A new vascular substitute : femoral artery angioplasty in dogs using sugarcane biopolymer membrane patch – hemodynamic and histopathologic evaluation

Background: Achievement of an ideal arterial substitute to be used in the reconstruction of smalland medium-caliber arteries is still the main objective of most scientific research studies developed in the area of vascular substitutes. Sugarcane biopolymer membrane could be extremely useful to perform vascular reconstruction in arteries with diameter smaller than 4 mm, allowing treatment of diseases that affect millions of people in Brazil and worldwide. Objective: To evaluate the hemodynamic and histopathologic behavior of sugarcane biopolymer membrane when used as a patch in femoral artery angioplasty in dogs. Method: Eight adult mongrel dogs, under general anesthesia, underwent percutaneous Doppler velocimetry of the left and right femoral arteries for preoperative control at the Health Sciences Center Experimental Research Laboratory of Universidade Federal de Pernambuco. After being disinfected, the dogs underwent femoral artery patch angioplasty using a sugarcane membrane biopolymer patch on the left side and an expanded PTFE (e-PTFE) patch on the right side. The dogs underwent daily clinical evaluation for the first week and weekly thereafter. Clinical evaluation consisted of an examination of femoral artery pulses, gait assessment and verification as to whether there was any pulsatile tumor, bruising, hemorrhage or surgical wound infection. One hundred and eighty days after the angioplasties, percutaneous Doppler velocimetry of the femoral arteries was performed under general anesthesia. Next, the dogs underwent dissection of the femoral arteries, measurement of the arterial diameter and perioperative Doppler velocimetry proximally and distally to the artery angioplasty. Laparotomy was then performed to expose the abdominal aorta for angiographic access. The femoral artery segments with patches were harvested for histopathologic analysis, and the animals were sacrificed under a toxic dosage of anesthetic. Results: At 180 days, no cases of surgical wound infection, aneurysm, rupture, pseudoaneurysm or thrombosis were observed in either group. A chronic inflammatory reaction with lymphocytes and neutrophils was seen in both groups on the outer surface of patches. Fibrosis was seen on the inner and outer surfaces of the patches. Invasion by fibroblasts of all e-PTFE patches occurred in the control group. Conclusions: Based on the results obtained with the experimental model employed, for a 180-day period, it may be concluded that the sugarcane biopolymer membrane is an adequate arterial substitute when used as a patch in femoral artery angioplasty in dogs.


Introduction
Closure of arteriotomies, using varied techniques, may cause reduction in vascular lumen, especially in small-diameter arteries.[3][4][5] The concept of patch angioplasty was first established by Carrel & Guthrie in 1906.]6 Several types of materials have been used as patches in angioplasties.Nowadays, the most widely used are autogenous veins, biological grafts of bovine pericardium and synthetic Dacron and expanded polytetrafluoroethylene (e-PTFE) patches.9][10][11] An extracellular polysaccharide produced by bacterial synthesis based on sugar cane molasses has been recently developed at Estação Experimental de Cana-de-Açúcar de Carpina of Universidade Federal Rural de Pernambuco. 12e membranes formed based on that polymer were processed for application as surgical graft at the Experimental Surgery Sector of the Center of Health Sciences at Universidade Federal de Pernambuco.They were evaluated by laboratory tests and presented flexibility, low toxicity, biocompatibility and resistance against rupture. 13ch membrane has been used in many areas of experimental surgery: healing of skin wounds; in ureteral reconstruction; as a substitute of tympanic membrane; among others.[16][17][18] Following the line of research using the sugarcane biopolymer membrane in several experiments of reconstructive surgery of organs and tissues, this study aims at evaluating, from the hemodynamic and histopathologic perspective, the behavior of that membrane as an arterial wall substitute as patches in the femoral artery of dogs.

Method
Between January 2005 and June 2006, eight dogs ( Canis familiaris) were used, one female and seven males, mean body weight of 15.4 kg.
The animals were kept in an experimental laboratory for 21 days before starting the study.They were given chow diet, rabies vaccine and vermicide.
Fasting for solid foods occurred over the 24 hours preceding the surgery for all animals.
Longitudinal 5-cm skin incisions were then performed in inguinal regions to expose the left and right femoral arteries.After exposure, arterial diameters were measured using a manual pachymeter.
Next, longitudinal arteriotomies were performed by removing an elliptic fragment of the arterial wall measuring 1.5 cm in diameter and a diameter equal to 1/3 of the arterial diameter.To correct arterial wall defects, femoral angioplasties using e-PTFE patches on the right side and sugarcane biopolymer membrane patches on the left side were performed using a cardiovascular polypropylene continuous 7-0 suture (Figure 1).
After anesthetic recovery, the animals were sent to independent cages in the experimental laboratory.Solid foods were released 8 hours after the surgery.
Over the first postoperative week, the dogs were submitted to daily and weekly clinical assessment starting on the eighth day.Clinical assessment consisted of the examination of femoral pulses, walking and presence of pulsatile tumor, hematoma or hemorrhage and surgical wound infection.Six months after the surgery, the dogs were submitted to a new surgery.Preoperative cares and anesthetic technique were repeated.A new percutaneous Doppler velocimetry of the femoral arteries was performed, as well as inguinal incisions to expose femoral arteries.Next, perioperative Doppler velocimetry of the femoral arteries in distal and proximal sites to the angioplasty were performed and the measurements of arterial diameter repeated using a manual pachymeter.
Next, the dogs were submitted to laparotomy for exposure and puncture of the abdominal aorta using an 18-gauge Jelco ® , followed by iodinated-contrast arteriographies (Conray ® ).After arteriography and macroscopic evaluation, all arterial segments having patches were removed for histopathologic tests.The material was stained with hematoxylin-eosin and picrosirius (Figure 2).
The experiments performed were distributed into two randomly established groups: Group I, eight arterial patches using the biopolymer (experimental group); Group II, eight arterial patches using e-PTFE (control group).In this study, each animal was its own control.
The research protocol was sent for appraisal of the Bioethics Committee at Universidade Federal de Pernambuco, which approved it without restrictions.
Significance level used for all statistical tests was 5%.The data were included in an Excel spreadsheet and analyzed using the software Statistical Package for the Social Sciences (SPSS) version 11.
Sample size of each group was determined considering the comparative objective of significant difference between groups as to mean variation in diameter (before and after arterioplasty).Significance level was set in 5.0% (test error).Test power had an 80% level.Minimal acceptable difference between both groups was 0.005 cm (or 0.5 mm) in relation to variation in diameter before and after arterioplasty.That is the difference based on which significant difference should be obtained.Ratio between groups was 1.00, aiming at having equal sample sizes in both groups.

Results
All animals survived the established period of 180 days for clinical observation and second surgery.There were no cases of infection, dilatation or aneurysm, rupture or pseudoaneurysm and thrombosis in any group (Figure 3).
In assessments before and after the arterioplasty, Doppler sign in the left and right femoral arteries was present and had three-phase wave flow.
Arteriography showed that the femoral arteries in the control and experimental group had no thrombosis.The main arterial flow occurred through those vessels, and there was no development of irregular pattern in collateral circulation.There were no dilatations or pseudoaneurysms in both groups.
Measurement of arterial diameters before and 180 days after angioplasties did not show statistically significant differences (p > 0.05, using the signed-rank Wilcoxon test) (Table 1).
Histological evaluation showed presence of a chronic inflammatory reaction in all patches of both groups mediated by lymphocytes, neutrophils with no statistically significant differences between groups (Fisher's exact test), as well as fibrosis in its external surface.There was formation of a new intimal layer continuing the receptive artery in the internal surface, consisted of fibrosis.The new adventitia and intimal layers were strongly attached to wall patches.In both groups it was not possible to confirm presence of endothelial cells in the internal surface, since the immunohistological test was not used (Figure 4).
In both groups there were no focuses of degenerative process or calcification areas.In the control group using e-PTFE patches, invasion by fibroblasts occurred in all cases (Figure 5).

Discussion
All current substitutes have major limitations.Autogenous veins are scarce, biological grafts have high rates of aneurysmal dilatation and calcification, and synthetic grafts are expensive and have low resistance to infection.Thus, search for an ideal substitute remains, especially for application in smalland medium-diameter arteries.9][10][11] Nowadays, many research studies have evaluated use of new materials for substitution of blood vessels.Some of them have been directed to the study of materials produced based on substances synthesized by microorganisms, such as bacterial cellulose, which was used in the production of tubular grafts by Klemm et al., in 2001, for substitution of the carotid artery in rats. 19[22][23][24][25][26][27][28][29] Such complications did not occur in the present study.The results were confirmed by clinical assessment and by Doppler velocimetry, as well as by arteriography.Absence of dilatation in the femoral arteries after implantation of the patches was also confirmed by measurement of femoral artery diameters at the patch implantation site.Association between Doppler flowmetry and arteriography allowed proper assessment of collateral circulation, since it allowed analysis both of morphology and characteristics of blood flow in femoral arteries.
Complications resulting from use of arterial patches can be caused by failure of material used as a patch, as observed by Pena & Husni, who reported 20% of thrombosis in femoral angioplasties with Dacron patches in dogs and by McCready et al., when using porcine intestinal submucosa as a patch in carotid angioplasties and obtaining 10% of pseudoaneurysms. 24,29milarly, Menon et al. found 35% of thrombosis in femoral and carotid angioplasties using Dacron patches compared with 25% of thrombosis using autogenous vein patches, showing superiority of the latter. 22e healing process of sugarcane membrane patches was characterized by presence of a chronic inflammatory reaction in the external surface mediated by lymphocytes and neutrophils, as well as fibrosis.In the internal surface of these patches, there was development of an intimal layer formed by fibrosis.1,28 Also similarly to e-PTFE patches, focuses of degenerative process with loss of integrity were not observed in sugarcane membrane patches, as in autogenous artery or vein patches.In both groups, rare presence of macrophages confirms low rate of antigens in those materials.There were no focuses of calcification, as observed in biological graft healing. 20,22,24,28fferently of e-PTFE patches, sugarcane membrane patches did not allow invasion by fibroblasts.Penetration of cells in e-PTFE patches occurs due to presence of micropores in their structure.That process has been described in the literature and is important for a complete healing of patches.,31

Conclusion
Based on the results obtained with the experimental model used, over a 180-day observation period, it can be concluded that the sugarcane biopolymer membrane is a proper arterial substitute when used as patches in the femoral artery of dogs.