Effects of heparin on vascularization of artificial skin grafts in rats

Artificial skin is a recent development in the clinical care of the severely burned patient. Its manufacture entails the covalent bonding of collagen and polysaccharide, followed by the coating of one surface with a thin layer of silicone rubber. Artificial skin was grafted onto rats and examined for neovascularization at 7 days. Vascular patency was shown by perfused yellow latex casts. Five percent of the patent vessels grew into the graft soaked in physiological buffered saline (PBS). When the graft was soaked in heparin, 1 mg/ml buffered saline solution, before grafting, 54% of the patent vessels in the grafted area had grown into the matrix. These experiments show that the local application of heparin promotes early ingrowth of blood vessels into the healing site. The vascularity of artificial skin can be modified by heparin, which promotes angiogenesis, and leads to earlier deposits of greater amounts of new connective tissue.     

小口径生物型人工血管移植后的血管内皮再生

背景：经过生物化改造的人工血管特性更接近人体血管,移植后自体化程度也较高,但人工血管的内皮再生是解决血管长期通畅的关键。 目的：观察新型小口径生物型人造血管移植后不同时期移植材料的组织相容性及移植血管壁内膜再生的组织病理学变化。 方法：建立犬颈总动脉-人造血管端端连续缝合的动物模型。 结果与结论：①光镜：移植后12周于吻合口处见新内膜表面有不连续的内皮细胞生长;移植后6个月通畅的整段管腔内面均可见内皮细胞生长;移植后1.5年管腔通畅,部分内膜组织呈慢性炎症表现。②电镜：移植后12周新生血管内皮细胞排列规则,从吻合口向移植血管中段爬行;移植后6个月内皮细胞从吻合口向移植血管中段爬行,移植血管中段呈跳跃式片状生长的内皮细胞群落,细胞排列更致密,形态更接近成熟血管内皮细胞;移植后1.5年整段血管内壁均有致密内皮细胞覆盖,部分内膜组织呈慢性炎症表现。说明新型小口径生物型人造血管新生内皮形成早,血管内膜重构能力强,生物相容和稳定性好。     

An analysis of the reflex systemic vasodilator response elicited by lung inflation in the dog

1. A maintained inflation of the lungs caused a reflex reduction in total systemic vascular resistance in anaesthetized dogs under conditions in which the systemic circulation was perfused at constant blood flow and the arterial blood P(O2) and P(CO2) were maintained constant.2. The fall in systemic arterial perfusion pressure evoked by inflation of the lungs was accompanied by an increase in blood flow to the lower limbs and a reduction in their calculated vascular resistance. Since the fall in resistance occurred when the limb was perfused either at constant pressure or at constant blood flow, it must be due to vasodilatation.3. Lung inflation caused vasodilatation in skin, muscle, and in the splanchnic vascular bed. The responses in vertebral circulation were, however, small and variable.4. The vasodilator responses in the vascular territories studied were reflex in nature, being abolished by cutting the cervical vagosympathetic nerves, in which run the afferent fibres, or by interrupting the sympathetic pathways to the blood vessels.5. In the intact limb, muscle, skin and splanchnic vascular bed, the vasodilator responses to lung inflation were unaffected by atropine or propranolol, but were abolished by hexamethonium, dibenyline and bretylium tosylate, indicating that they were due predominantly to a reduction in the activity in sympathetic adrenergic vasoconstrictor fibres.     

Development of a pre-vascularized 3D scaffold-hydrogel composite graft using an arterio-venous loop for tissue engineering applications

Hyaluronic acid (HA) and fibrin glue (FG) are effective hydrogels for tissue engineering applications as they support tissue in-growth, retain growth factors, and release them slowly with time. The scaffolds, in combination with a hydrogel, effectuate a successful graft. However, the survival of a graft entirely depends upon a functional vascular supply. Therefore, hydrogels must support the in-growing vasculature. To study and compare the vascular patterns, HA and FG hydrogel-containing PLDLLA-TCP-PCL scaffolds were implanted in the groin of male Lewis rats and supplied with a micro-surgically prepared arterio-venous (A-V) loop. The rats were perfused with a vascular contrast media after 4 and 8 weeks and sacrificed for further analysis. The specimens were scanned with micro-CT to find the vascular growth patterns. Corrosion casting of blood vessels followed by SEM demonstrated a high vascular density near the parent blood vessels. Histologically, HA and FG implanted animal groups showed significant angiogenetic activity, especially within the pores of the scaffold. However, formation of new blood vessels was more conspicuously observed at 4 weeks in FG than HA implants. Furthermore, by 8 weeks, the number and pattern of blood vessels were comparable between them. At this time, HA was still present indicating its slow degradation. The finding was confirmed by histomorphometric analysis. This experimental study demonstrates that HA containing composite scaffold systems permit stabile in-growth of blood vessels due to sustained degradation over 8 weeks. HA is a potential matrix for a tissue engineered composite graft.     

小口径生物型人工血管移植后的血管壁重构

背景：人工血管相对人体血管最大的优势就是来源丰富,经过生物化改造的人工血管,其特性更接近人体血管,移植后自体化程度也较高。 目的：观察新型小口径生物型人造血管移植后1.5年内不同时期实验犬的生存、生活状况,移植材料的组织相容性、移植血管壁重构的组织病理学变化。 方法：以猪血管为基材,经交联固定,多方位去抗原,共价结合肝素,以及偶联可黏附、富集生长因子的特定多肽等系列生化处理而制成的一种高抗凝的人造血管,管径3.5~4.5 mm。建立犬颈总动脉-人造血管端端连续缝合的动物模型,1.5年内不同时期切取标本,做病理组织学检查。 结果与结论：切取标本发现,移植血管与周围组织粘连少、疏松。病理组织学检查：移植后8周,镜下开始发现宿主组织通过人造血管孔隙长入血管腔内参与移植血管新内膜的形成,移植后12周,镜下于吻合口处,可见新内膜表面有不连续的内皮细胞生长,移植后6个月,通畅的人造血管整段管腔内面均可见内皮细胞生长。移植后12个月,移植血管管壁VG染色尚可见支架层内有大量胶原纤维和毛细血管生长,原先的支架结构已部分被宿主血管壁组织取代。移植后18个月,原先的支架结构已大部分被宿主血管壁组织取代。说明新型小口径生物型人造血管新内膜形成早且完整,自然内皮化相对满意,血管壁重构和血管支架的再生能力强,生物相容和稳定性好。     

保存真皮下血管网皮肤移植中血管重建的实验研究

本文采用家兔进行实验,在家兔动脉内灌注墨汁,观察保存真皮下血管网皮肤移植后不同时期墨汁在血管内的充盈状态,血运重建的过程。结果表明:(1)保留真皮下血管网皮肤移植后第2天血运开始建立;第3天血运增加;第7天血运基本完善。(2)保存真皮下血管网的游离皮肤移植后的血管重建过程,基本上通过移植片的真皮下血管网在切口周缘和基底部与受皮区的血管吻合。吻合处的血管为迂曲、扩张的薄壁血管。通过它们先与真皮内原有血管交通,并逐步在皮片内新生血管,建立起完整的血液循环。(3)皮片与受区随着吻合部位的不同,术后2天开始建立吻合,7天吻合最多,术后2周血管构筑基本完善。     

人造血管在治疗四肢大血管损伤中的初步应用

目的作者自1995～2002年采用人造血管移植修复四肢大血管损伤23例,进行回顾性研究人造血管在治疗四肢大血管损伤中的应用.方法23例中男19例,女4例.年龄21～47岁,平均34.7岁.手术清创和探查后,针对不同的情况选择人造血管移植修复方法.结果采用人造血管移植23例,肢体全部保存,肢体功能恢复较佳.结论四肢大血管损伤救治应快速诊断、快速救治,采用人造血管移植修复四肢主要血管损伤是一种快捷有效的方法.     

人造血管在治疗四肢大血管损伤中的初步应用

目的作者自1995-2002年采用人造血管移植修复四肢大血管损伤23例，进行回顾性研究人造血管在治疗四肢大血管损伤中的应用。方法23例中男19例，女4例。年龄：21～47岁，平均34．7岁。手术清创和探查后，针对不同的情况选择人造血管移植修复方法。结果采用人造血管移植23例，肢体全部保存，肢体功能恢复较佳。结论四肢大血管损伤救治应快速诊断、快速救治，采用人造血管移植修复四肢主要血管损伤是一种快捷有效的方法。     

Histological criteria for immunological rejection of mouse skin homografts.

The sequence of events in homograft rejection of skin in mice was studied by light microscopy in comparison to the reactions to isografts. Male C3H mice were used as donors and Balb/c mice were the recipients. In non-infected grafts no invasion of graft tissue by inflammatory cells occurred except for the marginal areas bordering on the graft bed. The most constant feature of immunological rejection was occlusion of blood vessels at the graft-host border with ischaemia of the graft. This indicated that skin homograft rejection is mainly a vascular phenomenon which can be best diagnosed by the presence of empty venules and capillaries. Leukocytic infiltration was prominent in presumably infected grafts. Destruction and inflammatory infiltration of the epidermis was not found to be a criterion for immunological rejection. In the graft bed adjoining the graft the granulation tissue contained basophilic and small lymphocytes, and macrophages. These cell tended to concentrate nearest to the graft and were surrounded on the outside by the lymphoid cells. It is probable that the macrophages were active in digestion of breakdown products.     

Experimental neovascularization in vivo: the early changes in a stable adult vasculature responding to angiogenic stimulation by a syngeneic neoplasm.

To examine the vascular changes that lead to the development of new vessel sprouts from differentiated vessels the early response of an uninjured, stable, adult vascular bed to the presence of neoplastic tissue has been studied. Small grafts of a squamous cell carcinoma were implanted above the cremaster muscle of host rats syngeneic with the rat in which the neoplasm arose. The vascular response was examined by light microscopy of whole cremaster preparations after intravenous injection of colloidal carbon to label leaky vessels, and by scanning electron microscopy of methyl methacrylate injection casts. Three to five days after implantation there was a dramatic increase in the number of visible blood vessels of the microcirculation adjacent to the graft as a result of altered blood flow through the existing microvasculature. Capillaries and post-capillary venules became widely distended, tortuous and variably permeable to the introduced colloidal marker. Capillary involvement was restricted to the area nearest the graft while post-capillary venules were affected in more remote regions. Networks of newly formed vascular channels developed from the extremities of the tortuous loops. Altered permeability within the pre-existing vessels was related to the distension and tortuosity, with a pattern of vascular labelling quite unlike that induced by inflammatory mediators or tumour secreted vascular permeability mediator (VPM). These changes are considered to be the result of altered inter-endothelial cell adhesion and cellular rearrangement, and represent important antecedent stages in the formation of the new vascular structures.     

Experimental neovascularization in vivo: the early changes in a stable adult vasculature responding to angiogenic stimulation by a syngeneic neoplasm

To examine the vascular changes that lead to the development of new vessel sprouts from differentiated vessels the early response of an uninjured, stable, adult vascular bed to the presence of neoplastic tissue has been studied. Small grafts of a squamous cell carcinoma were implanted above the cremaster muscle of host rats syngeneic with the rat in which the neoplasm arose. The vascular response was examined by light microscopy of whole cremaster preparations after intravenous injection of colloidal carbon to label leaky vessels, and by scanning electron microscopy of methyl methacrylate injection casts. Three to five days after implantation there was a dramatic increase in the number of visible blood vessels of the microcirculation adjacent to the graft as a result of altered blood flow through the existing microvasculature. Capillaries and post-capillary venules became widely distended, tortuous and variably permeable to the introduced colloidal marker. Capillary involvement was restricted to the area nearest the graft while post-capillary venules were affected in more remote regions. Networks of newly formed vascular channels developed from the extremities of the tortuous loops. Altered permeability within the pre-existing vessels was related to the distension and tortuosity, with a pattern of vascular labelling quite unlike that induced by inflammatory mediators or tumour secreted vascular permeability mediator (VPM). These changes are considered to be the result of altered inter-endothelial cell adhesion and cellular rearrangement, and represent important antecedent stages in the formation of the new vascular structures.     

兔预构血管化股骨血供重建的研究

目的　研究兔腹壁浅动静脉血管束植入股骨干中预构血管化骨的血运重建。方法　通过肉眼观察及血管墨汁灌注组织学检测方法观察血管植入后 ,股骨的血运重建。结果　术后 3周时、股骨骨质内血管周围可见较多迂曲扩张的小血管 ,5周时血管束周围及骨髓腔内可见大量毛细血管 ;5～ 6周时肌肉、骨膜及骨髓腔内充满丰富密集的毛细血管网。结论　兔腹壁浅动静脉束植入股骨干适于作血管化骨游离移植。     

骨髓间充质干细胞构建组织工程化小口径血管

目的采用在动物体外构建初级组织工程化血管、体内强化的方法,探讨构建小口径组织工程化血管的可能性.方法体外培养骨髓间充质干细胞(BMSC),用含全反式维甲酸(AT-RA)、双丁酰环磷酸腺苷(db-cAMP)的DMEM-LG培养液和含血管内皮细胞生长因子(VEGF)的培养液诱导BMSC分别向血管平滑肌样细胞和血管内皮样细胞分化.免疫荧光观察平滑肌样细胞β肌动蛋白的表达和内皮样细胞vWF的表达.电镜观察超微结构的改变.诱导的血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸(PGA)的复合支架表面,将细胞和支架复合体种植于动物皮下,于植入后第4、8周再次麻醉动物,取出植入皮下的组织工程化血管,行组织学检查、压力实验及免疫荧光检查.结果诱导14 d后,BMSC能够分化为血管平滑肌样细胞和血管内皮样细胞:β肌动蛋白和vWF呈阳性表达,电镜证实细胞出现了相应的形态学改变.人工血管组织学观察见管壁结构清晰.单纯支架组可承受100～150 mm Hg(1mm Hg=0.133 kPa)的血管腔内压力,实验组则均可承受200mm Hg的血管腔内压力不破裂.实验组皮下培养8周Brdu标记细胞的免疫荧光结果显示部分细胞核呈现明亮的黄绿色荧光.结论以动物皮下为生物反应器可构建出组织工程化血管,其大体结构和天然血管相似.     

Sustained vessel dilation induced by increased pulsatile perfusion of porcine carotid arteries in vitro.

Arterial pulse pressure (PP) increases with exertional stress and ageing, and can modify vessel diameter in smaller vessels. To test if PP must exceed a certain range to influence vessel diameter, and determine if such effects are endothelium-dependent or intrinsic to vascular viscoelasticity, eight fresh excised porcine carotid artery segments were perfused with modified Krebs-Henseleit by a servo-controlled system generating physiological arterial pressure waveforms. In a separate group of vessels (n = 10), the endothelium was mechanically removed. Vessel external diameter was measured by video edge-detection. Vessels partially preconstricted with noradrenaline were perfused at 9 mL min(-1) mean flow, at mean pressure of 90 or 120 mmHg, and zero PP. PP alone was then increased to 40, 70, or 120 mmHg at 1 Hz cycling rate for 5 min, then returned to zero and vessel diameter measured immediately thereafter. The protocol was repeated after 10-20 min stabilization. Mean vessel diameter rose proportionally with PP only once PP exceeded 40 mmHg, with maximal increases of 6-9% at a PP of 120 mmHg. Similar responses were obtained in vessels with and without a functional endothelium, at both mean pressures. Thus, when exposed to higher than normal resting PP, conduit arteries dilate owing to the stress-relaxation response of their viscoelastic wall. This mechanism of PP-mediated vascular dilatation may contribute to enhanced organ perfusion when small resistance arteries are already dilated.     

The challenge of small diameter vascular grafts

Symptomatic vascular disease involving blood vessels smaller than 6 mm internal diameter constitutes the majority of vascular disease cases. In many cases the only correction for the condition is vascular reconstruction, or bypass surgery. The only adequate replacement for these vessels at the present time are autologous arteries, or more commonly veins. The availability of a reliable small diameter artificial graft would greatly increase the number of patients that could be treated surgically. Three materials are used for present grafts, tanned natural vessels, and artificial vessels constructed of knitted Dacron or expanded Teflon. All three types eventually fail due to thrombosis, either because of their inherent thrombogenicity or because of encroachment of tissue (intimal hyperplasia) (IH) into the lumen of the graft at the point where the natural and prosthetic vessel join. The exact reasons for thrombosis and IH are poorly understood and constitute a major area of current research. Attempts to alleviate the problem involve: designing non-thrombogenic grafts, lining grafts with the living cells (endothelial cells) that form the inner surface of natural vessels, designing grafts that are elastic like natural vessels, providing biodegradable grafts that are replaced by tissue that reconstitutes a natural vessel, and producing living vessels in vitro. An overview of the problems and current research reveals some promising approaches to the solution, but all are still in the developmental stages.     

Quantitation of the changes in vascularity during arthritis in the knee joint of a mouse with a digital image analysis system

Many joint and bone diseases are caused by, or associated with vascular changes. Particularly in rheumatoid arthritis, vascular sprouting of synovial vessels plays a major role in the generation of joint pathology. To assess the effects of pharmaceuticals that are designed to inhibit neovascularization, we developed a quantitative procedure to measure vascular changes in cross-sections of the mouse knee joint during arthritic inflammation. Arthritis was induced in the knee joint of C57Black6 mice by a single subpatellar injection of methylated BSA after previous immunization. Total vascularity was visualized with a specific monoclonal rat anti-mouse antibody (9F1). Functional vessels were detected with the fluorescent perfusion marker Hoechst 33342. The localization of Hoechst and the vascular marker 9F1 were analyzed in separate images with an automated digital image processing system. By combining the two images, total vascularity and the perfusion status of the vessels during arthritis could be established. The digital image system measures synovial area (SA), number of all blood vessels (NBV) and the number of perfused blood vessels (NpBV). From these parameters the percentage of perfused vessels (perfusion fraction; PF), the vessel density (VD = NBV/SA) and the density of perfused vessels (VDp = NpBV/SA) can be calculated. The measurements showed that the area of synovial tissue had increased during arthritis. Moreover, both the number of blood vessels (NBV) and the number of perfused vessels (NpBV) in the synovial area had increased significantly on Days 4 and 7 after arthritis induction. This procedure enabled quantitation of total vascularity and of functional blood vessels in cross-sections of synovial tissue. It is expected to be a powerful tool, not only to analyze the effects of anti-angiogenic therapies in animal models of arthritis, but could also be applicable to study vascular and perfusion changes in vascular related diseases of the skeleton.     

Sustained vessel dilation induced by increased pulsatile perfusion of porcine carotid arteries in vitro

Arterial pulse pressure (PP) increases with exertional stress and ageing, and can modify vessel diameter in smaller vessels. To test if PP must exceed a certain range to influence vessel diameter, and determine if such effects are endothelium-dependent or intrinsic to vascular viscoelasticity, eight fresh excised porcine carotid artery segments were perfused with modified Krebs–Henseleit by a servo-controlled system generating physiological arterial pressure waveforms. In a separate group of vessels (n = 10), the endothelium was mechanically removed. Vessel external diameter was measured by video edge-detection. Vessels partially preconstricted with noradrenaline were perfused at 9 mL min^–1 mean flow, at mean pressure of 90 or 120 mmHg, and zero PP. PP alone was then increased to 40, 70, or 120 mmHg at 1 Hz cycling rate for 5 min, then returned to zero and vessel diameter measured immediately thereafter. The protocol was repeated after 10–20 min stabilization. Mean vessel diameter rose proportionally with PP only once PP exceeded 40 mmHg, with maximal increases of 6–9% at a PP of 120 mmHg. Similar responses were obtained in vessels with and without a functional endothelium, at both mean pressures. Thus, when exposed to higher than normal resting PP, conduit arteries dilate owing to the stress-relaxation response of their viscoelastic wall. This mechanism of PP-mediated vascular dilatation may contribute to enhanced organ perfusion when small resistance arteries are already dilated.     

Why does menopausal hormone therapy lead to irregular uterine bleeding? Changes to endometrial blood vessels

BACKGROUND: Abnormal bleeding is common in hormone therapy (HT) users. We aimed to determine how HT alters endometrial blood vessels and stromal factors known to regulate vascular growth and integrity. METHODS: Prospective observational study of 165 post-menopausal women in Western Australia. The following were measured in endometrial biopsies: vascular density (vessels/mm(2)), total vessel area (total area enclosed by peripheral vascular immunostaining for perivascular pericytes in mm(2)), total luminal area (mm(2)) and vessel wall area (total vessel area minus luminal area), stromal expression of matrix metalloproteinases (MMP) -1, -3, -9 and -14, their tissue inhibitors (TIMPs) -1-4 and vascular endothelial growth factor (VEGF) by immunohistochemistry. RESULTS: Total vessel area was greater during bleeding compared with HT users with no bleeding (P = 0.028) or with a prior irregular bleeding (P = 0.039). Total vessel area was greater in non-HT users compared with HT users with no bleeding (P = 0.021). In HT users, vessel luminal area was greater during bleeding compared with HT users with no bleeding (P = 0.030) and vessel wall area was also increased (P = 0.025). During bleeding there was an increase in stromal TIMP-2 staining (P = 0.044). No significant changes in endometrial MMP or VEGF were seen. CONCLUSIONS: Abnormal bleeding in HT users is associated with changes in endometrial vessel size and in stromal expression of factors known to regulate vascular growth and integrity. These changes may contribute to abnormal bleeding.     

Expression of the Vascular Endothelial Growth Factor C Receptor VEGFR-3 in Lymphatic Endothelium of the Skin and in Vascular Tumors

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.     

蛋白酶腐蚀法在保留肌腱的血管铸型中的应用研究

目的　使用蛋白酶消化技术制作保留肌腱的血管铸型,拓展解剖学标本类型,丰富肌腱相关的临床解剖学研究方法。 方法　分别使用浓度为0.5%、1.0%、1.5%和2.0%的蛋白酶混合液对掌长肌进行分解实验,选出适合分解肌组织的基础浓度;以丙烯酸树脂为填充剂,灌注新鲜前臂标本的血管,制作铸型标本。填充剂凝固后,将前臂标本浸泡于起始浓度为1.0%的蛋白酶溶液,每24 h在混合液里加入35 g碱性蛋白酶粉,腐蚀5 d后获得保留肌腱的前臂血管铸型标本。 结果　通过对掌长肌组织进行分解实验发现,浓度为1.0%的蛋白酶混合液在肌腱断裂前能更充分地分解肌腹组织;通过对保留肌腱的血管铸型观察发现,其皮肤、脂肪组织和肌组织的肌腹基本消失,而骨骼、肌腱和铸型血管基本保存完好,可以清晰地观察皮肤及浅层、肌腱和肌腹丰富的血供来源、走向及其分布,清晰地观察铸型血管与皮肤及浅层、肌腱和肌腹的立体关系。 结论　以1.0%为起始浓度,每24 h加入1次起始浓度所需溶质（碱性蛋白酶）剂量的50%所配制的蛋白酶混合液,是制作保留肌腱的血管铸型的合适腐蚀液;保留肌腱的血管铸型为新的解剖学标本类型,并丰富了肌腱相关的临床解剖学研究方法。