Characterization of biomaterial‐free cell sheets cultured from human oral mucosal epithelial cells

The purpose of this study was to report the characteristics of biomaterial‐free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal‐deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony‐forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki‐67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial‐free sheets. Biomaterial‐free sheets which had been detached with 1% dispase or biomaterial‐free sheets generated by fibrin support were transplanted to 12 limbal‐deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial‐free sheets were compared with those of sheets formed by fibrin‐coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial‐free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial‐free sheets were comparable to those of sheets cultured by fibrin support, in limbal‐deficient models. Both p63 and Ki‐67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal‐deficient models. Our results suggest that biomaterial‐free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal‐deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd.     

Endothelial colony‐forming cells for preparing prevascular three‐dimensional cell‐dense tissues using cell‐sheet engineering

Vascular‐derived endothelial cell (EC) network prefabrication in three‐dimensional (3D) tissue constructs before transplantation is useful for inducing functional anastomosis with the host vasculature. However, the clinical application of ECs is limited by cell isolation from the existing vasculature, because of the requirement for invasive biopsies and difficulty in obtaining a sufficient number of cells. Endothelial colony‐forming cells (ECFCs), which are a subtype of endothelial progenitor cells in the blood, have a strong proliferative and vasculogenic potential. This study attempted to fabricate prevascular 3D cell‐dense tissue constructs using cord blood‐derived ECFCs and evaluate the in vivo angiogenic potential of these constructs. Human umbilical vascular endothelial cells (HUVECs) were also used in comparison with ECFCs, which were sandwiched between two human dermal‐derived fibroblast (FB) sheets using a fibrin‐coated cell‐sheet manipulator. The inserted ECFCs in double‐layered FB sheets were cultured for 3 days, resulting in the formation of network structures similar to those of HUVECs. Additionally, when ECFCs were sandwiched with three FB sheets, a lumen structure was found in the triple‐layered cell‐sheet constructs at 3 days after co‐culture. These constructs containing ECFCs were transplanted into the subcutaneous tissue of immune‐deficient rats. One week after transplantation, ECFC‐lined functional microvessels containing rat erythrocytes were observed in the same manner as transplanted HUVEC‐positive grafts. These results suggest that ECFCs might become an alternative cell source for fabricating a prevascular structure in 3D cell‐dense tissue constructs for clinical application. Copyright © 2013 John Wiley & Sons, Ltd.     

How transfer rates generate Gd‐BOPTA concentrations in rat liver compartments: implications for clinical liver imaging with hepatobiliary contrast agents

Following the injection of hepatobiliary contrast agents, MRI detects all molecules included in a region of interest but cannot estimate true concentrations in sinusoids, interstitium, hepatocytes or bile canaliculi. The aim of the study was to measure true concentrations in hepatocytes and to show how transfer rates across sinusoidal and canalicular membranes generate these concentrations. We perfused livers isolated from normal rats with 200 μM Gd‐DTPA and Gd‐BOPTA and measured clearances from sinusoids to liver and from hepatocytes to bile canaliculi or back to interstitium. We detected Gd‐BOPTA with a gamma probe and determined true concentrations in each liver compartment knowing their liver volumes. No pharmacokinetic modelling was applied. Gd‐BOPTA clearance from sinusoids to liver (2.5 ± 0.4 mL/min) was 50 times higher than that of Gd‐DTPA (0.05 ± 0.02 mL/min) when portal flow rate was 30 mL/min (p < 0.0001). Gd‐BOPTA clearance from sinusoids to liver was always superior to hepatocyte clearance, and hepatocyte Gd‐BOPTA concentrations measured by the probe increased over time. Gd‐BOPTA concentrations reached 439 ± 83 μM in hepatocytes and 15 × 700 ± 3100 μM in bile canaliculi, while concentrations in sinusoids were 200 μM. Gd‐BOPTA true concentrations in hepatocytes depend on the simultaneous clearances from sinusoids to hepatocytes and from hepatocytes to bile canaliculi and back to sinusoids. The study better defines how signal intensities are generated when hepatobiliary contrast agents are injected in clinical imaging. Copyright © 2016 John Wiley & Sons, Ltd.     

Efficient generation of functional hepatocyte‐like cells from menstrual blood‐derived stem cells

In recent years, the advantages of menstrual blood‐derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum‐supplemented or serum‐free culture media, were also investigated. The sequential differentiation was monitored by real‐time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT‐4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin‐18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte‐specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte‐like cells. We recommend a complementary serum‐free differentiation protocol for enrichment of in vitro production of functional MenSC‐derived hepatocyte‐like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro vascularization of a combined system based on a 3D printing technique

A vital challenge in complex organ manufacturing is to vascularize large combined tissues. The aim of this study is to vascularize in vitro an adipose‐derived stem cell (ADSC)/fibrin/collagen incorporated three‐dimensional (3D) poly(d,l ‐lactic‐co‐glycolic acid) (PLGA) scaffold (10 × 10 × 10 mm³) with interconnected channels. A low‐temperature 3D printing technique was employed to build the PLGA scaffold. A step‐by‐step cocktail procedure was designed to engage or steer the ADSCs in the PLGA channels towards both endothelial and smooth muscle cell lineages. The combined system had sufficient mechanical properties to support the cell/fibrin/collagen hydrogel inside the predefined PLGA channels. The ADSCs encapsulated in the fibrin/collagen hydrogel differentiated to endothelial and smooth muscle cell lineage, respectively, corresponding to their respective locations in the construct and formed vascular‐like structures. This technique allows in vitro vascularization of the predefined PLGA channels and provides a choice for complex organ manufacture. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo vascularization of cell sheets provided better long‐term tissue survival than injection of cell suspension

Cell sheets have shown a remarkable ability for repairing damaged myocardium in clinical and preclinical studies. Although they demonstrate a high degree of viability as engrafted cells in vivo, the reason behind their survivability is unclear. In this study, the survival and vascularization of rat cardiac cell sheets transplanted in the subcutaneous tissue of athymic rats were investigated temporally. The cell sheets showed significantly higher survival than cell suspensions for up to 12 months, using an in vivo bioluminescence imaging system to detect luciferase‐positive transplanted cells. Terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL) assay also showed a smaller number of apoptotic cells in the cell sheets than in the cell suspensions at 1 day. Rapid vascular formation and maturation were observed inside the cell sheets using an in vivo imaging system. Leaky vessels appeared at 6 h, red blood cells flowing through functional vessels appeared at 12 h, and morphologically matured vessels appeared at 7 days. In addition, immunostaining of cell sheets with nerve/glial antigen‐2 (NG2) showed that vessel maturity increased over time. Interestingly, these results correlated with the dynamics of cell sheet mRNA expression. Genes related to endothelial cells (ECs) proliferation, migration and vessel sprouting were highly expressed within 1 day, and genes related to pericyte recruitment and vessel maturation were highly expressed at 3 days or later. This suggested that the cell sheets could secrete appropriate angiogenic factors in a timely way after transplantation, and this ability might be a key reason for their high survival. Copyright © 2014 John Wiley & Sons, Ltd.     

Transplantation of cancerous cell sheets effectively generates tumour‐bearing model mice

Tumour‐bearing mice were created by transplanting cancerous cell sheets onto the subcutaneous tissue of the dorsal region, using luciferase gene‐transfected mammary gland adenocarcinoma cells, 4T1‐luc2, to investigate the tumourigenicity of the cell sheet relative to a conventional injection of cell suspension. Contiguous breast cancerous cell sheets were harvested from temperature‐responsive culture dishes by reducing the temperature from 37 °C to 20 °C; the sheets were then transplanted onto the dorsal side of the mouse subcutaneous tissue, using a chitin‐based supporting membrane. Cell suspensions obtained by trypsin digestion were subcutaneously injected into the dorsal region of mice. The tumour growth of the transplanted cancer cells was evaluated by the tumour volume and by the bioluminescence from luciferase‐gene transfected cancer cells, using an in vivo imaging system. The cell sheet method improved the 4 T1‐luc2 engraftment efficiency in living mouse tissues at the initial stage by 13‐fold compared with that from injecting cell suspensions. On day 14 after the transplantation, the tumour formation at the transplanted area of cell sheet‐transplanted mice also accelerated, and the mean tumour volume became 1116 mm³, which was 10 times larger than that in cell suspension‐transplanted mice. The cell sheets engrafted on the recipient tissues efficiently due to the preserved extracellular matrix on their basal sides, such that cancer cells were supplied with sufficient oxygen and nutrients from the host tissues to develop tumour tissues. Therefore, cancerous cell sheet‐based transplantation is a promising method for efficiently creating cancer‐bearing mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Enhanced endothelial differentiation of adipose‐derived stem cells by substrate nanotopography

Adipose‐derived stem cells (ADSCs) have great potential as a cell source for tissue engineering and regenerative medicine because they are easier to obtain, have lower donor‐site morbidity and are available in larger numbers than stem cells harvested using bone marrow aspiration. Until now, little has been known about how nanotopography affects the proliferation and endothelial differentiation of ADSCs. In the present study, two nanograting substrates with a period (ridge and groove) of about 250 and 500 nm, respectively, were fabricated on quartz and their effect on ADSC fate was investigated. The results showed that proliferation of ADSCs on nanograting substrates decreased while cell attachment was not significantly affected compared to a flat substrate. Endothelial differentiation of ADSCs on both flat and nanograting substrates can be induced with vascular endothelial growth factor, as shown by immunofluorescent staining. Quantitative real‐time PCR analysis showed significantly enhanced upregulation of vWF, PECAM‐1 and VE‐cadherin at the gene level by ADSCs on the nanograting substrates. In vitro angiogenesis assay on Matrigel showed that nanograting substrates enhanced capillary tube formation. This study highlights the beneficial influence of nanotopography on the differentiation of ADSC into endothelial cells which play an important role in vascularization. Copyright © 2012 John Wiley & Sons, Ltd.     

Temporal profiling of the growth and multi‐lineage potentiality of adipose tissue‐derived mesenchymal stem cells cell‐sheets

Cell‐sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold‐free constructs. Adipose tissue‐derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow‐derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC‐CSs) is non‐existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC‐CSs development; and (b) the multipotentiality of ASC‐CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid‐supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC‐CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT–PCR and histology at multiple time points. ASC‐CSs remained healthy up to 3 weeks of hyperconfluent culture. One week‐old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non‐hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC‐CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.     

The influence of sustained dual‐factor presentation on the expansion and differentiation of neural progenitors in affinity‐binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co‐transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate–sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor‐2 (bFGF) were affinity‐bound to alginate–sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium‐crosslinked alginate. NPCs isolated from 18 day‐old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10–14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ. Copyright © 2013 John Wiley & Sons, Ltd.     

Three‐dimensional co‐culture of human hepatocytes and mesenchymal stem cells: improved functionality in long‐term bioreactor cultures

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short‐term viability in two‐dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co‐cultured with human bone marrow mesenchymal stem cells (BM‐MSCs) as spheroids in automated, computer‐controlled, stirred‐tank bioreactors with perfusion operation mode. A dual‐step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM‐MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co‐cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated‐dose toxicity testing. Moreover, the excretory activity was maintained in co‐cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM‐MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Scaffold‐free cell sheet injection results in bone formation

We previously reported a new cell transplantation method in which mesenchymal stem cells (MSCs) were cultured as cell sheets. The cultured MSC sheets showed high alkaline phosphatase (ALP) activities and osteocalcin (OC) contents. In the present study, we transplanted such sheets by injection to assess whether the injectable MSC sheets could form bone tissue at subcutaneous sites. At 4 weeks after the subcutaneous injection, the injected areas showed hard mass formation. Each mass consisted of newly formed bone, as evaluated by radiographic, histological and gene expression analyses as well as three‐dimensional computed tomography (3D‐CT). Histological analyses revealed extracellular bone matrix together with osteocytes and active osteoblasts. Real‐time PCR analyses showed high ALP and OC mRNA expressions. We also injected the cell sheets into dead bone to determine whether the lost osteogenic potential could be rescued, and histological analyses revealed that the injected cell sheets supplied osteogenic potential to the dead bone. The present study clearly indicates that osteogenic MSC sheets can be transplanted via injection through a needle and that bone formation results in the injected areas. Owing to its usage of a needle for fabrication of in vivo bone tissue, this injection method can be applied as a minimally invasive approach for hard tissue reconstruction. Copyright © 2010 John Wiley & Sons, Ltd.     

Three‐dimensional functional human myocardial tissues fabricated from induced pluripotent stem cells

The most radical treatment currently available for severe heart failure is heart transplantation; however, the number of donor hearts is limited. A better approach is to make human cardiac tissues. We developed an original cell sheet‐based tissue‐engineering technology to fabricate human cardiac tissue by layering myocardial cell sheets. Human induced pluripotent stem (iPS) cells were differentiated into cardiomyocytes to fabricate cardiomyocyte sheets. Initially, three‐layer human iPS cardiomyocyte (hiPSCM) sheets were transplanted on subcutaneous tissues of nude rats. Next, to fabricate thicker tissue, three‐layer sheets were transplanted on one day, then additional three‐layer sheets were transplanted onto them the following day, after the first sheets were vascularized. On day 3, the final three‐layer sheets were again transplanted, creating a nine‐layer graft (multi‐step transplantation procedure). In the last step, six‐layer sheets were transplanted on fat tissues of the inguinal portion, which were subsequently resected together with the femoral arteries and veins to make transplantable grafts with connectable vessels. They were then transplanted ectopically to the neck portion of other rats by anastomosing vessels with the host's jugular arteries and veins. Transplanted three‐layer hiPSCMs were beating and, histologically, showed a cardiac muscle‐like structure with vascular systems. Moreover, transplanted hiPSCMs proliferated and matured in vivo. Significantly thicker tissues were fabricated by a multi‐step transplantation procedure. The ectopically transplanted graft survived and continued to beat. We succeeded in fabricating functional human cardiac tissue with cell sheet technology. Transplanting this cardiac tissue may become a new treatment option for severe heart failure. Copyright © 2015 John Wiley & Sons, Ltd.     

Combination of platelet‐rich plasma within periodontal ligament stem cell sheets enhances cell differentiation and matrix production

The longstanding goal of periodontal therapy is to regenerate periodontal tissues. Although platelet‐rich plasma (PRP) has been gaining increasing popularity for use in the orofacial region, whether PRP is useful for periodontal regeneration is still unknown. The purpose of this study was to determine whether a mixture of periodontal ligament stem cell (PDLSC) sheets and PRP promoted bone regeneration, one of the most important measurement indices of periodontal tissue regenerative capability in vitro and in vivo. In this study, we evaluated the effects of different doses of PRP on the differentiation of human PDLSCs. Then cell sheet formation, extracellular matrix deposition and osteogenic gene expression in response to different doses of PRP treatment during sheet grafting was investigated. Furthermore, we implanted PDLSC sheets treated with 1% PRP subcutaneously into immunocompromised mice to evaluate their bone‐regenerative capability. The results revealed that 1% PRP significantly enhanced the osteogenic differentiation of PDLSCs. Based on the production of extracellular matrix proteins, the results of scanning electron microscopy and the expression of the osteogenic genes ALP, Runx2, Col‐1 and OCN, the provision of 1% PRP for PDLSC sheets was the most effective PRP administration mode for cell sheet formation. The results of in vivo transplantation showed that 1% PRP‐mediated PDLSC sheets exhibited better periodontal tissue regenerative capability than those obtained without PRP intervention. These data suggest that a suitable concentration of PRP stimulation may enhance extracellular matrix production and positively affect cell behaviour in PDLSC sheets. Copyright © 2014 John Wiley & Sons, Ltd.     

Regeneration of dental pulp/dentine complex with a three‐dimensional and scaffold‐free stem‐cell sheet‐derived pellet

Dental pulp/dentine complex regeneration is indispensable to the construction of biotissue‐engineered tooth roots and represents a promising approach to therapy for irreversible pulpitis. We used a tissue‐engineering method based on odontogenic stem cells to design a three‐dimensional (3D) and scaffold‐free stem‐cell sheet‐derived pellet (CSDP) with the necessary physical and biological properties. Stem cells were isolated and identified and stem cells from root apical papilla (SCAPs)‐based CSDPs were then fabricated and examined. Compact cell aggregates containing a high proportion of extracellular matrix (ECM) components were observed, and the CSDP culture time was prolonged. The expression of alkaline phosphatase (ALP), dentine sialoprotein (DSPP), bone sialoprotein (BSP) and runt‐related gene 2 (RUNX2) mRNA was higher in CSDPs than in cell sheets (CSs), indicating that CSDPs have greater odonto/osteogenic potential. To further investigate this hypothesis, CSDPs and CSs were inserted into human treated dentine matrix fragments (hTDMFs) and transplanted into the subcutaneous space in the backs of immunodeficient mice, where they were cultured in vivo for 6 weeks. The root space with CSDPs was filled entirely with a dental pulp‐like tissue with well‐established vascularity, and a continuous layer of dentine‐like tissue was deposited onto the existing dentine. A layer of odontoblast‐like cells was found to express DSPP, ALP and BSP, and human mitochondria lined the surface of the newly formed dentine‐like tissue. These results clearly indicate that SCAP‐CSDPs with a mount of endogenous ECM have a strong capacity to form a heterotopic dental pulp/dentine complex in empty root canals; this method can be used in the fabrication of bioengineered dental roots and also provides an alternative treatment approach for pulp disease. Copyright © 2013 John Wiley & Sons, Ltd.     

Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature‐responsive closed culture device

Temperature‐responsive culture surfaces make it possible to harvest transplantable carrier‐free cell sheets. Here, we applied temperature‐responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony‐forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature‐responsive closed culture devices. The results were similar to those obtained using temperature‐responsive culture inserts. These results indicate that the novel temperature‐responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets. Copyright © 2013 John Wiley & Sons, Ltd.     

Three‐dimensional polymer scaffolds for enhanced differentiation of human mesenchymal stem cells to hepatocyte‐like cells: a comparative study

Stem cell‐based tissue engineering has emerged as a promising avenue for the treatment of liver diseases and as drug metabolism and toxicity models in drug discovery and development. The in vitro simulation of a micro‐environmental niche for hepatic differentiation remains elusive, due to lack of information about crucial factors for the stem cell niche. For generation of functional hepatocytes, an in vivo three‐dimensional (3D) micro‐environment and architecture should be reproduced. Towards this, we fabricated three scaffolds as dextran–gelatin (DG1), chitosan–hyaluronic acid (CH1) and gelatin–vinyl acetate (GEVAC). Hepatic differentiation of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) was induced by culturing hUC‐MSCs on these scaffolds. The scaffolds support hepatic differentiation by mimicking the native extracellular matrix (ECM) micro‐environment and architecture to facilitate 3D cell–cell and cell–matrix interactions. The expression of hepatic markers, glycogen storage, urea production, albumin secretion and cytochrome P450 (CYP450) activity indicated the hepatic differentiation of hUC‐MSCs. The differentiated hUC‐MSCs on the 3D scaffolds formed hepatospheroids (3D hepatocyte aggregates), as illustrated by scanning electron microscopy (SEM), confocal microscopy and cytoskeleton organization. It was observed that the 3D scaffolds supported improved cell morphology, expression of hepatic markers and metabolic activities, as compared to Matrigel‐coated plates. To the best of our knowledge, this is the first report demonstrating the use of a well‐characterized scaffold (GEVAC) for enhanced differentiation of hUC‐MSCs to hepatocyte‐like cells (HLCs). Copyright © 2016 John Wiley & Sons, Ltd.     

Adipose‐ and bone marrow‐derived mesenchymal stem cells display different osteogenic differentiation patterns in 3D bioactive glass‐based scaffolds

Mesenchymal stem cells can be isolated from a variety of different sources, each having their own peculiar merits and drawbacks. Although a number of studies have been conducted comparing these stem cells for their osteo‐differentiation ability, these are mostly done in culture plastics. We have selected stem cells from either adipose tissue (ADSCs) or bone marrow (BMSCs) and studied their differentiation ability in highly porous three‐dimensional (3D) 45S5 Bioglass®‐based scaffolds. Equal numbers of cells were seeded onto 5 × 5 × 4 mm³ scaffolds and cultured in vitro, with or without osteo‐induction medium. After 2 and 4 weeks, the cell–scaffold constructs were analysed for cell number, cell spreading, viability, alkaline phosphatase activity and osteogenic gene expression. The scaffolds with ADSCs displayed osteo‐differentiation even without osteo‐induction medium; however, with osteo‐induction medium osteogenic differentiation was further increased. In contrast, the scaffolds with BMSCs showed no osteo‐differentiation without osteo‐induction medium; after application of osteo‐induction medium, osteo‐differentiation was confirmed, although lower than in scaffolds with ADSCs. In general, stem cells in 3D bioactive glass scaffolds differentiated better than cells in culture plastics with respect to their ALP content and osteogenic gene expression. In summary, 45S5 Bioglass‐based scaffolds seeded with ADSCs are well‐suited for possible bone tissue‐engineering applications. Induction of osteogenic differentiation appears unnecessary prior to implantation in this specific setting. Copyright © 2013 John Wiley & Sons, Ltd.     

In vivo 3D analysis with micro‐computed tomography of rat calvaria bone regeneration using periosteal cell sheets fabricated on temperature‐responsive culture dishes

Transplantable cell sheets containing osteoblasts were fabricated from periostea on temperature‐responsive culture dishes. This study demonstrated the time‐course of bone regeneration in living small animals. This continuous observation of bone regeneration was achieved by micro‐computed tomography (µCT), which assessed the osteogenic capability of periosteal cells without biodegradable scaffolds. Real‐time bone regeneration was non‐invasively monitored in a rat calvarial bone defect model, using µCT. Three‐dimensional (3D) images obtained over time by µCT clearly showed that two different bone regeneration modes, specific to the control and experimental groups, were observed. In the control group, bone was regenerated only from the periphery of the defect edges. In the experimental group, bone regeneration was observed in several small regions within the central portions of the defects that were covered by the transplanted cell sheets. However, bone regeneration observed after periosteal cell sheet transplantation was limited. The results of ALP staining and the time‐course observations concluded that periosteal cell sheets contained a small fraction of cells that could differentiate osteoblasts. Fibroblasts in transplanted cell sheets or from around subcutaneous tissues suppressed bone regeneration. The periosteal cell sheets had a capability to produce ectopic regenerated bones. Therefore, to increase the content of osteogenic cells in harvested cell sheets, the enrichment of cells that could produce osteoblasts was expected by the modification of the initial cell preparation and the culture conditions. With further possible improvements, scaffold‐free periosteal cell sheet fabricated on temperature‐responsive culture dishes will be a valuable method for inducing and accelerating bone regeneration. Copyright © 2010 John Wiley & Sons, Ltd.     

Middle ear mucosal regeneration with three‐dimensionally tissue‐engineered autologous middle ear cell sheets in rabbit model

The likelihood of recurrent retraction and adhesion of newly formed tympanic membrane is high when middle ear mucosa is extensively lost during cholesteatoma and adhesive otitis media surgery. If rapid postoperative regeneration of the mucosa on the exposed bone surface can be achieved, prevention of recurrent eardrum adhesion and cholesteatoma formation, for which there has been no definitive treatment, can be expected. Suture‐less transplantation of tissue‐engineered mucosal cell sheets was examined immediately after the operation of otitis media surgery in order to quickly regenerate middle ear mucosa lost during surgery in a rabbit model. Transplantable middle ear mucosal cell sheets with a three‐dimensional tissue architecture very similar to native middle ear mucosa were fabricated from middle ear mucosal tissue fragments obtained in an autologous manner from middle ear bulla on temperature‐responsive culture surfaces. Immediately after the mucosa was resected from middle ear bone bulla inner cavity, mucosal cell sheets were grafted at the resected site. Both bone hyperplasia and granulation tissue formation were inhibited and early mucosal regeneration was observed in the cell sheet‐grafted group, compared with the control group in which only mucosal removal was carried out and the bone surface exposed. This result indicates that tissue engineered mucosal cell sheets would be useful to minimize complications after the surgical operation on otitis media and future clinical application is expected. Copyright © 2013 John Wiley & Sons, Ltd.