Decellularization of human umbilical arteries / Descelularización de las arterias umbilicales humanas

SUMMARY: Arterial obstruction in small diameter (<6 mm) vessels are many times treated with grafts, however autologous aren't always available and synthetic have a high rate of complications. Decellularization of umbilical arteries may provide a solution, but the ideal method is debatable. We compare effectiveness between SDS and Triton X-100. Umbilical cords obtained from full term pregnancies with normal development and no evident complications in the newborn, were micro-dissected within 12 h and stored in phosphate buffered saline without freezing. Arteries were then processed for decellularization using 0.1 % and 1 % SDS, and 1 % Triton X100 protocols. Evaluation of cellular and nuclear material, collagen fibers, elastic fibers, and glycosoaminoglycans of the extracellular matrix (ECM) were evaluated as well as morphometric analysis under histological and immunohistochemical techniques. Triton X-100 was ineffective, preserving nuclear remains identified by immunofluorescence, had the most notable damage to elastic fibers, and decrease in collagen. SDS effectively eliminated the nuclei and had a less decrease in elastic fibers and collagen. Laminin was preserved in all groups. No significant differences were identified in luminal diameters; however the middle layer decreased due to decellularization of muscle cells. In conclusion, 0.1 % SDS decellularization was the most effective in eliminating cells and preserving the main components of the ECM.

RESUMEN: La obstrucción arterial en vasos de pequeño diámetro (<6 mm) se trata muchas veces con injertos, sin embargo, los autólogos no siempre están disponibles y los sintéticos tienen una alta tasa de complicaciones. La descelularización de las arterias umbilicales puede proporcionar una solución, pero el método ideal es discutible. Comparamos la efectividad entre los métodos SDS y Triton X-100. Cordones umbilicales obtenidos a partir de embarazos a término con evolución normal y sin complicaciones evidentes del recién nacido, se microdiseccionaron en 12 horas y se almacenaron en solución salina con fosfato sin congelación. Las arterias se procesaron luego para la descelularización usando los protocolos de SDS al 0,1 % y 1 %, y Triton X-100 al 1 %. Se realizó la evaluación de material celular y nuclear, fibras de colágeno, fibras elásticas y glucosoaminoglicanos de la matriz extracelular (MEC), así como el análisis morfométrico bajo técnicas histológicas e inmunohistoquímicas. Triton X-100 fue ineficaz, conservando los restos nucleares identificados por inmunofluorescencia, tuvo el daño más notable a las fibras elásticas y la disminución del colágeno. SDS efectivamente eliminó los núcleos y tuvo una disminución menor en las fibras elásticas y el colágeno. Laminina fue preservado en todos los grupos. No se identificaron diferencias significativas en los diámetros luminales; sin embargo, la capa media disminuyó debido a la descelularización de las células musculares. la descelularización con SDS al 0,1 % fue la más efectiva para eliminar células y preservar los principales componentes de la MEC.

Development of HLA-matched vascular grafts utilizing decellularized human umbilical artery.

Worldwide, there is a great need of small diameter vascular grafts that can be used in human disorders such as cardiovascular and peripheral vascular disease. Until now, severe adverse reactions are caused from the use of synthetic or animal derived grafts, while the use of autologous vessels is restricted only in a small number of patients. The limited availability of the vessels might be resolved by the use of HLA-matched vascular grafts utilizing the decellularized human umbilical arteries. In this study, human umbilical arteries were decellularized and then repopulated with Mesenchymal Stem Cells. The HLA-genotype of the repopulated grafts, analyzed by Next Generation Sequencing technology, indicated their successful production. The HLA-matched vascular grafts could be generated efficiently and might be used in personalized medicine.

Stem cells cardiac patch from decellularized umbilical artery improved heart function after myocardium infarction.

The construction of the high biocompatible biomaterials pretreated with MSC offers a promising strategy to improve the effects of stem cell therapy for the myocardial infarction (MI). However, assembling vascularized three-dimensional (3-D) myocardial tissues remains an enormous challenge. In this study, we optimized the decellularization protocol with the umbilical artery to construct microporous 3-D scaffold which is suitable for the stem cells (SC) proliferation. The SD rats underwent proximal left coronary ligation and a 5-mm diameter microporous SC patch was implanted directly on the infarct area (SC patch group). The LV contractile function, regional myocardial wall compliance, and tissue histology were assessed 4 weeks after patch implantation. The MSC patch integrated to the local heart tissue and the neo-vessels have been observed in the MSC patch. The vessels in the MSC patch were positive for the CD31 (marker for the mature endothelial cells). The left ventricle wall was thicker in the MSC patch group than the control group (p<0.05 vs. empty patch group). And the LVEF has been improved in the MSC patch group than empty patch group (59±6.7% vs. 31±4.5%, p<0.05). CONCLUSIONS: Our results showed that the implantation of the MSC patch improved cardiac contractile function in heart infarction rat model. The construction of artificial tissue from the decellularized umbilical artery and the MSC may open a promising perspective for the tissue therapy for MI.

Utilizing an Umbilical Ligament for Complex Ureteroneocystostomy.

INTRODUCTION: To overcome the tension of anastomosis between ureter and bladder for complex ureteroneocystostomy in children, we describe a novel technique utilizing an ipsilateral umbilical ligament (occluded umbilical artery) to fix the bladder. TECHNICAL CONSIDERATIONS: From July 1991 to December 2013, 18 patients (13 girls, 5 boys) underwent our technique for complex ureteroneocystostomy. Median age at surgery was 61 months. The main indications for surgery were ectopic ureter in 9 patients, primary obstructive megaureter in 3, and persistent vesicoureteral reflux after surgery in 2. The ipsilateral umbilical ligament was ligated. A new hiatus was created craniolateral to the original hiatus. The submucosal tunnel was created trans-trigonally. The umbilical ligament was sutured to the whole bladder muscle at the hiatus. Ureteroneocystostomy was then performed. Mean duration of postoperative follow-up was 62.6 months. Seventeen patients underwent unilateral ureteroneocystostomy with our technique. One patient underwent bilateral ureteroneocystostomy with our technique on one side. Vesicoureteral reflux was not shown in 92.3% of patients and no signs of obstruction developed in any patients. No complications were encountered with our procedure. CONCLUSIONS: Utilizing an umbilical ligament for ureteroneocystostomy to fix the bladder is an excellent option when the distal ureter creates tension in uretero-bladder anastomosis. This method seems to be applicable in many cases of complex ureteroneocystostomy in children.

Development of decellularized human umbilical arteries as small-diameter vascular grafts.

OBJECTIVE: Developing a tissue-engineered small-diameter (<6mm) vascular graft for reconstructive surgery has remained a challenge for the past several decades. This study was conducted to develop a decellularized umbilical artery and to evaluate its composition, endothelial cell compatibility, mechanical properties, and in vivo stability for potential use as a small-diameter vascular graft. METHODS AND RESULTS: Human umbilical arteries were isolated and decellularized by incubation in CHAPS and sodium dodecyl sulfate buffers followed by incubation in endothelial growth media-2. Decellularized umbilical arteries were completely devoid of cellular and nuclear material while retaining the integrity of extracellular collagenous matrix. The mechanical strength of the decellularized umbilical artery as assessed by its burst pressure in vitro showed no significant change from its native form. Decellularized umbilical arteries supported endothelial adherence as indicated by the re-endotheliazation with a monolayer of human umbilical vein endothelial cells. Furthermore, decellularized vessels that were implanted into nude rats as abdominal aorta interposition grafts remained mechanically intact and patent for up to 8 weeks. CONCLUSION: Decellularized human umbilical arteries preserved the extracellular matrix, supported endothelialization, and retained function in vivo for up to 8 weeks. These properties suggest the potential use of decellularized umbilical arteries as small-diameter vascular grafts.

[Biomechanical properties of human umbilical vessels at different gestational ages and their clinical significance].

To provide theoretical data on biomechanical properties for the clinical application of human umbilical artery and umbilical vein. The calculation of the compliance of fetal umbilical artery and umbilical vein was made on the basis of measuring the pressure-volume relationship of the umbilical artery and umbilical vein collected from 50 normal fetuses and newborns of abortion and normal labor whose gestational ages ranged from 24 weeks to 42 weeks. It was found that the P-V relationship of umbilical artery and umbilical vein corresponded to parabolic curve and the compliance increased gradually with the growth of the gestational age, but when the gestational age was beyond 39 weeks the compliance began to decrease gradually. The compliance of umbilical vein is twice or triple as large as that of umbilical artery. Human umbilical artery and umbilical vein are good substitute materials for arterial grafts. In transplantation, two factors should be considered, first, the correspondence between the compliance of umbilical vessels and that of host artery; second, the relationship between the compliance and gestational age. We suggest that it be better to use selectively the umbilical artery and umbilical vein with a gestational age from 37 weeks to 40 weeks as the transplanting material.

Use of autologous umbilical artery and vein for vascular reconstruction in the newborn.

Autologous umbilical artery and vein were evaluated as vascular conduits in newborn lambs. Eight newborn lambs were delivered transabdominally under sterile conditions at term. The umbilical artery and vein were dissected from the cord and stored in culture media. On the same day, each lamb underwent bilateral superficial femoral artery transection and reconstruction. Nine arteries were reconstructed with autologous umbilical vein interposition grafts, five with umbilical artery interposition grafts, and two by primary native artery anastomosis. After the birth weight of the lambs quadrupled (37 to 45 days), they were killed and all grafts and anastomoses were examined grossly and histologically. At the conclusion of the study, both native artery anastomoses (2/2) were patent. Five umbilical vein (5/9) and two umbilical artery (2/5) autografts were also widely patent. Patent autografts retained an intact endothelium supported by a viable media. The nonpatent autografts had become atrophic remnants displaying histologic signs of early closure. Graft failures are attributed to the extreme vasoactive nature of the umbilical vessels. These preliminary results suggest that umbilical vessels may be useful as a vascular autograft if the vasoactive nature of these vessels can be overcome during the immediate perioperative period.

Morphological studies of cellular responses to experimental arterial grafts.

Cross-carotid microvascular bypass grafts 2-3 mm in diameter were implanted using microsurgical techniques for end-to-end anastomosis in four dogs. One autograft control and one of three denatured human umbilical artery xenografts (HUAG) were patent at 5 weeks. One of the other two denatured HUAGs had thrombosed at 1 week, and the other was occluded at 5 weeks. Host and graft vessel specimens were evaluated histologically as well as with transmission electron microscopy after sacrifice. Results indicate that failure of reconstitution of a true endothelial layer, presence of a subintimal myofibroblast population, increased collagen deposition of the muscularis, and occlusion of the adventitial and mural microcirculation were observed in both the early and late nonpatent vessels but not in the patent specimens. Evidence for myointimal cell proliferation was present in both patent and occluded grafts. A cohesive intimal layer was observed in both patent cases. Microvascular occlusion, due to an excess of endothelial cells, of new vasa vasorum in one case seems related to increased fibrosis, which could have resulted in graft stenosis. The surgical procedures and subsequent morphological analyses were adequate and sufficient for use in a long-term study of the possible causes of graft failure.

[Comparative study of 3 implanted micrografts placed in the abdominal aorta of the rat]. / Etude comparative de trois microgreffes implantées en position aortique abdominale chez le rat.

A comparative study was conducted on three micrografts of 2 mm diameter: a frozen umbilical artery, a bovine heterograft and an inert P.T.F.E. material graft. These were implanted in the infrarenal aorta of 158 Wistar rats. Each graft was examined on eight occasions: after 4, 24 and 48 hours, one and two weeks, and 1, 2 and 6 months. Excluding 46 animals that died within 24 hours, the overall permeability rate for all grafts was 87.7% (88.9% for umbilical arteries, 82.8% for bovine heterografts and 85.4% for P.T.F.E.). All grafts were examined by optical and electron microscopy. The inert material showed excellent parietal stability. In contrast, alteration and resorption of the primary matrix were moderate in extent for bovine heterografts, and major for frozen umbilical arteries which appear to be a very unstable grafts.

Human umbilical artery for microvascular grafting. Experimental study in the rat.

As microvascular surgery has developed, the necessity for smaller arterial prostheses of less than 2 mm in internal diameter (ID) has increased markedly. Glutaraldehyde-stabilized human umbilical arteries with 1.5 to 2.0 mm ID are smaller than any other graft material currently available. This study was designed to determine whether this graft material has potential clinical application for microanastomosis. Twenty male albino rats, each weighing 400 to 500 gm, were used in this study. The abdominal aorta was exposed and a 3- to 4-mm segment was resected. A 10-mm interposition graft of glutaraldehyde-stabilized human umbilical artery was implanted by end-to-end anastomosis. The patency of the grafts was determined by repeated operative exploration at intervals of 1 week, and 1, 3, 9, and 12 months. Two grafts were found to be occluded each time at the 1-, 3-, and 9-month explorations. At each exploration time, five rats were sacrificed and histopathological studies conducted. All five remaining grafts were patent at 12 months. There was no evidence of endothelial proliferation on the luminal surface of the patent grafts as determined by scanning electron microscopy. The luminal surface and underlying region consisted of an amorphous proteinaceous-like material. Significant degeneration of the vessel wall was noted in all grafts surviving over 9 months. Central necrosis surrounded by a chronic inflammatory process that extended to and included the adventitia was observed in the occluded grafts. While the patency demonstrated was good, the tissue changes noted in the walls of the grafts indicate that further study is necessary before this material can be used as a graft in humans.

[Preserved human umbilical cord vessel as a small caliber vascular substitute: experimental and clinical study].

The author has studied glutaraldehyde stabilized human umbilical cord vessels (PHUV) both experimentally and clinically since Nov. 1976. The experimental findings showed that the PHUV is not organized and its patency depends on the blood compatibility of the fibrin net and blood flow. The histologic examination of the graft wall revealed a slight inflammatory response consisting of macrophages and occasional foreign body giant cells around the Dacron mesh but with very little reaction in the umbilical vein itself. Clinically, 72 reconstructions using Biografts were performed in 52 cases with peripheral arterial occlusions. These consisted of 14 femoropopliteal (FP) bypasses above the knee, 26 below the knee, 10 femorotibial or peroneal and 16 other miscellaneous procedures including aorto-femoral and extra-anatomic bypasses. The cumulative patency rate at 5 years was 63.0% for the total group, 62.9% for the F-P group and 20% for the femoro-tibial or peroneal group. Early failure was thought to be related to technical factors in most instances. The majority of the late failures were due to intimal hyperplasia at the anastomotic sites. Although it is reasonable to employ a Biograft at the popliteal level because of the encouraging results in its clinical use, it is important to study the mechanism of the intimal hyperplasia at the anastomotic sites in order to attain excellent long term results.

Arterial umbilical cord as tubal replacement. A preliminary report.

In ten white New Zealand rabbits a one cm long human arterial cord graft was transplanted after resection of 0,5 cm of isthmus by microsurgical technique. The umbilical artery was previously prepared under microscope and treated for suppression of cord antigens. After two episodes of mating, no pregnancies were observed in the operated side, but total patency was demonstrated in 2 case (20%) and only distal occlusion in 2 more (20%). In 4 cases (40%) the growth of mucosa by apocrine metaplasia was histologically observed and in 5 cases (50%) the presence of foreign body reaction was detected. The hystological findings are presented and the results discussed.

Minute vascular replacements.

Glutaraldehyde-tanned, mesh-reinforced, mandrel-grown ovine collagen conduits were compared with tanned human umbilical arteries and polytetrafluoroethylene (PTFE) grafts in the aorta of rat recipients. All grafts had 100% patency and became lined by a cellular neointima. The mean maximum thickness of the neointima of the tanned human umbilical artery, ovine collagen graft, and the PTFE grafts was 68, 57, and 13 micrometer, respectively. Neointimal proliferation was complete for the two biosynthetic grafts, but none was seen in the center of the PTFE grafts as late as ten weeks. The mean increase in lumen cross-sectional area was 49% for the umbilical artery grafts, 23% for ovine collagen conduits, and 4% for the PTFE grafts. Longer follow-up periods will be required before unqualified support can be given to clinical trials of these small-diameter prostheses; however, long-term patency for synthetic grafts of such a small caliber is encouraging for future microvascular applications and for study of host-prosthesis interactions.