WO2005094575A1

WO2005094575A1 - Solution to preserve biological membranes and its use

Info

Publication number: WO2005094575A1
Application number: PCT/BR2005/000046
Authority: WO
Inventors: Ernesto Rodriguez Salas
Original assignee: Ernesto Rodriguez Salas
Priority date: 2004-04-02
Filing date: 2005-04-04
Publication date: 2005-10-13
Also published as: BRPI0400935A

Abstract

The present invention relates to a solution made up of water, electrolytes, the beta-lactamic, aminoglycoside, and polyene antibiotics, to be used in the preservation of a biological membrane, protecting it from microorganisms, so that the same may be conveniently used in surgical procedures, such as in the implementation and support of surgical procedures, like in surgeries to repair the muscular wall, as a result of hernias, and implants in tissue loss areas, caused by either traumatisms and/or post-operatory accidents.

Description

SOLUTION TO PRESERVE BIOLOGICAL .MEMBRANES AND ITS USE. Field of the Invention The present invention relates to a solution to preserve a biological membrane, more specifically, said solution is used to preserve a bovine pericardial biological membrane, keeping it free of microorganisms so that it may be conveniently used in surgical procedures, such as implants . Background of the Invention The use of biological membranes and their conservation have been studied for a very long time, with the purpose of preserving and maintaining biological membranes, for use in surgical procedures. Development of treatment methods for surgical affections. Said studies have aimed not only at proposing solutions for the conservation of those biological structures, but also at enabling their subsequent use on humans. In the I960' s, the research on biological prostheses was born, out of the need to treat pathologies in the cardiac valves. Among the biological prostheses invented at that time were natural valves taken from human cadavers

(homografts) , valves taken from animals (xenografts) and valves from the same patient (autograft) (MURRAY, 1956;

ROSS, 1962; BARRAT-BOYES, 1964 and 1965). Felippozi (1967) used a homologous aortic valve preserved in alcohol at 70% in the surgical treatment of the aortic valve. Zerbini, in that same year, registered good results with the use of aortic homografts preserved in formaldehyde. In an experimental study, Pigossi (1967) used glycerin at 0.98% to preserve dura mater. Considering the facility in obtaining these animal structures for human consumption, because of their anatomic features, antigenicity aspects, size, availability, swine structures have been the most widely used (BINET, et al., 1968; McINTOSCH, et. al . , 1975; RIVERA, et al. 1980; JANUSZ, et al . , 1982; REECE, et al . , 1986 and t

DAVID, et al., 1988). Carpentier, et al . , (1969) introduced the use of low concentration glutaraldehyde for the preservation of biological tissues. So, they proposed a new terminology, calling them bio-prostheses, provided that one of the most widely used membranes in the production of prostheses from living tissues has been the bovine pericardium. Stopiglia (1978), in the partial reconstitution of the thoracic wall in dogs, used an equine pericardial membrane preserved in glycerin. Glycerol at 98% has been used in the preservation of many biological materials, for its fast-working fixative and dehydrating effect, acting like an anti-septic (ALVARENGA, 1992) . Petropoulus & Kleinhenz (1962) , in the partial substitution of the wall of the thoracic esophagus in dogs, performed an implant of homologous dura mater, in natural temperature, and preserved in a cooled saline solution, containing penicillin and streptomycin, for a period ranging from two to five days. Andretto et al., (1975); Andreto, et al.(1976^a) and Andretto et al . , (1976^b) used a pericardial membrane from a dog preserved in glycerin, in the surgical correction of the abdominal aorta, correction of the trachea and the biliferous tract in dogs. Alvarenga (1978) used a homologous pericardial membrane preserved in glycerin, to partly substitute a segment of the choledoch in dogs. Alvarenga et al., (1982) used a flap of equine pericardium preserved in glycerin, to perform an esophagoplasty in a dog, after the resection of approximately six by three centimeters of that tissue to remove a Spirocerca lupi node at the level of the distal portion of the thoracic esophagus. In other studies, we have Ranzani (1986) who, using a pericardial membrane preserved in glycerin, replaced a segment of the diaphragmatic muscular section in dogs, and Daleck (1986) , who performed a cervical esophagoplasty using an autologous and homologous pericardial membrane preserved in glycerin. Lantzman (1986), in experimental surgical procedures on the duodenum of dogs, used an equine pericardial membrane preserved in glycerin. Daleck (1986), in an experimental study, used an autologous or homologous peritoneum preserved in glycerin in an esophagoplasty performed on a dog. Daleck (1992) used a bovine peritoneum preserved in glycerin in the correction of perineal hernia on dogs. Freud et al. (1993) used lyophilized dura mater, in the form of a flap or tube, in the surgical correction of defects created through experiments on the esophagus of dogs. Almeida (1996), in an experimental study, used a caprine pericardium preserved in glycerin at 98% or cooled in a saline solution at 0.9% in the reconstruction of the esophagus of dogs. Pigatto et al . (1996^b) performed an esophagoplasty using a homologous graft of the auricular cartilage preserved in glycerin, experimentally, after the resection of a segment of one by two centimeters of a canine esophagus. Araujo, et al. (1998) used different techniques for the preservation of bovine pericardium to be used in reconstructive surgery. Cunrath (1999), in an experimental work, used glutaraldehyde in the treatment of the bovine pericardial membrane to repair an incisional hernia. Araύjo et al. (2000) experimented with a number of different techniques for the conservation of bovine pericardium for use in reconstructive surgery, including glycerin at 98%. Mazzanti et al. (2001) proceeded to correct the canine diaphragm using a homologous orthoptic muscular segment preserved in a supersaturated sugar solution. Rappeti, et al. (2003) performed an experimental reconstitution of the thoracic wall in cats, with the use of a heterogeneous implant of auricular cartilage preserved in glycerin at 98%. In the prior art, among the alternatives to address the problem of preservation of biological membranes, glycerin has been widely acknowledged. Observations by researchers and surgeons have prompted general acceptance by the research community. So, the main purpose of the present invention is to present a preservation solution to maintain pericardial membranes in the proper condition to be used in bio- prostheses, and to determine the minimal inhibitory concentration (CIM) in anti-microbial substances, so that it will remain free of microorganisms. Description of the invention The present invention consists of a solution made up of water and electrolytes, distributed in concentrations that afford an isotonic environment, with an osmolarity very similar to plasma, and to the beta-lactamic, aminoglycoside and polyene antibiotics. Electrolytes are the elements comprising it, the osmolarity refers to the characteristic of the isotonic environment, similar to plasma, which is a component of human blood, and the beta-lactamic, aminoglycoside antibiotics prevent bacterial growth, while the polyene antibiotic prevents the growth of fungi. From the moment when the bovine pericardial membrane (MPB) was collected, until it reached the laboratory where the test was performed, it was transported in an icebox to preserve it during transportation. The membrane was placed in the preservation solution consisting of water and electrolytes distributed in concentrations that afford an isotonic environment, with an osmolarity very similar to plasma, and beta-lactamic, aminoglycoside and polyene antibiotics . The calculation of the antimicrobial agents that were added to the MPB preservation solution was based on the weight of the pre-treated membrane (RIZZO et.al., 1983;

HUBER, 1988), starting with a stock solution, where the antimicrobial agents were diluted in 10 ml of sterilized bi- distilled water. Upon determination that the preservation environment was in fact sterile, with its inoculation in Infusio Heart Brain (BHI) and Agar Saboraud, a bacterioscopy was performed with the Gram method (KONEMAN, et al., 2001), determining that it was sterile, after which the MPB was placed in a sterilized glass flask, and kept cool under a temperature of 12 to 14°C for 15 to 30 days, after which time a new bacterioscopy was performed on each flask. The initial concentrations of antimicrobial agents were then doubled and the bacterioscopy was repeated within the following intervals: T-15 and T-30, thus determining the CIM. After quite some time holding trials and experimental studies to test the substances that are appropriate to preserve membranes capable of being used in surgical procedures, a conclusion was reached as to the types of electrolytes and the quantities required therefor. The components of the solution that is the subject of this invention are electrolytes required to maintain the organic homeostasis and to process the physiological ionic exchanges, to wit: sodium (129.0 mEq) , chloride (109.0 mEq) , lactate (26.8 mEq), potassium (4.0 mEq) and calcium (2.7 mEq) and bi-distilled water q.s.p.(100 ml). Additional components of this environment are the antibiotics benzatin benzylpenicillin (3,234 U.I./mL), streptomycin sulphate (0.035 mg/mL) and amphotericin B (0.035 mg/mL) , so as to keep the solution free and clear of microorganisms. The preparation process involves sterilized equipment (glass) , working with vertical laminar-flow workbenches and high-precision electronic scales, to meet the rules and specifications for pharmaceutical procedures. After the applicable weighing procedures, the electrolytes were placed in a graduated balloon, to which the antimicrobial agents were added and, finally, the water, thereafter proceeding with the proper homogenizing process. Afterward, the membranes collected in sanitary slaughterhouse facilities were submitted to the removal of adjacent fat, blood vessels, delimited and cut with a plate measuring (13 x 20 cm), provided that 260 cm² is the area of the membrane that can be used in surgical procedures. The membrane: environment ratio was estimated on the basis of 1.0 ml (milliliter) : 1.0 cm². RESULTS The first bacterioscopic results suggested the existence of gram-positive bacillus in the liquid environment and plate, as well as a fungus compatible with the morphology of the Aspergillus sp and Corvularia spp colonies, and after the CIM was computed, no further microbial growth was observed to this date, which corresponds to 120 days. The example narrated herein was executed on bovine biological membranes, however, it is understood that the solution described may be used in the preservation of any membrane of animal origin. All biological membranes can and should be considered, without limitation to the bovine membrane . Minor changes and appropriate adjustments of a variety of conditions and parameters regularly found in the preparation of a solution for the preservation of biological membranes, to inhibit the presence of microorganisms are deemed to be obvious to those familiar with the prior art and fall within the scope of the present invention.

Claims

CLAIMS 1. Solution for the preservation of biological membranes, CHARACTERIZED by consisting of: electrolytes such as sodium at 129.0 mEq, chloride at 109.0 mEq, lactate at 26.8 mEq, potassium at 4.0 mEq and calcium at 2.7 mEq, bi-distilled water q.s.p. 100 ml; and the antibiotics benzatin benzylpenicillin at 3,234 U.I./mL, streptomycin sulphate at 0.035 mg/mL and amphotericin B at 0.035 mg/mL. 2. Solution, according to claim 1, CHARACTERIZED by the function of preserving and/or maintaining the structures-fiber-arrangement of the membrane unchanged or in good order, thus preserving their original structure, as well as its living cells. 3. Use of the solution in the preservation of biological membranes, as defined in claim 1, CHARACTERIZED by the fact that it is applied in the preservation of biological membranes to be used in surgical procedures.