delivery carriers to improve the efficacy of cell transplantation due to the lack of adequate scaffolds. Herein, we report an injectable scaffold composed of functionalized gelatin for application in cell transplantation. Injectable functionalized gelatin-based hydrogels crosslinked with reversible hydrogen bonding based on supramolecular chemistry were designed. The hydrogel exhibited thixotropy, enabling single syringe injection of cell-encapsulating hydrogels. Highly biocompatible and cell-adhesive hydrogels provide cellular scaffolds that promote cellular adhesion, spreading, and migration. The in vivo degradation study revealed that the hydrogel gradually degraded for seven days, which may lead to prolonged retention of transplanted cells and efficient integration into host tissues. In volumetric muscle loss models of mice, cells were transplanted using hydrogels and proliferated in injured muscle tissues. Thixotropic and injectable hydrogels may serve as cell delivery scaffolds to improve graft survival in regenerative medicine.

Postoperative barriers have been widely used to prevent adhesions. However, there are currently few barriers that satisfy clinical requirements, such as tissue adhesion, operability, and biocompatibility. Inspired by the adhesion system of living organisms, we report a liquid-liquid phase-separated hydrogel as a single-syringe hotmelt-type postoperative barrier. Mixing polyethylene glycol with gelatin formed liquid-liquid phase-separated hydrogels through segregative liquid-liquid phase separation. Incorporation of a liquid-liquid phase-separated system into gelatin can enhance the sol-gel transition temperature to give a hotmelt-adhesive property to hydrogels. Hotmelt-adhesive hydrogels became a sol phase and cohered into tissue gaps when warmed and solidified at body temperature to adhere to soft tissues. The hydrogels exhibited tissue adhesion to large intestine tissues and showed improved mechanical strength, gelation time, and shear-thinning properties. In rat cecum-abdominal adhesion models, it was confirmed that the resulting hydrogels prevented abdominal adhesion and did not prevent tissue regeneration. Hotmelt-adhesive hydrogels with high tissue adhesive properties, operability, and biocompatibility have enormous potential as barriers to prevent postoperative complications.

Psoriasis is an incurable, immune-mediated inflammatory disease characterized by the hyperproliferation and abnormal differentiation of keratinocytes. To study in depth the pathogenesis of this disease and possible therapy options suitable, pre-clinical models are required. Three-dimensional skin equivalents are a potential alternative to simplistic monolayer cultures and immunologically different animal models. However, current skin equivalents lack long-term stability, which jeopardizes the possibility to simulate the complex disease-specific phenotype followed by long-term therapeutic treatment. To overcome this limitation, the cell coating technique was used to fabricate full-thickness human skin equivalents (HSEs). This rapid and scaffold-free fabrication method relies on coating cell membranes with nanofilms using layer-by-layer assembly, thereby allowing extended cultivation of HSEs up to 49 days. The advantage in time is exploited to develop a model that not only forms a disease phenotype but can also be used to monitor the effects of topical or systemic treatment. To generate a psoriatic phenotype, the HSEs were stimulated with recombinant human interleukin 17A (rhIL-17A). This was followed by systemic treatment of the HSEs with the anti-IL-17A antibody secukinumab in the presence of rhIL-17A. Microarray and RT-PCR analysis demonstrated that HSEs treated with rhIL-17A showed downregulation of differentiation markers and upregulation of chemokines and cytokines, while treatment with anti-IL-17A antibody reverted these gene regulations. Gene ontology analysis revealed the proinflammatory and chemotactic effects of rhIL-17A on the established HSEs. These data demonstrated, at the molecular level, the effects of anti-IL-17A antibody on rhIL-17A-induced gene regulations. This shows the physiological relevance of the developed HSE and opens venues for its use as an alternative to

An implantable scaffold-based vaccination system is a promising platform to generate robust immune responses by modulating the immune system. However, establishment of an effective vaccine using a biodegradable, cell-infiltrative scaffold remain challenging. Here we demonstrate a biodegradable, nanocellulose-based immune scaffold capable of sustainably activating immune cells to elicit cellular immunity. Cell-infiltrative nanocellulose hydrogels were used as a delivery carrier and cellular scaffold microenvironment. Nanofibrous hydrogels allowed for high cell infiltration and delivery of antigen-loaded nanocellulose while cells degraded the hydrogel matrix. Importantly, antigen-loaded nanocellulose hydrogels exhibited sustained activation of macrophages in vitro compared to free antigen and collagen scaffold. Histological observation revealed infiltration of macrophages and dendritic cells into the nanocellulose scaffold subcutaneously implanted in mice. In vivo fluorescence imaging indicated that the implanted scaffold released antigens at a zero-order release profile without burst diffusion. Antigen-loaded nanocellulose hydrogels increased interferon-γ-producing cells compared to free antigen injection, suggesting the enhancement of cellular immunity. Thus, nanocellulose immune scaffold may serve as a sustained-immunostimulatory vaccine platform by providing favorable microenvironments for immune cells thus enhancing vaccine efficacy.

Complete resection of early-stage cancer and subsequent wound care of the resection site are both important factors for successful endoscopic submucosal dissection (ESD). Although many submucosal injection materials (SIMs) have been developed to lift up lesions and completely remove cancers, a lack of materials with multiple functions, such as stable submucosal cushion formation, emergency perforation closure, and blood coagulation and wound sealing abilities, still exists. In this study, an adhesive submucosal injection material based on nonanal group-modified poly­(vinyl alcohol) (C9-PVA) and α-cyclodextrin (α-CD) was developed to solve clinical problems associated with ESD. Focusing on the inclusion ability of α-CD for alkyl groups, a water-soluble α-CD/C9-PVA inclusion complex (IC) was prepared using water-insoluble C9-PVA in aqueous solutions. The IC of 2.5 mol % nonanal group-modified PVA (2.5C9-PVA), with 53 mM α-CD, showed good gel–sol reversibility and high injectability. Additionally, the α-CD/2.5C9-PVA IC performed well at submucosal cushion formation, with the increased height reaching 5.90 ± 0.38 mm. The complex also adhered well to the submucosa, successfully sealing wounds for over 7 days even in an aqueous environment. Furthermore, following incubation in physiological saline (1.80 ± 0.37 kPa), the burst strength of the α-CD/2.5C9-PVA IC was 1.5-fold higher compared with that of the commercial fibrin sealant, illustrating the possibility of emergency perforation closure. Finally, the complex promoted blood coagulation when mixed with fresh porcine whole blood. These results indicate that the multifunctional α-CD/2.5C9-PVA IC could be an ideal material for ESD.

Post-surgical treatment with tissue adhesives enables the closure of wounds and promotes tissue regeneration, which contributes to minimally invasive surgery. Although there are many clinically available tissue adhesives, compromises are made on either the tissue adhesion strength or biocompatibility due to inefficient material design. Here, we report a facile and versatile approach to engineer an injectable, tough tissue adhesive by reinforcing hydrogels with nanocellulose (NC). NC is a class of nanomaterial possessing unique structural features, such as high aspect ratio and superior mechanical properties. NC-reinforced hydrogels have improved mechanical strength depending on the NC concentration. The tissue adhesion strength of collagen casings and porcine-derived aorta and stomach tissues was drastically enhanced by the NC reinforcement of the hydrogels. This facile approach was applied to a variety of tissue adhesives and hydrogels, including poly(ethylene glycol), fibrin, protein-glutaraldehyde, and collagen-based matrix components. NC-reinforced hydrogels subcutaneously implanted into rats showed biocompatibility and degradability. This approach has enormous potential to improve the tissue adhesion strength of conventional medical materials and contribute to minimally invasive surgery.

Postoperative adhesion is a serious and frequent complication, but there is currently no reliable anti-adhesive barrier available due to low tissue adhesiveness, undesirable chemical reactions, and poor operability. To overcome these problems, we report a single-syringe hotmelt tissue adhesive that dissolves upon warming over 40 °C and coheres at 37 °C as a postoperative barrier. Tendon-derived gelatin was conjugated with the ureidopyrimidinone unit to supramolecularly control the sol-gel transition behavior. This functionalization improved bulk mechanical strength, tissue-adhesive properties, and stability under physiological conditions through the augmentation of intermolecular hydrogen bonding by ureidopyrimidinone unit. This biocompatible adhesive prevented postoperative adhesion between cecum and abdominal wall in adhesion models of rats. This hotmelt tissue adhesive has enormous potential to prevent postoperative complications and may contribute to minimally invasive surgery. STATEMENT OF SIGNIFICANCE: There is a strong need to develop medical tissue adhesives with high biocompatibility, tissue adhesiveness, and operatability to prevent postoperative complications. In this report, single syringe, hotmelt-type tissue adhesive was developed by controlling sol-gel transition behavior of gelatin through supramolecular approach. The functionalization of gelatin with quadruple hydrogen bonding improved key features necessary for anti-adhesive barrier including bulk mechanical strength, tissue adhesive property, stability under physiological conditions, and anti-adhesive property. The hotmelt tissue adhesive can be used for a sealant, hemostatic reagent, and wound dressing to prevent postoperative complications including delayed bleeding, perforation, and inflammation and contribute to minimally invasive surgery.

Topical hemostatic agents have been widely used to stop intra-/postoperative bleeding from wounds and resected tissue surfaces. However, hemostatic agents that can accelerate blood coagulation and promote wound healing have not been established because of their poor tissue adhesiveness under wet conditions. Here, we report a colloidal wound dressing of hemostatic and tissue-adhesive hydrophobized microparticles (hMPs) functionalized by UV irradiation. hMPs were prepared by adding ethanol to hydrophobically modified Alaska pollock gelatin and applying the thermal cross-linking method. The hMPs were then subjected to simple UV irradiation to introduce hydrophilic groups. The UV-irradiated hMPs improved water/blood absorption and exerted hemostatic property in a rat model of liver injury. On the other hand, the hMPs maintained tissue adhesiveness even after UV irradiation owing to the cohesion force generated by hydrophobic interactions between the hMPs. Moreover, the hMPs did not show undesirable adhesion to other tissues after swelling. This colloidal wound dressing can be used to promote tissue regeneration in intra-/postoperative wounds through hemostasis and protection from postoperative adhesion.

Injectable gels have been used in minimally invasive surgery for tissue regeneration and treatment of inflammatory diseases. However, polymeric hydrogels often fail in cell infiltration, because of the presence of dense, cross-linked molecular networks and a lack of bioactivity, which causes delayed tissue remodeling. Here, we report a thixotropic, cell-infiltrative hydrogel of biofunctionalized nanocellulose that topologically enhances cell infiltration and biochemically upregulates cellular activity for the promotion of tissue remodeling. Biodegradable, sulfonated nanocellulose forms a nanofibrous hydrogel, mimicking cellular microenvironments through cross-linking between nanocellulose and gelatin. Resulting nanocellulose hydrogels showed thixotropy, allowing for single syringe injection. Nanofiber-based hydrogels possess high molecular permeability, which is due to nanoporous structures. Sulfonate groups on nanocellulose increase protein adsorption and induce cellular extension in vitro. Highly sulfonated nanocellulose hydrogels enhanced cell infiltration and vascularization upon implantation into rats. Macrophage polarization to M2 was observed in nanocellulose hydrogels, which may be involved in tissue remodeling. Injectable, biofunctionalized nanocellulose gels have enormous potential as artificial biomatrices to heal inflammatory diseases through manipulation of the immune system and promotion of tissue remodeling.

Despite the success of minimally-invasive endoscopic submucosal dissection (ESD) for the treatment of early gastrointestinal cancer, additional symptoms after ESD, including contracture, perforation, bleeding, and esophageal stricture remain. Conventional wound dressings were ineffective in preventing stricture because of poor stability of underwater-adhesives on living tissues. Here, we present a microparticle-based wound dressing with underwater adhesive stability for the treatment of gastrointestinal tract wound healing after ESD. Monodisperse microparticles composed of hydrophobically-modified Alaska pollock gelatin were prepared by self-assembly of gelatin in water-ethanol mixed solvents and thermal crosslinking. Hydrophobic modification of gelatin with aliphatic aldehydes increased adhesion strength to gastric and esophageal submucosal tissues through hydrophobic interaction with living tissues and cohesion force. Optimal hydrophobic modification drastically improved underwater stability of microparticles compared to that of non-modified gelatin and formed a thick, integrated hydrogel layer on tissues. Histological observation of rat skin wound healing models showed that hydrophobically-modified gelatin microparticles decreased the expression levels of α-smooth muscle actin in the dermis layer and could suppress fibrosis and inflammation after ESD. The microparticle wound dressing with high underwater-adhesive stability has enormous therapeutic potential to promote wound healing in the gastrointestinal tract and prevent additional symptoms after ESD. STATEMENT OF SIGNIFICANCE: The goal of this study was to develop wound dressing with strong tissue-adhesive property to living tissues for promoting wound healing after ESD treatment. Monodisperse microparticles composed of hydrophobically-modified Alaska pollock gelatin were prepared by self-assembly of gelatin in water-ethanol mixed solvents and thermal crosslinking. Hydrophobic modification of gelatin with aliphatic aldehydes enhanced adhesion strength to gastric and esophageal submucosal tissues through hydrophobic interaction with living tissues and cohesion force. Optimal hydrophobic modification drastically improved underwater stability of microparticles. The in vivo studies were performed to evaluate the ability of this colloidal wound dressing to suppress fibrosis. This new biomaterial has enormous potential to promote wound healing after ESD.

Biomedical devices trigger immune responses when implanted in the body, as they are treated as foreign bodies. To avoid inflammatory responses and enhance the biocompatibility of biomedical devices, advanced coating technology that can modulate immune responses is essential. As a part of the immune response in the body, autologous cells evade attack from macrophages using CD47 ligands that function as markers for self. Inspired by this self-recognition system, we developed a camouflage coating for biomaterial surfaces using cell membrane vesicles that could suppress inflammatory responses. In this study, we used monocyte-derived cell membrane vesicles expressing CD47 for coating nanocellulose-coated substrates. Our data showed that presentation of CD47 to macrophages elicited negative signal transduction for immunosuppression. Further, for coating, we used cell membrane vesicles and plant-derived nanofibers. We observed that the supporting layer of cellulose nanofibers physically fixed cell membrane vesicles and provided hydrophilic surfaces to the polystyrene substrate. Based on CD47 signaling, cell membrane vesicle coating suppressed the inflammatory responses of stimulated macrophages. Camouflaging biomaterial surfaces with cell-derived components might serve as an advanced coating platform to suppress inflammatory responses and enhance tissue integrity for biomedical devices after implantation.

An injectable bone may serve as a minimally invasive therapy for large orthopedic defects and osteoporosis and an alternative to allografting and surgical treatment. However, conventional bone substitutes lack the desirable biodegradability, bioresponsibility, and functionality to regulate the bone regeneration process. Here, we report an injectable, bioresponsive bone composed of bisphosphonate-modified nanocellulose (pNC) as a bone substitute for bone regeneration. Composites composed of nanofibrillated cellulose and β-tricalcium phosphate (β-TCP) mimic bone structures in which apatite reinforces collagen fibrils. Bisphosphonate groups on nanocellulose provide reversible, physical cross-linking with β-TCP, apatite formation, binding property to bone, and pH responsiveness. When the pH drops to ∼4.5, which corresponds to an osteoclast-induced pH decrease, pNC-β-TCP composite degrades and releases pNC. pNC suppresses osteoclast formation and pit formation. This osteoclast-responsive property allows for controlling the degradation rate of the composite. Moreover, the composite of pNC, α-tricalcium phosphate (α-TCP), and β-TCP enhances osteoblast differentiation. This injectable bone substitute of pNC that regulates osteoclast/osteoblast activity has enormous potential for the treatment of bone diseases and prevention of locomotive syndrome.

Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.

We report a highly stretchable hydrogel based on the crosslinking structure between calcium cations and alendronates (ALN) conjugated with poly-γ-glutamate (γ-PGA), a typical biodegradable polymer. γ-PGA with ALN (γ-PGA-ALN) forms the hydrogel in the aqueous solution containing CaCl

Preventing cancer metastasis requires a thorough understanding of cancer cell invasion. These phenomena occur in human 3-D living tissues. To this end, we developed a human cell-based three-dimensional (3-D) cultured tissue constructs that imitate in vivo human tissue organization. We investigated whether our 3-D cell culture system can be used to analyze the invasion of human oral squamous cell carcinoma (OSCC) cells. The 3-D tissue structure consisted of five layers of normal human dermal fibroblasts along with human dermal lymphatic endothelial cell tubes and was generated by the cell accumulation technique and layer-by-layer assembly using fibronectin and gelatin. OSCC cells with different lymph metastatic capacity were inoculated on the 3-D tissues and their invasion through the 3-D tissue structure was observed. Conventional methods of analyzing cell migration and invasion, that is, 2-D culture-based transwell and Matrigel assays were also used for comparison. The results using the 3-D cultured tissue constructs were comparable to those obtained using conventional assays; moreover, use of the 3-D system enabled visualization of differential invasion capacities of cancer cells. These results indicate that our 3-D cultured tissue constructs can be a useful tool for analysis of cancer cell invasion in a setting that reflects the in vivo tissue organization. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 292-300, 2019.

Tumour models mimicking in vivo three-dimensional (3D) microenvironments are of increasing interest in drug discovery because of the limitations inherent to current models. For example, preclinical assays that rely on monolayer or spheroid cell cultures cannot easily predict 3D cancer behaviours because they have no vasculature. Furthermore, there are major differences in cancer behaviour between human and animal experiments. Here, we show the construction of 3D blood/lymph-vascularized human stromal tissues that can be combined with cancer cells to mimic dynamic metastasis for real-time throughput screening of secreted proteinases. We validated this tool using three human carcinoma cell types that are known to invade blood/lymph vessels and promote angiogenesis. These cell types exhibited characteristic haematogenous/lymphogenous metastasis and tumour angiogenesis properties. Importantly, these carcinoma cells selectively secreted different matrix metalloproteinases depending on their metastasis stage and target vasculature, suggesting the possibility of developing drugs that can target each secreted proteinase. We conclude that the 3D tissue tool will be a powerful throughput system for predicting cancer cell responses and time-dependent secretion of molecules in preclinical assays.

Targeted delivery of antigens to immune cells using micro/nanocarriers may serve as a therapeutic application for vaccination. However, synthetic carriers have potential drawbacks including cytotoxicity, low encapsulation efficiency of antigen, and lack of a morphological design, which limit the translation of the delivery system to clinical use. Here, we report a carrier-free and three-dimensional (3D)-shape-designed antigen nanoparticle by multiphoton lithography-based 3D-printing. This simple, versatile 3D-printing approach provides freedom for the precise design of particle shapes with a nanoscale resolution. Importantly, shape-designed antigen nanoparticles with distinct aspect ratios show shape-dependent immune responses. The 3D-printing approach for the rational design of nanomaterials with increasing safety, complexity, and efficacy offers an emerging platform to develop vaccine delivery systems and mechanistic understanding.

Advanced biology 5(8), 2000427 (2021). doi:10.1002/adbi.202000427, Published by Wiley-VCH, Weinheim

In vitro reconstruction of an alveolar barrier for modeling normal lung functions and pathological events serve as reproducible, high-throughput pharmaceutical platforms for drug discovery, diagnosis, and regenerative medicine. Despite much effort, the reconstruction of organ-level alveolar barrier functions has failed due to the lack of structural similarity to the natural basement membrane, functionalization with specific ligands for alveolar cell function, the use of primary cells and biodegradability. Here we report a bipolar cultured alveolar-capillary barrier model of human primary cells supported by a basement membrane mimics of fully synthetic bifunctional nanofibers. One-step electrospinning process using a bioresorbable polyester and multifunctional star-shaped polyethylene glycols (sPEG) enables the fabrication of an ultrathin nanofiber mesh with interconnected pores. The nanofiber mesh possessed mechanical stability against cyclic expansion as seen in the lung in vivo. The sPEGs as an additive provide biofunctionality to fibers through the conjugation of peptide to the nanofibers and hydrophilization to prevent unspecific protein adsorption. Biofunctionalized nanofiber meshes facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and showed higher permeability for molecules compared to microporous films. This nanofiber mesh for a bipolar cultured barrier have the potential to promote growth of an organ-level barrier model for modeling pathological conditions and evaluating drug efficacy, environmental pollutants, and nanotoxicology.

High-throughput screening of drug diffusion and cell transports from the blood-/lymph-capillary (BC/LC) networks to the peripheral cells in 3D engineered tissues using a microplate would make a powerful tool for in vitro pharmacokinetic assessments. Here, perfusable BC/LC networks embedded in 3D-tissues inside a 24-microplate using a cell-coating technology are reported which allows location control of cell layers. Arrangement of an endothelial cell layer at the top, middle, and bottom of dermal fibroblast tissues provides an interconnected BC/LC networks possessing open pores on both surfaces. When fluorescently labeled dextran, microparticles, and red blood cells are applied to the top surfaces, diffusion and penetration through the networks are observed depending on the size of the substances. Moreover, BC networks mimick a series of in vivo processes of cancer metastasis, extravasation, growth, and growth suppression with drug treatment. The perfusable networks existing in 3D-tissues show great potential for in vitro pharmacokinetic studies.

In vitro development of three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has long been desired in tissue regeneration and pharmaceutical assays. In particular, in vitro construction of 3D-iPSC–CM tissues with blood capillary networks have attracted much attention because blood capillaries are crucial for nutrient and oxygen supplies for CMs. Blood capillaries in 3D-iPSC–CM tissues will also be important for in vitro toxicity assay of prodrugs because of the signaling interaction between cardiomyocytes and endothelial cells. Here, we report construction of vascularized 3D-iPSC–CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique for cells, instead of our previous centrifugation-LbL technique. The filtration-LbL allowed us to fabricate nanometer-sized extracellular matrices (ECM), fibronectin and gelatin (FN–G), films onto iPSC–CM surfaces without any damage and with high yield, although centrifugation-LbL induced physical stress and a lower yield. The fabricated FN–G nanofilms interacted with integrin molecules on the cell membrane to construct 3D-tissues. We found that the introduction of normal human cardiac fibroblasts (NHCFs) into the iPSC–CM tissues modulated organization and synchronous beating depending on NHCF ratios. Moreover, co-culture with normal human cardiac microvascular endothelial cells (NHCMECs) successfully provided blood capillary-like networks in 3D-iPSC–CM tissues, depending on NHCF ratios. The vascularized 3D-iPSC–CM tissues indicated significantly different toxicity responses as compared to 2D-iPSC–CM cells by addition of doxorubicin as a model of a toxic drug. The constructed vascularized 3D-iPSC–CM tissues would be a promising tool for tissue regeneration and drug development. Statement of Significance In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC–CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN–G) films onto iPSC–CM surfaces. The FN–G nanofilms induce cell–cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC–CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering.

For the treatment of cardiac arrhythmia, electronic pacemakers are often employed. However, they have issues such as bio-incompatibility and battery limitations. Recently, the use of cells expressing hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels for use as pacemaker cells instead of electronic pacemakers has attracted increasing attention. However, the cell transplantation treatment was not sufficiently effective because of the low engraftment rate of the transplanted cells and the risk of inflammatory reactions. Here, in order to overcome these issues, we constructed 3D-pacemaker tissues composed of mouse-embryonic-cell-derived cardiomyocytes (mESC-CMs) in which the HCN4 gene had been introduced by the cell accumulation technique. The obtained tissues beat faster than control tissues and beats per minute (BPM) increased clearly with tissue thickness. This is the first report suggesting the relation between BPM and tissue thickness. Moreover, the pacemaker tissue could control the beating of the patched tissue.

Modulation of living cell surfaces by chemical and biological engineering and the control of cellular functions has enormous potential for immunotherapy, transplantation, and drug delivery. However, traditional detection techniques have limitations in the identification of physical properties of viscoelastic films and interaction with living cells in real time. Here, we present the structural analysis of extracellular matrix (ECM) based nanofilms and their interaction with living cells using a quartz crystal microbalance (QCM) with dissipation (QCM-D), multiple parameter surface plasmon resonance (SPR), and flow cytometry measurements. QCM-D measurements according to the Voigt-based viscoelastic model allowed for the evaluation of the kinetic adsorption of extracellular matrix (ECM) proteins and physical parameters of viscoelastic ECM-nanofilms in a swelled state. These results reflected the characteristics of viscoelastic films as compared to Sauerbrey's equation. Moreover, we found that gelatin molecules played a crucial role as a binder to build up layered films and control their properties. Using the multiple parameter SPR approach, we confirmed the interaction between FN-G nanofilms and living cells from signal response in real time which was different from the gold substrate-protein signal. Moreover, flow cytometry analysis supported the importance of the domain interaction between the RGD sequence in FN and integrin as a driving force to form the films on cell surfaces. The use of three different analyses supported clarification of the contribution of the protein-protein interaction and viscoelastic properties of ECM films and investigation of the interaction between films and living cells. The knowledge regarding protein-protein and protein-cell interaction in real time would make a contribution to biomaterial design by using protein interactions for modulating the living cell surfaces in biomedical applications.

When bio-molecular recognition between nanofilms and proteins occurs on cell surfaces, rapid cellular assembly takes place. Cells coated with layer-by-layer nanofilms composed of fibronectin and heparin form aggregates after centrifugation and nanofilms induce attractive forces between cells through bio-molecular recognition between membrane proteins and heparin. Cell aggregates display network structures of cells as seen in colloidal gels with high viscosity. Cell-cell crosslinking allows for the construction of 3D-tissues with rich glycosaminoglycan. This cell-cell crosslinking process that uses a layer-by-layer technique has enormous potential for in vitro tissue applications in regenerative medicine and cell signaling assays.

Endoscopic submucosal dissection (ESD) provides strong therapeutic benefits for early gastrointestinal cancer as a minimally invasive treatment. However, there is currently no reliable treatment to prevent scar contracture resulting from ESD which may lead to cicatricial stricture. Herein, a multifunctional colloidal wound dressing to promote tissue regeneration after ESD is demonstrated. This sprayable wound dressing, composed of hydrophobized microparticles, exhibits the multifunctionality necessary for wound healing including tissue adhesiveness, blood coagulation, re-epithelialization, angiogenesis, and controlled inflammation based on hydrophobic interaction with biological systems. An in vivo feasibility study using swine gastric ESD models reveals that this colloidal wound dressing suppresses fibrosis and accelerates wound healing. Multifunctional colloidal and sprayable wound dressings have an enormous therapeutic potential for use in a wide range of biomedical applications including accelerated wound healing after ESD, prevention of perforation, and the treatment of inflammatory diseases.

Functional 3D-engineered tissues are successfully harvested from a substrate using stimuli-responsive hydrogel films with dynamic nano-interface. The dynamic wettability control at the interfaces allows cellular detachment, leading to tissue harvesting without serious damage and remaining polymers. This method can be applied to various types of organs and used for tissue transplantation in regenerative medicine.

The in vitro fabrication of vascularized tissue is a key challenge in tissue engineering, but little is known about the mechanisms of blood-capillary formation. Here we investigated the mechanisms of in vitro vascularization using precisely-controlled 3D-microenvironments constructed by a sandwich culture using the cell-accumulation technique. 3D-microenvironments controlled at the single layer level showed that sandwich culture between more than 3 fibroblast-layers induced tubule formation. Moreover, the secretion of angiogenic factors increased upon increasing the number of sandwiching layers, which induced highly dense tubular networks. We found that not only angiogenic factors, but also the 3D-microenvironments of the endothelial cells, especially apical side, played crucial roles in tubule formation in vitro. Based on this knowledge, the introduction of blood and lymph capillaries into mesenchymal stem cell (MSC) tissues was accomplished. These findings would be useful for the in vitro vascularization of various types of engineered organs and studies on angiogenesis.

Living tissues dynamically modulate their structure and functions through physical and biochemical interactions in the three-dimensional (3D)-microenvironment for their homeostasis or the developmental process of an embryo. However, the manipulation of cellular functions in vitro is still challenging due to the lack of a dynamic material system that can vary the 3D-cellular microenvironment in time and space. Here, we show an in situ 3D-printing technique based on multiphoton lithography using a biocompatible photoresist, bio-ink. The bio-ink composed of protein-photosensitizer conjugates has the ability to cause singlet oxygen and cross-linking reaction to fabricate protein gels with submicrometer-scale precision. Remarkably, the conjugates substantially improve the cytocompatibility and the efficiency of gelation due to the stealth effect of rose bengal (RB) and efficient transfer of singlet oxygen to bovine serum albumin (BSA). 3D-printing in the presence of cells allows for the microfabrication of a protein scaffold and controlled single-cell behavior. This dynamic material system to direct cell fate may offer emerging applications for drug discovery and regenerative medicine.