BG109565A - Multilayer bioresorbable membrane for guided tissue regeneration - Google Patents

Abstract

The invention relates to biomaterial consisting of a biocompatible and bioresorbable membrane and finding application in surgery, dentistry in particular, as well as in face jaw surgery and oral implantology. It may be used as a mechanical barrier for ensuring guided tissue regeneration in the reconstruction of bone defects. The biomaterial contains a porous layer of lyophilized microfibrillar collagen, a collagen membrane of chemically and enzymically treated pig derma and an adhesive gelatin-based layer.

Description

Technical field

The present invention relates to the composition and method of manufacturing a multilayer bioresorbable membrane consisting of microfibrillar collagen, chemically and enzymatically treated porcine dermis and an adhesive layer based on gelatin. The finished product of this invention can be used as a mechanical barrier to provide guided tissue regeneration in surgery and is particularly intended for use in the reconstruction of bone defects in dentistry and maxillofacial surgery and in oral implantology.

BACKGROUND OF THE INVENTION

The principle of directing the growth of one's own bone into a given bone defect involves sealing the anatomic area (osteogenesis zone) with mechanical barriers. New bone has been found to form in the presence of blood clot, vital osteoblasts and contact with living tissues. Covering bone defects with a barrier membrane interferes with the ingrowth of the soft tissue into the defect area and creates better conditions for complete bone healing, while the control defects are filled with fibrous connective tissue and a minimal amount of new bone (Dahlin et al., 1988). . As mechanical barriers to the provision of guided periodontal and bone regeneration, various synthetic and natural biocompatible materials have been studied and applied in medical practice (Caffesse et al., 1997).

Membranes for directional bone regeneration from expanded polytetrafluoroethylene (e-GTTPE) are known. This material is highly biological and chemical inert (US Pat. 5,197,882). Its main disadvantage is that it is not absorbed, which requires a second operation to remove it. Barrier membranes that are absorbed into the body are more suitable. Many synthetic and semi-synthetic materials are known in this group - polymers and copolymers of glycolic and lactic acid, polycaprolactone, polyglycine, modified cellulose and the like. (U.S. Pat. 4,961,707; U.S. Pat. 6,350,284), as well as naturally occurring membrane materials - bone plates, collagen membranes, dura mater, and the like. Membranes of biological origin are better tolerated, but their major drawback is the rapid absorption of tissues. As complete bone regeneration requires bone defect isolation for at least 2-3 months, the basic requirement for implanted barrier material is to remain stable for this period of time.

Patent BG 61596 B1 discusses multilayer membranes for directional tissue regeneration based on materials of natural origin - esters of high molecular weight fractions of hyaluronic acid or mixtures thereof with other polymers, which are films combined with a reinforcing mesh of the same material. These products are bioresorbable and biocompatible, but the multistage technology of obtaining the corresponding hyaluronic acid derivatives involves a large number of operations for the isolation, purification and chemical modification of the raw material, which affects the cost-effectiveness of the process.

Tissue regeneration membranes combining synthetic and natural polymers are known. In US Pat. No. 5,607,590 describes compositions containing collagen membranes and an inner layer between them - a mesh with a pore size of 150 to 500µm, constructed of various synthetic materials: 1) absorbable (polymers and copolymers of glycolic and lactic acid, polydioxanone, copolymers of glycolic acid and trimethylene carbonate, etc.) and 2) non-absorbable (silicone, expanded polytetrafluoroethylene, polyethylene, polypropylene, polyethylene terephthalate, polyurethane, polyvinyl alcohol, etc.). As mentioned above, membranes containing non-absorbable synthetic polymers require a second operation to remove the membrane, and the absorbent may cause secondary irritation and inflammation due to the degradation of the monomer products.

In this respect, collagen-based materials have undoubted advantages in terms of biocompatibility, cost and accessibility.

_β ·· ···· · _te Collagen is a major protein in mammals, aerwjsw · composition! £ ..

The structure is similar in species of mammals, which is the reason for the poor allergenicity and immunogenicity of collagen products, regardless of the raw material from which they are derived. Purified collagen with wholly or partially preserved native structure has long been used in various fields of medicine, including as a covering material for wounds and soft tissue implants. For the industrial production of collagen in various forms, the most commonly used are the skins, tendons, cartilage and other by-products of a number of farm animals treated by various methods (EP127067; US Pat. 4,412,947; US Pat 4,980,403; US Pat 5,885,634;).

Collagen products break down in the body most often in about 4-5 weeks, but this indicator varies greatly depending on the type of collagen, the way it is produced and the type of product - film, lyophilized porous sponge, collagen fibers and more. The rate of absorption in the body may be further delayed by chemical modification and addition of crosslinking and enhancing agents (US Pat. 5,885,634; US Pat. 5,206,028; US Pat. 5,837,278).

SUMMARY OF THE INVENTION

The essence of the invention is that a method for producing a multilayer membrane containing collagen type I has been devised in which the finished product (with a pore size of 0.005 to 0.10 pm and a thickness of 0.4 to 1.0 mm) has better elasticity and strength in dry and moist condition than conventional collagen films. Combining the porous layer of microfibrillar collagen with insoluble collagen with a preserved native structure, allows for controlled and delayed absorption in the body.

In general, the technology for producing the collagen product of this invention involves three main steps:

1. Preparation of a compressed porous sheet 0.1 to 0.4 mm thick composed of microfibrillar collagen, preferably type I, comprising the following operations: a) production of atelopeptic collagen dispersion c. , '.........

a certain concentration; b) partial networking; c) freeze the vacuum -:., freeze drying the aqueous dispersion and obtain a collagen sponge ^ d) '* compressing the collagen sponge under certain conditions;

2. Preparation of an insoluble collagen type I membrane, by the following operations: a) preparation of the raw material - sections 0.2-0.6 mm thick are separated from the endodermal layer of pig skin from which the fat has been removed; b) enzymatic and chemical treatment of the raw material - removal of collagen proteins and telopeptides; c) networking; (e) washing to remove unreacted cross-linking agent.

3. Preparation of the final product - the intermediates of steps 1 and 2 are bonded together by an adhesive layer of gelatin-glycerine gel and then frozen and lyophilized. The multilayer membrane is brought to a certain residual humidity and compressed to the desired thickness.

The collagen membrane of the present invention is intended for use in medical practice, having, in comparison with conventional collagen products of this group, improved dry and wet strength properties, is tear-resistant and has a controlled rate and rate of absorption. The porous layer of microfibrillar collagen has good haemostatic properties and can be further used as a pharmaceutical carrier. Various pharmacologically active substances, such as antibiotics, heparin, glucosaminoglycans, cytokinins, osteoinductive substances, etc., may be included in the treatment according to the needs.

The main purpose of the membrane is in surgery, and in particular in dentistry and maxillofacial surgery, where it can be used as a roofing barrier to ensure complete bone repair for bone defects due to: removal of benign tumors, cysts, etc .; in osteomyelitis, plasticity of cranial defects, after auto-grafting, etc .; and in oral implantology - to prepare implant sites and stabilize implants.

. . . , .......

Barrier layer of insoluble native collagen. is; no- ^ Zvnr: ... the absorbent part of the diaphragm and enables complete and sufficient 'continuous isolation of the defect area. Due to the multi-stage process of enzymatic and chemical treatment of the raw material, the collagen product of the present invention is purified from the accompanying proteins and other artifacts and is atelopeptide, which is a prerequisite for its very good tolerance and the absence of allergic and toxic reactions when applied.

Detailed description of the invention

For the preparation of the porous part of the multilayer collagen membrane, a dispersion of microfibrillar atelopeptide collagen obtained by standard technology is used, with known modifications proposed by us. All available sources of collagen type I - hides (preferably pigs), tendons, bones, etc. can be used as feedstock. The raw material is purified from fats and other artifacts, washed with distilled water and lyophilized, then ground and subjected to enzymatic treatment with trypsin, chymotrypsin, pepsin, bromelin, etc., preferably with a combined enzyme product with proteolytic, lipolytic amylolytic activity in an amount of 0.5 to 1.0% w / w relative to the amount of protein. The enzyme treatment is continued for 5 to 20 hours, preferably 8 hours, at a temperature of 30-32 ° C and at an optimum pH for the enzyme concerned. Treatment with a combined enzyme product (preferably pancreatin) provides a more complete cleansing of collagen in the skin from the by-products.

Q Under the action of the enzyme complex, globular proteins and lipids are simultaneously degraded and the telopeptides released, which must be removed when purifying collagen products (US Pat. 4,412,947). The lipase and amylase in the enzyme complex help to better purify collagen from by-products.

The dissolution of collagen from the enzyme-treated feedstock is carried out in a dilute solution of acetic, lactic, tartaric or citric acid with a pH of 3.0-4.0 (preferably 3.5) for 72-96 hours at 15. .......

18 ° C. The resulting dilute colloidal solution of acidic collagen: is: centrifuged 3x: .. _in * .. ·· .. · separation of undissolved particles and other impurities.

The acidic dilute solution of collagen is neutralized to reach the isoelectric point (pH 4.6-6.0), resulting in spontaneous fibrillation. The resulting dispersion was concentrated by centrifugation or filtration to a concentration of 1 -2% (preferably 1.4%). The collagen microfibrillar dispersion is crosslinked partially with a dilute solution of formaldehyde, glutaraldehyde or glyoxal (preferably glutaraldehyde) in an amount of 0.04 to 0.06%, frozen at temperatures from -30 to -50 ° C (preferably -45 ° C). ) and dried in a vacuum-sublimation installation for 20-30b (for O preferably 24 h).

The lyophilized collagen sponge with a thickness of 7 to 10 mm and a residual humidity of 6 to 10% is placed in a closed chamber with a relative humidity of 75 to 90%, at a temperature of 25 ° C for 20 to 80 min (up to a residual humidity of the material 15-20%) and pressed to a thickness of 0.1 - 0.35 mm (preferably 0.2-0.25 mm). Moisturizing the collagen sponge facilitates its compression to the desired thickness, since the absorbed moisture provides better cohesion between the collagen fibrils. With more residual moisture than necessary, the structure of the resulting product changes and resembles that of a collagen film.

The insoluble collagen type I membrane is obtained from the hairless and degreased pork skin from which the epidermal layer has been removed. The raw material is cut into sections 0.2-0.6 mm thick (preferably 0.4 mm) and immersed in 0.5-2% sodium hydroxide solution (preferably 1%) for 2 to 10 hours (preferably 3h). After washing to pH 7.5, the alkaline treated dermis is enzymatically exposed to trypsin, chymotrypsin, pepsin, pancreatin, alkaline and neutral protease, etc. (preferably trypsin) for 30-40 min at 35 ° C. . It is washed to remove the enzyme and immersed in dilute hydrochloric acid solution (preferably at a concentration of '0.2 to 0.6%) for 1-3 h, then repeatedly washed with

... ........

distilled water to a negative reaction for chlorides. At. chemical form.

·· ·· ·· * * ··· * _in * _# * · raw material are removed residual fat (saponified by sodium hydroxide), alkaline and acid-soluble proteins, mucopolysaccharides and proteoaminoglikanite. The enzymatic effect facilitates the elimination of collagen proteins and telopeptides.

The purified derm, in which the collagen fibers are hydrated, is crosslinked with a suitable crosslinking agent, preferably 0.5% formaldehyde solution, and then washed thoroughly with distilled water to remove unreacted formaldehyde. The resulting collagen membrane is stretched on glass or Teflon sheets. A gelatin-glycerin gel adhesive layer is applied to it and a compressed collagen sponge is placed. It is frozen at temperatures from -30 to -50 ° C (preferably -45 ° C) and dried in a vacuum sublimation installation for 10-20h (preferably 15 h).

The invention is illustrated by the following examples:

Example 1 - Preparation of microfibrillar collagen

100 g of lyophilized pigskin are ground to a particle size of 1 mm and transferred to a stirred glass reactor. 2.5 g of Pancreatin (Merck), 1 g of Polysorbate 80 and 4 l of distilled water are added sequentially. The pH of the solution was adjusted to 7.0-7.5 with the addition of 10% w / v sodium hydroxide solution or 10% w / v citric acid solution dropwise. Hot water was supplied to the reactor jacket, providing a temperature of 30-32 ° C. The enzyme treatment was continued for 8 h with occasional stirring. The mixture was then centrifuged at 2000 rpm for 10 min and the supernatant discarded.

The enzyme-treated feedstock is mixed with 20 I 0.1% aqueous citric acid solution and transferred to a stirred reactor. The mixture was allowed to stand for 72-96 h at 20-25 ° C with occasional stirring.

The resulting diluted colloidal solution of acidic collagen was centrifuged to remove the undissolved particles and brought to pH 4.6-6.0 by the addition of small portions of 20% sodium hydroxide solution. Upon reaching the isoelectric point, a spontaneous recovery occurs (reconstruction)

... ··. ·· .....

of the fibrillar structure of collagen. After adding 100.Ο110.75%: pa3jbp {) and?: * ··.

· Glutaraldehyde, the mixture was concentrated by centrifugation (2500 rpm / 20m1ft). "

The centrifuge was discarded and the precipitated collagen suspension was analyzed for dry matter content, final concentration 1.4%.

Example 2 - Preparation of porous sheets of microfibrillary collagen The collagen suspension of Example 1 was frozen at -45 ° C and subjected to vacuum freeze-drying. The lyophilization is carried out in an automatic mode at preset temperature and time parameters as follows: initial temperature in the drying chamber - (-35 ° C to -45 ° C); sample residence time at this temperature - 4 hours; raising the temperature to 0 ° C - 2 hours; from 0 ° C to 25 ° C - 8 hours; drying at 25 ° C for 12 hours. Received ©

a collagen sponge 8 mm thick was placed in a humidity controlled chamber (85%) at 25 ° C for 60min and immediately compressed to produce sheets 0.2-0.25mm thick.

Example-1r Preparation of a three-layer collagen valvate

Cutaneous and defatted pigskin (500 g) from which the epidermal layer has been removed is cut into pieces of about 10 x 20 cm in size and 0.4 ± 0.1 mm thick and treated with 1% sodium solution for three hours hydrox. Wash with distilled water and fill with 1.5 I trypsin solution at a concentration of 0.1% at pH 7.5. Incubate at 35 ° C for 30-40 min.

The enzyme-treated feedstock was immersed in a dilute hydrochloric acid solution (0.5%) and then washed with distilled water to the negative for chlorides. Transfer to a bath containing formaldehyde solution (0.2-0.5%) and allow to stand for 60 min at 20 ° C. The resulting partially cross-linked collagen membrane was washed with distilled water for 1 hour and stretched on glass plates. An adhesive layer of 5-8% gelatin gel is applied to it and adhered to the compressed collagen sponge. It was frozen at (-40 ° C) and lyophilized. The lyophilisate was brought to a residual humidity of 15% and pressed. The finished product is cut into sheets of 2.0 x 4.0 cm, packed and sterilized with gamma rays at a dose rate of 25 kGy.

.. ..; ···. ·

Example 4 - Characteristics of the multilayer membrane $ n%:. :: ··· ..; ····. ·· ·· * ·· * ··· * * ·· * ^e f *

The multilayer membrane obtained according to Example 3 has the following “characteristics: dimensions - 2.0 x 4.0 cm, thickness - 0.35 mm; water absorbency 7.8 times the mass of the membrane; density - 0.90 g / cm ³ ; pore size Ο, ΟΙΟμηη; residual moisture - 10.30%; residual lipids - 1.83%.

Literature

1. U.S. Pat. No. 4,412,947; 1983, Cioca.

2. U.S. Pat. 4,961,707; 10/1990, Magnusson

3. US Pat 4,980,403,1990, Teman et al.

4. U.S. Pat. 5,197,882; 03/1993, Jernberg et al.

5. U.S. Pat. 5,206,028; 04/1993, Shu-Tung U, Kiova Ter.

6. U.S. Pat. 5,607,590; 03/1997, Shimizu.

7. U.S. Pat. 5,676,699; 10/1997

8. U.S.Pat.5,837,278; 1998, Geistlich et al.

9. US Pat 5,885,634; 1999, Areso.

10. U.S. Pat. 6,350,284; 02/2002, Tormala et al.

11. EP1270672; 2003, Visser P R.

12. Chen C.C. et al. Evaluation of a collagen membrane with and within bone grafts in the treatment of periodontal intrabony defects. J. Perodontol 1995, Oct; 66 (10): 838-847.

13. Dahlin C. et al. Healing of bone defects by guided tissue regeneration. Plast Reconstr © Surg 1988; 71: 672

14. Caffesse RG, Mota LF, Qunones CR, Morrison EC. Clinical comparison of resorbable and non-resorbable barriers for guided periodontal tissue regeneration. SO J. Clin Perodontol 1997, Oct. ; 24 (10): 747-752.

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Multilayer bioresorbable guide membrane: ··· .. · ···, ·· ** ·. · * ·· * ·· ♦ * * · · · Tissue regeneration ””

Claims (3)

Claims A multilayer bioresorbable membrane comprising microfibrillar, atelopeptide collagen, a collagen membrane of chemically and enzymatically treated porcine dermis and an adhesive layer based on gelatin. The multilayer membrane according to claim 1, wherein said microfibrillar collagen is derived from porcine skin. The multilayer membrane according to claim 1, wherein said microfibrillar collagen is derived from bovine tendons. 4. A method of producing a compressed porous sheet of lyophilized microfibrillar collagen, characterized in that it comprises obtaining a dispersion of atelopeptide microfibrillar collagen, vacuum-freeze-drying said dispersion and obtaining a collagen sponge, compressing mushrooms. 0.1 to 0.4 mm ^v 5. A method for the preparation of a collagen membrane comprising the enzymatic and chemical treatment of porcine dermis for the removal of fat, collagen proteins and telopeptides, omre harvesting of the resulting membrane and vacuum-freeze-drying. _v 6. Process for preparing a multilayer bioresorbable membrane of claim 1, characterized in that the products of claims 4 and 5 are glued by an adhesive layer of gelatin-glycerol gel, lyophilized and compressed to a thickness of 0.4 to 1, Omm.