Long-term engraftment of bone marrow-derived cells in the intimal hyperplasia lesion of autologous vein grafts

Intimal hyperplasia of autologous vein grafts is a critical problem affecting the long-term patency of many types of vascular reconstruction. Within intimal hyperplasia lesions, smooth muscle cells are a major component, playing an essential role in the pathological process. Given that bone marrow-derived cells may differentiate into smooth muscle cells in the neointima of injured arteries, we hypothesized that the bone marrow may serve as a source for some of the smooth muscle cells within intimal hyperplasia lesions of vein grafts. To test this hypothesis, we used an established mouse model for intimal hyperplasia in wild-type mice that had been transplanted with bone marrow from a green fluorescent protein (GFP+/+) transgenic mouse. High-resolution confocal microscopy analysis performed 2 and 8 weeks after grafting demonstrated expression of GFP in 5.4 +/- 0.8% and 11.9 +/- 2.3%, respectively, of smooth muscle cells within intimal hyperplasia lesions. By 16 weeks, GFP expression in smooth muscle cells was not detected by immunohistochemistry; however, real-time PCR revealed that 20.2 +/- 1.7% of the smooth muscle cells captured from the neointima lesion by laser capture microdissection at 16 weeks contained GFP DNA. Our results suggest that bone marrow-derived cells differentiated into smooth muscle cells within the intimal lesion and may provide a novel clinical approach for decreasing intimal hyperplasia in vein grafts.     

In vitro and in vivo studies of ePTFE vascular grafts treated with P15 peptide

The purpose of this study is to evaluate the effectiveness of P15 cell-binding peptide treated ePTFE vascular grafts in vitro and in vivo. The P15 peptide was covalently immobilized onto ePTFE vascular grafts by an atmospheric plasma coating method. In vitro cell growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). X-ray photoelectron spectroscopy and amino-acid analysis were used to analyze the surface characteristics of the peptide treated and untreated grafts. The cell growth study showed that the P15 peptide effectively promoted the adhesion and proliferation of endothelial cells. 700% more endothelial cells were proliferated on the P15-treated ePTFE grafts compared to the untreated ePTFE controls. In contrast, the P15 peptide was significantly less effective for promoting the adhesion and proliferation of smooth muscle cells than endothelial cells; only about 100% more smooth muscle cells proliferated on the P15-treated samples compared to the untreated control samples. The sheep model was used in the in vivo study. The amount of neointimal hyperplasia present at the arterial and venous sides of the anastomosis and the degree of endothelialization on the luminal surface of the grafts were assessed. Four P15-treated grafts and two control grafts were implanted as arteriovenous grafts between the femoral artery and vein or the carotid artery and jugular vein in two sheep (n = 6). The in vivo study showed that the thickness of the neointimal hyperplasia of untreated grafts was 3-times thicker than that of P15-treated grafts (P < 0.05) at the venous side of the anastomosis. P15-treated grafts also had a higher degree of endothelialization on the graft lumen.     

In vitro and in vivo studies of ePTFE vascular grafts treated with P15 peptide

The purpose of this study is to evaluate the effectiveness of P15 cell-binding peptide treated ePTFE vascular grafts in vitro and in vivo. The P15 peptide was covalently immobilized onto ePTFE vascular grafts by an atmospheric plasma coating method. In vitro cell growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). X-ray photoelectron spectroscopy and aminoacid analysis were used to analyze the surface characteristics of the peptide treated and untreated grafts. The cell growth study showed that the P15 peptide effectively promoted the adhesion and proliferation of endothelial cells. 700% more endothelial cells were proliferated on the P15-treated ePTFE grafts compared to the untreated ePTFE controls. In contrast, the P15 peptide was significantly less effective for promoting the adhesion and proliferation of smooth muscle cells than endothelial cells; only about 100% more smooth muscle cells proliferated on the P15-treated samples compared to the untreated control samples. The sheep model was used in the in vivo study. The amount of neointimal hyperplasia present at the arterial and venous sides of the anastomosis and the degree of endothelialization on the luminal surface of the grafts were assessed. Four P15-treated grafts and two control grafts were implanted as arteriovenous grafts between the femoral artery and vein or the carotid artery and jugular vein in two sheep (n = 6). The in vivo study showed that the thickness of the neointimal hyperplasia of untreated grafts was 3-times thicker than that of P15-treated grafts (P < 0.05) at the venous side of the anastomosis. P15-treated grafts also had a higher degree of endothelialization on the graft lumen.     

自体移植兔静脉细胞增殖与钙激活钾通道的变化

目的: 研究静脉移植后钙激活钾通道(K_Ca)的变化,并探讨其病理生理意义。方法: 将兔的双侧股静脉倒置移植于同侧股动脉缺损之间,采用离体血管灌流的方法,测定移植静脉环的张力。分别以图像分析和四唑盐(MTT)比色法检测移植静脉内膜增厚的程度以及K_Ca的阻断剂盐酸四乙胺(tetraethylammonium chloride, TEA)对培养的家兔股静脉血管平滑肌细胞(vascular smooth muscle cells, VSMCs)增殖的影响。进而采用膜片钳技术记录K_Ca电流。 结果: 移植后1 week, 静脉的收缩性和内膜相对厚度没有显著变化。静脉移植后2 weeks,收缩性和内膜相对厚度显著大于对照组(P＜0.05, n=8)。移植后4 weeks静脉的收缩性和内膜相对厚度进一步大于对照组(P＜0.01, n=8)。TEA(2-8 mmol/L)显著增加培养VSMCs的MTT吸光值,并且有剂量依赖性(P＜0.05, n=8)。膜片钳实验结果显示,指令电位在(30-60)mV时,移植(1-4) weeks静脉VSMCs的K_Ca 电流密度均显著低于对照组(P＜0.05, n=5),指令电位在20 mV时,只有移植4 weeks静脉VSMCs的K_Ca电流密度显著低于对照组(P＜0.05, n=5)。结论: 自体移植静脉VSMC的K_Ca受到抑制,可能是血管收缩性增强、VSMCs异常增殖的原因,从而导致自体移植静脉痉挛和狭窄。     

Time course of the regression of intimal hyperplasia in experimental vein grafts.

A previous study in which vein grafts were removed from the arterial circulation and reimplanted into the venous circulation of the same animal demonstrated regression of vein graft intimal hyperplasia and medial thickening within 14 days. The present study was designed to characterize the kinetics of the morphological and ultrastructural changes over this 14-day period. Twenty-one male New Zealand White rabbits received a reversed vein interposition bypass graft of the right common carotid artery. Fourteen days after the procedure, 21 vein grafts were isolated, removed, and reimplanted into the contralateral external jugular venous system as veno-venous interposition bypass grafts (reversal grafts). The grafts were harvested at 60 minutes, 1 day, 3 days, 5 days, 7 days, and 14 days after reversal. Before insertion into the venous circulation, the vein graft had a confluent endothelial cell surface with multiple layers of smooth muscle cells representing intimal hyperplasia. After 1 hour, the reversal graft retained an intact endothelial cell layer with no evidence of tissue edema or cellular disruption. By 24 hours, there were a few blood cells on the endothelial cell surface. There was no inflammatory infiltrate seen in the subendothelium, and the smooth muscle cells were unaltered. At 3 days, the endothelial cell lining remained intact with no polymorphonucleocytes in the subendothelium or within the graft wall. Underlying smooth muscle cells at this time were noted to contain cytoplasmic vacuoles. At 5 days, there were no inflammatory cells seen on the surface or within the vein graft wall, but many of the underlying smooth muscle cells within the intimal hyperplasia were noted to be fragmented and to have clumping of chromatin. After 7 days, the endothelial cells remained intact and there was widespread evidence of apoptosis beneath the subendothelium with highly fragmented smooth muscle cells, some of which were histologically in the process of breaking up. At 14 days, the grafts retained uniform endothelial cell surfaces. Most of the smooth muscle cells that composed the intimal hyperplasia seen before implantation as a reversal graft were gone. Areas of newly laid down collagen could be observed. There were no acute inflammatory cells but for some mast cells seen in the graft wall. This study demonstrates that in this model, regression of intimal hyperplasia was associated with apoptosis of the smooth muscle cells and the deposition of collagen. There was no evidence that this process is mediated by an acute inflammatory response. Regression therefore appears to be due to induction of smooth muscle cell apoptosis by either a reduction in pressure or flow or a combination of both factors. The findings will enable a systematic cellular and molecular analysis of the biology of regression, which may afford clues to better understand the biology of the developing intimal hyperplasia.     

CD4+ mononuclear cells induce cytokine expression, vascular smooth muscle cell proliferation, and arterial occlusion after endothelial injury.

Studies of T cell-deficient or immunosuppressed animals undergoing arterial endothelial denudation have yielded conflicting results as to the contribution of the immune system to neointimal vascular smooth muscle cell accumulation and proliferation. We investigated the cell types and cytokine expression associated with intimal hyperplasia occurring 14 days after balloon angioplasty of the carotid artery in Sprague-Dawley rats. Immunohistological studies using monoclonal antibodies showed that the carotid luminal occlusion observed was associated with smooth muscle cell proliferation and neointimal accumulation of large numbers of CD4+, ED1+ mononuclear cells but no T cells. There was also wide-spread staining for the inflammatory cytokine interleukin-1B (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and IL-8, as well as dense expression of the potent smooth muscle mitogens platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), and protein S. The relationship of smooth muscle cell proliferation to monocyte/macrophage accumulation and cytokine expression was tested by daily intraperitoneal administration for 14 days of a rat CD4-specific monoclonal antibody, BWH-4 (500 micrograms/day). Morphometric analysis at day 14 showed that the intimal area of animals treated with CD4 monoclonal antibody comprised 7% +/- 4% of the arterial wall compared with 50% +/- 6% in control animals (n = 6/group, P < 0.001). In addition, immunohistological studies showed that CD4 monoclonal antibody treatment markedly reduced the intimal accumulation of mononuclear and smooth muscle cells and essentially abrogated expression of the cytokines PDGF-BB, TGF-beta, IL-1 beta, TNF-alpha, and IL-8, plus the anticoagulant molecule, protein S. Our results document the extensive expression in vivo of cytokines that in vitro promote vascular smooth muscle cell proliferation, and suggest that CD4+ mononuclear cells or their secreted products play a key role in the pathogenesis of intimal hyperplasia after endothelial injury. Furthermore, these observations may have clinical relevance in the development of novel strategies to prevent arteriosclerosis.     

Adaptive remodelling of smooth muscle in the neo-intima of vein-to-artery grafts in rats: a detailed morphometric analysis

Summary End-to-end autogenous vein-to-artery grafts in rats have been used extensively as a model for neointimal thickening (hyperplasia), which develops over the first 6 weeks after grafting. This study employed computerised morphometric techniques to analyse 16 grafts, in order to quantitate precisely how the neointima develops. Two important features were described that have not been identified previously, due to the extensive variation in neo-intimal thickness inherent in vein grafts. Firstly, the proximal region of the graft was significantly thicker than the distal region, up until 6 months after grafting. The smooth muscle cells in the graft may have developed more rapidly in the proximal region, due to the altered haemodynamics within the graft. Secondly, within the central region of the graft the characteristic focal nature of neo-intimal hyperplasia was evident throughout the period of the study, but by 6 months the neo-intima tended to be distrubuted more evenly. By 6 months remodelling of smooth muscle throughout the graft neo-intima had occurred, and the neo-intima had matured to a thickness equivalent to that of the intima plus media of the adjacent iliac artery.     

Vascular tissue adaptations in end-to-end autologous arterial grafts in rats: a morphometric analysis

Autologous vein grafts are employed extensively to bypass stenoses in the arterial circulation. More recently arterial segments have been used for such bypass surgery. In this study the adaptation of regenerating vascular tissues in experimental autologous artery grafts (4 mm long and 1 mm in diameter) in 20 adult male Wistar rats was analysed. At 1, 2, 4, 8 and 16 wk after insertion, 4 grafts per time interval were removed, processed for high resolution light microscopy and the thicknesses of the media and neointima, as well as the area fractions of smooth muscle cells, were analysed morphometrically. All grafts were reendothelialised by 2 wk. Neointimal hyperplasia (a subendothelial layer of smooth muscle cells) developed in all grafts and reached its maximal thickness (40.4±4.7 μm) at 2 wk. The area fraction of smooth muscle cells in the neointima of the artery grafts did not change significantly at any time from 2 to 16 wk. The media underlying the neointima of the artery grafts remained relatively constant throughout the 16 wk duration of the experiment. Whilst the total wall thickness of the grafts reduced significantly between 2 and 4 wk after insertion, at all times the grafts were thicker than the host artery.     

Mouse Model of Venous Bypass Graft Arteriosclerosis

Saphenous vein grafts are widely used for treatment of severe atherosclerosis via aortocoronary bypass surgery, a procedure often complicated by later occlusion of the graft vessel. Because the molecular mechanisms of this process remain largely unknown, quantitative models of venous bypass graft arteriosclerosis in transgenic mice could be useful to study this process at the genetic level. We describe herein a new model of vein grafts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Autologous or isogeneic vessels of the external jugular or vena cava veins were end-to-end grafted into carotid arteries of C57BL/6J mice. Vessel wall thickening was observed as early as 1 week after surgery and progressed to 4-, 10-, 15-, and 18-fold original thickness in grafted veins at age 2, 4, 8, and 16 weeks, respectively. The lumen of grafted veins was significantly narrowed because of neointima hyperplasia. Histological and immunohistochemical analyses revealed three lesion processes: marked loss of smooth muscle cells in vein segments 1 and 2 weeks after grafting, massive infiltration of mononuclear cells (CD11b/18⁺) in the vessel wall between 2 and 4 weeks, and a significant proliferation of vascular smooth muscle cells (α-actin⁺) to constitute neointimal lesions between 4 and 16 weeks. Similar vein graft lesions were obtained when external jugular veins or vena cava were isografted into carotid arteries of C57BL/6J mice. Moreover, no significant intima hyperplasia in vein-to-vein isografts was found, although there was leukocyte infiltration in the vessel wall. Thus, this model, which reproduces many of the features of human vein graft arteriosclerosis, should prove useful for our understanding of the mechanism of vein graft disease and to evaluate the effects of drugs and gene therapy on vascular diseases.     

Molecular mechanisms in intimal hyperplasia

Intimal hyperplasia is the process by which the cell population increases within the innermost layer of the arterial wall, such as occurs physiologically during closure of the ductus arteriosus and during involution of the uterus. It also occurs pathologically in pulmonary hypertension, atherosclerosis, after angioplasty, in transplanted organs, and in vein grafts. The underlying causes of intimal hyperplasia are migration and proliferation of vascular smooth muscle cells provoked by injury, inflammation, and stretch. This review discusses, at a molecular level, both the final common pathways leading to smooth muscle migration and proliferation and their (patho)-physiological triggers. It emphasizes the key roles played by growth factors and extracellular matrix-degrading metalloproteinases, which act in concert to remodel the extracellular matrix and permit cell migration and proliferation.     

T cell differentiation in athymic nude rats (rnu/rnu): demonstration of a distorted T cell subset structure by flow cytometry analysis

The expression of T cell-associated antigens was analyzed on extrathymically differentiated T cells from athymic nude (rnu/rnu) rats. Two-color flow cytometry and monoclonal antibodies were used to compare the subset structure of the rnu/rnu T cell population with that of normal T cells. In adult nude rats CD4+ and CD8+ cells were found which co-expressed the OX19 antigen (CD5) clearly defining them as T cells. Double-positive (CD4+CD8+) cells were not detected in nylon wool-enriched rnu/rnu T cells. The ratio of CD4+ to CD8+ cells in nude rat lymph node cells did not significantly differ from normal T cells. However, the composition of the nude rat CD4+ and CD8+ subsets [as identified by the subset-dividing monoclonal antibodies OX22 and P4/16 (anti-RT6)] differed from that found in control rats. The most striking observation among the rnu/rnu CD4+ subset was an inversion in the ratio of OX22+ to OX22- cells (0.5) compared to normal rats (2.3). Only 61% of the nude rat CD4+ cells were found to be RT6+ compared to 85% of rnu/+ CD4+ cells. In the rnu/rnu CD8 subset the proportion of RT6 co-expressing cells was also markedly reduced (38% vs. 77% of the CD8+ subset). Furthermore, the nude rat CD8+ subset contained a substantial number of OX22- cells which were nearly absent in normal rats. The demonstration of cells bearing T cell markers in adult athymic rats further confirms the existence of an extrathymic pathway of T cell differentiation. The unusual T cell subset composition found in athymic rats, however, indicates that T cell subset generation and/or maintenance along this pathway differs from normal T cell development.     

阿托伐他汀对大鼠自体移植静脉内膜增生的影响

目的： 探讨新型降脂药阿托伐他汀对自体移植静脉内膜增生的影响。 方法： 将Wistar大鼠颈外静脉移植于腹主动脉,建立大鼠自体静脉移植模型,实验分为3组：假手术组、移植对照组和移植实验组。自术后第1 d起,对移植实验组大鼠经胃管灌注给予阿托伐他汀(5 mg·kg-1·d-1)处理。干预4周后取移植静脉组织标本,制备4 μm厚组织切片,行病理组织学观察分析移植静脉内膜增生情况,行免疫组化染色分析新生内膜细胞SMα-actin和PCNA的表达情况。 结果： 移植对照组和实验组移植静脉内皮下层SMα-actin染色阳性平滑肌细胞大量增生,导致静脉内膜显著增厚,血管管腔明显狭窄。新生内膜定量分析显示移植实验组移植静脉内膜增生受到明显抑制,其新生内膜面积及新生内膜/中膜面积比均显著低于对照组(P<0.01);并且实验组移植静脉新生内膜细胞PCNA标记指数显著低于对照组(P<0.01)。 结论： 阿托伐他汀通过抑制新生内膜平滑肌细胞的增殖能有效抑制自体移植静脉内膜增生的发生发展,在防治血管重建术后再狭窄方面显示出良好的应用前景。     

Rapid development of vein graft atheroma in ApoE-deficient mice

Several animal models manifesting lesions resembling neointimal hyperplasia of human vein grafts have been developed, but no spontaneous atheromatous lesions in their vein grafts have been observed. We developed and here characterize a new animal model of vein graft atheroma, a maturated atherosclerotic plaque, in apoE-deficient mice. The lesion displayed classical complex morphological features and heterogeneous cellular compositions and consisted of a fibrous cap, infiltrated mononuclear cells, foam cells, cholesterol crystal structure, necrotic core with calcification, and neovasculature. Cell component analysis revealed smooth muscle cells (SMCs) localized in the cap region, macrophages which made up a large portion of the lesions, and CD4+ T cells scattered under the cap. Importantly, apoptotic/necrotic cells determined by TUNEL assay in vein grafts into apoE-/- mice were significantly higher than wild-type mice, although a similar number of proliferating cell nuclear antigen-positive cells in both types of lesions was found. Interestingly, vascular SMCs cultivated from aortas of apoE-deficient mice showed a high rate of spontaneous apoptosis/necrosis and a higher rate of cell death stimulated by a nitric oxide donor, sodium nitroprusside, H(2)O(2), and oxidized low density lipoprotein (LDL), although no difference in proliferation of both SMCs incubated with platelet-derived growth factor, angiotensin II, LDL, and oxidized LDL was seen. Thus, the pathogenic mechanisms of vein graft atheroma involve increased intimal cell death initiated by biomechanical stress and amplified by hypercholesterolemia, which leads to continuous recruitment of blood mononuclear cells to constitute atheromatous lesions. This mouse model resembling human vein graft disease has many advantages over other animal models.     

Foam cell replication and smooth muscle cell apoptosis in human saphenous vein grafts

Occlusion of saphenous vein grafts is a major problem after coronary artery bypass grafting. Segments of occluded and suboccluded implanted aortocoronary grafts were obtained during re-intervention bypass grafting in 47 patients yielding a total of 80 vein grafts. The grafts were studied by immunohistochemistry for smooth muscle cells (ÉL-SMC actin), macrophages (HAM56), cell replication (PCNA, Ki-67) and transmission and scanning electronmicroscopy (TEM, SEM). In 81% of the examined grafts the (sub)occlusion was due to a myo-intimal thickening and an associated luminal accumulation of foam cells and mural thrombi. The foam cells were constantly found at the luminal site of the myo-intimal thickening and within the luminal part of adherent thrombi. Transmission electronmicroscopy demonstrated phagocytosis of platelets and platelet fragments by the foam cells. A significant fraction of the foam cells demonstrated nuclear immunoreactivity for Ki-67 and PCNA. The myo-intimal thickening of the vein grafts was composed of smooth muscle cells lying in a fibrous tissue matrix. The smooth muscle cells were surrounded by prominent basal lamina and showed ultrastructural features of apoptosis. Our results support the hypothesis that phagocytosis of lipid rich platelets by monocytes set up a mechanism for foam cell formation and replication in human saphenous vein grafts. The transformation of a smooth muscle cell rich myo-intimal thickening towards a fibrous, cell poor intimal thickening could be induced by progressive smooth muscle cell loss through apoptosis.     

Transplantation of Cultured Thymic Fragments in Congenitally Athymic and Euthymic Rats Culture with Deoxyguanosine or Cyclosporin A does not influence the Histologic Characteristics and Outcome after Transplantation in Syngeneic and Allogeneic Combinations

Cultured thymic fragments (CTF) from WAG/CPB (RT1u) and DA/01a (RT1a) rats were prepared in the presence or absence of 2'deoxyguanosine or cyclosporin A, and subsequently transplanted under the kidney capsule of congenitally athymic and euthymic WAG/CPB recipients. The rationale of the culture supplements was that these may affect the disappearance of medullary dendritic cells, with subsequent induction of allotolerance. However, the immunohistology of the CTF showed more RT1 class II-positive cells than keratin-positive cells, indicative of the maintenance of dendritic cells. Grafts in athymic animals showed the recovery of the original thymic architecture within 6 weeks after transplantation. The influx of host-derived lymphocytes was accompanied by an influx of dendritic cells in the medulla-like area and macrophages in the cortex. A similar recovery was observed for syngeneic CTF in euthymic recipients. In addition lymphocytic infiltration was seen in the connective tissue surrounding the epithelial areas. Allogeneic grafts in euthymic animals were rejected within 3 weeks after transplantation. This outcome of the transplanted CTF under different conditions was not affected by the supplementation of the thymic culture before transplantation with 2'deoxyguanosine or cyclosporin A. We conclude that there is no tolerance induction after transplantation in euthymic allogeneic rats of CTF prepared in the presence of 2'deoxyguanosine. This conclusion is in contrast to data in the mouse, which may be explained by the maintenance of dendritic cells during culture. A chimaeric state of donor-derived epithelium and host-derived dendritic cells is obtained by transplantation of allografts in athymic rats.     

Balloon catheter de-endothelialization of the nude rat carotid. Response to injury in the absence of functional T lymphocytes.

The development of an intimal proliferative lesion after balloon catheter de-endothelialization was studied in congenitally athymic nude rats lacking T lymphocytes. Significant intimal thickening was observed in both the homozygous (nu/nu) and euthymic heterozygous (nu/+) animals 6 days after injury, which increased further after 10 days. There was no significant difference in mean intimal:medial cross-sectional area between the nu/nu and nu/+ animals at either time. Approximately 1% of the cells in the neointima of both groups of animals were leukocytes (OX-1 positive); 0.7% were macrophages (ED-1 positive). In neither nu/nu nor nu/+ animals did T lymphocytes (OX-19-positive cells) constitute more than 0.1% of the neointimal cell population. These data suggest that T lymphocytes do not play a significant role in the accumulation of neointimal cells. The presence of macrophages within the lesions raises the possibility that they may be involved in the recruitment and proliferation of smooth muscle cells. In vitro characterization of nu/nu carotid medial smooth muscle cells demonstrated approximately 500,000 binding sites for platelet-derived growth factor (PDGF)-BB and few PDGF-AA binding sites (less than 10,000). The mitogenic and chemotactic responses of these cells to the three dimeric forms of PDGF correlated with this receptor subunit distribution. Platelet-derived growth factor accounted for approximately 50% of the mitogenic activity of a rat platelet releasate. Platelet-derived growth factor-BB and PDGF-AB were both potent chemotactic agents for the nude rat carotid smooth muscle cells with a peak response at approximately 10 ng/ml. In contrast, PDGF-AA, transforming growth factor beta, and basic fibroblast growth factor were only weak chemoattractants for these cells.     

原位静脉动脉化与静脉动脉间置后静脉的实验形态学研究

目的：探讨原位静脉动脉化与静脉动脉间置后，静脉的组织学和超微结构变化。在１８只犬后肢设计原位大隐静脉动脉化，静脉动脉间置实验模型，对原位大隐静脉动脉化和静脉动脉间置后不同时间（２、４、８、１６ｗ）静脉管壁的变化，进行了实验形态学观察。结果：①原位静脉动脉化后早期内皮细胞损伤较轻微，中、晚期主要表现为管腔内皮细胞损伤较轻微，管腔的扩张和中膜平滑肌的增生，肥厚；②静脉动脉间置早期内皮细胞有广泛脱落，中膜平滑肌细胞肿胀，细胞间积液，中晚期变化主要为内皮不规则增生，肥厚，中膜平滑肌不同程度的增生与纤维化，使血管腔呈现不同程度的狭窄。结论：原位静脉动脉化后静脉呈现结构上的“动脉化”倾向，并且较静脉动脉间置更有利于保持血管的通畅。     

Tissue engineered vascularized bone formation using in vivo implanted osteoblast-polyglycolic acid scaffold

Repair of skeletal defects with vascularized bone grafts has many advantages over non-vascularized free grafts, but the availability of these grafts is extremely limited. This study was designed to determine whether new vascularized bone could be engineered by transplantation of osteoblasts around existing vascular pedicles using biodegradable, synthetic polymer as a cell delivery vehicle. Cells were isolated from the periosteum of fetal bovine humerus, and then seeded onto non-woven multifilament, polyglycolic acid polymer. The polymers provided three dimensional support during in vitro culture. The cell-polymer constructs were maintained in vitro for two weeks and then implanted around the right femoral vessels of twelve athymic nude rats. The polymer templates without the cells were implanted around left femoral vessels of each mouse as a control. Twelve rats were sacrificed at the following intervals: three rats at six,and nine rats at nine weeks. New bone formation was evident in 10 out of the 12 periosteal-derived cell seeded implants. At six weeks, the tissue was primarily composed of what appeared both grossly and histologically to be cartilage enveloping small islands of osteoid. The degree of osteoid and bone formation progressed with time, as blood vessels invaded the tissue. This tissue ultimately underwent morphogenesis to become an organized trabeculated bone with a vascular pedicle. We believe that this technique may prove to be useful in the reconstruction of bony defect.     

细胞在小口径组织工程血管中的抗钙化作用

背景：小口径组织工程血管的远期结果研究极少,尚未见到研究组织工程血管分子水平、离子水平远期结果和平滑肌细胞与钙化关系的报道。 目的：利用脱细胞猪股动脉基质作为支架和犬血管壁细胞作为种子细胞体外构建小口径组织工程血管,植入种子细胞供体犬股动脉部位6个月,观察植入物中层平滑肌细胞与钙化的关系。 方法：12只实验犬被随机分为支架组(n=6)和再细胞化组(n=6),自体股动脉被作为对照组;支架组犬接受猪股动脉经脱细胞后的基质支架植入双侧股动脉,再细胞化组犬接受受体血管壁细胞共同培养、联合种植于脱细胞的猪股动脉基质并体外预适应后植入血管壁细胞供体双侧股动脉位置;6个月后测定植入物和对照组股动脉组织钙含量、平滑肌密度和病理学变化。 结果与结论：小口径组织工程血管植入后6个月检查见2组植入物无明显狭窄和扩张,扫描电镜示内表面均已完全内皮化,有管壁僵硬和局部钙化斑块形成,以上改变以支架组植入物更明显。支架组管道组织钙含量显著高于再细胞化组和自体股动脉(P < 0.01),再细胞化组植入物组织钙含量亦显著高于自体股动脉(P < 0.01);病理学检查示再细胞化组植入物平滑肌密度高于支架组(P < 0.01),再细胞化组和支架组植入物平滑肌密度均低于对照组(P < 0.01);超声检查见2组管道植入术后即刻与6个月后舒缩幅度较邻近自体股动脉舒缩幅度小,有部分管道无舒缩功能。结果提示,猪股动脉常规脱细胞方去获得的基质作为支架体外构建组织工程血管时,平滑肌细胞难于迁移至支架中层,中层平滑肌密度低,植入体内6个月后中层平滑肌细胞密度仍低,平滑肌细胞有抗血管钙化作用。