| This study was funded by grants from both National 863 High-TechnologyProjects (No.2006AA03Z441) and Tackled Projects of Guangdong Province (No.2006B35850001).Objective This study was conducted to evaluate a novel biologicalsmall-diameter vascular graft surgically, biologically and histologically for up topostoperative eighteen months through graft patency and graft intimal membranehistological examination.Method: Artificial vascular grafts were implanted end-to-end in the carotidposition in experimental dogs by a continuous suture. Bedside Color DopplerSonography and Digital Subtraction Arteriography (DSA) were performed to monitorgraft patency at different time points for a period of eighteen months. Graft sampleswere collected after each DSA exam and subjected to further optical and SEM studiesfor the histopathological analysis of endothelial cell proliferation, intimal hyperplasiaand host tissue growth on the internal surface of the grafts.Experimental Procedure: The artificial vascular graft (provided by GuanghaoBiotech, Guangdong, China) was derived from porcine vessels and processed byepoxy fixation, coupling with special polypeptide that facilitates the enrichment andattachment of growth factors, surface anticoagulation and charging the inner surfacenegatively. The exhaustive depletion of antigen was achieved through blockage or alteration of antigen determinants instead of decellularition.28 healthy mongrel dogs underwent unilateral operations in this study. Thevascular grafts with an inner diameter of 4mm were used to replace a 3 cm segmentof the common carotid artery using two end-to-end anastomosises. Then totally 28operated common carotid arteries, each in one animal, were randomly divided into sixgroups according to predetermined check times (four arteries for 1-week group, fourfor 8-week group, six for 12-week group, five for 6-month group, five for 12-monthgroup and four for 18-month group, respectively). The graft patency was monitoredby Color Doppler Sonography, then graft samples were harvested and furtheranalyzed by light microscope (with both HE and VG staining) and SEM aftermeasurement of the patency rate by DSA examination. Bedside Color DopplerSonography examination demonstrated that all 28 vascular grafts were patent at oneto two weeks after grafting.Preoperative DSA examination was performed on three randomly selectedcommon carotid arteries in three dogs. Postoperative DSA examinations, via femoralarterial cannulation or common carotid artery directly (in only three arteries), wereconducted at 3 weeks, 8 weeks, 12 weeks, 6 months, 12 months and 18 months,respectively, after a regular screening by bedside Color Doppler Sonography. Allexperimental dogs of the first three groups were confirmed by DSA examination.Within postoperative 6 months, the dogs of 6 and 12 months groups were onlychecked by bedside Color Doppler Sonography. Additional DSA was required whenan obstruction or stenosis was revealed. All dogs of 18 months group were performedDSA examination via femoral arterial catheter. All specimens were processed, splitopened longitudinally and then subjected to histopathological and SEM examinations.For obstructed vascular grafts, only histopathologic examination was performed.Result Nervous system complications, such as hemiparalysis and aphonia, were notoccurred in all 28 dogs. In 2 dogs (one for 12-week group, one for 6-month group),regional bumps at operative site were found at postoperative day 11 and day 5,respectively, identified as infection by local puncturation and finally healed byantibiotics therapy with liquor extraction in 3 weeks. All wounds achieved goodhealing.Of all 28 blood vessels, the rest blood vessels were patent with the exception ofsix arteries (one of 12-week group, one of 6-month group, two of 12-month groupand two of 18-month group). However, various degrees of delayed visualization andfilling defect were observed in patent blood vessels of 12-month and 18-monthgroups. The patency rates of vascular grafts within postoperative 3 months, between3 and 12 months, and after 12 months were 96.15%, 80% and 50%, respectively.During the sampling procedure, the grafts were found stiffened and tightlyadhered to surrounding tissue excluding samples of 1-week group whichdemonstrated a loose adhesion. By contrast, the grafts in both 12 and 18 months werefound loosely adhered to surrounding tissues and formation of new blood vesselswere observed in the distal end of the grafts. When longitudinally slit open and laidflat fore examine, the graft luminal surface of one-week samples showed a nearlycomplete coverage of neointimal layer with different thickness and thrombus-free.The neointima became denser and uniform during the period of postoperative 8 to 12weeks. Between 6 and 18 months, obstruction was observed in a total of five grafts.None of the grafts had thrombus formation.Histopathology and SEM demonstrated that at 1 week post grafting, the graftswere wrapped by a layer of connective tissue adventia and blood clots were observedin the perivascular space. The internal surface of stoma demonstrated almostcomplete fibrous neointima healing but did not show endothelial cells. The rest portion of the luminal surface was covered by a thin layer of thrombus mainlycomposed of blood cells. The connective tissue surrounding the grafts had beenthoroughly organized up to postoperative 8 weeks. The neointima of the graft wasuniform than before, consisting of abundant smooth muscle fibers and a littlecollagenous fibers, however had no signs of hyperplasia. Nourishing vessels hadformed and penetrated the poros of grafts and protruding into the lumen. Endothelialcell was not yet observed. At 12 weeks, the graft had a highly denser but nohyperplased intima, on its surface endothelial cells growing discontinuously both assingle cell from each anastomosis and in many cell island in the middle portion. Inaddition, the closer to the stoma, morphologically cells were in closer proximity tonormal endothelial cell.In one sample of 6-month group, intimal fibrous hyperplasia could be observedin the proximal end of the stoma but the graft still maintained partly patent.Endothelial cells were presented on the whole surface of the graft and continuousendothelial growth could be observed on the surface of the neointima close to thestoma. Various degrees of intimal hyperplasia were presented and noncellularconnective tissue lined the inner surface of grafts from animals of both 12 and18-month groups. Formation of complete granulation tissue could be observed at thearea of stoma in some samples while endothelial cell morphology was absent on thesurface of the neointima. In addition, VG staining of the graft wall revealed that thegraft stent had been replaced by large amount of newly grown collagen fibers andblood capillaries.The result of SEM also demonstrated a persistently alterative procedure of themorphology of vascular grafts. At 1 week post grafting, the anastomosis site waswrapped by a newly grown layer of film with different thickness, but the stoma wasstill visible. Close to the stoma, a uniform and pyknotic fibrous film coverage was growing finger-likely toward the anastomosis, however the morphology character ofnewly grown endothelial cells was not observed. At 8 weeks, the graft outer surfacebecame completely covered by oval cells of various sizes and had a sort of"cobblestone morphology". The stoma in the luminal surface disappeared entirely.The anastomosis site of the grafts was covered by fragmentized islands of spindlecells which was slightly different from normal endothelial cells in morphology. Laterat 12 weeks, endothelial cells were sparsely distributed throughout the wall near thestoma; the luminal surface of the stoma was smoother and more regular than before.At 6 months, cells morphologically resembling normal ECs were migrating from thetwo anastomosis sites towards the middle portion of the grafts for about 1.1 cm. Thegrafts also displayed endothelialization in many islands in the mid-segment of thegraft and other parts of the graft.Conclusion Collectively the present data indicated that this new biologicalgraft could be used for the repair of a 3cm-defect of common carotid artery in dog.The neointima formation occurred acutely and completely. Proliferation ofendothelial cells was observed at 12-week after grafting. Within postoperative 6months the procedure of natural endothelialization was relatively satisfactory.Although intimal hyperplasia and growth of noncellular tissue were presented at12-month, taken together the good anti-infective and anti-thrombus capacities,biocompatibility and stability, and regeneration capacity of the intravascular stentconvincingly presented a promising application of this prothesis material. Furtherinvestigation was required to improve the outcome achieved over 12 months postgrafting, which was characterized by marked intima hyperplasia, unmatched vesselwall compliance, and also the caliber unicity of the grafts. |
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