RESUMO
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.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Integrina alfa4/metabolismo , Células-Tronco Mesenquimais/citologia , Polímeros/farmacologia , Adipócitos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA Complementar/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
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.
Assuntos
Tecido Adiposo , Óxido Nítrico/fisiologia , Engenharia Tecidual , Animais , HumanosRESUMO
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.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Doadores de Óxido Nítrico/administração & dosagem , Óxido Nítrico/administração & dosagem , Nitritos/administração & dosagem , Administração Cutânea , Adulto , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Cultura em Câmaras de Difusão , Histocitoquímica , Humanos , Técnicas In Vitro , Linimentos/administração & dosagem , Linimentos/química , Linimentos/farmacocinética , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/sangue , Óxido Nítrico/química , Óxido Nítrico/farmacocinética , Doadores de Óxido Nítrico/química , Doadores de Óxido Nítrico/farmacocinética , Nitritos/química , Nitritos/farmacocinética , Compostos Nitrosos/análise , Compostos Nitrosos/sangue , Pele/química , Pele/metabolismo , Absorção CutâneaAssuntos
Tecido Adiposo/efeitos dos fármacos , Antipsicóticos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Tecido Adiposo/metabolismo , Proliferação de Células/efeitos dos fármacos , Humanos , Técnicas In Vitro , Esquizofrenia/tratamento farmacológico , Células-Tronco/metabolismoRESUMO
BACKGROUND AIMS: Obesity is correlated with chronic low-grade inflammation. Thus the induction of inflammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of inflammation, 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 confluence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30-150 microm) 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, specific 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 significantly 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 pre-adipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a significant 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 inflammatory condition could be regulated to the desired level.
Assuntos
Adipogenia/efeitos dos fármacos , AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Óxido Nítrico/farmacologia , Células-Tronco/efeitos dos fármacos , Inibidores de Adenilil Ciclases , Adipócitos/citologia , Adipócitos/fisiologia , Adulto , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , DDT/análogos & derivados , DDT/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/análogos & derivados , Óxido Nítrico/metabolismo , Oxidiazóis/farmacologia , Quinoxalinas/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Gordura Subcutânea/citologia , Engenharia TecidualRESUMO
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.
Assuntos
Adipogenia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Humanos , Modelos Biológicos , Espécies Reativas de Oxigênio/química , Transdução de SinaisRESUMO
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.
Assuntos
Gordura Abdominal/transplante , Adipócitos/citologia , Ácido Hialurônico , Células-Tronco/citologia , Engenharia Tecidual/métodos , Gordura Abdominal/citologia , Animais , Diferenciação Celular , Separação Celular , Células Cultivadas , Géis , Ácido Hialurônico/análogos & derivados , Ácido Hialurônico/química , Projetos Piloto , Suínos , Transplante AutólogoRESUMO
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.
Assuntos
Amidas/química , Materiais Biocompatíveis/química , Poliésteres/química , Engenharia Tecidual/métodos , Adipatos/química , Animais , Butileno Glicóis/química , Caprolactama/química , Linhagem Celular , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Teste de Materiais , Camundongos , Espectrometria por Raios X , TêxteisRESUMO
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.
Assuntos
Materiais Biocompatíveis/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/toxicidade , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Análise por Conglomerados , Feminino , Temperatura Alta , Humanos , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de VarreduraRESUMO
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.
Assuntos
Arginina/uso terapêutico , Artéria Femoral/efeitos dos fármacos , Preservação de Órgãos/métodos , Refrigeração/métodos , Túnica Íntima/efeitos dos fármacos , Grau de Desobstrução Vascular/efeitos dos fármacos , Animais , Arginina/administração & dosagem , Arginina/sangue , Pressão Sanguínea/efeitos dos fármacos , Constrição Patológica/diagnóstico por imagem , Constrição Patológica/prevenção & controle , Artérias Epigástricas/cirurgia , Artéria Femoral/diagnóstico por imagem , Artéria Femoral/transplante , Sobrevivência de Enxerto , Hiperplasia , Imuno-Histoquímica , Coelhos , Fluxo Sanguíneo Regional/fisiologia , Transplante de Pele/patologia , Retalhos Cirúrgicos/irrigação sanguínea , Fatores de Tempo , Transplante Homólogo , Túnica Íntima/diagnóstico por imagem , Ultrassonografia DopplerRESUMO
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.
Assuntos
Adipócitos/citologia , Tecido Adiposo/fisiologia , Diferenciação Celular , Células Endoteliais/citologia , Células-Tronco/fisiologia , Tecido Adiposo/química , Tecido Adiposo/citologia , Meios de Cultura , Células Endoteliais/química , Humanos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Células-Tronco/química , Células-Tronco/citologia , Células Estromais/citologia , Células Estromais/fisiologiaRESUMO
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.
Assuntos
Adipogenia/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Quimiocina CCL2/farmacologia , Óxido Nítrico/farmacologia , Obesidade/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/metabolismo , Engenharia Tecidual/métodos , Zimosan/farmacologiaRESUMO
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.
Assuntos
Adipócitos/citologia , Tecido Adiposo , Materiais Biocompatíveis/química , Células-Tronco/citologia , Engenharia Tecidual/métodos , Tecido Adiposo/química , Tecido Adiposo/citologia , Animais , Biopolímeros/química , Diferenciação Celular , Humanos , Hidrogéis/químicaRESUMO
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.
Assuntos
Adipócitos/citologia , Adipócitos/fisiologia , Corantes Fluorescentes , Engenharia Tecidual/métodos , Adipócitos/efeitos dos fármacos , Adulto , Carbocianinas/toxicidade , Técnicas de Cultura de Células/métodos , Fluoresceínas/toxicidade , Corantes Fluorescentes/toxicidade , Humanos , Compostos Orgânicos/toxicidade , Succinimidas/toxicidadeRESUMO
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.
Assuntos
Citocinas/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Primers do DNA , Nitritos/metabolismo , RNA Mensageiro/metabolismo , RatosRESUMO
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 microm 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.
Assuntos
Adipócitos/citologia , Adipócitos/transplante , Tecido Adiposo/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Regeneração Tecidual Guiada/métodos , Ácido Hialurônico/química , Engenharia Tecidual/métodos , Adipócitos/fisiologia , Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/citologia , Adolescente , Adulto , Idoso , Animais , Materiais Biocompatíveis/química , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Estudos de Viabilidade , Regeneração Tecidual Guiada/instrumentação , Humanos , Implantes Experimentais , Teste de Materiais , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neovascularização Fisiológica/fisiologia , PorosidadeRESUMO
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.
Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Animais , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , Oligonucleotídeos Antissenso/genética , TransfecçãoRESUMO
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.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/citologia , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Consumo de Oxigênio/fisiologia , Células 3T3 , Adulto , Animais , Contagem de Células/métodos , Proliferação de Células , Respiração Celular , Células Cultivadas , Humanos , Camundongos , Soro/metabolismo , Estatísticas não Paramétricas , Fatores de Tempo , Engenharia TecidualRESUMO
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.
Assuntos
Adipócitos/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Tecido Adiposo/citologia , Adulto , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Meios de Cultura , Fibronectinas/química , Fibronectinas/metabolismo , Humanos , Pessoa de Meia-Idade , Soro , Fatores de TempoRESUMO
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 degrees 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.