RESUMEN
Diabetic patients suffer from impaired wound healing, characterized by only modest angiogenesis and cell proliferation. Stem cells may stimulate healing, but little is known about the kinetics of mobilization and function of bone marrow progenitor cells (BM-PCs) during diabetic wound repair. The objective of this study was to investigate the kinetics of BM-PC mobilization and their role during early diabetic wound repair in diabetic db/db mice. After wounding, circulating hematopoietic stem cells (Lin(-)c-Kit(+)Sca-1(+)) stably increased in the periphery and lymphoid tissue of db/db mice compared to unwounded controls. Peripheral endothelial progenitor cells (CD34(+)VEGFR(+)) were 2.5- and 3.5-fold increased on days 6 and 10 after wounding, respectively. Targeting the CXCR4-CXCL12 axis induced an increased release and engraftment of endogenous BM-PCs that was paralleled by an increased expression of CXCL12/SDF-1α in the wounds. Increased levels of peripheral and engrafted BM-PCs corresponded to stimulated angiogenesis and cell proliferation, while the addition of an agonist (GM-CSF) or an antagonist (ACK2) did not further modulate wound healing. Macroscopic histological correlations showed that increased levels of stem cells corresponded to higher levels of wound reepithelialization. After wounding, a natural release of endogenous BM-PCs was shown in diabetic mice, but only low levels of these cells homed in the healing tissue. Higher levels of CXCL12/SDF-1α and circulating stem cells were required to enhance their engraftment and biological effects. Despite controversial data about the functional impairment of diabetic BM-PCs, in this model our data showed a residual capacity of these cells to trigger angiogenesis and cell proliferation.
Asunto(s)
Células de la Médula Ósea/citología , Diabetes Mellitus Tipo 2/complicaciones , Células Madre/fisiología , Cicatrización de Heridas/fisiología , Animales , Proliferación Celular , Quimiocina CXCL12/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/citología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores CXCR4/metabolismo , Heridas y Lesiones/complicaciones , Heridas y Lesiones/terapiaRESUMEN
BACKGROUND: Natural Orifice Transluminal Endoscopic Surgery (NOTES) is a developing, minimally invasive surgical approach whose potential benefits are being investigated. Little is known about secure access site closure and early healing kinetics of transvisceral access. STUDY DESIGN: Transvisceral access incisions were created in the colon (C-NOTES, n = 8) and stomach (G-NOTES, n = 8) for peritoneal exploration. Incisions were closed primarily with endoloops, endoclips, or t-tags. Macroscopic and histologic analyses performed on postoperative day 7 assessed gross appearance, granulation tissue, inflammation, ulceration, and complications. RESULTS: Macroscopically, incisions appeared closed without intraperitoneal spillage. Incisions closed by endoloop and t-tags showed intense granulation tissue fill of defect despite partial (G-NOTES, n = 3) and transmural ulceration (C-NOTES, n = 8; G-NOTES, n = 3). Of the 30 t-tags applied, 40% broke or deployed into the peritoneal cavity. Endoclip closures (C-NOTES, n = 1; G-NOTES, n = 1) did not show histologic mucosal continuity. Healing complications included transmural necrosis (C-NOTES, n = 1; G-NOTES, n = 1), foreign body material (C-NOTES, n = 3; G-NOTES, n = 2), and microabscesses (G-NOTES, n = 1). CONCLUSIONS: This study provides a reproducible model to assess noninvasive repair of planned visceral perforations. Of investigated technologies, endoloop closure was favored for transcolonic incisions, and t-tags with omental patch for transgastric incisions, although these have significant limitations. Endoclips were inadequate for primary closure, but may be useful as an adjunctive closure modality. Additional studies are needed to examine visceral repair at later time points, as they will help determine the quality and kinetics of repair of a variety of incision closure strategies. This study demonstrates the need for improved technologies to more reliably close visceral transluminal defects.
Asunto(s)
Endoscopía Gastrointestinal , Laparoscopía , Cicatrización de Heridas , Animales , Colonoscopía , Duodenoscopía , Femenino , Gastroscopía , Tejido de Granulación , Inflamación , Microscopía , Sus scrofa , Técnicas de SuturaRESUMEN
Tension is a principal force experienced by skin and serves a critical role in growth and development. Optimal tension application regimens may be an important component for skin tissue engineering and dermatogenesis. In this study, we designed and tested a novel servo-controlled skin-stretching device to apply predetermined tension and waveforms in mice. The effects of static and cyclical stretching forces were compared in 48 mice by measuring epidermal proliferation, angiogenesis, cutaneous perfusion, and principal growth factors using immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and hyperspectral imaging. All stretched samples had upregulated epidermal proliferation and angiogenesis. Real-time reverse transcriptase-polymerase chain reaction of epidermal growth factor, transforming growth factor beta1, and nerve growth factor demonstrated greater expression in cyclically stretched skin when compared to static stretch. Hypoxia-induced factor 1alpha was significantly upregulated in cyclically stretched skin, but poststretch analysis demonstrated well-oxygenated tissue, collectively suggesting the presence of transient hypoxia. Waveform-specific mechanical loads may accelerate tissue growth by mechanotransduction and as a result of repeated cycles of temporary hypoxia. Further analysis of mechanotransduction signaling pathways may provide additional insight to improve skin tissue engineering methods and optimize our device.
Asunto(s)
Piel/crecimiento & desarrollo , Animales , Secuencia de Bases , Proliferación Celular , Cartilla de ADN , Análisis de Elementos Finitos , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/irrigación sanguínea , Piel/citología , Resistencia a la TracciónRESUMEN
INTRODUCTION: Platelets partake in hemostasis, wound healing, and tumor growth. Although platelet-rich-plasma (PRP) has been used in surgery for several years, its mechanism of action and application methods are still poorly characterized. MATERIALS AND METHODS: A single unit of human platelets obtained by plateletpheresis was diluted in plasma and divided into three equal volumes. One volume was stored at room temperature as fresh platelets (RT), another volume was frozen by storage at -80 degrees C (FZ), and the third volume was frozen at -80 degrees C with 6% DMSO (FZ6). Plasma (PL) was used as control. Using flow cytometry, platelets were tested for platelet glycoprotein GPIb and annexin V binding, as survival and activation markers, respectively. Hemostatic function was assessed by thromboelastometry. In vivo, platelets were topically applied on 1 cm,(2) full-thickness wounds on db/db mice (n = 10/group) and healing was staged microscopically and macroscopically. RESULTS: All platelet preparations showed hemostatic ability. RT platelets were GPIb positive (nonactivated-quiescent platelets) and stimulated angiogenesis by threefold, and cell proliferation by fourfold in vivo. FZ platelets were positive for annexin V, indicating activated platelets and, in vivo, increased only wound granulation. FZ6 platelets contained 30% nonactivated-quiescent and 50% activated platelets and stimulated granulation, angiogenesis, cell proliferation, and promoted re-epithelialization in vivo. CONCLUSIONS: Platelets showed distinct mechanisms to induce hemostasis and wound healing. Quiescent platelets are required to induce angiogenesis in vivo. Platelets stored at room temperature and frozen with 6% DMSO and stored at -80 degrees C achieved optimal wound healing in diabetic mice.
Asunto(s)
Plaquetas , Complicaciones de la Diabetes/terapia , Neovascularización Fisiológica , Plasma Rico en Plaquetas , Cicatrización de Heridas , Animales , Criopreservación , Crioprotectores , Dimetilsulfóxido , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: The vacuum-assisted closure device is a widely used mechanical modulator of wound healing; however, the optimal time kinetics of application have not been determined. The objective of the study was to optimize the kinetics of vacuum-assisted closure application. METHODS: Full-thickness wounds in seven diabetic mice per study group were treated with either an occlusive dressing alone, the vacuum-assisted closure device for 6 or 12 hours, or the vacuum-assisted closure device periodically for 4 hours every other day or continuously for 7 days. Wound closure and tissue response were evaluated by macroscopic, histologic, and immunohistochemical analyses on day 7. RESULTS: Wound closure was significantly faster after short initial vacuum-assisted closure (6-hour and 12-hour groups) when compared with continuous treatment. Increased granulation tissue formation was seen in the 12-hour group (2.4-fold increase) and in those treated periodically for 4 hours every other day (3.2-fold increase) compared with the dressing-alone controls. Significant stimulation of cell proliferation was seen after all vacuum-assisted closure patterns (3.6- to 5.3-fold increase), whereas angiogenesis was augmented only after the device was applied for either three times for 4 hours (4.3-fold) or continuously (4.7-fold) when compared with dressing-treated wounds. Treatment three times for 4 hours showed a superior angiogenic effect also when compared with short initial applications (6-hour and 12-hour groups). CONCLUSIONS: Short vacuum-assisted closure treatment induced an extended biological response in the wound. A total of 12 hours of periodically applied vacuum-assisted closure reached a similar wound tissue response as continuously applied vacuum-assisted closure for 7 days. These findings suggest new clinical approaches for mechanical wound-healing devices.
Asunto(s)
Tejido de Granulación , Terapia de Presión Negativa para Heridas , Cicatrización de Heridas , Animales , Biomarcadores/metabolismo , Proliferación Celular , Diabetes Mellitus Experimental , Tejido de Granulación/metabolismo , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Cinética , Ratones , Terapia de Presión Negativa para Heridas/instrumentación , Neovascularización Fisiológica , Apósitos Oclusivos , Factores de Tiempo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND: The vacuum-assisted closure device is widely used clinically, yet its mechanisms of action are incompletely understood. In this study, the authors designed a partially splinted full-thickness murine vacuum-assisted closure model to better understand the mechanism of action of the vacuum-assisted closure device. METHODS: Full-thickness wounds (n = 10 per group) were excised in diabetic mice and treated with the vacuum-assisted closure device or its isolated components: an occlusive dressing, subatmospheric pressure at 125 mmHg (suction), and a polyurethane foam without and with downward compression. Results were quantified with a two-dimensional immunohistochemical staging system based on blood vessel density (CD31) and cell proliferation (Ki67) 7 days after wounding. Microscopic strain was measured by fixing in situ all dressing modalities. RESULTS: Wounds exposed to polyurethane foam in compressed and uncompressed dressings or to the vacuum-assisted closure device showed a 2-fold increase in vascularity compared with the occlusive dressing group (p < 0.05). The vacuum-assisted closure device in addition stimulated cell proliferation, with up to 82 percent Ki67-positive nuclei, compared with the other groups. Direct measurements of wound surface deformations showed significant microstrains in the vacuum-assisted closure and foam in compressed dressing groups (60 percent and 16 percent, respectively) compared with all other groups. CONCLUSIONS: These data provide profound insights into the mechanism of action of the vacuum-assisted closure device, providing an explanation for the increases in wound bed vascularity and cell proliferation based on its components. Results suggest that the vascular response is related to the polyurethane foam, whereas tissue strains induced by the vacuum-assisted closure device stimulated cell proliferation.
Asunto(s)
Terapia de Presión Negativa para Heridas , Heridas y Lesiones/terapia , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Moléculas de Adhesión Celular/análisis , División Celular , Diabetes Mellitus Experimental/complicaciones , Inmunohistoquímica , Cinética , Masculino , Ratones , Terapia de Presión Negativa para Heridas/instrumentación , Apósitos Oclusivos , Poliuretanos , Cicatrización de Heridas/fisiologíaRESUMEN
The incidence and prevalence of chronic and diabetic wounds are increasing and clinical treatments to tackle these epidemics are still insufficient. In this study, we tested the ability of freeze-dried platelet-rich plasma (PRP) and an allogenic micronized acellular dermal matrix alone and in combination to modulate diabetic wound healing. Therapeutic materials were applied to 1.0 cm(2) excisional wounds on genetically diabetic (db/db) mice. Wound-healing kinetics and new tissue formation were studied at 9 and 21 days posttreatment. Quantitative immunohistochemistry was used to study vascularity and cellular proliferation (days 9 and 21), and collagen deposition was evaluated 21 days postwounding. In vitro, micronized allogenic dermis, when combined with PRP, absorbed nearly 50% of original platelet-derived growth factor, transforming growth factor-beta, vascular endothelial growth factor, and epidermal growth factor from platelets and stimulated fibroblast proliferation. In vivo, micronized dermis increased the formation of vascularized wound tissue by day 9. Freeze-dried PRP alone or in combination with micronized dermis increased wound tissue revascularization and proliferation compared with spontaneous healing. The increase in cell proliferation persisted until day 21 only when freeze-dried PRP was used in combination with micronized dermis. These results indicate that micronized allogenic dermis may be used to provide a dermal matrix to stimulate tissue formation and the combination with PRP may confer additional beneficial growth factors to chronic or diabetic wounds.
Asunto(s)
Dermis/trasplante , Diabetes Mellitus Experimental/fisiopatología , Liofilización , Plasma Rico en Plaquetas , Cicatrización de Heridas , Animales , Proliferación Celular , Colágeno/metabolismo , Matriz Extracelular , Masculino , Ratones , Ratones Endogámicos , Piel/fisiopatología , Trasplante Homólogo , Cicatrización de Heridas/fisiologíaRESUMEN
The increased number of chronic nonhealing wounds mirrors the epidemic of type 2 diabetes. Diabetic animal models may allow for better understanding of the pathophysiology of wound healing and may lead to the pre-clinical testing of a variety of therapeutic modalities for this patient group. The authors present an overview of the literature on excisional wound mouse models and focus on the authors' experience with the db/db mouse. Excisional wounds in wild type mice heal quickly due primarily to wound contraction, which is delayed in the db/db mouse. In this animal model it is possible to study and quantify the main mechanisms of healing and produce highly reproducible information. Differences in methodologies, infection control, as well as fine details such as the dressing option, partially explain heterogeneous results in the literature. Given the increase of the diabetic population, the db/db mouse model provides a powerful tool to study the effects of therapeutics for improving wound healing. The standardization of this animal model represents an important aspect to improve in the wound care field.