Continuous synthesis of octacalcium phosphate

Abstract

The present innovation relates to biomaterials, particularly, to the synthesis method of octacalcium phosphate. The method can be applied in the commercial manufacturing of biomaterials for bone tissue regeneration or dental restoration, such as bioinks for 3D printing, calcium phosphate cements, bioceramics, composites and drug delivery systems. The current invention developed a rapid, instant, controllable as well as reproducible procedure for the continuous synthesis of octacalcium phosphate.

Description

[0001] CONTINUOUS SYNTHESIS OF OCTACALCIUM PHOSPHATE

[0002] DESCRIPTION

[0003]

[0001] The invention relates to the field of biomaterials, particularly the method to synthesize octacalcium phosphate (OCP). This process can be applied in the commercial manufacturing of biomaterials for bone tissue regeneration or dental restoration, such as bioinks for 3D printing, calcium phosphate cements, bioceramics, composites and drug delivery systems.

[0004] Background of the art

[0005]

[0002] The biological occurrence of OCP has been reported in calcifying dentine. The presence of OCP in human dentine crystal indicated that the nucleation of non-stoichiometric hydroxyapatite (HAp) crystals as well as OCP occurs simultaneously at the same site of mineralization [1], Thus, OCP is known as a precursor of biological apatite crystals in hard tissues.

[0003] Several bioceramics such as HAp, dicalcium phosphate (DCP), amorphous calcium phosphate (ACP) and octacalcium phosphate (OCP) were implanted in mouse calvaria [2], Amongst all, OCP promoted bone tissue formation within one week whereas DCP, ACP (3 weeks) and HAp (5 weeks) revealed delayed tissue appearance. This confirmed synthetic OCP possesses enhanced bone formation ability.

[0006]

[0004] The most common routes for the synthesis of OCP are precipitation and hydrolysis. It has been reported that the large-scale synthesis of OCP is very challenging [3], The synthesis of OCP is influenced by Gibbs free energy, supersaturation, common ion effect, molarity, pH, temperature, and mixing order / rate. A minor alteration in any of these reaction parameters affects the purity of OCP [4],

[0007]

[0005] A method for the synthesis of OCP is reported. Calcium acetate solution was added dropwise into sodium dihydrogen phosphate solution using a peristaltic pump. The speed of the peristaltic pump was maintained at 58 rpm and the reaction was performed at 67.5 °C for 5 hours. The reaction mixture was stirred at 350 rpm. The resultant precipitate was filtered, washed with double distilled water, and dried for 5 days in an incubator at 55 °C [5], The flow rate of calcium solution into phosphate solution was kept very low which consumed about 5 hours for the synthesis of OCP. As it was identified that a faster dose rate leads to the formation of undesirable phases along with OCP.

[0008]

[0006] The large-scale synthesis of OCP by dropwise addition of calcium solution (0.04 M) into phosphate solution (0.03 M) is known. The calcium solution was added to phosphate solution at a dose rate of 116 mL / min. The reaction was performed at 67.5 °C and the final pH was 4.88. The end product was composed of HAp and OCP mixture [3], The rapid flow rate was found to influence the reaction and compromises the purity of OCP. The faster dose rate of calcium solution assisted in creating a high local concentration in the area of the drop in the phosphate solution. This caused a temporary increase in the Ca / P ratio in this area and resulted in the formation of HAp instead of OCP. Thus, it is recommended to maintain a lower dose rate for a good precipitation reaction large-scale synthesis.

[0009]

[0007] To date, numerous attempts have been made for the synthesis of OCP but no reports are found in the literature about the continuous synthesis of OCP. The methodologies used for the preparation of OCP are time-consuming and the entire reaction process takes from hours to days to complete. Moreover, the findings are unable to report precise reaction conditions for the preparation of OCP (Table 1).

[0010]

[0008] Therefore, there is still a great need for continuous synthesis of OCP for large-scale production and to meet the demands of the biomaterials market.

[0011] Table 1. An Overview of OCP synthesis conditions reported in the literature

[0012] *NA: Information not available

[0013] The object of the invention

[0014]

[0009] An object of the present invention is to develop a method having the ability to produce OCP in continuous process.

[0015]

[0010] Searching for a new synthesis process, we unexpectedly discovered that the use of the wet route in combination with optimum reaction parameters leads to conditions for controlled continuous synthesis of OCP.

[0016] Detailed description of the invention

[0017] [Oi l] OCP is prepared by the wet precipitation method. Stock solutions are prepared separately in deionized water.

[0018]

[0012] The term “stock solution” refers to a solution made from calcium source and phosphate sources.

[0019]

[0013] The term “calcium source” refers to calcium acetate hydrate.

[0020]

[0014] The term “phosphate source” refers to sodium dihydrogen phosphate dihydrate or potassium dihydrogen phosphate.

[0021]

[0015] The term “heated” refers to atemperature of 60 - 80 °C.

[0022]

[0016] The Ca / P ratio for the synthesis of OCP is ranging from 1 to 2, preferably from 1.2 to 1.4 or more preferably 1.3. The calcium source and phosphate source are heated separately. Both sources are pumped separately concurrently continuously using a system (P) providing continuous flow such as a peristaltic pump or automatic liquid dosing system or continuous flow equipment or gravitational flow system, through pipe 1 and pipe 2 respectively, and mixed together in mixing chamber (3) and precipitated OCP suspension is released through pipe (4) as shown in Fig. 1. The volume ratio of both solutions can be 1: 1. The reaction is carried out at a temperature ranging from 60 °C to 80 °C, preferably 60 °C to 70 °C, pH ranging from 4.0 to 6.0, preferably from 4.5 to 5.0.

[0017] Further, from the obtained mixture the precipitate is separated by vacuum fdtration or centrifugation or gravity fdtration. The sample obtained after filtration can be dried in an incubator or lyophilization or hot air oven or at room temperature. The selected reaction conditions assist in obtaining OCP bioceramic.

[0023]

[0018] The term “mixing” refers to any type of mixing of liquids to blend the components evenly throughout the mixture, ensuring uniformity and consistency.

[0024]

[0019] The term “pipes” refers to hollow channels used to transport liquids from one location to another within a system. The pipes can be made of materials such as metal, alloy, plastic, or rubber and are designed to facilitate the efficient and controlled movement of liquids, from a source to a destination.

[0025]

[0020] The term “pumped” refers to the action of using a pump to move or transfer liquid from one point to another. It involves creating a pressure difference, which forces the fluid to move through a system.

[0026]

[0021] The term “separately” refers to the movement or transfer of liquids in a manner where each liquid flows independently, without combining or mixing with each other. This could involve using distinct pipes, tubes, channels, or conduits for each liquid to maintain their individuality throughout the process.

[0027]

[0022] The term “concurrently” refers to the movement or flow of multiple fluids through separate channels in a system or the parallel movement of liquid streams in a netw ork of pipes / tubes.

[0028]

[0023] The term “continuous” refers to the process that occurs without interruption, pause, or break. It describes the reaction that proceeds without stopping.

[0029]

[0024] The term “continuous flow” refers to the uninterrupted movement of liquid. It implies a continuous movement or constant flow of liquid maintaining a consistent rate and direction over time.

[0030]

[0025] The term “peristaltic pump” refers to any pump used to move fluids and operates on the principle of peristalsis. The peristaltic pump maintains precise flow rates of liquids.

[0031]

[0026] The term “automatic liquid dosing system” refers to a system that can precisely dispense liquid substances automatically.

[0032]

[0027] The term “continuous flow equipment” refers to equipment used to perform operations continuously, without interruption. Continuous flow7equipment maintains a steady and uninterrupted flow7of liquids resulting in a continuous production of desired material.

[0028] The term "gravitational flow system" refers to the movement of fluids under the influence of gravity. This flow is driven by the potential energy stored in the fluid due to its elevation. When the fluid is allowed to flow freely, it moves from higher elevations to lower elevations, following the path of least resistance.

[0033]

[0029] A method for the synthesis of octacalcium phosphate (OCP) comprises the following steps: a) heating separately calcium and phosphate source; The heating temperature is in the range from 60 to 80 °C; b) pumping continuously, concurrently and separately calcium and phosphate sources through pipes (1,2) using a system (P) providing continuous flow; c) mixing calcium and phosphate source in mixing chamber (3), wherein the Ca / P molar ratio is from 1 to 2, a temperature range is from 60 to 80 °C with pH range from 4.0 to 6.0; The mixing is performed for a duration of less than 5 minutes, preferably less than 3 minutes, or even less than 1 minute. d) releasing the octacalcium phosphate precipitated in step c) through pipe (4); e) drying the octacalcium phosphate obtained in step d).

[0034]

[0030] Temperature plays a key role in controlling the phase purity, morphology, crystallinity, and rate of precipitation of octacalcium phosphate. Heating of reactants before mixing enhances the ion mobility, supersaturation, and decreases the solubility of OCP, leading to the rapid precipitation of OCP within minutes.

[0035]

[0031] Brief description of the drawings:

[0036] Fig.l. System used for the continuous synthesis of OCP: (P- system providing continuous flow; 1- inlet pipe; 2- inlet pipe; 3- mixing chamber; 4- releasing pipe)

[0037] Fig.2. X-ray diffraction pattern (a) and scanning electron microscopy image (b) of sample synthesized at 60 °C

[0038] Fig.3. X-ray diffraction pattern (a) and scanning electron microscopy image (b) of sample synthesized at 70 °C.

[0039] Fig. 4. X-ray diffraction pattern (a) and scanning electron microscopy image (b) of sample synthesized at 60 °C

[0040] Fig.5. X-ray diffraction pattern (a) and scanning electron microscopy image (b) of sample synthesized at 70 °C.

[0041] Examples

[0042]

[0032] The following examples are intended to illustrate certain preferred embodiments of the invention and are not limiting in nature.

[0033] Example 1. Sodium dihydrogen phosphate dihydrate solution (500 mL, 80 mM) and calcium acetate hydrate solution (500 mL, 106.4 mM) was heated separately at 60 °C. Calcium acetate hydrate solution and sodium dihydrogen phosphate dihydrate solution were mixed together using peristaltic pumps as shown in Fig. 1. The speed of the peristaltic pumps is 250 rpm and the reaction was performed at pH 4.7 to 4.9. Both the solutions are mixed simultaneously and the precipitate was fdtrated directly. The resultant precipitate was fdtered using a vacuum fdter, washed with distilled water, frozen in liquid nitrogen, and lyophilized. The sample obtained after lyophilization was analyzed by powder X-ray diffraction (XRD: Panalytical, Aeries, Netherlands) and scanning electron microscope (SEM: Tescan, Bmo, Czech Republic). X-ray diffraction pattern (Fig. 2a.) revealed pure OCP (98 %) phase. The characteristic diffraction maxima observed in XRD pattern matched with the standard octacalcium phosphate ICDD card no. 00-026-1056. This confirmed that the diffraction maxima corresponds only to OCP and no other phases were identified. The scanning electron micrograph (Fig. 2b) showed plate-like morphology.

[0043]

[0034] Example 2. Sodium dihydrogen phosphate dihydrate solution (500 mL, 80 mM) and calcium acetate hydrate solution (500 mL, 106.4 mM) was heated separately at 70 °C. Calcium acetate hydrate solution and sodium dihydrogen phosphate dihydrate solution were mixed together using a peristaltic pumps as shown in Fig. 1. The speed of peristaltic pump is 250 rpm and the reaction was performed at pH 4.7 to 4.9. Both the solutions are mixed simultaneously and the precipitate was filtrated directly. The resultant precipitate was filtered using a vacuum filter, washed with distilled water, frozen in liquid nitrogen and lyophilized. The characterization of the sample obtained after lyophilization was performed as mentioned in Example 1. X-ray diffraction pattern (Fig. 3a.) revealed pure OCP (98%) phase. The characteristic diffraction maxima observed in XRD pattern matched with the standard octacalcium phosphate ICDD card no. 00-026-1056. This confirmed that the diffraction maxima corresponds only to OCP and no other phases were identified. The scanning electron micrograph (Fig. 3b) showed plate-like morphology.

[0044]

[0035] Example 3. Potassium dihydrogen phosphate solution (500 mL, 80 mM) and calcium acetate hydrate solution (500 mL, 106.4 mM) was heated separately at 60 °C. Calcium acetate hydrate solution and potassium dihydrogen phosphate solution were mixed together using a peristaltic pumps as shown in Fig. 1. The speed of peristaltic pump is 250 rpm and the reaction was performed at pH 4.7 to 4.9. Both the solutions are mixed simultaneously and the precipitate was filtrated directly. The resultant precipitate was filtered using a vacuum filter, washed with distilled water, frozen in liquid nitrogen and lyophilized. The characterization of the sample obtained after lyophilization was performed as mentioned in Example 1 . X-ray diffraction pattern (Fig. 4a.) revealed pure OCP (98 %) phase. The characteristic diffraction maxima observed in XRD patern matched with the standard octacalcium phosphate ICDD card no. 00-026-1056. This confirmed that the diffraction maxima corresponds only to OCP and no other phases were identified. The scanning electron micrograph (Fig. 4b) showed plate-like morphology.

[0045]

[0036] Example 4. Potassium dihydrogen phosphate solution (500 mL, 80 mM) and calcium acetate hydrate solution (500 mL, 106.4 mM) was heated separately at 70 °C. Calcium acetate hydrate solution and potassium dihydrogen phosphate solution were mixed together using a peristaltic pumps as shown in Fig. 1. The speed of peristaltic pump is 250 rpm and the reaction was performed at pH 4.7 to 4.9. Both the solutions are mixed simultaneously and the precipitate was filtrated directly. The resultant precipitate was filtered using a vacuum filter, washed with distilled water, frozen in liquid nitrogen and lyophilized. The characterization of the sample obtained after lyophilization was performed as mentioned in Example 1 . X-ray diffraction patern (Fig. 5a.) revealed pure OCP (98 %) phase. The characteristic diffraction maxima observed in XRD patern matched with the standard octacalcium phosphate ICDD card no. 00-026-1056. This confirmed that the diffraction maxima corresponds only to OCP and no other phases were identified. The scanning electron micrograph (Fig. 5b) showed plate-like morphology.

[0046] References

[0047] [1] Bodier-Houlle, P., Steuer, P., Voegel, J.-C., Cuisinier, F.J.G., 1998. First Experimental Evidence for Human Dentine Crystal Formation Involving Conversion of Octacalcium Phosphate to Hydroxyapatite. Acta Crystallogr D Biol Crystallogr 54, 1377-1381.

[0048] [2] Suzuki, O., Nakamura, M., Miyasaka, Y., Kagayama, M., Sakurai, M., 1991. Bone Formation on Synthetic Precursors of Hydroxyapatite. Tohoku J. Exp. Med. 164, 37-50.

[0049] [3] O’Sullivan, R. and Kelly, D., 2020, Synthesis methodologies options for large-scale manufacturer of octacalcium phosphate, Octacalcium Phosphate Biomaterials Understanding of Bioactive Properties and Application, Suzuki O and Insley G. (eds), Woodhead Publishing, United Kingdom, 147-176.

[0050] [4] Chow LC, Eanes ED. (eds): Octacalcium Phosphate. Monographs in Oral Science. Basel, Karger, 2001, 18, 94-111.

[0051] [5] Murray K. A., Nicola Dobelin, N., Albadarin, A. B., Sadlo, J., Ren, G., Maurice N. Collins, M. N., O’Neill, C., 2020, The influence of sterilization on octacalcium phosphate for clinical applications, Octacalcium Phosphate Biomaterials Understanding of Bioactive Properties and Application, Suzuki O and Insley G. (eds), Woodhead Publishing, United Kingdom, 55-79. [6] LeGeros, R.Z., 1985, ‘Preparation of octacalcium phosphate (OCP): A direct fast method’, Calcified Tissue International, 37(2), 194-197.

[0052] [7] Saengdet, P. (Milin) & Ogawa, M., 2021, ‘Directional growth of octacalcium phosphate using micro-flow reactor mixing and subsequent aging’, RSC Advances, 11(26), 15969- 15976.

[0053] [8] Graham, S. & Brown, P.W., 1993, ‘The low temperature formation of octacalcium phosphate’, Journal of Crystal Growth, 132(1-2), 215-225.

[0054] [9] Bigi, A., Boanini, E., Borghi, M., Cojazzi, G., Panzavolta, S., Roveri, N., 1999. Synthesis and hydrolysis of octacalcium phosphate: effect of sodium polyacrylate. Journal of Inorganic Biochemistry 75, 145—151. https: / / doi.org / 10. 1016 / S0162-0134(99)00047-l

[0055]

[0010] Arellano-Jimenez, M.J., Garcia-Garcia, R. & Reyes-Gasga, J., 2009, ‘Synthesis and hydrolysis of octacalcium phosphate and its characterization by electron microscopy and X- ray diffraction’, Journal of Physics and Chemistry of Solids, 70(2), 390-395.

[0056]

[0011] Bigi, A., Boanini, E., Walsh, D., Mann, S., 2002. Morphosynthesis of Octacalcium Phosphate Hollow Microspheres by Polyelectrolyte-Mediated Crystallization This work was supported by MURST, the University of Bologna (Funds for Selected Research Topics), and the EPSRC (UK). Angew. Chem. Int. Ed. 41, 2163. https: / / doi.org / 10.1002 / 1521-3773(20020617)41: 12<2163::AID-ANIE2163>3.0.CO;2-G.

[0057]

[0012] Forte, L., Torricelli, P., Boanini, E., Gazzano, M., Fini, M. & Bigi, A., 2017, ‘Antiresorptive and anti-angiogenetic octacalcium phosphate functionalized with bisphosphonates: An in vitro tri-culture study’, Acta Biomaterialia, 54, 419-428.

[0058]

[0013] Bigi, A., Bracci, B., Panzavolta, S., Iliescu, M., Plouet-Richard, M., Werckmann, J., Cam,

[0059] D., 2004. Morphological and Structural Modifications of Octacalcium Phosphate Induced by Poly- 1 -Aspartate. Crystal Growth & Design 4, 141-146. https: / / doi.org / 10.1021 / cg034078d

[0060]

[0014] Shelton, R.M., Liu, Y., Cooper, P.R., Gbureck, U., German, M.J. & Barralet, J.E., 2006, ‘Bone marrow cell gene expression and tissue construct assembly using octacalcium phosphate microscaffolds’, Biomaterials, 27(14), 2874—2881.

[0061]

[0015] Temizel, N., Girisken, G. & Tas, A.C., 2011, ‘Accelerated transformation of brushite to octacalcium phosphate in new biomineralization media between 36.5°C and 80°C’, Materials Science and Engineering: C, 31(5), 1136—1143

[0062]

[0016] Heydari, Z., Mohebbi-Kalhori, D. & Afarani, M.S., 2017, ‘Engineered electrospun polycaprolactone (PCL) / octacalcium phosphate (OCP) scaffold for bone tissue engineering’, Materials Science and Engineering: C, 81, 127-132.

[0017] Sugiura, Y., Munar, M.L., Ishikawa, K., 2018. Fabrication of octacalcium phosphate foam through phase conversion and its histological evaluation. Materials Letters 212, 28-31. https: / / doi.Org / 10.1016 / j.matlet.2017.10.051

[0063]

[0018] Sugiura, Y., Ishikawa, K., 2018. Effect of Calcium and Phosphate on Compositional Conversion from Dicalcium Hydrogen Phosphate Dihydrate Blocks to Octacalcium Phosphate Blocks. Crystals 8, 222. https: / / doi.org / 10.3390 / crvst8050222

[0064]

[0019] Sugiura, Y., Ishikawa, K., 2019. Fabrication of pure octacalcium phosphate blocks from dicalcium hydrogen phosphate dihydrate blocks via a dissolution-precipitation reaction in a basic solution. Materials Leters 239, 143-146. htps: / / doi.Org / 10.1016 / j.matlet.2018.12.093

[0065]

[0020] Sugiura, Y., Munar, M.L., Ishikawa, K., 2018. Fabrication of octacalcium phosphate block through a dissolution-precipitation reaction using a calcium sulphate hemihydrate block as aprecursor. J Mater Sci: Mater Med 29, 151. htps: / / doi.org / 10.1007 / sl0856-018-6162-l

[0066]

[0021] Tripathi, G. & Miyazaki, T., 2021, ‘Spontaneous fabrication of octacalcium phosphate: synthesis conditions and basic characterizations’, Bulletin of Materials Science, 44(2), 163.

[0067]

[0022] Teterina, A.Y.; Smirnov, I.V.; Fadeeva, I.S.; Fadeev, R.S.; Smirnova, P.V.; Minaychev, V.V.; Kobyakova, M.I.; Fedotov, A.Y.; Barinov, S.M.; Komlev, V.S. Octacalcium Phosphate for Bone Tissue Engineering: Synthesis, Modification, and In Vitro Biocompatibility Assessment. Int. J. Mol. Sci. 2021, 22, 12747. htps: / / doi.org / 10.3390 / ijms222312747.

Claims

CLAIMS1. A method for the synthesis of octacalcium phosphate (OCP) comprises the following steps: a) heating both phosphate and calcium source separately in a range from 60 to 80 °C; b) pumping continuously, concurrently and separately calcium and phosphate sources through pipes (1,2) using a system (P) providing continuous flow; c) mixing calcium and phosphate source in a mixing chamber (3), wherein the mixing is performed for a duration of less than 5 minutes, wherein the Ca / P molar ratio is from 1 to 2, a temperature range is from 60 to 80 °C with pH range from 4.0 to 6.0; d) releasing the octacalcium phosphate precipitated in step c) through pipe (4).

2. The method according to Claim 1, wherein the mixing in step c) is performed for a duration, preferably, of less than 3 minutes, or even less than 1 minute.

3. The method according to Claim 1 wherein the calcium source is calcium acetate hydrate.

4. The method according to Claim 1 wherein the phosphate source is selected from the group consisting of sodium dihydrogen phosphate dihydrate or potassium dihydrogen phosphate.

5. The method according to Claim 1 wherein the system (P) of step b) is selected from a peristaltic pump or automatic liquid dosing system or continuous flow equipment or gravitational flow system.

6. The method according to Claim 1 wherein the pH of step c) is in the range from 4.5 to 5.0.

7. The method according to Claim 1 wherein the Ca / P ratio of step c) is in the range from 1.2 to 1.4.

8. The method according to any of the previous Claims wherein the OCP obtained in step d) is dried.

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