The present invention regards a method of manufacturing composite bone implants containing synthetic hydroxyapatite and fibroin, a method of manufacturing a powdered raw material for such composite bone implants, a powdered raw material for implants of this kind, and a composite bone implant containing synthetic hydroxyapatite and fibroin.

One of the concerns of regenerative medicine are durable and biocompatible materials for bone implants. Optimally, such material should be resorbable and have properties similar to the natural bone. The cortical bone tissue is characterized by compressive strength between 100 and 230 MPa, bending strength between 50 and 150 MPa and Young's modulus in the range from 7 to 30 GPa. These data are disclosed in Jan Henkel et al. „Bone Regeneration Based on Tissue Engineering Conceptions - A 21st Century Perspective" [Bone Research (2013) 1 , pp. 216-248]. A bone implant consisting entirely of synthetic hydroxyapatite has a high biocompatibility and bioactivity, but at the same time has a low bending strength and too high Young's modulus. Therefore, in manufacuring of such implants, a composite consisting of crystalline hydroxyapatite and a polymer is often used, both synthetic and natural, for example a natural silk building protein, i.e. fibroin. Fibroin is biocompatible and resorbable. In addition, fibroin naturally occurs in the form of fibres characterized by high mechanical strength. The use of fibrous structure for purposes of bone tissue regeneration is also beneficial from a biological point of view. Composite structures containing fibroin and synthetic hydroxyapatite have been the subject of a number of patent applications. Publication WO 2009/100280 discloses a method of manufacturing mixtures consisting of silk and hydroxyapatite, from which when in a liquid form solid structures of required shapes can be cast or which can be used in a form of special paste for treating bones and teeth. The hydroxyapatite content of such a mixture can vary from 0.1% to 90%. The preparation of the mixture starts with boiling of silkworm cocoons in an aqueous salt solution to remove the second silk building protein, i.e. sericin, from silk fibres. The silk which has been purified from the serine is dissolved in the lithium bromide solution (LiBr) and then it is separated from the solution by dialysis and centrifugation. The thus-separated fibroin is freeze-dried and then dissolved in hexafluoro isopropanol (HFIP). The synthetic hydroxyapatite is added to the fibroin solution in the desired proportion and a liquid substance is obtained which is suitable for casting during which a solid form is obtained by evaporation. The material thus produced, however, has a low mechanical strength, and its average Young's modulus during compression for the material with 30% by weight of hydroxyapatite is 84.2 MPa.

Publication CN 02058907 discloses a method of manufacturing bone implants containing hydroxyapatite. In the first step, a wollastonite suspension is chemically prepared, then dried to powder. Fibroin is dissolved in calcium nitrate solution. To the mixture of solutions: fibroin with calcium nitrate, sodium phosphate and sodium hydroxide a water-diluted wollastonite is added. During 48 to 72 hours the suspension precipitates, which is then separated from the solution and dried. The obtained powder contains from 0 to 30% by weight of wollastonite, from 25 to 35% of fibroin and from 40 to 60% of hydroxyapatite. The powder is then cold pressed isostatically. The compressive strength of the thus obtained composite is 90 MPa. Publication CN 102000362 discloses a process of manufacturing porous scaffolds containing fibroin and nano-hydroxyapatite in an amount of 30 to 90% by weight. In this process, fibroin is first dissolved in a three-component system of calcium chloride-ethanol-water, and then diammonium phosphate, ammonium hydroxide and ammonia are added to the solution. The resulting precipitate, containing fibroin and hydroxyapatite, is dried and grinded. The powder is mixed with water to form a paste, to which the carboxymethyl cellulose is added and then by lyophi- lisation a porous structure is obtained. The thus-obtained scaffold is characterized by an open porosity of 40%, but at the same time has a very low compressive (from 3 to 3.69MPa) and bending (from 3 to 4.75MPa) strength.

The aim of the invention was to obtain composite bone implants containing hydroxyapatite and fibroin with a higher mechanical strength than previously known.

This aim is reached by a method of manufacturing implants, which includes a step of preparation a raw material (consisting of the synthetic hydroxyapatite and fibroin), and a step of forming the implant from such a raw material by pressing. The method of manufacturing implants according to the invention consists of using the raw material in a powdered form, and in the raw material preparation step a composite powder is produced by simultaneous cryogenic grinding and mixing of fragmented natural silk fibres and powdered synthetic nanometric hydroxyapatite. The proportion of hydroxyapatite in the obtained raw material is from 70% to 95% by weight.

In one of variants of the method of manufacturing implants according to the invention a nanometric hydroxyapatite with a particle size of not more than 80 nm is used. In another variant of the method of manufacturing implants according to the invention the composite powder, obtained by cryogenic mixing and grinding, is dried, advantageously at a temperature of not more than 160°C, until the powder has lost at least 2% of the initial weight.

In next variant of the method of manufacturing implants according to the invention the cryogenic grinding and mixing is carried out at a temperature no higher than -150°C.

In next variant of the method of manufacturing implants according to the invention, during the step of implant formation the raw material in a form of composite powder is pressed at a pressure of not less than 0.8 GPa and not more than 1.5 GPa.

In next variant of the method of manufacturing implants according to the invention hydroxyapatite is used with a molar ratio of calcium to phosphorus in the range from 1.57 to 1.65.

In next variant of the method of manufacturing implants according to the invention the cryogenic grinding and mixing is carried out until a powder with a homogenous appearance is obtained. ln another variant of the method of manufacturing implants according to the invention the drying of the composite powder is carried out in a vacuum, after which the powder is purged with an inert gas.

In yet another variant of the method of manufacturing implants according to the invention the pressing of the composite powder is carried out at a temperature not lower than 20°C and not higher than 160°C.

The method of manufacturing a powdered raw material for bone implants according to the invention consists of cryogenic grinding of sections of natural silk fibre with a length not exceeding 20 mm and mixing them with powdered synthetic nanometric hydroxyapatite in an amount from 70% to 95% by weight.

In one of variants of the method of manufacturing a raw material according to the invention a nanometric hydroxyapatite with a particle size of not more than 80 nm is used.

In next variant of the method of manufacturing a raw material according to the invention the composite powder, obtained by cryogenic grinding and mixing, is dried, preferably at a temperature not higher than 160°C, until the powder has lost at least 2% of the initial mass.

In next variant of the method of manufacturing a raw material according to the invention the cryogenic grinding and mixing is carried out at a temperature no higher than -150°C.

In next variant of the method of manufacturing a raw material according to the invention hydroxyapatite is used with a molar ratio of calcium to phosphorus in the range of 1.57 to 1.65.

In another variant of the method of manufacturing a raw material according to the invention a cryogenic grinding and mixing are carried out until a powder with a homogenous appearance is obtained.

In yet another variant of the method of manufacturing a raw material according to the invention the drying of the composite powder is carried out in a vacuum, after which the powder is purged with an inert gas.

The powdered raw material according to the invention contains a synthetic hydroxyapatite and fibroin and is characterized by containing from 70% to 95% by weight of hydroxyapatite with a particle size of not more than 80 nm and a molar ratio of calcium to phosphorus in the range from 1.57 up to 1.65. In one of variants of the raw material according to the invention it contains fibroin in the form of natural silk fibres.

In another variant of the raw material according to the invention the fibroin fibres have a length of not more than 1000 pm and a diameter of not more than 20 pm. In another variant of the raw material according to the invention at least 50% of the fibroin fibre surface is coated with hydroxyapatite particles adhering to it.

In yet another variant of the raw material according to the invention it can be manufactured by the above described method according to the invention.

The implant according to the invention, consisting of a homogeneous mixture of synthetic hydroxyapatite and fibroin, has a content of 70% to 95% by weight of nanometric hydroxyapatite with a particle size of not more than 80 nm and a molar ratio of calcium to phosphorus from 1.57 to 1.65, Young's modulus in the range from 7.5 to 9.5 GPa, total porosity in the range from 10 to 20%, bending strength not less than 30 MPa and compressive strength not less than 100 MPa.

In one of variant of the implant according to the invention it consists fibroin in the form of a component of a natural silk fibre.

In next variant of the implant according to the invention the fibroin fibres have a length of not more than 1000 pm and a diameter of not more than 20 pm.

In next variant of the implant according to the invention it has an isotropic micro- pattern in areas of 500 pm x 500 pm x 500 pm.

In yet next variants of the implant according to the invention is made by the methods according to the invention described above or from the raw material according to the invention described above.

The invention presents a very simple way for obtaining bone implants containing hydroxyapatite and fibroin with much higher strength parameters than those previously known. This is achieved without the chemical reaction stage and without introducing other substances into the composite. Furthermore, the implants according to the invention have fibrous structures that improve the biocom- patibility of the implant.

The examples of the embodiments of the invention are shown on the drawings, in which Fig.1 presents an SEM image of the implant from the first example, Fig.2 presents an SEM image of the implant from the second example, Fig.3 presents an SEM image of the implant from the third example, and Fig.4 presents an SEM image of the implant from the fourth example. Fig.5A, Fig.5B, Fig.5C i Fig.5D present microtomographic images of the implant section from the first example. Fig.6 and Fig.7 show, in two different magnifications, the SEM images of the composite powder from which the implant of the first and third examples has been made, Fig.8 and Fig.9 show the composite powder of the second and fourth examples,

The invention will be described in further detail in the following embodiments: Example 1

In order to obtain an implant containing 15% of natural silk and 85% of hydroxyapatite, 0.3 g of natural silk fibres made by Bombyx Mori silkworms with an average thickness of 10 m were weighed on the analytical balance. Fibres used were of the so-called de-gummed silk, i.e. having sericin removed. The weighed fibres were cut with scissors into fragments not longer than 5 mm. An amount of 1.7 g of synthetic hydroxyapatite known as GoHAP type I, which is offered by the Instytut Wysokich Cisnien PAN, was also weighed. The selected hydroxyapatite powder was characterized by a particle size ranging from 7 to 9 nm, a specific surface area ranging from 225 to 275 m ² /g, a density of 2.87 g/cm ³ and a molar ratio of calcium to phosphorus from 1.57 to 1.65. The hydroxyapatite powder and cut silk fibres were placed in a container of a cryogenic mill type 6775, SPOEX SamplePrep. The mill grinds substances in a heat-conducting container immersed in liquid nitrogen. In the container, apart from the given substance, a ferromagnetic grinding impactor is placed, which is moved inside the container by a variable magnetic field. In the cryogenic mill described above, a temperature of -190°C was generated and the frequency of impacts of the impactor was set at 2/s. After 15 minutes of cooling the contents of the container, the mill was turned on three times for one minute, with six-minute intervals for cooling the contents of the container to the starting temperature between the subsequent periods of operation. The parameters of the cryogenic treatment of the described components were selected for to obtain a powder with a homogeneous appearance when assessed by the unaided eye. The two-component (composite) powder thus obtained was kept for three hours in a vacuum oven at 100°C. The degree of powder dryness was evaluated using STA thermal analysis. It was assumed the powder has dried after reaching at least 2% weight loss due to evaporation of water, the weight loss observed during the drying process described above ranged from 2 to 6%. After drying was completed, the powder was purged with nitrogen. Surprisingly, it was noticed that in the powder obtained the hydroxyapatite agglomerates adhere closely to the silk fibres, which was documented using a high resolution scanning microscope (SEM) and shown in a magnification of 500x in Fig.6, and in 5000x magnification in Fig.7. In Fig. 7 a single fibre is shown in a close-up. Dried composite powder, a raw material for the production of composite implants, was cast into a matrix, with rectilinear walls measuring 20 mm x 4 mm, and a matching stamp, in which temperature of 80°C was maintained. In the hot matrix the composite powder was left for 10 minutes to equilibrate the temperatures, after which the stamp was pressed in order obtain a pressure of 1 GPa in the matrix, which was maintained for one minute. After removing the load, the matrix was disassembled to extract the finished implant. The material of the obtained implant had homogeneous fibres evenly distributed in the hydroxyapatite matrix, a total porosity of 14.1%, a bending strength of 86 MPa and a compressive strength of 276 MPa. During further examination of implants breakthroughs, it turned out that the agglomerates of hydroxyapatite still adhere closely to the silk fibres, which was registered with a high-resolution scanning electron microscope SEM in a magnification of 500x and is shown in Fig.1. The composite implant was also subjected to X-ray microimaging to examine its structure. Surprisingly, it has been found that the structure of the implant is isotropic, i.e. the silk fibres are uniformly distributed in all directions, as can be seen in the microtomogramms. Fig.5A shows a three- dimensional model of the distribution of fibroin fibres in a 500 μηη x 500 μηη x 1000 m rectangular breakthrough of the implant. Fig.5B shows a tomographic image of the implants microstructure in the XY plane of Fig.5A, Fig.5C shows a tomographic image of the implants microstructure in the XZ plane of Fig,5A, and Fig.5D shows an image of the implants microstructure in the YZ plane of Fig.5A. None of these three figures reveal the directional orientation of silk fibres. Example 2

In order to obtain an implant containing 5% of silk and 95% of hydroxyapatite, 0.15 g of silk fibres from the first example were weighed and cut as previously. To the container of the described above cryogenic mill, apart from silk, also 2.85 g of hydroxyapatite (same as in the first example) were added and the frozen container was immersed in liquid nitrogen in the mill chamber. After fifteen minutes of cooling the contents of this container, the contents were grinded as described in the first example, and then the powder obtained was dried in the same way. Fig.8 shows a SEM image of the powder in which, at 500x magnification, short pieces of fibres closely covered with a hydroxyapatite layer can be seen, whereas Fig.9 shows a SEM image of the same powder in which, at 10000x magnification, a single silk fibre is visible with closely adhering hydroxyapatite. The powder was pressed in the same way as before, but the matrix was not heated. The material of the obtained implant, shown in Fig.2, had silk fibres evenly distributed in the hydroxyapatite matrix, total porosity of 15.3%, and a bending strength of 32 MPa. Fig.2 shows a SEM image in which implants breakthrough is visible at a magnification of 5000x, as a result of breakage the fibre has been broken, which confirms the good adhesion of hydroxyapatite to silk fibres.

Example 3

As in the previous examples, an implant containing 85% by weight of hydroxyapatite and 15% silk was subjected to cold pressing under pressure of 1 GPa. This implant has been made of the powdered composite raw material described in the first example. Fig.3 shows in the SEM image, at 1000x magnification, a homogenous distribution of the silk fibres embedded in the hydroxyapatite matrix of this implant. The material of the obtained implant was characterized by a total porosity of 16.8% and a bending strength of 56 MPa.

Example 4

Similarly to the previous examples an implant made of a powdered composite material same as described in the second example was prepared, i.e. containing 95% by weight of hydroxyapatite and 5% of silk fibres, wherein pressing of this material at 1 GPa pressure was made in this case in a matrix heated to a temperature of 80°C. Fig.3 is an 1000x magnified SEM image of this implant structure. The image shows silk fibres in a morphologically unchanged form and adhering well to the hydroxyapatite matrix. The obtained implant was characterized by a total porosity of 11.3% and a bending strength of 46 MPa.