RESUMO
A combined strategy using bioluminescence imaging, bone densitometry and histology was used to analyze the bone regeneration capacity of human bone marrow (hBMSC) and adipose tissue (hAMSC) mesenchymal stem cells, seeded in an osteoconductive arginine-glycine-aspartate (RGD) crosslinked hydrogel scaffold, implanted in a mouse calvarial bone defect. We show that firefly luciferase labeled stem cells can be monitored in vivo through a prolonged 90 days period, during which hBMSCs survive better than hAMSCs and that the density of scaffold bearing defects increased significantly more than that of defects without scaffolds.
Assuntos
Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Osteogênese , Crânio/citologia , Crânio/fisiologia , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Expressão Gênica , Genes Reporter/genética , Humanos , Medições Luminescentes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regeneração , Crânio/anormalidadesRESUMO
Recent developments in stem cell research have promoted a flourishing of new biomaterials and scaffolds for tissue repair. However, there is a scarcity of procedures to monitor the performance of scaffold-stem cell combinations implanted in live animals, avoiding the inherent artefacts associated with in vitro assay conditions. We report the implementation of a procedure based on the use of the luciferase gene as a cell proliferation tracer to monitor, by in vivo non-invasive imaging, the performance of stem cell-biomaterial combinations used for tissue regeneration. In a model system using immunodepressed mice we show preference of a mouse embryonic mesenchymal cell line (C3H/10T1/2) for specific implantation sites and biomaterials during a prolonged in vivo growth period (3 months). Moreover, we analyzed the safety of implanted cells using a sensitive luminometric procedure and showed that the implanted cells did not spread to other organs. Our results demonstrate the utility of this simple and resource-saving procedure in the development and screening of biomaterials for tissue engineering.
Assuntos
Materiais Biocompatíveis , Células-Tronco Embrionárias/citologia , Imageamento Tridimensional/métodos , Fótons , Animais , Movimento Celular , Proliferação de Células , Estabilidade Enzimática , Fluorescência , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Luciferases/metabolismo , Luminescência , Camundongos , Camundongos Nus , Microesferas , Músculo Esquelético/citologia , Próteses e ImplantesRESUMO
BACKGROUND: In healing wounds, rising levels of vascular endothelial growth factor (VEGF) induce a period of robust angiogenesis. The levels of pro-angiogenic factors in the wound begin to decline just before a period of vascular regression, suggesting that these mediators are necessary to sustain vessel density. The purpose of this study was to determine if the maintenance of pro-angiogenic stimuli in the wound would prevent physiological vessel regression. MATERIALS AND METHODS: A standard subcutaneous sponge wound model was modified by the addition of a mini-osmotic pump, allowing manipulation of the wound milieu by the addition of exogenous growth factors. After initial characterization of this model, exogenous VEGF (10 microg/mL), FGF (10 microg/mL), PDGF (10 microg/mL), or VEGF (10 microg/mL) plus FGF (10 microg/mL) were delivered to wounds and blood vessel density analyzed by immunohistochemistry. RESULTS: VEGF administration resulted in a transient increase in wound vessel density (P < 0.05). None of the pro-angiogenic growth factors (VEGF, FGF, PDGF, VEGF/FGF) were able to prevent vascular regression (P = NS). CONCLUSIONS: These findings suggest that the anti-angiogenic signals that mediate physiological vascular regression in wounds are strongly dominant over pro-angiogenic stimuli during the later phases of wound healing. Clinical manipulation of anti-angiogenic signals in addition to the currently used pro-angiogenic targets may be needed to achieve therapeutic modulation of blood vessel density.
Assuntos
Indutores da Angiogênese/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Cicatrização/fisiologia , Análise de Variância , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Feminino , Fatores de Crescimento de Fibroblastos/farmacologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Acute ethanol exposure represents an increased risk factor for morbidity and mortality associated with surgical or traumatic injury. Despite clinical observations suggesting that ethanol exposure before injury alters tissue repair processes, little direct evidence about the mechanism by which ethanol affects the wound healing process is available. In this study, excisional wounds from female BALB/c mice with or without circulating ethanol levels of 100 mg/dl were used to assess wound closure, angiogenesis, and collagen content. Ethanol exposure resulted in a significant but transient delay in wound closure at day 2 postwounding (28 +/- 4% vs. 17 +/- 1%). In addition, total collagen content was significantly reduced by up to 37% in wounds from ethanol-treated mice compared with controls. The most significant effect of ethanol exposure on wounds was on vascularity because angiogenesis was reduced by up to 61% in wounds from ethanol-treated mice. The reduction in vessel density occurred despite near-normal levels of proangiogenic factors VEGF and FGF-2, suggesting a direct effect of ethanol exposure on endothelial cell function. Further evidence for a direct effect was observed in an in vitro angiogenesis assay because the exposure of endothelial cells to ethanol reduced angiogenic responsiveness to just 8.33% of control in a cord-forming assay. These studies provide novel information regarding the effect of a single dose of ethanol on multiple parameters of the wound healing process in vivo and suggest a potential mechanism by which ethanol impairs healing after traumatic injury.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Queimaduras/fisiopatologia , Divisão Celular/efeitos dos fármacos , Colágeno/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pele/irrigação sanguínea , Pele/lesões , Pele/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor (VEGF-A), a potent stimulus for angiogenesis, is up-regulated in the skin after wounding. Although studies have shown that VEGF is important for wound repair, it is unclear whether this is based solely on its ability to promote angiogenesis or if VEGF can also promote healing by acting directly on non-endothelial cell types. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was detected in murine keratinocytes during wound repair and in normal human epidermal keratinocytes (NHEKs). The presence of VEGF receptors on NHEKs was verified by binding studies with 125I-VEGF. In vitro, VEGF stimulated the proliferation of NHEKs, an effect that could be blocked by treatment with neutralizing VEGFR-1 antibodies. A role for VEGFR-1 in keratinocytes was also shown in vivo because treatment of excisional wounds with neutralizing VEGFR-1 antibodies delayed re-epithelialization. Treatment with anti-VEGFR-1 antibodies also reduced the number of proliferating keratinocytes at the leading edge of the wound, suggesting that VEGF sends a proliferative signal to these cells. Together, these data describe a novel role for VEGFR-1 in keratinocytes and suggest that VEGF may play several roles in cutaneous wound repair.
Assuntos
Queratinócitos/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Cicatrização/fisiologia , Animais , Linhagem Celular , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Isótopos de Iodo/metabolismo , Queratinócitos/química , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis, the formation of new capillaries from existing vasculature, plays an essential role in tissue repair. The rapid onset and predominance of proangiogenic factors optimizes healing in damaged tissues. One factor that directly mediates wound vessel angiogenesis is vascular endothelial growth factor (VEGF). Although much is known about the biology of VEGF and its cognate receptors, VEGFR1 and VEGFR2, the role of a recently identified co-receptor for VEGF, neuropilin-1, is not well understood. Using a murine model of dermal wound repair, we found that neuropilin-1 was abundantly expressed on new vasculature in healing wounds. Moreover, mice treated with anti-neuropilin-1 antibodies exhibited a significant decrease in vascular density within these wounds (67% decrease, P = 0.0132). In in vitro assays, VEGF induced formation of endothelial cord-like structures on collagen gel and endothelial cell migration toward VEGF was inhibited by antibodies directed against neuropilin-1. These results provide both in vitro and in vivo evidence for a critical role of neuropilin-1 in wound angiogenesis.