The power of fat and its adipose‐derived stromal cells: emerging concepts for fibrotic scar treatment

Lipofilling or lipografting is a novel and promising treatment method for reduction or prevention of dermal scars after injury. Ample anecdotal evidence from case reports supports the scar‐reducing properties of adipose tissue grafts. However, only a few properly controlled and designed clinical trials have been conducted thus far on this topic. Also, the underlying mechanism by which lipofilling improves scar aspect and reduces neuropathic scar pain remains largely undiscovered. Adipose‐derived stromal or stem cells (ADSC) are often described to be responsible for this therapeutic effect of lipofilling. We review the recent literature and discuss anticipated mechanisms that govern anti‐scarring capacity of adipose tissue and its ADSC. Both clinical and animal studies clearly demonstrated that lipofilling and ADSC influence processes associated with wound healing, including extracellular matrix remodelling, angiogenesis and modulation of inflammation in dermal scars. However, randomized clinical trials, providing sufficient level of evidence for lipofilling and/or ADSC as an anti‐scarring treatment, are lacking yet warranted in the near future. © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd     

Vascular endothelial growth factor‐transfected adipose‐derived stromal cells enhance bone regeneration and neovascularization from bone marrow stromal cells

Vascular endothelial growth factor (VEGF)‐transfected adipose‐derived stromal cells (ADSCs^VEGF) were devised to promote bone regeneration and neovascularization of bone marrow stromal cells (BMSCs). ADSCs^VEGF were added to BMSCs and cocultured in variable proportions. ADSCs^VEGF alone or ADSCs^VEGF with BMSCs (BMSCs:ADSCs^VEGF ratio of 1:0.025–0.5) induced significantly greater tube formation in human umbilical vein endothelial cells than untransfected ADSCs. The cocultures of BMSCs and ADSCs^VEGF at ratios of 1: 0.025–0.1 showed significantly greater osteogenic differentiation and mineralization than BMSCs alone in vitro. Osteogenic markers COL1A1, OCN and BSP were most effectively induced at the BMSC: ADSC^VEGF ratio of 1:0.05. Of angiogenesis‐related genes, upregulation of cathepsin Z and downregulation of early growth response 1 were observed while two osteogenesis‐related genes, osteoactivin and tetranectin, were upregulated in BMSCs/ADSCs^VEGF compared to BMSCs/ADSCs. When critical size calvarial defects in rats were implanted with mixture of BMSCs and ADSCs^VEGF along with hydroxyapatite/β‐tricalcium phosphate granules, BMSCs and ADSCs^VEGF at the ratio of 1:0.05 showed better bone regeneration that BMSCs alone. The cotransplantation of ADSCs^VEGF with BMSCs significantly increased neovascularization on the regenerated bone of the repaired defect than BMSCs alone. In conclusion, ADSCs^VEGF added in small proportion to BMSCs effectively promote bone regeneration and neovascularization. Copyright © 2017 John Wiley & Sons, Ltd.     

Optimal administration routes for adipose‐derived stem cells therapy in ischaemic flaps

Improvement of flap survival represents an ongoing challenge in reconstructive surgery. The angiogenic potential of adipose‐derived stem cells (ASCs) offers a promising approach to improve the viability of random pattern flaps. Recently, to maximize the therapeutic effects of ASCs, increasing focus is being placed on how to deliver the stem cells to target lesions. The purpose of the present study was to compare the effectiveness of different administration routes of ASCs to improve the viability of the random pattern skin flap. ASCs labelled with PKH26 were applied via four methods to the cranially‐based random pattern skin flaps of rats: (a) intravenous injection; (b) subcutaneous injection; (c) application with collagen sponge seeding; and (d) application with fibrin glue seeding. ASCs led to a significant increase in flap viability in the subcutaneous injection group and the collagen sponge group. Cutaneous blood flow was increased in the intravenous injection, subcutaneous injection and collagen sponge groups. Capillary density in the intravenous injection group and collagen sponge group was significantly greater than in the control group (no treatment). PKH26‐positive cells via the collagen sponge were distributed more densely within the flap than in other groups. This study demonstrated that the collagen sponge method delivered ASCs most effectively within the flap and increased flap vascularity. The clinical therapeutic effects of ASCs can therefore be maximized when the optimal delivery route is chosen. Copyright © 2012 John Wiley & Sons, Ltd.     

Adipose‐derived stromal cells for the reconstruction of a human vesical equivalent

Despite a wide panel of tissue‐engineering models available for vesical reconstruction, the lack of a differentiated urothelium remains their main common limitation. For the first time to our knowledge, an entirely human vesical equivalent, free of exogenous matrix, has been reconstructed using the self‐assembly method. Moreover, we tested the contribution of adipose‐derived stromal cells, an easily available source of mesenchymal cells featuring many potential advantages, by reconstructing three types of equivalent, named fibroblast vesical equivalent, adipose‐derived stromal cell vesical equivalent and hybrid vesical equivalent – the latter containing both adipose‐derived stromal cells and fibroblasts. The new substitutes have been compared and characterized for matrix composition and organization, functionality and mechanical behaviour. Although all three vesical equivalents displayed adequate collagen type I and III expression, only two of them, fibroblast vesical equivalent and hybrid vesical equivalent, sustained the development of a differentiated and functional urothelium. The presence of uroplakins Ib, II and III and the tight junction marker ZO‐1 was detected and correlated with impermeability. The mechanical resistance of these tissues was sufficient for use by surgeons. We present here in vitro tissue‐engineered vesical equivalents, built without the use of any exogenous matrix, able to sustain mechanical stress and to support the formation of a functional urothelium, i.e. able to display a barrier function similar to that of native tissue. Copyright © 2013 John Wiley & Sons, Ltd.     

Chondrogenic potential of human dermal fibroblasts in a contractile,soft, self‐assembling,peptide hydrogel

The present paper describes a simple approach to obtain three‐dimensional (3D) cartilage constructs using human normal dermal fibroblasts (hNDFs) cultured in a self‐assembling peptide nanofibre scaffold. During the first days of culture, the 3D constructs underwent morphological changes consisting of a substantial contraction process that ended in a small compact structure. During this process the system became sensitive to induction with standard chondrogenic medium, evidenced by the expression of specific markers of mature cartilage. First, it was detected that the samples become highly stained with toluidine blue dye over time (40–50 days), indicating that the system produced significantly high amounts of glycosaminoglycans. By quantitative PCR, it was confirmed that the system significantly upregulated the expression of the proteoglycan aggrecan, a good indicator of cartilage commitment. Moreover, collagen type II was upregulated at protein level, confirming that the system differentiated to a chondrocyte‐like construct. Additionally, during the first days of culture in control medium analysed hNDFs proliferation capacity in this 3D system was analysed. This platform could be used in the future to obtain an autologous source of cells from a simple patient skin biopsy, which could be easily translated into a low‐cost and effective regenerative therapy. 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.     

Collagen modules for in situ delivery of mesenchymal stromal cell‐derived endothelial cells for improved angiogenesis

Modular tissue engineering is a strategy to create scalable, self‐assembling, three‐dimensional (3D) tissue constructs. This strategy was used to deliver endothelial‐like cells derived from bone marrow mesenchymal stromal cells (EL‐MSCs) to locally induce vascularization. First, tissue engineered modules were formed, comprising EL‐MSCs and collagen‐based cylinders. Seven days of module culture in a microfluidic chamber under continuous flow resulted in the formation of interstices, formed by random packing of the modules, which served as channels and were lined by the EL‐MSCs. We observed maintenance of the endothelial phenotype of the EL‐MSCs, as demonstrated by CD31 staining, and the cells proliferated well. Next, collagen modules covered with EL‐MSCs, with or without embedded MSCs, were implanted subcutaneously in immune‐compromised SCID/Bg mice. After 7 days, CD31‐positive vessels were observed in the samples. These data demonstrate the feasibility of EL‐MSCs coated collagen module as a strategy to locally stimulate angiogenesis and vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

Effective expansion of human adipose‐derived stromal cells and bone marrow‐derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles‐coated substratum with human platelet‐rich plasma

Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet‐rich plasma (PRP). In this study, we investigated the capability of F/P NP‐coated plates to act as a substratum for the proliferation of human adipose‐derived stromal cells (ASCs) and bone marrow‐derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP‐coated plates and grew optimally, with a doubling time of 30 and 32 h in low‐concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor‐2 (FGF‐2) on the F/P NP‐coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF‐2 on F/P NP‐coated plates pretreated with PRP and FGF‐2 in a concentration‐dependent manner. Thus, F/P NP‐coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low‐concentration PRP medium supplemented with FGF‐2. No xenogeneic serum is required. Copyright © 2012 John Wiley & Sons, Ltd.     

Using minimalist self‐assembling peptides as hierarchical scaffolds to stabilise growth factors and promote stem cell integration in the injured brain

Neurotrophic growth factors are effective in slowing progressive degeneration and/or promoting neural repair through the support of residual host and/or transplanted neurons. However, limitations including short half‐life and enzyme susceptibility of growth factors highlight the need for alternative strategies to prolong localised delivery at a site of injury. Here, we establish the utility of minimalist N‐fluorenylmethyloxycarbonyl (Fmoc) self‐assembling peptides (SAPs) as growth factor delivery vehicle, targeted at supporting neural transplants in an animal model of Parkinson's disease. The neural tissue‐specific SAP, Fmoc‐DIKVAV, demonstrated sustained release of glial cell line derived neurotrophic factor, up to 172 hr after gel loading. This represents a significant advance in drug delivery, because its lifetime in phosphate buffered saline was less than 1 hr. In vivo transplantation of neural progenitor cells, together with our growth factor‐loaded material, into the injured brain improved graft survival compared with cell transplants alone. We show for the first time the use of minimalist Fmoc‐SAP in an in vivo disease model for sustaining the delivery of neurotrophic growth factors, facilitating their spatial and temporal delivery in vivo, whilst also providing an enhanced niche environment for transplanted cells.     

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.     

Effects of resveratrol on enrichment of adipose‐derived stem cells and their differentiation to osteoblasts in two‐and three‐dimensional cultures

The goal of this study was to develop a method for increasing the yield of multipotent adipose‐derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271⁺, CD73⁺ and CD45^–) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11⁺ and OCN⁺. Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µ m ) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two‐dimensional (2D) cultures as a function of alkaline phosphatase‐specific activity and osteocalcin production. In addition, cells were seeded onto three‐dimensional (3D) poly‐ε‐caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro‐CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol‐treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Cultured cell‐derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells

Cell‐derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro‐environment. Therefore, cultured cell‐derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro‐environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell‐derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)‐derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.     

Influence of self‐assembling peptide nanofibre scaffolds on retinal differentiation potential of stem/progenitor cells derived from ciliary pigment epithelial cells

Aim of the study was to investigate the influence of the self‐assembling peptide nanofibre scaffolds (SAPNs) on the growth, proliferation and retinal neuronal differentiation of the stem/progenitor cells (SCs) derived from the ciliary pigment epithelium (CPE) of human cadaveric eye. Here SAPNs (RADA16‐I, PM), which is well described in previous studies, commercially available and xeno‐free. The CPE cells isolated were cultured in DMEM/F12 supplemented with N2 and growth factors such as basic fibroblast growth factor and epidermal growth factor, encapsulated in the scaffolds. The entrapped SCs actively expanded and formed clone‐like clusters in the scaffolds. Many cells in the cluster were proliferating, as revealed by 5‐bromo‐2‐deoxyuridine uptake and could be maintained for up to 6 days and expressed neural progenitor markers such as β‐III tubulin, Nestin, Pax6 and Musashi1. Upon differentiation of these cells in conditioned medium, the cells exhibited retinal neuronal markers such as s‐Opsin, rhodopsin and Recoverin. The RT² profiler polymerase chain reaction array experiments showed selective gene expression, possibly involved in neural stem/progenitor cell adhesion and differentiation. These findings suggest the suitability of the three‐dimensional culture system for the proliferation and maintenance of CPE stem/progenitor cells (CPE‐NS) and for possible use in ex vivo studies of small molecules, drug deliveries for retinal diseases and for use in combination with directed stem/progenitor cell differentiation. and ultimately for tissue replacement therapies. Copyright © 2014 John Wiley & Sons, Ltd.     

Matrix‐directed differentiation of human adipose‐derived mesenchymal stem cells to dermal‐like fibroblasts that produce extracellular matrix

Commercially available skin substitutes lack essential non‐immune cells for adequate tissue regeneration of non‐healing wounds. A tissue‐engineered, patient‐specific, dermal substitute could be an attractive option for regenerating chronic wounds, for which adipose‐derived mesenchymal stem cells (ADMSCs) could become an autologous source. However, ADMSCs are multipotent in nature and may differentiate into adipocytes, osteocytes and chondrocytes in vitro, and may develop into undesirable tissues upon transplantation. Therefore, ADMSCs committed to the fibroblast lineage could be a better option for in vitro or in vivo skin tissue engineering. The objective of this study was to standardize in vitro culture conditions for ADMSCs differentiation into dermal‐like fibroblasts which can synthesize extracellular matrix (ECM) proteins. Biomimetic matrix composite, deposited on tissue culture polystyrene (TCPS), and differentiation medium (DM), supplemented with fibroblast‐conditioned medium and growth factors, were used as a fibroblast‐specific niche (FSN) for cell culture. For controls, ADMSCs were cultured on bare TCPS with either DM or basal medium (BM). Culture of ADMSCs on FSN upregulated the expression of differentiation markers such as fibroblast‐specific protein‐1 (FSP‐1) and a panel of ECM molecules specific to the dermis, such as fibrillin‐1, collagen I, collagen IV and elastin. Immunostaining showed the deposition of dermal‐specific ECM, which was significantly higher in FSN compared to control. Fibroblasts derived from ADMSCs can synthesize elastin, which is an added advantage for successful skin tissue engineering as compared to fibroblasts from skin biopsy. To obtain rapid differentiation of ADMSCs to dermal‐like fibroblasts for regenerative medicine, a matrix‐directed differentiation strategy may be employed. Copyright © 2014 John Wiley & Sons, Ltd.     

Increased survival of human free fat grafts with varying densities of human adipose‐derived stem cells and platelet‐rich plasma

The high absorption rate of transplanted fat has limited the application of autogenous fat grafts in the clinical setting. Therefore, this study aimed to evaluate the effects of platelet‐rich plasma (PRP) and adipose‐derived stem cells (ASCs) on fat regeneration by investigating the impact of PRP and conditioned medium on the biological characteristics of ASCs. Fat grafts were prepared with ASCs at densities of 10⁷/ml, 10⁶/ml, 10⁵/ml, 10⁴/ml and 0/ml with and without PRP and injected subcutaneously into nude mice. Liquid overflow method, haematoxylin and eosin staining, and immunohistochemical analyses were used to examine the fat grafts. The residual fat volume of the 10⁵/ml ASC + PRP group was significantly higher than that of other treatment conditions after 90 days. Furthermore, histological examination revealed that in 10⁵/ml ASCs‐treated grafts normal adipocyte area and capillary formation were increased dramatically compared with other treatment conditions. It is concluded that fat grafts consisting of PRP and 10⁵/ml ASCs constitute an ideal transplant strategy, which may result in decreased absorption and accelerated fat regeneration. This simple and reliable method could provide a valuable and needed tool in plastic and reconstructive surgery. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparing the osteogenic potential of bone marrow and tendon‐derived stromal cells to repair a critical‐sized defect in the rat femur

Despite significant advancements in bone tissue‐engineering applications, the clinical impact of bone marrow stromal cells (BMSCs) for the treatment of large osseous defects remains limited. Therefore, other cell sources are under investigation for their osteogenic potential to repair bone. In this study, tendon‐derived stromal cells (TDSCs) were evaluated in comparison to BMSCs to support the functional repair of a 5 mm critical‐sized, segmental defect in the rat femur. Analysis of the trilineage differentiation capacity of TDSCs and BMSCs cultured on collagen sponges revealed impaired osteogenic differentiation and mineral deposition of TDSCs in vitro, whereas chondrogenic and adipogenic differentiation was evident for both cell types. Radiographic assessment demonstrated that neither cell type significantly improved the healing rate of a challenging 5 mm segmental femoral defect. Transplanted TDSCs and BMSCs both led to the formation of only small amounts of bone in the defect area, and histological evaluation revealed non‐mineralized, collagen‐rich scar tissue to be present within the defect area. Newly formed lamellar bone was restricted to the defect margins, resulting in closure of the medullary cavity. Interestingly, in comparison to BMSCs, significantly more TDSC‐derived cells were present at the osteotomy gap up to 8 weeks after transplantation and were also found to be located within newly formed lamellar bone, suggesting their capacity to directly contribute to de novo bone formation. To our knowledge, this is the first study investigating the in vivo capacity of TDSCs to regenerate a critical‐sized defect in the rat femur. Copyright © 2015 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.     

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.     

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.