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

Avaliação biomecânica de membranas de celulose microbiana (Zoogloea sp.) e de politetrafluoretileno expandido como implantes no reparo de defeitos produzidos na parede abdominal em ratos

ORIGINAL ARTICLE

MATERIALS TESTING

Biomechanical evaluation of microbial cellulose (Zoogloea sp.) and expanded polytetrafluoroethylene membranes as implants in repair of produced abdominal wall defects in rats¹

Avaliação biomecânica de membranas de celulose microbiana (Zoogloea sp.) e de politetrafluoretileno expandido como implantes no reparo de defeitos produzidos na parede abdominal em ratos

Suyiene Cordeiro Falcão^I; Antônio Roberto de Barros Coelho^II; Joaquim Evêncio Neto^III

^IPhD, Veterinary Science - Department of Veterinary Medicine - Federal Rural University of Pernambuco, Brazil

^IIPhD, Associated Professor- Department of Surgery- Federal University of Pernambuco, Brazil

^IIIPhD, Associated Professor - Department of Animal Morphology and Physiology - Federal Rural University of Pernambuco, Brazil

^{Correspondence}

ABSTRACT

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 Group- 30 animals) or extended polytetrafluoroethylene (ePTFE) (ePTFE Group- 30 animals). Each group was subdivided in 14^th POD, 28^th POD and 60^th POD Subgroups. Under anesthesia, animals were submitted to euthanasia at 14^th POD, 28^th POD and 60^th POD for evaluation of Load of Rupture.

RESULTS: Load of Rupture levels were significantly elevated (p<0, 05) among 14^th, 28^th and 60^th 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.

Key words: Membranes, Artificial. Zoogloea. Polytetrafluoroethylene. Implants, Experimental. Abdominal wall. Tensile Strength. Rats.

RESUMO

OBJETIVO: Avaliar a Carga de Ruptura de implantes de membranas de celulose microbiana (Zoogloea sp.) e de politetrafluoretileno expandido em defeitos agudos produzidos na parede abdominal de ratos.

MÉTODOS: Sessenta ratos machos Wistar, com média de peso de 437,7g ± 40,9, anestesiados com uma mistura de cetamina (5mg/100g) e xilazina (2mg/100g), foram submetidos à excisão retangular (2x3cm) na parede ventral do abdômen, incluindo fáscia, músculo e peritônio. Subseqüentemente, foram tratados com implante de membranas de celulose microbiana (CM) (Grupo CM - 30 animais) ou de politetrafluoretileno expandido (PTFEe) (Grupo PTFEe - 30 animais). Cada grupo foi ainda subdividido nos Subgrupos 14º DPO, 28º DPO e 60º DPO. Os animais foram submetidos à eutanásia com doses letais de tiopental no 14º DPO, 28º DPO e 60º DPO, para avaliação da Carga de Ruptura na área do implante.

RESULTADOS: Os níveis da Carga de Ruptura foram significativamente elevados (p<0,05) entre os Subgrupos 14º DPO, 28º DPO e 60º DPO de cada grupo estudado. Quando comparados entre Grupos, os valores da Carga de Ruptura foram significativamente maiores (p<0,05) no Grupo PTFEe do que no Grupo CM.

CONCLUSÃO: A interface implante/hospedeiro apresentou maior resistência a tração no Grupo PTFEe do que no Grupo CM.

Descritores: Membranas artificiais. Zoogloea. Politetrafluoretileno. Implantes experimentais. Parede abdominal. Resistência à tração. Ratos.

Introduction

The best method for the treatment of muscle-aponeurotics 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 reconstruction¹. 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 cellulose³. 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 biocellulose³.

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 g rays^ª. This process propitiates membranes with pores of mean diameter of 0, 07µm (70nm) ⁵.

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 g 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 muscle-aponeurotic 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.

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 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 ePTFE Group are shown in graphic form (Figure 7).

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).

Infiltration of host tissue within implants was observed only in ePTFE Group (Figures 9 and 10)

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 nerves⁷; 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; threads¹¹; agent for increases soft tissue, reconstruction of the pelvic floor, suspension of bladder, repair of hernias and patch for inguinal hernias⁹.

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 nerves¹⁴; duraplasty¹⁵; healing of epithelial lesions of cornea¹⁶; healing of duodenal lesions ¹⁷; substitute of blood vessels¹⁸; cuffs for of reconstruction of micronerves¹⁸; reconstruction of the retroperitoneum¹⁹; and technical training in microsurgery¹⁸.

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 vessels²⁶; repair of tympanic lesions²⁷; suburethral slings for treatment of urinary incontinence²⁸; urethroplasty²⁹; reconstruction of tunica albuginea of the pennis³⁰; treatment of vesico-ureteral reflux³¹; and dressing after surgical correction of hypospadia³².

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 nerves³³; healing of experimental wounds of bovine mammary teats ³⁴; healing of experimental tegument wounds in equine³⁵ and swine³⁶; prophylaxis of the formation of membrane post laminectomy in dogs³⁷ and healing of incisional experimental lesions of the cornea in dogs³⁸. 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 repair⁴³.

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 3µm¹⁸. 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 host⁴⁵. 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 organism⁴⁶. 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 Cana-de-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.

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43- Bellón JM, Contreras LA, Buján J, Carrera-SanMartin A Experimental assay of Dual Mesh® polytetrafluoroethylene prosthesis (non-porous on one side) in the repair of abdominal wall defects. Biomaterials. 1996;17(24):2367-72.

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Received: August 29, 2007

Review: November 12, 2007

Accepted: December 18, 2007

Conflict of interest: none

Financial source: none

1

Research performed at Laboratory of Experimental Surgery-Department of Surgery- Federal University of Pernambuco, Brazil.

a

Departamento de Energia Nuclear da UFPE

b

GoreTex®, W. L. Gore & Associates, Inc. ,Newark, DE.

c

Labina, Laboratório Nestle/Purina/PetCare Company, São Lourenço da Mata PE.

d

Protocol for anesthesia in rodents adopted at the Núcleo de Cirurgia Experimental

e

Prolene®, Ethicon , Johnson & Johnson Comércio e Distribuição Ltda.

f

Mononylon®, Ethicon , Johnson & Johnson Comércio e Distribuição Ltda.

g

Lutron FG-20 kg- Rs232- Taiwan, Impac Instrumentos de Medição - BR.

h

Thiopentax, Laboratório Cristália-BR

Publication Dates

• Publication in this collection
  26 Mar 2008
• Date of issue
  Apr 2008

History

• Accepted
  18 Dec 2007
• Reviewed
  12 Nov 2007
• Received
  29 Aug 2007