Description:[001] The present invention relates to the field of tissue engineering and regenerative medicine. More particularly, it pertains to a bone graft material comprising calcium-deficient apatite (CDA) doped with divalent cations, specifically strontium (Sr²⁺) and magnesium (Mg²⁺), for enhanced osteogenesis and angiogenesis. The invention further relates to 3D-printed scaffolds incorporating this material and methods of preparing the same.
BACKGROUND OF THE INVENTION
[002] Bone regeneration is heavily reliant on vascularization to ensure nutrient transport and promote healing. Traditional bone graft methods—including autografts, allografts, and xenografts—carry risks of donor site morbidity, immune rejection, and poor integration. While synthetic grafts mitigate some issues, they often lack adequate osteo-inductive and angiogenic properties.
[003] Recent advancements focus on ion-doped calcium phosphates to enhance both osteogenesis and angiogenesis. Calcium-deficient apatite (CDA), comprising hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) phases, exhibits favourable resorption kinetics and mechanical properties. Doping with Sr²⁺ and Mg²⁺ ions further improve cellular proliferation, angiogenesis, and bone integration. Sr²⁺ stabilizes the HA phase and promotes osteoblast proliferation, while Mg²⁺ enhances angiogenesis by stimulating nitric oxide production.
[004] 3D printing allows for patient-specific scaffold fabrication, overcoming shape mismatch and improving post-operative outcomes. Porosity can be tailored to match cortical or cancellous bone requirements, balancing mechanical strength with vascular infiltration.
SUMMARY OF THE INVENTION
[005] The present invention provides a bone graft material comprising a calcium-deficient apatite (CDA) doped with at least one divalent cation selected from Sr²⁺ and Mg²⁺. This composition enhances both osteogenesis and angiogenesis, overcoming the limitations of existing bone graft technologies. The invention also includes a 3D-printed scaffold incorporating the bone graft material with controlled porosity, further promoting bone regeneration and vascularization.

[006] One of the advantages of the invention is the enhanced osteogenesis and angiogenesis provided by the doped CDA material. The incorporation of Sr²⁺ and Mg²⁺ ions into the CDA material stimulates bone formation and blood vessel growth, leading to improved bone regeneration outcomes. Another advantage is the use of a 3D-printed scaffold with controlled porosity. This scaffold provides a structural framework for bone regeneration and allows for the controlled release of the doped CDA material, further enhancing its effectiveness.

[007] Yet another advantage of the invention is the method for preparing the doped CDA material. The controlled precipitation and calcination process ensures the consistent production of high-quality bone graft material with the desired composition and properties. This method is scalable and reproducible, making it suitable for commercial production. Additionally, the invention has broad clinical applications in various medical fields, including periodontology, orthopaedics, and tissue engineering. This versatility makes it a valuable tool for addressing a wide range of bone regeneration challenges
FIGURES:
[008] Figure 1: This figure illustrates the X-ray diffraction patterns of the synthesized CDA powders with different concentrations of Sr²⁺ and Mg²⁺ ions. It demonstrates the presence of both HA and β-TCP phases in the CDA material.

[009] Figure 2: This figure shows the Fourier Transform Infrared (FTIR) spectra of the synthesized CDA powders. It confirms the presence of characteristic functional groups associated with HA and β-TCP.

[010] Figure 3: This figure illustrates the capability of fabricating 3D scaffolds with various internal architectures, including linear, round, oval, and 100% infill, using 3D printing technology.

[011] Figure 4: This figure shows fluorescent images of cells seeded on the 3D scaffolds, stained with fluorescein diacetate (FDA) and propidium iodide (PI). It demonstrates the biocompatibility of the scaffolds and the ability to support cell attachment and proliferation.

[012] Figure 5: This figure presents the results of the Alamar blue assay, which measures cell metabolic activity. It shows the increase in metabolic activity of cells on the scaffolds over time, indicating cell viability and proliferation.

[013] Figure 6: This figure illustrates the in vitro tube formation assay, which assesses the ability of the scaffolds to induce angiogenesis. It demonstrates the formation of capillary-like structures by endothelial cells in a 3D matrix, indicating their angiogenic potential.

[014] Figure 7: This figure shows the quantitative results of the tube formation assay, which measures the number of nodes per field and the total capillary length per field. It demonstrates the ability of different compositions to promote vascularization

[015] Figure 8: This figure illustrates the quantitative mRNA expression of angiogenic genes (CD31, ANGPT1, ANGPT2, vWF) and osteogenic genes (RUNX2, OPN, OCN, ALP) in endothelial cells (ECs) and mesenchymal stem cells (MSCs) cultured for 7 days using the eluted media collected from different samples (CS0, CSM0.03, and CSM0.06).

[016] Figure 9: This figure represents the radiographic defect fill at baseline, 3 months, and 6 months for the test group (using the bone graft material of the invention) and the control group (Demineralized and freeze-dried bone allograft)
DETAILED DESCRIPTION OF FIGURES:
[017] Figure 1: This figure illustrates the X-ray diffraction patterns of the synthesized CDA powders with different concentrations of Sr²⁺ and Mg²⁺ ions. The X-ray diffraction analysis was conducted to identify the crystalline phases present in the synthesized powders. The results confirm the presence of both hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) phases in the CDA material, as evidenced by the characteristic peaks corresponding to each phase. The relative intensities of the HA and β-TCP peaks vary with the concentration of Sr²⁺ and Mg²⁺ ions, indicating that the incorporation of these ions influences the phase composition of the CDA material. This figure highlights the ability to tailor the phase composition of the CDA material by adjusting the dopant concentrations, which can be used to optimize its bioactivity and resorption properties for specific bone regeneration applications.

[018] Figure 2: This figure shows the Fourier Transform Infrared (FTIR) spectra of the synthesized CDA powders. The FTIR spectroscopy was performed to analyze the functional groups present in the synthesized powders. The spectra exhibit characteristic absorption bands associated with phosphate groups, confirming the presence of both HA and β-TCP in the CDA material. The intensity of the absorption bands varies with the concentration of Sr²⁺ and Mg²⁺ ions, suggesting that the incorporation of these ions affects the chemical composition and bonding characteristics of the CDA material. This figure provides further evidence for the successful synthesis of CDA material with tunable properties through the incorporation of Sr²⁺ and Mg²⁺ ions.

[019] Figure 3: This figure illustrates the capability of fabricating 3D scaffolds with various internal architectures, including linear, round, oval, and 100% infill, using 3D printing technology. The scaffolds were fabricated using an extrusion-based 3D bioprinter, which allows for precise control over the internal structure and porosity of the scaffolds. The SEM images demonstrate the successful fabrication of scaffolds with different internal architectures, which can be tailored to match the specific requirements of the bone defect being treated. This figure highlights the versatility of the 3D printing approach for creating customizable scaffolds with controlled porosity and internal structure, which can enhance bone regeneration and vascularization.

[020] Figure 4: This figure shows fluorescent images of cells seeded on the 3D scaffolds, stained with fluorescein diacetate (FDA) and propidium iodide (PI). The FDA/PI staining was performed to assess the viability and proliferation of cells on the scaffolds. Live cells are stained green by FDA, while dead cells are stained red by PI. The images demonstrate that the cells are able to attach, proliferate, and remain viable on the scaffolds, indicating the biocompatibility of the CDA material and its ability to support cell growth. This figure provides evidence for the cytocompatibility of the scaffolds and their potential to promote bone regeneration by providing a suitable environment for cell attachment and growth.

[021] Figure 5: This figure presents the results of the Alamar blue assay, which measures cell metabolic activity. The Alamar blue assay was conducted to quantify the metabolic activity of cells on the scaffolds. The results show a significant increase in metabolic activity over time, indicating that the cells are actively proliferating and producing energy. This increase in metabolic activity is further evidence of the scaffolds' ability to support cell growth and viability. The Alamar blue assay provides a quantitative measure of cell viability and proliferation, confirming the biocompatibility of the scaffolds and their potential to promote bone regeneration.

[022] Figure 6: This figure illustrates the in vitro tube formation assay, which assesses the ability of the scaffolds to induce angiogenesis. The in vitro tube formation assay was performed to evaluate the angiogenic potential of the scaffolds. The images show the formation of capillary-like structures by endothelial cells on the scaffolds, indicating that the scaffolds are able to induce angiogenesis. This angiogenic potential is crucial for bone regeneration, as it ensures the formation of new blood vessels to supply oxygen and nutrients to the growing bone tissue. This figure provides evidence for the ability of the scaffolds to promote vascularization, which is essential for successful bone regeneration.

[023] Figure 7: This figure shows the quantified results of the invitro tube formation assay using image j software. Image j analysis was performed at selected random locations. The quantification results indicate a significant difference in the tube formation at 2 hours in CSM0.06 samples when compared with control and other sample groups. There is no significant difference between CSM0, CSM0.03, and control at 2 hours. At 4 hours and 6 hours, the number of nodes and tube length of the control group is significantly different from CSM0.03 and CSM0. The number of nodes and tube length of the CSM0.06 sample group is significantly higher when compared with the control and all other sample groups. This figure quantifies the ability of the prepared material compositions to induce neovascularization.

[024] Figure 8: This figure presents the quantitative mRNA expression of angiogenic genes (CD31, ANGPT1, ANGPT2, vWF) and osteogenic genes (RUNX2, OPN, OCN, ALP) in endothelial cells (ECs) and mesenchymal stem cells (MSCs) cultured for 7 days using the eluted media collected from different samples (CS0, CSM0.03, and CSM0.06). The data is normalized with CSM0 as the control group. The figure demonstrates the upregulation of both angiogenic and osteogenic genes in cells cultured with the eluted media from the doped CDA samples, indicating the ability of these materials to promote both angiogenesis and osteogenesis, which are crucial processes for bone regeneration.

[025] Figure 9: This figure shows the radiographic defect fill at baseline, 3 months, and 6 months for the test group (using the bone graft material of the invention) and the control group. The radiographic images demonstrate the progressive bone regeneration and defect fill in both groups over time. The figure highlights the superior performance of the test group compared to the control group, as evidenced by the greater radiographic bone fill at both 3 and 6 months. This observation supports the clinical efficacy of the bone graft material in promoting bone regeneration and defect repair.

DETAILED DESCRIPTION OF THE INVENTION
[026] The present invention is not limited to the specific embodiments described herein, but encompasses all modifications and variations that fall within the scope of the appended claims

[027] In one of the embodiments, the present invention offers a significant advancement in the field of bone regeneration by providing a novel bone graft material with enhanced osteogenic and angiogenic properties. This invention overcomes the limitations of current bone graft technologies, such as donor site morbidity, risk of disease transmission, and poor vascularization. The invention also includes a 3D-printed scaffold with controlled porosity, further promoting bone regeneration and vascularization. Additionally, the invention provides a method for preparing the doped CDA material via controlled precipitation and calcination. This innovative bone graft material has broad clinical applications in periodontology, orthopedics, and tissue engineering.

[028] The bone graft material of the present invention is composed of calcium-deficient apatite (CDA) doped with at least one divalent cation selected from Sr²⁺ and Mg²⁺. The CDA comprises both hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) phases. The incorporation of Sr²⁺ and Mg²⁺ ions into the CDA material enhances both osteogenesis and angiogenesis, promoting bone regeneration and vascularization. The molar ratio of (Ca + Mg + Sr) / P in the CDA can be between 1.60 and 1.80. This unique composition and controlled molar ratio contribute to the enhanced osteogenic and angiogenic properties of the bone graft material.

[029] The 3D-printed scaffold of the present invention incorporates the bone graft material with controlled porosity. This scaffold provides a structural framework for bone regeneration and allows for the controlled release of the doped CDA material. The controlled porosity of the scaffold facilitates cell attachment, proliferation, and vascularization, further enhancing the effectiveness of the bone graft material. The 3D printing process allows for the fabrication of scaffolds with various internal architectures, including linear, round, oval, and 100% infill, providing flexibility in matching the specific requirements of the bone defect being treated.

[030] The method for preparing the doped CDA material involves controlled precipitation and calcination. The controlled precipitation process ensures the consistent production of high-quality CDA powder with the desired composition and properties. The calcination process further refines the material and enhances its bioactivity. This method is scalable and reproducible, making it suitable for commercial production. The sintering of the 3D scaffold at a temperature of about 1150°C enhances its mechanical properties, ensuring its stability and integrity during implantation.

[031] The bone graft material of the present invention has broad clinical applications in various medical fields, including periodontology, orthopedics, and tissue engineering. In periodontology, the material can be used to treat periodontal defects and promote bone regeneration around teeth. In orthopedics, the material can be used to repair bone fractures and defects, and to promote the integration of bone implants. In tissue engineering, the material can be used to create scaffolds for the growth and differentiation of bone cells, leading to the development of new bone tissue. The versatility of this material makes it a valuable tool for addressing a wide range of bone regeneration challenges.

[032] The present invention offers several advantages over existing bone graft technologies. The doped CDA material enhances both osteogenesis and angiogenesis, leading to improved bone regeneration outcomes. The 3D-printed scaffold with controlled porosity provides a structural framework for bone regeneration and allows for the controlled release of the doped CDA material. The method for preparing the doped CDA material is scalable and reproducible, ensuring the consistent production of high-quality bone graft material. Additionally, the invention has broad clinical applications in various medical fields, making it a valuable tool for addressing a wide range of bone regeneration challenges.

Composition:
[033] The bone graft material consists of calcium-deficient apatite doped with Sr²⁺ and Mg²⁺. The CDA structure features HA and β-TCP phases to balance mechanical stability with bio resorption.

Table 1:
[034] Table 1 discloses the compositions of the prepared CDA powder samples, including varying concentrations of Sr²⁺ and Mg²⁺ dopants, Ca/P ratios, and corresponding phases.

[035] Further, Table 1 provides a summary of the prepared sample powder compositions, detailing the molar concentrations of precursors used in the synthesis process and the resulting Ca/P and (Ca + Mg + Sr)/P molar ratios. The table includes three sample codes: CSM 0, CSM 0.03, and CSM 0.06, representing different compositions with varying concentrations of Mg²⁺ and Sr²⁺ dopants.

[036] Further, the data in Table 1 demonstrates the ability to control the composition of the CDA material by adjusting the molar concentrations of the precursors. This control allows for the fine-tuning of the material's properties, such as its bioactivity and resorption rate, to meet the specific needs of various bone regeneration applications. The varying Ca/P and (Ca + Mg + Sr)/P molar ratios achieved through these different compositions highlight the versatility of the synthesis process in tailoring the material's properties.
Molar Ratios:
Ca/P: 1.60 to 1.80
(Ca + divalent cations)/P: 1.50 to 2.00
Dopant Concentrations:
Sr²⁺ and Mg²⁺: 0.03 M to 0.06 M

Characterization:
[037] XRD Analysis: Reveals increased HA peak intensities with higher Sr²⁺ and Mg²⁺ doping concentrations.

[038] FTIR Spectroscopy: Confirms the presence of PO₄³⁻ bands characteristic of both HA and β-TCP phases.

[039] SEM Imaging: Displays crack-free, uniformly printed scaffolds with customizable internal architectures (linear, round, and oval porosity patterns).

3D-Printed Scaffold:
[040] Extrusion-based 3D printing utilizes a ceramic ink composed of CDA powder and hydroxypropyl methylcellulose. Parameters:

Printing Conditions:
[041] Infill density: 50% to 100%
[042] Scaffold dimensions: Customizable to patient-specific defects (e.g., 10 × 10 × 5 mm)
[043] Porosity: Designed to support cell migration and vascularization.
[044] Post-processing: Controlled sintering at 1150°C for elimination of the polymer binder and increase the mechanical integrity.

Method of Preparation:
In one embodiment, the method for preparing a doped calcium-deficient apatite (CDA) material comprises the following steps:
[045] Solution Preparation: Dissolving calcium nitrate, strontium nitrate, and magnesium nitrate in distilled water to form a precursor solution.

[046] Phosphate Addition: Introducing a phosphate solution dropwise into the precursor solution under continuous stirring at approximately 300 rpm, while maintaining a reaction temperature of about 90°C.

[047] pH Adjustment: Adjusting the pH of the reaction mixture to approximately 9 using 0.5 M ammonium hydroxide (NH₄OH) to facilitate controlled precipitation.

[048] Precipitate Isolation: Isolating the precipitate by centrifugation at 5000 rpm, followed by sequential washing and drying at 120°C to remove residual impurities.

[049] Calcination: Subjecting the dried precipitate to a calcination process at a temperature of approximately 1150°C for 4 hours, thereby forming the doped calcium-deficient apatite (CDA) powder with enhanced structural and compositional properties.

[050] 3D Printing and Scaffold Fabrication: Preparing a ceramic ink from the synthesized CDA powder, followed by 3D printing of scaffolds with a controlled porosity architecture. The printed structures are then subjected to a sintering process to achieve the desired mechanical strength and bioactivity.

Examples:
[051] The examples demonstrate the various embodiments of the invention and the methods for preparing and using the bone graft material.
Example 1 – Composition Synthesis:
[052] A CDA sample with a Ca/P ratio of 1.70 and dopant levels of 0.05 M Sr²⁺ and Mg²⁺ was synthesized via a controlled precipitation method. The resulting material exhibited enhanced osteoblast activity and vascular endothelial cell proliferation, as evidenced by in vitro assays. This example demonstrates the ability to tailor the composition of the CDA material to optimize its biological properties for bone regeneration.
Example 2 – In Vitro Cytocompatibility:
[053] Human mesenchymal stem cells were cultured on the 3D-printed scaffold incorporating the bone graft material. The cells demonstrated improved cell adhesion and proliferation over seven days, as observed through microscopic imaging. Live/dead staining and Alamar Blue assays confirmed cell viability and metabolic activity, indicating the biocompatibility of the scaffold and its ability to support cell growth. This example highlights the cytocompatibility of the scaffold and its potential to promote bone regeneration by providing a suitable environment for cell attachment and growth.
Example 3 – Clinical Evaluation:
[054] The bone graft material was evaluated in a clinical study for the treatment of periodontal defects. The following clinical outcomes were observed at 6 months:
[055] Probing pocket depth (PPD) reduction: 4.53 mm (test group) vs. 3.93 mm (control group)
[056] Clinical attachment level (CAL) gain: 4.27 mm (test group) vs. 3.93 mm (control group)
[057] Intra-bony defect fill: 5.12 mm (test group) vs. 3.22 mm (control group)
[058] These results demonstrate the clinical efficacy of the bone graft material in promoting periodontal regeneration. The significant improvements in PPD reduction, CAL gain, and intra-bony defect fill highlight the potential of this material for clinical use in treating periodontal defects.

Clinical Applications
[059] The bone graft material of the present invention has broad clinical applications in various medical fields, including periodontology, orthopedics, and tissue engineering.

[060] In periodontology, the material can be used to treat periodontal defects and promote bone regeneration around teeth. In orthopedics, the material can be used to repair bone fractures and defects, and to promote the integration of bone implants. In tissue engineering, the material can be used to create scaffolds for the growth and differentiation of bone cells, leading to the development of new bone tissue.

Advantages of the Invention
[061] The present invention offers several advantages over existing bone graft technologies. The doped CDA material enhances both osteogenesis and angiogenesis, leading to improved bone regeneration outcomes. The 3D-printed scaffold with controlled porosity provides a structural framework for bone regeneration and allows for the controlled release of the doped CDA material. The method for preparing the doped CDA material is scalable and reproducible, ensuring the consistent production of high-quality bone graft material. Additionally, the invention has broad clinical applications in various medical fields, making it a valuable tool for addressing a wide range of bone regeneration challenges.

, Claims:1. A 3D-printed bone graft scaffold, comprising:
a calcium-deficient apatite (CDA) doped with at least one divalent cation;
wherein the CDA comprises both hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) phases;
wherein the scaffold is fabricated using additive manufacturing to achieve a controlled porosity internal architecture; and
wherein the scaffold is customized for bone grafting applications, exhibiting enhanced osteogenesis and angiogenesis to promote bone regeneration and vascularization.
2. A method of preparing the bone graft material of claim 1, comprising: (a) preparing an aqueous solution containing calcium nitrate, strontium nitrate, and magnesium nitrate; (b) adding a phosphate precursor solution dropwise under continuous stirring; (c) adjusting the pH to approximately 9; (d) isolating the resulting precipitate; and (e) calcining the precipitate to form the doped CDA powder.
3. The bone graft material of claim 1, wherein the molar ratio of calcium to phosphate (Ca/P) is between 1.60 and 1.80.
4. The bone graft material of claim 1, wherein the concentrations of Sr²⁺ and Mg²⁺ are each between 0.03 M and 0.06 M.
5. The 3D-printed scaffold of claim 2, wherein the infill density ranges from 50% to 100%.
6. The 3D-printed scaffold of claim 2, is sintered to 300°C at a heating rate of 2 to 5°C per minute for removing the binder and sintered to 1150°C ranging from 2 to 4 hours at a heating rate of 5°C to 10°C per minute.
7. The method of claim 3, wherein the stirring rate is maintained at approximately 300 rpm and the temperature at about 90°C.
8. The method of claim 3, further comprising sintering the synthesized and dried powder at approximately 1150°C ranging from 2 to 4 hours.
9. The bone graft material of claim 1, wherein at least one divalent cation is selected from the group consisting of Sr²⁺, Mg²⁺, or combinations thereof.
10. The bone graft material of claim 1, wherein the molar ratio of (Ca + divalent cations) to phosphorus [(Ca + divalent cations)/P] is between 1.50 and 2.00.