Search

Due to an increasing number of cardiovascular diseases, artificial heart valves and blood vessels have been developed. Although cardiovascular applications using decellularized tissue have been studied, the mechanisms of their functionality remain unknown. To determine the important factors for preparing decellularized cardiovascular prostheses that show good in vivo performance, the effects of the luminal surface structure of the decellularized aorta on thrombus formation and cell behavior were investigated. Various luminal surface structures of a decellularized aorta were prepared by heating, drying, and peeling. The luminal surface structure and collagen denaturation were evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining, and scanning electron microscopy (SEM) analysis. To evaluate the effects of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting tests and recellularization of endothelial cells and smooth muscle cells were performed. The results of the blood clotting test showed that the closer the luminal surface structure is to the native aorta, the higher the anti-coagulant property. The results of the cell seeding test suggest that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation, and functional expression accordingly. These results provide important factors for preparing decellularized cardiovascular prostheses and will lead to future developments in decellularized cardiovascular applications., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: The field of regenerative medicine has developed promising techniques to improve current neobladder strategies used for radical cystectomies or congenital anomalies. Scaffolds made from molecularly defined biomaterials are instrumental in the regeneration of tissues, but are generally confined to small flat patches and do not comprise the whole organ. We have developed a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold, mimicking the shape of the whole bladder, and with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized, with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. Human and porcine bladder urothelial and smooth muscle cells were able to attach to the scaffold and maintained their phenotype in vitro. The closed luminal side and the porous outside of the scaffold facilitated the formation of an urothelial lining and infiltration of smooth muscle cells, respectively. The cells aligned according to the provided scaffold template. The technology used is highly adjustable (shape, size, materials) and may be used as a starting point for research to an off-the-shelf medical device suitable for neobladders., Statement of Significance: In this study, we describe the development of a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold mimicking the shape of the whole bladder with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. The closed luminal surface and the porous exterior of the scaffold facilitated the formation of a urothelial lining and infiltration of smooth muscle cells, respectively. The applied technology is highly adjustable (shape, size, materials) and can be the starting point for research to an off-the-shelf medical device suitable for neobladders., (Copyright © 2016. Published by Elsevier Ltd.)

Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor-BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers α-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-α (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of α-SMA and SM22-α, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure.

Ureteral replacement by tissue engineering might become necessary following tissue loss after excessive ureteral trauma, after retroperitoneal cancer, or even after failed reconstructive surgery. This need has driven innovation in the design of novel scaffolds and specific cell culture techniques for urinary tract reconstruction. In this study, compressed tubular collagen scaffolds were evaluated, addressing the physical and biological characterization of acellular and cellular collagen tubes in a new flow bioreactor system, imitating the physiological pressure, peristalsis, and flow conditions of the human ureter. Collagen tubes, containing primary human smooth muscle and urothelial cells, were evaluated regarding their change in gene and protein expression under dynamic culture conditions. A maximum intraluminal pressure of 22.43 ± 0.2 cm H2O was observed in acellular tubes, resulting in a mean wall shear stress of 4 dynes/cm(2) in the tubular constructs. Dynamic conditions directed the differentiation of both cell types into their mature forms. This was confirmed by their gene expression of smooth muscle alpha-actin, smoothelin, collagen type I and III, elastin, laminin type 1 and 5, cytokeratin 8, and uroplakin 2. In addition, smooth muscle cell alignment predominantly perpendicular to the flow direction was observed, comparable to the cell orientation in native ureteral tissue. These results revealed that coculturing human smooth muscle and urothelial cells in compressed collagen tubes under human ureteral flow-mimicking conditions could lead to cell-engineered biomaterials that might ultimately be translated into clinical applications.

Purpose: To compare human urothelial and smooth muscle cells attachment and proliferation using three different matrices; poly lactic-co-glycolic acid (PLGA), PLGA/collagen and human amniotic membrane (hAM)., Materials and Methods: Human urothelial and smooth muscle cells were cultured and examined for expression of urothelium (pancytokeratin and uroplakin III) and smooth muscle cells [desmin and alpha smooth muscle actin (α-SMA)] markers. Cells were cultured on three scaffolds; PLGA, PLGA/collagen and hAM. Thereafter, they were analyzed for cell growth on days 1, 3, 7, 14 and 21 after seeding by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Scaffolds were fixed and processed for hematoxylin and eosin (H&E) staining and immunohistochemistry against their cell specific markers after 7 and 14 days of culture., Results: MTT assay results revealed that collagen has improved cell attachment features of PLGA and led to significant increase of MTT signal in PLGA/collagen compared to PLGA (P < .001) and hAM (P < .001). hAM was a weaker matrix for both cell types as demonstrated in MTT assay and scanning electron microscope (SEM) images. SEM micrographs showed normal phenotype and distribution on PLGA and PLGA/collagen. In the same line, cells formed a well-developed layer either on PLGA or PLGA/collagen, which maintained expression of their corresponding markers., Conclusion: Our findings demonstrated significant improvement of cell attachment and growth achieved by collagen coating (PLGA/collagen) compared to PLGA and hAM. hAM despite of its natural entity was a weaker matrix for bladder engineering purposes.

Endothelial cells (ECs) are aligned longitudinally under laminar flow, whereas they are polygonal and poorly aligned in regions of disturbed flow. The unaligned ECs in disturbed flow fields manifest altered function and reduced survival that promote lesion formation. We demonstrate that the alignment of the ECs may directly influence their biology, independent of fluid flow. We developed aligned nanofibrillar collagen scaffolds that mimic the structure of collagen bundles in blood vessels, and examined the effects of these materials on EC alignment, function, and in vivo survival. ECs cultured on 30-nm diameter aligned fibrils re-organized their F-actin along the nanofibril direction, and were 50% less adhesive for monocytes than the ECs grown on randomly oriented fibrils. After EC transplantation into both subcutaneous tissue and the ischemic hindlimb, EC viability was enhanced when ECs were cultured and implanted on aligned nanofibrillar scaffolds, in contrast to non-patterned scaffolds. ECs derived from human induced pluripotent stem cells and cultured on aligned scaffolds also persisted for over 28 days, as assessed by bioluminescence imaging, when implanted in ischemic tissue. By contrast, ECs implanted on scaffolds without nanopatterning generated no detectable bioluminescent signal by day 4 in either normal or ischemic tissues. We demonstrate that 30-nm aligned nanofibrillar collagen scaffolds guide cellular organization, modulate endothelial inflammatory response, and enhance cell survival after implantation in normal and ischemic tissues., (Published by Elsevier Ltd.)

Bioengineered tissue equivalents should provide physiologically relevant biochemical and mechanical cues to support the growth and differentiation of seeded cells. Herein, tubular dense collagen constructs (TDCCs) with collagen content comparable to native extracellular matrix were used to investigate the effect of shear stress alone (i.e. under laminar fluid flow), and shear stress in combination with circumferential strain (i.e. under pulsatile fluid flow) on the proliferation, alignment, and phenotype of three-dimensionally (3D) seeded airway smooth muscle cells (ASMCs). In addition, the effect of ASMC-mediated remodelling on TDCC matrix morphological and mechanical properties was investigated. Compared to static culture, pulsatile flow increased seeded ASMC growth by 70%, improved the homogeneity of cell distribution within the TDCCs and induced differential cellular alignment depending on the primary stimuli. Specifically, within the inner wall, where shear stress is predominant, ASMCs were aligned parallel to fluid flow direction, while within the outer wall ASMCs were aligned parallel to the circumferential strain (perpendicular to fluid flow). In contrast, under laminar flow, ASMCs were aligned parallel to fluid flow direction within both walls. Compared to laminar flow, pulsatile flow resulted in increased positive staining for α-smooth muscle actin, and in up-regulated typical ASMC contractile markers suggesting that circumferential strain modulates ASMC differentiation. Pulsatile flow also caused a 60 and 30% increase in collagen density within both acellular and cellular TDCCs, respectively, which was reflected in an increased apparent modulus. Compared to static culture, pulsatile stimulation of cellular constructs resulted in 70% higher circumferential strength. The TDCCs provide ASMC niche for greater insight into the responses of 3D seeded SMCs to physiologically equivalent in vitro dynamic conditioning., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

There is an essential demand for tissue-engineered autologous small-diameter vascular grafts, which offer temporary supports and guides for vascular tissue organization, repair and remodeling. This study reports on the effect of collagen/smooth muscle cells (SMCs) mixtures under dynamic cultures and SMC-endothelial cell (ECs) co-culture on cell proliferation, uniform cell distribution, extracellular matrix deposition, and endothelial cells monolayer formation in tissue-engineered tubular arterial constructs of 4 mm inner diameter. Rabbit aortic SMCs were infiltrated with collagen solution in poly(L-lactide-co-ϵ-caprolactone) (PLCL) scaffolds under vacuum to form collagenous gel and subjected to dynamic strain by culturing them in a dynamic perfusion bioreactor. The construct lumen was subsequently seeded with ECs and experiments were completed to create ECs-SMCs co-culture constructs. The collagen/SMCs incorporated elastic scaffold cultured under dynamic culture conditions promoted matrix deposition, leading to the development of tissue-engineered vascular constructs, and induced SMC to have more uniform cell distribution. Scanning electron microscopic examination and von Willebrand Factor staining demonstrated the presence of ECs spread over the lumen. Quantitative analysis of elastin contents demonstrated that the engineered vessels acquired similar elastin contents as native arteries. The collagen/SMCs/ECs incorporated PLCL scaffolds under dynamic culture conditions can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.

Two major requirements for a tissue-engineered vessel are the establishment of a continuous endothelium and adequate mechanical properties. In this study, a novel tubular collagen scaffold possessing nanopatterns in the form of channels (with a 650 nm periodicity) on both sides was designed and examined after seeding and co-culturing with vascular cells. Initially, the exterior of the tube was seeded with human vascular smooth muscle cells (VSMCs), cultured for 14 days, and then human internal thoracic artery endothelial cells (HITAECs) were seeded on the inside of the tube and cultured for a further week. Microscopy revealed that nano-scale patterns could be reproduced on collagen with high fidelity and preserved during incubation in vitro. The VSMCs were circumferentially orientated with the help of these nanopatterns and formed multilayers on the exterior, while HITAECs formed a continuous layer on the interior, as is the case in natural vessels. Both cell types were observed to proliferate and retain their phenotypes in the co-culture., (Copyright © 2010 John Wiley & Sons, Ltd.)

Matrix remodeling plays a fundamental role in physiological and pathological processes, as well as in tissue engineering applications. In this paper, optical coherence tomography (OCT), a non-destructive optical imaging technology, was used to image collagen gel remodeling by smooth muscle cells (SMCs). The optical scattering properties of collagen-SMC gels were characterized quantitatively by fitting OCT data to a theoretical model. Matrix remodeling over 5 days produced a 10-fold increase in the reflectivity of the collagen gels, corresponding to a decrease in scattering anisotropy from 0.91 to 0.46. The increase in reflectivity was corroborated in confocal mosaic images. Blocking matrix degradation in collagen-SMC gels with doxycycline, a non-specific matrix metalloproteinases (MMPs) inhibitor, impeded the decrease in scattering anisotropy and resulted in few macroscopic signs of remodeling. Causing matrix degradation in acellular gels with a 3 h treatment of MMP-8 (collagenase 2) partially mimicked the decrease in anisotropy measured in collagen-SMC gels after 5 days. These results suggest that the decrease in scattering anisotropy in the collagen-SMC gels was due to MMP activity that degrades collagen fibrils into smaller fragments., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo-tissue level. This controlled, cell-independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell-seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation.

Biodegradable tubular scaffolds have been developed for vascular graft application. This study was focused to improve the adhesion and proliferation of vascular smooth muscle cells (SMCs) in a tubular scaffold. Tubular scaffolds (ID 4 mm, OD 6 mm) were fabricated from a biodegradable elastic polymer, poly(L-lactide-co-epsilon-caprolactone) (PLCL) (50:50, M(n) 1.58 x 10(5)), by an extrusion/particulate leaching method. SMCs suspended in a collagen solution were infiltrated in tubular PLCL scaffolds under vacuum and incubated for 1 h at 37 degrees C to form a collagenous gel. Results from SEM image analysis showed that collagen was infiltrated into the inside of the scaffolds. Cell adhesion and proliferation rate increased in collagen/SMC-incorporated tubular PLCL scaffolds as compared with the scaffolds in which only SMCs were seeded. From SEM image and histological analysis, we further found that SMCs grew on the inside as well as on the surface of collagen/SMCs-incorporated scaffolds and the cells continued to grow as a monolayer on collagen fibers. In particular, cell proliferation and elastin contents were the highest in a PLCL scaffold with 50-100 microm pore size than any other scaffolds used in this experiment. A collagen/SMC-incorporated PLCL scaffold may support SMC growth and functions and can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation., ((c) Koninklijke Brill NV, Leiden, 2009)

Elastin synthesis and physiologic compliance are significant challenges in blood vessel tissue engineering. Here, we report that a biocompatible elastomeric scaffold can support the co-expression of elastin and collagen, which likely yielded the physiologic compliance in the constructs. A biodegradable elastomer, poly(glycerol sebacate), was fabricated into highly porous tubular scaffolds. Primary baboon arterial smooth muscle cells (SMCs) were seeded in the lumen of the scaffolds followed by a 1-week culture under gentle perfusion. Circulating endothelial progenitor cells (EPCs) isolated from baboon peripheral blood was seeded directly on the smooth muscle layer in the lumen on day 8. The constructs were perfused using a pulsatile flow system for another 2 weeks before characterization. In another set of experiments, the SMCs were cultured for 7 weeks and were co-cultured for 1 week with the EPCs. Constructs obtained using either set of culture conditions contained elastin and collagen: Masson's trichrome stain showed a circumferential collagen band in the constructs, and elastin was evident from its characteristic autofluorescence, Verhoff's stain, and amino acid analysis of insoluble remnants after hot alkali digestion. All constructs had a confluent cellular lumen with cells well-dispersed throughout the scaffolds. At physiologic pressures, the compliance of the 8-week construct was comparable to human arteries as observed in pressure-diameter testing. Combination of elastomeric scaffolds, co-culture of EPC and SMC, and mechanical conditioning appears to encourage the expression of a more natural extracellular matrix and lead to physiologically-relevant compliance; both are major challenges in blood vessel tissue engineering.

Microvascular remodeling is known to depend on cellular interactions with matrix tissue. However, it is difficult to study the role of specific cells or matrix elements in an in vivo setting. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local collagen matrix remodeling by single smooth muscle cells. We combined a motorized microscopic setup and time-lapse video microscopy with a new cross-correlation based image analysis algorithm to enable automated recording of cell-induced matrix reorganization. This method rendered 60-90 single cell studies per experiment, for which collagen deformation over time could be automatically derived. Thus, the current setup offers a tool to systematically study different components active in matrix remodeling.

Our objective in this work was to develop a flexible, biodegradable scaffold for cell transplantation that would incorporate a synthetic component for strength and flexibility and type I collagen for enzymatic lability and cytocompatibility. A biodegradable poly(ester urethane)urea was synthesized from poly(caprolactone), 1,4-diisocyanatobutane, and putrescine. Using a thermally induced phase separation process, porous scaffolds were created from a mixture containing this polyurethane and 0%, 10%, 20%, or 30% type I collagen. The resulting scaffolds were found to have open, interconnected pores (from 7 to >100 um) and porosities from 58% to 86% depending on the polyurethane/collagen ratio. The scaffolds were also flexible with breaking strains of 82-443% and tensile strengths of 0.97-4.11 MPa depending on preparation conditions. Scaffold degradation was significantly increased when collagenase was introduced into an incubating buffer in a manner that was dependent on the mass fraction of collagen present in the scaffold. Mass losses could be varied from 15% to 59% over 8 weeks. When culturing umbilical artery smooth muscle cells on these scaffolds higher cell numbers were observed over a 4-week culture period in scaffolds containing collagen. In summary, a strong and flexible scaffold system has been developed that can degrade by both hydrolysis and collagenase degradation pathways, as well as support cell growth. This scaffold possesses properties that would make it attractive for future use in soft tissue applications where such mechanical and biological features would be advantageous.

Biodegradable polycaprolactone and collagen nanofibers were produced by electrospinning, with fiber diameters of around 300-700nm and features similar to the extracellular matrix of natural tissue. Human coronary artery smooth muscle cells (SMCs) seeded on nanofibrous matrices tend to maintain normal phenotypic shape and growth tends to be guided by the nanofiber orientation. The SMC and nanofibrous matrix interaction was observed by SEM, MTS assay, trypan blue exclusion method and laser scanning confocal microscopy. The results showed that the proliferation and growth rate of SMCs were not different on polycaprolactone (PCL) nanofibrous matrices coated with collagen or tissue culture plates. PCL nanofibrous matrices coated with collagen showed that the SMCs migrated towards inside the nanofibrous matrices and formed smooth muscle tissue. This approach may be useful for engineering a variety of tissues in various structures and shapes, and also to demonstrate the importance of matching both the initial mechanical properties and degradation rate of nanofibrous matrices to the specific tissue engineering.

During pathologic vessel remodeling, vascular smooth muscle cells (VSMCs) embedded within the collagen-rich matrix of the artery wall mobilize uncharacterized proteolytic systems to infiltrate the subendothelial space and generate neointimal lesions. Although the VSMC-derived serine proteinases, plasminogen activator and plasminogen, the cysteine proteinases, cathepsins L, S, and K, and the matrix metalloproteinases MMP-2 and MMP-9 have each been linked to pathologic matrix-remodeling states in vitro and in vivo, the role that these or other proteinases play in allowing VSMCs to negotiate the three-dimensional (3-D) cross-linked extracellular matrix of the arterial wall remains undefined. Herein, we demonstrate that VSMCs proteolytically remodel and invade collagenous barriers independently of plasmin, cathepsins L, S, or K, MMP-2, or MMP-9. Instead, we identify the membrane-anchored matrix metalloproteinase, MT1-MMP, as the key pericellular collagenolysin that controls the ability of VSMCs to degrade and infiltrate 3-D barriers of interstitial collagen, including the arterial wall. Furthermore, genetic deletion of the proteinase affords mice with a protected status against neointimal hyperplasia and lumen narrowing in vivo. These studies suggest that therapeutic interventions designed to target MT1-MMP could prove beneficial in a range of human vascular disease states associated with the destructive remodeling of the vessel wall extracellular matrix.

Carbon nanotubes (CNT) are attractive for use in fiber-reinforced composite materials due to their very high aspect ratio, combined with outstanding mechanical and electrical properties. Composite materials comprising a collagen matrix with embedded CNT were prepared by mixing solubilized Type I collagen with solutions of carboxylated single-walled carbon nanotubes (SWNT) at concentrations of 0, 0.2, 0.4, 0.8, and 2.0 weight percent. Living smooth muscle cells were incorporated at the time of collagen gelation to produce cell-seeded collagen-CNT composite matrices. Constructs containing 2.0 wt % CNT exhibited delayed gel compaction, relative to lower concentrations that compacted at the same rate as pure collagen controls. Cell viability in all constructs was consistently above 85% at both Day 3 and Day 7, whereas cell number in CNT-containing constructs was lower than in control constructs at Day 3, though statistically unchanged by Day 7. Scanning electron microscopy showed physical interactions between CNT and collagen matrix. Raman spectroscopy confirmed the presence of CNT at the expected diameter (0.85-1.30 nm), but did not indicate strong molecular interactions between the collagen and CNT components. Such collagen-CNT composite matrices may have utility as scaffolds in tissue engineering, or as components of biosensors or other medical devices., (Copyright (c) 2005 Wiley Periodicals, Inc.)

Background: The resistance to stretch and the elasticity of the vein wall depend on the collagen and elastic fibers, respectively. Contradicting evidence exists, however, on the connective tissue concentration in varicose veins., Methods: A prospective, comparative study was conducted at Asir Central Hospital and the College of Medicine in Abha, Saudi Arabia. Twenty-three vein specimens collected from both the proximal thigh long saphenous vein (LSV) and the distal calf blowouts in 10 primary varicose vein patients and from the normal, proximal thigh LSV in 3 young vascular trauma patients were examined. Paraffin sections stained with hematoxylin and eosin, Verhoeff von Gieson (VVG) and Masson's Trichrome stains were examined under the light microscope. Ultra thin sections were examined under the transmission electron microscope (TEM)., Results: Compared with the normal control LSV, varicose vein sections showed increased diameter of the lumen and hypertrophy of the wall, mainly of the intima, due to increased amounts of collagen fibers. This marked fibrous infiltration disrupted the normal palisade arrangement of the intimal and the regular sheet-like arrangement of the medial smooth muscle cells. Collagen fibers also lost their normal pattern and showed abnormal forms. Elastic fibers lost their regular laminar arrangement and formed clumps or scattered fragments., Conclusions: Varicose veins showed increased collagenosis and distortion of the elastic fibers. The presence of abnormal collagen to elastin ratio and the loss of the regular collagen/elastic lattice of the vein wall may play a major role in the pathogenesis of varicose veins.