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.     

Platelet‐rich fibrin‐based matrices to improve angiogenesis in an in vitro co‐culture model for bone tissue engineering

In the context of prevascularization strategies for tissue‐engineering purposes, co‐culture systems consisting of outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs) have been established as a promising in vitro tool to study regeneration mechanisms and to identify factors that might positively influence repair processes such as wound healing or angiogenesis. The development of autologous injectable platelet‐rich fibrin (PRF), which can be generated from peripheral blood in a minimal invasive procedure, fulfils several requirements for clinically applicable cell‐based tissue‐engineering strategies. During this study, the established co‐culture system of OECs and pOBs was mixed with injectable PRF and was cultivated in vitro for 24 h or 7 days. The aim of this study was to analyse whether PRF might have a positive effect on wound healing processes and angiogenic activation of OECs in the co‐culture with regard to proinflammatory factors, adhesion molecules and proangiogenic growth factor expression. Histological cell detection revealed the formation of lumina and microvessel‐like structures in the PRF/co‐culture complexes after 7 days of complex cultivation. Interestingly, the angiogenic activation of OECs was accompanied by an upregulation of wound healing‐associated factors, as well as by a higher expression of the proangiogenic factor vascular endothelial growth factor, which was evaluated both on the mRNA level as well as on the protein level. Thus, PRF might positively influence wound healing processes, in particular angiogenesis, in the in vitro co‐culture, making autologous PRF‐based matrices a beneficial therapeutic tool for tissue‐engineering purposes by simply profiting from the PRF, which contains blood plasma, platelets and leukocytes.     

Human endothelial colony‐forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold‐based tissue engineering

One of the major challenges in tissue engineering is to supply larger three‐dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)‐derived endothelial colony‐forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue‐culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo‐expanded ECFCs are a novel, very potent cell source for scaffold‐based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

TLR4 stimulation by LPS enhances angiogenesis in a co‐culture system consisting of primary human osteoblasts and outgrowth endothelial cells

The development of new approaches leading to fast and successful vascularization of tissue‐engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS‐EB) significantly enhances microvessel formation in a co‐culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS‐EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT–PCR have shown that the level of two adhesion molecules (ICAM‐1 and E‐selectin), two cytokines (IL‐6 and IL‐8) and two growth factors (VEGF and PDGF‐BB) related to angiogenesis increase significantly after LPS‐EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Undifferentiated and differentiated adipose‐derived stem cells improve nerve regeneration in a rat model of facial nerve defect

Autologous nerve grafting is the current procedure used for repairing facial nerve gaps. As an alternative to this method, tissue engineering cell‐based therapy using induced pluripotent stem cells, Schwann cells and bone marrow‐derived mesenchymal stem cells has been proposed. However, these cells have major problems, including tumorigenesis in induced pluripotent stem cells and invasiveness and limited tissue associated with harvesting for the other cells. Here, we investigated the therapeutic potential of adipose‐derived stem cells (ASCs), which can be harvested easily and repeatedly by a minimally invasive liposuction procedure. The ASCs had characteristics of mesenchymal tissue lineages and could differentiate into Schwann‐like cells that were relatively simple to isolate and expand in culture. In an in vivo study, a silicone conduit containing undifferentiated ASCs, differentiated ASCs or Schwann cells were transplanted, embedded in a collagen gel and the efficacy of repair of a 7 mm‐gap in the rat facial nerve examined. Morphometric quantification analysis of regenerated facial nerves after a regeneration period of 13 weeks showed that undifferentiated ASCs, differentiated ASCs, and Schwann cells had similar potential for nerve regeneration. Furthermore, the functional recovery of facial nerve regeneration using a rat facial palsy scoring system in the three groups was close to that in autologous nerve graft positive controls. These findings suggest that undifferentiated and differentiated ASCs may both have therapeutic potential in facial nerve regeneration as a source of Schwann cells in cell‐based therapy performed as an alternative to autologous nerve grafts. Copyright © 2014 John Wiley & Sons, Ltd.     

In vitro analysis of the interactions between preadipocytes and endothelial cells in a 3D fibrin matrix

The volume‐persistent survival of transplanted adipose tissue in vivo relies on early vascularization, due to an otherwise early induction of apoptosis of the centrally located cells. Thus, one way to enable the early formation of a capillary network resulting in a sufficient perfusion of the transplanted construct might be the co‐transplantation of autologous preadipocytes with endothelial cells. To investigate preadipocyte–endothelial cell interaction, three‐dimensional proliferation‐ and angiogenesis assays were performed in vitro. Proliferation rates of co‐cultured endothelial cells and preadipocytes suspended in a fibrin matrix were elucidated by Alamarblue assays. The spheroid angiogenesis model was applied for analyzing the effects of vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) (produced by preadipocytes) as well as the impact of cell‐cell interaction between preadipocytes and endothelial cells and fibrin matrix on endothelial cell migration. Preadipocytes proliferated in fibrin glue, whereas endothelial cells underwent apoptosis. By co‐culturing, both cell types demonstrated an increased proliferation rate. Preadipocytes provoked migration of endothelial cells. Blocking bFGF and/or VEGF led to a significant decrease of migration. Changes in fibrin structure were followed by migration of single cells instead of sprouting. An appropriate fibrin matrix as well as already differentiated endothelial cells are necessary for preadipocytes to develop their angiogenic activity via bFGF and VEGF.     

Co‐transplantation of Wharton's jelly mesenchymal stem cell‐derived osteoblasts with differentiated endothelial cells does not stimulate blood vessel and osteoid formation in nude mice models

A major challenge in bone tissue engineering is the lack of post‐implantation vascular growth into biomaterials. In the skeletal system, blood vessel growth appears to be coupled to osteogenesis—suggesting the existence of molecular crosstalk between endothelial cells (ECs) and osteoblastic cells. The present study (performed in two murine ectopic models) was designed to determine whether co‐transplantation of human Wharton's jelly mesenchymal stem cell‐derived osteoblasts (WJMSC‐OBs) and human differentiated ECs enhances bone regeneration and stimulates angiogenesis, relative to the seeding of WJMSC‐OBs alone. Human WJMSC‐OBs and human ECs were loaded into a silicate‐substituted calcium phosphate (SiCaP) scaffold and then ectopically implanted at subcutaneous or intramuscular sites in nude mice. At both subcutaneous and intramuscular implantation sites, we observed ectopic bone formation and osteoids composed of host cells when WJMSC‐OBs were seeded into the scaffold. However, the addition of ECs was associated with a lower level of osteogenesis, and we did not observe stimulation of blood vessel ingrowth. in vitro studies demonstrated that WJMSC‐OBs lost their ability to secrete vascular endothelial growth factor and stromal cell‐derived factor 1—including when ECs were present. In these two murine ectopic models, our cell‐matrix environment combination did not seem to be optimal for inducing vascularized bone reconstruction.     

纤维蛋白凝胶与大鼠嗅鞘细胞的生物相容性

背景:纤维蛋白凝胶已被证实为优质的生物材料,其在神经组织工程中亦得到应用,既往研究表明纤维蛋白凝胶可以做为一些移植细胞的支架材料.目的:观察纤维蛋白凝胶与大鼠嗅鞘细胞的生物相容性.设计、时间与地点:体外细胞学对比观察,于2007-08/2008-02在南通大学神经生物学研究所完成.材料:将纤维蛋白单体与催化剂混合制成纤维蛋白凝胶;取SD大鼠嗅球,行嗅鞘细胞原代培养.方法:实验分为对照组(单纯培养嗅鞘细胞)和纤维蛋白凝胶组(嗅鞘细胞与纤维蛋白凝胶联合培养).培养1周后冰冻切片,行免疫荧光抗体标记,显微镜下观察及检测.主要观察指标:嗅鞘细胞形态学,细胞计数、胞体面积及细胞周长.结果:嗅鞘细胞接种到纤维蛋白凝胶后,大多数嗅鞘细胞能在纤维蛋白凝胶内生长,悬浮在纤维蛋白凝胶之中,以下层为主,接种7 d后细胞胞体呈梭形或三角形,多为双极或三极.纤维蛋白凝胶组的嗅鞘细胞数明显多于对照组(P<0.05),且胞体相对较大(P<0.05),两组细胞周长差异无显著性意义(P>0.05).结论:纤维蛋白凝胶与嗅鞘细胞具有良好的生物相容性,嗅鞘细胞能够在纤维蛋白凝胶内存活和增殖.     

Clinical‐scale expansion of adipose‐derived stromal cells starting from stromal vascular fraction in a single‐use bioreactor: proof of concept for autologous applications

Adipose‐derived stromal cells (ASCs) are adult multipotent cells increasingly used for cell therapy due to their differentiation potential, their paracrine effect and their convenience. ASCs are currently selected from stromal vascular fractions (SVFs) of adipose tissue and expanded in 2D flasks following good manufacturing practices. This process is limited in surface area, labour‐intensive and expensive, especially for autologous applications requiring selection and expansion steps for every patient. Closed and automated bioreactors offer an alternative for scalable and cost‐effective production of ASCs. This study investigated a single‐use stirred‐tank bioreactor that can expand ASCs from SVFs on microcarriers. A preliminary microcarrier screening in static and spinner flask conditions was performed to evaluate the best candidate for adhesion, amplification and harvest. The selected microcarrier was used for process development in the bioreactor. The first experiments showed poor selectivity and growth of the ASCs from the SVF (n  =  2). The process was then adjusted by two means: (1) decreasing the platelet lysate in the medium for enhancing cell adherence; and (2) adding a shear protectant (Pluronic F68). Following these modifications, we demonstrated that the number of population doublings of ASCs from SVFs was not significantly different between the bioreactor and the 2D controls (n  =  3). In addition, the ASC characterization after culture showed that cells maintained their clonogenic potential, phenotype, differentiation potential and immunosuppressive capacities. This study provides the proof of concept that isolation and amplification of functional ASCs from SVFs can be performed in a stirred‐tank bioreactor combined with microcarriers. Copyright © 2016 John Wiley & Sons, Ltd.     

eNOS transfection of adipose‐derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering

This study evaluates the durability of a novel tissue engineered blood vessel (TEBV) created by seeding a natural vascular tissue scaffold (decellularized human saphenous vein allograft) with autologous adipose‐derived stem cells (ASC) differentiated into endothelial‐like cells. Previous work with this model revealed the graft to be thrombogenic, likely due to inadequate endothelial differentiation as evidenced by minimal production of nitric oxide (NO). To evaluate the importance of NO expression by the seeded cells, we created TEBV using autologous ASC transfected with the endothelial nitric oxide synthase (eNOS) gene to produce NO. We found that transfected ASC produced NO at levels similar to endothelial cell (EC) controls in vitro which was capable of causing vasorelaxation of aortic specimens ex vivo. TEBV (n = 5) created with NO‐producing ASC and implanted as interposition grafts within the aorta of rabbits remained patent for two months and demonstrated a non‐thrombogenic surface compared to unseeded controls (n = 5). Despite the xenograft nature of the scaffold, the TEBV structure remained well preserved in seeded grafts. In sum, this study demonstrates that upregulation of NO expression within adult stem cells differentiated towards an endothelial‐like lineage imparts a non‐thrombogenic phenotype and highlights the importance of NO production by cells to be used as endothelial cell substitutes in vascular tissue engineering applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Alteration of the extracellular matrix and alpha‐gal antigens in the rat lung scaffold reseeded using human vascular and adipogenic stromal cells

Regenerated organs are expected to solve the problem of donor organ shortage in transplantation medicine. One approach to lung regeneration is to decellularize the organ and reseed it with selected cells. An advantage of the procedure is reduced immunogenicity, because all cells can be theoretically replaced by autologous cells. However, little is known regarding the extracellular matrix (ECM) damage during decellularization and ECM reconstruction process in the organ regeneration. We aimed to evaluate ECM damage and reconstruction of the decellularized–recellularized rat lung, including the removal of alpha‐gal xenoantigens. Rat lungs were perfused with sodium dodecyl sulfate and Triton X‐100 via the pulmonary artery, after which the decellularized scaffold was reseeded with rat or human endothelial cells and adipose‐derived stem cell (ASCs). The ECM and alpha‐gal antigen were evaluated using immunohistochemistry, western blotting, and a glycosaminoglycan assay. Alcian blue staining revealed increased production of proteoglycan following the addition of ASCs to the rat lung recellularized with rat lung microvascular endothelial cells. Glycosaminoglycan levels decreased in the decellularized lung and increased in the recellularized lung, especially in the ASC‐treated group. Immunohistochemical expression of the alpha‐gal protein was decreased to an undetectable level in the decellularized lung tissue and disappeared after recellularization with human cells. In western blot analysis, the bands of alpha‐gal protein almost disappeared after recellularization with human cells. In conclusion, characteristics of the regenerated ECM might depend on the species and type of cells used for recellularization. Therefore, alpha‐gal antigen might be eliminated after a prolonged culture, when using human cells.     

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.     

Targeted delivery of adipose‐derived stem cells via acellular dermal matrix enhances wound repair in diabetic rats

Cell‐based therapeutic intervention has emerged as a new approach to accelerate wound closure. Adipose‐derived stem cells (ASCs), as a fascinating cell source, have received much attention in tissue repair and regeneration. In this study we evaluated the potential of acellular dermal matrix (ADM) scaffold serving as a carrier for the delivery of ASCs and investigated its therapeutic effects on wound healing. First, ASCs were isolated and characterized for multidifferentiation potential. ASCs–ADM grafts were then prepared, and ADM scaffold was shown to support the in vitro growth and proliferation of ASCs. Next, we analysed paracrine factors in conditioned medium and found that ASCs–ADM grafts secreted various cytokines, including VEGF, HGF, TGFβ and bFGF. Moreover, ASCs–ADM conditioned medium notably stimulated the migration and proliferation of fibroblasts. In vivo, we established an excisional wound model in diabetic rats which received phosphate‐buffered saline (PBS), ADM or ASCs–ADM grafts, respectively. Our results demonstrated that implantation of ASCs–ADM significantly enhanced tissue regeneration and increased epithelialization, resulting in accelerated wound closure. Immunofluorescence analysis further indicated that capillary density was evidently increased in the ASCs–ADM group compared with the control or ADM group. In addition, western blot analysis showed that ASCs–ADM significantly increased the expression of angiogenic factors, which was consistent with in vitro data. Taken together, our results suggest that targeted delivery of ASCs via ADM scaffold accelerate diabetic wound healing through a paracrine mechanism, with enhanced granulation tissue formation and increased re‐epithelialization and neovascularization. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid vascularization of starch-poly(caprolactone) in vivo by outgrowth endothelial cells in co-culture with primary osteoblasts

The successful integration of in vitro-generated tissues is dependent on adequate vascularization in vivo. Human outgrowth endothelial cells (OECs) isolated from the mononuclear cell fraction of peripheral blood represent a potent population of circulating endothelial progenitors that could provide a cell source for rapid anastomosis and scaffold vascularization. Our previous work with these cells in co-culture with primary human osteoblasts has demonstrated their potential to form perfused vascular structures within a starch-poly(caprolactone) biomaterial in vivo. In the present study, we demonstrate the ability of OECs to form perfused vascular structures as early as 48 h following subcutaneous implantation of the biomaterial in vivo. The number of OEC-derived vessels increased throughout the study, an effect that was independent of the OEC donor. This finding of rapid and thorough OEC-mediated scaffold vascularization demonstrates the great potential for OEC-based strategies to promote vascularization in tissue engineering. OECs have the potential to contribute to host-derived scaffold vascularization, and formed vascular structures at a similar density as those arising from the host. Additionally, immunohistochemical evidence demonstrated the close interaction between OECs and the co-cultured osteoblasts. In addition to the known paracrine activity osteoblasts have in modulating angiogenesis of co-cultured OECs, we demonstrate the potential of osteoblasts to provide additional structural support for OEC-derived vessels, perhaps acting in a pericyte-like role.     

Combined treatment with platelet‐rich plasma and insulin favours chondrogenic and osteogenic differentiation of human adipose‐derived stem cells in three‐dimensional collagen scaffolds

Osteochondral lesions due to injury or other pathology commonly result in the development of osteoarthritis and progressive joint destruction. Bioengineered scaffolds are widely studied for regenerative surgery strategies in osteochondral defect management, also combining the use of stem cells, growth factors and hormones. The utility in tissue engineering of human adipose‐derived stem cells (ASCs) isolated from adipose tissue has been widely noted. Autologous platelet‐rich plasma (PRP) represents an alternative strategy in regenerative medicine for the local release of endogenous growth factors and hormones. Here we compared the effects of three‐dimensional (3D) collagen type I scaffold culture and combined treatment with PRP and human recombinant insulin on the chondro‐/osteogenic differentiation of ASCs. Histochemical and biomolecular analyses demonstrated that chondro‐/osteogenic differentiation was increased in ASC‐populated 3D collagen scaffolds compared with two‐dimensional (2D) plastic dish culture. Chondro‐/osteogenic differentiation was further enhanced in the presence of combined PRP (5% v/v) and insulin (100 nm ) treatment. In addition, chondro‐/osteogenic differentiation associated with the contraction of ASC‐populated 3D collagen scaffold and increased β1/β3‐integrin expression. Inhibition studies demonstrated that PRP/insulin‐induced chondro‐/osteogenic differentiation is independent of insulin‐like growth factor 1 receptor (IGF‐1R) and mammalian target of rapamycin (mTOR) signalling; IGF‐R1/mTOR inhibition even enhanced ASC chondro‐/osteogenic differentiation. Our findings underline that 3D collagen scaffold culture in association with platelet‐derived growth factors and insulin favour the chondro‐/osteogenic differentiation of ASCs, suggesting new translational applications in regenerative medicine for the management of osteochondral defects. Copyright © 2016 John Wiley & Sons, Ltd.     

Multilineage co‐culture of adipose‐derived stem cells for tissue engineering

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue‐engineering strategies with adipose‐derived stem cells (ASCs), the focus of this study was on developing indirect co‐culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co‐culture with each of the mature cell populations was shown to successfully induce or enhance lineage‐specific differentiation of the ASCs. In general, a more homogeneous but lower‐level differentiation response was observed in co‐culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non‐canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF‐1 and DKK‐1 on multilineage differentiation in co‐culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co‐culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.     

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume‐persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self‐assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co‐culture spheroids combining adipose‐derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary‐like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT‐qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary‐like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high‐throughput (pre‐)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co‐culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular‐like structures within engineered tissues in vitro.     

Development of volume‐stable adipose tissue constructs using polycaprolactone‐based polyurethane scaffolds and fibrin hydrogels

Adipose tissue engineering aims at the restoration of soft tissue defects and the correction of contour deformities. It is therefore crucial to provide functional adipose tissue implants with appropriate volume stability. Here, we investigate two different fibrin formulations, alone or in combination with biodegradable polyurethane (PU) scaffolds as additional support structures, with regard to their suitability to generate volume‐stable adipose tissue constructs. Human adipose‐derived stem cells (ASCs) were incorporated in a commercially available fibrin sealant as well as a stable fibrin hydrogel previously developed by our group. The composite constructs made from the commercially available fibrin and porous poly(ε‐caprolactone)‐based polyurethane scaffolds exhibited increased volume stability as compared to fibrin gels alone; however, only constructs using the stable fibrin gels completely maintained their size and weight for 21 days. Adipogenesis of ASCs was not impaired by the additional PU scaffold. After induction with a common hormonal cocktail, for constructs with either fibrin formulation, strong adipogenic differentiation of ASCs was observed after 21 days in vitro. Furthermore, upregulation of adipogenic marker genes was demonstrated at mRNA (PPARγ, C/EBPα, GLUT4 and aP2; qRT–PCR) and protein (leptin; ELISA) levels. Stable fibrin/PU constructs were further evaluated in a pilot in vivo study, resulting in areas of well‐vascularized adipose tissue within the implants after only 5 weeks. Copyright © 2013 John Wiley & Sons, Ltd.     

Adipose stromal cells differentiation toward smooth muscle cell phenotype diminishes their vasculogenic activity due to induction of activin A secretion

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell‐contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC‐lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor β1 (TGFβ₁) or activin A has induced expression of SMC markers in ASCs. Analysis performed after treatment withdrawal revealed that secretome of pre‐differentiated ASCs had a reduced potency to support EC survival and these ASCs had diminished ability to support EC vasculogenesis in vitro. Vascularization of subcutaneous implants carrying a mixture of ECs and ASCs was 50% lower when, instead of control, pre‐differentiated ASCs were used. Pre‐differentiated ASCs had an inferior mitogenic response to EC‐produced factors. Differentiation of ASCs was accompanied by upregulation of vascular endothelial growth factor and a decrease in hepatocyte growth factor (HGF) production; however, addition of HGF to the co‐culture incubation media did not improve vasculogenesis. In parallel, ASC treatment with TGFβ₁ induced secretion of activin A. Augmenting co‐culture incubation media with anti‐activin A IgG restored the ability of pre‐differentiated ASCs to support vasculogenesis to the same degree as control ASCs. The present study suggests that TGFβ₁ or activin A‐induced ASC commitment to SMC phenotype negatively affects the ability of ASCs to support EC vasculogenesis in applications based on EC and ASC co‐injection into target tissues. Copyright © 2016 John Wiley & Sons, Ltd.     

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM‐MSCs) combined with calcium phosphate microparticles and enveloped in a platelet‐rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose‐derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM‐MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM‐MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM‐MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un‐aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM‐MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose‐derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.