WO2008079034A2

WO2008079034A2 - A biomaterial composed of microbiological cellulose for internal use, a method of producing the biomaterial and the use of the biomaterial composed of microbiological cellulose in soft tissue surgery and bone surgery

Info

Publication number: WO2008079034A2
Application number: PCT/PL2007/000083
Authority: WO
Inventors: Stanislaw Bielecki; Alina Krystynowicz; Marek Kolodziejczyk; Justyna Bigda; Maciej Smietanski; Jerzy Jankau
Original assignee: Politechnika Lodzka; Akademia Medyczna
Priority date: 2006-12-24
Filing date: 2007-12-21
Publication date: 2008-07-03
Also published as: PL381388A1; WO2008079034A3

Abstract

The subject of the present invention are a biomaterial composed of microbiological cellulose for internal use, a method of producing the biomaterial as well as the use of the biomaterial composed of microbiological in soft tissue surgery and bone surgery. The goal of the present invention is the indication of a novel method of manufacturing and application of a biotechnological product, microbiological cellulose, as a material for use in internal body cavities or as an implantable wound dressing for lesions of the lining of the abdominal or thoracic cavity, including the hernia treatment.

Description

A biomaterial composed of microbiological cellulose for internal use, a method of producing the biomaterial and the use of the biomaterial composed of microbiological cellulose in soft tissue surgery and bone surgery

It is particularly significant in the reconstructive treatment of hernias that there is no other method than conventional or laproscopic surgery due to the extensive damage to the abdominal cavity wall, particularly in the case of extensive herniation, exceeding 10 cm in diameter [I]. These treatments make use of allogenic prostheses from synthetic compounds (Dallon, Marlex, Mersilene, PTFE, Polygalacttine 910, Vicryl), and more recently biomaterials (SurgiSIS mesh, AlloDerm, ShellHigh). Attempts have also been made to use autogenic tissue (skin patches and strips and others) or homogenous tissue (renal fascia, dura mater) [2]. Various types of implants are used, depending on the location and extent of the hernia.

The role of the prosthesis is to strengthen the weakened area (herniation site) and to prevent its possible recurrence [3, 4, 9]. A synthetic implant, being a foreign body, must be highly biocompatible and its structure should be amenable to infiltration by growing connective tissue, fusion with the peritoneum and/or fascia to produce a homogenous, durable scar. When using this method, the size and the recurrence of the hernia are irrelevant. Li most cases, the mesh is implanted on the inside of the fascia, and the material is thus pressed against the lips of the wound by the tonal pressure, the very same which causes herniation [5, 6].

The type of synthetic material used is important, because it influences any complications such as intestinal punctures or fusion [7]. The use of synthetic mesh may also cause excessive seroma accumulation and this also depends on the type of mesh used. Seroma can gather most copiously around politetrafluoroethylene (PTFE) mesh, more rarely around polypropylene and occasionally polyester. In order to prevent fusion between the synthetic implant and abdominal organs, the materials used are coated on one side with teflon, silicon or fibrin. Alternatively, an absorbable mesh is used on this side, which covers the non-absorbable mesh [8] .

Since the use of polypropylene or polyester meshes leads to the risk of intestinal fusion or punctures, it is necessary to separate the remaining synthetic implant from the organs using any conserved peritoneum of the hernia. In order to prevent fusion between the synthetic implant and abdominal organs, the materials used are coated on one side with teflon, silicon or fibrin or the non-absorbable mesh is covered by absorbable material.

The choice of mesh is based on the type of hernia, its location, the patient overall physical condition, any additional illnesses and body mass [9].

In the case of extensive herniation, regardless of the type of operation, heavy meshes are used (Dacron, Mersilene), since they make it possible to reconstruct the shape of the abdominal cavity. The newly formed alloplastic-tissue layer gives the mesh enough strength, even at high pressures inside the cavity.

Light polypropylene meshes are used most frequently to dress small hernias, or alternatively partially absorbable meshes of increased elasticity [6]. Synthetic meshes used in laproscopic hernia surgery are usually attached to the abdominal wall using titanium staples.

Surgical methods of repairing hernias using synthetic prostheses entail a danger of infection, like all surgical interventions. Presently, local antibiotic therapy or initial saturation of the prosthesis with antiseptics facilitate the treatment of infections without the need to remove the implant [6, 5, 10, H]. This excludes polytetrafluoroethylene implants, which always need to be removed upon infection.

The need and frequency of using synthetic implant in the surgery of abdominal fascia and hernia operations, as well as the arising imperfections of the products available on the market, continue to inspire research on new materials which would reduce the risk of complications dangerous to the patient's health or even his life.

The most frequent complaints pertain to increased scarring (much greater than that described by the manufacturer), but also mesh shrinkage. The former property leads to tissue fusion between abdominal organs, usually the small and/or large intestine and the mesh, as well as the staples which affix the mesh to the peritoneum. This results in stoppages of the gastric tract after a few years, often needing emergency surgery. The latter property, implant shrinkage, causes hernia recurrence when after several months or years the mesh placed on the defect does not cover its entire extent. Despite the multitude of materials available on the market, there are none which would fully satisfy the surgeon, and in effect the patient.

Microbiological cellulose may be the material which could eliminate all problems entailed by the use of synthetic implants. In vitro and in vivo research on microbial cellulose confirms that due to its biological and physical characteristics it is a "medical quality" material [8, 12].

Microbiological cellulose consists of β-1,4 glucan chains, and is chemically identical to plant cellulose. Microbiological cellulose is a highly crystalline cellulose rich in the Ia fraction and is synthesized in a reaction catalysed by the cellulose synthase in the active UDPG form and the allosteric activator c-di-GMP. The cellulose synthase operon is known, as are the functions of the proteins encoded by the genes contained therein [13, 14, 10, 15]. It is a nanoproduct, since it consists of microfibrils some 3 nm across, which form a fibril, known as a strand, which is some 100 nm across. In contrast to phytocellulose, microbiological cellulose is of very high purity, as it is accompanied by no other substances. Basic research regarding glucagon chain polymerization, crystallization and the molecular regulation of synthesis is accompanied by technological studies aimed at optimising the production conditions of the cellulose material using various culture methods, depending on the final use of the product [1, 16, 17].

Cellulose strands made by many bacterial cells form an intricately intertwined web, which forms an elastic, highly hydrated membrane. Said membrane gathers on the surface of the medium in stationary culture [13]. The texture of thusly formed material is reminiscent of the fibrous structure of muscle. The efficiency of the biosynthesis process is dependent on the activity of the producing strain, the composition of the growth medium and the culture conditions.

Detailed in vivo research by Helenius et al. [8,12] proved that cellulose material implanted subdermally in Wistar rats did not cause inflammation during the study period (12 weeks) and integrated well with host tissues.

Patents describing methods of surgical treatment of abdominal hernias using synthetic implants most often give examples of the use of polyester, polypropylene, or polytetrafluoroethylene (PTFE) meshes, in combination with biocompatible polymers: proteins or polysaccharides. Patent description WO9603165 (publ. 1996-02-08) presents an application of a gelatine- covered polyester mesh, whereas description G/32406522 (publ. 2005-04-06) describes implants in which the absorbable layer is made of starch or cellulose gel. Patent descriptions US2003/100955 (publ. 2003-05-29), WO00143789 (publ. 2001-06- 21) present bilayer implants: a non-absorbable mesh and an absorbable mesh composed of urea derivatives, hyaluronic acid or cellulose derivatives (CMC). The latter is meant to prevent fusion with tissues.

Patent description JP2003062062 (publ. 2003-03-04) describes a method of producing heterogenous implants, composed of non-biodegradable materials and synthetic degradable polymers (i.e. poliglycolic acid, polyhydroxyalkanic acid and their copolymers), and natural polymers such as chitin, cellulose and collagen.

Patent descriptions WO9951163 (publ. 1999-10-14), US2006083767 (publ. 2006-04- 20), WO200602922 (publ. 2006-02-23) discuss the merits of the use of bilayer prostheses composed of materials differing in biodegradability, particularly useful in modelling abdominal fascia. The more durable layer construes reinforcement facilitating the formation of stronger scars, whereas the more rapidly degraded material in contact with the internal organs prevents tissue fusion.

Implants used in the surgical treatment of hernias may also contain: local analgesic preparations (US 2005015102 (publ. 2005-01-20)), tissue adhesives to prevent prosthesis displacement (US200501239 publ. 2005-01-13), substances preventing tissue fusion such as hyaluronic acid (JP5124968 publ. 1993-05-21), and antibacterial substances (WO9603165 publ. 1996-02-08).

Patent descriptions PL171952 (publ. 1995-02-06) and application P-317139 (filed 1996-11-20) as well as WO2005003366 (publ. 2005-01-13) relate to a method of producing microbiological cellulose in the form of membranes via surface cultures of Acetobacter xylinum. The solution is based on the isolated bacterial strain of Acetobacter xylinum P23 of the genus Acetobacter, an oval rod shaped organism. This bacteria is a typically aerobic organism, with growth optima at 28-30°C, pH 4,0 - 6,5 on solid media containing glucose, yeast extract, peptone and agar where it forms colonies. In liquid media it forms a slimy, dense membrane and is characterised by the ability to produce acids via the incomplete oxidation of most carbohydrates and alcohols, where the acids produced as final or temporary products are secreted into the medium. In the presented solution the P23 strain of Acetobacter xylinum is incubated in a liquid medium. The method according to said invention produces cellulose membranes containing α-cellulose at 90 - 97%. Such membranes may be used as wound dressings in surgery and dermatology. Patent description WO8602095 (publ. 1986-04-10) relates to a method of producing cellulose membranes using microbiological synthesis, used as artificial skin for grafting. Patent descriptions US4788146 (publ. 1988-11-29) and US45884000 (publ. 1986-05-13) relate to methods of forming cellulose membranes in cultures of Acetobacter xylinum, methods of their purification and then subsequently their application in the treatment of burns and other lesions.

Patent descriptions JP03165774, JP08126697, JP03272772, JP63205109, EP0369344, US2003013163 (publ. 2003-01-16), and WO0161026 (publ. 2001-08-23) present methods of manufacturing cellulose materials in the form of tubules (and others) and attempts to use said materials in microsurgery as vasoprostheses or prostheses of other internal conduits. Examples of composites of microbiological cellulose in flake form with a layer of calcium phosphate are given by patents JP7132214 (publ. 1995-05-23) and JP8229115 (publ. 1996-09-10). The material produced is characterised by a high degree of biocompatibility and elasticity. Used as an internal prosthesis, it prevents the formation of punctures in the intestines and tissue fusion in the abdominal cavity. Patent descriptions CA2506691 (publ. 2004-06-03) and US2006147612 (publ. 2006-07- 06) describe an endoprosthesis and methods of producing it. They describe a metal mesh structure coated by a biomaterial, more precisely a cylindrical endoprosthesis coated with a biosynthetic cellulose membrane for the treatment of atherosclerosis, meant to be placed in the lumen of an artery.

Despite the described methods of the reconstruction, completion and reinforcement of soft and bone tissues using synthetic endoprosthetic implants and their composites with polymers there still exists the risk of complications since every kind of implant has drawbacks due to biological, physico-chemical and mechanical properties. Due to the enormous demand for synthetic implants for soft-tissue and bone surgery there is still a real need to seek prostheses facilitating the limitation of complications, particularly in the form of tissue fusion and intestinal punctures.

The goal of the present invention is to yield the means of producing implants composed of microbiological cellulose in the form of heterogeneous cellulose membrane or its composites of medical quality.

The embodiment of such a stated goal and the solution of the problems described in the state of the technology connected with the search for new materials, which would lessen the risk of complications entailed by increased tissue fusion between abdominal organs and the mesh, as well as the staples affixing the mesh to the peritoneum, and in effect bowel obstructions, and implant shrinkage causing recurrences of the hernia have been resolved in the present invention. The subject of the present invention is a biomaterial composed of microbiological cellulose for internal use, characterized in that when introduced into an organism it becomes covered with a structure of connective tissue, and that it retains the characteristics of a native membrane and biocompatibility with surrounding tissue. Preferentially, the biomaterial is produced in the form of a composite with the fibres of other substances.

Preferentially, the microfibrils of the microbiological cellulose encompass the fibres of a mesh of other synthetic material, preferentially a mesh of synthetic or natural polymers, wherein a homogenous layer of membrane forms in the eyelets of the mesh, which seals the mesh and does not separate from it. Preferentially, its absorbability, ranging from 10 - 90%, is proportional to its degree of dehydration and the culture conditions, particularly the source of carbon used in the medium.

Preferentially, the material constitutes a carrier of chemical compounds exhibiting bacteriocidal or bacteriostatic properties for internal use. Preferentially, the material constitutes a carrier for a substance which alters local or systemic blood clotting.

Preferentially, the cellulose material constitutes the reinforcing material, or coating material or a reinforcement for another implant material. Preferentially, it is meant to serve as a protectant for blood vessel connections, preferentially in the connection of the vessels of a donor and recipient in the case of soft organs, and haemostatic for internal bleeding in the case of resection or defect of soft tissues, preferentially the spleen, liver or kidney, both autogenic and transplanted, or haemostatic as a dressing material, or as a dressing which may be a carrier for antibiotics for use at sites of organ removal.

Preferentially, the biomaterial contains anticoagulant substances, and in the case of vascular prostheses serves as a protectant for said prostheses.

The next subject of present invention is a method of producing a biomaterial with a structure composed of bacterial cellulose microfibrils and fibres of another polymer, characterized in that after a shaken culture, in which cellulose fibres pervade the mesh of another polymer, a stationary culture is carried out where the mesh is penetrated and the cellulose accretes on its surface. Preferentially, a mesh of artificial or natural polymers is used, with an eyelet size of 0.05 x 0.05 to 0.4 x 0.4 cm, in which additional holes are cut out, with diameters of 0.5 cm to 1.5 cm at intervals of 1.0 cm to 3.0 cm, wherein these holes a homogenous layer of membrane is formed which encloses the mesh and produces a perforated cellulose material. Preferentially, following culturing and cleaning, the cellulose membrane is perforated using a cutter punching holes with diameters of 0.08 to 0.5 cm at 0.7 — 2.5 cm intervals. Preferentially, the level of medium in the bioreactor is no higher than the thickness of the composite.

The next subject of present invention is an use of the biomaterial composed of microbiological cellulose, which consists of a material composed of a meshwork of microbiological cellulose microfibrils and/or consists of a composite of microbiological cellulose and the fibres of another polymer, where the biomaterial becomes encased in a connective tissue structure, and retains the characteristics of a native membrane and biocompatibility with surrounding tissues, in the surgery of soft and hard tissues. Preferentially, the biomaterial is supplemented with an indicator of the same or other material which facilitates orientation within the abdominal cavity and its installation. Preferentially, the biomaterial is designed to cover sites of resection or defect of soft tissues and/or other organs, bones and/or cartilage, or other structures of bodily cavities, preferentially the following membranes: peritoneum, pleura, diaphragm, in the reconstruction of lesions of the diaphragm resulting from diaphragmatic hernias, both innate and acquired.

Preferentially, the biomaterial constituting a carrier for chemical compounds exhibiting bacteriocidal properties is designed to combat deep infections such as abscesses, phlegmonous cellulitis and/or other infections inside the bodily cavities, or sub- and epifascial infections, adipose tissue infections including epiperitoneal adipose tissue, retroperitoneal space, periorganic spaces and as absorbent pads to be used in abscesses and at infected sites, including perianal abscesses and perianal fistula. Preferentially, the biomaterial is used in the reconstruction of previously infected tissues, implanted into the infected environ and/or with another accompanying infective disorder of the abdominal cavity, particularly when the biomaterial is used in the reconstruction of previously removed infected or necrotic tissue, or implanted into an excised tissue fragment.

The attached figures facilitate better understanding of the nature of the present invention.

Figure 1 , A mesh implanted subperitoneally.

Figure 2. A method of evaluating intraperitoneal adhesions.

Figure 3. After 3, adhesions evaluated as being at most 1 on the Hooker scale.

Given below are example embodiments of present invention defined above. Examples of producing cellulose membranes and composites with synthetic materials (Examples 1 — 7)

Example 1.

Pl inoculation medium containing by mass: 20 parts glucose, 5 parts yeast extract, 5 parts peptone, 2.5 parts MgSO₄x7H₂O, 2.7 parts Na₂HPO₄, 1.15 parts citric acid, 10 parts ethanol, to 1000 parts distilled water, is inoculated with 5% (v/v) of Gluconacetobacter suspension (5xlO⁷ U/ml) maintained on this medium in 50 ml batches, for no longer than 7 days at 4°C, and incubated for 2 days at 30°C, resulting in a culture, which following intensive mixing is used to inoculate the production medium of identical composition at a rate of 5% (v/v) and the whole mixture is preincubated for 1 day at 30°C. Thereafter the mixture is transferred into bioreactors of an appropriate surface area, such that the surface area/volume ratio (S/V) is from 0.4 to 0.8 cm^"1. The membranes formed after 7 days (7 to 10 mm thick) are cleaned by pressing out the culture medium (from 50 - 60% of their initial mass), rinsing in hot tap water, whereafter the membranes are treated with 1% NaOH at 100⁰C for Ih, or for 20 - 24 h at RT. After this, the material is rinsed again in tap water, wrung out to 40 to 50% of its initial mass, treated with 1% acetic acid for 20 to 24 h, rinsed again in tap water and then in distilled water. Well cleaned membranes should be white, translucent, and should have a neutral pH. Excess water is wrung out, whereafter the membranes, containing ca. 90% water in relation to dry mass, are packed into sealed foil bags and sterilized thermally or by irradiation.

Example 2 The culture was carried out as described in Example 1, with the change that animal peptone was not included.

The culture was carried out as in Example 1, except that at the bottom of the reactor we placed a sterile plate, into which we placed vertical glass rods 0.5 cm spaced out some 1.5 to 2.0 cm, or 0.08 cm in diameter, spaced out at 1.5 cm intervals. The tips of these rods protruded 1 cm above the surface of the medium. After culturing for 7 days the perforated membranes were cleaned, and sterilized as in Example 1.

Example 3

The cellulose membrane process was carried out as in Example 1, except that after wringing out the cleaned membranes they were perforated using a cutter, to produce holes of 0.5 cm or 0.08 cm at 1.5 cm intervals. The material produced was packed and sterilized as in Example 1.

Example 4.

In the culture medium as in Example 1, glucose was replaced with fructose and the process continued as in Example 1. Example 5.

A stationary culture was carried out as in Example 1, using a bioreactor with a surface area of 133 cm². 0.05 dm³ of culture medium were prepared. After this medium was inoculated as in Example 1, the production culture was carried out on a medium thickness of about 0.38 cm. Prior to the addition of the medium, a sterile natural or synthetic fibre mesh was placed at the bottom of the reactor 8 cm wide by 10 cm long, and a stationary culture was maintained for 7 days. After the initial 2 days of culturing, when a cellulose membrane 0.2 to 0.3 cm thick had formed on the mesh, it was turned over, and the culture was continued adding 0.5 ml of the medium daily for the next 5 days. The thickness of medium in the reactor should not exceed the thickness of the composite. The composite was sterilized and cleaned as in Example 1.

Example 6. Using a medium like in Example 1, 2 or 4, an agitated culture was carried out under the following conditions: a bioreactor 24 cm x 17 cm x 7 cm, in it a polypropylene mesh affixed horizontally at 1.0 to 1.5 cm from the bottom, was filled with medium such that the mesh was 0.3 - 0.5 cm above the medium surface, and the oscillating shaker was set to 50 - 70 RPM. The culture was maintained for 4 - 5 days at 3O⁰C. During such a culture, the cellulose accretes on the surface of the mesh, and within its eyelets. Over the next 3 to 4 days of culture the cellulose continues to accrete on the surface of the mesh. Cleaning and purification of the material was carried out as in Example 1.

Example 7.

Under conditions as in Example 1, a polyester mesh was used (mesh density of 0.2 x 0.2 cm) with additional 1 cm eyelets every 1.5 cm - 2.0 cm. A homogenous layer of membrane formed in these eyelets, which sealed the mesh.

Physico-chemical properties of cellulose membranes (Example 8 - 10)

Example 8.

The membranes produced according to Examples 1 and 5 were used in research meant to ascertain their physical and mechanical characteristics. The research was carried out using a tensile testing machine TMM-I 111, with the following parameters set for all of the samples:

- distance between the tensile testing machine jaws 10 cm

- width of the membrane strip tested 2 cm - thickness of the membrane strip tested 2 - 5 mm

- tensile speed lO cm/min

- test force range 100 N

- recording paper tape speed 50 cm/min

- relative air humidity RH 20°C The following parameters were measured: tensile force (F), tensile stress (G), relative tensile elongation (ε), modulus of elasticity (E), longitidudinal tearing force value (F₁-). The tensile strength of the membranes grows as the degree of wringing out grows. The characteristics of this increase differs for membranes grown in media containing glucose and fructose. The increase in tensile strength along with the degree to which the membrane was wrung out is much greater in membranes produced on fructose media than, despite a similar cellulose content prior to wringing out. When comparing the ultimate elongation, the membrane produced on the fructose medium is advantageous. This membrane is also characterised by a higher tensile force value.

Table 1. Indicators of resistance and distortion of cellulose membranes as a function of wringing out

Example 9.

Bacterial cellulose 4 cm x 4 cm x 0,3 cm, obtained as in Examples 1, 2 and 3, was tested for its ability to resist hernia. An INSTRON model 1112 testing device was used setting the following parameters for all samples:

- external diameter of the ring for testing a hernia force 68,7 mm

- internal ring diameter 20,0 mm

- piercing ball bearing diameter 18,0 mm

- horizontal cross-bar speed 5,0 mm/min.

- recording paper tape speed 50,0 mm/min.

- relative air humidity RH 20⁰C

The results are presented in a table:

The results (table 2) show that the full membrane composed of z microbiological cellulose is the most resistant material to herniation, and the weakest membranes are those perforated with larger diameter holes. Among the perforated membranes, regardless of perforation size, membranes in which holes have been cut are stronger than those in which holes were formed during culturing. Table 2. Resistance of cellulose samples to hernia

Example 10.

The effect of the degree of wringing out on the absorbability of the membranes. Cellulose membranes produced as in Example 1 were used to test for their ability to absorb water depending on how well they had been dehydrated. The masses of the samples in question were weighed, membranes dehydrated by 10-90% in relation to their initial mass were used. It was determined that the absorbability of the membrane produced on the fructose medium (CeIF) is lower than that produced on the glucose medium (CeIG). Thus, for example, a CeIF sample having lost 86% of its water will absorb around 27%, whereas a CeIG sample dried to an identical extent will absorb some 40%.

Biological comparative studies in vivo of tested cellulose nanomeshes

Example 11. The technique and conditions of implantation during research

Comparative in vivo analyses of meshes were carried out on Wistar rats with body masses of about 250 g. hi the research we used a nanomesh of bacterial cellulose obtained and prepared as in Example 1, and a polypropylene mesh admitted for medical use. The animals were divided into 4 groups: 1. Experimental group - 15 animals. A cellulose mesh was implanted and left inside the peritoneum for 21 days.

2. Experimental group - 15 animals. A cellulose mesh was implanted and left inside the peritoneum for 90 days.

3. Control group - 1 animal. A polypropylene mesh was implanted and left inside the peritoneum for 21 days. 4. Control group - 1 animal. A polypropylene mesh was implanted and left inside the peritoneum for 90 days.

The rats were anaesthetized prior to implantation of the mesh using intraperitoneal ketamine at 50 mg/kg and xylazine at 10 mg/kg. After anaesthesia, the rats were placed horizontally on their dorsal side. After shaving the incision site and disinfection (2 x 72% ethanol in water), the abdominal fascia were exposed using a lengthwise incision about 3 cm long. Laparotomy was performed on lengths of 2 — 2.5 cm. The implants, 1.6 x 2.6 cm in each group of animals, were sutured in place with single unabsorbable sutures (Prolene 3-0, Eticon Inc.) under the subperitoneally and additionally covered with the external tendon layer of the front abdominal wall (Fig. 1). Subdermal tissue was sutured with a absorbable suture (Vicryl 4-0, Ethicon Inc.), and the skin with non- degradable single sutures (Prolene 3-0, Eticon Inc.). Meshes were implanted to all animals subperitoneally, according to the internationally used IPOM {intraperitoneal onlay mesh) protocol. Example 12.

Sampling techniques and evaluation of material cultured in vivo

In order to collect the implants after 21 and/or 90 days following implantation (depending on the experimental group) the animals were anaesthetized as in Example 13, and then sacrificed using overdose of trichloroethanediol. The abdominal cavities of killed animals were opened using a lateral cut some 3 cm from the centre line, in order to visualise the implant. Slowly lifting the lips, the mesh was exposed and any attached structures as well. Under tension, the adhesions formed in peritoneum were examined according to the Hooker descriptive scale, where: 0 - no adhesions; 1 - adhesions sites easily removed, blunt tool; 2 - moderately difficult to remove adhesions zones, sharp tool; 3 - difficult to remove adhesions sites, need for sharp tools (Figs. 2 and 3).

The above were compared with the results of the implantation of an ordinary polypropylene mesh (control group).

Bacteriological examination:

In all cases in which symptoms of infection were observed at the site of surgery, material was collected for bacteriological examination (a swab or mesh fragment) in order to culture and determine the degree of colonization of the mesh following surgery. The identification was designed to show the origin of the bacteria. The presence of intestinal flora would indicate intestinal damage during implantation or anaesthesia, whereas Acinetobacter spurium could indicate accidental infection during the procedure.

Results: 1. No mesh shrinkage.

2. Minimal adhesions formation = degrees 0 or 1 on the Hooker scale (Fig. 3).

3. No bedsores nor lesions of the serosa or peritoneum under the mesh.

4. No intestinal fistulas observed (Fig. 2).

5. No seroma formation (potential for abscess formation following infections of serum reservoirs) (Fig. 2).

6. Maintenance of biological and mechanical characteristics of the implant 3 weeks and 3 months following the implant (Fig. 3).

7. Weak inflammatory titre (no tissue titre for the material).

8. No infective characteristics suggests the bacteria come from the "manufacturing" process.

Final conclusions:

The physico-chemical characteristics of the membrane composed of microbiological cellulose make it possible for it to be used in the treatment of abdominal integument internal defects, particularly in the treatment of hernias due the limitation of adhesions, ischaemic spaces and fistulas (facilitating direct contact with all organs of the abdominal cavity), using both classical surgery and laparoscopy. The use of microbiological cellulose in the form of a perforated sheet facilitates its more extensive overgrowth with a layer of connective tissue, through easier penetration of the entire structure. This method of applying cellulose membranes is particularly due to: biocompatibility of the material, its mechanical resistance, elasticity, chemical purity, as well as the possibility of producing biomaterial of a desired surface area and shape, as well as its ease of sterilization. Use of this material as an implant would improve the quality and effectiveness of treatment, and its production would satisfy social needs.

The cellulose contained in the membranes is characterized by a high α-cellulose content (above 90%) with a crystalline content of above 60%. The porosity of the material is a product of the spatial structure of the intricately intertwined nanofibers. Pores with diameters less than 3 μm cover 50-93% of the surface area. The thinness of the nanofibers ensures that the internal surface of the material is highly developed and this is essential to its large absorptive ability. Another important characteristic of materials composed of bacterial cellulose, significant to its use in medicine, is its high purity, absence of irritant and allergenic properties as well as its biocompatibility.

Claims

Patent Claims

1. A biomaterial composed of microbiological cellulose for internal use, characterized in that when introduced into an organism it becomes covered with a structure of connective tissue, and that it retains the characteristics of a native membrane and biocompatibility with surrounding tissue.

2. A biomaterial according to Claim 1, characterized in that the biomaterial is produced in the form of a composite with the fibres of other substances.

3. A biomaterial according to Claims 1 or 2, characterized in that the microfibrils of the microbiological cellulose encompass the fibres of a mesh of other synthetic material, preferentially a mesh of synthetic or natural polymers, wherein a homogenous layer of membrane forms in the eyelets of the mesh, which seals the mesh and does not separate from it.

4. A biomaterial according to Claims 1 or 2 or 3, characterized in that its absorbability, ranging from 10 - 90%, is proportional to its degree of dehydration and the culture conditions, particularly the source of carbon used in the medium.

5. A biomaterial, according to Claims 1 or 2 or 3, characterized in that constitutes a carrier of chemical compounds exhibiting bacteriocidal or bacteriostatic properties for internal use.

6. A biomaterial, according to Claim 1 or 2 or 3, characterized in that the material constitutes a carrier for a substance which alters local or systemic blood clotting.

7. A biomaterial according to Claim 1 or 2 or 3, characterized in that the cellulose material constitutes the reinforcing material, or coating material or a reinforcement for another implant material.

8. A biomaterial according to Claim 1 or 2 or 3, characterized in that it is meant to serve as a protectant for blood vessel connections, preferentially in the connection of the vessels of a donor and recipient in the case of soft organs, and haemostatic for internal bleeding in the case of resection or defect of soft tissues, preferentially the spleen, liver or kidney, both autogenic and transplanted, or haemostatic as a dressing material, or as a dressing which may be a carrier for antibiotics for use at sites of organ removal.

9. A biomaterial according to Claim 1 or 2 or 3, characterized in that the biomaterial contains anticoagulant substances, and in the case of vascular prostheses serves as a protectant for said prostheses.

10. A method of producing a biomaterial with a structure composed of bacterial cellulose microfibrils and fibres of another polymer, characterized in that after a shaken culture, in which cellulose fibres pervade the mesh of another polymer, a stationary culture is carried out where the mesh is penetrated and the cellulose accretes on its surface.

11. A method according to Claim 10, characterized in that a mesh of artificial or natural polymers is used, with an eyelet size of 0.05 x 0.05 to 0.4 x 0.4 cm, in which additional holes are cut out, with diameters of 0.5 cm to 1.5 cm at intervals of 1.0 cm to 3.0 cm, wherein these holes a homogenous layer of membrane is formed which encloses the mesh and produces a perforated cellulose material.

12. A method according to Claim 10 or 11, characterized in that following culturing and cleaning, the cellulose membrane is perforated using a cutter punching holes with diameters of 0.08 to 0.5 cm at 0.7 - 2.5 cm intervals.

13. A method, according to Claim 10 or 11, characterized in that the level of medium in the bioreactor is no higher than the thickness of the composite.

14. Use of the biomaterial composed of microbiological cellulose, which consists of a material composed of a meshwork of microbiological cellulose microfibrils and/or consists of a composite of microbiological cellulose and the fibres of another polymer, where the biomaterial becomes encased in a connective tissue structure, and retains the characteristics of a native membrane and biocompatibility with surrounding tissues, in the surgery of soft and hard tissues.

15. Use according to Claim 14, characterized in that the biomaterial is supplemented with an indicator of the same or other material which facilitates orientation within the abdominal cavity and its installation.

16. Use according to Claim 14, characterized in that the biomaterial is designed to cover sites of resection or defect of soft tissues and/or other organs, bones and/or cartilage, or other structures of bodily cavities, preferentially the following membranes: peritoneum, pleura, diaphragm, in the reconstruction of lesions of the diaphragm resulting from diaphragmatic hernias, both innate and acquired.

17. Use according to Claim 14, characterized in that the biomaterial constituting a carrier for chemical compounds exhibiting bacteriocidal properties is designed to combat deep infections such as abscesses, phlegmonous cellulitis and/or other infections inside the bodily cavities, or sub- and epifascial infections, adipose tissue infections including epiperitoneal adipose tissue, retroperitoneal space, periorganic spaces and as absorbent pads to be used in abscesses and at infected sites, including perianal abscesses and perianal fistula.

18. Use according to Claim 14 or 17, characterized in that the biomaterial is used in the reconstruction of previously infected tissues, implanted into the infected environ and/or with another accompanying infective disorder of the abdominal cavity, particularly when the biomaterial is used in the reconstruction of previously removed infected or necrotic tissue, or implanted into an excised tissue fragment.

Literature:

1. Thoman D. S., Philips E.H.: Current status of laparoscopic ventral hernia repair. Surgical Endoscopy and other Intervetional Techniques (2002)16: 939 - 942.

2. Rutkow I.M., Robbins A.W.,: The mesh plug technique for recurrent groin herniorrhaphy: a nine -year experience of 407 repairs. Surgery, 1998,124,844.

3. Prywiήski S. i wsp.: Wyniki leczenia duzych przepuklin w bliznie. Wybrane zagadaysenia z Chir., 1999, t. IV,366. 4. Awad S., Shwan P., Fagan S.: Current approaches to ingual hernia repair. Am. J. Surg. 188,(suppl., to December 2004)9S-16S

5. Read R.C.: The contributors of Usherand others to the elimination of tension from groin herniorrhaphy. Hernia, 2005, 10:s 10029-005-0322-1

6. Wantz G.E., i wsp..: Incisional hernia: the problem and the cure. J.Am. Coll. Surg., 1999, 188,429.

7. Solorzano C.C.i wsp.: Prospective evaluation of the giant prosthetic reinforcement of the visceral sac for recurrent and complex bilateral inguinal hernias. Am. J. Surg., 1999,117,19.

8. Adamonis W. i wsp. Alloplastyka przepuklin brzusznych w bliznie pooperacyjnej. Wybrane Zagadaysenia z Chir., 1999, t.IV,264

9. Read R.C.: Inguinal herniation in the adult, defect or disease: a surgeon odyssey. Hernia,2004;8:296-299.

10. Celdran A., FrieyroO., de Ia Pinta J.C. i wsp. The role of antibiotic prophylaxis on wound infection after mesh hernia repair under local anesthesia on an ambulatory basis. Hernia, 2004,8,20-22.

11. Kuzu M.A., Haiznedaroglu S., Dolalan S. i wsp.: Prevention of surgical site infection after open prosthetic inguinal hernia repair: Efficacy of paretnteral versus oral prophylaxis with amoxicilin- clavoulonic acid in randomized clinical trial. World J. Surg.,2005,29,794-799 12. Helenius G., Backdahl H., Bodin A., Nannmrak U., Gathenholm P., Risberg B.jn vivo biocompatibility of bacterial cellulose., Wiley Periodicals,Ine.,2005,431-438

13. Bielecki S., Krystynowicz A., Turkiewicz M., Kalinowska H.: Bacterial cellulose.; Biopolymers,2002,5(3),37-90.

14. Asvang E., Kethlet H.,: Surgical management of chronic pain after inguinal hernia repair. Br. J. Surg.,2005,92.885-889

15. Saxena M., Brown R.M.Jr.: Cellulose biosynthesis., Annals of Botany 96, 2005,9- 21.

16. Krystynowicz A., Czaja W., Bielecki S.: Biosynteza i mozliwosci wykorzystania cellulose bakteryjnej. Zywnosc .;1999, 3(20), 22-34.

17. Krystynowicz K., Koziotkiewicz M., Wiktorowska-Jezierska A., Bielecki S., Klemensa A., Masny A., Plucienniczak A.:Molecular basis of cellulose biosynthesis disapperance in submerged culture of Acetobacter xylinum; Acta Biochemica Polonica, 52,3,2005,691- 698