Porous composite materials based on chitosan for filling of bone defects

FIELD: medicine.

SUBSTANCE: invention is related to the field of medicine and is related to composite materials for plastic reconstruction of damaged bone tissues. Invention represents porous composite material on the basis of chitosan for filling of bone defects, which contains chitosan, tricalcium phosphate and differs by the fact that it contains chitosan with molecular weight of more than 300000 g/mole, additive of ammonium carbonate, and calcium-phosphate fillers used are substances in the form of powder or granules with particle size of 1-1000 mcm, selected from the following group: brushit, monetite, tetracalcium phosphate, hydroxyapatite, carbonate hydroxyapatite, or their mixtures, at the same time components of material are available in a certain ratio.

EFFECT: invention provides for filling of bone defects of various shape and size, and calcium-phosphate fillers contained in sponge together with chitosan create favourable conditions for formation of natural human bone tissue.

11 ex, 2 tbl, 1 dwg

The invention relates to medicine, namely for plastic reconstruction of damaged bone tissue.

Chitosan is a biocompatible and biodegradiruemym natural polymer, which allows its use in various fields of medicine, including for rapid wound healing, different etymology (Chitin and Chitosan. Production, properties and application. /Under the editorship of academician of the RAAS KG Scriabin. The science. 2002. 365 pages). Especially widely used chitosan materials in the form of a plastic porous sponges. Due to the plasticity and porosity of these materials are easily deformed to the desired size (bone defect) and after placing them in a compressed state in the bone defect straightened (due to the inverse deformation of the filling volume of the defect. By their nature, chitosan is a polysaccharide nutrient that promotes the formation of bone tissue. However, pure chitosan sponges do not contain such important for bone formation elements such as phosphorus and calcium. So when resorption of clean sponge in the bone defect is formed mainly of cartilage (chondroitine).

To create favorable conditions for the formation of natural bone tissue is possible with the use of composite materials based on chitosan sponges containing calcium phosphate fillers.

N is the most closest technical solution and the achieved effect is a composite sponge chitosan-based (CHG), containing tricalcium phosphate (Yong-Moo Lee, Yoon Jeong Park et al. Tessie Engineered Boon Formation Used Chitosan/Tricalcium Phosphate Sponges // J. Calls, vol.71, No. 3, 2000). Composite sponge was obtained by mixing powder of tricalcium phosphate with a solution of chitosan. Then from the solution was removed excess water method of drying and freezing. When freezing of the initial solution in the form of particles of ice crystallizes aqueous solution, which is then removed by drying in vacuum. As a result, the remote location of the ice formed pore size of about 100 microns. The drawback received CHG is the use of special equipment (freeze drying), and the polymerization (crosslinking) of the jaws of environmentally harmful solution of sodium tripolyphosphate.

The technical result of the invention is obtaining a porous composite sponge chitosan and calcium phosphate filler formed at physiological temperatures, does not contain harmful substances.

The technical result is achieved in that the porous composite material based on chitosan for filling bone defects containing chitosan, tricalcium phosphate, according to the invention contains chitosan with molecular weight of more than 300,000 g/mol, additive ammonium carbonate, as well as calcium phosphate fillers used substances in the form of powder or granules with a size of 1-1000 μm selected from the group of brushite, monetic, tetrachloroferrate, hydroxyapatite, carbomethoxyamino or mixtures thereof, in the following ratio, wt.%:

The composition CHG unknown. To obtain KG in a solution of ethanoic acid are dissolved high-molecular chitosan at pH of 6 to 6.5. After complete dissolution of chitosan was added with stirring calcium phosphate fillers in an amount up to 90 wt.%. and powder additives ammonium carbonate to 60 wt.%.

As a result of interaction of the acid contained in the original solution, and ammonium carbonate foaming due to the release of carbon dioxide and the formation of a porous spongy structure with simultaneous hardening of the resulting sponge. The resulting sponge is then washed from excess water and residual acid in ethanol and dried. The result is a composite sponge with a porosity of from 50 to 98% depending on the composition. When the calcium content of the filler is greater than 90 wt.% sponges become fragile and break easily during deformation. If you increase the mass content of the additive is more than 60 wt.% in ubke formed numerous large pores larger than 2 mm, in the result, the structure becomes loose and brittle, which leads to the destruction of the jaws during deformation. By reducing the amount of ammonium carbonate less than 5 wt.% no hardening of the jaws when they are foaming. The reduction of the filler is less than 2 wt.% allows you to get a sponge with a uniform distribution of the components by volume.

Example 1. Powder of high molecular weight chitosan (molecular weight 450000-500000 g/mol) in an amount of 1 g (33.3 wt.%) dissolved in a solution of ethanoic acid. Then under stirring was added 1 g (33.3 wt.%) granules of hydroxyapatite (filler) with granule size of 100-300 μm and 1 g (33.3 wt.%) powder of ammonium carbonate. The resulting sponge is washed with ethanol and air-dried to remove ethanol. She got plastic composite sponge with a porosity of 85%.

Example 2. The blood samples were obtained analogously to example 1. The difference is the use of chitosan with molecular weight of 300000-350000 g/mol in an amount of 0.5 g (5 wt.%) and filler powder tricalcium phosphate with a particle size of 1-5 μm in the amount of 9 g (90 wt.%) and supplements of ammonium carbonate in the amount of 0.5 g (5 wt.%). She got plastic composite sponge with a porosity of 50%.

Example 3. The blood samples were obtained analogously to example 1. The difference is the use of chitosan with molecular weight of more than 500,000 g/mol in an amount of 2 g (20 wt.%) and filler powder is of carbonitesetuplite with a particle size of 1-5 μm in a quantity of 1 g (10 wt.%) and brushite powder with a particle size of 5-6 microns in quantities of 1 g (10 wt.%) and supplements of ammonium carbonate in the amount of 6 g (60 wt.%). She got plastic composite sponge with a porosity of 95%.

Example 4. The blood samples were obtained analogously to example 1. The difference is the use of chitosan with molecular weight of 400000-500000 g/mol in an amount of 6.0 g (60 wt.%) and filler powder carbonitesetuplite with a particle size of 15-30 nm in the amount of 1 g and tetrachlorophthalic granules with a size of 100-200 μm in the amount of 0.5 g (5 wt.%) and tricalciumphosphate granules with a size of 200-500 μm in the amount of 0.5 g (5 wt.%) and supplements of ammonium carbonate in the amount of 3.0 g (30 wt%). She got plastic composite sponge with a porosity of 70%.

Example 5. Powder of high molecular weight chitosan (molecular weight 450000 500000 g/mol) in an amount of 1 g (33 wt.%) dissolved in a solution of ethanoic acid. Then, with stirring, was added a mixture of 0.5 g (16.5 wt.%) monetite and 0.5 g (16.5 wt.%) the brushite with a size of 10-15 μm and 1 g (33 wt.%) powder of ammonium carbonate. The resulting sponge is washed with ethanol and air-dried to remove ethanol. She got plastic composite sponge with a porosity of 80%.

Example 6. The blood samples were obtained analogously to example 1. The difference is the use of chitosan with molecular weight of 300000-350000 g/mol in an amount of 0.5 g (5 wt.%) the mixture of filler granules tricalcium phosphate 6 g (60 wt.%) with the size of 500-700 μm and carbonitesetuplite 3 g (30 wt%) with the size of 800-1000 MK and supplements ammonium carbonate in the amount of 0.5 g (5 wt.%). She got plastic composite sponge with a porosity of 70%,

Example 7. The blood samples were obtained analogously to example 1. The difference is the use of chitosan with molecular weight of more than 500,000 g/mol in an amount of 3.0 g (30 wt.%) and filler powder tetrachloroferrate with a particle size of 50-60 microns in the amount of 0.5 g (5 wt.%) and hydroxyapatite powder with a particle size of 1-2 μm in the amount of 0.5 g (5 wt.%) and supplements of ammonium carbonate in the amount of 6 g (60 wt.%). She got plastic composite sponge with a porosity of 92%.

Example 8. The blood samples were obtained analogously to example 1. The difference is the use of chitosan with molecular weight of 400000-500000 g/mol in an amount of 5.0 g (50 wt.%) and filler powder carbonitesetuplite with a particle size of 15-30 nm in the amount of 1.5 g (15 wt.%) and carbonitesetuplite granules with a size of 300-500 μm in a quantity of 1.5 g (15 wt.%) and supplements of ammonium carbonate in the amount of 2.0 g (20 wt%). She got plastic composite sponge with a porosity of 70%.

In accordance with the examples were also produced samples of ceramics having compositions within the stated limits, and defines their properties in comparison with the prototype. The results obtained are summarized in table 1.

Data composites evaluated toxicity and bioactivity.

Example 9. In vitro cytotoxicity clicks scow porous composite material, obtained using: high-molecular chitosan - 60 wt.%, powder of hydroxyapatite (HA) - 20 wt.% and supplements of ammonium carbonate and 20 wt.% or chitosan - 20 wt.%, carbonitesetuplite the CCA - 20 wt.% and supplements of ammonium carbonate and 60 wt.% (MTT-test, model-culture immortalized human fibroblasts).

Before beginning the experiment, all the materials sterilized by γ-irradiation (20 KGy). On the eve of the experiment sterile samples were placed in 96-well card (in triplets), poured full growth medium (ORS) and left overnight in CO₂thermostat. On the day of the experience before making cells from each well decantation entire free volume of ORS was added 100 μl of a fresh portion of ORS, and at the last stage of cell suspension at a concentration of 70,000 cells/ml in a volume of 100 μl. The Board was placed for 24 hours in CO₂the incubator (37°C, 5% CO₂). All other cell manipulation, staging MTT-test and calculation of the results was carried out according to standard techniques. After 24 hours of cultivation was determined acute cytotoxicity of materials, calculating the value of the pool of viable cells (Agrocomplex) percent in any given period as the ratio of the rate of optical density (OD) of the solution formazan in the experiment to measure the optical density of the solution formazan in control. The sample is considered non-toxic in the amount the e indicator Agrocomplex> 70%.

It is shown that the samples are highly porous materials based on chitosan containing powders HA or the CCA, is not toxic to cells, the magnitude of the Agrocomplex data samples amounted to 91.8-106,4% (table 2).

Example 10 differs from the previous ones that were used pellets in the following ratio: high molecular weight chitosan 30 wt.%, granules HA - 10 wt.% and the addition of ammonium carbonate to 60 wt.% or are you homomolecular chitosan 5 wt.%, granules of the CCA - 50 wt.% and the addition of ammonium carbonate to 45 wt.% (MTT-test, model-culture immortalized human fibroblasts).

Estimated acute cytotoxicity of the samples of porous composite materials based on chitosan with HA granules or the CCA. Sample preparation samples, production MTT-test and the calculation of Agrocomplex was carried out as in example 9. Detected, these samples are also non-toxic in relation to the culture of fibroblasts:

the value of Agrocomplex 24 hours joint incubation was 72-97% of control (table 3).

In General, the presented material as the samples of example 9, improve adhesion and respectiveiy fibroblasts in a time comparable with the control.

Example 11. Medical-biological evaluation of porous composite sponge on the basis of high molecular weight chitosan - 33.3 wt.%, pellet carbonitesetuplite - 33.3 wt.% with the size of 200-500 μm and supplements ammonium carbonate in the amount of 33 wt.%.

The biocompatibility of this biocomposite was assessed after 2, 4 and 8 weeks after subcutaneous transplantation to mice BDF₁(sterilization by γ-irradiation of 25 kGy). It is shown that already in the early stages after surgery in preimplantational zone and a thin connective tissue capsule, within which is poorly visualized chitosan component. 2 weeks after subcutaneous transplantation around the granules loose patterns are formed headbands extracellular substance of bones. Themselves granules of the CCA submitted that the observation chaotically arranged cylindrical/rod-shaped structures (certej and paint hematoxylin-eosin, uvelj).

In the next term (4 weeks) shows the increase vnekletochnogo matrix of bone around the granules. While the structure of the granules becomes rarefied, in the peripheral portion and is of plantat in the spaces between the granules, in addition to the intercellular substance of bone tissue begins to form connective tissue in which are localized osteoclasts (certi,b). To 8 weeks, the amount of intercellular substance of bone is growing, especially around zapustevshih granules. In the fields of view are osteoclasts and malacitana connective tissue (certes,in).

Thus, the data of porous composite materials based on chitosan containing filler - pellets the CCA, have, on the one hand, biocompatibility, since neither of the drugs found no signs of inflammatory reaction and, on the other hand are truly osteoconductive potency as contribute to ectopic formation of new bone. The results of this study, these biomaterials are very promising as implants (independently), or osteogenesis 3D materials-matrices for engineering bone defects.

The porous composite material based on chitosan for filling bone defects containing chitosan, tricalcium phosphate, characterized in that it contains chitosan with molecular weight of more than 300,000 g/mol, additive ammonium carbonate, as well as calcium phosphate fillers used substances in the form of powder or granules with a particle size of 1-1000 μm, selected from the group bruchi is, monetic, tetrachloroferrate, hydroxyapatite, carbomethoxyamino, or mixtures thereof in the following ratio, wt.%: