RU2758551C1 - Method for determining the adsorption capacity of granular osteoconductive bone graft material - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to maxillofacial surgery, and discloses a method for determining the adsorption capacity of granular osteoconductive osteoplastic material (GOOM). The method is characterized by the fact that dust and air bubbles are removed from the GOOM 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. Next, the difference between the initial liquid volume and the liquid volume after passive degassing is calculated, as well as the difference between the liquid volumes before and after active degassing, then the indicated values are summed up, the resulting sum calculated for 1 ml of GOOM is the adsorption capacity of granular osteoconductive material. The method is aimed at optimizing the reparative regeneration of bone tissue by the method for directed bone regeneration. The use of the method for directed bone regeneration to eliminate a bone defect implies the use of bone-grafting materials as a matrix for reconstruction with external-internal surfaces and spaces available for the adsorption of bone growth factors.

EFFECT: method allows, due to the proposed degassing method, increasing the adsorption capacity of the original GOOM, optimizing the biotransformation of the osteoplastic material placed in the recipient bed into native bone, which will optimize the treatment time, avoid postoperative complications and repeated surgical interventions.

1 cl, 1 ex, 1 tbl

Description

The invention relates to the field of medicine, namely, maxillofacial surgery, and is aimed at optimizing the reparative regeneration of bone tissue 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, P 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 removal of air from pores and channels is not carried out 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 hydrodynamic flap created by air bubbles in the pores and canals, which prevents their filling with blood elements, the germination of blood vessels and the osteogenic cell pool inside the graft, is not removed;

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 the granular osteoplastic material is not calculated, which does not allow determining the preparation of a sufficient volume of bone growth factors for placement on the 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 well-known method for determining the influence of the geometry of hydroxyapatite as a cell substrate on bone formation was taken as a prototype (Q M Jin 1, H Takita, T Kongo, K Atsumi, H Itoh, Y 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 take into account the presence of a hydrodynamic valve created by air bubbles in the pores and channels, preventing their filling with blood elements, the germination of 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.

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

The essence of the method consists in performing sequential actions aimed at determining the adsorption capacity of granular osteoconductive osteoplastic material by determining the available area and volume of the external-internal spaces of the graft. To do this, the GOKM is subjected to passive and active degassing, the difference in liquid volumes before and after each degassing stage is determined, these differences are summed up, and the value of the adsorption capacity of granular osteoconductive osteoplastic material is obtained.

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

For the correct determination of the adsorption capacity index, 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 bone-grafting material production. Therefore, before introducing the osteoplastic material into the recipient bed, it is necessary to carry out actions aimed at determining its adsorption capacity.

For this purpose, it was proposed to remove coarse crumbs, fine dust and air bubbles from the cavities and channels of the CeraBone GOKM using the degassing method developed by us, 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 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 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).

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. 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 removing the hydrodynamic shutters took place due to the combination of small acid residues, the fraction of the material was immersed in a 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). 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, the volume of 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 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 for other osteoplastic materials was found out: Maxresorb, BioOSS, Xenograft Collagen, Osteon II, the data for which are given in Table 1.

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 determining the adsorption capacity of GOKM using the above technique is new in comparison with the described analogues and prototype, it allows to determine and increase the adsorption capacity of GOKM and to 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 , to avoid postoperative complications and repeated surgical interventions.

CLINICAL EXAMPLE

To develop the method, we conducted a study of 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 result of the method is achieved by the following conditions for performing the 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 by the 3D X-ray image of the skull bones, the boundaries of the restoration of the external contours are designed The surface relief of the bone defect is drawn and a blank of the profile template of the external relief of the restoration is drawn. The southern part of the pattern is 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 its adsorption capacity by removing coarse crumbs, fine dust and air bubbles from the cavities and channels of the CeraBone GOKM. 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 liquid volume and after passive degassing was 0.38 ml (2 ml - 1.62 ml = 0.38 ml).

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 material 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 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). 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, 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 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 makes it possible to accurately determine the adsorption capacity of the GOKM and the required volume of PRK.

Claims (1)

A method for determining the adsorption capacity of granular osteoconductive osteoplastic material (GOKM), including the removal of air bubbles and dust from its channels and cavities, characterized in that dust and air bubbles are removed by the degassing method, which includes the stages of passive and active degassing, and passive degassing is carried out in physiological solution, for which in the thermostat the temperature of the saline solution is brought to + 37 ° C, then 1 ml of the GOKM granules is placed in a test tube in the thermostat and filled with saline in a ratio of 1: 2, that is, 2 ml of saline is added ^{to 1 cm 3 of the GOKM,} immersed for 20 minutes GOKM in physiological solution at + 37 ° C, then separate the physiological solution from the material using a pipette dispenser, place it in an eppendorf and determine the difference between the initial volume of liquid and the volume of liquid after passive degassing, then proceed to the stage of active degassing, for this, a fraction of the material is poured citric acid solution with pH1 at + 37 ° C for 10 minutes, then place the material for ultrasonic treatment in an ultrasonic bath UZU-0.25 with a frequency of 18 kHz, a power of 250 W and an exposure time of 60 s, the ultrasonic generators are started 10 minutes after the presence of GOKM in a citric acid solution, while the release of gases and free-lying fine dust from the granules of the material into the washing liquid, then the difference between the volumes of the liquid before and after active degassing is measured, then the indicated differences in the volumes of the liquid after two stages of degassing are summed up, the resulting sum, calculated for 1 ml of GOKM, is the adsorption capacity of granular osteoconductive material.