RU2758570C1 - Method for degassing granular osteoconductive bone graft material - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, namely maxillofacial surgery, and discloses a method for degassing granular osteoconductive osteoplastic material (GOOM). The method is characterized in that dust and air bubbles from the GOOM are removed by the degassing method, which includes the stages of passive and active degassing using physical and chemical methods, namely, heating in a thermostat in physiological solution and sonication using a citric acid solution.

EFFECT: method allows optimizing the reparative regeneration of bone tissue by the method for directed bone regeneration, allows increasing the adsorption capacity of the initial GOOM and obtaining a carrier matrix for bone tissue reconstruction with external-internal surfaces and spaces available for the adsorption of bone growth factors.

1 cl, 1 tbl, 1 ex

Description

The invention relates to medicine, namely to maxillofacial surgery, and is aimed at optimizing reparative bone tissue regeneration by the method of directed bone regeneration. The use of the method of guided bone regeneration for the elimination of a bone defect implies the use of osteoplastic materials as a matrix for reconstruction with external-internal surfaces and spaces available for the adsorption of bone growth factors.

The necessary and sufficient volume of bone growth factors (/ hereinafter PRK), which can adsorb granular osteoconductive osteoplastic material (hereinafter GOKM), depends on its adsorption capacity. The adsorption capacity of granular osteoconductive material is an indicator of the ability of a fraction of osteoplastic material to place the maximum possible amount of PRK on its outer-inner surfaces and spaces. The adsorption properties of GOKM have limitations associated with the presence of air and fine crumbs in the pores and channels of the fraction granules that penetrate into the material at the stages of the production cycle, which leads to the emergence of hydrodynamic and mechanical obstacles to the migration of PRK and the germination of vessels from the recipient bed into the granules.

There is a known model for predicting changes in the substance of a blood clot under the action of shear forces, similar to those that would occur in vivo under conditions of spatial heterogeneity of a blood clot and its environment, affecting the process of its contraction and fixation on the surrounding passive viscoelastic material of the carrier (Valerie Tutwiler, Hailong Wang , Rustem I. Litvinov, John W. Weisel, Vivek B. Shenoy. Interplay of Platelet Contractility and Elasticity of Fibrin / Erythrocytes in Blood Clot Retraction. Biophysical Journal. Article vollume 112, issue 4, Ρ 714-723, February 28, 2017 . DOI: https: //doi.org/10.1016/j.bpj.2017.01.005). The presented model does not allow:

1.determine the effect of the pores and channels of the underlying matrix of the carrier substrate on the ability of a blood clot and other blood components to move inside the carrier;

2. the adsorption capacity of the underlying substrate and its influence on the material's ability to adsorb a sufficient volume of bone growth factors are not determined;

3. to determine the volume of blood transformed into a clot, which can be placed on its accessible surfaces by the substrate, the carrier, including the surfaces of pores and channels;

4. The adsorption capacity of GOKM is not increased by removing air from pores and channels when placing a blood clot on a substrate in vitro.

A known method for assessing the relationship between porosity and pore size of biomaterials used for bone regeneration (Karageorgiou V., Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Journal of Biomaterials.

Published online 7 April 2005 in ScienceDirect (www.sciencedirect.com). DOI: 10.1016 / j.biomaterials.2005.02.002). The authors examined the influence of these morphological features on osteogenesis in vitro and in vivo. Determined that in vitro, lower porosity stimulates osteogenesis by suppressing cell proliferation and causing cell aggregation. In contrast, it has been demonstrated in vivo that high porosity and pore sizes> 300 μm promote direct osteogenesis both by recruiting osteoblastic cells, which are stimulated to migrate into the scaffold, and by vascularization, which promotes favorable new bone formation.

The key disadvantages of this method are:

1.the adsorption capacity of the GOKM is not increased by removing the hydrodynamic flap created by air bubbles in the pores and canals, which prevents them from filling with blood elements, the germination of blood vessels and the osteogenic cell pool inside the graft;

2. the ratio of pore volume / total volume of internal spaces in the granules of osteoplastic materials is not taken into account, which does not allow calculating the available area for the placement of bone growth factors in the graft;

3. the adsorption capacity of granular osteoplastic material is not calculated and does not increase, which does not allow determining the preparation of a sufficient volume of bone growth factors for placement on an osteoconductive carrier;

4. Calculation of the mass volume of cultivated growth factors without taking into account the adsorption capacity of the osteoconductive carrier leads to an unreasonable increase in the costs of material and human resources of the institution.

A known method for determining the influence of the geometry of hydroxyapatite as a cellular substrate on bone formation (Q Μ Jin 1, Η Takita, Τ Kohgo, K Atsumi, Η Itoh, Υ Kuboki. Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation. Journal of Biomedical materials research 2000 Dec 15; 52 (4): 491-9). The authors compared three different types of porous hydroxyapatite with a pore size of 100-200 μm in diameter in terms of their ability to induce osteogenesis by subcutaneous implantation of recombinant human BMP-2 in rats and extraction after 1, 2, 3 and 4 weeks. Found that pore geometry affects bone regeneration: long canals and interconnected spheroidal pores promote cell colonization and bone growth, while curved, poorly interconnected pores on the scaffold surface (e.g. superficial pits) prevent osteoblast progenitor cell penetration and capillary infiltration , which allows bone formation only on the surface of the framework.

The disadvantage of this method is that it does not increase the adsorption capacity of GOKM, does not take into account the presence of a hydrodynamic valve created by air bubbles in the pores and channels, which prevents their filling with blood elements, the germination of blood vessels and osteogenic cell pool inside the graft; the ratio of pore volume / total volume of internal spaces in granules of osteoplastic materials is not taken into account, which does not allow calculating the available area for placing bone growth factors in the graft; it is impossible to calculate the adsorption capacity of the granular osteoplastic material, which does not allow the preparation of a sufficient volume of bone growth factors for placement on the osteoconductive carrier; Calculation of the volume of the mass of cultivated growth factors without taking into account the adsorption capacity of the osteoconductive carrier leads to an unreasonable increase in the costs of material and human resources of the institution.

Closest to the claimed invention is a method for studying the physicochemical characteristics of mineral-based biomaterials, which are often used in dentistry (bovine: BioOss and PepGen; porcine: OsteoBiol and coral: Biocoral). The selected materials are presented in the form of granules of various biological origin: cattle, pig and coral (M. Figueiredo, J Henriques, G Martins, F Guerra, F Judas, Η Figueiredo. Physicochemical Characterization of Biomaterials Commonly Used in Dentistry as Bone Substitutes-Comparison with Human Bone. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 92 B: 409-419, 2010. In Wiley InterScience (www.interscience.wiley.com. DOI: 10.1002 / jbm.b.31529).

In addition to the classical rationalization of chemical composition and crystallinity, the main focus is on the measurement of various morphostructural properties such as particle size, porosity, density and specific surface area. It is noted that these properties are critical to obtain a complete interpretation of in vivo performance. The disadvantages of this method is that in this study there is no characteristic of natural pollution and air congestion on the outer-inner surfaces of cavities and canals, their influence on the osteogenic properties of bone-plastic material is not analyzed. No method has been developed for removing contaminants and air from the outer-inner surfaces of the cavities and channels of granules, aimed at determining and increasing the adsorption capacity of the graft and improving its osteogenic characteristics.

The objective of the invention is to eliminate these disadvantages and create a method for degassing granular osteoconductive bone-plastic material to increase its adsorption capacity.

The essence of the method consists in performing sequential actions for degassing the GOKM, aimed at increasing the adsorption capacity of granular osteoconductive osteoplastic material by increasing the available area and volume of the external-internal spaces of the graft and removing air bubbles and fine fractional crumbs from the canals and cavities of the GOKM. For this, the GOKM is subjected to passive and active degassing.

The essence of the method is shown on the example of the CeraBone GOKM.

To increase the adsorption capacity of GOKM, air bubbles and fine fractional crumbs were removed from its channels and cavities, which create mechanical obstacles to migration and adsorption of bone growth factors on the surface of the outer-inner spaces. Fine dust and air bubbles are deposited on the outer-inner surfaces of the cavities and channels of the granules at the stages of production of bone-plastic material and thereby reduce the adsorption capacity of the GOKM and worsen the results of surgical treatment. Therefore, before introducing osteoplastic material into the recipient bed, it is necessary to carry out actions aimed at increasing its adsorption capacity, that is, degassing.

For this purpose, it is proposed to remove coarse crumbs, fine dust and air bubbles from the cavities and channels of the CeraBone GOKM using the developed degassing method, which includes the stages of passive and active degassing. The task of passive degassing is to free the outer surfaces of the material from coarse crumbs and air. The task of active degassing is to free the inner surfaces of channels, pores and voids of the material from fine dust and residual gases.

Passive degassing is carried out in physiological solution, since its osmotic pressure (0.9%) corresponds to the osmotic pressure of blood plasma. In a thermostat, the temperature of the saline solution was brought to + 37 ° C, which corresponded to the temperature of the recipient bed and reduced the stress experienced by the cellular elements located in the tissues surrounding the graft. In a thermostat, HOCM was placed in a test tube and it was poured with physiological solution in a ratio of 1: 2 (2 ml of physiological solution was added to 1 cm of HOCM).

It has been experimentally established that at the stage of passive degassing, when the GOKM is immersed for 20 minutes in a physiological solution at + 37 ° C, gas bubbles and coarse fractional crumbs are released from the GOK. In 20 minutes after the passive degassing stage, the physiological solution was separated from the material using a pipette dispenser and placed in an Eppendorf. It was determined that after passive degassing, the volume of the recovered liquid was 1.62 ml. The difference between the initial liquid volume and after passive degassing was 0.38 ml (2 ml - 1.62 ml = 0.38 ml).

Next, proceed to the stage of active degassing. For this, a fraction of the material is poured with a citric acid solution (pH1) of 1.62 ml. at + 37 ° С for 10 minutes. Then, active degassing is carried out in an ultrasonic bath UZU - 0.25 with a frequency of 18 kHz, a power of 250 W and an exposure time of 60 sec. The ultrasonic generators are launched 10 minutes after the GOKM is in the citric acid solution. The effect of cavitation arose, which initiated water shocks on the outer-inner surfaces of the granules. The unevenness of these surfaces led to the appearance of interference and diffraction of waves, as a result of which gases and free-lying fine dust were released from the granules of the material into the washing liquid. A stable process of removal of hydrodynamic dampers took place due to the combination of small air bubbles into large ones, followed by their collapse and release of the material from the channels into the solution.

At the end of the stage of active degassing, using a pipette dispenser, we again took the liquid and placed it in the eppendorf to determine its residual volume, which was 1.44 ml. To remove acid residues, a fraction of the material was immersed in physiological solution at a temperature of + 37 ° C and treated in an ultrasonic bath UZU - 0.25 with a frequency of 18 kHz, a power of 250 W and an exposure time of 60 sec. The difference between the liquid volumes before and after active degassing was 0.18 ml (1.62 ml - 1.44 ml = 0.18 ml). The results of the difference in liquid volumes after two stages of degassing were summed up, the sum was 0.56 ml (0.38 ml + 0.18 ml = 0.56 ml). Next, we proceeded to calculate the difference between the initial volume of liquid and the final volume after two stages of degassing. Thus, after carrying out two stages of degassing, there was a decrease in the volume of liquid by 0.56 ml (2.0 ml - 1.44 ml = 0.56 ml) and an increase in its adsorption capacity by 0.56 ml or 28%. Consequently, the adsorption capacity of the granular osteoconductive material CeraBone as a result of degassing was 0.56 ml and increased from the initial one by 28%.

It has been experimentally revealed that the value of the adsorption capacity can be used to determine the volume of bone growth factors adsorbed by the graft, which the material is able to place on its surfaces. Accordingly, in this case, to place 1 ml of granular osteoconductive osteoplastic material CeraBone on the external-internal surfaces prepared by the proposed method and in free spaces, it is enough to prepare 0.56 ml of PRK. Thus, by determining the adsorption capacity of GOCM, the value of the adsorption capacity is obtained and the volume of free spaces of the material available for adsorption of PRK is increased, and, if it is used as a PRK carrier, it is determined what volume of PRK will be sufficient to be prepared for placement on the carrier.

Similarly, the adsorption capacity and the percentage of its increase for other osteoplastic materials were found out: Maxresorb, BioOSS, Xenograft Collagen, Osteon II, the data for which are given in table 1. Table # 1. Indicators of adsorption capacity, and the percentage of its increase in granular bone-plastic materials of animal and synthetic origin.

The thus increased volume of the outer-inner spaces of the GOKM becomes available for greater diffusion of cells, tissue fluid, blood plasma and vascular germination, due to the displacement of air bubbles, coarse crumbs and fine dust. The increased internal volume of the graft and, accordingly, the purified area of its surfaces, increase the diffusion of cells, tissue fluid, blood plasma and vascular invasion into the granules of the material and make it possible to achieve oppositional reparative osteogenesis.

The method for increasing the adsorption capacity of GOKM using the above-described degassing technique is new in comparison with the described analogs and prototype, it allows to increase the adsorption capacity of GOKM and achieve a new result: to optimize the biotransformation of the osteoplastic material placed in the recipient bed into the native bone, which will optimize the treatment time, avoid postoperative complications and repeated surgical interventions.

CLINICAL EXAMPLE

To develop a degassing method, a study was carried out on GOKM from different manufacturers with the same type of characteristics specified by the manufacturer in the instructions for use (BioOSS, CeraBone, Maxresorb, Osteon II, Xenograft Collagen, and registered in Russia for use (Table 1). The essence of the method is described using the example of CeraBone GOKM.

The technical result of the method is achieved by the following conditions for carrying out actions. In order to plan the elimination of a complex bone defect in a computer program, the topography, design, composition of the segments and the volume of a complex bone defect in the axial, frontal and sagittal planes are determined in a computer program using a 3D X-ray image of the bones of the skull, a template for the external relief of the restoration. Then, the volumes of the defect segments are calculated within the boundaries of the planned restoration and the perimeter of the template for the insulating membrane is measured. The vestibular and axial parts of the pattern are combined along the lower border of the vestibular part of the alveolar process and a general contour for cutting the pattern of the insulating membrane template is obtained.

After calculating the required volume of granular osteoconductive osteoplastic material, before placing bone growth factors on it, actions were taken to determine and increase its adsorption capacity by removing coarse crumbs, fine dust and air bubbles from the cavities and channels of the CeraBone GOKM due to degassing. At the stage of passive degassing, the outer surfaces of the GOKM were freed from coarse crumbs and air. At the stage of active degassing, the inner surfaces of the channels, pores and voids of the GOKM were freed from fine dust and residual gases.

Passive degassing was carried out in physiological solution, since its osmotic pressure (0.9%) corresponds to the osmotic pressure of blood plasma. In a thermostat, the temperature of the saline solution was brought to + 37 ° C, which corresponded to the temperature of the recipient bed and reduced the stress experienced by the cellular elements located in the tissues surrounding the graft. In a thermostat, HOCM was placed in a test tube and it was poured with physiological solution in a ratio of 1: 2 (2 ml of physiological solution was added ^{to 1 cm 3 of HOCM).}

It has been experimentally established that at the stage of passive degassing, when the GOKM is immersed for 20 minutes in a physiological solution at + 37 ° C, gas bubbles and coarse fractional crumbs are released from the GOK. In 20 minutes after the passive degassing stage, the physiological solution was separated from the material using a pipette dispenser and placed in an Eppendorf. It was found that after passive degassing, the volume of the recovered liquid was 1.62 ml. The difference between the initial volume of liquid and after passive degassing was 0.38 ml (2 ml - 1.62 ml = 0.38 ml), that is, the adsorption capacity of the material increased by 19% (0.38: 2.0 = 0.19 ).

Further, we proceeded to the stage of active degassing. For this, a fraction of the material was poured with a citric acid solution (pH1) of 1.62 ml. at + 37 ° С for 10 minutes. Then, active degassing was carried out in an ultrasonic bath UZU - 0.25 with a frequency of 18 kHz, a power of 250 W and an exposure time of 60 sec. The ultrasonic generators were launched 10 minutes after finding the GOKM in a citric acid solution. The effect of cavitation arose, which initiated water shocks on the outer-inner surfaces of the granules. The unevenness of these surfaces led to the appearance of interference and diffraction of waves, as a result of which gases and free-lying fine dust were released from the granules of the material into the washing liquid. There was a stable process of combining small air bubbles into large ones, followed by their collapse and release from the channels of the material into the solution.

At the end of the stage of active degassing, using a pipette dispenser, we again took the liquid and placed it in the eppendorf to determine its residual volume, which was 1.44 ml. To remove acid residues, a fraction of the material was immersed in physiological solution at a temperature of +37 degrees C and sonicated in an ultrasonic bath UZU - 0.25 with a frequency of 18 kHz, a power of 250 W and an exposure time of 60 sec. The difference between the liquid volumes before and after active degassing was 0.18 ml (1.62 ml - 1.44 ml = 0.18 ml), that is, the adsorption capacity of the material increased by 9% (0.18: 2.0 = 0 , 09).

The results of the difference in liquid volumes after two stages of degassing were summed up, the sum was 0.56 ml (0.38 ml + 0.18 ml = 0.56 ml), that is, the adsorption capacity of the GOKM increased by 28% (19% + 9% = 28 %). Next, we proceeded to calculate the difference between the initial volume of liquid and the final volume after two stages of degassing. Thus, after carrying out two stages of degassing, the volume of the liquid decreased by 0.56 ml (2.0 ml - 1.44 ml = 0.56 ml) and its adsorption capacity increased by 0.56 ml. Therefore, the adsorption capacity of the CeraBone granular osteoconductive material is 0.56 ml.

It has been experimentally revealed that the value of the adsorption capacity can be used to determine the volume of bone growth factors adsorbed by the graft, which the material is able to place on its surfaces. Accordingly, in this case, to place 1 ml of granular osteoconductive osteoplastic material CeraBone on the external-internal surfaces prepared by our method and in free spaces, it is enough to prepare 0.56 ml of PRK. Thus, by determining the adsorption capacity of GOCM, the value of the adsorption capacity available for the adsorption of PRK is obtained, and, if it is used as a PRK carrier, we find out what volume of PRK will be sufficient to prepare for placement on the carrier.

The invention allows due to the proposed degassing method to increase the adsorption capacity of the original GOKM and to accurately determine the required volume of PRK.

Claims (1)

A method for degassing granular osteoconductive osteoplastic material (GOKM), including removing air bubbles and dust from its channels and cavities, characterized in that dust and air bubbles are removed by degassing, which includes the stages of passive and active degassing, and passive degassing is carried out in physiological solution, in the thermostat, the temperature of the saline solution is brought to + 37 ° C, the HOCM is placed in the thermostat in a test tube and it is poured with saline in a ratio of 1: 2, that is, 2 ml of physiological solution is added to 1 cm of the HOCM, and the HOCM is kept in a thermostat for 20 minutes, through 20 minutes after the passive stage of degassing, the physiological solution is separated from the GOKM using a pipette dispenser, then the stage of active degassing is carried out, for this, a fraction of the material is poured with a citric acid solution with pH1 at + 37 ° C for 10 minutes, then ultrasonic treatment is carried out in an ultrasonic bath. - 0.25 with a frequency of 18 kHz, a power of 250 W and an exposure time of 60 seconds, the ultrasonic generators are launched 10 minutes after the GOKM is in a citric acid solution, a cavitation effect is created, after which a fraction of the material is immersed in a physiological solution at a temperature of + 37 ° C and re-processed in an ultrasonic bath USU - 0.25 with a frequency of 18 kHz, a power of 250 W and an exposure time of 60 sec.