Method for preparing 3D printing ink by extracting hydroxyapatite from skeletal cells

Abstract

The invention discloses a method for preparing 3D printing ink from skeleton-derived hydroxyapatite, efficient extraction and printing application of natural HA are realized through skeleton pretreatment, decellularization, nanocrystallization and composite ink preparation technologies, and the method has environmental protection property, emotional value and artistic innovation.

Description

Technical Field

[0001] The present invention belongs to the cross - field of biocompatible material preparation and 3D printing technology, and particularly relates to a method for extracting hydroxyapatite (HA) from bone cells and preparing it into 3D printing ink for manufacturing personalized human figures, commemorative items and bio - artworks. Background Art

[0002] 1. Limitations of the prior art:

[0003] Traditional 3D printing materials (such as plastics and resins) have problems such as poor biocompatibility, non - degradability, and lack of ecological significance.

[0004] Hydroxyapatite is the main inorganic component of bone, with excellent biocompatibility and mechanical properties. However, existing technologies mostly rely on chemical synthesis or mineral extraction, which are costly and lack personalized sources.

[0005] Currently, there is no technology that uses the bones of the deceased or animals to extract HA and directly apply it to 3D printing, which limits its application in the fields of souvenirs and artworks.

[0006] 2. Market demand:

[0007] The demand for personalized souvenirs is increasing, and an environmentally friendly material with emotional value is needed;

[0008] Bio - artworks need to combine natural aesthetics and sustainability, and animal bone resources can be utilized on a large scale.

[0009] Object of the Invention

[0010] 1. Provide a method for extracting natural hydroxyapatite from bone cells to achieve efficient utilization of waste bone resources.

[0011] 2. Develop an HA - based ink suitable for 3D printing for manufacturing personalized human figures, commemorative items and bio - artworks.

[0012] 3. Promote the development of environmentally friendly 3D printing materials through the biocompatibility and degradability of natural HA. Summary of the Invention

[0013] 1. Technical Solution

[0014] The present invention provides a systematic method for extracting hydroxyapatite (HA) from bone cells and preparing 3D printing ink, covering key steps such as bone pretreatment, HA purification, nanosizing treatment, ink formulation design and printing process optimization. The following is a detailed description of the technical solution:

[0015] The core of the present invention includes the following steps:

[0016] Bone pretreatment: Clean the bone samples (from human or animal sources), removing soft tissues and impurities;

[0017] Decellularization and HA extraction: Remove organic components through a chemical-enzymatic method, retaining high-purity hydroxyapatite;

[0018] Nanometerization treatment: Grind the HA particles to the nanoscale (particle size 50 - 200 nm) to improve printing accuracy;

[0019] Ink formulation: Compound the nano-HA with biocompatible polymers (such as chitosan, gelatin), adjusting the rheological properties to meet the requirements of 3D printing;

[0020] Printing and post-treatment: Form through an extrusion 3D printer, and enhance the structural stability by low-temperature curing or sintering.

[0021] 2. Bone pretreatment

[0022] Purpose: Remove organic residues, impurities and potential contaminants in the bone samples to ensure the purity of HA extraction.

[0023] Specific steps (chemical cleaning):

[0024] Immerse the bone (from human or animal sources) in 5% hydrogen peroxide solution (H2O2) for 24 hours to degrade residual soft tissues and fat;

[0025] Use 0.5M NaOH solution for secondary treatment for 2 hours to neutralize acidic residues and remove pigments.

[0026] Mechanical treatment:

[0027] Use an ultrasonic cleaner (frequency 40 kHz, power 100 W) for 30 minutes to remove microscopic impurities on the bone surface;

[0028] Mechanically crush the bone into 1 - 3 mm particles to increase the contact area for subsequent reactions.

[0029] Drying:

[0030] Place the cleaned bone particles in a vacuum freeze dryer (-50 °C, 0.1 mbar) for 48 hours to avoid damaging the HA structure at high temperatures.

[0031] 3. Decellularization and HA extraction

[0032] Purpose: Thoroughly remove organic components (such as collagen) to obtain high-purity HA.

[0033] Core process (high-temperature sintering method):

[0034] Place the pretreated bone particles in a muffle furnace and heat them at a rate of 5 °C / min to 800 °C. Keep them at this temperature for 2 hours to completely carbonize the organic components.

[0035] After cooling, white porous HA powder (purity > 98%) is obtained.

[0036] Chemical-assisted purification:

[0037] Immerse the HA powder in 0.1 M dilute hydrochloric acid (HCl) (pH = 4) for 30 minutes to dissolve the residual carbonate impurities.

[0038] Use a centrifuge (12000 rpm, 30 minutes) to separate the HA precipitate, and wash it with deionized water until neutral.

[0039] Alternative method (for heat-sensitive samples):

[0040] Enzymatic hydrolysis-chemical combined method:

[0041] Treat with trypsin (0.25% concentration, 37 °C) for 24 hours to degrade collagen.

[0042] Combine with ethylenediaminetetraacetic acid (EDTA, 5% concentration) to chelate metal impurities other than calcium ions.

[0043] This step aims to remove the organic components (such as collagen and lipids) in the bone and extract high-purity hydroxyapatite (HA). The specific process is carried out according to the following levels:

[0044] 3.1 Core process: High-temperature sintering method

[0045] 3.1.1 Bone pretreatment

[0046] Degreasing treatment:

[0047] Immerse the crushed bone particles (1-3 mm) in a chloroform-methanol mixture (volume ratio 2:1), and shake them in a water bath at 60 °C for 6 hours to remove the residual fat.

[0048] Centrifuge (8000 rpm, 10 minutes) to separate the bone particles, and wash them 3 times with deionized water.

[0049] Deproteinization pretreatment:

[0050] Immerse the bone particles in a 1% sodium dodecyl sulfate (SDS) solution for 24 hours (stir at room temperature) to degrade the surface proteins.

[0051] 3.1.2 High-temperature sintering process

[0052] Sintering parameters:

[0053] Place the pretreated bone particles in an alumina crucible and put it into a muffle furnace.

[0054] Heat up to 800 °C at a rate of 5 °C / min and hold for 2 hours to ensure complete carbonization of the organic components;

[0055] Cool naturally to room temperature to obtain white porous HA powder.

[0056] Sintering optimization:

[0057] For heat-sensitive samples (such as thin bone slices), use stepwise heating (pre-burn at 200 °C for 1 hour → 500 °C for 1 hour → 800 °C for 1 hour) to reduce thermal stress cracks.

[0058] 3.1.3 Chemical-assisted purification

[0059] Pickling for impurity removal:

[0060] Immerse the HA powder in 0.1 M dilute hydrochloric acid (HCl) (pH = 4) for 30 minutes to dissolve the residual calcium carbonate (CaCO3);

[0061] Centrifuge (12000 rpm, 15 minutes) to collect the precipitate, and wash with deionized water until neutral (pH = 7 ± 0.2).

[0062] Alkaline washing for neutralization:

[0063] Use 0.5 M NaOH solution to neutralize the residual acid solution, and wash with water again after centrifugation.

[0064] 3.2 Alternative process: Enzymolysis-chemical combined method

[0065] 3.2.1 Enzymatic cell detachment

[0066] Enzymolysis conditions:

[0067] Immerse the bone particles in 0.25% trypsin solution (pH = 7.4, containing 5 mM CaCl2), and oscillate at a constant temperature of 37 °C for 48 hours;

[0068] Replace the enzymolysis solution every 12 hours to ensure sufficient degradation of collagen.

[0069] Terminate the reaction:

[0070] Add 10% fetal bovine serum (FBS) to terminate the enzyme activity, and centrifuge to remove the supernatant.

[0071] 3.2.2 Chemical demineralization and HA retention

[0072] Selective demineralization:

[0073] Treat the bone particles with 0.5 M EDTA solution (pH = 8.0) for 24 hours to chelate non-HA minerals (such as magnesium, strontium);

[0074] Centrifugally retain the precipitate (HA framework) and wash it 3 times with deionized water.

[0075] Ultrasonic-assisted purification:

[0076] Treat with 40 kHz ultrasonic waves for 30 minutes to separate loosely bound impurities.

[0077] 3.3 Quality control and testing

[0078] 3.3.1 Purity analysis

[0079] X-ray diffraction (XRD):

[0080] Detect the HA crystal phase structure, compare with the standard PDF card (JCPDS 09-0432), and calculate the crystallinity > 90%;

[0081] The intensity of impurity peaks (such as β-TCP, CaO) should be < 5%.

[0082] Thermogravimetric analysis (TGA):

[0083] The mass loss rate of residual organic matter is < 2% (heating to 1000 °C at a rate of 10 °C / min).

[0084] 3.3.2 Morphology and composition characterization

[0085] Scanning electron microscopy (SEM):

[0086] Observe the morphology of HA particles, and the porosity should be > 50% (a porous structure is beneficial for ink compounding);

[0087] The Ca / P molar ratio detected by energy dispersive spectrometer (EDS) is 1.67 ± 0.02.

[0088] Fourier transform infrared spectroscopy (FTIR):

[0089] Confirm the PO4 3- Characteristic peaks (560 cm -1 、600 cm -1 、1040 cm -1 ), and there is no collagen amide band (1640 cm -1 ).

[0090] 3.4 Process adaptability design

[0091] 3.4.1 Human bone treatment

[0092] Ethical compliance:

[0093] It is necessary to obtain the written consent of the donor and adopt double anonymous coding (separating the sample ID from the donor information);

[0094] Final product sterilization treatment (γ-ray irradiation, 25 kGy).

[0095] 3.4.2 Large-scale extraction of animal bones

[0096] Batch processing optimization:

[0097] Use a continuous centrifuge (processing capacity 50 L / h) to replace traditional batch centrifugation;

[0098] Establish a bone source database to record the Ca / P ratio differences (±0.05) of HA from bovine, porcine, and fish bones.

[0099] 3.4.3 Technical effects

[0100] Extraction efficiency: The extraction rate of HA by the high-temperature sintering method is >98% (human bones) and 95% (animal bones);

[0101] Energy consumption control: The stepped heating process reduces energy consumption by 15%;

[0102] Safety: The final HA powder is sterile (<1 CFU / g), and the heavy metal residues (Pb, Cd) are <1 ppm.

[0103] 4. Purpose of HA nanosizing treatment: Refine HA particles to the nanoscale (50 - 200 nm) to improve printing resolution and material uniformity.

[0104] Method:

[0105] Wet ball milling:

[0106] Mix HA powder with zirconia grinding balls (diameter 3 mm) at a mass ratio of 1:10, and add anhydrous ethanol as a dispersion medium;

[0107] The ball mill runs at 300 rpm for 24 hours to obtain a nano-HA suspension.

[0108] Spray drying:

[0109] Prepare the suspension into dry nano-HA powder through a spray dryer (inlet temperature 180 °C, outlet temperature 80 °C). Quality control:

[0110] Use a dynamic light scattering instrument (DLS) to detect the particle size distribution to ensure that 90% of the particles are within the target range;

[0111] Observe the particle morphology by scanning electron microscopy (SEM) to avoid agglomeration.

[0112] This step aims to refine the extracted hydroxyapatite (HA) particles to the target size (nanoscale or microscale) to meet the resolution and performance requirements of different 3D printing applications. The specific process is carried out according to the following levels:

[0113] 4.1 Core processes of nanosizing treatment

[0114] 4.1.1 Wet ball milling for refinement

[0115] Equipment and parameters:

[0116] Use a planetary ball mill equipped with a zirconia grinding pot and grinding balls (diameter 3 - 5 mm);

[0117] Grinding medium: anhydrous ethanol (solid - liquid ratio 1:5) to prevent high - temperature denaturation of HA;

[0118] Rotation speed: 300 rpm, time 24 - 48 hours (adjusted according to the initial particle size).

[0119] Process control:

[0120] Pause for 10 minutes every 6 hours to avoid overheating (temperature in the pot < 40°C);

[0121] Adopt a circulating cooling system to maintain a constant grinding temperature.

[0122] 4.1.2 Spray drying to prepare nano - powder

[0123] Process parameters:

[0124] Input the ball - milled HA suspension into a centrifugal spray dryer;

[0125] Inlet temperature: 180 - 200°C, outlet temperature: 80 - 90°C;

[0126] Rotation speed of the atomizing disk: 15000 - 20000 rpm, controlling the droplet particle size to 20 - 50 μm.

[0127] Product characteristics:

[0128] Obtain dry nano - HA powder with a particle size range of 50 - 200 nm;

[0129] Specific surface area > 80 m 2 / g (measured by the BET method).

[0130] 4.1.3 Dispersion and stabilization treatment

[0131] Addition of dispersant:

[0132] Add 0.5% ammonium polyacrylate (PAA) during the ball - milling stage to prevent nanoparticle agglomeration;

[0133] Alternative: 0.3% sodium hexametaphosphate (SHMP).

[0134] Ultrasonic - assisted dispersion:

[0135] The spray-dried powder was resuspended in deionized water and sonicated for 30 minutes using 40 kHz ultrasound.

[0136] 4.2 Micronization Alternatives

[0137] 4.2.1 Jet Milling Method

[0138] Equipment Selection:

[0139] A fluidized bed jet mill was used with a compressed air pressure of 0.8 - 1.0 MPa;

[0140] The classifier wheel speed was 6000 - 8000 rpm, controlling the particle size to be 5 - 50 μm.

[0141] Energy Saving Optimization:

[0142] The milling air flow was recycled, reducing energy consumption by 20% (compared to traditional mechanical milling).

[0143] 4.2.2 Screening and Classification Process

[0144] Multi-stage Vibrating Screening:

[0145] Pass through 400 mesh (38 μm) and 800 mesh (18 μm) stainless steel sieves in sequence;

[0146] Collect HA particles in the target particle size range (such as 18 - 38 μm).

[0147] Electrostatic Precipitation:

[0148] Use a high-voltage electrostatic separator to remove ultrafine dust (<5 μm), improving the purity of the micron-sized product.

[0149] 4.2.3 Surface Modification (Optional)

[0150] Treatment with Silane Coupling Agent:

[0151] Immerse micron-sized HA in a 3-aminopropyltriethoxysilane (APTES) ethanol solution (1% concentration) and react at 60 °C for 2 hours;

[0152] Enhance the interfacial bonding force with the polymer matrix.

[0153] 4.3 Quality Control and Detection

[0154] 4.3.1 Detection of Nanoscale HA

[0155] Dynamic Light Scattering (DLS):

[0156] Measure the particle size distribution, requiring D90 < 200 nm and the polydispersity index (PDI) < 0.3;

[0157] Transmission Electron Microscopy (TEM):

[0158] Observe the grain morphology and confirm that there is no grain boundary fusion or abnormal growth.

[0159] 4.3.2 Micron-sized HA Detection

[0160] Laser diffraction method:

[0161] Use a Malvern particle size analyzer to detect the volume average particle size (D50) and span ((D90 - D10) / D50 < 1.5);

[0162] Tap density test:

[0163] The tap density of micron HA > 1.2 g / cm 3 (Compare with 0.6 - 0.8 g / cm of nano HA 3 )

[0164] 4.3.3 General Performance Indicators

[0165] Purity verification:

[0166] The proportion of the main peak intensity of HA detected by XRD > 95%;

[0167] Biological safety:

[0168] Cytotoxicity test of nano / micron HA extract (ISO 10993 - 5), cell viability > 85%.

[0169] 4.4 Process Adaptability Design

[0170] 4.4.1 Nano-Prioritized Scenario

[0171] High-precision printing:

[0172] For human facial details (50μm resolution) or microporous structures (pore diameter < 100μm);

[0173] Suitable for precision printers with nozzle diameters of 0.2 - 0.4 mm.

[0174] 4.4.2 Micron-Economical Solution

[0175] Large-scale art production:

[0176] Printing layer thickness of 0.3 - 0.5 mm, saving 30 - 50% of material cost;

[0177] Suitable for the large-scale production of HA for animal bones (the cost of bovine bone micron HA < 300 / kg).

[0178] Technical Effects

[0179] 1. Nano-sized HA:

[0180] The minimum feature size for printing is 50 μm, and the compressive strength is increased by 20% (compared with the micron level);

[0181] The specific surface area is increased by 3 times, enhancing the interfacial force of the polymer composite.

[0182] 2. Micron-sized HA:

[0183] The fluidity is improved (the angle of repose < 30°), suitable for high-speed printing (20 - 30 mm / s);

[0184] The environmental humidity sensitivity is reduced (the moisture absorption rate < 5%, compared with 15% for the nanoscale).

[0185] This hierarchical coding system clarifies the technical details of the dual paths of nanosizing and micronizing. Through multi-level process control and detection methods, it ensures the size accuracy and application suitability of HA powder, while covering the requirements of high precision and economy.

[0186] 5. Purpose of formulating 3D printing ink: To prepare printable ink with suitable rheological properties, mechanical strength, and biocompatibility.

[0187] Formulation design:

[0188] Substrate selection:

[0189] Natural polymers: Chitosan (excellent film-forming property), sodium alginate (fast ionic cross-linking), gelatin (low-temperature gelation);

[0190] Plasticizer: Glycerol (5 - 10% by volume) to adjust flexibility;

[0191] Cross-linking agent: Glutaraldehyde (0.1 - 0.5%) or Ca 2+ solution (for sodium alginate).

[0192] Composite process:

[0193] Mix the nano-HA powder and the polymer solution in a mass ratio of 1:2 to 1:4, and stir magnetically for 2 hours;

[0194] Add 0.5% defoaming agent (such as polydimethylsiloxane), and degas under vacuum for 30 minutes to avoid printing bubbles.

[0195] Rheological property optimization:

[0196] Test the viscosity through a rotational rheometer and adjust it to 500 - 1500 mPa·s (25 °C, shear rate 10 s-1) to suit the requirements of extrusion printing.

[0197] This step aims to compound nano-hydroxyapatite (HA) with a biocompatible polymer to form a 3D printing ink with suitable rheology, mechanical strength, and stability. The specific process unfolds in the following levels:

[0198] 5.1 Substrate Selection and Pretreatment

[0199] 5.1.1 Natural Polymer Screening

[0200] Chitosan Substrate:

[0201] Use chitosan powder with a deacetylation degree ≥ 85%, dissolve it in a 1-3% acetic acid solution (pH = 4.5-5.5), and prepare a 3% concentration colloid;

[0202] Advantages: Excellent film-forming property, and the curing speed can be regulated by humidity.

[0203] Sodium Alginate Substrate:

[0204] Dissolve food-grade sodium alginate in deionized water to form a 2-4% concentration solution (magnetic stirring for 2 hours, 60 °C);

[0205] Advantages: Fast ionic crosslinking, suitable for low-temperature printing.

[0206] Gelatin Substrate:

[0207] Use cold-water fish-derived gelatin (Bloom strength 250-300), and dissolve it into a 10% solution at 40-50 °C;

[0208] Advantages: Low-temperature gelation, suitable for bioactive loading.

[0209] 5.1.2 Preparation of Auxiliary Additives

[0210] Plasticizer:

[0211] Add 5-10% glycerol (volume fraction) to reduce the rigidity of polymer chains and improve the flexibility of the ink;

[0212] Alternative: Polyethylene glycol (PEG-400, 3-5% mass fraction).

[0213] Crosslinking Agent:

[0214] Chemical crosslinking: Glutaraldehyde (0.1-0.5% volume fraction), used for covalent crosslinking of chitosan-based inks;

[0215] Ionic crosslinking: Premix 5% CaCl2 solution (for post-treatment of sodium alginate-based inks).

[0216] Dispersant:

[0217] Add 0.5% sodium polyacrylate to prevent nano-HA particles from agglomerating.

[0218] 5.2 Composite Process and Homogenization Treatment

[0219] 5.2.1 HA / Polymer Mixing

[0220] Quality Ratio Control:

[0221] High-precision Formula: Nano-HA and polymer solution are mixed at a mass ratio of 1:2 to 1:4 (e.g., 1g HA + 3g chitosan colloid);

[0222] Gradient Test: Balance the compressive strength (5 - 15 MPa) and printing fluidity, and preferably select a ratio of 1:3.

[0223] Mixing Method:

[0224] Use a planetary mixer (rotation speed 200 rpm, 30 minutes) to ensure uniform dispersion of HA;

[0225] Operate light-sensitive materials (such as gelatin) using light-proof containers.

[0226] 5.2.2 Rheological Property Regulation

[0227] Viscosity Adjustment:

[0228] Add 0.1 - 0.3% carboxymethyl cellulose (CMC) to control the viscosity at 500 - 1500 mPa·s (25°C, shear rate 10s -1 );

[0229] Testing Instrument: Rotational rheometer (cone-plate mode, gap 0.1 mm).

[0230] Thixotropy Optimization:

[0231] Enhance the shear-thinning property through 0.2% fumed silica to ensure rapid shape recovery after ink extrusion.

[0232] 5.2.3 Defoaming and Filtration

[0233] Vacuum Defoaming:

[0234] Place the mixed slurry in a vacuum environment of -0.1 MPa for 20 minutes to remove microbubbles;

[0235] Detection Standard: No visible bubbles by visual inspection, or no cavitation signal by ultrasonic detection (40 kHz).

[0236] Fine Filtration:

[0237] Use a 200-mesh nylon filter to remove undispersed HA agglomerates and impurities.

[0238] 5.3 Ink Performance Verification

[0239] 5.3.1 Rheological Property Testing

[0240] Steady Shear Testing:

[0241] Measure the shear stress - shear rate curve and verify that it conforms to the Herschel - Bulkley model (flow index n < 1);

[0242] Target value: Yield stress 50 - 200 Pa, suitable for extrusion printing.

[0243] Oscillation Frequency Scanning:

[0244] Evaluate the elastic modulus (G’) and viscous modulus (G”) of the ink to ensure G’ > G” (dominated by solid - state behavior).

[0245] 5.3.2 Printing Compatibility Testing

[0246] Extrusion Experiment:

[0247] Use a standard 0.4 mm nozzle to test the continuous extrudability in the speed range of 5 - 15 mm / s;

[0248] Acceptance Criteria: No flow interruption, wire drawing or blockage.

[0249] Inter - layer Bonding Strength:

[0250] Print multi - layer test specimens (layer thickness 0.1 mm) and evaluate the inter - layer bonding strength (> 1 MPa) through tensile tests.

[0251] 5.3.3 Biocompatibility Detection

[0252] Cytotoxicity Testing:

[0253] According to the ISO 10993 - 5 standard, co - culture the ink extract with fibroblasts, and the cell survival rate > 90%;

[0254] Key Control Point: Glutaraldehyde residue < 0.01% (detected by HPLC).

[0255] 5.4 Ink Storage and Stability

[0256] Storage Conditions:

[0257] Chitosan - based ink: Store at 4℃ in the dark, with a shelf life of 7 days (adding 0.02% NaN3 for antibacterial);

[0258] Sodium alginate - based ink: Store sealed at room temperature, with a shelf life of 30 days (pH = 7.0 - 7.5).

[0259] Stability Monitoring:

[0260] Detect the viscosity change rate (within ±10%) weekly and observe the dispersion state of HA by SEM.

[0261] Technical effects:

[0262] Printing resolution: Nano-HA enables the minimum feature size to reach 50 μm (such as the details of a portrait's face);

[0263] Mechanical properties: The compressive strength after curing is 8 - 12 MPa (chitosan-based), 10 - 15 MPa (calcium alginate cross-linking);

[0264] Ecological compatibility: The natural degradation rate is >70% within 6 months (under soil burial conditions).

[0265] 6. 3D Printing and Post-processing

[0266] Printing parameters:

[0267] Equipment: Twin-screw extrusion 3D printer (nozzle diameter 0.2 - 0.6 mm);

[0268] Temperature control:

[0269] Chitosan-based ink: 25 - 30 °C (to prevent premature gelation);

[0270] Sodium alginate-based ink: 15 - 20 °C (requires Ca 2+ post-crosslinking).

[0271] Printing speed: 5 - 15 mm / s, layer thickness 0.1 - 0.3 mm.

[0272] Post-processing process:

[0273] Curing:

[0274] Chitosan ink: Place in an environment with 90% humidity for 24 hours to promote the self-assembly of polymer chains;

[0275] Sodium alginate ink: Immerse in 5% CaCl2 solution for 10 minutes to form an ionic crosslinking network.

[0276] Sintering enhancement (optional):

[0277] Perform low-temperature sintering on the pure HA structure (600 °C, 1 hour) to increase the compressive strength to over 20 MPa.

[0278] 7. Application Scenario Customization

[0279] Souvenirs: Combine the HA from the bones of the deceased to print miniature portraits or reliefs on urns, retaining the biological symbolic meaning,

[0280] Biological artworks:

[0281] Print a porous scaffold, implant plant seeds or microorganisms to form a "life-material" symbiont.

[0282] Utilize the osteoconductivity of HA to create degradable outdoor sculptures (such as gradually being absorbed by the soil).

[0283] 8. Summary of Technical Advantages

[0284] 8.1. Full-chain controllability: A complete process chain from bone processing to ink formulation ensures material traceability.

[0285] 8.2. High-precision adaptation: The synergy of nano-HA and natural polymers achieves a printing resolution of 50 μm.

[0286] 8.3. Green sustainability: The resource utilization of waste bones reduces the energy consumption of synthetic HA (carbon emissions reduced by 60%).

[0287] This technical solution solves the pain points of traditional 3D printing materials lacking emotional value and ecological friendliness through interdisciplinary innovation, providing a new solution for the fields of personalized souvenirs and bio-art.

[0288] 9. Originality

[0289] The present invention has significant innovation in technical path, material application and product design, specifically reflected in the following aspects: Source personalization: HA can be directly extracted from the bone cells of the deceased, endowing souvenirs with special emotional value.

[0290] Large-scale extraction: HA is extracted in batches from animal bones (such as cow and pig bones) to reduce material costs.

[0291] Composite ink design: The synergy of HA and natural polymers takes into account both printing fluidity and the mechanical strength of the finished product.

[0292] Application in bio-artworks: Utilize the biological activity of HA to create degradable or plant-symbiotic art installations.

[0293] 9.1. Raw material source and personalized design

[0294] Pioneering commemorative application of bone-derived HA:

[0295] It is first proposed to extract HA from the bones of the deceased and use it for 3D printing personalized souvenirs (such as miniature portraits, urn reliefs), endowing the material with emotional value and biological symbolic meaning, and solving the problem that traditional commemorative materials (stone, metal) lack life connection.

[0296] Through an ethical and compliant anonymization process and donor authorization mechanism, ensure that the technology can be legally promoted.

[0297] High-value utilization of animal bones on a large scale:

[0298] Develop a continuous processing process (defatting-demineralization-grading) for discarded bones from slaughterhouses, reducing the cost to 20% of chemically synthesized HA, breaking through the bottleneck of biomaterial economics.

[0299] 9.2. Process Innovation and Interdisciplinary Integration

[0300] Nano / micro dual-path precise control:

[0301] An innovative parallel technical solution of wet ball milling-spray drying (nanoscale) and air flow crushing-electrostatic classification (micrometer scale) is proposed to adapt to different printing precision requirements and improve the processing efficiency by 40% compared with a single particle size.

[0302] APTES surface modification was introduced to enhance the interfacial bonding between micron HA and polymer, solving the technical problem of easy sedimentation of large particle fillers.

[0303] Bio-artwork design:

[0304] It is the first to combine HA porous scaffolds with living organisms (moss, microorganisms), realize the dynamic artistic expression of "growth-decay" through controlled degradation, and expand the ecological interactivity of 3D printing materials.

[0305] 9.3. Breakthroughs in Ink Formulation and Printing Technology

[0306] Natural polymer composite system:

[0307] The innovative use of chitosan / sodium alginate-HA composite ink, through glycerol plasticization and ionic cross-linking to synergistically regulate rheological properties, takes into account both printing smoothness (viscosity 500-1500mPa·s) and mechanical strength of the finished product (>8MPa).

[0308] Compared with traditional light-curing resins, its biodegradability is improved by 100% (natural degradation rate within 6 months> 65%).

[0309] Low temperature curing process:

[0310] A humidity-induced chitosan self-assembly curing method (instead of high-temperature sintering) was proposed to avoid deformation of heat-sensitive structures. The finished product resolution reached 50μm, which is better than similar bio-inks (usually >100μm).

[0311] 10. Beneficial effects

[0312] 10.1. Technical performance advantages

[0313]

[0314] 10.2. Application Scenario Expansion

[0315] Emotional Value Area:

[0316] Provide high - value - added personalized souvenir solutions for the funeral industry. Market research shows that the user's willingness to pay has increased by 300% (compared with ordinary urns).

[0317] Sustainable art field:

[0318] Biological artworks can be applied to public art installations, such as "living sculptures" formed after HA scaffolds are implanted with moss.

[0319] Medical compatibility:

[0320] After sterilization treatment, the biocompatibility of human - sourced HA souvenirs meets the requirements of ISO 10993 - 5 standard, with potential expansion to bone repair auxiliary devices (such as personalized bone defect models).

[0321] 10.3. Environmental and social benefits

[0322] Resource recycling:

[0323] It can process over 100,000 tons of waste bones from slaughterhouses every year, reduce landfill pollution, and the carbon footprint is reduced by 60% (compared with the fossil raw material dependence of synthetic HA).

[0324] Cultural innovation:

[0325] Reshape the connotation of souvenirs through the concept of "life materials" and promote the cultural transformation of "green funerals".

[0326] Through the full - chain innovation of bone - sourced HA extraction - nano / micron dual - mode treatment - biocomposite ink design, the present invention solves the core pain points of traditional 3D printing materials in terms of emotional value, ecological friendliness and cost. Its originality lies in the revolutionary application of raw material sources, interdisciplinary optimization of processes and breakthroughs in product scenarios. The technical effects have both performance improvement and market differentiation, with significant commercial potential and social value. Specific implementation methods

[0327] Example 1: Preparation of souvenir ink from human - sourced HA

[0328] 11.1. Material preparation and pretreatment

[0329] 11.1.1 Source of bone samples and ethical compliance

[0330] Sample acquisition:

[0331] Use teeth or bone fragments provided by donors (approved by the ethics committee and anonymized);

[0332] Sample quality: The single - treatment amount is 10 - 50g, and it is stored dry at - 20°C.

[0333] Sterilization treatment:

[0334] Autoclaving (121 °C, 15 minutes) is adopted to ensure biological safety.

[0335] 11.1.2 Preparation of chemical reagents

[0336] Degreasing solution: Chloroform-methanol mixture (2:1 volume ratio), prepared and used immediately;

[0337] Decellularization reagent: 5% hydrogen peroxide (H2O2) solution, pH = 3.0 (adjusted with hydrochloric acid);

[0338] Pickling solution: 0.1M HCl solution (pH = 4.0), prepared using ultrapure water.

[0339] 11.2. Bone decellularization and HA extraction

[0340] 11.2.1 Degreasing and deproteinization

[0341] 11.2.1.1. Degreasing treatment:

[0342] Bone fragments are immersed in 200 mL of degreasing solution and shaken in a water bath at 60 °C for 6 hours;

[0343] Centrifuge (8000 rpm, 10 minutes), discard the supernatant, and repeat 3 times until the solution is colorless.

[0344] 11.2.1.2. Deproteinization treatment:

[0345] Transfer to 5% H2O2 solution and treat with ultrasonic assistance (40 kHz, 100 W) for 2 hours;

[0346] Rinse with deionized water until neutral and vacuum freeze-dry for 48 hours.

[0347] 2.2. High-temperature sintering purification

[0348] 11.2.2.1. Sintering parameters:

[0349] Muffle furnace program: 25 °C → 200 °C (5 °C / min), hold for 1 hour → 800 °C (5 °C / min), hold for 2 hours;

[0350] Cooling rate: ≤2 °C / min to prevent cracking of the HA lattice.

[0351] 11.2.2.2. Product collection:

[0352] The white HA powder is sieved through a 200-mesh sieve, weighed, and the extraction rate is recorded (expected > 95%).

[0353] 11.2.3. Chemical purification

[0354] 11.2.3.1. Pickling and impurity removal:

[0355] The HA powder is mixed with 0.1M HCl solution (solid-liquid ratio of 1:10) and magnetically stirred for 30 minutes;

[0356] Centrifuge (12,000 rpm, 15 minutes), and the supernatant is tested for Ca 2+ concentration (ion chromatography, <10 ppm).

[0357] 11.2.3.2. Neutralization and drying:

[0358] The precipitate is neutralized with 0.5M NaOH to pH = 7.0 and dried in a vacuum drying oven (60 °C, 24 hours).

[0359] 11.3. HA nanosizing treatment

[0360] 11.3.1 Wet ball milling process

[0361] 11.1. Ball milling parameters:

[0362] Tank body material: zirconia; grinding ball diameter 3 mm (ball-to-material ratio 10:1);

[0363] Medium: anhydrous ethanol (covering the powder surface by 2 cm), rotation speed 300 rpm, time 36 hours;

[0364] Temperature control: The circulating water cooling system maintains the temperature in the tank ≤ 35 °C.

[0365] 11.2. Dispersion and stability:

[0366] Add 0.5% PAA dispersant and perform ultrasonic treatment (40 kHz, 30 minutes) to eliminate soft agglomerates.

[0367] 11.3.2. Spray drying

[0368] 1. Drying parameters:

[0369] Feed rate: 5 mL / min, atomization pressure 0.3 MPa;

[0370] Inlet temperature 180 °C, outlet temperature 85 °C, collect nano-HA powder (water content < 2%).

[0371] 2. Particle size detection:

[0372] DLS test: D50 = 120 ± 20 nm, PDI = 0.25;

[0373] SEM observation: Sphericity > 90%, no hard agglomerates.

[0374] 4. Ink formulation and printing

[0375] 4.1 Ink formulation design

[0376] 1. Substrate selection:

[0377] Chitosan solution (3% concentration, dissolved in 1% acetic acid), viscosity reference value 800 mPa·s;

[0378] Nano-HA addition amount: 25% mass fraction (HA:chitosan = 1:3).

[0379] 2. Additives:

[0380] Plasticizer: 8% glycerol (volume fraction);

[0381] Defoamer: 0.5% polydimethylsiloxane.

[0382] 4.2 Mixing and homogenization

[0383] 1. Mixing process:

[0384] Mix HA and chitosan solution with a planetary mixer (200 rpm, 1 hour);

[0385] Vacuum degassing (-0.1 MPa, 30 minutes), filtering through a 200-mesh filter screen.

[0386] 2. Rheological properties:

[0387] Test with a rotational rheometer: viscosity at a shear rate of 10 s -1 is 950 ± 50 mPa·s;

[0388] Thixotropic recovery rate > 95% (test with 3 shear cycles).

[0389] 4.3 3D printing parameters

[0390] 1. Equipment configuration:

[0391] Extrusion printer (model: Bioplotter 3D), nozzle diameter 0.4 mm;

[0392] Printing temperature 25°C, cooling the platform to 10°C to prevent collapse.

[0393] 2. Printing path:

[0394] Layer thickness 0.1 mm, filling density 80%, speed 8 mm / s;

[0395] Support structure: water-soluble PVA material, removed by post-treatment dissolution.

[0396] 4.4 Post-curing treatment

[0397] 1. Humidity curing:

[0398] The printed material was placed in an environment with 90% humidity (saturated KNO3 solution) for 24 hours;

[0399] Compressive strength test: 12.5 ± 1.2 MPa (ASTM D695 standard).

[0400] 2. Surface treatment:

[0401] Spray a 0.1% chitosan coating and dry it at 60 °C to enhance the surface gloss.

[0402] Example 2: Preparation of art ink from animal bone-derived HA

[0403] 1. Large-scale extraction process

[0404] 1.1 Raw material pretreatment

[0405] 1. Source of animal bones:

[0406] Bovine femurs (waste from slaughterhouses), crushed into 1 - 3 cm 3 fragments;

[0407] Softened by high-pressure steaming (100 °C, 2 hours) and mechanically crushed into 1 - 3 mm particles.

[0408] 1.2 Continuous degreasing and demineralization

[0409] 1. Degreasing production line:

[0410] Processed by a continuous centrifuge (model: GEA QES-510), and the chloroform-methanol solution is recycled;

[0411] The processing capacity is 50 kg / h, and the fat residue is < 0.5%.

[0412] 2. Acid demineralization:

[0413] Dynamically soak in 0.5 M HCl (pH = 1.5, 4 hours) to remove non-HA minerals;

[0414] Online pH monitoring, automatically add acid solution to maintain the reaction efficiency.

[0415] 2. Preparation of micronized HA

[0416] 2.1 Jet milling and classification

[0417] 1. Milling parameters:

[0418] Jet pressure 0.9 MPa, classifier wheel speed 7000 rpm;

[0419] The product D50 = 25 ± 5 μm, span < 1.2 (laser diffraction method).

[0420] 2. Surface modification:

[0421] Soak in APTES ethanol solution (1% concentration) for 2 hours and dry at 60°C;

[0422] Contact angle test: Decrease from 75° to 45°, improving hydrophilicity.

[0423] 2.2 Screening and dust removal

[0424] 1. Vibration screening:

[0425] Use 400 mesh (38μm) and 800 mesh (18μm) in combination to collect particles with a size of 18 - 38μm;

[0426] Yield > 85%, tapped density 1.35 g / cm 3 .

[0427] 2. Electrostatic dust removal:

[0428] Adsorb ultrafine dust with a high - voltage electric field (15 kV), emission concentration < 1 mg / m 3 .

[0429] 3. Ink formulation and artistic application

[0430] 3.1 Sodium alginate - based ink

[0431] 1. Formula optimization:

[0432] Sodium alginate concentration 2.5%, micron HA addition amount 40% (mass ratio);

[0433] Cross - linker: Premix 0.1 M CaCl2 microspheres (particle size 50μm, slow - release cross - linking).

[0434] 2. Print adaptability:

[0435] Viscosity = 1200 ± 100 mPa·s (20°C), suitable for freeze printing (-10°C platform);

[0436] Layer thickness 0.3 mm, speed 15 mm / s, self - supporting design of the support structure.

[0437] 3.2 Biologized artistic design

[0438] 1. Porous structure printing:

[0439] Pore diameter 300 - 500μm, porosity 70%, implant moss spore suspension;

[0440] Cultivation conditions: Humidity > 80%, light 12 hours / day, survive in 7 days.

[0441] 2. Dynamic degradation test:

[0442] Exposed outdoors for 6 months, the mass loss rate is 65% (partial degradation of HA into Ca 2+ , PO4 3- ).

[0443] Quality control and verification data

[0444] 1. Key performance indicators

[0445]

[0446] 2. Basis of detection methods

[0447] Purity: XRD semi - quantitative analysis (JCPDS 09 - 0432);

[0448] Strength: Universal material testing machine (ASTM D695);

[0449] Degradation rate: Mass loss method (ISO 20200);

[0450] Biocompatibility: CCK - 8 cytotoxicity test (ISO 10993 - 5).

[0451] Technical advantages of the implementation method

[0452] 1. Precise control: Step - by - step sintering and real - time pH monitoring ensure high purity of HA;

[0453] 2. Flexible adaptation: Nano / micron dual paths meet different precision and cost requirements;

[0454] 3. Ecological closed - loop: Waste bone source → HA ink → Degradable artworks, sustainable throughout the chain.

[0455] This technology description clarifies the parameters and verification methods of each step through a hierarchical coding system, ensuring the industrial reproducibility of the process. At the same time, it covers ethical compliance, large - scale production, and innovative application scenarios, strengthening the practicality and protection scope of the patent.

[0456] Technical effects

[0457] 1. The purity of the extracted HA > 98%, and the printing accuracy of the ink can reach 50 microns;

[0458] 2. The compressive strength of the finished product reaches 10 - 15 MPa, meeting the usage requirements of daily souvenirs;

[0459] 3. The cost of extracting from animal bones is reduced to 1 / 5 of that of chemically synthesized HA, with commercial potential. Description of the drawings

[0460] Figure 1 : Flow chart of bone pretreatment

[0461] The figure shows the steps of bone samples undergoing mechanical crushing, source classification (human or animal), different pretreatment methods (ethical anonymization coding or batch cleaning and disinfection), degreasing, deproteinization, and vacuum freeze-drying, etc.

[0462] Figure 2 : Flow chart of HA extraction and purification

[0463] It presents the process flow of extracting HA from dry bone particles by high-temperature sintering or enzymatic hydrolysis method, then undergoing pickling purification, centrifugal washing to neutrality, and finally obtaining high-purity HA powder verified by XRD.

[0464] Figure 3 : Flow chart of HA particle size control

[0465] The process flow of HA powder, according to different target particle sizes (nanoscale or microscale), respectively adopting wet ball milling or air jet milling and other processes, and controlling the particle size through spray drying or electrostatic classification, and finally conducting particle size detection.

[0466] Figure 4 : Flow chart of ink formulation

[0467] The process of formulating ink after nano- or micro-HA is compounded with polymers and then enters the chitosan-acetic acid system or sodium alginate-water system, and undergoes steps such as adding glycerol or premixing CaCl2 microspheres, vacuum degassing, filtration, and viscosity testing.

[0468] Figure 5 : Flow chart of 3D printing application

[0469] The process path of HA ink, according to different application types (souvenirs or artworks), adopting different nozzles, printing temperatures, or cross-linking methods, and finally making products such as bone-derived portraits or biological scaffolds.

Claims

1. A method for preparing 3D printing ink by extracting hydroxyapatite (HA) from bone cells, characterized in that, It includes the following steps: Provide bone samples and perform degreasing and deproteinization pretreatment; Extract high-purity hydroxyapatite (HA) by high-temperature sintering or enzymatic-chemical combined method; Perform nanosizing or microsizing treatment on the extracted HA to obtain HA powder with the target particle size; Compound the HA powder with a biocompatible polymer to prepare 3D printing ink; Form by extrusion 3D printing technology and perform post-curing treatment.

2. The method according to claim 1, characterized in that The bone samples are derived from human or animal bones, and human bones need to be processed in accordance with ethical compliance and anonymously coded.

3. The method according to claim 1, wherein The process conditions of the high-temperature sintering method are: Raise the temperature to 800 °C at a heating rate of 5 °C / min, hold for 2 hours, and then cool naturally; The sintered HA powder is purified by pickling with 0.1M HCl to remove residual carbonate impurities.

4. The method according to claim 1, wherein The enzymatic-chemical combined method includes: Use 0.25% trypsin solution to enzymatically hydrolyze for 48 hours at 37 °C to remove collagen; Use 0.5M EDTA solution for selective demineralization to retain the HA framework structure.

5. The method according to claim 1, wherein The nanosizing treatment uses a wet ball milling process, and the specific parameters are: Zirconia grinding balls, ball-to-material ratio of 10:1, rotation speed of 300 rpm, grinding time of 24 - 48 hours; Prepare nano-HA powder by spray drying, with a particle size range of 50 - 200 nm.

6. The method according to claim 1, wherein The microsizing treatment uses a jet milling method, and the specific parameters are: Compressed air pressure of 0.8 - 1.0 MPa, classifier wheel rotation speed of 6000 - 8000 rpm; The final micron HA particle size ranges from 5 to 50 μm, and the tapped density > 1.2 g / cm 3 .

7. The method according to claim 1, characterized in that, The biocompatible polymer is selected from at least one of chitosan, sodium alginate or gelatin, and a plasticizer (5 - 10% volume fraction of glycerol) and a crosslinking agent (glutaraldehyde or CaCl2) are added.

8. The method according to claim 1, characterized in that, The viscosity of the 3D printing ink is 500 - 1500 mPa·s (25 °C, shear rate 10 s -1 ), suitable for extrusion printing, and the nozzle diameter is 0.2 - 0.6 mm.

9. The method according to claim 1, characterized in that The post-curing treatment includes: The chitosan-based ink is cured by humidity (90% humidity, 24 hours); The sodium alginate-based ink is crosslinked ionically (soaked in 5% CaCl2 solution for 10 minutes).

10. A 3D printed article, characterized in that, Printed with the HA-based ink prepared by the method described in any one of claims 1 - 9, and the products include personalized souvenirs, biological artworks or medical models. The key point to be protected is that the various functions of this patent cannot be split and disposed of by the separation method to simulate the achievable effect environment of this patent. The implementation manners of the present invention are not limited by the above embodiments. Any changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention. The specific implementation manners of the composite formulation of the present invention are not exhaustive, and any transformation by those skilled in the art without creative labor belongs to the scope of the protection of the rights of the present invention.

Images