The agent stimulates the regeneration of hard tissues

 

(57) Abstract:

The invention relates to the use of chitosan and fixed it polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and dextransucrase, as agent, capable of providing stimulated the regeneration of hard tissues. Also discovered a way to stimulate and/or accelerate the regeneration of hard tissue by applying a specified agent to the appropriate section of the cloth. The third object is the implant to integrate into a solid fabric. At least part of the implant, designed to integrate coated with the specified agent. The invention provides growth stimulation of cells of solid tissues, the formation of regenerated bone tissue predominantly lamellar, compact type. 3 C. 17.p. f-crystals.

The present invention relates to a new method of stimulating or accelerating the regeneration of hard tissues in connection with the so-called osseointegration, for example, effect, sought after implantation of alien implants in hard tissue, in particular bone tissue. This invention relates also to a method of performing such osseointegration using this new technique and bone or any other need in the bone tissue.

The background to the invention

Implants intended for fixation of hard tissue, in particular bone tissue, more and more widely used in dentistry, orthopedics, neurosurgery, hand surgery, and plastic and reconstructive surgery. Long-term fixation of the implant in the bone is made of titanium, and should take into account that materials based on titanium and in the future will be widely used as the material of choice for implants and prostheses.

Clinical problem related to the so-called osseointegration, is that the implant cannot be load until, until it reaches sufficient bone fixation, and it can take 6 to 9 months. Therefore, the clinic is extremely important to accelerate the healing process by providing the Association stimulated bone formation and implant. There is also a need of bone regeneration for connection of the defects or areas where there has been bone loss, for example in the toothless parts of the jaws, or where bone has been lost due to, for example, trauma, tumors or surgery.

Sosnoski bone cells, so that the volume of solid tissue increased.

Another objective of the present invention is to provide a method that provides the formation of regenerated bone tissue predominantly lamellar, compact type, while the extensive formation of runaway tissue is minimized.

Another objective of the present invention is the creation of a new agent, is able to provide, together with implants in solid fabric, stimulated the regeneration of this hard tissue such as bone.

Another objective of the present invention is to provide a method of improved osseointegration in connection with implantation of alien implants in solid tissue with simultaneous stimulation of tissue regeneration.

Another objective of the present invention is the creation of implants treated with an agent that stimulates the regeneration of hard tissues.

For these and other purposes, which can be seen from the further description, the present invention is provided a new use of chitosan and fixed it polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and doctranslate. This new application refers to segeneiti maybe for example, be provided in connection with implants in hard tissue such as bone.

The polysaccharide can be fixed on chitosan matrix in several ways, for example by ionic bonds, covalent links with many sites or end-site or by means of mechanical fixation on chitosan matrix due to precipitation from solution. Ionic bonds and covalent bonds are the preferred methods of fixation.

Agent, stimulating the solid fabric of the present invention can be in various physical forms, for example, in the form of a membrane, powder, gel, granules or solution. In the case of using the implant, the part of him that should be integrated into the solid tissue may be immersed in the agent for application to the implant. This agent, of course, can be placed in a solid fabric pez se, for example, in the cavity created in the bone tissue.

Preferred implant material is titanium, but can also be used and other materials for implantation.

Chitosan is a linear 1,4-polysaccharide built from units of D-glucosamine. Chitosan receive through N-dataretention is obtained from the shells of crabs and shrimp, representing a fish processing waste production. By regulating the processing of chitin with alkali can get chitosans with different degrees of N-acetylation. When processing of chitin with concentrated alkali, usually sodium hydroxide, is N-dezazetilirovanie, i.e., acetamide group pre rotate in the amino group, resulting in the formation of chitosan.

Physical properties of chitosan determine its suitability depends on the degree of N-acetylation, molecular weight and homogeneity. Chitosan is a biodegradable substance such as due to the chitin of the digestive system, and at the expense of lysozyme and other enzymes of the body.

In connection with the application of the present invention preferably chitosan had a degree of N-acetylation of not more than about 90% and, preferably, not more than approximately 50%. Particularly preferably, the degree of N-acetylation was less than approximately 25%.

The preferred polysaccharide for fixing to the chitosan matrix is heparin or heparansulfate. A special method of attaching heparin covalent bond to the matrices containing an amino group described in Patera USA N 4613665. the DOI fabric, for example, in connection with the so-called osseointegration. This method is characterized by the fact that prior to implantation of the implant and/or solid fabric cause the specified agent derived from chitosan, and fixed on him of a polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and dextransucrase, in a quantity sufficient to stimulate. Osseointegration is the best form of long-term fixation in hard tissue, in particular bone tissue, the implant from Neutrogena and various alien materials.

When implementing this method, the agent can be applied in the form of a powder, a solution, a gel, granules, films or membranes. Alternatively, the agent may be applied by dipping the part of the implant that is designed for integration into a solid fabric in the solution of the corresponding components of chitosan and fixed it polysaccharide.

The present invention also includes implants, designed for integration into solid tissue, particularly bone tissue. In these implants that part which is intended to integrate, cover agent, stimulates the regeneration of hard tissue, which includes chitosan and poly is translate. Particularly preferably, the implant is comprised of titanium.

Examples of preferred embodiments of the present invention

The present invention is further illustrated by the non-restrictive examples. In these examples, percentages and parts are by weight, unless otherwise specified.

Example 1

Application of chitosan coating on titanium screw

Titanium screws were immersed in a solution of chitosan in 2% acetic acid (1 weight. %/volume) and left in the specified solution for 15 minutes. Chitosan was a drug Sea Cure 313, Pronova Biopolymer, 15% N-acetylation. The treated titanium screws were then dried in a dry-heat Cabinet at 70oC for 16 hours, and then neutralized 1 M NaOH and repeatedly rinsed in distilled water, and then dried in a dry-heat Cabinet. The screws were placed in the packaging such as "peel open" and sterilized with ethylene oxide.

Example 2

Titanium screws with heparin attached ionic bond

The screws are covered with chitosan, as described in example 1, was transferred to a solution consisting of 125 mg of heparin (Pig Mucosa Kabivitrum) in 500 ml of distilled water, and left for approximately 16 hours, after which it was rinsed in distilled and home. The number of fixed heparin was approximately 2 mg/cm2.

Example 3

Titanium screws with heparin attached covalent bond joining end area)

Heparin was dissolved in water (300 ml). This solution was cooled to 0oC in ice water and maintained in a cold condition. First, to the solution under stirring was added sodium nitrite (NaNO3, 10 mg), and then acetic acid (2 ml). The reaction mixture was maintained at 0oC for 2 hours, dialyzed and lyophilized. The output was 0.7 g

Titanium screws coated with chitosan, as described in example 1, was transferred to a solution consisting of 125 mg described above is decomposed by nitrite heparin, 15 mg NaCNBH3in 500 ml of distilled water, and brought the pH to 3.9 using 0.1 M HCl. The reaction mixture is maintained at room temperature for 16 hours. The screws were then washed in distilled water and dried at room temperature. The treated titanium screws were placed in the packaging such as "peel open" and sterilized with ethylene oxide. The number of fixed heparin was approximately 1.5 mg/cm2.

Example 4

Titanium screws with heparine the military periodates sodium, received as follows: one gram of periodate sodium NaIO4, was dissolved in 200 ml of distilled water. To the solution was added to ten grams of heparin-sodium and stirred overnight in the dark. The resulting solution, after adding 10 ml of glycerin and mixing for two hours, dialyzed against water. The water is changed every hour. The result is a solution containing heparin, oxidized periodate, at a concentration of approximately 19 mg/ml

Titanium screws coated with chitosan, as described in example 1, was transferred to a solution consisting of 125 mg of the above-described oxidized periodata heparin, 15 mg NaCNBH3in 500 ml of distilled water. The reaction is then carried out as described in example 3.

Example 5

Fabrication of chitosan films

5 g of the hydrochloride (50% degree of acetylation, Pronoya) was dissolved in distilled water (0.5 l, 1 vol.%/weight). 10 ml of the obtained solution was transferred into a Petri dish and received chitosan film by evaporation and drying in a dry-heat Cabinet at 70oC for 24 hours. The resulting film is then neutralized by the addition of a phosphate buffer, 0.2 M, pH of 9.0. The film was left in a Petri dish in this buffer at room Teniente attached heparin (accession limit area)

To the neutralized chitosan film prepared as described in example 1 was added 20 ml of a solution containing 125 mg nitrite is decomposed by heparin, prepared as described in example 3, dissolved in 0.5 l of water containing 4.4 g of NaCI. To the solution was added 15 mg of laborgerate sodium. the pH of the solution was brought to 3.9 using 0.5 M chloride-hydrogen acid or another acid. The solution containing chitosan film, left to stand at room temperature for 14 hours and the treated film is then 3-4 times washed with water and left to dry.

Example 7

Films with heparin attached ionic bond

To the neutralized chitosan film prepared as described in example 1 was added 20 ml of a solution containing 125 mg of heparin dissolved in 0.5 l of water and 4.4 g of NaCI. The solution containing chitosan film, left to stand at room temperature for 14 hours and the treated film is then 3-4 times washed with water and left to dry. The resulting film can be ground into powder for applying for osseointegration of the present invention.

Example 8

Biological tests

As an experimental animal apoplast knee joints of rabbits was removed coat.

Produced distal skin incision length 35-40 mm in the area of the knee joint against the tibial proximal region Epifanio cartilage. Cut the periosteum and cooling continuously supplied sterile saline phosphate buffer was drilled 3.5mm drill at low speed a hole through the compact substance of the bone to the bone marrow cavity. Then in the proximal portion at a distance of 4 mm threaded titanium screw with a hexagonal head and distally at a distance of 6 mm was placed another titanium screw, both with a diameter of 3.5 mm Pascalou and skin wound was sewn up single joints. In these experiments, in addition to processed as described in examples 1-3, titanium screws, also used raw titanium screws.

After 4 and 12 weeks, respectively, rabbits again produced anesthesia. In the area of the knee joint was vestigal coat and produced a skin incision distal to the knee joint in the direction of the tibia. The screws were stripped and indecipherable, as well as to determine the moment of extraction of the bone of the proximal screws. The distal screws were prepared for light microscopy, and the implant remained in the bone jn s is of chitosan on the titanium powder was performed, in most aspects, as described in example 1, and fixation was performed as described in example 3.

Fibula on each hind foot was exposed and her muscle tissue was taken away millimetre longitudinally at a distance of approximately 10 mm in the midline of the body bones. Segment Nude fibula length b mm was removed together with the periosteum and the defect between the bone ends were filled with titanium powder. To connect the edges of the defect and prevent the invasion of granulation tissue around the wrapped membrane filter made of PTFE. On the one hand titanium powder was with heparin coating, and on the other titanium powder without heparin coating. Then the wound was closed. The rats were given the ability to move without restriction within 3 weeks before the second study and ameridream animals. The study of defects fibula on each side showed that bone, spongy and plate, connected region in both defects. However, along heparinised titanium powder was observed more extensive bone formation. In addition, along heparinised titanium powder was observed more plate, i.e., well-organized bone.

Thus, titanium n the Itza.

Example 10

Osteogenic activity of the membranes of chitosan with heparin attached ionic bond, according to the results of method substitution defect of fibula

In rats under anesthesia on each hind foot has exposed the fibula and its muscle tissue was taken away millimetre longitudinally at a distance of approximately 10 mm in the midline of the body bones. Segment Nude fibula length of 6 mm was removed together with the periosteum and the defect between the bone ends were wrapped chitosan film.

To the left was placed chitosan membrane, prepared as described in example 5 (chitosan with 15% acetylation), and the defect of the right fibula was placed heparinized chitosan membrane, prepared as described in example 7 (chitosan with 15% acetylation).

In order to avoid spadine tube formed by the membranes and connecting edges 6 mm bone defect, along the defect was placed small fragments of bone.

Study three weeks later showed that both animals were first observed more pronounced formation of bone matrix and bone, i.e. where used heparinized chitosan film.

Example 11

Osteogenic activity HepB
Long been established that if the bone defects exceed a certain size, their healing occurs with the formation of the membrane of fibrous scar tissue connecting the edges of the bone defects. A critical size defect in the cranial vault adult rats is 8 mm, i.e., hole 8 mm or larger diameter is not closed bone tissue.

Rats were made pyramidally skin incision from nasofrontal region to the outer protrusion of the occipital bone. The skin and underlying tissue, including a large part of the temporal muscle each side, was taken away millimetre. To create a 8 mm hole in the skull on both sides used a specially made trepan. Took extraordinary precautions not to damage the meninges and the brain.

First on the Dura was placing the membrane of chitosan with heparin attached ionic bond, prepared as described in example 9, it was placed multiple bone fragments and the skull was placed identical additional membrane. Then in exactly the same way on the left was placed nerepressivnyh chitosan membrane, prepared as described in example 5, with intermediate bone fragments. After the but, that on the right side of the defect veil more asteroidea and newly formed bone than on the left side. In addition, experienced less severe inflammatory reaction in heparinized chitosan membrane.

Example 12

Preparation of chitosan granules coated doctranslate or heparin or chondroitin-4-sulfate

Dropwise from a syringe was added aqueous solutions of chitosan (2% wt./volume, 18% acetylation) to a solution of dextransucrase, or heparin or chondroitin-4-sulfate (0.1 weight. %/volume) in tripolyphosphates buffer. The resulting granules were placed on a glass filter, washed with water (1 l) and dried at 30oC during the night.

Example 13

Osteogenic activity of chitosan granules according to the results of bone formation after subperiostal Deposit on the skull

The location of the test in relation to the osteogenic activity of the compounds under the periosteum of the skull is a well-known technique.

Pellets made from chitosan and heparin, chitosan-coated doctranslate, and chitosan coated with chondroitin-4-sulfate, as described in example 12, was placed subperiosteally on the frontal bone of adult rats. One taconova granules were osteogenic, as evidenced by the formation of osteoid and bone tissue in the frontal bone. Chitosan granules, coated with chondroitin sulfate and chitosan granules coated doctranslate, also demonstrated osteogenic activity. Was also determined for cells of inflammation in various, usually small, amounts.

These experiments showed that chitosan in combination with certain polysaccharides demonstrate osteogenic activity.

It may be possible to achieve even better stimulate healing by combining the present invention with growth factors.

In vitro experiments with CFRP labeled with iodine-125 (acidic fibroblast growth factor, Bachem California), showed significantly higher specific binding of growth factor from heparinized screw compared to najprimitivniji screw. Even if you do not restrict the present invention to any specific theory, it seems likely that endogenous growth factors accumulate at the boundary between the implant and surrounding bone when the screw is provided with a coating of chitosan-heparin, resulting in stimulation of bone regeneration.

The present invention is not limited to the integration of a solid fabric, in particular in the bone tissue. Thus, the present invention is applicable in all areas, for example, dentistry, orthopedics, neurosurgery, hand surgery and plastic and reconstructive surgery. Also the present invention is quite suitable for use in the field of dentistry.

1. Chitosan and fixed it polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and dextransucrase, as agent, capable of providing stimulated the regeneration of hard tissues.

2. Application under item 1, characterized in that stimulated the regeneration of hard tissues is created in connection with implants in hard tissue such as bone.

3. Application under item 1 or 2, characterized in that the polysaccharide immobilized on chitosan by ionic bonds.

4. Application under item 1 or 2, characterized in that the polysaccharide immobilized on chitosan by covalent bonds.

5. The use according to any one of the preceding paragraphs, characterized in that the polysaccharide is heparin or heparansulfate.

6. The use according to any one of the preceding paragraphs, characterized in that the agent is in the form of powder or RA is 2">

8. The use according to any one of paragraphs.1 to 5, characterized in that the agent is in the form of granules.

9. The use according to any one of the preceding paragraphs, characterized in that the chitosan has a degree of N-acetylation of not more than about 90% and preferably not more than approximately 50%.

10. Application under item 9, characterized in that the chitosan has a degree of N-acetylation of less than about 25%.

11. The use according to any one of paragraphs.2 to 10, characterized in that the implant comprises titanium.

12. Ways to stimulate and/or accelerate the regeneration of hard tissue, wherein the agent containing chitosan and fixed it polysaccharide selected from heparin, heparan sulfate, chondroitin sulfates and dextransucrase, in a quantity sufficient to stimulate, put on the appropriate section.

13. The method according to p. 12, characterized in that the regeneration of hard tissues associated with osseointegration.

14. The method according to p. 13, characterized in that osseointegration is performed in connection with implants in hard tissue such as bone.

15. The method according to any of paragraphs.12 to 14, characterized in that the specified agent is applied in powder form.

16. The method according to any of the 2 - 16, characterized in that the polysaccharide is a heparin or heparansulfate.

18. The method according to any of paragraphs.13, 14 or 17, characterized in that the agent is applied by dipping the part of the implant, which is designed for integration into a solid fabric in a solution of chitosan and the specified polysaccharide.

19. The implant is designed for integration into solid tissue, characterized in that at least part of the implant, which is designed to integrate coated with an agent that stimulates the regeneration of hard tissue, comprising chitosan and fixed it the polysaccharide is selected from heparin, heparan sulfate, chondroitin sulfates and doctranslate.

20. The implant according to p. 19, characterized in that it comprises titanium.

 

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