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.     

Functional analysis in vivo of engineered valved venous conduit with decellularized matrix and two bone marrow‐derived progenitors in sheep

Tissue engineering has been considered a promising approach for creating grafts to replace autologous venous valves. Here, ovine bone marrow‐derived endothelial progenitor cells (EPCs) and multipotent adult progenitor cells (MAPCs) were harvested and then loaded into decellularized venous matrix to create tissue‐engineered (TE) valved vein. Subsequently, the ovine femoral veins containing the valve were removed and replaced by TE grafts or acellular matrix only. The morphology and function were analysed for up to 1 year by ultrasonography, angiography, H&E staining and scanning electron microscopy (SEM). The differentiation of seeded cells was traced immunofluorochemically. The results showed that decellularized venous matrix could initially and feebly attract endogenous cells, but failed afterwards and were insufficient to restore valve function. On the contrary, the seeded cells differentiated into endothelial cells (ECs) in vivo and formed a monolayer endothelium, and smooth muscle cells within the scaffold therefore produced TE grafts comparable to the native vein valve. This TE graft remained patent and sufficient after implantation into the venous circuit of the ovine lower extremity for at least 6 months. Unfortunately, cells seeded on the luminal surface and both sides of the leaflets lost their biological functions at 12 months, resulting in thrombosis formation and leading to complete occlusion of the TE grafts and impotent venous valves. These findings suggest that this TE valved venous conduit can function physiologically in vivo in the medium term. Before translating this TE venous valve into clinical practice, the durability should be improved and thrombogenicity should be suppressed. Copyright © 2016 John Wiley & Sons, Ltd.     

An additive manufacturing‐based PCL–alginate–chondrocyte bioprinted scaffold for cartilage tissue engineering

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self‐assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three‐dimensional (3D) cell‐printed scaffolds using layer‐by‐layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell‐encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell‐based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL–alginate gel constructs. PCL–alginate gels containing transforming growth factor‐β (TGFβ) showed higher ECM formation. The 3D cell‐printed scaffolds of PCL–alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL–alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell‐printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.     

Co‐culture of adipose‐derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co‐implantation of adipose‐derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co‐cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell–fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co‐implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary‐like structures by co‐implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary‐like structures near the top of the construct, indicating that perfusion of the cell–fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

Poly‐ε‐caprolactone scaffold and reduced in vitro cell culture: beneficial effect on compaction and improved valvular tissue formation

Tissue‐engineered heart valves (TEHVs), based on polyglycolic acid (PGA) scaffolds coated with poly‐4‐hydroxybutyrate (P4HB), have shown promising in vivo results in terms of tissue formation. However, a major drawback of these TEHVs is compaction and retraction of the leaflets, causing regurgitation. To overcome this problem, the aim of this study was to investigate: (a) the use of the slowly degrading poly‐ε‐caprolactone (PCL) scaffold for prolonged mechanical integrity; and (b) the use of lower passage cells for enhanced tissue formation. Passage 3, 5 and 7 (P3, P5 and P7) human and ovine vascular‐derived cells were seeded onto both PGA–P4HB and PCL scaffold strips. After 4 weeks of culture, compaction, tissue formation, mechanical properties and cell phenotypes were compared. TEHVs were cultured to observe retraction of the leaflets in the native‐like geometry. After culture, tissues based on PGA–P4HB scaffold showed 50–60% compaction, while PCL‐based tissues showed compaction of 0–10%. Tissue formation, stiffness and strength were increased with decreasing passage number; however, this did not influence compaction. Ovine PCL‐based tissues did render less strong tissues compared to PGA–P4HB‐based tissues. No differences in cell phenotype between the scaffold materials, species or cell passage numbers were observed. This study shows that PCL scaffolds may serve as alternative scaffold materials for human TEHVs with minimal compaction and without compromising tissue composition and properties, while further optimization of ovine TEHVs is needed. Reducing cell expansion time will result in faster generation of TEHVs, providing more rapid treatment for patients. Copyright © 2013 John Wiley & Sons, Ltd.     

Bioactive coating of decellularized vascular grafts with a temperature‐sensitive VEGF‐conjugated hydrogel accelerates autologous endothelialization in vivo

No ideal small‐diameter vascular graft for widespread clinical application has yet been developed and current approaches still suffer from graft failure because of thrombosis or degeneration. Decellularized vascular grafts are a promising strategy as they preserve native vessel architecture while eliminating cell‐based antigens and allow for autologous recellularization. In the present study, a functional in vivo rodent aortic transplantation model was used in order to evaluate the benefit of bioactive coating of decellularized vascular grafts with vascular endothelial growth factor (VEGF) conjugated to a temperature‐sensitive aliphatic polyester hydrogel (HG). Luminal HG‐VEGF coating persistence up to 4 weeks was confirmed in vivo by rhodamine‐labelling. Doppler‐sonography showed that the grafts were functional for up to 8 weeks in vivo. Histological and immunohistochemical analysis of the explanted grafts after 4 weeks and 8 weeks in vivo demonstrated significantly increased endothelium formation in the HG‐VEGF group compared with the control group (luminal surface covered with single‐layered endothelium, 4 weeks: 64.8 ± 7.6% vs. 40.4 ± 8.3%, p = 0.025) as well as enhanced media recellularization (absolute cell count, 8 weeks: 22.1 ± 13.0 vs. 3.2 ± 3.6, p = 0.0039). However, HG‐VEGF coating also led to increased neo‐intimal hyperplasia, resulting in a significantly increased intima‐to‐media ratio in the perianastomotic regions (intima‐to‐media ratio, 8 weeks: 1.61 ± 0.17 vs. 0.93 ± 0.09, p = 0.008; HG‐VEGF vs. control). The findings indicate that HG‐VEGF coating has potential for the development of engineered small‐diameter artificial grafts, although further research is needed to prevent neo‐intimal hyperplasia. Copyright © 2016 John Wiley & Sons, Ltd.     

Shear stress with appropriate time‐step and amplification enhances endothelial cell retention on vascular grafts

Endothelial cells (ECs) are sensitive to changes in shear stress. The application of shear stress to ECs has been well documented to improve cell retention when placed into a haemodynamically active environment. However, the relationship between the time‐step and amplification of shear stress on EC functions remains elusive. In the present study, human umbilical cord veins endothelial cells (HUVECs) were seeded on silk fibroin nanofibrous scaffolds and were preconditioned by shear stress at different time‐steps and amplifications. It is shown that gradually increasing shear stress with appropriate time‐steps and amplification could improve EC retention, yielding a complete endothelial‐like monolayer both in vitro and in vivo. The mechanism of this improvement is mediated, at least in part, by an upregulation of integrin β1 and focal adhesion kinase (FAK) expression, which contributed to fibronectin (FN) assembly enhancement in ECs in response to the shear stress. A modest gradual increase in shear stress was essential to allow additional time for ECs to gradually acclimatize to the changing environment, with the goal of withstanding the physiological levels of shear stress. This study recognized that the time‐steps and amplifications of shear stress could regulate EC tolerance to shear stress and the anti‐thrombogenicity function of engineered vascular grafts via an extracellular cell matrix‐specific, mechanosensitive signalling pathway and might prevent thrombus formation in vivo. Copyright © 2016 John Wiley & Sons, Ltd.     

Osteogenesis of adipose‐derived stem cells on polycaprolactone–β‐tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I

The current study aimed to fabricate three‐dimensional (3D) polycaprolactone (PCL), polycaprolactone and β‐tricalcium phosphate (PCL–TCP) scaffolds via a selective laser‐sintering technique (SLS). Collagen type I was further coated onto PCL–TCP scaffolds to form PCL–TCP–COL scaffolds. The physical characters of these three scaffolds were analysed. The osteogenic potential of porcine adipose‐derived stem cells (pASCs) was compared among these three scaffolds in order to find an optimal scaffold for bone tissue engineering. The experimental results showed no significant differences in pore size and porosity among the three scaffolds; the porosity was ca. 75–77% and the pore size was ca. 300–500 µm in all three. The compressive modulus was increased from 6.77 ± 0.19 to 13.66 ± 0.19 MPa by adding 30% β‐TCP into a 70% PCL scaffold. No significant increase of mechanical strength was found by surface‐coating with collagen type I. Hydrophilicity and swelling ratios showed statistical elevation (p < 0.05) after collagen type I was coated onto the PCL–TCP scaffolds. The in vitro study demonstrated that pASCs had the best osteogenic differentiation on PCL–TCP–COL group scaffolds, due to the highest ALP activity, osteocalcin mRNA expression and mineralization. A nude mice experiment showed better woven bone and vascular tissue formation in the PCL–TCP–COL group than in the PCL group. In conclusion, the study demonstrated the ability to fabricate 3D, porous PCL–TCP composite scaffolds (PCL:TCP = 70:30 by weight) via an in‐house‐built SLS technique. In addition, the osteogenic ability of pASCs was found to be enhanced by coating COL onto the PCL–TCP scaffolds, both in vitro and in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Directional migration of endothelial cells towards angiogenesis using polymer fibres in a 3D co‐culture system

Development of an in vitro prevascularized scaffold is of great importance to produce vascularization in tissue‐engineered devices and for other clinical purposes. To this aim, polymer fibres covered with human umbilical vein endothelial cells (HUVECs) were used to induce directional ‘angiogenesis’ in a 3D co‐culture system. Gelatin or RGD peptides were immobilized on surface‐modified polymer fibres [100 µm diameter poly(ethylene terephthalate) monofilaments] via N‐hepthylamine plasma polymer and carboxy‐methyl‐dextran interlayers. Fibres fully covered with HUVECs were then embedded in a fibrin gel, following a parallel alignment pattern, in the presence of fibroblasts. Tube‐like structures occurred along the fibres and a network was formed between neighbouring fibres. These events were promoted with increased incubation times. Biomolecule‐grafted fibres created a guidance pathway that facilitated coated endothelial cells to form lumens and, from them, sprouting processes. However, there were no significant differences between the different surface modifications on fibres in terms of promoting tube‐like structures. Thus, different stages of angiogenesis can be initiated and guided using HUVECs precovered polymer fibres embedded in a soft supportive matrix, such as fibrin, which can be further applied to the development of in vitro prevascularized tissue‐engineered scaffolds. Copyright © 2010 John Wiley & Sons, Ltd.     

Cyclophosphamide plus granulocyte‐colony stimulating factor for hematopoietic stem cell mobilization in patients with multiple myeloma

We retrospectively reviewed the results of cyclophosphamide (3 g/m²), doxorubicin and dexamethasone plus granulocyte‐colony stimulating factor (G‐CSF) (ID‐CY/DOX group), low‐dose cyclophosphamide (2 g/m²) plus G‐CSF (LD‐CY group) and G‐CSF alone (G‐CSF group) for stem cell mobilization in patients with multiple myeloma. A total of 89 patients with 93 mobilizations were included. Apheresis was started when total white blood cell (WBC) count >10 × 10⁹/L for ID‐CY/DOX and LD‐CY groups and after eight doses of G‐CSF (5 μg/kg twice daily) for G‐CSF group. For five mobilizations in ID‐CY/DOX group, the rate of successful mobilization (≥4.0 × 10⁶/kg CD34+ cells) was 80%. For 78 mobilizations in LD‐CY group, the successful rate was 80.8%. For 10 mobilizations in the G‐CSF group, the successful rate was 50%. The mean yield of CD34+ cells was higher in ID‐CY/DOX and LD‐CY groups as compared with that in G‐CSF group (P = 0.026 and 0.020, respectively). There was no difference in the yield of CD34+ cells between ID‐CY/DOX and LD‐CY groups (P = 0.831). After autologous stem cell transplantation, the days to neutrophil and platelet engraftment were similar in these three groups (P = 0.713 and 0.821, respectively). In conclusion, we observed that ID‐CY/DOX and LD‐CY plus G‐CSF for stem cell mobilization resulted in a higher successful rate and higher stem cell yields than G‐CSF alone and their engraftment time were similar. Total WBC count >10 × 10⁹/L can be used as a guide to start apheresis in CY‐based stem cell mobilization. J. Clin. Apheresis 31:423–428, 2016. © 2015 Wiley Periodicals, Inc.     

Micropatterned three‐dimensional hydrogel system to study human endothelial–mesenchymal stem cell interactions

The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell types and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (a) bone marrow aspirates; (b) perivascular cells from the umbilical cord vein; and (c) perivascular cells from the umbilical cord artery. Cell populations were isolated and identified as MSCs according to their phenotypes and differentiation potential. Human umbilical vein endothelial cells (HUVECs) were used as a standard for endothelial cells. A novel co‐culture system was developed to study cell–cell interactions in a spatially controlled three‐dimensional (3D) fibrin hydrogel model. Using microfluidic patterning, it was possible to localize hydrogel‐encapsulated HUVECs and MSCs within separate channels spaced at 500, 1000 or 2000 µm. All three MSC populations had similar expression profiles of mesenchymal cell markers and similar capacity for osteogenic and adipogenic differentiation. However, bone marrow‐derived MSCs (but not umbilical vein or artery derived MSCs) showed strong distance‐dependent migration toward HUVECs and supported the formation of stable vascular networks resembling capillary‐like vasculature. The presented approach provides a simple and robust model to study the cell–cell communication of relevance to engineering vascularized tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Cell‐free artificial implants of electrospun fibres in a three‐dimensional gelatin matrix support sciatic nerve regeneration in vivo

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell‐free, three‐dimensional scaffold for axonal guidance in long‐distance nerve repair. Sub‐micron scale fibres of biodegradable poly‐ε‐caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15‐mm‐long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell‐free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.     

In vitro and in vivo co‐culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL–PEG–PCL hydrogels enhances cartilage formation

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co‐culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL–PEG–PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co‐cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co‐culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co‐culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co‐culture system in rabbit models. The co‐culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co‐culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Expression of platelet‐bound stromal cell‐derived factor‐1 in patients with non‐valvular atrial fibrillation and ischemic heart disease

Summary. Aims: Blood cell infiltration and inflammation are involved in atrial remodelling during atrial fibrillation (AF) although the exact mechanisms of inflammatory cell recruitment remain poorly understood. Platelet‐bound stromal cell‐derived factor‐1 (SDF‐1) is increased in cases of ischemic myocardium and regulates recruitment of CXCR4⁺ cells on the vascular wall. Whether platelet‐bound SDF‐1 expression is differentially influenced by non‐valvular paroxysmal or permanent atrial fibrillation (AF) in patients with stable angina pectoris (SAP) or acute coronary syndrome (ACS) has not been reported so far. Methods and results: A total of 1291 consecutive patients with coronary artery disease (CAD) undergoing coronary angiography were recruited. Among the patients with SAP, platelet‐bound‐SDF‐1 is increased in patients with paroxysmal AF compared with SR or to persistent/permanent AF (P < 0.05 for both). Platelet‐bound SDF‐1 correlated with plasma SDF‐1 (r = 0.488, P = 0.013) in patients with AF and ACS, which was more pronounced among patients with persistent AF (r = 0.842, P = 0.009). Plasma SDF‐1 was increased in persistent/permanent AF compared with SR. Patients with ACS presented with enhanced platelet‐bound‐SDF‐1 compared with SAP. Interestingly, among patients with ACS, patients with paroxysmal or persistent/permanent AF presented with an impaired platelet‐bound SDF‐1 expression compared with patients with SR. Conclusions: Differential expression of platelet‐bound and plasma SDF‐1 was observed in patients with AF compared with SR which may be involved in progenitor cell mobilization and inflammatory cell recruitment in patients with AF and ischemic heart disease. Further in vivo studies are required to elucidate the role of SDF‐1 in atrial remodeling and the atrial fibrillation course.     

Peritoneal cell sheets composed of mesothelial cells and fibroblasts prevent intra‐abdominal adhesion formation in a rat model

Postoperative intra‐abdominal adhesions remain an unsolved problem despite significant progress in the surgical procedures themselves. They often lead to small‐bowel obstruction, chronic abdominal and pelvic pain, as well as female infertility. The loss of mesothelial cells and several components of the inflammatory system following injury to the peritoneum results in fibrin formation and angiogenesis. The remaining fibrin matrix and angiogenesis lead to replacement by fibroblasts and fibrous band formation. The aim of this study was to develop a new therapeutic method of preventing intra‐abdominal adhesions. We fabricated transplantable peritoneal cell sheets from the rat peritoneum by cell sheet engineering using a temperature‐responsive culture system. The peritoneal cell sheets developed were composed of an upper monolayer of mesothelial cells and underlying multilayered fibroblasts, similar to the peritoneum in vivo. Transplantation of peritoneal cell sheets prevented tissue adhesion, fibrin deposition and angiogenesis, and, moreover, lymphangiogenesis and macrophage infiltration in a rat caecum cauterization adhesion model. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Melatonin promotes survival of nonvascularized fat grafts and enhances the viability and migration of human adipose‐derived stem cells via down‐regulation of acute inflammatory cytokines

Nonvascularized fat grafting is a valuable technique for soft tissue reconstruction but poor survival of fat in the host environment remains a problem. A process known as cell‐assisted transfer is used to enhance fat graft retention by adding stromal vascular fraction, an adipose‐derived stem cell (ASC) rich content to lipoaspirate. We have recently shown that the use of melatonin, a reactive oxygen species scavenger, protects human ASCs from hydrogen peroxide‐induced oxidative stress and cell death in vitro but its role as a pharmacological adjunct in clinical fat grafting has not been studied. Herein, the effect of melatonin was examined on human ASCs in vitro using survival and functional assays including the MTT assay, CellTox Green assay, monolayer scratch assay as well as a human cytokine chemoluminescence, and tumour necrosis factor‐α assay. Further, the effect of melatonin‐treated fat grafts was tested in vivo with a murine model. Haematoxylin and eosin staining, perilipin and CD31 immunostaining were performed with morphometric analysis of adipose tissue. The results demonstrate that, in vitro, the addition of melatonin to ASCs significantly improved their cell‐viability, promoted cell migration and preserved membrane integrity as compared to controls. In addition, it induced a potent anti‐inflammatory response by downregulating acute inflammatory cytokines particularly tumour necrosis factor‐α. For the first time, it is demonstrated in vivo that melatonin enhances fat graft volume retention by reducing inflammation and increasing the percentage of adipose volume within fat grafts with comparable volumes to that of cell‐assisted lipotransfer. Based on these novel findings, melatonin may be a useful pharmacological adjunct in clinical fat grafting.     

Asymmetrical seeding of MSCs into fibrin–poly(ester‐urethane) scaffolds and its effect on mechanically induced chondrogenesis

Mesenchymal stem cells (MSCs) are currently being investigated as candidate cells for regenerative medicine approaches for the repair of damaged articular cartilage. For these cells to be used clinically, it is important to understand how they will react to the complex loading environment of a joint in vivo. In addition to investigating alternative cell sources, it is also important for the structure of tissue‐engineered constructs and the organization of cells within them to be developed and, if possible, improved. A custom built bioreactor was used to expose human MSCs to a combination of shear and compression loading. The MSCs were either evenly distributed throughout fibrin‐poly(ester‐urethane) scaffolds or asymmetrically seeded with a small proportion seeded on the surface of the scaffold. The effect of cell distribution on the production and deposition of cartilage‐like matrix in response to mechanical load mimicking in vivo joint loading was then investigated. The results show that asymmetrically seeding the scaffold led to markedly improved tissue development based on histologically detectable matrix deposition. Consideration of cell location, therefore, is an important aspect in the development of regenerative medicine approaches for cartilage repair. This is particularly relevant when considering the natural biomechanical environment of the joint in vivo and patient rehabilitation protocols. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.     

Enhanced cell‐associated plasminogen activator pathway but not coagulation pathway activity contributes to motility in metastatic breast cancer cells

Summary. Background: Activation of tumor cell‐associated coagulation and plasminogen activator pathways occurs in malignant disease processes, including breast cancer, and may promote metastatic activity. Objectives/Methods: To compare the coagulation and plasminogen activator pathways of normal and metastatic cells, we examined two cell lines from the MCF‐10 family of breast cells: near‐normal immortalized MCF‐10A cells, and metastatic MCF‐10CA1 cells. Results: MCF‐10CA1 cell motility was significantly increased as compared with that of MCF‐10A cells. The two cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. MCF‐10A cells produced a stable fibrin network, whereas MCF‐10CA1 cells lysed the surrounding fibrin network within 24 h of network formation. Importantly, fibrin located proximal to (within 10 μm) the MCF‐10CA1 cell surface lysed substantially faster than fibrin located 100 μm from the surface. MCF‐10CA1 cells supported significantly increased plasmin generation rates as compared with MCF‐10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF‐10CA1 cells had increased expression (mRNA and protein) levels of urokinase plasminogen activator (u‐PA) and decreased levels of plasminogen activator inhibitor‐1 as compared with MCF‐10A cells. Blocking u‐PA activity with the active site‐directed protease inhibitor amiloride substantially decreased MCF‐10CA1 cell motility. Phosphorylated Akt levels were elevated in MCF‐10CA1 cells, which partially explains the increased u‐PA expression. Conclusions: These results suggest that the tumor‐associated plasminogen activator pathway, not the coagulation pathway, is a key distinguishing feature between metastatic MCF10‐CA1 cells and normal MCF‐10A cells.