RU2333010C1 - Material for closing osteal defects at reparative-plastic operations - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: described is material for closing osteal defects at reparative-plastic operations, manufacturing osteal implants, replacement of defects at various osteal pathologies. The material is made of calcium phosphate, represents particles of carbon-replaced hydroxyapatite with the general formula: Ca₁₀(PO₄)_x(OH)_y(CO₃)_z where 5<X<6, 0<Y<2, 0<Z<1, contains of 0.6 to 6.0 wt % CO₃ ^2- groups, with an adjustable nuclear ratio of calcium/phosphorus of 1.5 to 2.1. The material is made in the form of porous spherical granules with diameter of 100 to 1000 microns, having a rough microrelief of an external surface, with the size of pores from 0.5 to 15.0 mcm at the general open porosity of 50 to 80% and a specific surface of 0.3 to 0.6 m²/g. The material possesses the following properties: high adhesion in relation to cells; combination of osteoconductivity properties and osteoinductance; affinity of chemical and phase structure of implanted material to structure of replaced tissue; adjustability of rate of dissolution at its replacement by an osteal tissue; possibility of three-dimensional uniform filling of the osteal defect repeating its form. The material can be saturated with autologous mesenchymal stem cells.

EFFECT: improved properties of the material.

2 cl, 1 tbl, 6 dwg

Description

The invention relates to medicine, in particular to phosphorus-containing porous materials intended for the manufacture of bone implants and / or replacement of defects in various bone pathologies.

Currently, a number of composite materials based on calcium phosphate ceramics are known that are used as matrices for cellular technologies for the repair of damaged bone tissues.

A well-known example is the use of granular hydroxyapatite (hereinafter - HA) as a bone tissue implant with partial (1.2 and 2.05%) substitution with carbonate hydroxyapatite (hereinafter - KHA), which is comparable with the claimed material in terms of carbonate groups. The grain size of this material is 0.33 and 0.24 μm, respectively (Journal of materials science: materials in medicine 16 (2005) 899-907). An in vivo experiment on sheep is described. The disadvantages of the known material when used as a bone tissue implant include the fact that KHA particles are obtained by sintering ceramic bodies and subsequent grinding of the compact, which leads to a sharp decrease in the strength of the implant. In addition, the small grain size and the absence of internal pores is a significant obstacle to the process of osteogenesis.

The closest analogue to the claimed invention is KGA, which is a porous body obtained by impregnating cellulose sponges with KGA powder, followed by sintering. Bone implant testing was conducted in vivo in New Zealand white rabbits (Journal of the European Ceramic Society 23 (2003) 2931-2937). Adopted for the prototype. This material, including KGA, has an interconnected system of micro- and macropores with a total porosity of up to 45%. The Ca / P ratio in the known KGA is 2.01 ± 0.02. Such material, used as a bone substitute, showed in the experiment the properties of bioresorption and acceleration of the osteogenesis process in comparison with a material consisting of pure HA. However, as follows from the content of the prototype, the implantable material is placed in the bone defect in the form of ceramic particles of irregular shape, which can cause significant difficulties in filling defects of complex configuration and injure the surrounding tissue due to irregular particle shape, causing inflammation and other negative consequences. In addition, the ratio of Ca / P in the prototype is fixed equal to 2.01 ± 0.02, which does not allow to vary the chemical properties of the proposed KGA within the required limits.

The claimed invention is directed to solving the problem of creating a synthetic inorganic composite material designed to replace various bone defects.

When using the claimed invention, the following technical and therapeutic results can be achieved:

- biological activity of the implanted material, including high adhesive properties with respect to cells;

- a combination of the properties of osteoconductivity and osteoinductance;

- the proximity of the chemical and phase composition of the implanted material to the composition of the replaced tissue;

- adjustable dissolution rate when it is replaced by bone tissue:

- the possibility of three-dimensional uniform filling of bone defecg, repeating its shape.

The indicated technical and therapeutic results during the implementation of the invention are achieved due to the fact that the material for closing bone defects during reconstructive plastic surgery is a particle of KHA with a system of interconnected micro- and macropores.

A feature of the invention is that the KGA has the general formula Ca ₁₀ (PO ₄ ) _× (CO ₃ ) _y (OH) _z , where 5 <X <6, 0 <Y <2, 0 <Z <1, contains from 0 , 6 to 6.0 wt.% Groups CO ₃ ^2- , with an adjustable atomic ratio of calcium / phosphorus from 1.5 to 2.1. The material is made in the form of porous spherical granules with a diameter of 100 to 1000 microns, having a rough microrelief of the outer surface, and pore sizes of 0.5 to 15.0 microns with a total open porosity of 50 to 80% and a specific surface of 0.3 up to 0.6 m ² / g;

The invention consists in the following.

The main requirement for the materials used as matrices for cell technology for the restoration of damaged bone tissue is their biological activity. The term biological activity means the ability of a synthetic material to actively interact with surrounding tissues with the formation of a direct connection with them, exhibiting osteoconductivity and / or osteoinductance. Osteoconductivity is the ability of a material to adhere and bind osteogenic cells, ensure biological fluxes, neovascularization and maintain the processes of proliferation and differentiation of cells from surrounding living tissue with the formation of a direct connection with bone tissue and the gradual replacement of newly formed tissue. Osteoinductance is the ability of a material to induce on its surface the differentiation of cells from surrounding non-bone tissues into osteo-forming cells (chondrocytes, osteoblasts). A combination of osteoconductive and osteoinductive behavior is possible provided that the material affects the functions of the cells, for example, increasing the efficiency of differentiation of mesenchymal stem cells into osteoblast when cultivating the former on bioactive material. The inventive material is fully consistent with these requirements, which is confirmed by the results of in vitro and in vivo experiments, given below.

When developing materials for bone tissue reconstruction, it is advisable to achieve maximum closeness of the chemical and phase composition of the implanted material to the composition of the replaced tissue. From this point of view, KGA are of particular interest for use as biologically active material. It has been established that biological apatite of bone and dental tissues contains significant amounts of carbonate groups. Depending on the age of the person, the content of CO ₃ ^2- groups in bone tissues reaches from 2.3 to 8.0 wt.%. It is known that the mineral component of enamel, dentin and bone tissue contains, respectively, 3.5, 5.6 and 7.4 wt.% Groups of CO ₃ ^2- . Carbonate groups create lattice distortions, microstresses, and defects in the crystal lattice, which contributes to an increase in the resorbability of the material and an increase in the ability to osseointegration. The inventive material contains from 0.6 to 6.0 wt.% Groups of CO ₃ ^2- .

An important criterion when choosing a material for an implant is the kinetics of ceramic dissolution by extracellular body fluids (bioresorption kinetics). It has been established that the optimal kinetics of bioresorption, corresponding to the kinetics of osteogenesis during the replacement of the matrix with newly formed bone tissue, occurs at an atomic ratio of calcium / phosphorus from 1.5 to 2.1. The developed technology allows you to adjust the specified atomic ratio, which, ultimately, allows you to achieve the technical effect of the adjustable speed of bioresorption.

The inventive material is made in the form of granules having a spherical shape with a rough outer surface, a diameter of from 100 to 1000 microns, and pore sizes from 0.5 to 15.0 microns with a total open porosity of 50 to 80% and a specific surface of 0.3 up to 0.6 m ² / g. Granules have a system of open interconnected pores.

The use of spherical granules having the indicated dimensions allows for a uniform three-dimensional filling of the bone defect, repeating its shape. Granules of a smaller size (less than 100 μm) are a powder, the particles of which, when introduced into a bone defect, stick together with each other, forming an impenetrable mass. Excessive size of granules (more than 1000 microns) reduces the area of contact with the surrounding biological tissue, causing difficulties in their placement in defects of irregular shape.

Porosity, especially the presence of an interconnected pore system, enhances the adhesive properties of the material. The presence of small pores with a diameter of ~ 5.0 μm creates the conditions for the penetration of extracellular fluid into the implant from surrounding tissues. An interconnected system of larger pores with sizes of 10.0-15.0 μm creates optimal conditions for the replacement of the matrix with osteogenic cells, neovascularization and maintenance of proliferation and differentiation of cells from surrounding living tissue with the formation of a direct connection with bone tissue and the gradual replacement of newly formed tissue. At the stage of pre-preparation of the bioimplant, including the cultivation of mesenchymal stem cells (hereinafter referred to as MCT), the granule porosity acquires special significance, since with the increase in porosity the matrix cultural surface increases. The roughness of the outer surface of the granules provides an increase in their adhesive properties with respect to osteo-forming cells. A specific surface of less than 0.3 m ² / g is ineffective for adhesion; at a specific surface of more than 0.6 m ² / g, the granules are small and prone to the formation of agglomerates and dense mass, which makes it difficult to fill them with a bone defect.

Thus, due to the three-dimensional laying of porous ceramic granules in the bone defect, an optimal regime of reparative osteogenesis is achieved. Moreover, the surface roughness of individual granules significantly increases the total surface area, and therefore, the area for active cell expansion. Thus, the surface roughness of the granules provides them with matrix qualities - it facilitates the migration of osteoblasts with the colonization of individual granules and the formation of mature bone tissue. Porosity creates the conditions for effective neovasculogenesis.

The inventive material fully meets the biocompatibility requirements for materials for implantation, that is, the absence of cytotoxic and other negative reactions in contact with living tissue. The inventive material has an osteogenic effect, immune inertness.

The indicated qualities of the claimed material are confirmed by experimental results carried out in vitro and in vivo.

In vitro experiments to assess the dynamics of cell growth on the samples of the claimed material were performed on a model of the cell line of immortalized normal human fibroblasts (hereinafter referred to as PS), obtained from the Collection of Typical Cell Cultures of the Medical Genetic Research Center of the Russian Academy of Medical Sciences. The viability of the PS in the dynamics of the experiment was evaluated using the MTT method. In the study of acute toxicity of CAA with 0.6% and 6.0% substitution by CO ₃ ^2– anions, the absence of toxic manifestations of these materials with respect to transplantable culture of FC was found - after culturing for one day culture of FC survived 70-85% of the cell population. The inventors also investigated the matrix properties of these KGAs during long-term cultivation of PS on them. It was found that with a regular change in the growth medium twice a week, the amount of PS on these materials monotonically increased up to the 28th day of observation. Given that a day after incubation with these materials survive 70-85% from an initial pool of cells, the end of the experimental population on the FF samples increased 0.6% and 6.0% of replacement anions CO ₃ ^2- in 9 and 7 times, respectively.

In in vivo experiments with subcutaneous transplantation into mice of the claimed bioceramic materials, it was found that, at the studied observation time, the bioceramic materials studied did not cause an inflammation reaction, macro- and micro-signs of rejection, which indicates their biocompatibility. Two weeks after subcutaneous transplantation, it was found that KHA granules, having a spherical shape of different sizes and a rough outer surface, are surrounded by mature connective tissue. The inner space of the granules is sparse and has numerous cavities that are actively populated by fibroblasts. In parallel with the encapsulation process of each KHA granule and the granule conglomerate as a whole, the process of neovascularization is actively observed. Fibroblasts begin to form around them connective tissue with deposition of the intercellular matrix. In these islands of "young" connective tissue, the process of neovascularization also begins.

Thus, the results of a study of the matrix qualities of KHA granules with varying degrees of substitution with CO ₃ ^2- anions indicate their suitability for replacing bone defects (see table).

Biomedical indicators of KHA granules depending on their physicochemical characteristics Sample No. The size of the granules, microns The content of CO ₃ ^2- , wt.% Ca / P ratio Porosity,% Pore size, microns Result one <100 3.0 1.8 70 0.5-10 Adhesion and agglomeration of granules 2 one hundred 3.0 1.8 65 0.5-10 Satisfactory result * 3 500 3.0 1.8 74 0.5-10 Satisfactory result * four 1000 3.0 1.8 80 0.5-10 Satisfactory result * 5 1000 0.1 1.8 59 0.5-10 Low resorption rate 6 1000 7.0 2,5 60 Resorption rate too high 7 1000 3.0 1.8 68 0.5 Inadequate adhesive properties 8 1000 3.0 1.4 80 > 10 Narrow surface roughness and low cell growth dynamics 9 1000 4,5 2.2 65 0.5-10 Satisfactory result * 10 1000 3.0 1.8 83 0.5-10 Granules are fragile eleven 1000 3.0 1.7 47 0.5-10 Effective cell filling does not occur. 8 > 1000 3.0 1.7 65 Bone defect filling is ineffective * - according to the results of in vitro tests, the expressed matrix qualities of the surface of the samples.

Currently, a concept based on the use of stromal stem cells, the proliferation and differentiation of which provides tissue regeneration, has been developed. For the implementation of this technology, the parameters and properties of the matrices for the cultivation of cells with their subsequent implantation in a tissue defect are of great importance.

To obtain a bioimplant, KHA granules were saturated with autologous MSCs. For this, a culture of MSC 1-3 passports certified by immunophenotype, morphology, and proliferative activity was transferred to a culture vessel containing KHA granules in complete growth medium in an amount of 5-50 thousand cells per cm ² . The amount of cell mass was determined by the specific surface area of the ceramic material and the estimated volume of the tissue defect. It was found that after 5-8 days of co-cultivation, the bioimplant (MSC immobilized on the KGA matrix) can be used for intraoperative replacement of a bone defect of any configuration and volume.

Thus, the above information indicates that the claimed material for closing bone defects during reconstructive-plasmatic operations meets the criteria of patentability and has significant advantages compared with known materials of the same purpose.

The authors also conducted in vivo studies regarding the possibility of closing the bone defect of the lower leg of the rat with granular KHA ceramics, and ceramics saturated with autologous MSCs. For this, 3 groups of animals were formed:

1 g - shin defect - a control group to study the spontaneous closure of the defect;

2 gr. - implantation of KGA granules pre-saturated with a complete growth medium into the defect;

3 gr. - closing of the tibia defect in the rat with bioimplants - KHA granules with immobilized autologous MSCs of passage III.

To create a bone defect, male Wistar rats under general anesthesia formed a fenestrated defect of the upper cortical layer of the lower leg 5-7 mm long. The content of the bone channel under the defect was removed to the lower cortical layer. Then the defect was completely filled with granular KGA bioceramics. To prevent periosteal physiological regeneration, the periosteum was cut off along the edges of the defect. The operation was completed by layering the wound. In terms of 3, 7, 9, 12 weeks, 6, 9 and 12 months, 3 animals of each group were removed from the experiment. Tissue defect area tissues with surrounding tissues were used to prepare histological specimens.

The dynamics of the replacement of the tibia defect in rats with KHA granules is shown in Figs. 1a - 1 g (enlarged × 100), color - hematoxylin-eosin.

It was found that already 3 weeks after the injury, KHA granules without autologous MSCs stimulated osteo- and chondrogenesis. (Figa), and these processes proceeded in parallel with the bioresorption of the substance of the granules. As a result, in the area of the medullary canal and in place of the upper cortical layer, the granules of the reticular structure were “embedded” in the young bone tissue with foci of hematopoiesis, and in the area of the emerging periosteum, they were surrounded by fibrous connective tissue. By the 7th-9th weeks, the process of osteogenesis was growing (Fig. 1b, c), by 13 weeks the remains of the granules were walled up in mature bone tissue (Fig. 1d). Thus, the granular KHA showed pronounced osteoconductive properties: even in a large hemopoiesis focus, the remains of the granules were surrounded by the rim of the forming bone tissue.

In the 3rd group of animals (closing the defect with KHA granules saturated with autologous MSCs), the processes of reparative osteogenesis proceeded more actively than in the 2nd experimental group: 2 weeks after implantation, active ossification was observed with foci of hematopoiesis between the “sparse” granules, which testified to the formation of cancellous bone tissue, and these processes by the 7th - 9th weeks were visualized inside the resorbable granules (Figa, b). At the same time, the process of physiological remodeling of bone tissue was noted:

accumulations of multinucleated osteoclasts around the remains of individual granules in the inner layer of the periosteum.

Thus, it was found that porous KHA granules without autologous MSCs and loaded with autologous MSCs have osteoconductive and osteoinductive potencies. It is important to note that this biomaterial does not inhibit the morphogenetic potentials of the surrounding tissues and stimulates reparative regeneration.

Claims (2)

1. Material for closing bone defects during reconstructive plastic surgery based on calcium phosphates, which is a carbonate-substituted hydroxyapatite particle with a system of interconnected micro- and macropores, characterized in that carbonate-substituted hydroxyapatite of the general formula Ca ₁₀ (PO ₄ ) × (OH) _y ( СО ₃ ) _z , where 5 <X <6, 0 <Y <2, 0 <Z <1, contains from 0.6 to 6.0 wt.% CO ₃ ^2- groups with an adjustable atomic ratio of calcium / phosphorus from 1 , 5 to 2.1, while the material is made in the form of porous spherical granules with a diameter of from 100 to 1000 microns, having a rough m krorelef outer surface and having a pore size of from 0.5 to 15.0 microns for a total open porosity of 50 to 80% and a specific surface area of 0.3 to 0.6 m ² / g. 2. The material according to claim 1, characterized in that it is additionally saturated with autologous mesenchymal stem cells.

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