Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

1. TITLE OF THE INVENTION:
MULTI-PHASIC SYNTHETIC BONE GRAFT AND METHODS THEREOF

2. APPLICANT:
Name : Meril Medical Innovations Private Limited
Nationality : Indian
Address : Survey No 1574 (Old No 135/139), Bilakhia House, Muktanand Marg, Chala, Vapi, Valsad, Gujarat, 396191 India

3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:
 
FIELD OF INVENTION
[1] The present disclosure relates to a synthetic graft. More particularly, the present disclosure relates to a multi-phasic synthetic bone graft and methods thereof.
BACKGROUND OF INVENTION
[2] Bone defects caused by trauma, tumors, or degenerative diseases often require synthetic bone graft. The synthetic bone graft should be biocompatible and support osteogenesis (i.e., new bone formation). The synthetic bone graft provides temporary mechanical support and maintains structural integrity at the defect site. The synthetic graft undergoes resorption progressively as new bone tissue forms.
[3] However, conventional synthetic bone graft (or graft) made of mineral phase(s) may fail to offer controlled resorption, sufficient cohesion, predictable bone regeneration and/or improved handling characteristics (such as, moldability and injectability). For example, some grafts resorb very slowly, which may interfere with the natural rate of bone remodeling. In contrast, few grafts resorb very quickly before sufficient new bone tissue is formed, resulting in premature loss of structural support. Certain grafts resorb non-uniformly, leading to uneven healing and void formation within the regenerated bone.
[4] Few grafts rely on collagen or polymer matrices that act primarily as structural carriers to provide improved handling and bioactivity. But, they fail to provide sufficient mechanical strength due to hindered/weak interaction between the organic binders (i.e., the collagen) and the mineral phases (i.e., the calcium phosphates) of the graft leading to reduced osteo-conductivity.
[5] Moreover, the conventional methods to prepare multi-phasic graft rely on a single precipitation, sintering, or co-precipitation process where multiple calcium phosphate phases form within a single reaction environment. These conventional methods provide limited control over the phase composition as the calcium phosphate phases can transform depending on temperature, pH, and ionic environment. The resulting graft may have inconsistent variation in crystallinity and particle size distribution as multiple phases nucleate and grow simultaneously. Further, during formation of the graft, multiple transient intermediate phases (such as, amorphous calcium phosphate or octacalcium phosphate) are formed, which may subsequently convert into other apatite structures. Even when organic binders are introduced in the graft, the organic binder is simply mechanically blended resulting in weak mineral-organic interface.
[6] Thus, there rises a need for a synthetic bone graft that provides controlled resorption while maintaining balanced mechanical support, thereby enabling reliable and predictable bone regeneration.
OBJECTS OF INVENTION
[7] The principal object of this invention is to provide a multi-phasic synthetic bone graft,
[8] Another object of this invention is to provide a tri-phasic calcium phosphate,
[9] A further object of this invention is to provide an organic binder, etc.
SUMMARY OF INVENTION
[10] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings; however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.
[11] In an embodiment, the present disclosure relates to a method to prepare a multi-phasic synthetic bone graft. hydroxyapatite (HAp) is prepared having calcium to phosphate (Ca/P) ratio ranging from 1.60 to 1.67. The hydroxyapatite (HAp) includes macro-scale particles having particle size distribution between 5 µm and 30 µm. Calcium-deficient apatite (CDA) is prepared having a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55. The calcium-deficient apatite (CDA) includes nano-scale particles having particle size distribution of less than 1 µm. β-tricalcium phosphate (β-TCP) is prepared having a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55. The β-tricalcium phosphate (β-TCP) includes micro-scale particles having particle size distribution between 50 µm and 150 µm. The hydroxyapatite (HAp), the calcium-deficient apatite (CDA), and the β-tricalcium phosphate (β-TCP) is mixed at a pre-defined ratio ranging from 1:1:1 to 4:1:4 to obtain a tri-phasic calcium phosphate. An organic binder is prepared. The organic binder includes at least one branching element and at least one interface element. A pre-defined amount of the tri-phasic calcium phosphate is added in per unit volume of the organic binder to obtain the multi-phasic synthetic bone graft.
[12] In another embodiment, the present disclosure relates to a multi-phasic synthetic bone graft including an organic binder, and 7.5 g to 8 g of a tri-phasic calcium phosphate per unit volume of the organic binder. The organic binder including 4% (w/v) to 6% (w/v) of a branching element, and 1% (w/v) to 3% (w/v) of an interface element. The tri-phasic calcium phosphate including 33.3% (w/w) to 44.4% (w/w) of hydroxyapatite (HAp), 11.1% (w/w) to 33.3% (w/w) of calcium-deficient apatite (CDA), and 33.3% (w/w) to 44.4% (w/w) of a β-tricalcium phosphate (β-TCP). The hydroxyapatite (HAp) has a calcium to phosphate (Ca/P) ratio ranging from 1.60 to 1.67. The hydroxyapatite (HAp) includes macro-scale particles having particle size distribution between 5 µm to 30 µm. The calcium-deficient apatite (CDA) has a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55. The calcium-deficient apatite (CDA) includes nano-scale particles having particle size distribution of less than 1 µm. The β-tricalcium phosphate (β-TCP) has a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55. The β-tricalcium phosphate (β-TCP) includes micro-scale particles having particle size distribution between 50 µm to 150 µm.
BRIEF DESCRIPTION OF DRAWINGS
[13] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the apportioned drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentality disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.
[14] Fig. 1 depicts a method 100 of preparing a synthetic bone graft, according to an embodiment of the present disclosure.
[15] Fig. 1a depicts a method 100a of preparing hydroxyapatite (HAp), according to an embodiment of the present disclosure.
[16] Fig. 1b depicts a method 100b of preparing calcium-deficient apatite (CDA), according to an embodiment of the present disclosure.
[17] Fig. 1c depicts a method 100c of preparing β-tricalcium phosphate (β-TCP), according to an embodiment of the present disclosure.
[18] Fig. 1d depicts a method 100d of preparing an organic binder, according to an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE DRAWINGS
[19] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like. Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.
[20] Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean “one or more but not all embodiments” unless expressly specified otherwise. The terms “including,” “comprising,” “having,” and variations thereof mean “including but not limited to” unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and/or mutually inclusive, unless expressly specified otherwise. The terms “a,” “an,” and “the” also refer to “one or more” unless expressly specified otherwise.
[21] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.
[22] Furthermore, the described includes, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific includes or advantages of a particular embodiment. In other instances, additional includes and advantages may be recognized in certain embodiments that may not be present in all embodiments. These includes and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.
[23] This current disclosure pertains to a multi-phasic synthetic bone graft (hereinafter, referred to as graft) and methods thereof. In an embodiment, the graft includes a multi-phasic ceramic/mineral composition in combination with an organic binder that provides a biomimetic regeneration platform. In an embodiment, the graft includes tri-phasic calcium phosphate (as the multi-phasic ceramic composition) and a combination of a branching element and an interface element (as the organic binder) to achieve staged and controlled resorption during bone regeneration.
[24] In an exemplary embodiment, the tri-phasic calcium phosphate is formed of calcium-deficient apatite (CDA), β-tricalcium phosphate (β-TCP), and hydroxyapatite (HAp) in a predefined ratio. The CDA includes nano-scale and low-crystallinity particles that provides rapid ionic release to simulate early osteogenic differentiation. The β-TCP includes crystalline and micro-scale particles that enables controlled intermediate resorption and vascularized remodeling. The HAp includes highly crystalline macro-scale particles that maintains long-term mechanical/structural integrity. This staged mineral degradation profile of the tri-phasic calcium phosphate mimics the physiological bone maturation rate during osteogenesis.
[25] The method to prepare the tri-phasic calcium phosphate involves independent synthesis of the CDA, β-TCP, and HAp. Since each phase is already crystallographically established while being synthesized in respective controlled reaction environments, the resulting tri-phasic calcium phosphate maintains three distinct calcium phosphate phases. The respective controlled reaction environments prevent formation of additional calcium phosphate phases (such as, amorphous calcium phosphate or octacalcium phosphate) in the resulting tri-phasic calcium phosphate. The coexistence of these phases introduces controlled and predictable crystallinity, calcium to phosphate (Ca/P) ratio, lattice structure, and particle morphology of the tri-phasic calcium phosphate.
[26] In an embodiment, the organic binder provides a chemically coordinated mineral-organic interface that enhances cohesion, protein/drug adsorption, and cellular interaction. The collagen (an exemplary branching element) of the organic binder improves handling characteristics (such as, yield stress, washout resistance, moldability and injectability), allowing the graft to conform to irregular defect geometries. The phosphoserine (an exemplary interface element) of the organic binder is capable of chelating calcium ions within the tri-phasic calcium phosphate to create a biologically active extracellular matrix-like (ECM-like) interface for improved osteo-conductivity supporting improved cellular attachment and growth factor retention. In an embodiment, the combination of collagen–phosphoserine of the organic binder facilitates incorporation and sustained release of bioactive molecules, including osteogenic (such as bone morphogenic proteins (BMPs)), antimicrobial, or angiogenic factors.
[27] The graft may be used in a variety of orthopedic, dental, and regenerative medicine applications. The graft may be provided in versatile forms, such as, an injectable putty, granules, or a porous scaffold depending upon the application and requirements.
[28] For example, the graft may be used for filling bone defects arising from trauma, tumor resection, infection, degenerative conditions, or congenital deformities. Due to its moldability and injectability, the graft may easily conform to irregular geometries of bone defect. The graft may further be used in spinal fusion procedures and orthopedic surgeries involving long bones, where temporary mechanical support and gradual resorption are required to facilitate bone union. The graft may be used to fabricate porous scaffold for bone tissue engineering applications, enabling in vitro cell seeding, incorporation of growth factors, or controlled delivery of bioactive agents. For dental and maxillofacial applications, the graft may be used for alveolar ridge augmentation, sinus lifts, and periodontal bone regeneration. Additionally, the graft may be utilized in research and preclinical models for evaluating in vitro or in vivo studies related to bone regeneration, resorption characteristics, and osteoconductive performance.
[29] Now referring to figures, Fig. 1 depicts a method 100 of preparing a multi-phasic synthetic bone graft (hereinafter, referred to as graft). The method 100 commences at step 102 by preparing hydroxyapatite (HAp).
[30] Fig. 1a depicts an exemplary method 100a of preparing HAp. At step 102a, a predefined amount of a first calcium salt is dissolved in a predefined amount of at least one first solvent to obtain a first solution. The concentration of the first calcium salt in the first solution may range between 8% (w/v) and 12% (w/v). The first calcium salt is at least one of calcium nitrate tetrahydrate, calcium chloride dihydrate, calcium acetate monohydrate, etc. The first solvent is at least one of water, deionized water, etc. In an embodiment, 9.8% (w/v) of calcium nitrate tetrahydrate is dissolved in water to obtain the first solution.
[31] At step 102b, a predefined amount of a first phosphate salt is dissolved in a predefined amount of at least one first solvent to obtain a second solution. The concentration of the first phosphate salt in the second solution may range between 6% (w/v) and 10% (w/v). The first phosphate salt is at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, phosphoric acid, etc. The first solvent is at least one of water, deionized water, etc. In an embodiment, 8.2% (w/v) of diammonium hydrogen phosphate is dissolved in water to obtain the second solution.
[32] At step 102c, the second solution obtained from step 102b is gradually added to the first solution obtained from step 102a at a pre-defined ratio under alkaline condition while maintaining a predefined temperature to obtain a third solution. The alkaline condition is defined by a predefined pH ranging between 7 and 10. The predefined temperature may range from 60°C to 65°C. The pre-defined volumetric ratio of the second solution and the first solution in the third solution ranges from 1:1 to 1:2. In an embodiment, the second solution is added to the first solution at a ratio of 1:1.5, while maintaining the temperature of the first solution at 60°C and pH 8.5. In an embodiment, the second solution is added dropwise to the first solution under continuous stirring at 600 rpm using a magnetic stirrer. In an embodiment, while the second solution is being added to the first solution, the pH of the first solution is continuously monitored using a pH meter and adjusted by adding ammonium hydroxide (NH₄OH) solution (having a pH of 11-13) to the first solution if the pH deviates towards acidic conditions (i.e., less than 6.5).
[33] Although the above step is described with example of adding the second solution to the first solution, the first solution may be added to the second solution instead and the same is within the scope of the teachings of the present disclosure.
[34] At step 102d, the third solution obtained from step 102c is continuously stirred to allow uniform mixing of the first solution with the second solution at a pre-defined temperature and a pre-defined speed for a predefined time period. The third solution is stirred using at least one of a magnetic stirrer, overhead mechanical stirrer, etc. The predefined temperature may range from 60°C to 65°C. The third solution is stirred at the predefined speed ranging from 600 rpm to 800 rpm. The predefined time period may range between 2 hours to 3 hours. In an embodiment, the third solution is stirred at 60°C and 800 rpm for 3 hours.
[35] At step 102e, the third solution obtained from step 102d is allowed to rest undisturbed for a predefined time period to facilitate precipitation of crude hydroxyapatite (cHAp). The predefined time period ranges ranging from 6 hours to 8 hours. In an embodiment, the third solution is allowed to rest for overnight which allows the cHAp to precipitate out (i.e., settle down). After the cHAp has settled down, the supernatant is decanted. An exemplary precipitation reaction of cHAP is illustrated below:
10Ca2+ + 6PO43− + 2OH− → Ca10(PO4)6(OH)2 ↓
[36] At step 102f, the cHAp obtained from step 102e is washed for two or more cycles using at least one of milli q water, deionized water, ethanol, etc. In an embodiment, the cHAp is washed using milli q water. In an embodiment, the cHAp is washed 3 times. The washing of cHAp helps to remove unreacted components.
[37] In an embodiment, during a single wash cycle, 200 mL of milli q water is added to 2000 mg of cHAp and then mixed vigorously to obtain a suspension. Thereafter, the suspension is allowed to rest for a predefined time period to allow the cHAp to settle down. The predefined time period may range between 12 hours and 24 hours. In an embodiment, the suspension is allowed to rest for 24 hours. During washing, the unreacted components and water-soluble impurities partition into the supernatant phase, while the cHAp is allowed to precipitate. The supernatant is then decanted while retaining the precipitate.
[38] Additionally, or optionally, while washing the cHAp in the last cycle, the suspension is not allowed to rest and is subjected to centrifugation at a predefined force for a predefined time period. The predefined force may range from 2000 x g to 8000 x g. The predefined time period may range from 5 minutes to 15 minutes. In an embodiment, the suspension is centrifuged at 2000 x g force for 10 minutes. Centrifuging the suspension helps in rapid separation of precipitate (cHAP) from supernatant and efficient removal of fine impurities.
[39] At step 102g, the cHAp obtained from step 102f is filtered using at least one of a centrifugation technique, vacuum filtration, etc. In an embodiment, the cHAp is filtered using a filter member centrifuged at a predefined force for a predefined time period ranging from 2000 x g to 8000 x g, and 5 minutes to 15 minutes, respectively. The filter member has a plurality of pores having a predefined pore size ranging between 0.2 µm and 5 µm. In an embodiment, the filter member has a pore size of 0.45 µm. Filtering the cHAp helps to remove unreacted excess chemical.
[40] In an embodiment, the cHAp obtained from step 102e is filtered using an assembly of a filter member placed at least partially within a centrifuge tube. The cHAp is placed within the filter member and then centrifuged at 2000 x g for 10 min, thus obtaining a first residue on the filter member containing cHAp.
[41] At step 102h, the first residue (having the cHAp) obtained from step 102g is dried at a predefined temperature to remove moisture. The predefined temperature may range from 80°C to 100°C. In an embodiment, the cHAp is dried at 60°C.
[42] At an optional step 102i, the cHAp obtained from step 102h may be subjected to calcination at a predefined temperature and a predefined pressure for a predefined time period. The predefined temperature may be less than or equal to 600°C. The predefined pressure may be less than or equal to 1 atm. The predefined time period ranges from 2 hours to 3 hours. In an embodiment, the cHAp is subjected to calcination at 600°C for 3 hours under 1 atm pressure. Calcination helps to enhance crystallinity of cHAp. In an embodiment, the crystallinity of cHAP ranges from 70% to 90%, as measured by X-ray diffraction (XRD).
[43] At step 102j, the cHAp is subjected to milling to obtain macro-scale particles of the HAp having a pre-defined particle size. In an embodiment, the cHAp is milled using a milling apparatus, such as, planetary ball mill machine (procured from laboteck, model: LABMV2S), though other functionally equivalent apparatus may be employed to achieve similar results. In an embodiment, the particle size distribution of HAp after milling ranges between 5 µm and 30 µm. The HAp particle, sized from 5 µm to 30 µm, provides optimized HAp overcoming limitations of conventional single-size particles and demonstrates improved clinical performance in bone regeneration applications. In an embodiment, the particle size of the Hap is validated by laser diffraction technique.
[44] The HAp obtained from step 102 has a pre-defined calcium to phosphate (Ca/P) ratio ranging from 1.60 to 1.67. In an exemplary embodiment, the Ca/P ratio of the HAp is 1.67. The HAp has the following chemical formula:
Ca10(PO4)6(OH)2
[45] The macro-scale particles of HAp improve resorption control, mineralization, osteoconductivity, ionic bioavailability and mechanical stability of the tri-phasic calcium phosphate.
[46] At step 104 of method 100, calcium-deficient apatite (CDA) is prepared. Fig. 1b depicts an exemplary method 100b of preparing CDA. The CDA is relatively less crystalline than the HAp obtained from step 102, because the CDA has lower Ca/P ratio compared to HAp. The relatively less crystallinity of the CDA helps to maintain fast resorption.
[47] At step 104a, a predefined amount of a second calcium salt is dissolved in a predefined amount of at least one second solvent to obtain a fourth solution. The concentration of the second calcium salt in the fourth solution may range between 8% (w/v) and 12% (w/v). The second calcium salt is at least one of calcium nitrate tetrahydrate, calcium chloride dihydrate, calcium acetate monohydrate, etc. The second solvent is at least one of water, deionized water, etc. In an embodiment, 9% (w/v) of calcium nitrate tetrahydrate is dissolved in water to obtain the fourth solution.
[48] At step 104b, a predefined amount of a second phosphate salt is dissolved in a predefined amount of at least one second solvent to obtain a fifth solution. The concentration of the second phosphate salt in the fifth solution may range between 6% (w/v) and 10% (w/v). The second phosphate salt is at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, phosphoric acid, etc. The second solvent is at least one of water, deionized water, etc. In an embodiment, 9% (w/v) of diammonium hydrogen phosphate is dissolved in water to obtain the fifth solution.
[49] At step 104c, the fifth solution obtained from step 104b is gradually added to the fourth solution obtained from step 104a at a pre-defined ratio under alkaline condition while maintaining a predefined temperature to obtain a sixth solution. The alkaline condition is defined by a predefined pH ranging between 9 and 11. The predefined temperature may range from 40°C to 60°C. The pre-defined volumetric ratio of the fifth solution and the fourth solution in the sixth solution ranges from 1:1 to 1:1.5. In an embodiment, the fifth solution is added to the fourth solution at a ratio of 1:1.5, while maintaining the temperature of the fourth solution at 40°C and pH 10. In an embodiment, the fifth solution is added dropwise to the fourth solution under continuous stirring at 400 rpm using a magnetic stirrer. In an embodiment, while the fifth solution is being added to the fourth solution, the pH of the fourth solution is continuously monitored using a pH meter and adjusted by adding ammonium hydroxide (NH₄OH) solution (having a pH of 11-13) to the fourth solution if the pH deviates towards acidic conditions (i.e., less than 8.5).
[50] Although the above step is described with example of adding the fifth solution to the fourth solution, the fourth solution may be added to the fifth solution instead and the same is within the scope of the teachings of the present disclosure.
[51] At step 104d, the sixth solution obtained from step 104c is continuously stirred to allow uniform mixing of the fourth solution with the fifth solution at a pre-defined temperature and a pre-defined speed for a predefined time period. The sixth solution is stirred using at least one of a magnetic stirrer, overhead mechanical stirrer, etc. The predefined temperature may range from 40°C to 60°C. The sixth solution is stirred at the predefined speed ranging from 600 rpm to 850 rpm. The predefined time period may range between 2 hours to 3 hours. In an embodiment, the sixth solution is stirred at 40°C, and 850 rpm for 3 hours.
[52] At step 104e, the sixth solution obtained from step 104d is allowed to rest undisturbed for a predefined time period to facilitate precipitation of crude calcium-deficient apatite (cCDA). The predefined time period ranges ranging from 6 hours to 8 hours. In an embodiment, the sixth solution is allowed to rest for overnight which allows the cCDA to precipitate out (i.e., settle down). After the cHAp has settled down, the supernatant is decanted. An exemplary precipitation reaction of cCDA is illustrated below:
(10−x)Ca2+ + (6−x)PO43− + xHPO42− + (2−x)OH− → Ca10−x(HPO4)x(PO4)6−x(OH)2−x ↓
[53] At step 104f, the cCDA obtained from step 104f is washed for two or more cycles using at least one of milli q water, deionized water, ethanol, etc. In an embodiment, the cCDA is washed using milli q water. In an embodiment, the cCDA is washed 3 times. The washing of cCDA helps to remove unreacted components.
[54] In an embodiment, during a single wash cycle, 200 mL of milli q water is added to 2000 mg of cCDA and is then mixed vigorously to obtain a suspension. Thereafter, the suspension is allowed to rest for a predefined time period to allow the cCDA to settle down. The predefined time period may range between 12 hours and 24 hours. In an embodiment, the suspension is allowed to rest for 24 hours. During washing, the unreacted components and water-soluble impurities partition into the supernatant phase, while the cCDA is allowed to precipitate. The supernatant is then decanted while retaining the precipitate.
[55] Additionally, or optionally, while washing the cCDA in the last cycle, the suspension is not allowed to rest and is subjected to centrifugation at a predefined force for a predefined time period. The predefined force may range from 3000 x g to 8000 x g. The predefined time period may range from 5 minutes to 15 minutes. In an embodiment, the suspension is centrifuged at 5000 x g force for 10 minutes. Centrifuging the suspension helps in efficient separation of cCDA precipitate and removal of residual soluble impurities.
[56] At step 104g, the cCDA obtained from step 104e is filtered using at least one of centrifugation technique, vacuum filtration, etc. In an embodiment, the cCDA is filtered using a filter member centrifuged at a predefined force for a predefined time period ranging from 2000 x g to 8000 x g, and 5 minutes to 15 minutes, respectively. The filter member has a plurality of pores having a predefined pore size ranging between 0.2 µm and 5 µm. In an embodiment, the filter member has a pore size of 0.45 µm. Filtering the cCDA helps to remove unreacted excess chemical.
[57] In an embodiment, the cCDA obtained from step 104e is filtered using an assembly of a filter member placed at least partially within a centrifuge tube. The cCDA is placed within the filter member and then centrifuged at 2000 x g for 10 min, thus obtaining a second residue on the filter member containing cCDA.
[58] At step 104h, the cCDA obtained from step 104g is dried at a predefined temperature to remove moisture. The predefined temperature may range from 50°C to 60°C. In an embodiment, the cCDA is dried at 60°C to preserve the poor crystalline structure of the cCDA.
[59] At step 104i, the cCDA is subjected to milling to obtain nano-scale particles of the CDA having a pre-defined particle size. In an embodiment, the cCDA is milled using a milling apparatus, such as, planetary ball mill machine (procured from laboteck, model: LABMV2S), though other functionally equivalent apparatus may be employed to achieve similar results. In an embodiment, the particle size distribution of CDA after milling is less than 1 µm. In an embodiment, the particle size of the CDA is validated by BET surface area technique.
[60] The CDA obtained from step 104 has a pre-defined calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55. In an exemplary embodiment, the Ca/P ratio of the CDA is 1.5. The CDA has the following chemical formula:
Ca10-x(HPO4)x(PO4)6-x(OH)2-x
[61] Compared to HAp, CDA has missing calcium ions (Ca2+) in the crystal lattice leading to poor/low crystallinity. To maintain charge neutrality, these missing positive charges of the calcium ions (Ca2+) are compensated by addition of protons (forming HPO42- instead of PO43-) and a loss of hydroxide ions (OH-).
[62] The nano-sized calcium-deficient apatite (CDA) particles (<1 µm) enhance the biological performance of tri-phasic calcium phosphate (CAP) due to their high surface area-to-volume ratio. This increased surface area accelerates the dissolution of CDA under physiological conditions, leading to an elevated release of calcium (Ca²⁺) and phosphate (PO₄³⁻) ions, which are critical for bone mineralization and the formation of biological apatite. Simultaneously, the enlarged surface of CDA promotes greater protein adsorption, providing more binding sites for adhesion proteins and growth factors that facilitate improved cell attachment, proliferation, and differentiation. These combined effects stimulate a rapid osteogenic response by encouraging osteoprogenitor cells to differentiate into bone-forming osteoblasts, thereby accelerating early-stage bone regeneration. Furthermore, within the tri-phasic CAP system, nano-sized CDA acts as the fast-resorbing phase, dissolving early to release bioactive ions and create space for new bone ingrowth, while the other phases resorb more slowly to provide sustained structural support. This staged resorption behavior ensures a balanced and efficient transition from biomaterial to natural bone, ultimately enhancing overall bone healing and integration.
[63] At step 106 of method 100, β-Tricalcium phosphate (β-TCP) is prepared. Fig. 1c depicts an exemplary method 100c of preparing β-TCP.
[64] At step 106a, a predefined amount of a calcium precursor and a pre-defined amount of a phosphate precursor is mixed to obtain a first mixture. The calcium precursor is at least one of calcium carbonate (CaCO3), calcium hydroxide (Ca(OH)₂), calcium oxide (CaO), etc. The phosphate precursor is at least one of phosphoric acid (H3PO4), ammonium dihydrogen phosphate (NH₄H₂PO₄), etc. The predefined amount of the calcium precursor in the first mixture may range between 55% (w/w) to 65% (w/w). The predefined amount of the phosphate precursor in the first mixture may range between 45% (w/w) to 35% (w/w). In an embodiment, 60% (w/w) of calcium carbonate (CaCO3) is mixed with 40% (w/w) of phosphoric acid to obtain the first mixture.
[65] At step 106b, the first mixture obtained from step 106a is subjected to thermal solid-state reaction at a predefined temperature for a predefined time period under a predefined pressure. This facilitates calcination, thereby forming crude β-Tricalcium phosphate (cβ-TCP). The predefined temperature may range from 800°C to 900 °C. The predefined time period may range from 2 hours to 3 hours. The predefined pressure may be less than or equal to 1 atm pressure. In an embodiment, the first mixture is calcinated to 850°C for 2.5 hours at 1 atm. An exemplary reaction to obtain cβ-TCP is illustrated below:
3CaCO3 + 2H3PO4 → Ca3(PO4)2 + 3CO2↑ + 3H2O
[66] At step 106c, the cβ-TCP obtained from step 106b is subjected to milling to obtain micro-scale particles of β-TCP having a pre-defined particle size. In an embodiment, the β-TCP is grinded using a milling apparatus, such as, planetary ball mill machine (procured from laboteck, model: LABMV2S), though other functionally equivalent apparatus may be employed to achieve similar results. In an embodiment, the particle size distribution of β-TCP after grinding ranges between 50 µm and 150 µm.
[67] At an optional step 106d, the β-TCP obtained from step 106c, is subjected to sieving. The sieve has a pore size which is less than or equal to 150 µm. This helps in obtaining particles of β-TCP within the desired particle size range and to remove oversized particles/aggregates. In an embodiment, the particle size of the β-TCP is validated by XRD phase verification technique.
[68] The β-TCP obtained from step 106 has a pre-defined calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55. In an exemplary embodiment, the Ca/P ratio of the β-TCP is 1.5. The β-TCP has the following chemical formula:
Ca3(PO4)2
[69] The β-tricalcium phosphate (β-TCP) particles in the size range of 50–150 µm plays a crucial role in optimizing the structural and biological performance of tri-phasic calcium phosphate (CAP). In the given size, β-TCP exhibits a surface area that allows for controlled, intermediate resorption, meaning it degrades neither too rapidly nor too slowly, thereby maintaining scaffold integrity during the critical phases of bone healing. In an embodiment, β-TCP in the tri-phasic Calcium phosphate can last for six to eighteen months after implantation inside the body.
[70] The relatively larger particle size provides macro-porosity to the graft that facilitate cell migration, vascularization, and new bone ingrowth. In addition, these particles enhance the mechanical stability of the scaffold by providing a supportive framework that can withstand physiological loads while still being gradually replaced by natural bone.
[71] The 50–150 µm range further enables optimal packing behavior, ensuring sufficient inter-particle spacing for tissue infiltration without compromising structural cohesion. Altogether, β-TCP serves as the intermediate-resorbing phase in CAP, bridging the gap between the fast-resorbing nano-sized components and the more stable phases, thereby supporting staged degradation and promoting effective bone regeneration.
[72] Although the method 100 is described with sequentially preparing the HAp, CDA, and β-TCP as an example, the same may be prepared in any order or simultaneously and the same is within the scope of the teachings of the present disclosure.
[73] At step 108 of the method 100, a tri-phasic calcium phosphate (CAP) is obtained by physically blending/mixing the HAp, CDA, and β-TCP at a pre-defined ratio which defines the multi-phasic ceramic/mineral composition of the graft. In an embodiment, step 108 does not involve any chemical precipitation reactions. In an embodiment, the HAp, CDA, and β-TCP is physically blended using a milling apparatus, such as, planetary ball mill machine (procured from laboteck, model: LABMV2S), though other functionally equivalent apparatus may be employed to achieve similar results. The pre-defined ratio between the HAp, CDA, and β-TCP ranges from 1:1:1 to 4:1:4. The ratio may be adjusted based on resorption rates required for a particular application, preferably, to match the rate of new bone formation. In an embodiment, the HAp, CDA, and β-TCP is physically blended at 2:1:2 ratio. In an embodiment, the homogeneity and bulk density of the tri-phasic calcium phosphate is verified using scanning electron microscopy (SEM) and tapped density measurement techniques, respectively. The bulk density of the tri-phasic calcium phosphate may range from 0.8 g/cm³ to 1.5 g/cm³. In an exemplary embodiment, the bulk density of the tri-phasic calcium phosphate is 1.1 g/cm³.
[74] Since each phase is already crystallographically established while being synthesized in respective controlled reaction environments (steps 102, 104, 106 of method 100), the resulting tri-phasic calcium phosphate maintains three distinct calcium phosphate phases. The respective controlled reaction environments prevent formation of additional calcium phosphate phases (such as, amorphous calcium phosphate or octacalcium phosphate) in the resulting tri-phasic calcium phosphate. The coexistence of these phases introduces controlled and predictable crystallinity, calcium to phosphate (Ca/P) ratio, lattice structure, and particle morphology of the tri-phasic calcium phosphate.
[75] Additionally, or optionally, the tri-phasic calcium phosphate is subjected to bioactive ion doping for additional osteogenic, antibacterial properties, and/or controlled resorption behavior. The bioactive ions may be one of Sr²⁺, Zn²⁺, etc. In an embodiment, while independently preparing HAp, CDA, and β-TCP, the calcium ions may be partially substituted with the bioactive ions in a concentration ranging from 1 mol% to 10 mol%. In an embodiment, the calcium ions in HAp are partially substituted with the bioactive ions when the third solution is allowed to rest at step 102e. In an embodiment, the calcium ions in CDA are partially substituted with the bioactive ions when the sixth solution is allowed to rest at step 104e. In an embodiment, the calcium ions in β-TCP are partially substituted with the bioactive ions during the calcination step 106b.
[76] At an optional step 110 of the method 100, the tri-phasic calcium phosphate obtained from step 108 is sealed inside a vial and subjected to sterilization. The tri-phasic calcium phosphate may be sterilized using at least one of ethylene oxide (EtO) gas, or Gamma radiation. In an embodiment, the tri-phasic calcium phosphate is sterilized using gamma radiation of 25 kGy. In an embodiment, the vial includes 10g of the tri-phasic calcium phosphate.
[77] At step 112 of method 100, an organic binder of the graft is prepared. Fig. 1d depicts an exemplary method 100d to prepare the organic binder. In an embodiment, the organic binder includes a pre-defined amount of at least one branching element and a pre-defined amount of at least one interface element.
[78] At step 112a, the predefined amount of the branching element is dissolved in a predefined amount of at least one third solvent to obtain a seventh solution. The branching element may be at least one of collagen, gelatin, silk fibroin, fibrin, elastin, keratin, chitosan, alginate, and modified hyaluronic acid. The third solvent is at least one of diluted acetic acid, lactic acid, phosphate buffered saline (PBS), etc. The predefined amount of branching element in the seventh solution may range from 4% (w/v) to 6% (w/v). In an embodiment, 5% (w/v) of type I collagen is dissolved in 0.5%(v/v) acetic acid solution to obtain the seventh solution. The branching element is configured to form a cohesive matrix and is capable of interacting with the interface element(s) and calcium ions of the tri-phasic calcium phosphate.
[79] At step 112b, the predefined amount of interface element is dissolved in the seventh solution obtained from the step 112a to obtain the organic binder. The interface element may be at least one of phosphoserine, phosphorylated amino acids, casein phosphopeptides, phytic acid, glycerophosphates, bisphosphonates, polyphosphates, or carboxylate-containing polymers, etc. The predefined amount of interface element in the organic binder ranges from 1% (w/v) to 3% (w/v). The amount of interface element added influences the cohesion and/or bioactivity of the graft. In an embodiment, 2% (w/v) of phosphoserine is dissolved in the seventh solution while continuously stirring at 500 rpm using a magnetic stirrer for a predefined time period. The predefined time period may range from 1 hour to 2 hours. In an embodiment, the organic binder is stirred for 1.5 hours. The interface element is capable of interacting with calcium ions and forming a stable mineral–organic interface.
[80] In an embodiment, phosphoserine contains phosphate groups (-PO43-) and carboxyl groups (-COO-). The phosphate groups are capable of interacting with calcium (Ca2+) ions present on the surface of calcium phosphate particles via ionic interactions. The carboxyl groups are capable of interacting with collagen via ionic interactions and hydrogen bonding. Because of the said interactions, phosphoserine is capable of creating a bridging interface between the inorganic (tri-phasic calcium phosphates) and the organic (collagen) fractions.
[81] At an optional step 112c, a pH of the organic binder is adjusted if the pH of the organic binder deviates from 6.8 (towards acidic conditions). In an embodiment, the pH of the organic binder is adjusted to 6.8 by continuously monitoring (with the help of a pH meter) and adding a predefined amount of ammonium hydroxide (NH₄OH) solution.
[82] At an optional step 112d, a pre-defined amount of a crosslinking agent may be added to the organic binder obtained from step 112c. The cross-linking agent is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), genipin, glutaraldehyde, (N,N')-dicyclohexylcarbodiimide (DCC), PEG-crosslinkers, transglutaminase, riboflavin, etc. In an exemplary embodiment, 5 mol% to 20 mol% of EDC and 2.5mol% to 10 mol% of NHS is added to the organic binder. The crosslinking agent helps in mild carbodiimide (covalent) crosslinking of the collagen (i.e., the branching element) which stabilizes the collagen particles and improves structural cohesion of the graft.
[83] At an optional step 112e, a pre-defined amount of a bioactive molecule may be added to the organic binder, as per requirements. The bioactive molecule may be at least one of osteogenic factor (such as bone morphogenic proteins (BMPs)), antimicrobial, or angiogenic factors, etc., or a combination thereof.
[84] At step 112f, the organic binder is sterilized and then sealed inside a syringe. The organic binder is sterilized using one of filtration, gamma irradiation, ethylene oxide (EtO) gas, etc. In an embodiment, the organic binder is sterilized using a 0.22 µm filter. In an embodiment, the syringe includes 3mL of organic binder.
[85] Although the method 100 is described with sequentially preparing the tri-phasic calcium phosphate and organic binder as an example, the same may be prepared in any order or simultaneously and the same is within the scope of the teachings of the present disclosure.
[86] At step 114, prior to application of the graft, a pre-defined amount of the tri-phasic calcium phosphate is added in per unit volume of the organic binder to obtain the graft in the form of a cohesive, moldable putty. In an embodiment, the tri-phasic calcium phosphate is gradually mixed with the organic binder using a magnetic stirrer at 300 RPM to 600 RPM. The amount of tri-phasic calcium phosphate added per unit volume of organic binder ranges from 7.5 g/mL to 8 g/mL. In an embodiment, 20 g of the tri-phasic calcium phosphate is added to 2.5 mL of the organic binder, under continuous mixing at 440 RPM using a magnetic stirrer, to obtain the graft. Exemplary chemical interactions in the graft between the tri-phasic calcium phosphate (calcium ions) and the organic binder (phosphate and carboxyl groups) is illustrated below:
Ca2++R−PO43−→Ca−O−PO3−R
Ca2++R−COO−→Ca−OOC−R
[87] Additionally, or optionally, a pre-defined amount of sterile water may be added to the graft to adjust the viscosity of the graft, if required. The amount of sterile water to be added to the graft depends upon the application where the graft is to be used and/or at the discretion of the medical practitioner.
[88] Additionally, or optionally, the graft may be used to prepare porous scaffolds for bone tissue engineering applications. In an embodiment, the graft is freeze dried at −40°C followed by sublimation under 0.01–0.1 mbar to obtain a porous scaffold (50% to 90% porosity). In another embodiment, the graft is extruded using a 3D printer to obtain a porous scaffold (pore size ranges from 50 µm to 300 µm).
[89] The graft obtained from the method 100 includes a pre-defined amount of the tri-phasic calcium phosphate per unit volume of the organic binder. The amount of tri-phasic calcium phosphate in the graft ranges from 7.5 g/mL to 8 g/mL. In other words, the graft includes 7.5 g to 8 g of tri-phasic calcium phosphate in per unit volume (in mL) of the organic binder.
[90] In an embodiment, the tri-phasic calcium phosphate includes a pre-defined amount of HAp, a pre-defined amount of CDA, and a pre-defined amount of β-TCP, which defines the multi-phasic ceramic/mineral composition of the graft. The amount of HAp in the tri-phasic calcium phosphate ranges from 33.3% (w/w) to 44.4% (w/w). The amount of CDA in the tri-phasic calcium phosphate ranges from 11.1% (w/w) to 33.3% (w/w). The amount of β-TCP in the tri-phasic calcium phosphate ranges from 33.3% (w/w) to 44.4% (w/w). In an embodiment, the amount of HAp, CDA, and β-TCP in the tri-phasic calcium phosphate are 40% (w/w), 20% (w/w), and 40% (w/w), respectively.
[91] Additionally, or optionally, the tri-phasic calcium phosphate may be doped with bioactive ions for additional osteogenic or antibacterial properties. The ions may be one of Sr²⁺, Zn²⁺, etc. In an embodiment, in each of the HAp, CDA, and β-TCP, the calcium ions may be partially substituted with the bioactive ions in a concentration ranging from 1 mol% to 10 mol%.
[92] In an embodiment, the organic binder includes a predefined amount of branching element and a pre-defined amount of interface element is dissolved in per unit volume of a third solvent. The third solvent may be at least one of diluted acetic acid, lactic acid, phosphate buffered saline (PBS), etc. The branching element may be at least one of collagen, gelatin, silk fibroin, fibrin, elastin, keratin, chitosan, alginate, and modified hyaluronic acid. The branching element is configured to form a cohesive matrix and is capable of interacting with the interface element(s) and calcium ions of the tri-phasic calcium phosphate. The interface element may be at least one of phosphoserine, phosphorylated amino acids, casein phosphopeptides, phytic acid, glycerophosphates, bisphosphonates, polyphosphates, or carboxylate-containing polymers, etc. The predefined amount of branching element in the organic binder may range from 4% (w/v) to 6% (w/v). The predefined amount of interface element in the organic binder ranges from 1% (w/v) to 3% (w/v). In an embodiment, the organic binder includes 5% (w/v) of type I collagen and 2% (w/v) of phosphoserine dissolved in 0.5% (v/v) acetic acid solution.
[93] Additionally, or optionally, the branching element in the organic binder has mild carbodiimide crosslinking via at least one crosslinking agent. The cross-linking agent is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), genipin, glutaraldehyde, (N,N')-dicyclohexylcarbodiimide (DCC), PEG-crosslinkers, transglutaminase, riboflavin, etc. In an exemplary embodiment, 5–20 mol% of EDC and 2.5–10 mol% of NHS is used for mild crosslinking of the collagen in the organic binder.
[94] Additionally, or optionally, a pre-defined amount of a bioactive molecule may be added to the organic binder. The bioactive molecule may be at least one of osteogenic (such as bone morphogenic proteins (BMPs)), antimicrobial, or angiogenic factors, etc., or a combination thereof.
[95] Now the graft of the present disclosure will be described with the help of following examples.
[96] Example 1: Preparation of conventional grafts
[97] Example 1a: A bi-phasic calcium phosphate composition was prepared with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) in a weight ratio of 60:40. The biphasic calcium phosphate was then physically blended with a non-phosphoserine collagen binder in a weight ratio of 8:1 to obtain a graft ‘A’. The non-phosphoserine collagen binder includes 5% (w/v) type I collagen dissolved in 0.5% (v/v) acetic acid solution. The non-phosphoserine collagen binder has a pH of 6.8.
[98] Example 1b: A graft ‘B’ was prepared with non-phosphoserine collagen binder, without any calcium phosphate component. The non-phosphoserine collagen binder includes 5% (w/v) type I collagen dissolved in 0.5% (v/v) acetic acid solution. The graft ‘B’ has a pH of 6.8.
[99] Example 1c: A graft ‘C’ was prepared by physically blending (single phasic) hydroxyapatite (HAp) with non-phosphoserine collagen binder in a weight ratio of 8:1. The non-phosphoserine collagen binder includes 5% (w/v) type I collagen dissolved in 0.5% (v/v) acetic acid solution. The non-phosphoserine collagen binder has a pH of 6.8.
[100] Example 1d: A graft ‘D’ was prepared by dissolving 1 g of α-calcium sulfate hemihydrate (α-CSH) per 0.35 mL of sterile water to form a setting paste.
[101] Example 2: Preparation of the graft of the present disclosure.
[102] A first solution was prepared by dissolving 14.8g of calcium nitrate tetrahydrate in 150mL of water. A second solution was prepared by dissolving 8.2g of diammonium hydrogen phosphate in 100 mL of water. The second solution was dropwise stirred in to the first solution to obtain a third solution, while maintaining the temperature at 60°C and pH at 8.5 (with the help of ammonium hydroxide solution). Thereafter, the third solution was stirred at 600 rpm and 60°C for 2.5 hours and then left undisturbed for overnight to obtain a precipitate of the crude HAp (or cHAp). The cHAp was washed three times, filtered and then dried at 60°C. The cHAp was then subjected to calcination at 600°C for 2 hours under 1 atm to enhance crystallinity. After calcination, the cHAp was milled to obtain 8g of HAp having particle size distribution ranging from 5 µm and 30 µm.
[103] A fourth solution was prepared by dissolving 14g of calcium nitrate tetrahydrate in 150mL of water. A fifth solution was prepared by dissolving 8.5g of diammonium hydrogen phosphate in 100 mL of water. The fifth solution was dropwise stirred in to the fourth solution to obtain a sixth solution, while maintaining the temperature at 40°C and pH at 10 (with the help of ammonium hydroxide solution). Thereafter, the sixth solution was stirred at 600 rpm for 2.5 hours and then left undisturbed for overnight to obtain a precipitate of the crude CDA (or cCDA). The cCDA was washed three times, filtered and then dried at 55°C. The cCDA was milled to obtain 4g of CDA having particle size distribution of less than 1 µm.
[104] A first mixture was prepared by mixing 12g of calcium carbonate and 8g of phosphoric acid. The first mixture was subjected to thermal solid-state reaction at 850°C for 2.5 hours under 1 atm to obtain crude β-TCP (or cβ-TCP). The cβ-TCP was subjected to grinding and sieving to obtain 8g of β-TCP having particle size distribution ranging from 50 µm and 150 µm.
[105] The tri-phasic calcium phosphate was prepared by physically blending 8 g of HAp, 4 g of CDA, and 8 g of β-TCP. The tri-phasic calcium phosphate was sterilized using gamma radiation of 25 kGy.
[106] A seventh solution was prepared by dissolving 2.5g of type I collagen (procured from Sigma-Aldrich (Merck)) in 50 mL of 0.5% (v/v) acetic acid solution. Thereafter, 1g of phosphoserine (procured from Sigma-Aldrich (Merck)) was dissolved in the seventh solution and stirred at 500 rpm for 1.5 hours to obtain the organic binder. The pH of the organic binder was adjusted to 6.8 (with the help of NaOH solution) and then filtered using a 0.22µm filter.
[107] To prepare the graft of the present disclosure, 20g of the tri-phasic calcium phosphate was added to 2.5 mL of the organic binder which formed a cohesive, moldable putty.
[108] Example 3: Comparative study of graft of the present disclosure obtained from example 2 with respect to conventional grafts (obtained from example 1a-1c) on the basis of mechanical and handling properties of the graft. Metrics, such as, cohesion strength, washout loss in simulated body fluid (SBF), yield stress and moldability score, were measured. For measurements of the metrics, all the samples were prepared in three batches, and metrics for each sample was measured 5 times per batch.
[109] Cohesion strength was determined using a universal testing machine under uniaxial compression. Samples included cylindrical specimens (10 mm × 10 mm) tested at a crosshead speed of 1 mm/min. The peak stress at failure was recorded as cohesion strength.
[110] Washout loss was determined by immersing graft samples in SBF at 37°C for 24 hours. SBF mimics human plasma and is used to evaluate the in vitro stability and bioactivity of the grafts. The initial and final weights were recorded using an analytical balance and washout loss was calculated as the percentage mass loss after immersion.
[111] Yield stress was determined using a rotational rheometer. A shear stress sweep (0.1–1000 Pa) was performed at 25°C using a parallel plate geometry. The yield stress was identified as the stress at which the material transitioned from solid-like to flow behavior.
[112] Moldability was subjectively assessed using a semi-quantitative scoring system on a 1–5 scale through manual evaluation by trained assessors. The evaluation considered ease of shaping, material cohesion, and shape retention after molding.
[113] The means and coefficient of variation (CV) were calculated from the measurements and then observations are tabulated in Table 1 below.
Table 1: Mechanical and handling properties
Metric Graft of the present disclosure Graft ‘A’ Graft ‘B’ Graft ‘C’ Graft ‘D’
Cohesion strength (in kPa) 42.6 (CV = 6.7%) 26.8 9.4 21.2 32.0
Washout loss in SBF (in % after 24 h) 4.8 (CV = 15.6%) 14.6 38.9 18.2 22.0
Yield stress (in Pa) 325 (CV = 9.5%) 190 65 155 230
Moldability score 4.8/5 3.9/5 2.1/5 3.6/5 3.5/5

[114] It is evident from the observations tabulated above that the graft of the present disclosure demonstrated 59% improvement in cohesion strength, 71% improvement in yield stress, and 67% reduction in washout loss (compared to the conventional calcium phosphate-based graft). The moldability score of the graft of the present disclosure represents how easy it is for the medical practitioner to work with the graft of the present disclosure when applying the graft over uneven and complex anatomies.
[115] Example 4: The graft of the present disclosure obtained from example 2 was studied for its structural integrity after being subjected to different sterilization techniques.
[116] X-ray diffraction (XRD) analysis of three different samples of calcium-deficient apatite (CDA) (before and after sterilization) were performed using an X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å). The scan was performed over a 2θ range of 10° to 60° with a step size of 0.02°. The analysis was used to confirm phase composition and crystallinity. Broad diffraction peaks indicated an apatite structure with low crystallinity.
[117] Fourier Transform Infrared (FTIR) spectroscopy analysis of three organic binder samples (before and after sterilization) were carried out using an FTIR spectrometer (ATR mode). The spectra were recorded over a wavenumber range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹. Each sample was scanned 16–32 times per measurement. Characteristic amide bands confirmed the structural integrity of collagen in the organic binder.
[118] Cohesion loss was determined by immersing three graft samples in simulated body fluid (SBF) at 37°C under gentle agitation (50–60 rpm). SBF mimics human plasma and is used to evaluate the in vitro stability and bioactivity of the grafts. The initial and final weights were measured five times using an analytical balance, and cohesion loss was calculated as the percentage mass loss after sterilization.
[119] The observations are tabulated in Table 2 below.
Table 2: Structural integrity
Sterilization XRD CDA FTIR collagen Cohesion loss
Gamma (25 Gy) Preserved Preserved −6%
EtO Preserved Preserved −4%

[120] Example 5: The organic binder of the present disclosure obtained from example 2 and the graft ‘B’ obtained from example 1b was studied for its ability to bind protein.
[121] BSA adsorption was determined by incubating three organic binder samples with a 1 mg/mL BSA solution in phosphate buffer saline (PBS) at 37°C for 2 hours. Protein concentration was measured three times at 280 nm using a UV–Vis spectrophotometer. Adsorption was calculated from the difference between initial and final concentrations.
[122] Growth factor retention was determined by incubating three organic binder samples with 100 ng/mL BMP-2 solution at 37°C for 2 hours. The unbound growth factor in the supernatant was quantified using Enzyme-Linked Immunosorbent Assay (ELISA) at 450 nm. Retention efficiency was calculated from the difference between initial and residual concentrations.
[123] The observations are tabulated in Table 3 below.
Table 3: Protein binding performance
Binder Type BSA Adsorption (in µg/mg) Growth factor retention (after 24 h, in %)
Organic binder of the present disclosure 185 70
Graft ‘B’ 90 34

[124] It is evident from the observations tabulated above that the protein binding ability of the organic binder of the present disclosure is more than twice than that of the conventional graft/binders.
[125] Example 5: An in-vitro study between the individual calcium phosphate phases, and the tri-phasic calcium phosphate obtained from example 2 the bi-phasic calcium phosphate, and the α-CSH obtained from example 1. Each of the aforesaid calcium phosphate phases were evaluated for calcium ion (Ca2+) release, phosphate ion (PO43-) release, and mass loss/resorption.
[126] Calcium ion release was determined by immersing the samples in simulated body fluid at 37°C. The sample size was three, and each sample was measured three times. Ca²⁺ concentration was measured at predetermined time intervals using inductively coupled plasma optical emission spectroscopy (ICP-OES), based on calibration with standard solutions.
[127] The observations are tabulated in Tables 4, 5, and 6 below.
Table 4: calcium ion release (in mg/L)
Time α-CSH from example 1d β-TCP from example 2 HAp from example 2 bi-phasic calcium phosphate (HAp + β-TCP) from example 1a tri-phasic calcium phosphate (HAp + CDA + β-TCP) from example 2
1 week 85 18 5 22 62
2 weeks 120 30 8 38 95
4 weeks 150 48 12 60 135
8 weeks 155 65 18 82 165
12 weeks 158 72 22 95 182

[128] Based on the observations tabulated in Table 4 above, it is evident that the tri-phasic calcium phosphate of the present disclosure demonstrated an initial calcium burst from CDA followed by sustained release governed by β-TCP resorption and long-term stabilization by HAp. This confirms the staged ionic release synergy of the tri-phasic calcium phosphate of the present disclosure.
[129] Phosphate ion release was measured by immersing the samples in simulated body fluid at 37 °C. The sample size was three, and each sample was measured three times. Phosphate concentration was quantified at predetermined time intervals using the molybdenum blue method at ~880 nm. Results were calculated using a standard calibration curve.
Table 5: Phosphate ion release (in mg/L)
Time β-TCP from example 2 HAp from example 2 bi-phasic calcium phosphate (HAp + β-TCP) from example 1a tri-phasic calcium phosphate (HAp + CDA + β-TCP) from example 2
1 week 12 3 14 18
2 weeks 22 5 26 35
4 weeks 38 8 42 58
8 weeks 55 12 60 78
12 weeks 62 15 70 92

[130] β-TCP showed moderate ion release, while HAp exhibited the slowest and most limited release. The biphasic calcium phosphate (HAp + β-TCP) demonstrated intermediate dissolution behavior between the two phases. The tri-phasic calcium phosphate (HAp + CDA + β-TCP) showed the highest and most sustained release over time. The release profile indicated controlled dissolution behavior, contributing to enhanced bioactivity and mineralization potential of the tri-phasic calcium phosphate (CAP).
[131] Mass loss (resorption) was evaluated by immersing the samples in simulated body fluid (SBF) at 37 °C and measuring weight loss at predetermined time intervals using an analytical balance. The sample size was three, and each sample was measured three times (triplicate measurements). The percentage mass loss was calculated from the difference between initial and final weights.
Table 6: mass loss/resorption (in %)
Time α-CSH from example 1d β-TCP from example 2 HAp from example 2 bi-phasic calcium phosphate (HAp + β-TCP) from example 1a tri-phasic calcium phosphate (HAp + CDA + β-TCP) from example 2
1 week 48% 12% 2% 15% 25%
2 weeks 72% 25% 4% 28% 40%
4 weeks 88% 45% 6% 45% 62%
8 weeks 95% 55% 8% 55% 75%
12 weeks 48% 12% 2% 15% 25%

[132] The mass loss profiles of all formulations showed time-dependent degradation. The tri-phasic calcium phosphate (CAP) exhibited the highest overall resorption, followed by the biphasic calcium phosphate, β-TCP, and hydroxyapatite (HAp). This behavior reflects staged degradation, where CDA contributes to early resorption, β-TCP drives intermediate degradation, and HAp provides long-term structural stability.
[133] Example 6: In-vitro study.
[134] Two in vitro cell viability studies were conducted using MG-63 cells and human mesenchymal stem (hMCS) cells each, to evaluate the cytocompatibility of the graft of the present disclosure obtained from example 2. The MG-63 cells were procured from American Type Culture Collection (ATCC). 1 × 10⁵ number of MG-63 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (procured from Gibco, Thermo Fisher Scientific) in a first culture plate. The media was supplemented with 10% fetal bovine serum (FBS, procured from Gibco, Thermo Fisher Scientific) and 1% penicillin–streptomycin (procured from Gibco, Thermo Fisher Scientific). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂, with media replacement every 2–3 days until 80–90% of confluency was reached in the first culture plate.
[135] The graft from Example 2, single-phasic (HAp) graft ‘C’, and bi-phasic (HAp + β-TCP) graft ‘A’ were sterilized and added to respective wells of a second culture plate. The grafts were pre-wetted with phosphate buffer saline (PBS) for 30 minutes prior to cell seeding. 1 × 10⁵ numbers of cells (obtained from first culture plate) were seeded onto each graft provided in the respective wells of the second culture plate. The cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO₂. A control group consisting of 1 × 10⁵ number of cells cultured under identical conditions without exposure to any graft material was used as a baseline for comparison.
[136] The above protocol was repeated for human mesenchymal stem cells (hMSCs). For hMSCs, instead of DMEM, α - Minimum Essential Medium (α-MEM, procured from Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin was used as culture media.
[137] The above protocols were repeated three times and the observations for each metric (described below) were recorded in triplicates. The data was averaged and compared with single-phasic (HAp) graft ‘C’ and bi-phasic (HAp + β-TCP) graft ‘A’ obtained from examples 1c and 1a above.
[138] Example 6a: Cell viability study.
[139] Cells were incubated with the graft materials (in the second culture plate) as described above in example 6. Cell viability was determined using a 3-(4,5-di methyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and observations were recorded at predefined intervals. Cell viability was expressed as a percentage relative to the control group. Absorbance was measured at 570 nm using a microplate reader, and cell viability was calculated relative to control samples. The observations were averaged and then tabulated in Table 7 below.
Table 7: Cell viability (in %)
Time Control group Graft ‘C’ Graft ‘A’ Graft of the present disclosure
1 day 100 98 105 112
3 day 100 102 115 128
7 day 100 110 125 148

[140] Example 6b: Cell adhesion study.
[141] Cells were incubated with the graft materials (in the second culture plate) as described above in example 6. Cell adhesion on the grafts were evaluated to determine the ability of the graft to support initial cellular attachment. After 6 hours of incubation, the non-adherent cells were removed by gently washing the samples with PBS (3 times). The cells adhered to the graft were fixed using 4% paraformaldehyde for 15 minutes at room temperature. The fixed cells were stained using 0.1% (w/v) crystal violet. The stained cells were solubilized using 10% (v/v) acetic acid, and the absorbance of the resulting solution was measured at 570 nm using a UV–Visible spectrophotometer. The absorbances were compared and the cell adhesion was expressed as a percentage relative to the control group. The observations were averaged and then tabulated in Table 8 below.
Table 8: Cell adhesion (in %)
β-TCP from example 2 (Control group) Graft ‘C’ Graft ‘A’ Graft of the present disclosure
Adhesion (at 6 hours of incubation) 65 72 80 90

[142] Example 6c: Alkaline phosphatase (ALP) activity.
[143] Cells were incubated with the graft materials (in the second culture plate) as described above in example 6. ALP activity was determined using a p-nitrophenyl phosphate (pNPP)-based colorimetric assay. The enzymatic conversion of pNPP to p-nitrophenol was recorded at 405 nm using a microplate reader, and results were normalized to total protein content. The observations are tabulated in Table 9 below.
Table 9: ALP activity (in U/mg of protein)
Control group Graft ‘C’ Graft ‘A’ Graft of the present disclosure
ALP activity (at Day 14 of incubation) 1 U/mg protein 1.6 U/mg protein 2.2 U/mg protein 3.1 U/mg protein

[144] It is evident from the observations tabulated above that the graft of the present disclosure demonstrated 41% improvement and 94% improvement in ALP activity compared to bi-phasic and single-phasic grafts, respectively. ALP activity serves as an early indicator of osteogenic differentiation and plays a critical role in mineralization by generating inorganic phosphate ions required for hydroxyapatite formation, thereby reflecting the bone-forming potential of the graft material.
[145] Example 6d: Mineralization study.
[146] Cells were incubated with the graft materials (in the second culture plate) as described above in example 6. After 24 hours of incubation, to allow cell attachment, the culture medium was replaced with osteogenic induction medium. The osteogenic medium was prepared by supplementing fresh culture medium with 50 µg/mL of ascorbic acid, 10 mM of β-glycerophosphate, and 100 nM of dexamethasone. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for a period of 21 days with osteogenic induction medium replacement every 2-3 days to maintain optimal differentiation conditions.
[147] After 21 days of incubation, the grafts were gently washed with PBS to remove non-adherent cells and residual media. The cells adhered to the graft were fixed using 4% paraformaldehyde for 15 minutes at room temperature. The fixed cells were then washed with deionized water and stained with 2% (w/v) Alizarin Red S solution (pH adjusted to 4.2) for 25 minutes to visualize calcium-rich mineralized nodules under an optical microscope. Excess stain was removed by repeated washing with deionized water until a clear background was obtained. The bound Alizarin Red S dye was extracted by adding 10% (w/v) cetylpyridinium chloride solution to each well and incubating for 30 minutes to solubilize the calcium-bound dye. The resulting solution was transferred to a microplate and the absorbance was measured at 562 nm using a UV–Visible spectrophotometer. The extent of mineralization was determined based on the absorbance values, wherein higher absorbance indicated increased calcium deposition and enhanced osteogenic mineralization potential of the graft. The observations are tabulated in Table 10 below.
Table 10: Mineralization activity (in absorbance @562 nm)
Control group Graft ‘C’ Graft ‘A’ Graft of the present disclosure
Mineral deposition (at Day 21 of incubation) 0.32 0.58 0.75 1.05
[148] Example 6e: Osteogenic gene expression study.
[149] Cells were incubated with the graft materials (in the second culture plate) as described above in example 6. Gene expression was determined using quantitative real-time polymerase chain reaction (qPCR) and expressed as a fold change relative to the control group. Total RNA was extracted, followed by reverse transcription to complementary DNA (cDNA). Quantitative analysis was carried out using SYBR Green chemistry on a real-time PCR system. Relative gene expression levels were determined using the 2⁻ΔΔCt method, normalized against GAPDH as the housekeeping gene. The observations are tabulated in Table 10 below.
Table 11: Osteogenic gene expression (in fold change)
Marker genes Control group Graft ‘A’ Graft of the present disclosure
Runx2 1.0 2.3 3.8
Osteocalcin 1.0 2.0 3.5

[150] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. , Claims:WE CLAIM
1. A method (100) to prepare a multi-phasic synthetic bone graft, the method (100) comprising:
a. preparing hydroxyapatite (HAp) having calcium to phosphate (Ca/P) ratio ranging from 1.60 to 1.67, the hydroxyapatite (HAp) includes macro-scale particles having particle size distribution between 5 µm and 30 µm;
b. preparing calcium-deficient apatite (CDA) having a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55, the calcium-deficient apatite (CDA) includes nano-scale particles having particle size distribution of less than 1 µm; and
c. preparing β-tricalcium phosphate (β-TCP) having a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55, the β-tricalcium phosphate (β-TCP) includes micro-scale particles having particle size distribution between 50 µm and 150 µm;
d. mixing the hydroxyapatite (HAp), the calcium-deficient apatite (CDA), and the β-tricalcium phosphate (β-TCP) at a pre-defined ratio ranging from 1:1:1 to 4:1:4 to obtain a tri-phasic calcium phosphate;
e. preparing an organic binder including at least one branching element and at least one interface element;
f. adding a pre-defined amount of the tri-phasic calcium phosphate in per unit volume of the organic binder to obtain a multi-phasic synthetic bone graft.
2. The method (100) as claimed in claim 1, wherein the step of preparing the hydroxyapatite (HAp) includes:
a. dissolving 8% (w/v) and 12% (w/v) of a first calcium salt in a first solvent to obtain a first solution, the first calcium salt is at least one of calcium nitrate tetrahydrate, calcium chloride dihydrate, and calcium acetate monohydrate;
b. dissolving 6% (w/v) and 10% (w/v) of a first phosphate salt in the first solvent to obtain a second solution, the first phosphate salt is at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and phosphoric acid;
c. adding the second solution to the first solution at 60°C to 65°C under alkaline condition to obtain a third solution;
d. stirring the third solution at 60°C to 65°C for 2 hours to 3 hours;
e. allowing the third solution to rest for 6 hours to 8 hours to precipitate out crude hydroxyapatite (cHAp);
f. washing, filtering, and drying the crude hydroxyapatite (cHAp);
g. subjecting the crude hydroxyapatite (cHAp) to calcination at less than or equal to 600°C for 2 hours to 3 hours; and
h. subjecting the crude hydroxyapatite (cHAp) to milling to obtain the macro-scale particles of hydroxyapatite (HAp).
3. The method (100) as claimed in claim 1, wherein the step of preparing the calcium-deficient apatite (CDA) includes:
a. dissolving 8% (w/v) and 12% (w/v) of a second calcium salt in a second solvent to obtain a fourth solution, the second calcium salt is at least one of calcium nitrate tetrahydrate, calcium chloride dihydrate, and calcium acetate monohydrate;
b. dissolving 6% (w/v) and 10% (w/v) of a second phosphate salt in the second solvent to obtain a fifth solution, the second phosphate salt is at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, and phosphoric acid;
c. adding the fifth solution to the fourth solution at 40°C to 60°C under alkaline condition to obtain a sixth solution;
d. stirring the sixth solution at 40°C to 60°C for 2 hours to 3 hours;
e. allowing the sixth solution to rest for 6 hours to 8 hours to precipitate out crude calcium-deficient apatite (cCDA);
f. washing, filtering, and drying the crude calcium-deficient apatite (cCDA); and
g. subjecting the crude calcium-deficient apatite (cCDA) to milling to obtain the nano-scale particles of calcium-deficient apatite (CDA).
4. The method (100) as claimed in claim 1, wherein the step of preparing the β-tricalcium phosphate (β-TCP) includes:
a. mixing 55% (w/w) to 65% (w/w) of a calcium precursor with 45% (w/w) to 35% (w/w) of a phosphate precursor to obtain a first mixture,
i. the calcium precursor is at least one of calcium carbonate, calcium hydroxide and calcium oxide, and
ii. the phosphate precursor is at least one of phosphoric acid, and ammonium dihydrogen phosphate;
b. subjecting the first mixture to a thermal solid-state reaction at 800°C to 900 °C for 2 hours to 3 hours to obtain crude β-tricalcium phosphate (cβ-TCP); and
c. subjecting the crude β-tricalcium phosphate (cβ-TCP) to milling to obtain the micro-scale particles of β-tricalcium phosphate (β-TCP).
5. The method (100) as claimed in claim 1, wherein the step of preparing an organic binder includes:
a. dissolving 4% (w/v) to 6% (w/v) of the branching element in a third solvent to obtain a seventh solution, the branching element is at least one of collagen, gelatin, silk fibroin, fibrin, elastin, keratin, chitosan, alginate, and modified hyaluronic acid;
b. dissolving 1% (w/v) to 3% (w/v) of the interface element in the seventh solution to obtain the organic binder, the interface element is at least one of phosphoserine, phosphorylated amino acids, casein phosphopeptides, phytic acid, glycerophosphates, bisphosphonates, polyphosphates, and carboxylate-containing polymers.
6. The method (100) as claimed in claim, wherein the step of preparing an organic binder includes adding a pre-defined amount of a crosslinking agent to the organic binder, the crosslinking agent is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), genipin, glutaraldehyde, (N,N')-dicyclohexylcarbodiimide (DCC), PEG-crosslinkers, transglutaminase, and riboflavin.
7. The method (100) as claimed in claim 1, wherein the step of preparing an organic binder includes adding a pre-defined amount of a bioactive molecule to the organic binder, the bioactive molecule is at least one of osteogenic factors, antimicrobial, and angiogenic factor.
8. The method (100) as claimed in claim 1, wherein the step of adding a pre-defined amount of the tri-phasic calcium phosphate per unit volume of the organic binder includes adding 7.5 g/mL to 8 g/mL of the tri-phasic calcium phosphate in the organic binder under continuous mixing.
9. The method (100) as claimed in claim 1, wherein the method (100) includes adding a pre-defined amount of sterile water to the multi-phasic synthetic bone graft.
10. A multi-phasic synthetic bone graft, comprising:
a. an organic binder including:
i. 4% (w/v) to 6% (w/v) of a branching element, and
ii. 1% (w/v) to 3% (w/v) of an interface element;
b. 7.5 g to 8 g of a tri-phasic calcium phosphate per unit volume of the organic binder, the tri-phasic calcium phosphate including:
i. 33.3% (w/w) to 44.4% (w/w) of hydroxyapatite (HAp) having a calcium to phosphate (Ca/P) ratio ranging from 1.60 to 1.67, the hydroxyapatite (HAp) includes macro-scale particles having particle size distribution between 5 µm and 30 µm,
ii. 11.1% (w/w) to 33.3% (w/w) of calcium-deficient apatite (CDA) having a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55, the calcium-deficient apatite (CDA) includes nano-scale particles having particle size distribution of less than 1 µm, and
iii. 33.3% (w/w) to 44.4% (w/w) of β-tricalcium phosphate (β-TCP) having a calcium to phosphate (Ca/P) ratio ranging from 1.45 to 1.55, the β-tricalcium phosphate (β-TCP) includes micro-scale particles having particle size distribution between 50 µm and 150 µm.
11. The multi-phasic synthetic bone graft as claimed in claim 10, wherein the branching element is at least one of collagen, gelatin, silk fibroin, fibrin, elastin, keratin, chitosan, alginate, and modified hyaluronic acid.
12. The multi-phasic synthetic bone graft as claimed in claim 10, wherein the interface element is at least one of phosphoserine, phosphorylated amino acids, casein phosphopeptides, phytic acid, glycerophosphates, bisphosphonates, polyphosphates, and carboxylate-containing polymers.
13. The multi-phasic synthetic bone graft as claimed in claim 10, wherein the organic binder includes a pre-defined amount of a crosslinking agent, the crosslinking agent is at least one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS), genipin, glutaraldehyde, (N,N')-dicyclohexylcarbodiimide (DCC), PEG-crosslinkers, transglutaminase, and riboflavin.
14. The multi-phasic synthetic bone graft as claimed in claim 10, wherein the organic binder includes a pre-defined amount of a bioactive molecule, the bioactive molecule is at least one of osteogenic factors, antimicrobial, and angiogenic factor.
15. The multi-phasic synthetic bone graft as claimed in claim 10, wherein the tri-phasic calcium phosphate defines a bulk density ranging from 0.8 g/cm³ to 1.5 g/cm³.