A 3D printable wild animal bone composite

Abstract

The application discloses a kind of composite materials for 3D printing wild animal skeleton, which is composed of composite nanoparticles of calcium phosphate and hydroxyapatite, and is prepared by a combined method of biomimetic in-situ synthesis-solvent thermal phase inversion-spray drying method.The composite material has high specific surface area and excellent fluidity, and can be used for 3D printing wild animal skeleton, with broad market application prospect.

Description

Technical Field

[0001] This invention relates to the field of materials, and more specifically to a composite material that can be used for 3D printing of wildlife skeletons. Background Technology

[0002] Wildlife populations are a vital component of Earth's ecosystems, and their survival and health directly impact the maintenance of biodiversity and the stability of ecological balance. However, with the continuous expansion of human activity, intensified habitat fragmentation, and the effects of climate change, the survival threats faced by wild animals are becoming increasingly severe. Especially for rare and endangered species, skeletal damage often means reduced hunting ability, limited mobility, and even permanent disability. Without timely and effective intervention, it can directly lead to individual death and irreversible loss of genetic resources. However, currently, there is a severe shortage of materials for repairing skeletal defects in wild animals. Clinically, bone repair materials from the human medical field are often directly borrowed, such as metal implants or commercial bioceramics. While these materials can meet the needs for morphological filling to some extent, they differ significantly from the mechanical properties, metabolic characteristics, and healing cycles of wild animal bones. Metal implants are prone to stress shielding effects, leading to atrophy of the bone tissue around the implant; traditional commercial ceramic materials have uncontrollable degradation rates, failing to match the vastly different bone regeneration rates among wild animal species, and their insufficient bioactivity makes efficient osseointegration difficult. More importantly, the morphology of skeletal defects in wild animals is complex and varied, making it impossible to achieve personalized matching with standardized implants. While the introduction of 3D printing technology has made personalized customization possible, existing printing powder materials generally suffer from poor flowability, insufficient mechanical properties after sintering, and limited bioactivity, making it difficult to meet the stringent requirements of implants for wild animals' high activity levels and complex living environments. Therefore, developing a novel bone repair material that combines high printability, controllable degradation behavior, and excellent bioactivity has become an urgent need for the medical treatment and protection of wild animals.

[0003] In recent years, with the deep integration of nanotechnology and additive manufacturing in the biomedical field, significant progress has been made in the research of calcium phosphate-based biomaterials. Hydroxyapatite, as a major component of the inorganic phase of bone, is widely used in bone repair due to its excellent biocompatibility and osteoconductivity; however, its slow degradation in vivo often hinders the ingrowth and remodeling of new bone tissue. While biodegradable calcium phosphate materials such as tricalcium phosphate have relatively fast degradation rates, their bioactivity is relatively limited, and their degradation products may cause local pH fluctuations. To overcome the limitations of single components, researchers have begun to explore the combination of hydroxyapatite and biodegradable calcium phosphate to achieve synergistic regulation of degradation rate and osteogenic activity. Meanwhile, the introduction of nanotechnology has opened up new pathways for improving the bioactivity of materials: nanoscale calcium phosphate materials have higher specific surface area and surface energy, which can significantly enhance protein adsorption and cell adhesion. Their nanoscale topology can also simulate the physical signals of the natural bone matrix, activating osteogenic signaling pathways. However, the high surface energy of nanomaterials also brings the prominent problem of poor fluidity, making them difficult to directly apply to high-precision 3D printing. Therefore, preparing composite powders with high specific surface area, excellent flowability, adjustable degradation rate and high osteogenic activity, and applying them to personalized bone repair in wild animals, remains a core challenge that urgently needs to be overcome. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the prior art by proposing a composite material that can be used for 3D printing of wild animal skeletons.

[0005] A nanocomposite material that can be used for 3D printing of wild animal bones is characterized in that: the nanocomposite material is composed of composite nanoparticles of calcium phosphate and hydroxyapatite, and is prepared by a combined method of in-situ biomineralization synthesis, solvothermal phase inversion and spray drying.

[0006] The preparation method of this nanocomposite material is as follows:

[0007] (1) Preparation of calcium phosphate / bacterial complex by in-situ biomineralization synthesis

[0008] Tryptone (purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 91079-40-2), yeast extract (purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 8013-01-2), sodium chloride, and deionized water were weighed and mixed evenly to prepare a bacterial culture medium. The bacterial culture medium was then placed in an oven for heat sterilization to obtain a sterilized bacterial culture medium. Bacillus subtilis was then weighed and placed in the sterilized bacterial culture medium and cultured in a constant temperature shaker. After the culture was completed, the bacterial cells were collected by centrifugation and washed with deionized water to obtain pure bacterial cells.

[0009] Weigh out calcium chloride, sodium hydrogen phosphate, magnesium chloride, and deionized water and mix them evenly to prepare a mineralization medium. Then, place the pure bacterial cells in the pre-cooled mineralization medium and incubate the reaction with shaking at low temperature. After the incubation reaction is completed, collect the calcium phosphate / bacterial cell complex by centrifugation and then freeze-dry it in a freeze dryer to obtain the pure calcium phosphate / bacterial cell complex.

[0010] (2) Preparation of calcium phosphate / hydroxyapatite composite oxide by solvothermal phase inversion method

[0011] The pure calcium phosphate / bacterial complex, calcium hydroxide, sodium hydroxide and deionized water obtained in step (1) are mixed evenly to form a mixed slurry. Then the mixed slurry is placed in a high-pressure hydrothermal reactor for hydrothermal reaction. After the reaction is completed, it is centrifuged, then washed with deionized water and dried. Finally, it is placed in a muffle furnace for low-temperature calcination to obtain calcium phosphate / hydroxyapatite composite oxide.

[0012] (3) Preparation of nanocomposite materials by spray drying

[0013] The calcium phosphate / hydroxyapatite composite oxide, chitosan, and deionized water obtained in step (2) are mixed and magnetically stirred to form a mixed slurry. Finally, the mixed slurry is granulated by spray dryer and dried to obtain a nanocomposite material.

[0014] In the above preparation method: the purchase number of Bacillus subtilis mentioned in step (1) is CGMCC 1.8801, and the mass ratio of tryptone, yeast extract, sodium chloride, Bacillus subtilis and deionized water is 1: (0.3~0.7): (0.5~2): (0.5~1): (600~800).

[0015] In the above preparation method: the temperature for heating sterilization in step (1) is 110~130℃, and the heating sterilization time is 30~90min; the culture temperature is 36~38℃, the culture time is 3~7d, and the shaking speed during culture is 100~200r / min; the centrifugation speed is 6000~9000r / min, and the centrifugation time is 10~20min.

[0016] In the above preparation method, the mass ratio of calcium chloride, sodium hydrogen phosphate, magnesium chloride, pure bacterial cells and deionized water in step (1) is 1: (2~3): (0.3~0.5): (3~6): (100~150).

[0017] In the above preparation method: the pre-cooling temperature of the mineralized culture medium in step (1) is 2~4℃, the temperature of the shaking incubation reaction is 2~4℃, the speed of the shaker during the shaking incubation reaction is 5~10r / min, the shaking incubation reaction time is 24~48h, the freeze-drying temperature is -50~-40℃, and the freeze-drying time is 48~96h.

[0018] In the above preparation method, the mass ratio of the pure calcium phosphate / bacterial complex, calcium hydroxide, sodium hydroxide and deionized water in step (2) is 1: (0.3~0.6): (0.1~0.3): (60~80).

[0019] In the above preparation method: the hydrothermal reaction temperature in step (2) is 180~200℃, the hydrothermal reaction time is 2~8h, the drying temperature is 80~100℃, the drying time is 6~8h, the low-temperature calcination temperature is 300~350℃, and the low-temperature calcination time is 1~2h.

[0020] In the above preparation method: the mass ratio of calcium phosphate / hydroxyapatite composite oxide, chitosan and deionized water in step (3) is 1:(0.05~0.10):(50~100); the magnetic stirring speed is 100~200r / min and the magnetic stirring time is 6~12h.

[0021] In the above preparation method: the inlet temperature of the spray dryer during granulation in step (3) is 160~180℃, the drying temperature is 80~100℃, and the drying time is 2~4h.

[0022] Beneficial effects:

[0023] (1) In order to ensure the uniform bonding of nano-calcium phosphate on the template and lay the structural basis for subsequent reactions, thereby avoiding the problem of uneven morphology of amorphous calcium phosphate prepared by conventional methods when synthesizing hydroxyapatite, this invention first selects Bacillus subtilis to expand in conventional culture medium, and then biomineralizes it in mineralized culture medium. The negatively charged carboxyl functional groups on the cell surface of Bacillus subtilis adsorb positively charged calcium ions in the mineralized culture medium. When the phosphate ions in the mineralized culture medium diffuse to the cell surface, the local concentration of calcium ions and phosphate ions will exceed their solubility product, thereby forming amorphous calcium phosphate in situ using the cell surface as a template for heterogeneous nucleation.

[0024] (2) Since calcium phosphate degrades too quickly in vivo and cannot provide long-term support, and hydroxyapatite degrades too slowly and can easily affect bone growth, this invention uses a solvothermal phase inversion method to prepare calcium phosphate / hydroxyapatite composite oxide. By coupling the synergistic effect of calcium phosphate and hydroxyapatite, the problem of mismatch between degradation rate and osteogenic rate cannot be solved by a single component. Therefore, this invention mixes pure calcium phosphate / bacterial complex, calcium hydroxide, sodium hydroxide and deionized water to form a mixed solution, and then performs a high-temperature hydrothermal reaction to form calcium phosphate / hydroxyapatite composite oxide. Calcium hydroxide mainly supplements the calcium ion concentration of calcium phosphate converted to hydroxyapatite, and sodium hydroxide mainly adjusts the pH value of the mixed solution. Finally, by controlling the composition of the mixed solution, the hydrothermal reaction temperature and the hydrothermal reaction time, the kinetics of dissolution of amorphous calcium phosphate and crystallization of hydroxyapatite are controlled, so as to realize the controllable degradation rate of nanocomposite material as 3D printed medical bone.

[0025] (3) Since nano-calcium phosphate and nano-hydroxyapatite are prone to problems such as poor flowability and uneven powder spreading, this invention uses spray drying technology to break the slurry containing nano-composite particles into droplets through an atomizer. In the drying tower, the droplets come into contact with hot air, and the surface moisture evaporates instantly. Under the action of surface tension, the solid particles in the droplets are compressed inward and spontaneously shrink to obtain micro / nano secondary particles, thereby solving the contradiction between high specific surface area and high flowability required for nano-composite powder as 3D printed medical bone.

[0026] (4) Traditional 3D printed bone materials are made by mechanically mixing pre-synthesized hydroxyapatite powder and calcium phosphate powder, which is difficult to distribute evenly. This invention achieves chemical composite of hydroxyapatite and calcium phosphate at the nanoscale through two steps: in-situ synthesis of biological template and solvothermal phase transformation. In addition, by precisely controlling the solvothermal conditions, the ratio of hydroxyapatite and calcium phosphate microstructure units is designed. Calcium phosphate is responsible for providing calcium phosphate ions and activating osteogenic formation, while hydroxyapatite is responsible for long-term support, thus achieving a match between the 3D printed medical bone and the regeneration rate of the patient's own bone.

[0027] Therefore, the nanocomposite material prepared by this invention has a high specific surface area and excellent flowability, which can be used for 3D printing bone materials, and can regulate the degradation rate of bone implants in vivo, thus having strong application and promotion value. Attached Figure Description

[0028] Figure 1 This is a field emission scanning electron microscope image of the nanocomposite material prepared in Example 1. Detailed Implementation

[0029] The present invention will be further described below with reference to the embodiments. The embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0030] Example 1

[0031] (1) Preparation of calcium phosphate / bacterial complex by in-situ biomineralization synthesis

[0032] 20.000g of tryptone, 6.000g of yeast extract, 10.000g of sodium chloride, and 16000.000g of deionized water were weighed and mixed evenly to prepare a bacterial culture medium. The bacterial culture medium was then placed in an oven and sterilized at 110℃ for 90min to obtain a sterilized bacterial culture medium. 10.000g of Bacillus subtilis was weighed and placed in the sterilized bacterial culture medium, and cultured in a constant temperature shaker at 36℃ at 100r / min for 3 days. After the culture was completed, the bacterial cells were collected by centrifugation at 6000r / min for 10min and washed with deionized water to obtain pure bacterial cells.

[0033] Weigh 5.000g calcium chloride, 10.000g sodium hydrogen phosphate, 1.500g magnesium chloride, and 500.000g deionized water and mix them evenly to prepare a mineralization medium. Then, place 15.000g of pure bacterial cells in the mineralization medium pre-cooled at 2℃ and incubate the reaction at 2℃ with shaking for 48h (the shaking speed of the shaker during the incubation reaction is 10r / min). After the incubation reaction is completed, collect the calcium phosphate / bacterial cell complex by centrifugation and then place it in a freeze dryer and freeze dry at -50℃ for 48h to obtain pure calcium phosphate / bacterial cell complex.

[0034] (2) Preparation of calcium phosphate / hydroxyapatite composite oxide by solvothermal phase inversion method

[0035] Weigh 5.000g of the pure calcium phosphate / bacterial complex obtained in step (1), 1.500g of calcium hydroxide, 0.500g of sodium hydroxide, and 300.000g of deionized water and mix them evenly to form a mixed slurry. Then, place the mixed slurry in a high-pressure hydrothermal reactor and hydrothermally react at 180℃ for 8h. After the reaction is completed, centrifuge, wash with deionized water, dry at 80℃ for 8h, and finally calcine at 300℃ for 2h in a muffle furnace to obtain calcium phosphate / hydroxyapatite composite oxide.

[0036] (3) Preparation of nanocomposite materials by spray drying

[0037] Weigh 5.000g of the calcium phosphate / hydroxyapatite composite oxide obtained in step (2), 0.250g of chitosan, and 250.000g of deionized water, mix them, and magnetically stir at 100r / min for 12h to form a slurry. Finally, granulate the mixture using a spray dryer at an inlet temperature of 160℃, and dry it at 80℃ for 4h to obtain the nanocomposite material (FE-SEM image of the nanocomposite material is shown in Figure 1). Figure 1 (as shown)

[0038] (4) Specific surface area test

[0039] The specific surface area of ​​the composite material was tested and analyzed using an ASAP 2020M V3.00H specific surface area analyzer from the United States. 0.1g of the composite material was weighed and vacuum-treated at 150℃ for 6h. After degassing, nitrogen gas was introduced, and then the degassed sample tube was quickly transferred to the analysis station and fitted into a Dewar flask filled with liquid nitrogen. The instrument automatically controlled the quantitative injection of nitrogen gas into the sample tube, and the equilibrium adsorption amount under different relative pressures (P / P0) was measured to calculate the specific surface area of ​​the sample.

[0040] (5) Liquidity test

[0041] The nanocomposite powder is placed in a funnel fixed at a height of 10 cm and allowed to fall freely onto a horizontal circular platform, allowing the nanocomposite powder to accumulate naturally until the tip of the accumulation touches the opening of the funnel, forming a cone. The height (H) and the base radius (R) of the cone are measured, and the angle of repose is calculated.

[0042] Example 2

[0043] (1) Preparation of calcium phosphate / bacterial complex by in-situ biomineralization synthesis

[0044] Weigh 20.000g of tryptone, 14.000g of yeast extract, 40.000g of sodium chloride, and 12000.000g of deionized water and mix them evenly to prepare a bacterial culture medium. Then, place the bacterial culture medium in an oven and sterilize it at 130℃ for 30 minutes to obtain a sterilized bacterial culture medium. Weigh 20.000g of Bacillus subtilis and place it in the sterilized bacterial culture medium. Incubate it in a constant temperature shaker at 38℃ at 200r / min for 7 days. After the incubation is completed, collect the bacterial cells by centrifugation at 9000r / min for 20 minutes and wash them with deionized water to obtain pure bacterial cells.

[0045] Weigh out 5.000g of calcium chloride, 15.000g of sodium hydrogen phosphate, 2.500g of magnesium chloride, and 750.000g of deionized water and mix them evenly to prepare a mineralization medium. Then, place 30.000g of pure bacterial cells in the mineralization medium pre-cooled at 4℃ and incubate the reaction at 4℃ with shaking for 24h (the shaking speed of the shaker during the incubation reaction is 5r / min). After the incubation reaction is completed, collect the calcium phosphate / bacterial cell complex by centrifugation and then place it in a freeze dryer and freeze dry at -40℃ for 96h to obtain pure calcium phosphate / bacterial cell complex.

[0046] (2) Preparation of calcium phosphate / hydroxyapatite composite oxide by solvothermal phase inversion method

[0047] Weigh 5.000g of the pure calcium phosphate / bacterial complex obtained in step (1), 3.000g of calcium hydroxide, 1.500g of sodium hydroxide, and 400.000g of deionized water and mix them evenly to form a mixed slurry. Then, place the mixed slurry in a high-pressure hydrothermal reactor and hydrothermally react at 200℃ for 2h. After the reaction is completed, centrifuge, wash with deionized water, dry at 100℃ for 6h, and finally calcine at 350℃ for 1h in a muffle furnace to obtain calcium phosphate / hydroxyapatite composite oxide.

[0048] (3) Preparation of nanocomposite materials by spray drying

[0049] Weigh 5.000g of the calcium phosphate / hydroxyapatite composite oxide obtained in step (2), 0.500g of chitosan, and 500.000g of deionized water, mix them, and magnetically stir at 200r / min for 6h to form a mixed slurry. Finally, granulate the mixture by spray drying at an inlet temperature of 180℃ and dry at 100℃ for 2h to obtain the nanocomposite material.

[0050] (4) Specific surface area test

[0051] The specific surface area of ​​the composite material was tested and analyzed using an ASAP 2020M V3.00H specific surface area analyzer from the United States. 0.1g of the composite material was weighed and vacuum-treated at 150℃ for 6h. After degassing, nitrogen gas was introduced, and then the degassed sample tube was quickly transferred to the analysis station and fitted into a Dewar flask filled with liquid nitrogen. The instrument automatically controlled the quantitative injection of nitrogen gas into the sample tube, and the equilibrium adsorption amount under different relative pressures (P / P0) was measured to calculate the specific surface area of ​​the sample.

[0052] (5) Liquidity test

[0053] The nanocomposite powder is placed in a funnel fixed at a height of 10 cm and allowed to fall freely onto a horizontal circular platform, allowing the nanocomposite powder to accumulate naturally until the tip of the accumulation touches the opening of the funnel, forming a cone. The height (H) and the base radius (R) of the cone are measured, and the angle of repose is calculated.

[0054] Table 1 shows the specific surface area data of the nanocomposites prepared in Examples 1-2.

[0055] sample <![CDATA[Specific surface area (m 2 / g)]]> Example 1 65.3 Example 2 57.6 .

[0056] Table 2 shows the flowability data of the nanocomposites prepared in Examples 1 and 2.

[0057] sample Angle of repose (°) Example 1 23.1 Example 2 26.4 .

Claims

1. A nanocomposite material that can be used for 3D printing of wild animal bones, characterized in that: This nanocomposite material is composed of composite nanoparticles of calcium phosphate and hydroxyapatite, and is prepared by a combined method of in-situ biomineralization synthesis, solvothermal phase inversion and spray drying. The preparation method of this nanocomposite material is as follows: (1) Preparation of calcium phosphate / bacterial complex by in-situ biomineralization synthesis Weigh out tryptone, yeast extract, sodium chloride, and deionized water, mix them evenly to prepare a bacterial culture medium, and then place the bacterial culture medium in an oven to sterilize it to obtain a sterilized bacterial culture medium. Weigh out Bacillus subtilis and place it in the sterilized bacterial culture medium, and then incubate it in a constant temperature shaker. After the incubation is completed, collect the bacterial cells by centrifugation and wash them with deionized water to obtain pure bacterial cells. Weigh out calcium chloride, sodium hydrogen phosphate, magnesium chloride, and deionized water, mix them evenly to prepare a mineralization medium, then place the pure bacterial cells in the pre-cooled mineralization medium and incubate the reaction with shaking at low temperature. After the incubation reaction is completed, collect the calcium phosphate / bacterial cell complex by centrifugation, and then freeze-dry it in a freeze dryer to obtain the pure calcium phosphate / bacterial cell complex. (2) Preparation of calcium phosphate / hydroxyapatite composite oxide by solvothermal phase inversion method The pure calcium phosphate / bacterial complex, calcium hydroxide, sodium hydroxide and deionized water obtained in step (1) are mixed evenly to form a mixed slurry. The mixed slurry is then placed in a high-pressure hydrothermal reactor for hydrothermal reaction. After the reaction is completed, the mixture is centrifuged, washed with deionized water and dried. Finally, it is placed in a muffle furnace for low-temperature calcination to obtain calcium phosphate / hydroxyapatite composite oxide. (3) Preparation of nanocomposite materials by spray drying The calcium phosphate / hydroxyapatite composite oxide, chitosan, and deionized water obtained in step (2) are mixed and magnetically stirred to form a mixed slurry. Finally, the mixed slurry is granulated by spray dryer and dried to obtain a nanocomposite material.

2. The nanocomposite material for 3D printing wild animal skeletons according to claim 1, characterized in that: The purchase number of Bacillus subtilis mentioned in step (1) is CGMCC 1.8801. The mass ratio of tryptone, yeast extract, sodium chloride, Bacillus subtilis and deionized water is 1: (0.3~0.7): (0.5~2): (0.5~1): (600~800).

3. The nanocomposite material for 3D printing wildlife skeletons according to claim 1, characterized in that: In step (1), the temperature for heating sterilization is 110~130℃ and the time for heating sterilization is 30~90min; the temperature for incubation is 36~38℃ and the time for incubation is 3~7d, and the speed of the shaker during incubation is 100~200r / min; the speed of centrifugation is 6000~9000r / min and the time for centrifugation is 10~20min.

4. The nanocomposite material for 3D printing wild animal skeletons according to claim 1, characterized in that: In step (1), the mass ratio of calcium chloride, sodium hydrogen phosphate, magnesium chloride, purified bacterial cells, and deionized water is 1:(2~3):(0.3~0.5):(3~6):(100~150).

5. The nanocomposite material for 3D printing wildlife skeletons according to claim 1, characterized in that: In step (1), the pre-cooling temperature of the mineralized medium is 2~4℃, the temperature of the shaking incubation reaction is 2~4℃, the shaking speed of the shaker during the shaking incubation reaction is 5~10r / min, the shaking incubation reaction time is 24~48h, the freeze-drying temperature is -50~-40℃, and the freeze-drying time is 48~96h.

6. The nanocomposite material for 3D printing wildlife skeletons according to claim 1, characterized in that: In step (2), the mass ratio of pure calcium phosphate / bacterial complex, calcium hydroxide, sodium hydroxide, and deionized water is 1:(0.3~0.6):(0.1~0.3):(60~80).

7. The nanocomposite material for 3D printing wildlife skeletons according to claim 1, characterized in that: In step (2), the hydrothermal reaction temperature is 180~200℃, the hydrothermal reaction time is 2~8h, the drying temperature is 80~100℃, the drying time is 6~8h, the low-temperature calcination temperature is 300~350℃, and the low-temperature calcination time is 1~2h.

8. The nanocomposite material for 3D printing wildlife skeletons according to claim 1, characterized in that: In step (3), the mass ratio of calcium phosphate / hydroxyapatite composite oxide, chitosan, and deionized water is 1:(0.05~0.10):(50~100); the magnetic stirring speed is 100~200 r / min, and the magnetic stirring time is 6~12 h.

9. The nanocomposite material for 3D printing wildlife skeletons according to claim 1, characterized in that: In step (3), the inlet temperature of the spray dryer during granulation is 160~180℃, the drying temperature is 80~100℃, and the drying time is 2~4h.

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