| Part ? Negative pressure wound therapy influence microvessel maturation by regulating the Ang/Tie-2 system in diabetic ratsObjective For investiaged the effect of negative pressure wound therapu promoting microvessel maturation, and further explore the potential molecular mechanism and relevant signal pathway.Methods 96 rats through intraperitoneal injection streptozotocin(Streptozotocin, STZ), and by measure blood glucose level>16.7 mmol/L to determine diabetic, after the diabetic model were successful created, and a 3.5 cm×3.5 cm area of skin and panniculus carnosus were removed tocreate a full-thickness diabetic wound in diabetic rats.96 diabetes mellitus rats were randomly divided into NPWT, NPWT+Tie, Gauze and Gauze+Tie groups. NPWT and NPWT+Tie groups included 48 diabetes mellitus rats whose wounds were covered with Duoderm foam, followed by therapy using a vacuum-assisted closure (VAC) device. Gauze and Gauze+Tie-2 groups included 48 diabetes mellitus rats that were treated with petrolatum gauze. In addition,48 diabetes mellitus rats in Gauze and Gauze+Tie-2 groups were injected intraperitoneally with Tie-2 kinase inhibitor. The immunofluorescence was applied to detect Microvessel Density (MVD), Proliferating Capillary Index (PCI), the number of pericyte and Microvessel Pericyte Coverage Index (MPI) at days 1,3,7,10; simultaneously immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) and western blot were applied to detect protein expression of angiotesin-1 (Ang-1), angiotesin-2 (Ang-2),?-smooth muscle actin (?-SMA) and phosphorylation levels of tyrosine kinase receptor-2 (pTie-2).Results Negative pressure wound therapycould increase the PCI, promote the mRNA and protein expression levels of Ang-2, decrease the expression ratios of Ang-1/Ang-2, decrease the expression levels of Ang-1 and pTie-2 at early stage (1-3 days) of wound healing. At later stage (7-10 days) of wound healing, negative pressure wound therapycould increase MVD, MPI and the number of pericyte, and promote the protein expression levels ofAng-1, ?-SMA and pTie-2, as well asincrease the expression ratiosof Ang-1/Ang-2, and decrease the mRNA and protein expression levels of Ang-2.Conclusion In the early stages of wound healing, NPWT could preferentially induced the occur of wound microvessel destabilized microenvironment, thereby increase initial angiogenesis in diabetic rats wound; and in later stages of wound healing, the NPWT could preferentially induce the occur of wound microvessel stabilized microenvironmen t, thereby promote microvascular reconstruction and maturation. Meanwhile Tie-2 inhibitors can mild affect angiogenesis in the early stages of wound healing, but it can significantly influence microvascular reconstruction and maturation process in the late stages of wound healing, and the wound reconstruction and maturation process were main regulated by Ang/Tie-2 signaling pathways in diabetic rats wound.Part II Negative pressure wound therapy:regulating blood flow perfusion and microvessel maturation through microvessel pericyteObjective For investigate whether the blood flow perfus ions were increased in mature microvessels after through negative pressure wound therapy, and further explore underlying molecular mechanism and signal pathway, as well as relationships between pericytes and collagen type IV.Methods 96 rats through intraperitoneal injection streptozotocin(Streptozotocin, STZ), and by measure blood glucose level>16.7 mmol/L to determine diabetic, after the diabetic model were successful created, and a 3.5 cmx3.5 cm area of skin and panniculus carnosus were removed tocreate a full-thickness diabetic wound in diabetic rats.96 diabetes mellitus rats were randomly divided into NPWT, NPWT+Tie, Gauze and Gauze+Tie groups. NPWT and NPWT+Tie groups included 48 diabetes mellitus rats whose wounds were covered with Duoderm foam, followed by therapy using a vacuum-assisted closure (VAC) device. Gauze and Gauze+Tie-2 groups included 48 diabetes mellitus rats that were treated with petrolatum gauze. In addition,48 diabetes mellitus rats in Gauze and Gauze+Tie-2 groups were injected intraperitoneally with Tie-2 kinase inhibitor. The microvascular blood flow perfusion was detected by a Laser Doppler Blood Perfusion Imager, and immunofluorescence was applied to detect microvessel density and pericyte coverage index at days 1,3,7,10; simultaneously RT-PCRwere applied to detect the mRNAexpression of Ang-1, a-SMAand collagen IV, as well as immunohistochemistry and western blot were applied to detect protein expression of Ang-1, a-SMA, pTie-2 and collagen IV.Results Negative pressure wound therapy could decrease expression of Ang-1 andpTie2 at early stage (1-3 days), and gradually increase MVD, MPI and the number of pericyte. At later stage of wound healing (7-10 days), negative pressure wound therapy could significantly increase the number of pericyte, MVD and MPI, as well as promote over expression of Ang-1, pTie-2, a-SMA and collagen type IV, smultaneously increase blood flow perfusion and wound healing speed.Conclusion Negative pressure wound therapy could preferentially promote microvascular maturation and increased wound capillary blood flow perfusion, and could promote the proliferation of pericytes. Pericyte serve as an important mature vascular structure, which not only can effectively change the wound blood flow perfusion, but also could regulatethe participate of collagen IV, prompte the basement membrane matrix gradual integral. Meanwhile NPWT could preferentially induce pericyte and collagen IV concertedly complete microvascular maturation process, and Ang/Tie-2 signaling pathway not only effect the proliferation of pericyte, and could regulate basement membrane degrade indirectly.Part III Clinical study of negative pressure wound therapy promotingwound blood vessel maturationObjective For investigate the potential effect of NPWT on angiogenesis and vessel maturation, and investigate relevant association between mature microvessels and wound prognosis, as well as the potential regulatory mechanisms in human wound.Methods Successive 48 patients were included in this study. The patients were randomly divided into experiment group and control group, and the patients were treated with negative pressure or gauze, and at 1,3,7,15 days the blood flow perfusion were detected through Laser Doppler Blood Perfusion Imager, immunofluorescence was applied to detected PCI, the number of pericyte, MVD and MPI, RT-PCR was applied to detected the mRNA expression levels of Ang-1, Ang-2, a-SMA and collagen type IV. As well as the Western boltting were applied to detected the protein expression level of Ang-I, pTie-2, Ang-2, a-SMA and collagen type IV.Results Negative pressure wound therapy could increase the expression of Ang-2, and decrease the expression of Ang-1 and ratios of Ang-1/Ang-2 in the early stage (1-3 days) of wound healing. However in the later stage (7-15 days), the expression level of Ang-1 and ratios of Ang-1/Ang-2, as well as the pTie-2 levels were upregualted by negative pressure wound thrapy. As the result of that, the number of microvessel pericyte elevated gradually and thus increasedblood flow perfusion at later stage of wound healing.Conclusion Negative pressure wound therapycould preferentially promote the production of destabilized microvessel microenvironment in the early stage of wound healing in patients. leading to the increase of the number of angiogenesis and vascular sprouting, but also NPWT could preferentially promote the production of stabilized microvessel microenvironment in the later stages of wound healing in patients, and thus promote vessel maturation and formation functional microvessels, thus affecting wound prognosis. Meanwhile the Ang/Tie-2 signaling pathway could modulate angiogenesis and maturation process in human wound. |
PDF Full Text Request