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Stem cells represent an ideal cell source for tissue engineering and regenerative medicine, because they can be readily isolated, expanded, differentiated and transplanted. For stem cell-based therapies, biomaterials are required to allow for a spatial distribution of the stem cells within a defined area in the body. In our recent studies, we analysed the interaction of a large panel of stem cell types with an array of biomaterials and demonstrated that a rational prediction of stem cell behaviour on a specific biomaterial is so far not possible. Interestingly, even ontogenetically related stem cell types, such as mesenchymal stem cells (MSCs), preadipocytes and dental pulp stem cells (DPSCs), exhibit distinct adhesion properties on the very same biomaterial surface. Therefore, we investigated integrin and extracellular matrix (ECM) protein expression of stem cells to relate gene expression to adhesion behaviour. MSCs, preadipocytes and DPSCs were cultured on selected synthetic polymers, such as Texin, a thermoplastic polyurethane, poly(dimethyl siloxane) (PDMS), poly-d,l-lactic acid (PDLLA) and l-lactic acid-trimehylene carbonate (Resomer® LT706). Integrins and ECM proteins were analysed by RT-PCR, real-time PCR and immunohistochemistry. Analysis of several adhesion molecules yielded that only one molecule, integrin α4, might play a significant role in differential adhesion on polymers for preadipocytes compared to DPSCs and MSCs. Thus, our studies on the molecular interactions of stem cells and polymers are expected to lead to a more profound understanding of the stem cell-biomaterial interactions to eventually allow for a rational biomaterial design.

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.

Background aims. Obesity is correlated with chronic low-grade infl ammation. Thus the induction of infl ammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of infl ammation, we investigated the effect of NO and its downstream signaling molecule guanosine 3 ′ ,5 ′ -cyclic monophosphate (cGMP) as well as adenosine 3′ ,5 ′ -cyclic monophosphate (cAMP) on preadipocytes in vitro . Methods. Preadipocytes were isolated from human subcutaneous adipose tissue, cultured until confl uence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30 – 150 μ m) was added during proliferation and differentiation. Additionally, cGMP/cAMP analogs 8-bromoguanosine 3 ′ ,5′ -cyclic monophosphate (8-Br-cGMP), 8-(4-chlorophenylthio)-guanosine 3′ ,5 ′ -cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3′ ,5 ′ -cyclic monophosphate (8-Br-cAMP), and the adenylyl cyclase activator forskolin, specifi c guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and adenylyl cyclase inhibitor 2′ -5 ′ -dideoxyadenosine (ddA), were applied. Proliferation and differentiation were evaluated. Results. DETA/NO in combination with the standard differentiation procedure signifi cantly enhanced maturation of precursor cells to adipocytes. Proliferation, in contrast, was inhibited in the presence of NO. The application of cGMP and cAMP, respectively, increased preadipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a signifi cant decrease in adipogenic conversion. Conclusions. Our results support the application of NO donors during transplantation of preadipocytes in a 3-dimensional setting to accelerate and optimize differentiation. The results suggest that, instead of the rather instable and reactive molecule NO, the application of cGMP and cAMP would be even more effective because these substances have a stronger adipogenic effect on preadipocytes and a longer half-life than NO. Also, by applying inhibitors of the underlying pathways, the induced infl ammatory condition could be regulated to the desired level.

Objective: An increasing body of evidence is emerging linking adipogenesis and inflammation. Obesity, alone or as a part of the metabolic syndrome, is characterized by a state of chronic low-level inflammation as revealed by raised plasma levels of inflammatory cytokines and acute-phase proteins. If inflammation can, in turn, increase adipose tissue growth, this may be the basis for a positive feedback loop in obesity. We have developed a tissue engineering model for growing adipose tissue in the mouse that allows quantification of increases in adipogenesis. In this study, we evaluated the adipogenic potential of the inflammogens monocyte chemoattractant protein (MCP)-1 and zymosan-A (Zy) in a murine tissue engineering model. Research Methods and Procedures: MCP-1 and Zy were added to chambers filled with Matrigel and fibroblast growth factor 2. To analyze the role of inducible nitric oxide synthase (iNOS), the iNOS inhibitor aminoguanidine was added to the chamber. Results: Our results show that MCP-1 generated proportionally large quantities of new adipose tissue. This neoadipogenesis was accompanied by an ingrowth of macrophages and could be mimicked by Zy. Aminoguanidine significantly inhibited the formation of adipose tissue. Discussion: Our findings demonstrate that low-grade inflammation and iNOS expression are important factors in adipogenesis. Because fat neoformation in obesity and the metabolic syndrome is believed to be mediated by macrophage-derived proinflammatory cytokines, this adipose tissue engineering system provides a model that could potentially be used to further unravel the pathogenesis of these two metabolic disorders.

To investigate the potential of inflammation to induce new adipose tissue formation in the in vivo environment.Using an established model of in vivo adipogenesis, a silicone chamber containing a Matrigel and fibroblast growth factor 2 (1 microg/ml) matrix was implanted into each groin of an adult male C57Bl6 mouse and vascularized with the inferior epigastric vessels. Sterile inflammation was induced in one of the two chambers by suspending Zymosan-A (ZA) (200-0.02 microg/ml) in the matrix at implantation. Adipose tissue formation was assessed at 6, 8, 12 and 24 weeks. ZA induced significant adipogenesis in an inverse dose-dependent manner (P0.001). At 6 weeks adipose tissue formation was greatest with the lowest concentrations of ZA and least with the highest. Adipogenesis occurred both locally in the chamber containing ZA and in the ZA-free chamber in the contralateral groin of the same animal. ZA induced a systemic inflammatory response characterized by elevated serum tumour necrosis factor-alpha levels at early time points. Aminoguanidine (40 microg/ml) inhibited the adipogenic response to ZA-induced inflammation. Adipose tissue formed in response to ZA remained stable for 24 weeks, even when exposed to the normal tissue environment.These results demonstrate that inflammation can drive neo-adipogenesis in vivo. This suggests the existence of a positive feedback mechanism in obesity, whereby the state of chronic, low-grade inflammation, characteristic of the condition, may promote further adipogenesis. The mobilization and recruitment of a circulating population of adipose precursor cells is likely to be implicated in this mechanism.

Extended soft tissue defects resulting from injuries or tumor resections are still an unresolved problem in plastic and reconstructive surgery because adequate reconstruction is difficult. Immature adipogenic precursor cells, called preadipocytes, which are located between mature adipocytes in adipose tissue, represent a powerful tool for soft tissue engineering because of their ability to proliferate and differentiate into adipose tissue after transplantation. In previous studies, we compared preadipocyte-loaded hyaluronan or collagen biomaterials and their applicability for adipose tissue engineering. Our findings demonstrated successful de novo formation of adipose tissue in vivo but pore size and stiffness were limiting factors not allowing for sufficient cell distribution in the construct. This study presents a nonwoven made of novel bioabsorbable co-poly(ester amide) based on e-caprolactam, adipic acid, and 1,4-butanediol in an innovative 3-dimensional architecture. The material was formed into nonwovens by textile manufacturing using an aerodynamic web formation process and a needle felting technique. Carriers were seeded with human preadipocytes and examined for cellular proliferation and differentiation. In addition, methods of preparing scaffolds for optimal cell interaction were evaluated. Our findings show that polyesteramide-derived nonwovens allow good adherence, proliferation, and differentiation of preadipocytes. These results are promising guidance toward an optimally designed scaffold for in vivo use.

To date, no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, tumor resections or in congenital defects is available. A biohybrid composed of viable adipose precursor cells and an optimised matrix could help towards a solution. Morphologically, preadipocytes resemble fibroblasts and have not yet built a large cytoplasmic lipid droplet as found in differentiated adipocytes. Additionally, preadipocytes are smaller than mature adipocytes allowing a quicker revascularization after transplantation. Furthermore, transplanted preadipocytes can form adipose tissue in vivo whereas the transplantation of mature adipocytes often gives poor results, i.e. oil cysts or shrinkage of the transplant. Since these observations point to differences in metabolic activity between preadipocytes and adipocytes, we investigated the oxygen consumption of preadipocytes stimulated to undergo differentiation, and fibroblasts, by measuring the respiration with a Clark-type oxygen electrode. Preadipocytes had a significantly lower oxygen consumption than mature adipocytes. This advantage in respiration and the better revascularization of undifferentiated adipose tissue cells allow the development of innovative transplants and point to preadipocytes as promising tool to improve transplantations in adipose tissue reconstruction.

The correction of soft-tissue defects by adipose tissue transplantation often produces poor and unpredictable results. The implantation of isolated and cultured preadipocytes offers a solution to this problem since these cells differentiate into adipocytes when implanted in vivo. A field of major interest is to maximize the yield of preadipocytes isolated from adipose tissue showing only low contamination with other cell types. Aspiration and excision are two concurrent clinical ways of harvesting adipose tissue for the isolation of preadipocytes. This tissue is usually discarded after surgery. In this study, the yield of preadipocytes obtained from liposuction material was compared to that of excised adipose tissue. Furthermore, we determined the loss of precursor cells if isolation of preadipocytes was delayed for 24 h. Preadipocytes were isolated from the stromal cell fraction of human subcutaneous adipose tissue samples. Harvesting of adipose tissue by suction was performed according to the Coleman procedure (manually applied negative pressure using a 10-ml syringe with a blunt tip cannula). Isolation was either carried out within 60 min after extraction or after storage for 24 h in culture medium at 4°C. Isolated preadipocytes were cultured for 24 h, trypsinized and counted in a Neubauer chamber. Our results show clearly that the yield of preadipocytes isolated from liposuction material (within 60 min after extraction and after 24 h of storage) is higher than the cell yield from excised adipose tissue. Overnight storage for 24 h leads to a significant loss of preadipocytes in excised tissue but not in liposuction material. The high yield of cells isolated from liposuction material proves that extraction by suction does not damage the stromal cell fraction in the adipose tissue. If cell isolation is not performed immediately after the operation, liposuction material is clearly the better alternative for storage.

Extended soft tissue defects after extensive deep burns or tumor resections are still an unresolved problem in plastic and reconstructive surgery. There is a clinical need for an adequate solution to this problem but currently, no adequate implant material is available for the correction of these defects. Since the autologous transplantation of mature adipose tissue gives poor results, this study explores the advantages of using human preadipocytes in collagen sponges for tissue reconstruction purposes. Human preadipocytes of young adults were isolated, cultured, seeded onto collagen sponges with uniform pore size, and implanted into immunodeficient mice. After 24 hours of incubation in vitro and after explantation at 3, 8, and 12 weeks, sponges were examined for macroscopic appearance, weight, thickness, histology, immuno-histochemistry, and ultrastructure. We find good penetration of cells into the scaffold, layers of adipose tissue, and new vessels on all grafts while controls appear unchanged. These results are promising for improving the reconstruction of soft tissue defects.

Background—Oxidative damage of vascular endothelium represents an important initiation step in the development of atherosclerosis. Recently, we reported about protection of inducible nitric oxide synthase (iNOS)-derived high-output NO in endothelial cells. Because iNOS activity critically depends on the availability of its substratel-arginine, the present study aims at elucidating iNOS-mediated effects on H2O2-induced apoptosis of cytokine-activated rat aortic endothelial cells (AECs) subject to mediuml-arginine concentrations.Methods and Results—In cytokine-activated AECs, iNOS activity was found to be half-maximal at 60 μmol/L arginine, which represents the medium serum level in rats but also in humans. Maximal activity is seen at and above 200 μmol/L arginine. Activated cells grown in the absence of arginine with minimal iNOS activity are highly sensitive toward H2O2-induced apoptosis, and increases in medium arginine concentrations result in increased cell survival. Moreover, competition experiments show that iNOS activity is completely dependent on cationic amino acid transporter-mediated arginine uptake. We also find that the arginine-dependent protection includes inhibition of endothelial lipid peroxidation and increases in the expression of vasoprotective stress response genes.Conclusions—Our data demonstrate that arginine concentrations corresponding to physiological serum levels do not allow for optimal endothelial iNOS activity. Thus, decreases in systemic arginine concentrations, or locally within atherosclerotic plaques, will impair the endothelial iNOS-mediated stress response and will significantly increase the risk of endothelial dysfunction.

Adipose tissue engineering aims to grow fat tissue for soft tissue reconstruction after tumour resection or trauma. However, insufficient progenitor cell differentiation and poor vascularization compromise the generation of clinically applicable adipose tissue. The desired process of neo-adipogenesis seems to be difficult to mimic, even though it takes place in all of us, inevitably and rapidly, as soon as we start consuming high-caloric diets. It has previously been proposed that inflammation and its key regulator, nitric oxide (NO), may play a relevant part in neo-adipogenesis. We here discuss how a controlled activation of the nitric oxide system on various levels may represent a cure for several current shortcomings in adipose tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.

Adipose tissue physiology strongly differs between body regions. Reasonable amounts of intact RNA are required to analyze the local differences on a molecular level. Therefore, we developed an optimized RNA isolation procedure from adipose tissue samples. Excised human subcutaneous adipose tissue was obtained from elective operations, and the RNA Stabilization Reagent (RNAlater) was tested for its effect on RNA integrity. Additionally, 3 different RNA isolation kits were evaluated for efficacy in isolating RNA from tissue samples. The samples displayed a significant loss in recoverable RNA and signs of RNA degradation 30 to 60 minutes after tissue excision. The application of RNAlater significantly delayed this degradation. Using the RNeasy Lipid Tissue Kit resulted in a significantly higher RNA yield compared to the RNeasy Mini Kit. Our results demonstrate that combining the RNAlater and RNeasy Lipid Tissue Kit results in a higher yield of RNA even from relatively small tissue samples with guaranteed RNA integrity.

Stem cells represent an ideal cell source for tissue engineering and regenerative medicine, because they can be readily isolated, expanded, differentiated and transplanted. For stem cell-based therapies, biomaterials are required to allow for a spatial distribution of the stem cells within a defined area in the body. In our recent studies, we analysed the interaction of a large panel of stem cell types with an array of biomaterials and demonstrated that a rational prediction of stem cell behaviour on a specific biomaterial is so far not possible. Interestingly, even ontogenetically related stem cell types, such as mesenchymal stem cells (MSCs), preadipocytes and dental pulp stem cells (DPSCs), exhibit distinct adhesion properties on the very same biomaterial surface. Therefore, we investigated integrin and extracellular matrix (ECM) protein expression of stem cells to relate gene expression to adhesion behaviour. MSCs, preadipocytes and DPSCs were cultured on selected synthetic polymers, such as Texin, a thermoplastic polyurethane, poly(dimethyl siloxane) (PDMS), poly-d,l-lactic acid (PDLLA) and l-lactic acid-trimehylene carbonate (Resomer® LT706). Integrins and ECM proteins were analysed by RT-PCR, real-time PCR and immunohistochemistry. Analysis of several adhesion molecules yielded that only one molecule, integrin α4, might play a significant role in differential adhesion on polymers for preadipocytes compared to DPSCs and MSCs. Thus, our studies on the molecular interactions of stem cells and polymers are expected to lead to a more profound understanding of the stem cell-biomaterial interactions to eventually allow for a rational biomaterial design.

Objective: Obesity and the Metabolic Sydrome are correlated with chronic low-grade inflammation characterized by increased plasma levels of proinflammatory mediators. Thus, the induction of inflammation could be used to stimulate adipose tissue formation in tissue engineering approaches. Since nitric oxide (NO) is a key regulator of inflammation and has been demonstrated to have lipogenic properties we investigated the effect of NO and its downstream targets cGMP and cAMP, respectively, on preadipocytes in vitro. Methods: Preadipocytes were isolated from human subcutaneous adipose tissue samples, cultured until confluence, and differentiated by insulin, pioglitazone, dexamethasone, isomethylbutylxanthine, and transferrin. DETA/NO (30–150 μM) was added during proliferation and differentiation. Additionally, the specific guanylyl cyclase inhibitor ODQ, the adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (ddA), the cGMP/cAMP-analogues 8-Br-cGMP, 8-pCPT-cGMP, and 8-Br-cAMP, and the adenylyl cyclase activator forskolin were applied. Proliferation was evaluated by microscopy and XTT-test, differentiation was assayed by glycerophosphate dehydrogenase (GDDH) activity. Results: DETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes (p

Preadipocytes are multipotent adipogenic precursor cells that can be isolated from mature adipose tissue. They have been receiving increasing attention in the context of obesity, type 2 diabetes, and other nutrition-associated diseases. Understanding the physiological and pathophysiological processes in fat neo-formation, energy homeostasis, and adipose tissue physiology is the basis for research on metabolic diseases and the respective pharmaceutical intervention. While the hormonal influence on intracellular signaling in adipogenesis has been intensively investigated, the effects of free radical formation and oxidative stress have just started to gain scientific attention. This review summarizes the present knowledge on the main molecular pathways in preadipocyte maturation and focuses on recent findings indicating that besides hormonal stimuli reactive oxygen species (ROS) and free radicals may also interact with preadipocyte differentiation.

Several studies have shown that atypical neuroleptics, e. g. clozapine, cause a significant weight gain. In the treatment with neuroleptics this increase in weight is an unwanted side effect. In tissue engineering of adipose tissue, in contrast, an immitation of the clozapine-induced mechanisms is a promising approach to overcome present hurdles, e. g. low volume. This in vitro study assessed the influence of the neuroleptic drug clozapine and the green tea extract epigallocatechine gallate (EGCG) on preadipocytes. Adipose tissue progenitor cells were isolated from excised human fat tissue, cultured, and differentiated in the presence of clozapine and/or EGCG. Adipogenic conversion was determined by measuring the activity of the glycerophosphate dehydrogenase (GPDH), a molecular marker for fat cell maturation. Intracellular superoxide formation was evaluated by ethidium bromide staining. Our results demonstrate that clozapine promotes preadipocyte conversion to fat cells by generating superoxid anions (GPDH-induktion by 20–30 %, p < 0.01). EGCG effectively inhibits clozapine-enhanced differentiation by 80 % (p < 0.001), in the presence of clozapine by 50–60 % (p < 0.01). The generation of oxidative stress seems to represent a potential pathway to promote the differentiation of preadipocytes on biomaterials. Taking the pathogenesis of obesity, it is especially the drinking of green tea or medication with EGCG which represents a suitable tool to avoid weight gain and neoadipogenesis.

An "off the shelf" vascular conduit for free flap surgery that remained patent until the flap is vascularized from the recipient bed would be useful to avoid autograft donor-site morbidity and reduce operation time. To date, no successful studies of microvascular prostheses in low-flow states exist. This study investigated the effects of L-arginine on neointimal hyperplasia and the overall patency of cold-stored femoral arterial allografts implanted into a low-flow arterial defect model in rabbits. A cutaneous island flap based on the inferior epigastric artery was raised, and the femoral artery was ligated distally. Immediately proximal to the inferior epigastric artery, the femoral artery was divided and a 1-cm long cold-stored femoral arterial allograft was inserted into the defect. Half of the animals received L-arginine as a subcutaneous injection. When harvested at 4 weeks, 9 out of 11 allografts of the L-arginine-treated group and 8 out of 14 grafts of the control group were patent. Intimal thickening and graft stenosis were significantly reduced in the L-arginine group. Our findings reveal the potential of a combined approach of an arterial allograft and the use of L-arginine as a short-term vein graft substitute.

Monocyte chemoattractant protein (MCP-1) plays a key role in the pathogenesis of obesity. It is an inflammogen that attracts monocytes to the adipose tissue and promotes their differentiation to macrophages. These tissue macrophages produce a plethora of pro-adipogenic cytokines, one of which is nitric oxide (NO). NO is produced by the inducible nictric oxide synthase (iNOS) and is suspected to be a key factor in neoadipogenesis. In this study, we mimicked the processes of fat formation in a murine adipose tissue engineering chamber model. The aim was to analyse how the pathology as found in obesity can be exploited for soft tissue reconstruction based on adipose tissue enigneering. MCP-1 and the yeast-derived inflammogen Zymosan A (Zy) were evaluated for their adipogenic potential while aminoguanidine (AG) was applied as specific iNOS inhibitor. A plastic chamber (5 mm lang, 42 µl) filled with Matrigelℳ and fibroblast growth factor 2 was implanted around a vascular pedicle but otherwise isolated from the surrounding tissue. The following conditions were tested: 1) control (Matrigelℳ, bFGF only), 2) Matrigelℳ, bFGF, MCP-1 (0.05 ng/ml), 3) Matrigelℳ, bFGF, Zy (0.02 og/ml), 4) Matrigelℳ, bFGF, Zy, AG (40 µg/ml). Constructs were harvested after 6 weeks. Absolute volumes of fat tissue were calculated from the proportion of fat (%) and the wet volume. Matrigel-FGF-2 matrix alone was not sufficient to drive substantial adipogenesis (16.15 ± 5.03 % and 1.55 ± 0.63 µl adipose tissue). Zy- and MCP-1-supplemented chambers revealed dose-dependent neo-adipogenesis. Zy-treated constructs produced 44.4 ± 5.3 % fat (p < 0.005) and volumes of 5.93 ± 1.05 µl adipose tissue (p < 0.005). MCP-1 induced 40.57 ± 3.65 % (p = 0.005) and 5.05 ± 0.92 µl adipose tissue (p = 0.0085). AG addition decreased percentage and volume of fat significantly (19.73 ± 3.13 % (p < 0.005), 2.32 ± 0.54 µl (p < 0.05)). Histologically, all explanted MCP-1 and Zy samples showed healthy adipose tissue with infiltration of macrophages. The model presented here reveals that mimicking of the processes in obesity enables neoadipogenesis in an adipogenic matrix. Key factors in this setting are macrophages and the inflammation which they generate through their NO production. A better understanding of the processes in obesity and the Metabolic Syndrome will help to develop and test new, promising approaches in adipose tissue engineering.

Biomaterials are used in tissue engineering with the aim to repair or reconstruct tissues and organs. Frequently, the identification and development of biomaterials is an iterative process with biomaterials being designed and then individually tested for their properties in combination with one specific cell type. However, recent efforts have been devoted to systematic, combinatorial and parallel approaches to identify biomaterials, suitable for specific applications. Embryonic and adult stem cells represent an ideal cell source for tissue engineering. Since stem cells can be readily isolated, expanded and transplanted, their application in cell-based therapies has become a major focus of research. Biomaterials can potentially influence e.g. stem cell proliferation and differentiation in both, positive or negative ways and biomaterial characteristics have been applied to repel or attract stem cells in a niche-like microenvironment. Our consortium has now established a grid-based platform to investigate stem cell/biomaterial interactions. So far, we have assessed 140 combinations of seven different stem cell types and 19 different polymers performing systematic screening assays to analyse parameters such as morphology, vitality, cytotoxicity, apoptosis, and proliferation. We thus can suggest and advise for and against special combinations for stem cell-based tissue engineering.

Recent research findings postulate that adipocytes and endothelial cells (EC) may share a common progenitor. However, the interlinking pathways between adipose tissue and endothelium, and the differentiation potential of cells to convert from one tissue into the other via progenitor cells have not been elucidated and are therefore the focus of this study. Stromal vascular fraction (SVF) cells were isolated from liposuction aspirates or excised adipose tissue and separated into CD31+ and CD31− populations by magnet-assisted cell sorting. Differentiation to fat tissue was induced in both CD31 fractions after expansion by insulin, dexamethasone, isobutylmethylxanthine, triiodothyronine, pioglitazone, and transferrin. Differentiation was assayed enzymatically and by cell counting. Maturation to endothelium was performed with vascular endothelial growth factor (VEGF), insulin-like growth factor-1 plus 2% fetal calf serum, and confirmed by flow cytometry and tube formation assays on Matrigel™. Our results show that the SVF contains a CD31−, S100+ cell type that can differentiate into adipocytes and EC. The SVF also comprises CD31+ cells that, although they have an endothelial phenotype, can be converted into mature adipocytes. These findings demonstrate the potency of SVF cells to perform both adipogenic and endothelial differentiation. Further, they reveal the plasticity of mature cells of mesenchymal origin to undergo conversion from endothelium to adipose tissue and vice versa.

Adipose tissue engineering with preadipocyte-loaded scaffolds requires adequate tracking of preadipocytes to allow evaluation and quantification of cell proliferation, expansion and differentiation in three-dimensional systems. To differentiate between graft and host cells, labeling of preadipocytes before implantation and tracking of these cells until harvest would be useful. Immunohistochemistry enables the differentiation between cells of different species but is time-consuming, expensive, elaborate, and not applicable for autologous transplantation. So far, there is no published method to use externally applied dyes for tracking of human preadipocytes in adipose tissue engineering. We tested the cell dyes PKH26, CM-DiI, and CFSE to analyze their applicability for labeling human preadipocytes. CM-DiI had toxicity levels of 45–70%, while 3–4% proliferating cells were stained on day 35. CFSE revealed clear cytoplasmic coloring in proliferating cells with 5–6% stained cells after 35 days and toxicity ranging from 55 to 90% dead cells. PKH26 demonstrates lowest levels of toxicity and best labeling results after 4 weeks in proliferating preadipocytes in monolayer. Although none of the dyes showed long-lasting labeling during proliferation, all three dyes demonstrated permanent staining in differentiated cells. The results reveal the problems of preadipocyte tracking with fluorescent dyes but justify the dye application for limited time periods.

Background There is a major clinical need for strategies for adequately reconstructing the soft tissue defects found after deep burns, tumor resection, or trauma. A promising solution is adipose tissue engineering with preadipocytes, stem-cell derived precursors of the adipose tissue, implanted within biomaterials. This pilot study evaluated hyaluronan gels mixed with autologous undifferentiated preadipocytes in a pig model for their potency to generate new fat. Materials and methods Preadipocytes were isolated from intra-abdominal pig fat by collagenase digestion, plated on fibronectin-coated culture dishes in Dulbecco’s modified Eagle medium/Ham’s F12 (Biochrom, Berlin, Germany) combined with 10% pig serum, expanded, and mixed with hyaluronan gel. Two types of gels with varying degrees of amidation of the carboxyl groups were tested (HYADD3, HYADD4). Cell-loaded gels and unseeded controls were injected subcutaneously into the ears of three pigs, explanted at 6 wk, and analyzed histologically. Results Both cell-loaded specimens were detected macroscopically. They demonstrated a slight volume effect with limited stability after 6 wk. Unloaded HYADD3 and HYADD4 controls could not be identified at the time of explantation. Histology of HYADD3 revealed islets of mature adipocytes and vessels embedded in fat tissue surrounded by gel. In contrast, no fat formation was found in HYADD4 gels when implanted in the ear. Conclusions Histological findings demonstrate that HYADD3 is a promising gel for generating adipose tissue. Even though HYADD3 might be a potential material for the reconstruction of small tissue defects, the question remains as to whether the adipose tissue within the gel is attributable to preadipocyte maturation or ingrowth from neighboring tissue.

There is high clinical need for an adequate reconstruction of soft tissue defects as found after tumor resections, deep burns or severe trauma. A promising solution for these defects is adipose tissue engineering, with adult stem cells of the adipose tissue, implanted on 3D biomaterials. These adipogenic precursor cells survive ischemia better than mature adipocytes and have the potency to proliferate and differentiate into fat cells after transplantation. They can be yielded from excised adipose tissue or liposuction material. When preadipocytes are seeded on carriers for the generation of adipose tissue, chemical composition, mechanical stability and 3D architecture of the construct are crucial factors. They ensure cellular penetration into the construct, sufficient proliferation on the material and full differentiation inside the construct after transplantation. In hydrogels, it is especially the use and combination of growth factors that determine the overall outcome of the applied biopolymer. Over recent years, in vivo trials in particular have allowed significant insights into the potential, the perspectives, but also the current difficulties and draw-backs in adipose tissue engineering. This review focuses on the main strategies in adipose tissue regeneration, compares the various materials that have been used as carrier matrices so far and considers them in light of the challenges they have yet to meet.

Biodegradable polyesters are established biomaterials in medicine due to their chemical characteristics and options for material processing. A main problem, however, is the release of acid degradation products during biodegradation with severe local pH-drops and inflammatory reactions. Polyesteramides, in contrast, show a less prominent pH-drop during degradation. In this study, we developed a simple, reproducible synthesis of the poly(ester amide) (PEA) type C starting from epsilon-caprolactame, 1,4-butanediol, and adipic acid in a one-batch two-step reaction and conducted the manufacturing of PEA-derived 3D textile scaffolds applicable for tissue engineering purposes. The thermal and mechanical properties of PEA-type C were analysed and the structural conformity of different batches was confirmed by NMR spectroscopy and size exclusion chromatography. The polymer was formed into nonwovens by textile manufacturing. Cytotoxicity tests and X-ray photoelectron spectroscopy (XPS) were used to analyze the effect of scaffold extraction before cell seeding. The manufactured carriers were seeded with human preadipocytes and examined for cellular proliferation and differentiation. The production of PEA type C successfully occurred via simultaneous ring-opening polymerization of epsilon-caprolactame and polycondensation with 1,4-butanediol and adipic acid at 250 degrees C under high-vacuum. Soxhlet extraction allowed optimal cleaning of nonwoven scaffolds. Extracted PEA-derived matrices were capable of allowing good adherence, proliferation, and differentiation of preadipocytes. These results are encouraging and guidance towards an optimally prepared nonwoven carrier applicable for clinical use.

The impact of nitric oxide (NO) synthesized after activation by proinflammatory cytokines and/or bacterial products by an inducible NO synthase (iNOS) is still contradictory. Various methods to inhibit iNOS expression or activity have been established. A relatively new approach to inhibit iNOS-derived NO production is the antisense (AS) technique, which theoretically provides a specific and efficient method for inhibiting gene expression and function. This chapter focuses on the application of iNOS-specific AS-oligodeoxynucleotide (ODN) and highlights some of the pitfalls that must be considered to use this technique effectively.

Insufficient differentiation of precursor cells and inadequate vascularization in central parts of three-dimensional constructs are major problems in adipose tissue engineering. This study aimed at finding a solution to these problems by analyzing the effects of nitric oxide (NO) on preadipocytes and endothelial cells in a setting as seen after transplantation of a three-dimensional biohybrid composed of viable endothelial cells and adipose precursor cells. NO is an important signalling molecule regulating physiological and pathophysiological functions throughout the body, including endothelial and adipose tissue activities. NO is generated by the enzyme family of NO synthases that metabolize arginine and molecular oxygen to citrulline and NO. Preadipocytes were isolated from human subcutaneous adipose tissue samples, cultured in DMEM/F12 with 10% fetal calf serum (FCS), and differentiated after 7–14 days of expansion by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of FCS. To evaluate the influence of NO on proliferation and differentiation, the NO donor molecule DETA/NO was added at various concentrations. Proliferation was evaluated by microscopy and the extent of differentiation was assayed after 15 days by cell counting and analysis of the enzyme glycerophosphate dehydrogenase highly expressed in mature adipocytes. In parallel, primary rat aorta endothelial cells (EC) were isolated, cultured in RPMI 1640/20% FCS and treated with DETA/NO to analyze the effect of NO on the stress response genes Bcl-2, vascular endothelial growth factor (VEGF), and heme oxygenase (HO)-1. We find that NO in combination with the conventionally used differentiation-inducing factors significantly enhances maturation of precursor cells to adipocytes whereas proliferation, in contrast, is inhibited in the presence of NO. Treatment of EC with DETA/NO causes the upregulation of the antiapoptotic protein Bcl-2, the pro-angiogenic VEGF, and the antioxidative HO-1. Our results emphasize the pivotal role of iNOS activity in regulating gene expression. Further, they are encouraging for applying NO-donors during transplantation of preadipocytes and EC in a three-dimensional setting since it helps optimizing differentiation of adipogenic precursor cells and stimulating angiogenesis by inducing proangiogenic VEGF.

This study aimed at developing an optimal protocol for proliferation and differentiation of preadipocytes that is a prerequisite for constructing an ideal biohybrid composed of viable adipose precursor cells in a three-dimensional matrix. Such an implant could represent an adequate solution for correcting soft tissue defects, e.g., extensive deep burns or tumor resections. Preadipocytes were isolated from human subcutaneous adipose tissue samples and cultured in Dulbecco's modified eagle medium (DMEM)/Ham's F12 medium (F12) or OPTIMEM medium with or without the addition of human serum (hS) or fetal calf serum (FCS). The advantages of fibronectin-coated culture dishes for preadipocyte yield after isolation and differentiation were evaluated. After culture expansion, differentiation was induced by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of serum. The extent of differentiation was assayed by measuring the activity of glycerophosphate dehydrogenase as well as counting of differentiated versus undifferentiated cells. Our results show that fibronectin coating does not only strongly increase the yield of preadipocytes after isolation from adipose tissue but also significantly enhances differentiation of precursor cells to mature adipocytes. For optimal cell expansion, DMEM/F12 is more promoting than OPTIMEM and culturing with FCS shows a slightly better proliferation compared with hS supplementation. Differentiation, in contrast, is significantly improved when hS is used instead of FCS during proliferation. Our results smooth the way for autologous preadipocyte culturing and show that hS for preadipocyte culturing opens new and promising perspectives for adipose tissue engineering by optimizing in vitro expansion in cell culture and inducing substantial differentiation.

The reconstruction of soft tissue defects following extensive deep burns or tumor resections remains an unresolved problem in plastic and reconstructive surgery since adequate implant materials are still not available. Preadipocytes, immature precursor cells found between mature adipocytes in adipose tissue, are a potential material for soft tissue engineering since they can proliferate and differentiate into adipose tissue after transplantation. In previous studies, we identified hyaluronan benzyl ester (HYAFF ® 11) sponges to be promising carrier matrices. This study now evaluates, in vitro and in vivo, a new sponge architecture with pores of 400 μm either made of plain HYAFF ® 11 or HYAFF ® 11 coated with the extracellular matrix glycosaminoglycan hyaluronic acid. Human preadipocytes were isolated, seeded onto carriers and implanted into nude athymic mice. Explants harvested after 3, 8, and 12 weeks were examined for macroscopical appearance, thickness, weight, pore structure, histology, and immunohistochemistry. Compared to previous studies, we found better penetration of cells into both types of scaffolds, with more extensive formation of new vessels throughout the construct but with only minor adipose tissue. Our encouraging results contribute towards a better seeded and vascularised scaffold but also show that the enhancement of adipogenic conversion of preadipocytes remains a major task for further in vivo experiments.

The impact of nitric oxide (NO) synthesized after activation by proinflammatory cytokines and/or bacterial products by an inducible NO synthase (iNOS) is still contradictory. Expression of iNOS in inflammatory reactions is often found predominantly in cells of epithelial origin, and in these cases NO may serve as a protective agent limiting pathogen spreading, downregulating local inflammatory reactions by inducing production of Th2-like responses in a classical feedback circle, or limiting tissue damage during stress conditions. However, an abundant amount of data on chronic human disorders with predominant proinflammatory Th1-like reactions points to a destructive role of iNOS activity calling for a specific inhibition. Various methods to inhibit iNOS have been established to elucidate a protective versus a destructive role of NO during various stresses. In this review, we focus on antisense (AS)-mediated gene knock-down as a relatively new method to inhibit NO production and summarize the techniques applied and their successes. At least in theory, it provides a specific, rapid, and potentially high-throughput method for inhibiting gene expression and function. We here discuss the opportunities of iNOS-directed AS-ODN, and extensively deal with limitations and experimental problems.

The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions.

We have investigated the impact of the widely used antifungal agent Amphotericin B (AmB) on cytokine activated aortic endothelial cells (AEC) and their inflammatory response as monitored by cytokine and inducible nitric-oxide synthase (iNOS) expression as well as high-output nitric oxide synthesis. Because both blood-borne infections and systemically administered drugs will first encounter vessel lining endothelial cells, this cell type represents an important participant in innate immune reactions against xenobiotics. Culturing cytokine-activated AEC in the presence of 1.25 microg/ml AmB, a concentration equivalent to serum levels during patient treatment, we find increases in iNOS promoter activity up to 120%, in iNOS mRNA or protein expressions by factors of up to 3.5 +/- 1.1, and in iNOS activity of up to 180% compared with cells with cytokines only. In parallel, a strong increase in endothelial interleukin (IL)-1beta-converting enzyme (ICE) and IL-1beta expression and activity was observed. Specific inhibition of ICE activity or IL-1beta functionality significantly reduces expression and activity of the iNOS to control values. Because ICE activity is essential for the endogenous synthesis of active IL-1beta, ICE overexpression represents the key signal in the AmB-induced and IL-1beta-mediated effects on iNOS activity. In summary, in endothelial cells, AmB strongly augments cytokine-induced iNOS expression and activity by increasing the expression and activity of the ICE. This adjuvant activity for augmented endogenous cytokine processing adds to the efficacy of the antimycotic activity of AmB. Furthermore, our data underline the relevance of the endothelial iNOS as a potent effector of the innate immune system.

A common side effect of antipsychotic treatment with atypical neuroleptics is a significant weight gain. This stabilizes in the short to medium term but with clozapine often continues beyond the first year leading to weight gains of up to 20–30 kg.1 This increase in weight is significantly correlated with clinical improvements, but the causes of treatment-emergent weight gain in schizophrenic patients are currently unknown. It is assumed that the perception of satiety is impaired, as patients experience a lower suppression of appetite after meals when consuming atypical neuroleptics.1 Supporting this hypothesis, clozapine has been found to increase serum leptin levels within days after starting treatment.2 Unfortunately, strict food reduction shows little success indicating that central pathways rather than peripheral actions of atypical neuroleptics or their mediators are involved in weight gain. Evaluation of the body composition revealed an increase in body fat under antipsychotic treatment with atypical antipsychotic drugs,3 and blockade of serotonin and histamine receptors by these drugs was previously shown to be associated with weight gain.4 In addition to enhanced lipid accumulation in adipocytes, enhanced differentiation of preadipocytes can increase body fat. Our study addresses the question of whether clozapine-induced weight gain is based on neo-formation of adipose tissue from precursor cells. All experiments had been approved by the Ethical Committee of the Aachen University of Technology.