RO131943B1 - Process for preparing a hydroxyapatite-based scaffold-type product for reconstruction of bone defects - Google Patents

Description

The invention relates to a process for obtaining a scaffold product based on hydroxyapatite, for the bone reconstruction of major bone defects, whose physical-chemical properties are predetermined by the control of phase parameters.

An ideal substitute for bone tissue must respect the structure and functions of natural tissue. It must also be biocompatible, available in a wide range of shapes and sizes, exhibit osteoinductive and osteoconductive properties, and be biodegradable. The substitution material must have a chemical composition similar to the original bone (for this reason, the presence of calcium orthophosphates in the material is recommended [1]), a porosity that allows vascularization and growth of new tissue, and suitable mechanical properties, which will it ensures the mechanical resistance together with the tissue in the process of regeneration. The degradation process of an ideal bone substitution material must depend on the rate of regeneration of the replaced tissue, and the substances released after degradation must be able to be metabolized by the human body without side effects. finally, the sterilization resistance, the ease of storage and the possibility of processing at low costs are required [2],

Biphasic calcium phosphates were developed as a result of the efforts of designing synthetic bone grafts with adapted resorption rate. The resorption and bioactivity of biphasic calcium phosphates can be controlled by varying the phase composition and crystallinity of the ceramic materials. Furthermore (although initially associated mainly with growth factors added to bone scaffolds), the osteoinductive character of a bone substitution material may be influenced by specific chemical or structural characteristics. This phenomenon also applies in the case of biphasic calcium phosphates which, by controlling the chemical composition, macroporosity and microporosity, have become able to promote osteogenesis in vivo, without the addition of growth factors [3-6].

A complementary hypothesis states that the mechanical properties of these compounds can also be improved as a result of the introduction of secondary phases in a ceramic matrix. The comparative evaluation of materials with different concentrations of hydroxyapatite and tricalcium phosphate has led to different results regarding biocompatibility. The differences are largely due to the wide range of precursors that can be used in the preparation, and to the different methods of obtaining biphasic compounds [7],

The researches in the field have materialized in the production of biphasic calcium phosphates by chemical synthesis methods based on the reaction between calcium salts and phosphate solutions [RU 2359708/2009; RU 2546539/2015; UK 255685/2015]. Although the proposed methods lead to obtaining nano- or submicrometric powders, the chemical synthesis presents the risk of contamination of the ceramic product with residues of the reaction between precursors.

The use of a natural precursor of the type of bovine bone tissue eliminates the risk of contamination (provided the thermal processing at temperatures above 850 ° C [8]), but the regulated [9] and standardized [10] methods use toxic substances as in deproteinization. of bone tissue. This aspect is solved by methods based on exclusively thermal deproteinization (fully provided for treatments performed at temperatures above 600 ° C) [8].

Deproteinization of bovine bone yields a non-stoichiometric hydroxyapatite type material, thermally stable up to about 1200 ° C [6, 8]. The conversion of this material into biphasic calcium phosphate is possible either by the heat treatment carried out in controlled atmospheres [11] or by chemical treatments (with non-toxic reagents) applied to the product in the last stages of manufacture.

RO 131943 Β1

The ceramic material obtained by the thermal treatment of the bone is easily processed by 1 grinding. The use of ball mills made of agate eliminates the risk of contamination of ceramic products while obtaining powders. After the milling, the powders can be sorted granulometrically, 3 as the control of the obtained powders is an essential component for the synthesis of ceramics. The use of spells with different particle sizes contributes 5 to the better packaging of the mixture, and influences the porosity of the sintered body [12],

The porosity requirements imposed on the product depend on its use / destination: Ί in the case of ceramic scaffolds, the porosity must allow adequate vascularization of the substitution material, in order to regenerate the bone tissue. The natural tissues are 9 nourished with nutrients and oxygen through a system of intensely branched blood vessels, which are divided into capillary tissues. The maximum distance between these 11 capillaries is about 200 pm, a value correlated with the oxygen diffusion limit [13, 14], in the case of tissues such as skin or cartilage, cells can be fed with 13 nutrients and oxygen through diffusion, from blood vessels that are located at a greater distance. This feature allowed the creation of artificial skin and cartilage, but almost all other types of tissue in the body (including bone tissue) are based on a vascular system with vessels less than 200 µm [15]. 17

There is a consensus that three-dimensional bone scaffolds must have a porosity of 40 ... 60%, in order to facilitate rapid nutrient diffusion and cell migration, the recommended 19 pore size varies between 50 ... 1000 pm for bone regeneration [ 16, 17], The size of the connections varies in the range 15 ... 50 pm [18, 19]. All these 21 values are affected to varying degrees by the uncertainty associated with the characterization methods. In this light, it is clear that the idea of ideal material must be adapted 23 to each application.

The porosity of the material can be further controlled by controlling the sintering parameters [11] and by adding porigens in the composition of the mixture [20]. The main sintering parameter that contributes to the control of the material properties is the heat treatment of the bone tissue, which induces changes in the chemical, morphological and structural characteristics. As these changes depend largely on the temperature of the heat treatment, the rigorous determination of the influence of the temperature on the properties of the products obtained from bovine bone tissue can be proved with an effective strategy of personalizing ceramics for bone regeneration.

The modification of the bovine bone tissue begins with the evaporation of the absorbed water at the level of its surface 33. The phenomenon occurs up to about 250 ° C. Collagen degradation begins in parallel with the evaporation of the absorbed water, and continues until complete removal of the organic component (approximately 500 ... 600 ° C). During degradation, the organic component will undergo complex changes, influenced by the degree of hydration, 37 the amount of organic substance in the bone and its type (compact or spongy).

Within the bone tissue, the organic component acts as a protective shield for calcium phosphate 39 which forms the mineral component [21]. For this reason, the mineral component does not undergo thermal transformations up to a temperature of 500 ... 600 ° C, when The organic phase of the bone tissue is completely degraded and eliminated; starting at 500 ° C, the non-stoichiometric hydroxyapatite existing in the bone tissue will undergo a series of 43 compositional and morphological changes and will eventually undergo a recrystallization process [22],

Increasing the treatment temperature above 750 ° C ensures biological decontamination 45 of the bovine bone tissue. around this temperature, hydroxyapatite will undergo a massive reduction in size, a phenomenon associated with the modification of the morphological appearance and parameters of the crystalline network, as well as with the removal of carbonate groups from the structure. After this,

RO 131943 Β1 it is assumed that hydroxyapatite will begin to decompose, but the phenomenon is strongly influenced by factors such as the chemical composition of the crude material, or the environment in which the heat treatment takes place. The main decomposition products of hydroxyapatite are different forms of oxiapatitis which will subsequently decompose into tricalcium phosphate of beta type or calcium oxide [6, 21-34],

Continuation of the heat treatment at temperatures higher than 1000 ° C leads to the appearance of hydroxyapatite crystals with granular morphology, embedded in a porous architecture [35], but by increasing the temperature to 1100 ... 1200 ° C, the porosity is reduced due to a process of densification of microstructure that continues up to 1300 ° C (when porosity disappears) [36]. The formation of other compounds, such as calcium oxide (CaO), was studied and did not allow XRD detection [28]. Other compounds identified in different studies performed at similar temperatures for heat treatment were tricalcium P-phosphate [6, 30] and tetracalcium phosphate [37],

The temperature and duration of heat treatment depend on the sample size, the amount of oxygen in the atmosphere and the preparation methods used prior to treatment [38]. In the deproteinization treatment a sample of 1 cm ³ of bone tissue, heat treated at temperatures between 600 and 1000 ° C, requires about 2 hours of maintenance, for the complete removal of the organic component [30]. In sintering treatments, carried out at temperatures higher than 1000 ° C, the increase of the maintenance time contributes to the densification of the microstructure of the ceramic material [39].

Collagen degradation, identified in the range 250 ... 500 ° C, is faster in air compared to other treatment media [28, 29, 32, 40, 41], in the range 500 ... 800 ° C, release of groups carbonate is deferred when heating takes place in an argon atmosphere [29]. whereas the release of carbonate groups from the hydroxyapatite structure leads to decreased thermal stability and the production of calcium oxide as a product of the mineral phase breakdown of bone tissue, which affects both the biocompatibility and mechanical properties of the formed materials [42, 43], this may be avoided by performing heat treatments in the atmosphere of carbon dioxide [43]. Heat treatment of bovine bone tissue in argon allows to obtain a two-phase HAP-β-TCP product at 1000 ° C. The content of β-TCP increases up to about 30% after heat treatment at 1200 ° C.

The control of the cooling conditions can contribute to the modification / maintenance of the calcium phosphate phase composition: the reaction of transformation of tricalcium beta-phosphate (β-TCP) into tricalcium alpha-phosphate (α-TCP) is reversible upon slow cooling. However, the preservation of tricalcium alpha-phosphate (resorbable) in the structure of heat-processed calcium phosphates may be possible by rapid cooling [44], the phenomenon is not valid for the super-alpha form of tricalcium phosphate (superα-TCP), which does not withstand hardening. at high temperatures [45, 46]. The influence of the cooling conditions is also confirmed by the morphological aspect of the bovine bone tissue samples, which suggests that the densification of the microstructure is accentuated when the water is cooled, while the Ca / P ratio decreases. The results suggest the acceleration of structural changes in the rapid cooling of the heat treated hard tissue, which could contribute to the efficiency of processing of calcium phosphates derived from bovine bone [11],

Powders obtained by heat treatment of bovine bone are suitable for use as precursors in additive / SFF / three-dimensional printing. Although some methods have already been patented for hydroxyapatite (US 8071007/2011 and US 6993406/2006), the field faces shortcomings such as dimensional errors caused by the accuracy of the equipment, lack of suitable binders to support ceramic powders, and anisotropy of properties [11] 47], starting from a material that satisfies the biological and mechanical requirements imposed by a specific medical application, it must also be adapted to the additive manufacturing method selected, following that after obtaining the three-dimensional structures the efforts will focus on improving their properties [48] .

RO 131943 Β1

The objective of the patent proposal is to obtain a scaffold 1 for the repair of major bone defects, obtained from monophasic products (HAP hydroxyapatite) or biphasic (hydroxyapatite + tricalcium phosphate of type a or β), which meet 3 mechanical requirements. and biocompatibility.

The technical problem solved by the invention consists in adapting the sequence of phases 5 and the phase parameters of a specific process for producing a scaffold-type support structure for the repair of major bone defects, from hydroxyapatite and tricalcium phosphate, which will allow the predictability and obtaining of mechanical porosity characteristics. and mechanical strength of the scaffold end product. 9

The process according to the invention for the production of a material for the reconstruction of bone defects, based on hydroxyapatite, solves this technical problem in that 11, in order to obtain a predetermined ratio between hydroxyapatite and tricalcium phosphate: HAP / TCP, after a preliminary phase of obtaining the hydroxyapatite by thermal deproteinization 13, by boiling, of the bovine bone, with mechanical removal of the protein material and heat treatment at 400 ... 500 ° C, 2 ... 4 h, in electric oven, in air atmosphere, with continuous ventilation 15, the heat treatment of the deproteinized bone in an electric oven, in an air atmosphere, at temperatures between 800 and 1200 ° C, for 2 ... 6 h, grinding in the mill with agate balls 17 for 2. .6 h and granulometric sorting by vibration in successive sieves with mesh size between 200 and 20 pm, in the next step, the obtained hydroxyapatite powders 19 are mixed and homogenized. Sizes with sodium pyrophosphate solution with a concentration of between 0.1 and 0.5 molar, at a solution / PAH ratio between 1/3 and 1/4, predetermined in correlation 21 with the desired HAP / TCP ratio, and then compacted by pressing in cylindrical shapes with diameters between 10 and 50 mm, with pressing forces between 10 and 500 MPa, depending on the 23 porosity and mechanical characteristics desired for the final product, which is obtained by drying the compacted product at room temperature, between 24 and 240 h, and its mechanical 25 processing by drilling with a drill, the diameter of the holes obtained varying between 1 and 3 mm, the product finally being sintered at 700 ... 1300 ° C for 2 ... 6 h, in an atmosphere of 27 air argon.

The holes are practiced in two directions: axially (perpendicular to the circular surface) 29 and in a direction perpendicular to the cylinder generator. The distance between any two extremities of the holes is between 1 and 3 mm (the maximum distance that allows 31 to maintain vascularization for bone regeneration), the value of the HAP / TCP ratio and the porosity being controlled by the concentration value of the sodium pyrophosphate solution, of 33 parameters. heat treatment and mechanical processing.

The process according to the invention has the advantage that it allows predictability and obtaining 35 mechanical porosity and mechanical strength characteristics of the final scaffold-type product as a function of the ratio: sodium pyrophosphate / hydroxyapatite solution and of the parameters 37 of the thermo-mechanical treatment.

The invention is presented in detail below, in connection with FIG. 1 ... 3, what 39 represents:

FIG. 1 a, b, c, graphical representation of the scaffolds presented in the table below 41 (positions 3, 5, 7);

FIG. 2, the variation of the phase composition of the ceramic material as a function of the concentration of 43 sodium pyrophosphate used in its production;

FIG. 3, concrete embodiment of a scaffold by the process according to the invention. According to the invention, the novelty element, in relation to existing patents and published articles, consists of two aspects: 47

- possibility of tuning the composition and crystallographic structure (HAP / TCP ratio variable between 70% HAP-30% TCP and 100% HAP-0% TCP limits) depending on the desired resorption rate 49;

RO 131943 Β1

- the way of obtaining the three-dimensional structure at macroscopic scale and the porosity at microscopic scale.

According to the procedure, in a preliminary phase, hydroxyapatite is obtained, by treating the bovine bone (central component of the femur from which the joints were removed) heat treated in two stages:

- strictly deproteinization by thermal means, to obtain the primary ceramic product (without organic components):

- boiling in water 2 ... 4 h;

- removal of soft tissue residues by mechanical procedures;

- boiling in distilled water 2 ... 4 h;

- heat treatment at 400 ... 500 ° C, 2 ... 4 h, in electric oven, in air atmosphere, with continuous ventilation;

- heat treatment of the deproteinized product in an electric oven, in an air atmosphere, at temperatures between 800 and 1200 ° C, 2 ... 6 h.

After obtaining the deproteinized components in massive form (wholly ceramic products with irregular, fragmented forms), powders with variable dimensions are obtained. The powder is obtained by grinding mills with agate balls in the agate chamber. The milling is performed 2 ... 6 h, with variable mill speeds. After grinding, the granulometric sorting is performed by vibrating in successive sieves, with the mesh size between 200 and 20 pm.

The obtained hydroxyapatite powders are mixed and homogenized with sodium pyrophosphate solution (concentration between 0.1 and 0.5 molar), and compacted by pressing into cylindrical shapes (with diameters between 10 and 50 mm), using pressing between 10 and 500 MPa, depending on the porosity and mechanical characteristics of the finished product.

After compaction, cylinders with diameters between 10 and 50 mm are obtained, and heights between 10 and 100 mm. These products are dried at room temperature (24 ... 240 h). After drying, the cylindrical products are machined mechanically by drilling with drills (diameters between 1 and 3 mm). The holes are practiced in two directions: axially (perpendicular to the circular surface) and in a direction perpendicular to the cylinder generator. The distance between any two ends of the holes is between 1 and 3 mm.

The ratio of HAP / TCP variable between the limits of 70% HAP-30% TCP and 100% HAP-0% TCP is obtained by changing the concentration of sodium pyrophosphate solution and the heat treatment parameters of sintering of the compacted product, achieved at the following parameters:

- period 2 ... 6 h;

- temperature 700 ... 1300 ° C;

- the atmosphere in the furnace (air-argon in proportions in the range 0 ... 100%).

The desired macroscopic porosity can also be obtained using the granulometric sort. The relationship between the size of the lots and the porosity is of direct proportionality.

The microscopic porosity is modified by choosing the heat treatment parameters:

- treatment time between 2 and 6 h;

- the treatment temperature between 700 and 1300 ° C.

After applying all the procedures, in the pre-determined order and with the preselected parameters depending on the specific surgical application, massive products are obtained, cylindrical in shape, bidirectionally perforated, with variable mechanical characteristics (compressive strength), depending on the composition, structure and dimensions of the macro- and micropores. These products can be mechanically processed intraoperatively, depending on the form of the surgical defect.

RO 131943 Β1

The mechanical characteristics of the products, regardless of the technological parameters used, allow the modification of the scaffold shape using the usual equipment, in the operating room, to obtain a custom implant, according to the needs of each individual case. To achieve this objective, the procedure for diagnosing and designing the custom implant involves the use of specific medical imaging techniques, and of three-dimensional reconstruction methods through mathematical modeling.

The choice of all the dimensions included in this patent is required by the biological, anatomical and functional characteristics necessary for the rapid reconstruction of the major bone defects, and for the recovery of the integral functionality of the resected tissue. The porosity of the final scaffold type product is given by the mechanical processing parameters and exemplified in the table presented below. The porosity given by the mechanical processing is complemented by the intrinsic porosity of the ceramic material derived from bone tissue.

Correlation between the porosity of the scaffold type products and the mechanical processing parameters

Nr. crt. Scaffold dimensions Nr. holes perpendicular Ρθ generator Nr. holes perpendicular to the radius Nr. total holes Pore size (holes) Theoretical porosity height [mm] Diameter [mm] Pore diameter [mm] Pore distance [mm] 1 30 30 182 145 327 1 1 27% 2 30 30 90 69 159 1 2 15% 3 30 30 49 37 86 1 3 8% 4 50 50 240 181 421 2 1 46% 5 50 50 132 97 229 2 2 27% 6 50 50 90 69 159 2 3 20% 7 100 50 264 87 351 3 1 54% 8 100 50 171 61 232 3 2 37% 9 100 50 112 45 157 3 3 27%

The graphical representation of the scaffolds presented in the table (positions 3, 5 and 7) are shown in fig. 1.

The variation of the phase composition of the products, given by the concentration of the sodium pyrophosphate solution used in the preparation, is shown in FIG. 2.

A concrete embodiment of a scaffold type product according to the invention is shown in FIG. 3.

Example

Pieces of bone tissue were cut from the central part of the bovine femur, which were deproteinized by boiling in water for 2 hours. After boiling, the soft tissue remnants were mechanically removed. Deproteinization of the raw material ended with a heat treatment performed in an electric oven, with continuous ventilation; the pieces of bone tissue were heat treated at 500 ° C, in air, for 2 hours.

The pieces of deproteinized bone tissue (ceramic material) were subjected to a heat treatment in an electric oven, with continuous ventilation; the ceramic material was heat treated at 1000 ° C, in air, for 6 hours. The pieces of ceramic material were ground in a ball mill, for 6 hours, and the resulting powder was sorted granulometrically, to ensure dimensions of the particles in the range 100 ... 200 pm.

Ί

RO 131943 Β1

The ceramic powder was homogenized and mixed with a solution of sodium pyrophosphate of 0.1 M concentration. The obtained paste was molded and pressed at 25 MPa, to obtain a cylindrical body with a diameter of 50 mm and a height of 50 mm . The cylindrical body was dried in air at room temperature for 120 hours.

The ceramic body was mechanically processed by drilling with a drill with 0 = 2 mm, keeping a distance d = 2 mm between any 2 holes drilled. Thus, 192 holes were drilled perpendicular to the generator (height) of the cylindrical body, and 97 holes were perpendicular to the diameter of the cylindrical body.

The mechanically processed ceramic body has undergone a heat treatment of synthesis in an electric oven, with continuous ventilation; the ceramic bodies were heat treated at 1000 ° C, in argon, for 2 hours.

A scaffold type product with biphasic composition in the proportions 70% HAP-30% β-TCP, with a theoretical porosity of 27% and pore size of 0.8 ... 2000 pm, was obtained by the process described above.

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Claims (2)

claims 1. Process for obtaining a scaffold product based on hydroxyapatite, for the reconstruction of major bone defects, with a predetermined ratio between hydroxyapatite and tricalcium phosphate HAP / TCP, including a preliminary phase of obtaining hydroxyapatite by thermal deprotection by boiling, of bovine bone, mechanical removal of protein material and heat treatment at 400 ... 500 ° C, 2 ... 4 h, in electric oven, in air atmosphere, with continuous ventilation, thermal treatment of bone deproteinized in electric oven, in air atmosphere, at temperatures between 800 and 1200 ° C, for 2 ... 6 h, grinding in the mill with agate balls for 2 ... 6 h and granulometric sorting by vibration in successive sieves with mesh size between 200 pm and 20 pm, characterized in that the hydroxyapatite powders obtained are mixed and homogenized with sodium pyrophosphate solution with a concentration of 0 , 1 and 0.5 molar, at a ratio: solution / HAP between 1/3 and 1/4, preset in correlation with the desired HAP / TCP ratio, and then compacted by pressing in cylindrical shapes with diameters between 10 and 50 mm , with pressing forces between 10 and 500 MPa, depending on the porosity and mechanical characteristics desired for the final product which is obtained by drying the compacted product, at room temperature, between 24 and 240 h, and its mechanical processing, by drilling with drill, the diameter of the holes obtained varying between 1 and 3 mm, the holes being drilled in axial directions, perpendicular to the circular surface, and in radial directions, with the distance between any two ends of the holes between 1 and 3 mm, the product thus obtained being finally synthesized at 700 ... 1300 ° C for 2 ... 6 hours in air-argon atmosphere. 2. Process for obtaining a hydroxyapatite-based scaffold product according to claim 1, characterized in that the mechanical processing of the HAP / TCP product is performed according to the following correlation table between the porosity of the scaffold products and the mechanical processing parameters: