Biomechanical evaluation of microbial cellulose ( Zoogloea sp . ) and expanded polytetrafluoroethylene membranes as implants in repair of produced abdominal wall defects in rats

Purpose: To evaluate the Load of Rupture of implants of membranes of microbial cellulose (Zoogloea sp.) and extended polytetrafuoroethylene in sharp defects of abdominal wall of rats. Methods: Sixty Wistar male rats, with a mean weight of 437,7g ± 40,9, anesthetized by a mixture of ketamine (5mg/100g) and xylazine (2mg/100g), were submitted to a rectangular (2x3cm) excision of the abdominal wall, including fascia, muscle and peritoneum, and treated with membranes of microbial cellulose (MC) (MC Group30 animals) or extended polytetrafluoroethylene (ePTFE) (ePTFE Group30 animals). Each group was subdivided in 14th POD, 28th POD and 60th POD Subgroups. Under anesthesia, animals were submitted to euthanasia at 14th POD, 28th POD and 60th POD for evaluation of Load of Rupture. Results: Load of Rupture levels were significantly elevated (p<0, 05) among 14th, 28th and 60th postoperative days from each Group. When compared between groups, values of Load of Rupture were significantly larger (p<0, 05) in ePTFE Group than in MC Group. Conclusion: Resistance to strength at implant/host interface was more pronounced in PTFEe Group than in MC Group.


Introduction
The best method for the treatment of muscleaponeurotics defects of the abdominal wall is the approach, without tension, of fibromuscular structures of the own patient's tissue.
However, in some circumstances the repair can be affected by the great distance among the edges of the defect or by the lack of tissue with characteristics for an appropriate approach.In these cases, synthetic and biological prostheses, or even muscular grafts, vascularized or free, can be used for reconstruction 1 .The synthetic prostheses are of high cost and vascularized or free muscle grafts frequently constitute complex and prolonged surgeries that imply in creation of weak sites in surrounding areas of defect, that could need repair 1,2 .
Certain bacteria, algae and fungi produce cellulose 3 .In the last years, because of its good mechanical properties and biocompatibility, there was an increasing interest in bacterial cellulose, produced by the Acetobacter xylinum and its application in medicine.Important examples include products for temporary skin substitution and tissue replacement.These activities have been accompanied by the isolation of new bacterial strain producing biocellulose 3 .
In Northeast of Brazil, cellulose membrane was produced in the Estação de Cana-de-Açúcar de Carpina-UFRPE, by the microorganism Zoogloea sp. 4 .
Finally, it is not of our knowledge reports of previous use of cellulose membranes produced by the microorganism Zoogloea sp. or by other bacterial species, as repair of muscle-aponeurotics defects of the abdominal wall, in experimental or clinical scope.
Our purpose was to study comparatively the Load of Rupture of implants using membranes of bacterial cellulose and polytetrafluoroethylene in sharp defects of abdominal wall produced in the rat.

Methods
The microbial cellulose membrane is constituted by an exopolyssacharide produced by the bacteria Zoogloea sp., isolated by the Instituto de Antibióticos da Universidade Federal de Pernambuco, obtained in static culture, having molasses of the sugar-cane as culture medium.The non soluble content in water reaches 88% of the components of the gross membrane of exopolyssacharide 4,5 .
A double layer of this microbial cellulose (MC) was used.It was compressed and then evaporated on the air during the dehydration process, conserved in isopropyl alcohol moisturized at 20%, conditioned in polypropylene envelopes and sterilized in γ rays a .This process propitiates membranes with pores of mean diameter of 0, 07µm (70nm)
The film of expanded polytetrafuorethyene (ePTFE) b was obtained from vascular prostheses of internal diameter of 8mm and wall thickness of 0,8mm, with pores of 25µm, sectioned in longitudinal direction, after removal of the external helical structure.Rectangles of 2x3cm were prepared, conditioned in polypropylene envelopes and submitted to the sterilization in γ rays.
Sixty male Wistar rats, with mean weight of 437,7g±40,9, were conditioned in appropriate cages, fed with specific food c and mineral water ad libitum, before they were submitted to the procedures considered in the project.
The animals were distributed in two groups: A) Microbial Cellulose Group (MC Group): composed of 30 animals submitted to a sharp defect of the ventral wall of the abdomen, including fáscia, muscle and peritoneum, being treated with membrane of microbial cellulose; B)Expanded Polytetrafluorethylene Group ( ePTFE Group): composed of 30 animals submitted to the same procedure of MC Group , being treated with membrane of expanded polytetrafluorethylene; Each group was subdivided in three subgroups of 10 rats, in agreement with the evaluation period, being denominated of 14 th POD, 28 th POD and 60 th POD Subgroups (Figure 1).
Anesthesia was accomplished with a mixture of cloridrate of ketamine (5mg/100g of weight) and cloridrate of xylazine (2mg/100g of weight), preceded by sulphate of atropine (0,044mg/kg) d , all administered by intramuscular route.
Under aseptic conditions, a middle incision of five cm was accomplished in the skin and subcutaneous layers of the abdominal ventral area of the animals.These plans were dissected from the aponeurotic layer of the corresponding area, followed by placement of superficial retractors, to allow the creation of an area for the production of muscle-apneurotic sharp defect .
Through a mold made of RX film, measuring 2cm of width for 3cm of length, the defect was produced, centered in the middle line of abdomen, 1 cm below the xifoid appendix.A full thickness wall involving fascia, musculature and peritoneum were excised.In the MC Group, membrane of microbial cellulose was sutured at the level of the muscleaponeurotic defect with polypropilene e 4-0 and continuous suture, anchored at the four angles of the rectangle.In the ePTFE Group the same surgical procedures adopted in the precedent group were accomplished (Figure 2).Then the skin was closed with thread of nylon f 4-0 through an interrupted suture.

FIGURE 1 -Distribution of groups and subgroups
A device for the biomechanical measurement of Load of Rupture at the implant/host interface was built in the Núcleo de Cirurgia Experimental (Figure 3).
The applied load to the tissue sample was provided by a system of distilled water pumping, with a constant flow of 500 ml /min, into a plastic bag, which weight exerts traction in the implant/host sample.
Sample was positioned in the top and bottom clamps and tightened (Figure 4).
Top clamp was attached to a Digital Dynamometer® g and the bottom clamp was connected to the plastic bag.The plastic bag, moved down by an increment of constant weight, applied paralel to the sample.The dinamometer expressed the Load of Rupture in Kgf (scale precision: ± 0.5%), (resolution: 0.01 kg).A serial interface RS-232 was connected between the dynamometer and a computer and a dedicated software displays results as curves and tables.
Animals were submitted to euthanasia with lethal doses of thiopental® h to obtain samples.
The sample was composed by implant and part of the abdominal musculature, free from suture, measuring 5x2 cm, including the segment inserted within clamps (1 cm in each end), so that the implant/tissue rectangle between clamps measured 3x2 cm (Figure 4).
For validation of biomechanical test, only ruptures occurring in the implant/host interface were considered, not being computed results where the rupture occurs out of these structures, (i.e.abdominal musculature).
The test of differences of means (two tailed Student's "t" test), with probability of error of p <0,05 were applied between the averages of obtained values of Load of Rupture (in Kgf): A) in 14 th POD, 28 th POD and 60 th POD Subgroups of the Microbial Cellulose Group (Group MC); B) in 14 th POD, 28 th POD and 60 th POD Subgroups of the Expanded Polytetrafluorethylene Group (ePTFE Group); C) in 14 th POD, 28 th POD and 60 th POD Subgroups of each Group, in corresponding periods (Figure 5).Observations were made on the presence infiltration within the implants of microbial cellulose and expanded polytetrafluoroethilene.

Results
Comparative analysis accomplished among the averages of the values of the Load of Rupture obtained in the animals of 14 th POD, 28 th POD and 60 th POD Subgroups of MC Group are shown in graphic form (Figure 6).Comparative analysis accomplished between the averages of the values of the Load of Rupture obtained in the animals of 14 th POD, 28 th POD and 60 th POD Subgroups of MC Group and those obtained in the animals of 14 th POD, 28 th POD and 60 th POD Subgroups of ePTFE Group, in corresponding periods, are shown in graphic form (Figure 8). the animals of 14 th POD, 28 th POD and 60 th POD Subgroups of ePTFE Group are shown in graphic form (Figure 7).

Discussion
The use of microbial cellulose (Acetobacter xilynum) has been suggested in different areas of medicine as: substitute of blood vessels and linfatics 6,7 ; substitute of hollow internal organs as ureter, trachea and digestive tract 6,7,8 ; cuff for reconstruction of nerves 7 ; substitute of duramater 9,10 ;substitute of the abdominal wall, skin, subcutaneous tissue, articulation, cartilage and reinforcement of areas of decreased resistance in the abdominal wall, esophagus and intestinal tube 8 ; threads 11 ; agent for increases soft tissue, reconstruction of the pelvic floor, suspension of bladder, repair of hernias and patch for inguinal hernias 9 .
In humans, microbial cellulose (Acetobacter xilynum) has been used on lesions of tegument (serious burns, skin graft ( in the donor and receiving areas), facial peeling, infectious dermolysis, abrasion of tattoos, chronic ulcers, Hanseníase of the distal members 12,13 .Experimental studies for clinical application have been accomplished with membrane produced by the Acetobacter xilynum in several conditions as: protective cover for reconstruction of nerves 14 ; duraplasty 15 ; healing of epithelial lesions of cornea 16 ; healing of duodenal lesions 17 ; substitute of blood vessels 18 ; cuffs for of reconstruction of micronerves 18 ; reconstruction of the retroperitoneum 19 ; and technical training in microsurgery 18 .
With the creation of a research group named biopolímero da cana de açúcar at the Federal University of Pernambuco, studies of biocompatibility of the microbial cellulose produced by Zoogloea sp 20,21,22,23 .The satisfied results authorize the accomplishment of experimental research for clinical application.Thus, studies were performed on: repair of stomach and bladder lesions 24,25 ; substitution and patch of blood vessels 26 ; repair of tympanic lesions 27 ; suburethral slings for treatment of urinary incontinence 28 ; urethroplasty 29 ; reconstruction of tunica  Infiltration of host tissue within implants was observed only in ePTFE Group (Figures 9 and 10) albuginea of the pennis 30 ; treatment of vesico-ureteral reflux 31 ; and dressing after surgical correction of hypospadia 32 .
Experimentally, in Veterinary Medicine, the reports consulted about use of biocelulose produced by the Acetobacter xylinum refer to few applications for: conduit for isolation in reconstruction of peripheral nerves 33 ; healing of experimental wounds of bovine mammary teats 34 ; healing of experimental tegument wounds in equine 35 and swine 36 ; prophylaxis of the formation of membrane post laminectomy in dogs 37 and healing of incisional experimental lesions of the cornea in dogs 38 .Clinically, gross (brute) membrane of bacterial cellulose produced by the Zoogloea sp. was firstly used in healing of natural wounds of dogs 39. 40 .
In this review, no reports (experimental or clinical) on the use of microbial cellulose membrane were found as repair of defects of the abdominal wall in humans or animals.
Increases of resistance in the subgroups of the MC Group (Figure 6) and ePTFE Group (Figure 7) along the time, may be related to the evolution of the healing process, predominantly inflammatory at the early phase and with prevalence of fibroblasts and collagen fibers in the remodeling process, allowing the biomaterial to be incorporating more firmly by the host, already mentioned in biocompatibility studies 41,42 .
Elevation of resistance in ePTFE Group as compared to MC Group, in corresponding periods (Figure 8) is indicative of better integration between polymeric synthetic material and host tissue.
The most important factor in the response to the implants by the host is the structure of the material, more specifically its porous structure.Materials that have reticular or macroporous structure , as polypropylene mesh, seem to produce better integration with organism of the receiver and better resistance to the traction in the zone of repair 43 .
The microbial cellulose is characterized by a structure of microfibrils net.The cellulose membrane produced by the Zoogloea sp.presents small pores (empty spaces among the fibrils) with diameters around 0, 07µm (70nm) 5 .To have an idea of the size of these pores, it is enough to compare them with a thrombocyte (platelet), one of the smallest components of the blood, with a diameter of about 3µm 18 .The microporosity of the microbial cellulose doesn't allow ingrowth of host in cellulose structure (Figure 9).
The size of pores of the microbial exopolysacharide, resultant of the application of method of manual compression and exposition to the air for dehydration of the membrane, is reduced.Such pores, defined as spaces among the structural elements of the MC, enter in collapse due to superficial tension, as a consequence of liquid removal 44 .
The expanded polytetrafluoroethylene (ePTFE) presents a microscopic structure composed of nodules interconnected each other by fibrils.The interior of the fibrils net is constituted by empty spaces with dimensions sufficiently large (up to 80 µm), to allow infiltration of tissue from host 45 .The porosity of ePTFE is an important characteristic in synthetic materials for implant that allows the infiltration of connective tissue into the material, propitiating better integration between the implant and the organism 46 .In the present work it was used implants of ePTFE with pore diameters of 25µm, therefore, dimension enough to allow the penetration within implants with cellular elements and host tissue (Figure 10), starting from 14 th POD.Reports indicate that starting of this process can be observed from 7 th POP 42 .
The multiperforated cellulose membrane, partially dehydrate membrane and the gel form (by ultrasonic fragmentation of the membrane) interposed between sheets of cellulose (sandwich) are being tested at Núcleo de Cirurgia Experimental associated with Laboratório de Canade-Açúcar da Universidade Federal Rural de Pernambuco, in the sense to obtain a product that allows better integration with host tissue.

Conclusion
In the experimentation conditions, and with a probability of error of 5%, the implant/host interface presented larger resistance in ePTFE Group as compared with MC Group.

FIGURE 3 -FIGURE 4 -
FIGURE 3 -Device for measurement of Load of Rupture

FIGURE 5 -FIGURE 2 -
FIGURE 5 -Diagram showing statistical analysis programmed for Load of Rupture

FIGURE 7 -
FIGURE 7 -Graph showing results of statistical analysis between mean values of Load of Rupture (Kgf) obtained in subgroups of ePTFE group

FIGURE 6 -
FIGURE 6 -Graph showing results of statistical analysis between mean values of Load of Rupture (Kgf) obtained in subgroups of MC group

FIGURE 8 -FIGURE 9 -
FIGURE 8 -Graph showing results results of statistical analysis between mean values of Load of Rupture (Kgf) obtained in MC Group compared to those obtained in ePTFE Group, in corresponding periods POST-OPERATIVE DAY