BIOMIMETIC BIOMATERIAL AND PRODUCTION METHOD THEREOF

Abstract

This invention relates to production method comprising processes of slip casting and freeze drying, which is a hybrid system, for developing hydroxyapatite-containing bio-ceramic developed by combined utilization of medical and engineering sciences in order to use on bone diseases, wherein it discloses a new hybrid system comprising process steps of preparing a first suspension containing powder ceramic, solvent and dispersant mixture by slip casting method, molding the first suspension mixture and allowing it to dry from outside to inside, pouring excessive (residual) slip (first suspension) out of the mold when it reaches to desired thickness, removing the material shaped to form compact part (6) of the bone cortical layer from the mold, preparation of the second suspension mixture comprising powder ceramic, solvent, dispersant and binder for the formation of the trabecular part (5) by freeze drying, cooling the second suspension until the liquid (1) is frozen so as to form trabecular part (5), obtaining the solid (2) by removing the free water in the substance to be dried in the first drying phase, removing the relative water to obtain vapor (3) in the second drying phase.

Claims

1. (canceled)

2. A bioceramic hybrid system production method comprising hydroxyapatite used on bone disease, the method comprising: utilizing a slip casting method to: prepare a first suspension mixture comprising powder ceramic, solvent and dispersant, pouring into a silicon or gypsum mold the first suspension mixture and allowing it to dry from outside to inside, pour excessive (residual) slip (first suspension) out of the mold when it reaches to desired thickness, remove the material shaped to form compact part (6) of the bone cortical layer from the mold, and utilizing a freeze-drying method to: prepare a second suspension mixture comprising powder ceramic, solvent, dispersant and binder, pour the second suspension into the slip casted first suspension (compact part (6)), cool the second suspension until the liquid (1) is frozen so as to form trabecular part (5), obtain the solid (2) by removing the free water in the substance to be dried in the first drying phase, and remove the relative water to obtain vapor (3) in the second drying phase, wherein the method further comprises a process step of preparing the first and the second suspensions comprising the mixture comprising powder nano-sized ceramic (hydroxyapatite) synthesized by the wet chemical precipitation method using orthophosphoric acid (H.sub.3PO.sub.4) of 80%-85% by concentration and calcium hydroxide (Ca(OH).sub.2) chemicals.

3. The production method of hydroxyapatite-containing bio-ceramic according to claim 2, wherein the method further comprises the process step of preparing the first suspension containing the mixture comprising powder ceramic in the ratio of 40%-70% by weight, solvent in the ratio of 30%-60%, dispersant in the ratio of 0.1%-1% by weight of powder ceramic.

4. The production method of hydroxyapatite-containing bio-ceramic according to claim 3, wherein the method further comprises the process step of preparing the first suspension containing the mixture comprising powder ceramic in the ratio of 50% by weight.

5. The production method of hydroxyapatite-containing bio-ceramic according to claim 2, wherein the method further comprises the process step of preparing the second suspension containing the mixture comprising powder ceramic in the ratio of 40%-70% by weight, solvent in the ratio of 30%-60%, dispersant in the ratio of 0.1%-10% by weight of powder ceramic, binder in the ratio of 1%-10% by weight of powder ceramic.

6. The production method of hydroxyapatite-containing bio-ceramic according to claim 2, wherein the method further comprises the process step of preparing the first suspension mixture comprising the mixture of calcium phosphate (hydroxyapatite) as powder ceramic, water and/or organic solvents as solvent, and sodium tripolyphosphate and/or ammonium polyacrylate as dispersant.

7. The production method of hydroxyapatite-containing bio-ceramic according to claim 2, wherein the method further comprises the process step of preparing the second suspension mixture comprising the mixture of calcium phosphate (hydroxyapatite) as powder ceramic, water and/or organic solvents as solvent, sodium tripolyphosphate and/or ammonium polyacrylate as dispersant, and polyvinyl alcohol (PVA) and/or carboxy methyl cellulose (CMC) as binder.

8. (canceled)

9. The production method of hydroxyapatite-containing bio-ceramic according to claim 2, wherein the method further comprises the process step of preparing the suspensions comprising the mixture comprising powder ceramic hydroxyapatite sintered at 1300° C. for tissue scaffold after synthesized by the wet chemical precipitation method.

Description

DESCRIPTION OF THE FIGURES

[0021] FIG. 1: illustrates the schematic representation of the slip casting method

[0022] FIG. 2: illustrates schematic representation of the freeze-drying method

[0023] FIG. 3: illustrates schematic representation of the biomimetic material produced

[0024] FIG. 4: illustrates the sintering temperature-time graphic

[0025] FIG. 5: illustrates the Hydroxyapatite FT-IR analysis graphic

[0026] FIG. 6: illustrates the hydroxyapatite particle size distribution analysis

[0027] FIG. 7: illustrates the hydroxyapatite XRD Analysis

[0028] FIG. 8: illustrates the PVA thermogravimetric diagram

[0029] FIG. 9: illustrates the SEM image of surface of hybrid tissue scaffold

[0030] FIG. 10: illustrates the pore distribution graphic of surface of hybrid tissue scaffold

[0031] FIG. 11: illustrates the SEM image of hybrid tissue scaffold with magnification of 50.23K X

[0032] FIG. 12: illustrates particle size distribution graphic of hybrid tissue scaffold at magnification of 50.23K X

DESCRIPTION OF REFERENCE NUMBERS

[0033]

TABLE-US-00001 No Description 1 Liquid 2 Solid 3 Vapor 4 Bone Marrow Cavity 5 Trabecular Part 6 Compact Part

Detailed Description of the Invention

[0034] The process used in the present invention is a hybrid system unlike the methods available in the state of the art. The novelty of this invention is the development of a new technique and biomaterial using slip casting and freeze-drying methods together. Thus, the formation of bone model having the best biomimetic properties will be provided.

[0035] The process steps of the production of biomimetic biomaterials comprise the following steps: [0036] Preparation of the first suspension which is an essential step for the slip preparation; is prepared with the powder ceramic, solvent and dispersant mixture that are the main construction elements. [0037] The prepared slip bone compact part (6) is the first suspension to be used as mimetic and having a certain viscosity to be molded in slip casting technique application. [0038] For designing the segment mimetic of the bone compact part (6), the technique which is based on leaving the slip for drying from out to inside after molding and removing excessive (residual) slip from mold when it reaches desired thickness is used. The portion that remain unremoved will function as the compact part (6) in the bone tissue scaffold. [0039] For designing the segment mimetic of the bone trabecular part (5); primarily the second suspension mixture containing powdered ceramic, solvent, dispersant and binder is prepared. [0040] The compact part (6) formed in the slip mold will be formed in the inner part. In use as tissue scaffold in bone tissue engineering, retention, proliferation, migration, nutrient and oxygen permeability, vascularization of the cells will be ensured. In the mold design, the innermost portion will be the bone marrow cavity (4). [0041] In slip casting method; the first suspension (slip) is casted into the mold, the water in the suspension is absorbed by the porous mold, the excess suspension is removed, and the formed material is taken out from the mold. Thus, bone cortical layer is formed. (FIG. 1) [0042] Freeze drying method is considered to be a good method for improving the long-term stabilization of colloidal nanoparticles. The end product having the best quality is obtained by this method when compared to other methods. The most important factor is that the surface in which sublimation is occurred by means of structural hardness is realized by the fact that it is frozen. The material form is not deformed also after drying process. It consists of three phases; 1) freezing, 2) first drying, 3) second drying. In the freezing phase, the suspension (slip) liquid (1) is cooled until it freezes. In the first drying phase, solid (2) is obtained by removing the free water in the material to be dried and, in the second freezing phase, steam (3) is obtained by removing relative water. (FIG. 2)

[0043] By the combination of these two methods, a structure mimicking the bone tissue will be developed.

[0044] In the exemplary embodiment of the subject matter product shown in FIG. 3, the bone marrow cavity (4), the trabecular part (5) obtained by freeze drying and the compact part (6) obtained by slip casting constitute the biomimetic material.

[0045] An embodiment of the inventive production method comprises the following process steps: [0046] By means of the slip casting method, [0047] preparation of the first suspension mixture comprising powder ceramic, solvent and dispersant, [0048] molding the first suspension mixture and allowing it to dry from outside to inside, [0049] pouring excessive (residual) slip (first suspension) out of the mold when it reaches to desired thickness, [0050] removing the material shaped to form compact part (6) of the bone cortical layer from the mold, [0051] By means of the freeze-drying method, [0052] preparation of the second suspension mixture comprising powder ceramic, solvent, dispersant and binder, [0053] cooling the second suspension until the liquid (1) is frozen so as to form trabecular part (5), [0054] obtaining the solid (2) by removing the free water in the substance to be dried in the first drying phase, [0055] removing the relative water to obtain vapor (3) in the second drying phase.

[0056] A further significant characterization of the inventive production method comprising the slip casting and freeze drying processes which is a hybrid system for developing bio-ceramic is that it comprises the process step of preparing the first suspension containing the mixture comprising powder ceramic in the ratio of 40%-70% by weight, solvent in the ratio of 30%-60%, dispersant in the ratio of 0.1%-1% by weight of powder ceramic. Furthermore, it also comprises the process step of preparing the second suspension containing the mixture comprising powder ceramic in the ratio of 40%-70% by weight, solvent in the ratio of 30%-60%, dispersant in the ratio of 0.1%-10% by weight of powder ceramic, binder in the ratio of 1%-10% by weight of powder ceramic.

[0057] The first suspension used to form the bone cortical layer, the compact part (6) by the slip casting method comprises powder ceramic, solvent, dispersant, and the second suspension used to form the trabecular part (5) by freeze drying method is obtained by mixing powder ceramic, solvent, dispersant and the binder. In the present invention, preferably, calcium phosphate as powder ceramic; water and/or organic solvent(s) as solvent; stabilizer(s), surfactant(s) and/or antifoam(s) are used as dispersants. In the invention, sodium tripolyphosphate and/or ammonium polyactylates as dispersants, polyvinyl alcohol (PVA) and/or carboxy methyl cellulose (CMC) are preferably used as binder.

[0058] What is more, the subject matter production method is a method applicable to industry. Consistency of the product and production method is ensured by means of the different technical effect shown by combining the two methods to be used as hybrid. This developed system also has the property to be produced industrially by designing the appropriate production processes.

[0059] What is more, the subject matter production method also comprises hydroxyapatite synthesis. Hydroxyapatite is a calcium phosphate-based bio-ceramic. In the production method, hydroxyapatite (powder ceramic) synthesis has been performed by the wet chemical precipitation using orthophosphoric acid (H.sub.3PO.sub.4) and calcium hydroxide (Ca(OH).sub.2) chemicals. In one embodiment of the present invention, the suspension is prepared with a mixture of orthophosphoric acid of 80-85% and calcium hydroxide. A further characterization of the production method is; nano-sized hydroxyapatite (powder ceramic) synthesizing by the wet chemical precipitation method. The prepared hydroxyapatite (calcium phosphate) will be used as the powdered ceramic in the ratio of 40-70% which constitutes the mixture in the process step of preparing the suspensions (first and second suspensions) in the subject matter production method.

[0060] The inventor has carried out works to observe the technical effect of the subject matter production method. During the works, the synthesized hydroxyapatite by the wet chemical precipitation method in nano-size and obtained the product as result of the subject matter production method by using slip casting and freeze-drying techniques on shaping as hybrid.

[0061] Wet chemical precipitation method was used in the production of powder hydroxyapatite. Moreover, the hybrid shaping method used in the literature for the very first time is the combined application of slip casting and freeze-drying techniques. The slip casting technique makes it possible to obtain reliable ceramic bodies at high density values and is used to mimic the bone compact part (6). Since pore structures connected to each other three dimensionally and being well defined by the freeze-drying method, it is used to mimic trabecular part (5). In the characterization of the scaffolds obtained FTIR, DLS, XRD, TG-DTA, BET, He Pycnometer and SEM analysis methods were used.

[0062] A summary of the work carried out by the inventor is given below. In the experimental work, primarily hydroxyapatite synthesis was realized. Then, polyvinyl alcohol solution was prepared, mold design and production were conducted. Subsequently, slip casting and freeze-drying process steps were applied and ended with sintering process.

[0063] Firstly, in hydroxyapatite synthesis, suspension was prepared with the mixture of orthophosphoric acid (H.sub.3PO.sub.4) of 80-85% and calcium hydroxide (Ca(OH).sub.2). 0.6 M (20.47 ml) orthophosphoric acid was weighed, and distilled water was added up to 500 ml. 0.5 mol (37.045 g) of calcium hydroxide (Ca(OH).sub.2) was weighed. The orthophosphoric acid was put in the magnetic stirrer and, during stirring at medium speed (450 rpm), calcium hydroxide was added to the acid-water mixture by means of a spatula. The addition process was continued until the calcium hydroxide was completely ran out.

[0064] When the addition was completed, the mixture had a white homogeneous appearance. And then, mixed by adjusting the pH level.

[0065] 6 tubes of 35 ml were centrifuged at 3000 rpm for 10 min at the washing process. They were placed in the centrifuge after vortexing. Then, synthesized hydroxyapatite within centrifuge tubes were left for drying. These steps were repeated twice for wet chemical precipitation. As result of this process, a total of 600.87 g of powder hydroxyapatite was obtained.

[0066] Polyvinyl alcohol solution was prepared as the secondary step. In hydroxyapatite-based bio-ceramics, PVA has been preferred to eliminate brittleness and to prolong material life by providing mechanical stabilization. 10.5 g of polyvinyl alcohol was weighed and dissolved in 200 ml of distilled water. In order to carry out dissolution, it was mixed in magnetic mixer at 85° C. degree for 4 hours. When the reaction was completed, a homogeneous PVA solution was obtained.

[0067] Two materials, as silicone and gypsum, were used for design and production of the mold.

[0068] In the processes of slip casting and freeze drying, while polyvinyl alcohol (PVA) was used as binder to make the synthesized hydroxyapatite powder form into slip, sodium tripolyphosphate (STPP) surfactant was used as dispersant.

[0069] Different hydroxyapatite-bearing suspensions were prepared to mimic the compact part (6) and trabecular part (5) of the bone for slip. For the compact part (6), casting is performed by preparing three different hydroxyapatite-bearing suspensions, which are respectively 28.5%, 37.5% and 50%, containing 20 g, 30 g, 50 g of hydroxyapatite in 50 ml of distilled water and wherein STPP at the amount of 0.32% of the powder ceramic (hydroxyapatite) was added to each suspension. For the trabecular part (5); suspension was prepared by using 30 ml of distilled water, 20 g of hydroxyapatite, 2 g (40 ml) of PVA (10% of hydroxyapatite), STPP at the amount of 10% of hydroxyapatite. The suspension containing 50% powder ceramic (hydroxyapatite) among the three different suspensions, showed optimum retention to mold and drying behavior.

[0070] Slip casting and freeze-drying processes performed separately were applied as hybrid. As so in the natural bone structure, the compact part (6) surrounding the spongy layer was made by slip casting primarily with the first suspension of hydroxyapatite+distilled water+STPP in order to obtain the mimetic structure. The material that was not dried after the first suspension was dried until it reached to a certain thickness around the gypsum mold was discharged back. The second suspension Hydroxyapatite+PVA+distilled water+STPP for the trabecular part (5) was cast into the gypsum mold, which was previously slip casted and the middle part of which was hallow. After the castings were completed, freeze drying technique was applied by lyophilizer. Ceramic molds were used for slip casting and freeze-drying processes performed. Silicone molds were cracked during retention at −80° C. for freeze drying process and they were considered to be inappropriate for this assembly.

[0071] During the sintering process step, the chemicals used in the wet chemical precipitation method to synthesize hydroxyapatite affect the sizes of formed hydroxyapatite particles. Hydroxyapatite synthesized with orthophosphoric acid and calcium hydroxide has larger particle sizes compared to hydroxyapatite particle sizes synthesized with calcium nitrate tetrahydrate and diammonium hydrogen phosphate. This increases the temperature degree required for sintering.

[0072] Firing was carried out at different temperatures as 900° C. and 1200° C. for the prepared hybrid hydroxyapatite tissue scaffolds, but it was observed that the sintering was not realized as expected. The optimum sintering temperature for the hybrid hydroxyapatite tissue scaffold was determined as 1300° C. (FIG. 4)

[0073] The inadequacy of the surface porosity of tissue scaffolds obtained by merely applying slip casting and the brittleness problem of tissue scaffolds obtained by merely applying freeze drying were overcome by the use of these two techniques. Thus, the disadvantages, indicated in the literature, such as being inadequate for load carrying bones, have been eliminated.

[0074] Hybrid shaping methods to be applied for the first time in the literature within the scope of developed production art are; the slip casting and the freeze-drying techniques. Slip casting technique provides higher density values and therefore ensures obtaining more reliable ceramic bodies. By means of the three-dimensional hybrid design, layers having a structure similar to natural bone will be formed. While high densities cause a brittle structure in other works, brittleness will be avoided as they are supported to form a spongiosis layer by the freeze-drying technique. Suitable environment will also be formed for the cells to retain. Furthermore, since the bone marrow cavity (4) is also formed by specific mold design as is natural bone form, mechanic resistance will also be appropriate for load carrying bones in the body.

[0075] In the characterization of the scaffolds obtained FTIR, DLS, XRD, TG-DTA, BET, He Pycnometer and SEM analysis methods were used.

[0076] Analysis was carried out in the range of 4000-400 cm.sup.−1 for HA powders synthesized by wet chemical method. HA particle measurements synthesized with orthophosphoric acid and calcium hydroxide chemicals are shown. (FIG. 5) Shows in general 3000-2800 cm.sup.−1 CH.sub.3 group (methyl) C—H bonds, 3300 cm.sup.−1-3600 cm.sup.−1 characteristic —OH ion band, 1200-1600 cm.sup.−1 CO.sub.3.sup.−2 band (carbonate ions), 1000-1100 cm.sup.−1 PO.sub.4.sup.−3 existence.

[0077] A particle size distribution analysis for HA was performed and the resulting graphic is shown in FIG. 6. The mean particle size for HA was measured as 137.8 nm and the PDI value as 0.59. According to the DLS results, the irregular distribution in the graphics shows the existence of regional agglomeration.

[0078] In order to characterize the phase composition and crystallinity of HA, XRD which is a routinely used analysis was utilized. XRD analysis of HA is shown in FIG. 7. In the XRD patterns of the material, increasing peaks around the planes (002), (211), (300), (202), (222), (310), (213) and (004) are observed.

[0079] (PVA) thermal analysis of poly(vinylalcohol) used as binder is shown in FIG. 8.

[0080] SEM image of tissue scaffold prepared as hybrid is shown in FIG. 9. It is clearly seen in this image that the cortical layer—the compact part (6), formation of which is expected to be done by the slip casting, and the spongy layer—the trabecular part (5), formation of which is expected to be done by freeze drying, are formed. The pore distribution graphic of the SEM image is shown in FIG. 10.

[0081] SEM image of hybrid tissue scaffold with magnification of 50.23K X is seen in FIG. 2. The particle size distribution graph of said image is shown in FIG. 12.

[0082] The specific surface area of the HA powder was measured to be 55.11 m.sup.2/g. Estimated equivalent particle size was computed as 34.5 nm. The theoretical density of hydroxyapatite is 3.16 g/cm.sup.3. Particle size measurement at smaller sizes are obtained when the particle sizes (34.5 nm) obtained by BET analysis are compared to DLS analysis (137.5). The reason of that can be agglomeration occurred in the HA suspension prepared for DLS measurement (Gervaso et al., 2012). According to XRD analysis lattice tendency, average value (46 nm) of particle size conducted with Scherrer equation supports the BET analysis result (34.5 nm).

[0083] He pycnometer was used to calculate the density of bio-ceramic tissue scaffold developed as hybrid. The mass was measured as 3.4694 g, the volume as 1.1032 cm.sup.3 and the density was calculated as 3.1450 g/cm.sup.3 for the hybrid tissue scaffold. This result for hybrid tissue scaffold shows that this tissue scaffold which was developed by expecting that it has bone tissue mimetic has a porous structure.

[0084] The bulk density value of the hybrid tissue scaffold was calculated as 2.19 g/cm.sup.3 and the bulk volume value as 1.579 cm.sup.3. When calculating the bulk density and volume, the standard deviation was found to be 0.1 as result of the radius (0.72 cm) and the height (0.97 cm) taken from different regions of the hybrid tissue scaffold.

[0085] While performing nutrient and waste diffusion, it is proved that tissue scaffold that can also support cell proliferation and vascularization is developed by means of the existence of micro and nano-sized pores obtained by the images. Furthermore, it is seen that the average particle size is 100 nm. Depending on the sintering process, it is also observed that merging occurs on the HA particles. The mechanical resistance of the tissue scaffold will be supported by these mergers.

[0086] HA particle size of hybrid tissue scaffold is 50-120 nm and the surface porosity values are in the range of 100-180 μm. These values indicate that the inner pore connections essential for vascularization have appropriate sizes. Distribution of pores at micro and nano-sizes proves that an appropriate tissue scaffold for nutrient-gas diffusion and waste elimination has been developed.

[0087] In general, it is concluded that a hybrid tissue scaffold has been developed, wherein vascularization can be achieved by the presence of appropriate sized surface porosity and internal pore connections as expected from the bone tissue scaffold; and which can support cell retention, migration and proliferation; and wherein the basic requirements such as nutrient diffusion and waste elimination are met.