WO2011000071A1

WO2011000071A1 - Method for manufacturing a biocompatible collagen membrane for use in medicine and dentistry, and resultant product

Info

Publication number: WO2011000071A1
Application number: PCT/BR2010/000221
Authority: WO
Inventors: Benedicto De Campos Vidal; Ariel Lenharo
Original assignee: Agência De Inovação-Inova
Priority date: 2009-07-01
Filing date: 2010-06-29
Publication date: 2011-01-06
Also published as: BRPI0902410B8; BRPI0902410B1; BRPI0902410A2

Abstract

The present invention relates to a method for manufacturing a collagen membrane with uses in the field of medicine and dentistry. This membrane, which is industrially produced by a process involving chemical and thermal treatment steps, preserves the collagen fibres, which are arranged so as to withstand traction upon the fibres and remove cells and fat from the original tissue.

Description

PATENT DESCRIPTION REPORT

INVENTION

"BIOCOMPATIBLE COLLAGENIC MEMBRANE MANUFACTURING PROCESS FOR MEDICAL-ODONTOLOGICAL APPLICATION AND PRODUCT OBTAINED"

FIELD OF INVENTION

The present invention relates to a process for obtaining a collagen membrane consisting of type I collagen for medical and dental application.

The obtained membrane acts as a barrier in dental surgeries, in which it is desired to form bone tissue, preferably to fibrous tissue. In addition, it can be used in surgeries where it is desired to protect bone graft material.

This membrane obtained from industrial processing, which involves chemical and thermal treatment steps, allows the preservation of collagen fibers, arranged to provide tensile strength and remove cells and fats from the original tissue.

This results in better cell surface adhesion, cell recognition, proliferation and differentiation. oriented. It features easy handling and sufficient strength to withstand the tension of sutures and to provide support for the healing process of the periodontal support and protective tissue healing.

In the treatment of skin burns and ulcerations, biomembranes perform the following functions: restore the vapor barrier and prevent wound dehydration; decrease evaporation by heat loss; prevent bacterial contamination by protecting the wound and the patient from septicemia; allow less painful dressing changes; facilitate wound debridement and improve the quality of healing.

In the medical field, the biomembrane can still be used in urogenital, colon and rectum, stomach, obstetrics and gynecology, otorhinolaryngology, plastic surgery, urology, vascular surgery,

BACKGROUND OF THE INVENTION

The application of the present invention may be used as a dressing to aid in the treatment of burns. It may also be used in cases of urogenital surgeries, such as for the placement of urethral bands for the treatment of urinary incontinence and pelvic reconstruction. repair of wall defects, including ventral and inguinal hernias.

In the dental area, it acts as a barrier in the guided tissue regeneration (RTG) and guided bone regeneration (ROG) techniques. In surgical cases, in the dental area, where tissue regeneration is desired, it is difficult to create conditions to prevent the growth and proliferation of other tissues in the area where bone regeneration is required.

Formation of new bone tissue following surgery is often hampered by infiltration of faster-growing connective tissue cells such as gingival tissue fibroblasts.

In periodontal RTG, a physical mechanical barrier is placed between the gingival flap and the instrumented root surface prior to flap repositioning and suturing (Quinones and Caffesse, 1995).

These principles are also used in the recovery of extensive bone defects, also called perennial bone defects, called Guided Bone Regeneration (ROG), whose action is to increase bone volume, using a membrane as a barrier, a term proposed by Buser. et. al. Dahlin et al. originally applied this technique to regenerate bone in bone loss, establishing the concept of "osteopromotion". This technique has been applied in the dental clinic in several cases, including dental implant therapy, in cases where bone volume at the implant site is insufficient.

Initially, non-resorbable barriers were used in RTG. The first commercially available RTG barrier was manufactured from expanded polytetrafluoroethylene (Gore-Tex ^® , Periodontal Material, WL Gore & Associates, Flagstaff).

However, the use of this type of material requires a second surgical procedure to remove the membrane. Four to eight weeks after the first surgery a second surgical procedure is required to remove the membrane.

Resorbable barriers have been evaluated regarding their safety and their role as a barrier in tissue regeneration, such as collagen, polylactic acid, polyglycolic lactic acid. Resorbable barriers are of great interest because they do not require a second surgical act to remove them. Avoiding this second surgical procedure has the benefit for the patient of not having a second surgery and eliminating surgical trauma in new and immature periodontal tissue that has been regenerated. (Wang and McNeil, 1998)

Among these resorbable materials, collagen has deserved prominence, as it constitutes one of the main components of the extra-cellular matrix of periodontal connective tissue. In addition, it is physiologically metabolized, fibroblast chemotactic, hemostatic, has poor immunogenicity, and functions as a carrier for cell migration (Blumenthal, 1988; Pitaru et al., 1887).

Collagen is the main structural protein of vertebrate tissues and the most abundant of connective tissue and can be found in the skin, bones, tendons and teeth. In bone tissue corresponds to 90% of the organic portion of this. Its main feature is the formation of insoluble fibers with high tensile strength.

Collagen is considered one of the most useful biomaterials. Due to its physicochemical properties (presence or addition of crosslinking or variable cross-linking controlling the degree of imbibition and resorption), physicomechanical (interlacing or fiber orientation) and biocompatibility (low allergenicity) collagen is widely used. as implant material both in the form pure as gel (Friess, 2001) or associated with other molecules such as polysaccharides, biological and microbial (De Paula et al, 2002).

Both collagen and its degradation products are recognized by various studies found in the literature as recognized by various cell types and thus leading to phenomena of cell adhesion, growth and cell differentiation. Another feature of collagen is that it can be associated with synthetic materials, proving to be a versatile protein in terms of chemical reactivity, allowing the introduction of quite varied chemical, physicochemical, biological and mechanical properties.

Other advantages of collagen include hemostatic function, surgical wound stabilization, semipermeability, allowing nutrient passage, natural enzymatic degradation, and fibroblast chemotactic ability (Postlethwaite et al., 1978).

Collagen membranes, obtained by the proposed procurement process, are mechanically malleable, adaptable and easy to manipulate, which is beneficial in clinical application. The collagen membrane for tissue barrier in RTG used in this study is obtained from the porcine and or bovine intestinal submucosa. Numerous research in various areas of medicine (urology, gastroenterology, dermatology, orthopedics, immunology, cardiology, dentistry, bioengineering of tissues) has shown that this is a versatile and efficient source, as well as not triggering antigenic responses in the receptor. able to function as a template by inducing regeneration of the native tissue in which it was implanted (Cayan et al., 2002).

The biomembrane manufacturing process, through chemical and thermal steps, allows to obtain a membrane with high tensile strength, allowing its placement in surgical sites with high demand for tensile strength and mechanical stress.

Morphological and birefringence analyzes of the biomembranes showed an extensive network of collagen fibers, organized in different directions, but preferably along the biomembrane axis. This is important information and it is in agreement with the results of the mechanical properties, where the biomembranes presented a great tensile strength (MPa) resistance. By comparison, Derwin and colleagues (2006) biomechanically tested five internationally traded biomembrane manufacturers and described values up to 10 Mpa.

Tensile tests, performed with biomembranes submitted to this process, reached average values of 117.8 MPa, while other commercial membranes reached values close to 10 Mpa. Thus, the structure-function relationship of the collagen and non-collagen components of biomembranes are not affected by the biomembrane preparation process, maintaining good resistance to tensile forces.

Biomembrane processing also allows the presence of 2 glycosaminoglycans (dermatan sulfate and, especially, heparan sulfate), which may aid in intrinsic bioactivity. This fact reflects a good adherence, migration, differentiation and response of cell culture.

In medicine, the present invention can be used in the treatment of skin lesions such as diabetic or vascular burns and ulcerations, acts as a barrier against microorganisms and water loss and contributes to the wound healing process. And also used in reconstruction of soft tissues in urogynecological procedures, acting as a support for cell growth during the tissue remodeling process. These biomembranes can still be prepared to adsorb drugs such as silver sulfadizine, which is used to treat skin lesions in burns. They may also incorporate growth and differentiation factors, such as rhBMP, enabling their release into the surgical site.

In addition, the collagenous membrane procurement process has a relatively low procurement cost, which makes it possible to have a lower market value than other similar materials manufactured in the international market, making it more accessible to patients requiring dental treatment in which it is produced. be used.

The biomembrane obtained, therefore, is manufactured by a unique technological process that preserves the collagenic structure of the original tissue and prior to use, there is gamma radiation sterilization.

This results in better cell surface adhesion, recognition, proliferation and targeted cell differentiation. Features easy handling and sufficient strength to withstand suture tension and provide support for the process of scar remodeling. The present membrane, of natural origin, is resorbable and, therefore, does not require a second surgical procedure for its removal.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the present invention describes a process for obtaining a collagen membrane obtained from intestinal submucosa of porcine origin.

This process permits the obtaining of a biocompatible collagen membrane of natural origin, through chemical and thermal processes, and there is no permanence of chemical residues from reagents used in the obtaining process. The permanence of these residues can lead to exacerbated inflammatory response and impairment of the tissue regeneration process. The costs involved in raw material procurement and membrane processing allow the product to be marketed for a lower value than other membranes on the market.

Therefore, an object of the present invention is a process for obtaining a collagen membrane comprising the steps of: (a) cleaning of the raw material, where cleaning is chosen from the group comprising:

(i) mechanical cleaning to remove coarse waste from other fabrics;

(ii) enzymatic treatment with lipase and detergent to promote the removal of fat from the source tissue;

(iii) enzymatic treatment with proteases and detergents to promote the removal of proteins other than collagen as well as fats; and

(iv) combinations of the items described above; b) stretch the raw material at temperatures ranging from 5 ° C to 65 ° C. c) treatment of biomembranes with hydrochloric acid to allow removal of cellular debris and genetic material.

In a preferred embodiment, the obtaining process has a preliminary step of preserving the raw material comprising immersion in salt water followed by washing with hypochlorite. In a preferred embodiment, the obtaining process has an additional finishing step involving membrane cutting and sterilization.

Optionally, the treated material may be lyophilized, and lyophilization, if present, occurs after step c) and before finishing, and is preceded by a stretching step similar to step b).

The chemical and thermal processes to which the membranes are submitted also allow to obtain higher tensile strength than that observed in similar products in the market. Tensile strength is important so that the membrane can be more easily manipulated and sutured in the required regions.

A further object of the present invention is a biomembrane obtained by the process described above.

These and other objects of the present invention will be better understood from the detailed description below. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 (a) - Schematic view of the "tubule" porcine intestinal submucosa - no cuts made longitudinally

Figure 1 (b) - Schematic view of porcine intestinal submucosa cut longitudinally

Figure 1 (c) - Schematic view of open porcine intestinal submucosa (after longitudinal cut)

DETAILED DESCRIPTION OF THE INVENTION

The examples described herein are intended merely to illustrate some of the numerous embodiments of the process of the present invention, and therefore should not be construed as restrictive.

Feedstock

The proposed processing to obtain a collagen membrane can be performed from tissues of natural origin, preferably from animals, especially cattle and swine, and the intestine of these animals can be used. However, collagen-rich tissues other than the intestine may be used and are within the scope of the present invention. Preferably the tissue used should be collagen-rich, where collagen is selected from the group comprising collagen type I, II, III, IV and combinations thereof.

All other types of collagen are also suitable for use in the present invention.

Process for obtaining the Collagen Membrane

The process of obtaining the collagen membrane is a process comprising the steps of: a) cleaning the raw material, where cleaning is chosen from the group comprising:

(i) mechanical cleaning to remove coarse waste from other fabrics;

The process may comprise a preliminary step of preserving the raw material. The raw material is normally preserved salty until the moment of biomembrane preparation, using the proposed manufacturing method.

During the preservation step, the raw material is then treated with sodium hypochlorite solution, preferably at a concentration of 0.01% for 30 minutes to eliminate possible contaminants. After this, the material is washed thoroughly with deionized water.

The material is then subjected to cleaning steps comprising mechanical and / or protease and / or lipase cleaning. These procedures are intended to clean the material, remove fats and proteins other than collagen.

Mechanical cleaning is performed with the aid of cutting instruments to remove coarse residue from other tissues such as fat. Then submits the detergent treatment material (0.1% to 0.5% v / v, preferably 0.3%) and proteases (0.01% to 0.05%, preferably 0.02%) for 2 to 4h .

The material should be washed again with deionized water until these chemical residues are removed.

The material should be treated with detergent solution (0.1% to 0.5% v / v, preferably 0.3%) with lipase, (0.01%) to 0.05%, preferably 0, 02%) and rinse again with plenty of deionized water.

The detergent used may be any detergent known in the art.

After the cleaning step the material should be stretched over regular surfaces and subjected to temperatures from 5 ^o C to 65 ° C, and this stretching step occurs in several substeps. The first sub step takes place at temperatures of 5 ° C to 10 ° C, preferably at 7 ° C for 8h to 16h, preferably 12h.

The regular surfaces to be used must not allow the release of residues and must not allow the stretched biomembranes to be firmly adhered to them so that they cannot be detached from them. The membranes stretched in the smooth surface, must be subjected again to a temperature 30 ° C to 40 ° C, preferably at 37 ⁰ C for 18h 30h to preferably 24h.

After these steps, the membranes should be kept stretched at 55 ° C to 65 ° C, preferably 60 ° C, for 8h to 16h, preferably 12h.

The membranes are then treated with 1N hydrochloric acid for 8 to 24 hours, preferably 16 hours.

Biomembranes should be rinsed again with deionized water until acidic solution is removed.

When non-lyophilized membranes are desired, after treatment with 1N HCl and washing with deionized water described above, they are dried again.

The material must again be stretched over regular surfaces and subjected to temperature of 5 ° C to 10 ° C, preferably 7 ° C for 8h to 16h, preferably 12h.

The membranes stretched at regular surface must be subjected again to the temperature of 30 ° C to 40 ° C , preferably 37 ⁰ C for 18h to 30h, preferably 24h. After these steps, the membranes should be stretched at 55 ° C to 65 ° C, preferably 60 ° C, for 8h to 16h, preferably 12h.

To obtain lyophilized membranes, after treatment with 1 N HCl and washing with deionized water, the lyophilization process is followed, in which first the biomembranes must be stretched again on a regular surface and frozen (-90 ° C to -55 ° C). ), preferably -80 ° C for 8 to 24h, preferably 12h.

They are placed in a lyophilizer apparatus and must remain in it and be subjected to water removal until the weight of the biomembranes remains constant.

The lyophilized or non-lyophilized membranes are then cut according to an established parameter (eg 2cm X 2cm), packaged and subjected to gamma radiation for sterilization.

After this process, the obtained membranes can be subjected to drug incorporation and / or growth and differentiation factors.

The biomembranes are then cut to desired pattern and sterilized by gamma radiation. EXAMPLE

Non-lyophilized membrane preparation used as a dressing for treating burns

The raw material should be washed in 0.1% sodium hypochlorite solution for 0.5h. After this period, the coarser greasy tissue that can be removed mechanically should be removed. The membranes are washed thoroughly in deionized water.

The porcine intestinal submucosa is cut longitudinally in order to open the tubular structure, characteristic of this type of structure (Figure 1).

The membranes are then placed in 0.002% lipase and 0.3% detergent enzyme solution for 2.5h. After this time, they are washed extensively with deionized water. They are then placed in 600 mL of another enzyme solution with 0.002% protease and 0.3% detergent. They are then again washed with extensively deionized water.

The detergent has the function of transforming the fat into smaller particles and thus facilitating the action of lipase. Lipase is an enzyme whose function is to catalyze the hydrolysis of fats. This facilitates the removal of fats present in the raw material.

They are then cut into strips and stretched on a flat Teflon surface. They are then placed under refrigeration at 4 C. The membranes undergo another step of heat treatment at 35 ° C for 24h. They are subsequently submitted for 12 hours at 60 ° C.

The membranes are removed from the Teflon surface and subjected to treatment in 1 N hydrochloric acid solution for 8h. They are then washed in deionized water for a period of 2h. Afterwards, the membranes are stretched again on a flat surface. They are then placed under refrigeration at a temperature of 7 ° C. The membranes undergo another heat treatment step at 37 ° C for 24h. They are subsequently submitted to a temperature of 60 ° C for 12h.

EXAMPLE 2

Dental lyophilized membrane preparation

The raw material should be washed in 600 mL of solution with 0.1% sodium hypochlorite for 0.5h. After this period, the coarser greasy tissue that can be removed mechanically should be removed. Wash the membranes with abundant deionized water.

The membranes are then placed in enzyme solution with 0.02% lipase and 0.3% detergent for 2.5h. After this time, they are washed extensively with deionized water. They are then placed in another enzyme solution with 0.03% protease and 0.4% detergent. They are then again washed with extensively deionized water. The proteases used are not specific for collagen proteins and therefore do not alter or denature them.

They are then cut into strips and stretched on a stainless steel surface. They are then refrigerated at 8 ° C for 14h. The membranes undergo another heat treatment step at 35 ° C for 20h. They are then subjected to a temperature of 55 ° C for 13h.

The membranes are removed from the stainless steel surface and treated with a solution of 0.1N hydrochloric acid for 24h. They are then washed in deionized water for 4 hours.

These membranes should be frozen in a biofreezer at -80 ° C for 10h. They are then transferred to a freeze dryer and should remain there until the mass remains constant.

The biomombles are then cut to desired pattern and gamma-sterilized.

Claims

1. Biocompatible collagen membrane manufacturing process characterized by using raw material chosen from tissues of natural origin rich in collagen, bovine and / or swine.

Process according to Claim 1, characterized in that it comprises the steps of:

(a) cleaning of the raw material, where cleaning is chosen from the group comprising:

(i) mechanical cleaning to remove coarse waste from other fabrics;

(iv) combinations of the items described above;

b) stretch the raw material at temperatures ranging from 5 ° C to 65 ° C.

c) treatment of biomembranes with hydrochloric acid to allow removal of cellular debris and genetic material.

Process according to Claim 2, characterized in that the raw material is the intestinal submucosa of cattle and / or swine.

Process according to claim 2, characterized in that it comprises a preliminary step of preserving the raw material.

Process according to Claim 4, characterized in that the preservation step comprises the steps of:

a) treating the raw material with a solution preferably of 0.01% v / v sodium hypochlorite for 30 minutes; and

b) abundant washing with deionized water.

Process according to Claim 2, characterized in that the mechanical cleaning is carried out with cutting instruments.

Process according to Claim 2, characterized in that the detergent and protease cleaning comprises from 0.1% to 0.5% v / v detergent and 0.01% to 0.05% v / v protease.

Process according to Claim 7, characterized in that the concentration of detergent and protease is preferably 0.3% and 0.02% v / v respectively.

Process according to Claim 2, characterized in that the detergent and lipase concentration comprises from 0.1% to 0.5% v / v detergent and 0.01% to 0.05% v / v lipase.

Process according to Claim 9, characterized in that the concentration of detergent and lipase is preferably 0.3% and 0.02% v / v respectively.

Process according to Claim 2, characterized in that after each cleaning step the material is washed with abundant deionized water.

Process according to Claim 2, characterized in that the raw material is stretched on a regular surface.

Process according to Claim 2, characterized in that the stretching step is subdivided into 3 steps:

i) stretching at temperatures of 5 ° C to 10 ° C for 8 to 16 hours;

ii) stretching at temperatures of 30 ° C to 40 ° C for 18 to 30 hours; and

iii) stretching at temperatures of 55 ° C to 65 ° C for 8 to 16 hours;

Process according to Claim 13, characterized in that the stretching step comprises 3 steps:

i) stretching is preferably at a temperature of 7 ° C for 12 hours;

ii) stretching is preferably at a temperature of 37 ° C for 24 hours; and

iii) stretching is preferably at temperatures of 60 ° C for 12 hours;

Process according to Claim 2, characterized in that the hydrochloric acid is at a concentration of IN.

Process according to Claim 2, characterized in that the hydrochloric acid treatment lasts from 8 to 24 hours.

Process according to Claim 16, characterized in that the treatment preferably lasts 16 hours.

Process according to Claim 2, characterized in that the raw material after step c) is again subjected to a drying step.

Process according to Claim 2, characterized in that the raw material after step c) is lyophilized.

Process according to Claim 19, characterized in that the lyophilization step comprises the stretching and freezing at temperatures between -90 ° C and -55 ° C for 8 to 24 hours.

Process according to Claim 20, characterized in that the lyophilization step preferably takes place at 80 ° C for 12 hours.

Process according to either of claims 18 or 19, characterized in that the biomembranes are cut and sterilized by gamma radiation.

Process according to either of claims 18 or 19, characterized in that drugs and / or growth and differentiation factors are incorporated into the biomembranes.

24. Use of porcine and / or bovine intestinal submucosa characterized in that it is in the preparation of a biocompatible collagen membrane for dental application.

25. Use of porcine and / or bovine intestinal submucosa characterized in that it is in the preparation of a biocompatible collagen membrane for medical application.

Biocompatible collagen membrane characterized in that it is lyophilized and obtained by a process as described in claims I to 17 and 19 to 23.

Biocompatible collagen membrane characterized in that it is obtained by a process as described in claims 1 to 18 and 22 to 23.

28. Biocompatible collagenous membrane characterized by being composed of clean, dry and sterile swine and / or bovine intestinal submucosa.

Membrane according to Claim 28, characterized in that it is lyophilized.