Cryopreservation moderates the thrombogenicity of arterial allografts during storage.

INTRODUCTION: Management of vascular infections represents a major challenge in vascular surgery. The use of cryopreserved vascular allografts could be a feasible therapeutic option, but the optimal conditions for their production and use are not precisely defined. AIMS: To evaluate the effects of cryopreservation and the duration of storage on the thrombogenicity of femoral artery allografts. METHODS: In our prospective study, eleven multi-organ-donation-harvested human femoral arteries were examined at five time points during storage at -80°C: before cryopreservation as a fresh native sample and immediately, one, twelve and twenty-four weeks after the cryopreservation. Cross-sections of allografts were perfused with heparin-anticoagulated blood at shear-rates relevant to medium-sized arteries. The deposited platelets and fibrin were immunostained. The thrombogenicity of the intima, media and adventitia layers of the artery grafts was assessed quantitatively from the relative area covered by fibrin- and platelet-related fluorescent signal in the confocal micrographs. RESULTS: Regression analysis of the fibrin and platelet coverage in the course of the 24-week storage excluded the possibility for increase in the graft thrombogenicity in the course of time and supported the hypothesis for a descending trend in fibrin generation and platelet deposition on the arterial wall. The fibrin deposition in the cryopreserved samples did not exceed the level detected in any of the three layers of the native graft. However, an early (up to week 12) shift above the native sample level was observed in the platelet adhesion to the media. CONCLUSIONS: The hemostatic potential of cryopreserved arterial allografts was retained, whereas their thrombogenic potential declined during the 6-month storage. The only transient prothrombotic change was observed in the media layer, where the platelet deposition exceeded that of the fresh native grafts in the initial twelve weeks after cryopreservation, suggesting a potential clinical benefit from antiplatelet therapy in this time-window.

An ultrastructural pathologist's views on fibroblasts, modified smooth muscle cells, wound healing, stenosing arteriopathies, Kawasaki disease, Dupuytren's contracture, and the stroma of carcinomas.

It wasn't until 1960 that the dense bodies of the peripheral actin arrays of fibroblasts were finally visualized, i.e., stress fibers (SFs). Mistakenly assumed that its SFs turned the fibroblast into a unique cell situated somewhere in a continuum between it and a smooth muscle cell (SMC), it was descriptively named a "myofibroblast" (MF). Automatically, spindle cells with SFs and/or smooth muscle actin by SMA IHC-staining, became MFs, although endothelial cells, pericytes, modified SMCs (mSMC), and myoepithelial cells all contain SFs. An invisible "intermediate" cell was hypothesized to exist somewhere between SMA-negative and positive fibroblasts, and named a "proto-myofibroblast". The sub-epithelial spindle cells of normal and malignant tumors of the GI, GU, and respiratory tracts are all fibroblasts with SFs. The second erroneous myofibroblast came from a 1971 rat wound healing study and its 1974 human counterpart. Updated analysis of the papers' TEMs proved that the cells are mSMCs and not fibroblasts (AKA: MFs). The pathognomonic cells of Dupuytren's contracture are mSMCs and fibroblasts and that of the stenosing arteriopathy of Kawasaki Disease and other similar arteriopathies are mSMCs. TEM remains a powerful tool.

Principles and development of collagen-mediated tissue fusion induced by laser irradiation.

The mechanism underlying tissue fusion mediated by laser irradiation remains unclear. We clarify the mechanisms underlying laser-mediated tissue fusion using a novel model. Microscopic examinations of morphological changes within the adventitia of a bovine carotid artery and a collagen sheet prepared from bovine dermis showed collagen fibril bundle loosening and collagen fibre swelling following heating at 46 °C. An incised bovine carotid artery covered with a collagen sheet to which pressure and laser heat of 40 °C-52 °C were applied created a structure that was pressure resistant to >300 mmHg. Microscopic analyses of the irradiation site showed collagen fibril interdigitation. Hence, low-temperature laser-mediated tissue fusion causes collagen fibril bundles to loosen and swell, and crimping causes the fibres to intertwine. As the temperature declines, the loosened and swollen fibrils and fibres tighten, and collagen fibre interdigitation is completed. This technology could be applied to fuse tissues during surgery.

Relating the mechanical properties of atherosclerotic calcification to radiographic density: A nanoindentation approach.

Calcification morphology can determine atherosclerotic plaque stability and is associated with increased failures rates for endovascular interventions. Computational efforts have sought to elucidate the relationship between calcification and plaque rupture in addition to predicting tissue response during aggressive revascularisation techniques. However, calcified material properties are currently estimated and may not reflect real tissue conditions. The objective of this study is to correlate calcification mechanical properties with three radiographic density groups obtained from corresponding Computed Tomography (CT) images. Seventeen human plaques extracted from carotid (n = 10) and peripheral lower limb (n = 7) arteries were examined using micro-computed tomography (µCT), simultaneously locating the calcified deposits within their internal structure and quantifying their densities. Three radiographic density groups were defined based on the sample density distribution: (A) 130-299.99 Hounsfield Units (HU), (B) 300-449.99 HU and (C) >450 HU. Nanoindentation was employed to determine the Elastic Modulus (E) and Hardness (H) values within the three density groups. Results reveal a clear distinction between mechanical properties with respect to radiographic density groups (p < 0.0005). No significant differences exist in the density-specific behaviours observed between carotid and peripheral samples. Previously defined calcification classifications indicate an association with specific radiographic density patterns. Scanning Electron Microscopy (SEM) examination revealed that density group A regions consist of both calcified and non-calcified tissues. Further research is required to define the radiographic thresholds which identify varying degrees of tissue calcification. This study demonstrates that the mechanical properties of fully mineralised atherosclerotic calcification emulate that of bone tissues (17-25 GPa), affording computational models with accurate material parameters. STATEMENT OF SIGNIFICANCE: Global mechanical characterisation techniques disregard the heterogeneous nature of atherosclerotic lesions. Previous nanoindentation results for carotid calcifications have displayed a wide range. This study evaluates calcification properties with respect to radiographic density obtained from Micro-CT images. This is the first work to characterise calcifications from peripheral lower limb arteries using nanoindentation. Results demonstrate a strong positive correlation between radiographic density and calcification mechanical properties. Characterising calcifications using their density values provides clarity on the variation in published properties for calcified tissues. Furthermore, this study confirms the hypothesis that fully calcified plaque tissue behaviour similar to that of bone. Appropriate material parameters for calcified tissues can now be employed in computational simulations.

Murine breast cancer feed arteries are thin-walled with reduced α1A-adrenoceptor expression and attenuated sympathetic vasocontraction.

BACKGROUND: Perfusion of breast cancer tissue limits oxygen availability and metabolism but angiogenesis inhibitors have hitherto been unsuccessful for breast cancer therapy. In order to identify abnormalities and possible therapeutic targets in mature cancer arteries, we here characterize the structure and function of cancer feed arteries and corresponding control arteries from female FVB/N mice with ErbB2-induced breast cancer. METHODS: We investigated the contractile function of breast cancer feed arteries and matched control arteries by isometric myography and evaluated membrane potentials and intracellular [Ca2+] using sharp electrodes and fluorescence microscopy, respectively. Arterial wall structure is assessed by transmission light microscopy of arteries mounted in wire myographs and by evaluation of histological sections using the unbiased stereological disector technique. We determined the expression of messenger RNA by reverse transcription and quantitative polymerase chain reaction and studied receptor expression by confocal microscopy of arteries labelled with the BODIPY-tagged α1-adrenoceptor antagonist prazosin. RESULTS: Breast cancer feed arteries are thin-walled and produce lower tension than control arteries of similar diameter in response to norepinephrine, thromboxane-analog U46619, endothelin-1, and depolarization with elevated [K+]. Fewer layers of similarly-sized vascular smooth muscle cells explain the reduced media thickness of breast cancer arteries. Evidenced by lower media stress, norepinephrine-induced and thromboxane-induced tension development of breast cancer arteries is reduced more than is explained by the thinner media. Conversely, media stress during stimulation with endothelin-1 and elevated [K+] is similar between breast cancer and control arteries. Correspondingly, vascular smooth muscle cell depolarizations and intracellular Ca2+ responses are attenuated in breast cancer feed arteries during norepinephrine but not during endothelin-1 stimulation. Protein expression of α1-adrenoceptors and messenger RNA levels for α1A-adrenoceptors are lower in breast cancer arteries than control arteries. Sympathetic vasocontraction elicited by electrical field stimulation is inhibited by α1-adrenoceptor blockade and reduced in breast cancer feed arteries compared to control arteries. CONCLUSION: Thinner media and lower α1-adrenoceptor expression weaken contractions of breast cancer feed arteries in response to sympathetic activity. We propose that abnormalities in breast cancer arteries can be exploited to modify tumor perfusion and thereby either starve cancer cells or facilitate drug and oxygen delivery during chemotherapy or radiotherapy.

Mechanisms of Arterial Calcification: The Role of Matrix Vesicles.

Vascular calcification is related to vascular diseases, for example, atherosclerosis, and its comorbidities, such as diabetes and chronic kidney disease. In each condition, a distinctive histological pattern can be recognised that may influence technical choices, possible intra-operative complications, and procedure outcomes, no matter if the intervention is performed by open or endovascular means. This review considers the classification and initiating mechanisms of vascular calcification. Dystrophic and metastatic calcifications, Monckeberg's calcification, and genetic forms are firstly outlined, followed by their alleged initiation mechanisms; these include (a) ineffective macrophage efferocytosis; (b) ectopic osteogenesis driven by modified resident or circulating osteoprogenitors. As in physiological bio-mineralisation, active calcification starts with the deposition of cell derived matrix vesicles into the extracellular matrix. To substantiate this belief, an in depth ultra-structural documentation of hydroxyapatite crystal deposition on such vesicles is provided in an ex-vivo human vascular cell model. Revealing the vesicle composition and phenotype in normal and pathological vascular conditions will be essential for the development of new therapeutic strategies, in order to prevent and treat vascular calcification.

Estrogenic vascular effects are diminished by chronological aging.

The beneficial role of estrogen in the vascular system may be due, in part, through reduction of peripheral vascular resistance. The use of estrogen therapy to prevent cardiovascular disease in post-menopausal women remains contentious. This study investigated the influence of aging and the menopause on the acute vasodilatory effects of estrogen using ex vivo human and murine resistance arteries. Vessels were obtained from young (2.9 ± 0.1 months) and aged (24.2 ± 0.1 and 28.9 ± 0.3 months) female mice and pre- (42.3 ± 0.5 years) and post-menopausal (61.9 ± 0.9 years) women. Aging was associated with profound structural alterations of murine uterine arteries, including the occurrence of outward hypertrophic remodeling and increased stiffness. Endothelial and smooth muscle function were diminished in uterine (and tail) arteries from aged mice and post-menopausal women. The acute vasodilatory effects of 17ß-estradiol (non-specific estrogen receptor (ER) agonist), PPT (ERα-specific agonist) and DPN (ERß-specific agonist) on resistance arteries were attenuated by aging and the menopause. However, the impairment of estrogenic relaxation was evident after the occurrence of age-related endothelial dysfunction and diminished distensibility. The data indicate, therefore, that chronological resistance arterial aging is a prominent factor leading to weakened vasodilatory action of estrogenic compounds.

Vascular Telocytes.

Telocyte, different to fibroblast and dendritic cell, is a novel type of interstitial cell, whose key features are their smaller cell body with very long prolongations of uneven caliber, termed telopodes. The telocytes have been continuously discovered to be present in many tissues and organs. Whether telocytes exist in the blood and vascular wall is not clear. Our research group, for the first time, testified that telocytes also exist in the blood and large sized arterial and venous wall under scanning and transmission electron microscope. In static condition, blood telocytes and their prolongations usually attach on endothelial surface. We speculate that the blood telocyte maybe come from the bone marrow, because most of formed element in the blood originated from bone marrow. The telocytes within arterial wall locate in the tunica adventitia and close to outer elastic lamina. And, the telocytes in venous wall commonly situate in the subendothelial layer. The morphological features of blood and vascular telocytes are consistent with the telocytes in other organs and tissues. Their real function of telocytes in cardiovascular system preserved to be further investigated.

A fiber-progressive-engagement model to evaluate the composition, microstructure, and nonlinear pseudoelastic behavior of porcine arteries and decellularized derivatives.

The theoretical fiber-progressive-engagement model was proposed to describe the pseudoelastic behavior of an artery pre- and post-decellularization treatments. Native porcine arteries were harvested and decellularized with 0.05% trypsin for 12 h. The uniaxial tensile test data were fitted to the fiber-progressive-engagement model proposed herein. The effects of decellularization on the morphology, structural characteristics, and composition of vessel walls were studied. The experimental stress-strain curve was fitted to the model in the longitudinal and circumferential direction, which demonstrated the adequacy of the proposed model (R2>0.99). The initial and turning strains were similar in the longitudinal and circumferential directions in the aorta, suggesting the occurrence of collagen conjugation in both directions. Discrepancies in the initial and turning strain and initial and stiff modulus in both directions in the coronary artery revealed the anisotropic features of this vessel. Decellularization induced a decrease in the initial and turning strains, a slight change in the initial modulus, and a substantial decrease in the stiffness modulus. The decrease in the initial and turning strain can be attributed to the loss of waviness of collagen bundles because of the considerable decrease in elastin and glycosaminoglycan contents. This simple non-linear model can be used to determine the fiber modulus and waviness degree of vascular tissue. Based on these results, this mechanical test can be used as a screening tool for the selection of an optimized decellularization protocol for arterial tissues. STATEMENT OF SIGNIFICANCE: Decellularized vascular graft has potential in clinical application, such as coronary artery bypass surgery, peripheral artery bypass surgery or microsurgery. An ideal decellularization protocol requires balance in cell removal efficiency and extracellular matrix preserving. Both biochemical and biomechanical properties are crucial to the success of scaffold in cell seeding and animal study. A comprehensive understanding of the composition, microstructure, and mechanical behavior of the arterial wall is the key to the development of decellularized vascular grafts. For this purpose, we proposed this "Fiber-Progressive-Engagement" model to evaluate the microstructure, composition and mechanical properties of porcine coronary artery. The model provides a new perspective regarding the non-linear behavior of arterial tissue and its decellularized derivatives. It can be widely applied to different types of tissues, as demonstrated in the aorta and coronary artery. This model has several advantages; it provides an improved fit of non-linear curves (R2>0.99), can be used to elucidate the pseudoelastic properties of porcine vascular tissues using the concept of fiber engagement, and can estimate an elastic modulus with greater accuracy (compared to the graphical estimation or calculation by simple linear fittings), as well as to plot typical stress-strain curves.

Hyperhomocysteinemia induced by excessive methionine intake promotes rupture of cerebral aneurysms in ovariectomized rats.

BACKGROUND: Hyperhomocysteinemia (HHcy) is associated with inflammation and a rise in the expression of matrix metalloproteinase-9 (MMP-9) in the vascular wall. However, the role of HHcy in the growth and rupture of cerebral aneurysms remains unclear. METHODS: Thirteen-week-old female Sprague-Dawley rats were subject to bilateral ovariectomy and ligation of the right common carotid artery and fed an 8 % high-salt diet to induce cerebral aneurysms. Two weeks later, they underwent ligation of the bilateral posterior renal arteries. They were divided into two groups and methionine (MET) was or was not added to their drinking water. In another set of experiments, the role of folic acid (FA) against cerebral aneurysms was assessed. RESULTS: During a 12-week observation period, subarachnoid hemorrhage due to aneurysm rupture was observed at the anterior communicating artery (AcomA) or the posterior half of the circle of Willis. HHcy induced by excessive MET intake significantly increased the incidence of ruptured aneurysms at 6-8 weeks. At the AcomA of rats treated with MET, we observed the promotion of aneurysmal growth and infiltration by M1 macrophages. Furthermore, the mRNA level of MMP-9, the ratio of MMP-9 to the tissue inhibitor of metalloproteinase-2, and the level of interleukin-6 were higher in these rats. Treatment with FA abolished the effect of MET, suggesting that the inflammatory response and vascular degradation at the AcomA is attributable to HHcy due to excessive MET intake. CONCLUSIONS: We first demonstrate that in hypertensive ovariectomized rats, HHcy induced by excessive MET intake may be associated with the propensity of the aneurysm wall to rupture.

Dimensions of compartments and membrane surfaces in the intact rabbit heart of importance in studies on intramyocardial transfer of blood-borne substances.

Cardiac studies on the uptake, storage and intramyocardial transfer of blood-borne substances require detailed information on the geometric ultrastructural dimensions of myocardial compartments and parts thereof, and the membranes separating these compartments. Such a specific ultrastructural set of data of the heart is yet lacking. In the present study, we quantitatively assessed these dimensions in glutaraldehyde-perfusion fixed rabbit hearts by means of histological and tailored mathematical techniques. We showed the true ellipsoid nature of the myocardial capillary cross section and estimated the mean capillary diameter dcap. After correction for the ellipsoid shape, dcap was found to be 5.21±1.41 µm. Effective widths of the endothelial cell and the pericapillary interstitium (is1), dimensions of importance in diffusion, amounted to 187±7 and 160±10 nm, respectively. The fractional volume of the large vessels (arteries and veins larger than 10 µm), capillaries, endothelium, is1, cardiomyocytes, non-pericapillary interstitium is2, t-tubular compartment and interstitial cells amounted on average to 5.92%, 9.36%, 1.83%, 1.94%, 73.07%, 5.97%, 0.95% and 0.96%, respectively, of total myocardial volume, defined as the cardiac tissue volume, the large blood vessels included. Normalized to total myocardial volume, the surface area of the luminal and abluminal endothelial membranes and of the cardiomyocyte membrane opposing the endothelial cells amounted to 75.2±5.5·10³, 82.2±6.0·10³ and 89.1±6.5·10³ m²/m³, respectively. The present study provides quantitative information about ultrastructural dimensions of the adult rabbit heart, among others, of importance for studies on cardiac uptake, and intramyocardial transfer and storage of blood-supplied substances.

Distribution and innervation of putative arterial chemoreceptors in the bullfrog (Rana catesbeiana).

Peripheral arterial chemoreceptors have been located previously in the carotid labyrinth, the aortic arch, and the pulmocutaneous artery of frogs. In the present study we used cholera toxin B neuronal tract tracing and immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH), and serotonin (5HT) to identify putative O2-sensing cells in Rana catesbeiana. We found potential O2-sensing cells in all three vascular areas innervated by branches of the vagus nerve, whereas only cells in the carotid labyrinth were innervated by the glossopharyngeal nerve. Cells containing either 5HT or TH were found in all three sites, whereas cells containing both neurotransmitters were found only in the carotid labyrinth. Cell bodies containing VAChT were not found at any site. The morphology and innervation of putative O2-sensing cells were similar to those of glomus cells found in other vertebrates. The presence of 5HT- and TH-immunoreactive cells in the aorta, pulmocutaneous artery, and carotid labyrinth appears to reflect a phylogenetic transition between the major neurotransmitter seen in the putative O2-sensing cells of fish (5HT) and those found in the glomus cells of mammals (acetylcholine, adenosine, and catecholamines).

The performance of cross-linked acellular arterial scaffolds as vascular grafts; pre-clinical testing in direct and isolation loop circulatory models.

There is a significant need for small diameter vascular grafts to be used in peripheral vascular surgery; however autologous grafts are not always available, synthetic grafts perform poorly and allografts and xenografts degenerate, dilate and calcify after implantation. We hypothesized that chemical stabilization of acellular xenogenic arteries would generate off-the-shelf grafts resistant to thrombosis, dilatation and calcification. To test this hypothesis, we decellularized porcine renal arteries, stabilized elastin with penta-galloyl glucose and collagen with carbodiimide/activated heparin and implanted them as transposition grafts in the abdominal aorta of rats as direct implants and separately as indirect, isolation-loop implants. All implants resulted in high patency and animal survival rates, ubiquitous encapsulation within a vascularized collagenous capsule, and exhibited lack of lumen thrombogenicity and no graft wall calcification. Peri-anastomotic neo-intimal tissue overgrowth was a normal occurrence in direct implants; however this reaction was circumvented in indirect implants. Notably, implantation of non-treated control scaffolds exhibited marked graft dilatation and elastin degeneration; however PGG significantly reduced elastin degradation and prevented aneurismal dilatation of vascular grafts. Overall these results point to the outstanding potential of crosslinked arterial scaffolds as small diameter vascular grafts.

[Vascular morphological and microdensity changes of corneal neovascularization induced by topical bevacizumab and sunitinib in an animal model]. / Cambios en la morfología y la microdensidad neovascular corneal inducidos tras la administración tópica de bevacizumab y sunitinib en un modelo animal.

OBJECTIVE: To evaluate the effects of topical bevacizumab and topical sunitinib on vascular microdensity and morphology of corneal neovascularization (NV). METHODS: A total of 33 rabbits were distributed into 3 groups: group 1 (control; n=11): saline; group 2 (n=11): bevacizumab 5mg/ml; and group 3 (n=11): sunitinib 0.5mg/ml. A corneal NV model was used, based on sutures in the right eye of each rabbit. Each treatment was administered topically 3 times daily for 14 days. Corneas were then processed for the study of vascular microdensity (6 eyes) and vascular morphology analysis (5 eyes) using enzymatic staining histological techniques RESULTS: The vascular response in group 3 was limited to small-sized tree formations with various vascular axes compared with the extensive, lush and directional corneal NV of group 1 and 2. In the histological sections near the limb, there were no differences in vascular microdensity studies between the three groups. However, the mean sectional area of vessels (MSAV) in group 3 was 41.88% lower than in group 1 and 19.19% lower than in group 2. In distal sections, there were no differences between groups 1 and 2. However, group 3 was characterized by absence of vessels. CONCLUSIONS: Bevacizumab produced no changes in the morphology of the vessels or the vascular microdensity. Sunitinib reduced the size of the new vessels and induced changes in the vascular tree.

The outer arterial wall layers are primarily affected in spontaneous cervical artery dissection.

OBJECTIVE: To evaluate the macroscopic and microscopic phenotype of the distal superficial temporal artery (STA) in patients with spontaneous cervical artery dissection (sCAD, n = 14). Arteries of accident victims, free of clinically apparent vascular disease, served as reference samples (n = 9). METHODS: Specimens of distal STA branches were obtained by biopsy or at autopsy. Their fine and ultrafine structure was documented by close-up photography of native STA branches, light microscopy, and electron microscopy in a case-control study. RESULTS: STA specimens from patients with sCAD revealed pathologic changes mainly in the adventitial and medial layers. In these areas, vacuolar degeneration and fissuring were associated with neoangiogenesis of capillaries and microscopic erythrocyte extravasation into the connective tissue. In addition, some specimens showed overt microhematomas close to the medial/adventitial border visible at low magnification. The reference arteries showed virtually no pathologic changes in the outer arterial layers. CONCLUSION: Bearing in mind that the STA is only a surrogate for the cervical arteries affected by sCAD, we propose the following pathogenetic model. We hypothesize that sCAD affects primarily the outer arterial layers. The process starts with degenerative changes at the medial-adventitial border associated with neoangiogenesis of capillary vessels branching from vasa vasorum in the adventitia. Leakage of neoangiogenetic capillaries releases blood cells into the connective tissue and leads to formation of microhematomas along the medial/adventitial border, as well as disintegration of the medial and adventitial texture. Microhematomas might then cause successive rupture of multiple neoangiogenetic capillaries and vasa vasorum, ultimately resulting in dissection.

Angioarchitecture of the bovine tunica albuginea vascular complex--a corrosive and histological study.

Histological and corrosive studies of 75 bovine testes were focused on the vascular complex of the tunica albuginea. Thanks to used MultiScanBase v.14.02 software arterial and venous vessels and also vascular anastomoses were analyzed preciously. The studies revealed the superficial layer containing vessels of smaller diameter and reduced wall structure and the deeper layer with wider, winding vessels and complete wall structure. The branches of the testicular artery and intra-tunical veins formed a vascular complex of the tunica albuginea. The intra-tunical veins were divided into two types. Type I veins ran parallel, drained intra-parenchymal veins and formed the origin of pampiniform plexus. Type II veins drained into type I intra-tunical veins. Indirect anastomoses among intra-tunical veins and arteries and venovenous connections were observed. The results of the study could contribute fuller description of the regulatory mechanism for testicular blood supply and optimization of the testicular biopsy to minimize bleeding risk.

Endocrine organs and laser scanning confocal microscopy (LSCM) imaging: vascular bed in human spleen.

OBJECTIVE: This work was aimed to utilize the precise method of laser confocal microscopy (LSCM) to depict the image of spatial relationships of the vessel network in the tissue structures of the human spleen. METHODS: With the use of serial paraffin or vibratome sections of more than 20 µm thickness infiltrated with eosin fluorescence dye the images of arterial and venous walls of different calibres, capillaries, and venous sinuses were morphologically revealed. RESULTS: Venous sinuses were frequently found to create mutually communicating branches and their lining projected into the lumen protruding cells with distinct spherically or ovally shaped nuclei, positioned on the brightly fluorescent and fragmented lamina basalis. The presence of lymphocytes was distinct in periarteriolar lymphoid sheath (PALS) and lymphatic follicles. Lining cells of the red pulp veins sporadically contained marked eosinophilic granules. CONCLUSION: The method of LSCM allowed: 1. to reveal two-dimensional and sharp image of the human spleen structures, 2. to investigate the vertical course of venous structures in the tissue, 3. to obtain serial optic sections in z axis to their maximum spatial projections. These data will also serve for the creation of three-dimensional images of vessel network in the human spleen in the future studies.

The digital venous drainage in the human embryonic hand.

The histogenesis and morphology of the digital venous drainage in human embryonic and fetal hands, aged from 6 to 12 weeks, were studied by light microscopy in 18 fingers. In the sixth week, capillaries could be identified around the cartilaginous models of the phalanges. By the ninth week, the neurovascular bundles were identifiable in the palmar part of the finger. In 12 week fetuses, all of the superficial and deep vascular venous system could be seen easily in the palmar aspect of the finger in positions similar to those in the adult hand. However, the arch systems, present on the dorsum of the finger in the adult hand, were not yet differentiated.

The development of a tissue-engineered artery using decellularized scaffold and autologous ovine mesenchymal stem cells.

Alternatives to using native arteries in vascular surgery are urgently needed. Vessels made from synthetic polymers have shortcomings such as thrombosis, rejection, intimal hyperplasia, calcification, infection, chronic inflammation and no growth potential. Tissue-engineered blood vessels (TEBV) may overcome these problems. We developed a tissue-engineered artery using autologous bone marrow derived mesenchymal stem cells (MSCs) and a decellularized arterial scaffold. Vascular smooth muscle cell (SMCs)-like cells and endothelial cell (ECs)-like cells were differentiated from MSCs in vitro. We constructed TEBV by seeding these autologous cells onto decellularized ovine carotid arteries and interposed into the carotid arteries in an ovine host models. The scaffold retained the main structural components of a blood vessel, such as collagen and elastin. The TEBVs were patent, anti-thrombogenic, and mechanically stable for 5 months in vivo, whereas non-seeded grafts occluded within 2 weeks. Histological, immunohistochemical, and electron microscopic analyses of the TEBVs demonstrated the existence of endothelium, smooth muscle and the presence of collagen and elastin both at 2 and 5 months, respectively. MSCs labeled with a fluorescent dye prior to implantation were detected in the harvested TE artery 2 months after implantation, indicating that the MSCs survived and contributed to the vascular tissue regeneration. Therefore, TEBVs can be assembled from autologous MSCs and decellularized bioscaffold.

Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening.

BACKGROUND: A number of adhesion-mediated signaling pathways and cell-cycle events have been identified that regulate cell proliferation, yet studies to date have been unable to determine which of these pathways control mitogenesis in response to physiologically relevant changes in tissue elasticity. In this report, we use hydrogel-based substrata matched to biological tissue stiffness to investigate the effects of matrix elasticity on the cell cycle. RESULTS: We find that physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial cells and vascular smooth muscle cells; subcellular analysis in these cells, mouse embryonic fibroblasts, and osteoblasts shows that cell-cycle control by matrix stiffness is widely conserved. Remarkably, most mitogenic events previously documented as extracellular matrix (ECM)/integrin-dependent proceed normally when matrix stiffness is altered in the range that controls mitogenesis. These include ERK activity, immediate-early gene expression, and cdk inhibitor expression. In contrast, FAK-dependent Rac activation, Rac-dependent cyclin D1 gene induction, and cyclin D1-dependent Rb phosphorylation are strongly inhibited at physiological tissue stiffness and rescued when the matrix is stiffened in vitro. Importantly, the combined use of atomic force microscopy and fluorescence imaging in mice shows that comparable increases in tissue stiffness occur at sites of cell proliferation in vivo. CONCLUSIONS: Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1.