Differentiation of human adipose-derived stem cells into beating cardiomyocytes.

Human adipose-derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5-azacytidine (5-aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co-culture with neonatal rat cardiomyocytes. 5-aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5- to 1.9-fold) and the number of cells co-expressing nkx2.5/sarcomeric alpha-actin (27.2% versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11-fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA-treated cells also stained positively for cardiac myosin heavy chain, alpha-actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non-contact co-culture showed no cardiac differentiation; however, ASCs co-cultured in direct contact co-culture exhibited a time-dependent increase in cardiac actin mRNA expression (up to 33-fold) between days 3 and 14. Immunocytochemistry revealed co-expression of GATA4 and Nkx2.5, alpha-actin, TropI and cardiac myosin heavy chain in CM-DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca(2+) transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell-to-cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co-culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.

In vivo tissue engineering of an adipose tissue flap using fat grafts and Adipogel.

For decades, plastic surgeons have spent considerable effort exploring anatomical regions for free flap design. More recently, tissue-engineering approaches have been utilised in an attempt to grow transplantable tissue flaps in vivo. The aim of this study was to engineer a fat flap with a vascular pedicle by combining autologous fat grafts and a novel acellular hydrogel (Adipogel) in an established tissue-engineering model comprising a chamber and blood vessel loop. An arteriovenous loop was created in the rat groin from the femoral vessels and positioned inside a perforated polycarbonate chamber. In Group 1, the chamber contained minced, centrifuged autologous fat; in Group 2, Adipogel was added to the graft; and in Group 3, Adipogel alone was used. Constructs were histologically examined at 6 and 12 weeks. In all groups, new tissue was generated. Adipocytes, although appearing viable in the graft at the time of insertion, were predominantly nonviable at 6 weeks. However, by 12 weeks, new fat had formed in all groups and was significantly greater in the combined fat/Adipogel group. No significant difference was seen in final construct total volume or construct neovascularisation between the groups. This study demonstrated that a pedicled adipose flap can be generated in rats by combining a blood vessel loop, an adipogenic hydrogel, and a lipoaspirate equivalent. Success appears to be based on adipogenesis rather than on adipocyte survival, and consistent with our previous work, this adipogenesis occurred subsequent to graft death and remodelling. The regenerative process was significantly enhanced in the presence of Adipogel.

Remote Control in Formation of 3D Multicellular Assemblies Using Magnetic Forces.

Cell constructs have been utilized as building blocks in tissue engineering to closely mimic the natural tissue and also overcome some of the limitations caused by two-dimensional cultures or using scaffolds. External forces can be used to enhance the cells' adhesion and interaction and thus provide better control over production of these structures compared to methods like cell seeding and migration. In this paper, we demonstrate an efficient method to generate uniform, three-dimensional cell constructs using magnetic forces. This method produced spheroids with higher densities and more symmetrical structures than the commonly used centrifugation method for production of cell spheroids. It was also shown that shape of the cell constructs could be changed readily by using different patterns of magnetic field. The application of magnetic fields to impart forces on the cells enhanced the fusion of these spheroids, which could be used to produce larger and more complicated structures for future tissue engineering applications.

Fate of Free Fat Grafts with or without Adipogenic Adjuncts to Enhance Graft Outcomes.

BACKGROUND: Free fat grafting is popular, but it is still unclear how it works. Although focusing on graft survival seems an obvious direction for improving clinical results, the authors' research suggests that long-term volume retention is in part attributable to new fat regeneration. Measures to facilitate adipogenesis may therefore be equally important. METHODS: To investigate the relative roles of survival and regeneration of fat grafts, the authors measured the fate of human lipoaspirate implanted into the scalps of immunodeficient mice, with and without stromal vascular fraction and a porcine extracellular matrix (Adipogel). Specifically, the authors were interested in volume retention, and the composition of implanted or regenerated tissue at 6 and 12 weeks. RESULTS: Free fat grafts exhibited poor volume retention and survival. Almost all of the injected human adipocytes died, but new mouse fat formed peripheral to the encapsulated fat graft. Adipogel and stromal vascular fraction improved proliferation of murine fat and human vasculature. Human CD34 stromal cells were present but only in the periphery, and there was no evidence that these cells differentiated into adipocytes. CONCLUSIONS: In the authors' model, most of the implanted tissue died, but unresorbed dead fat accounted substantially for the long-term, reduced volume. A layer of host-derived, regenerated adipose tissue was present at the periphery. This regeneration may be driven by the presence of dying fat, and it was enhanced by addition of the authors' adipogenic adjuncts. Future research should perhaps focus not only on improving graft survival but also on enhancing the adipogenic environment conducive to fat regeneration.

Macrophages play a key role in angiogenesis and adipogenesis in a mouse tissue engineering model.

We have previously described a mouse adipose tissue engineering model using a silicon chamber enclosing the superficial epigastric pedicle in a Matrigel based environment. We have shown that when Zymosan, a sterile inflammatory agent, is added to the chamber, angiogenesis and adipogenesis are significantly improved. As Zymosan interacts with toll-like receptors on macrophages, the role of macrophages in new tissue development in the tissue engineering chamber was assessed. Morphological and histological results showed that macrophages were presenting in high numbers at 2 weeks but had decreased significantly by 4 and 6 weeks in the chamber. Numerous immature new blood vessels had formed by 2 weeks, becoming more mature at 4 and 6 weeks. Immature adipocytes were visualized at 4 weeks and mature cells, at 6 weeks. To investigate the functional role of macrophages in the tissue engineering process, we knocked out the local macrophage population by inserting Clodronate liposomes in this chamber. This study shows for the first time that when macrophages are depleted, there is minimal new vascular and adipose tissue development. We propose a new theory for tissue engineering in which macrophages play a central role in both neovascularisation and adipogenesis.

An adipoinductive role of inflammation in adipose tissue engineering: key factors in the early development of engineered soft tissues.

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1ß are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

The in vitro preconditioning of myoblasts to enhance subsequent survival in an in vivo tissue engineering chamber model.

The effects of in vitro preconditioning protocols on the ultimate survival of myoblasts implanted in an in vivo tissue engineering chamber were examined. In vitro testing: L6 myoblasts were preconditioned by heat (42 °C; 1.5 h); hypoxia (<8% O(2); 1.5 h); or nitric oxide donors: S-nitroso-N-acetylpenicillamine (SNAP, 200 µM, 1.5 h) or 1-[N-(2-aminoethyl)-N-(2-aminoethyl)amino]-diazen-1-ium-1,2-diolate (DETA-NONOate, 500 µM, 7 h). Following a rest phase preconditioned cells were exposed to 24 h hypoxia, and demonstrated minimal overall cell loss, whilst controls (not preconditioned, but exposed to 24 h hypoxia) demonstrated a 44% cell loss. Phosphoimmunoblot analysis of pro-survival signaling pathways revealed significant activation of serine threonine kinase Akt with DETA-NONOate (p < 0.01) and heat preconditioning (p < 0.05). DETA-NONOate also activated ERK 1/2 signaling (p < 0.05). In vivo implantation: 100,000 preconditioned (heat, hypoxia, or DETA-NONOate) myoblasts were implanted in SCID mouse tissue engineering chambers. 100,000 (not preconditioned) myoblasts were implanted in control chambers. At 3 weeks, morphometric assessment of surviving myoblasts indicated myoblast percent volume (p = 0.012) and myoblasts/mm(2) (p = 0.0005) overall significantly increased in preconditioned myoblast chambers compared to control, with DETA-NONOate-preconditioned myoblasts demonstrating the greatest increase in survival (p = 0.007 and p = 0.001 respectively). DETA-NONOate therefore has potential therapeutic benefits to significantly improve survival of transplanted cells.

Seeding of pancreatic islets into prevascularized tissue engineering chambers.

Transplantation of islets into the portal vein of diabetic patients has emerged as a promising procedure for the treatment of type 1 diabetes. However, shortages of donors and adverse effects leading to graft impairment and/or rejection have prevented this procedure from achieving widespread clinical application. The aim of this study was to develop a method that could support the survival and function of transplanted islets using a prevascularized tissue engineering chamber. Islets were transplanted into tissue engineering chambers established on the epigastric pedicle in the groin of diabetic mice. Islets were transplanted at the time of chamber implantation or with 21 days prevascularization of the chamber. Transplantation of islets into prevascularized chambers into diabetic RIP-K(b) mice resulted in a significant reduction in blood glucose levels that became evident in the third week and improved glycemic control as measured by a glucose tolerance test. This study highlights that islet survival and function are potentiated by allowing a period of prevascularization within tissue engineering chambers before islet transplantation. This novel prevascularized chamber may be an improved method of islet transplantation. It can be easily accessed for islet seeding, easily retrieved, and transplanted to alternative anatomical sites by microvascular methods.