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Biotechnology

Cellulose on the skin

Bacterium produces substance used for treating burns and in the coating of bulletproof vests

Chemistry institute/UNESP - ARARAQUARAUnder the microscope, cellulose fibers produced by the bacteriumChemistry institute/UNESP - ARARAQUARA

A biocompatible bandage to replace, in a temporary way, human skin and a material for coating bulletproof vests are two new products that are about to reach the market. In common, they have the same origin, the cellulose produced by the bacterium Acetobacter xylinum, a microorganism found in nature, particularly in decomposing fruit. The most important component of wood, cellulose is known for its vegetable origin and for its use in making paper. But it is also obtained from bacteria, algae, and even from marine invertebrate animals. The advantage of the bacterial cellulose, particularly the kind produced by A. xylinum , is that after a series of industrial procedures, it comes to have great mechanical resistance and becomes impermeable to liquids, while maintaining its permeability to gases.

To get to know better the properties of this material and discover new ways of applying it, Bionext Produtos Biotecnológicos, a company from de São Paulo, signed up partnerships with researchers from the Chemistry Institute of the São Paulo State University (Unesp), on the Araraquara campus, and from the São Carlos Physics Institute (IFSC), of the University of São Paulo (USP).The company and the researchers started to study the characteristics and the best way of taking advantage of bacterial cellulose, a product already known to science, though very little used commercially.

It was known that the bacterium, in order to capture oxygen better, produces long microfibers of cellulose that stick on the surface of a liquid medium, forming a zoogloea, (a sort of cover with a gelatinous consistency formed by bacteria). After being removed from this medium, purified, dried and compacted, these microfibers are transformed into extremely thin cellulose films, with potential for use in multiple applications in the medical and industrial area.In the assessment of plastic surgeon and the director of Bionext’s medical area, Lecy Marcondes Cabral, the cellulose film can be used on burns and in other medical procedures in areas of cardiology, neurology, and dentistry. In skin losses caused both by mechanical traumas and by chronic ulcers, the film works like a temporary replacement for this tissue. “It is a biocompatible bandage that adds to our quest for an ideal skin replacement”, says Cabral.

The dressing, also called artificial skin, is placed on the lesion after asepsis. It sticks to the place and, when the new skin grows, falls off as if it were a scab. Both taking a shower and exposure to the sun can be allowed for patients under treatment. Permeable to gases and impermeable to liquids, the artificial skin forms a bacteriological barrier, letting the wound breathe. Another advantage is the possibility of attenuating or even of eliminating pain in these patients. According to the plastic surgeon, the dressing reduces the treatment timeand thereby reduces the cost of hospitalization of patients with burns or chronic wounds.

Armor plating and documents
To develop materials for the manufacture of armor plate and compounds used in bulletproof vests, Bionext can count on guidance from a company specialized in armor plating. “Ballistic tests have shown that the material is highly resistant”, says Professor Bernhard Joachim Mokross, from the IFSC, the coordinator of the project at the universities. Another kind of use is in the production of special papers that may contribute towards the preservation of historical documents. So far, physical tests have been carried out with the film, to assess the resistance and the transparency of the material. “To confirm whether it actually resists the action of time, the behavior of certain chemical processes has to be tested”, says Norma Cassares, a specialist in conserving and restoring paper, who is assessing the material for this application. It is only after being submitted to rapid aging tests to see how it behaves and being analyzed by laboratories specialized in paper that it can begin to be used to restorehistorical documents.

Applications in the medical area are not limited to acting as a dressing. Studies indicate that the bacterial cellulose has potential for replacing, in cases of trauma or tumors, the dura mater, the outermost, thick and fibrous membrane that covers the brain and the spinal cord. The researches started to be done in 1990 by Luís Renato Mello, from the Blumenau University Foundation, in Santa Catarina, when he was doing his thesis for a doctorate in neurosurgery at the Federal University of São Paulo (Unifesp).

At the time, he exchanged the dura mater of 21 dogs for cellulose film, and compared the reactions with the muscular fascia (a fibrous membrane that covers the muscles of the cranium), another replacement for the outermost membrane used in neurosurgery. “I obtained good results, and, with authorization from Unifesp’s Research Ethics Committee, I used it on 25 patients as a phase 1 clinical study”, he reports. The researches were interrupted because, at that time, he was using a similar membrane produced in a non-systematic way by a company called BioFill. As phase 1 is the first of the four stages necessary for studies with human beings, the tests have to be restarted. To do so, Mello enjoys the support of the researchers from Araraquara, who are in search of the best way of producing the replacement membrane for the dura mater.

The film is also being tested for covering stents, small metallic meshes used as a mechanical support to prevent an artery from closing again during angioplasty, a procedure used to unblock coronary arteries in a process of arteriosclerosis (the formation of plaques on the wall of an artery). “The bacterial cellulose is used with the objective of covering the metallic mesh”, says cardiologist Ronaldo Loures Bueno, a professor from the Federal University of Paraná, who is also taking part in the multidisciplinary research group. The stent is introduced, closed, into the coronary artery, and only when it reaches the exact spot of the stenosis (the place where the artery narrows) is the balloon inside it inflated. The stent wrapped in cellulose is thus released against the wall of the artery. Accordingly, it acts against one of the processes that cause restenosis (a later complication in the procedure), which is the migration of smooth muscle cells from the wall of the vessel to the inside of the artery.

Tests in vitro and also with rabbits and pigs were carried out over three months in the United States and Canada. Assessments were made of the inflammatory reaction and of hemocompatibility (compatibility with the blood). “The material has enormous potential for covering stents”, Bueno says. According to the cardiologist, if it is approved in all the preclinical and clinical tests for coronary circulation, it will be able to be used with safety in all kinds of vessel (aorta and arteries in the legs and the arms) and in the tubular system (digestive tube, tubular structures of the lungs, bronchial tubes, trachea, and urethra).

The applications for the material indicate the most varied areas. One of the most recent, studied by professors Younes Messaddeq and Sidney Ribeiro, from Unesp’s Chemistry Institute in Araraquara, are for using the cellulose membranes for flexible luminescent screens (which may serve for computers, televisions or DVDs), with the thickness of a sheet of paper. “We are assessing the physical and chemical properties of the membrane for different applications”, Messaddeq says. The studies involve the molecular structure of the material, using an electron microscope, X-ray diffraction, and thermal analysis, besides the mechanical properties (resistance to traction, hardness, and permeability to liquid and to gases), dielectric properties (insulators of electricity) and luminescent properties (emission of light).

Crystalline polymer
The versatility of the bacterial cellulose, in a comparison with the vegetable kind, is due to the fact that it is chemically pure. This means that it is not accompanied by any other organic compound, unlike vegetable cellulose. “The most complicated thing for the paper industry is to separate the cellulose from the lignin and from the hemicellulose”, Ribeiro says. Whereas the bacterial cellulose only has what is of interest, that is, cellulose. Furthermore, it is a crystalline polymer, which distinguishes it from other kinds of cellulose.

The research into bacterial cellulose involves the production process, and for this reason can count on the consultancy of a specialist in fermentation, chemical engineer Walter Borzani, from the Mauá Technology Institute, of São Caetano do Sul, in São Paulo. “We studied the influence of several factors on fermentation, on yield, on the speed of the process, and on the quality of the product”, Borzani says. To be found amongst these factors are the concentration of nutrients, the temperature, the acidity, and the supply of oxygen. Fermentation for the production of cellulose films is carried out in a liquid medium. Depending on the final product, the whole process, which involves fermentation, collecting the cellulose, washing and drying, takes up to two weeks.

All the products obtained so far are the result of pioneering studies carried out with the A. xylinum bacterium in Brazil by Luiz Fernando Farah, Bionext’s scientific director. The son of a doctor and a former student of law and psychology, Farah says that he identified this microorganism when he was working with ornamental plants. His studies in botany took him to microbiology, where he found citations of the cellulose producing bacterium as a laboratory curiosity. “It was a way of demonstrating bacterial activity”, he says.

According to Farah, the texts would say that cellulose was an abundant material in the vegetable kingdom, but that there was no economic interest in the bacterial product, in spite of it being the purest form found in nature. This contradiction whetted his curiosity. The first strains of the bacterium were brought from Germany by a friend. The culture of the organismwas done at home, because at the time he was not connected with any institutions or companies. Until he arrived at the membrane, he dedicated plenty of time and study to his project. To protect his discovery, since at the time Brazilian legislation did not include protection for medical and food products, the membrane was patented as a dressing, or a skin orthesis, in 1985.

Through BioFill, Farah’s researches received support from the Financier of Studies and Projects (Finep), of the Ministry of Science and Technology. As a recognition of his invention, in 1996 the researcher was given an award by the World Intellectual Property Organization (Wipo), headquartered in Geneva, in Switzerland.For some time, the dressing was manufactured by BioFill and sold to hospitals. On account of an association between the company and a multinational that did not have the product in its business focus, the production was discarded.

In 2002, Bionext came into the circuit and decided to invest, not only in the curative, but in all the possible applications of the membrane. Besides forming a multidisciplinary team, it developed equipment intended to perfect the product and to purify and to dry the membrane, now patented, in order to improve the productive process.The patent of the membrane has now been deposited in Brazil as well, and is being extended to the United States, Asia, and the European Community. Bionext’s plans show that they want to go much further. “We want to have the biggest and most efficient bacterial cellulose factory in the world”, says Nelson Luiz Ferreira Levy, a director of the company.

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