Degradable polymer-based bone repair 3D printing composite material and preparation method and application thereof

Abstract

The application discloses a degradable polymer-based bone repair 3D printing composite material and a preparation method and application thereof, and belongs to the technical field of bone repair material preparation.The raw material of the degradable polymer-based bone repair 3D printing composite material comprises a degradable polymer, a compatibilizer and hydroxyapatite and Mg(OH)2 co-loaded chitosan microspheres.The degradable polymer-based bone repair 3D printing composite material has good mechanical properties, biocompatibility, mineralization capacity, antibacterial and anti-inflammatory properties, can be used as a bone repair material, and is used for preparing a bone repair product through 3D printing after being prepared into a bone repair 3D printing wire material through extrusion molding, thereby providing a novel material solution for bone tissue regeneration and repair.The chitosan can adjust macrophage polarization and reduce the expression of inflammatory factors, so that the composite material has anti-inflammatory properties.

Description

Technical Field

[0001] This invention relates to the field of bone repair material preparation technology, and in particular to a biodegradable polymer-based bone repair 3D printing composite material, its preparation method and application. Background Technology

[0002] In recent years, bone degeneration, fractures, osteoporosis, and wound healing problems have become increasingly common in animals and humans, leading to a significant increase in demand for bone repair and biomimetic scaffold-related implant materials. Among these, natural materials, such as natural hydroxyapatite extracted from recycled animal remains, have received considerable attention as promising materials. These emerging materials possess good biocompatibility, antibacterial properties, cell proliferation, and osteogenic potential. However, the relatively limited formability of these natural materials restricts their practical application in product manufacturing. To improve their mechanical and processing properties, integrating them into natural biodegradable polymers, then using extrusion molding technology to produce fused deposition modeling (FDM) 3D filaments, and subsequently using 3D printing technology to prepare bone repair products is a feasible direction. Researchers have used natural biodegradable polymers and hydroxyapatite to fabricate bone scaffolds and tested their performance in vitro with human osteoblasts, as well as in vivo to test their repair of skull defects in mice. The results showed that this composite scaffold enhanced osteoblast proliferation in vitro and promoted bone tissue regeneration in mice with skull defects.

[0003] Although composite systems of hydroxyapatite and biodegradable polymers possess bone repair capabilities, their various properties still need improvement (such as mechanical properties, ability to promote osteoblast growth and mineralization, etc.). To further expand the application range of bone engineering materials and enhance their applicability, it is essential to develop high-performance novel bone repair materials, especially novel bone repair materials for 3D printing of bone repair products. Summary of the Invention

[0004] The purpose of this invention is to provide a biodegradable polymer-based bone repair 3D printing composite material, its preparation method, and its application, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a biodegradable polymer-based bone repair 3D printing composite material, which, by weight, comprises: 80-95 parts of biodegradable polymer, 3-12 parts of compatibilizer, and 5-20 parts of chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2.

[0006] This invention employs a water-in-oil (W / O) emulsification technique to encapsulate hydroxyapatite and Mg(OH)₂ within chitosan microspheres, synthesizing chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)₂. These microspheres are then mixed with a biodegradable polymer and a compatibilizer to obtain a bone repair 3D printing composite material. Hydroxyapatite releases calcium and phosphate ions in body fluids, enhancing osteoblast activity and achieving biomineralization; Mg… 2+ The introduction of [a substance] can regulate the activity balance between osteoclasts and osteoblasts, inhibiting osteoclast activity and promoting osteoblast proliferation, thereby enhancing bone regeneration capacity; hydroxyapatite and Mg 2+ Synergistically, this composite material can enhance osteoblast activity while inhibiting osteoclast activity, thereby improving the mineralization capacity of bone cells. Chitosan imparts antibacterial and anti-inflammatory properties to the composite material. The groups in the compatibilizer can bond with the polar groups in hydroxyapatite and chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2, thus enhancing interfacial adhesion, effectively improving the mechanical properties of the composite material, and thereby regulating its degradation behavior. Furthermore, the chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 can effectively control the release of calcium, magnesium, and phosphorus, achieving long-term stable repair of bone damage. The bone repair 3D printing composite material of this invention possesses excellent mechanical properties, biocompatibility, mineralization capacity, antibacterial properties, and anti-inflammatory properties, and can be used as a bone repair material for 3D printing bone repair products, providing a novel material solution for bone tissue regeneration and repair.

[0007] Preferably, by weight, the raw materials include: 85-90 parts of biodegradable polymer, 5-8 parts of compatibilizer, and 10-15 parts of chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2.

[0008] Further, the biodegradable polymer includes one or more of polyhydroxyalkanoates (nPHA), polycaprolactone (PL), polylactic acid (PLA), polybutylene succinate (PBS), polylactic-glycolic acid copolymer (PLGA), and polybutylene adipate-terephthalate copolymer (PBAT), preferably polyhydroxyalkanoates.

[0009] Furthermore, the compatibilizer is maleic anhydride-grafted polyhydroxy fatty acid ester (abbreviated as nPHA-g-MA).

[0010] Furthermore, the grafting rate of maleic anhydride in the maleic anhydride-grafted polyhydroxy fatty acid ester is 0.5-2.0 wt%, preferably 0.8-1.0 wt%.

[0011] Furthermore, the preparation steps of the maleic anhydride-grafted polyhydroxy fatty acid ester include: adding maleic anhydride and benzoyl peroxide to molten polyhydroxy fatty acid ester, mixing, extruding and cooling the resulting melt to obtain the maleic anhydride-grafted polyhydroxy fatty acid ester.

[0012] Further, the amount of maleic anhydride added is 0.5-3.0 wt% of the mass of the polyhydroxy fatty acid ester, preferably 1.0-2.0 wt%; the amount of benzoyl peroxide added is 0.1-1.0 wt% of the mass of the polyhydroxy fatty acid ester, preferably 0.3-0.6 wt%.

[0013] Furthermore, the mixing temperature is 150-175℃ (preferably 160-170℃), the rotation speed is 40-80 rpm (preferably 60 rpm), and the time is 5-10 min (preferably 8 min).

[0014] Further, the preparation steps of the chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 include: mixing chitosan, natural hydroxyapatite, acetic acid solution and water to obtain an aqueous phase solution; mixing an emulsifier and paraffin oil to obtain an oil phase solution; adding the aqueous phase solution dropwise to the oil phase solution, heating and stirring to react, then cooling to 5-6℃, and then adding a crosslinking agent to carry out a crosslinking reaction to obtain the chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 (abbreviated as CHM).

[0015] Furthermore, the degree of deacetylation of the chitosan is 70-85%, preferably 75-80%.

[0016] Furthermore, the ratio of chitosan, natural hydroxyapatite, acetic acid solution, and water is 1.5-3.0g:0.1-0.3g:5-15mL:30-60mL, preferably 2.2g:0.17g:10mL:40mL.

[0017] Furthermore, the concentration of the acetic acid solution is 0.5-2.0 wt%, preferably 1 wt%.

[0018] Furthermore, the emulsifier is Span 80.

[0019] Furthermore, the volume ratio of the emulsifier to the paraffin oil is 1:40-70, preferably 1:55.

[0020] Furthermore, the crosslinking agent is a tripolyphosphate.

[0021] Furthermore, the volume ratio of the aqueous phase solution to the oil phase solution is 1:30-60, preferably 1:50-55.

[0022] Furthermore, the concentration of the crosslinking agent (specifically, the concentration of the crosslinking agent in the mixed system after its addition) is 1-5 wt%, preferably 3 wt%.

[0023] Adding a cross-linking agent can improve the stability of microspheres and slow down the release of magnesium, calcium, and phosphorus ions.

[0024] Furthermore, the heating and stirring reaction is carried out at a temperature of 50-70°C for 2-5 hours and at a stirring speed of 300-500 rpm.

[0025] Furthermore, the crosslinking reaction takes 20-60 minutes, preferably 35 minutes.

[0026] Furthermore, the crosslinking reaction is carried out at 5-25°C, preferably at 5-10°C.

[0027] Furthermore, the process of mixing chitosan, natural hydroxyapatite, acetic acid solution, and water further includes a step of stirring at 60°C for 3 hours.

[0028] Furthermore, the process of mixing the emulsifier and paraffin oil further includes a step of stirring at 60°C for 40 minutes.

[0029] Furthermore, the process after the crosslinking reaction is completed includes centrifugation, washing, and freeze-drying.

[0030] Furthermore, the freeze-drying includes: a pre-freezing temperature of -60 to -90°C for 6-12 hours; and a freeze-drying temperature of -40 to -60°C, a vacuum degree of ≤20Pa, and a time of 24-48 hours.

[0031] Furthermore, the natural hydroxyapatite is fish scale hydroxyapatite (i.e., extracted from fish scales), and the preparation steps include: cleaning the fish scales, heat-treating the cleaned fish scales at 1050℃ for 3 hours, and then grinding and sieving to obtain the fish scale hydroxyapatite (abbreviated as HMP).

[0032] Natural hydroxyapatite derived from fish byproducts exhibits more stable quality while reducing the potential risk of virus transmission. Furthermore, it possesses excellent properties that promote osteoblast proliferation and mineralization.

[0033] Furthermore, the chitosan is shrimp shell chitosan (i.e., prepared from shrimp shells). The preparation steps include: repeatedly soaking the shrimp shells in acid + rinsing + soaking in alkali + rinsing to obtain chitin; immersing the chitin in NaOH solution and heating it at 80°C for 12 hours, then drying and grinding it to obtain the shrimp shell chitosan.

[0034] The second technical solution of the present invention: a method for preparing the above-mentioned biodegradable polymer-based bone repair 3D printing composite material, comprising the following steps: mixing the biodegradable polymer, a compatibilizer, and chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 to obtain the biodegradable polymer-based bone repair 3D printing composite material.

[0035] The third technical solution of the present invention: a bone repair 3D printing filament, which is obtained by extrusion molding of the above-mentioned biodegradable polymer-based bone repair 3D printing composite material.

[0036] Furthermore, the diameter of the bone repair 3D printing filament is 1.75±0.05mm.

[0037] The fourth technical solution of the present invention: the application of the above-mentioned bone repair 3D printing filament in the 3D printing preparation of bone repair products or antibacterial coatings.

[0038] Furthermore, the bone repair product includes a biomimetic bone scaffold.

[0039] Furthermore, the 3D printing parameters include: a printing height of 0.025±0.008 mm per layer, an X-axis printing speed of 40-140 mm / s, and a Z-axis printing speed of 15-30 mm / s.

[0040] This invention extracts and processes chitosan powder from shrimp shells and natural hydroxyapatite from fish scales. Chitosan is then combined with hydroxyapatite microparticles (HMP), Mg(OH)2, and the crosslinking agent tripolyphosphate (TPP) to synthesize microspheres (CHM) using a W / O emulsification method. A complete spherical structure with approximately 67% crosslinking degree was observed at a TPP concentration of 3 wt%, and the release of magnesium, phosphorus, and calcium ions was delayed. To enhance the biomedical application of CHM, it was blended with nPHA and a compatibilizer to synthesize CPHA / CHM composite samples. These composite samples were then extruded into 3D printing filaments and further printed into bone scaffolds. Their release, structure, mechanical properties, cell compatibility, and mineralization capacity were characterized. The tensile strength at break of the CPHA / CHM sample was approximately 30-90 MPa higher than that of the nPHA / HMP sample. Cell compatibility tests showed that both nPHA / HMP and CPHA / CHM samples were non-toxic to mouse embryonic cells. Both CPHA / CHM and nPHA / HMP samples significantly promoted osteoblast proliferation. However, the nPHA / HMP sample continued to enhance osteoclast activity, while the CPHA / CHM sample showed a significant inhibitory effect on osteoclast activity. In simulated body fluid tests, CPHA / CHM showed better mineralization capacity than the nPHA / HMP sample. The CPHA / CHM sample exhibited significant antioxidant, anti-inflammatory, and antimicrobial growth properties, while nPHA / HMP did not possess these characteristics. This novel multifunctional CPHA / CHM product demonstrates excellent tensile properties, free radical scavenging ability, mineralization, anti-inflammatory effects, and the ability to promote osteoblast growth and reduce osteoclast growth. These characteristics make it highly suitable for future applications in bone tissue repair.

[0041] The present invention discloses the following technical effects: (1) This invention develops a biodegradable polymer-based bone repair 3D printing composite material. The raw materials include biodegradable polymer, compatibilizer, and chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2. It has good mechanical properties, biocompatibility, mineralization ability, antibacterial properties and anti-inflammatory properties. It can be used as a bone repair material. After being extruded into 3D printing filaments, it can be used to prepare bone repair products in 3D printing, providing a new material solution for bone tissue regeneration and repair.

[0042] (2) The CHM prepared by the W / O emulsification technology of this invention can effectively release calcium, magnesium and phosphorus ions, and the CPHA / CHM composite material formed with nPHA and compatibilizer exhibits excellent interfacial adhesion. Compared with nPHA / HMP, the Young's modulus of CPHA / CHM is increased by about 30-90 MPa.

[0043] (3) The CPHA / CHM sample prepared in this invention exhibits a slower degradation rate in PBS, demonstrating better structural stability. Furthermore, in in vitro mineralization experiments, the Ca / P mineralization layer generated by the CPHA / CHM sample closely resembles that of human bone tissue (Ca / P ratio approximately 1.6-1.8), showcasing excellent bone repair potential.

[0044] (4) The CPHA / CHM sample prepared in this invention promotes the proliferation and differentiation of osteoblasts, effectively inhibits the activity of osteoclasts, and has significant antibacterial activity against E. coli and S. aureus.

[0045] (5) The CPHA / CHM composite material prepared by the present invention exhibits excellent comprehensive performance in bone repair engineering and has great application potential. It can be used in the fields of bone scaffolds and antibacterial coatings. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 The following are schematic diagrams of the preparation process of chitosan from shrimp shells (a), the preparation process of hydroxyapatite from fish scales (b), the preparation process of chitosan microspheres (CHM) co-loaded with hydroxyapatite and Mg(OH)2 (c), and the preparation process of bone repair 3D printing filaments and the 3D printing of bone repair products using bone repair 3D printing filaments (d). Figure 2 This is a schematic diagram of the chemical reactions involved in the preparation of CHM. Figure 3 A schematic diagram of the chemical reaction process in which MA is grafted onto molten nPHA to prepare CPHA / CHM samples; Figure 4 The following are the analytical results of the surface morphology, composition and elemental distribution of CHM prepared under different crosslinking agent concentrations in Example 1. (a) and (b) are optical microscope and SEM images of CHM prepared at crosslinking agent concentrations of 0 wt%, 1 wt%, 3 wt%, and 5 wt%, respectively; (c) shows the elemental composition of the surface of CHM prepared under different crosslinking agent concentrations; and (d) is a mapping diagram of the distribution of Ca, P and Mg elements on CHM. Figure 5 XRD patterns of CHM (at 3wt% crosslinking agent concentration), chitosan, and chitosan-hydroxyapatite prepared in Example 1; Figure 6 The particle size distribution (a), crosslinking degree (b), swelling ratio (c), degradability (d), Mg release behavior (e), and Ca release behavior (f) of CHM prepared under different crosslinking agent concentrations are shown in the test results. Figure 7 XPS and XRD spectra of CPHA, nPHA / HPM, CPHA / CHM and raw material nPHA are shown, where (a) is the XPS spectrum of CPHA, nPHA / HPM 10wt%, CPHA / CHM 10wt% and raw material nPHA, and (b) is the XRD spectrum of nPHA / HPM 10wt%, CPHA / CHM 10wt% and nPHA. Figure 8 The results are shown in the tensile fracture test. (a)-(e) are scanning electron microscope (SEM) images of the tensile fracture surface of the nPHA(a), nPHA / HMP 10wt%(b), nPHA / HMP 20wt%(c), CPHA / CHM 10wt%(d), and CPHA / CHM 20wt%(e), respectively. (f)-(h) are the effects of HMP or CHM content on Young's modulus (E), tensile strength at break (δ), and elongation at break (ε), respectively. Figure 9 Results of water absorption test (a), in vitro degradation test (b), polysaccharide content test (c), and antioxidant activity test (d) for nPHA, CPHA, nPHA / HMP (10wt% and 20wt%), and CPHA / CHM (10wt% and 20wt%) samples; Figure 10 The results show the cell adhesion, cell cycle, and cell viability assays. (a) SEM images of NIH3T3 cells after 3 days of culture on control, nPHA, nPHA / HMP, and CPHA / CHM samples; (b)-(c) cell viability assay results for NIH3T3 and RAW-264.7 cells on control, nPHA, nPHA / HMP, and CPHA / CHM samples, respectively; (d) alkaline phosphatase assay results; and (e) calcium deposition assay results. Figure 11The results of the simulated body fluid assessment are as follows: (a)-(e) show the EDS spectra and SEM images of crystal deposition in simulated body fluid after immersion in simulated body fluid for 30 days for nPHA, nPHA / HMP 10wt%, nPHA / HMP 20wt%, CPHA / CHM 10wt%, and CPHA / CHM 20wt% samples; (f) shows the Ca / P ratio of the crystals as shown by EDS spectral analysis; (g) and (h) show the changes in the content of calcium ions and phosphate ions in the simulated body fluid solution for each sample at different immersion times; and (i) shows the changes in the pH value of the simulated body fluid solution for each sample at different immersion times. Figure 12 The results show the antibacterial and anti-inflammatory properties, where (a) is the IL-6 content in the cell culture supernatant; (b) and (c) are the growth rates of Escherichia coli and Staphylococcus aureus, respectively; and (d) is a photograph of bacterial growth on agar medium. Detailed Implementation

[0048] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0049] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0050] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0051] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0052] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0053] The biodegradable polymers described in this invention primarily serve as structural carriers and processing matrices for composite materials, enabling functions such as filler dispersion, interfacial bonding, and 3D printing. The bioactivity, mineralization capacity, antibacterial properties, and bone repair-related properties of the composite materials mainly originate from the introduced inorganic components and the resulting composite microstructure system. Therefore, although the following specific embodiments only verify the use of polyhydroxyalkanoates as biodegradable polymers, those skilled in the art will understand that, without departing from the technical concept of this invention, the composite system of this invention is not only applicable to polyhydroxyalkanoates but also to other biodegradable polymer matrix materials with melt processing capabilities, including but not limited to: polyhydroxyalkanoates, polylactic acid, polybutylene succinate, polycaprolactone copolymer, polylactic acid-glycolic acid copolymer, polybutylene adipate-terephthalate copolymer, and blends or modified systems of the above materials. When using the above-mentioned alternative matrix materials, the composite system of this invention can still achieve the loading, ion-regulated release, and bone repair functions of hydroxyapatite and microsphere structures, without affecting the realization of the core technical effects of this invention.

[0054] Unless otherwise specified, the room temperature mentioned in the following embodiments and test examples of this invention refers to 20-30°C.

[0055] The following embodiments of the present invention illustrate the preparation process of shrimp shell chitosan as shown in the schematic diagram. Figure 1 As shown in (a) above, the preparation process of fish scale hydroxyapatite is illustrated in the following diagram. Figure 1 As shown in (b) of the diagram, the preparation process of chitosan microspheres (CHM) co-supported with hydroxyapatite and Mg(OH)2 is as follows: Figure 1 As shown in (c), the process diagram for preparing bone repair 3D printing filaments and using the bone repair 3D printing filaments to 3D print bone repair products is as follows. Figure 1 As shown in (d) in the figure.

[0056] A schematic diagram of the chemical reactions in the preparation of CHM is shown below. Figure 2 As shown, a schematic diagram of the chemical reaction during the grafting of MA onto nPHA to prepare CPHA / CHM samples is presented. Figure 3 As shown. Figure 2 As shown, chitosan is first mixed with hydroxyapatite microparticles and Mg(OH)2 to form a composite system, and then microspheres are formed through an oil-in-water (W / O) emulsification method. Subsequently, a crosslinking agent, tripolyphosphate (TPP), is added to induce ionic crosslinking between the chitosan molecular chains, thereby obtaining structurally stable chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2. Figure 3As shown, maleic anhydride is first grafted onto the nPHA molecular chain under the action of an initiator to form nPHA-g-MA; then nPHA-g-MA is blended with nPHA to obtain a modified polyhydroxy fatty acid ester matrix (CPHA), which is further compounded with CHM microspheres to form a CPHA / CHM composite material with good interfacial bonding properties.

[0057] Except for the compatibilizer, all raw materials used in the following embodiments and test examples of this invention are common commercially available products, wherein: Natural polyhydroxyalkanoate (nPHA, EM 5400F) was purchased from Shenzhen Aikman Biotechnology Co., Ltd. Tripolyphosphate (TPP) specifically refers to sodium tripolyphosphate. All reagents used were of analytical grade purity.

[0058] The grafting rate of the compatibilizer (maleic anhydride-grafted polyhydroxy fatty acid ester, abbreviated as nPHA-g-MA) was 0.88 wt%. The specific preparation method was as follows: polyhydroxy fatty acid ester (nPHA) was placed in a mixer and melted at a temperature of 165℃, a speed of 60 rpm, and a time of 3 min; then maleic anhydride (MA) and initiator benzoyl peroxide (BPO) were added to the molten nPHA, wherein the amount of MA added was 1.5 wt% of the mass of nPHA, and the amount of BPO added was 0.5 wt% of the mass of nPHA; then, the mixing was continued for 8 min under the above temperature and speed conditions to complete the grafting reaction and obtain molten nPHA-g-MA; then the obtained nPHA-g-MA melt was extruded, cooled, and pelletized. The pelleted sample was washed with acetone and then vacuum dried at 60℃ to constant weight to obtain the final nPHA-g-MA.

[0059] In the following test cases, unless otherwise specified, all tests were repeated 5 times, and the results were taken as the average value.

[0060] Example 1 The preparation steps of chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 are as follows: (1) Preparation of fish scale hydroxyapatite (the preparation process diagram is shown in the figure) Figure 1 (as shown in (b)) Tilapia scales (0.11-0.22 mm thick, 10-15 mm long) were collected from the fish market. First, they were washed with clean water to remove surface impurities. Then, they were placed in a 0.1 M HCl solution and stirred at 350 rpm for 5 hours at room temperature to remove residual organic matter. Afterward, they were washed with a 0.1 M NaOH solution and rinsed with deionized water until the pH of the washing solution reached 7.0. The cleaned tilapia scales were then transferred to a high-temperature furnace and heat-treated at 1050 °C for 3 hours. Finally, they were ground and sieved to obtain fish scale hydroxyapatite powder (HMP) with a particle size range of 0.5-2.5 μm.

[0061] (2) Preparation of shrimp shell chitosan (the preparation process diagram is shown in the figure) Figure 1 (as shown in (a)) The shells of Litopenaeus vannamei were collected and first washed with deionized water to remove surface impurities. The washed shells were then soaked in a 2M HCl solution for 24 hours to remove minerals, followed by rinsing with deionized water until the pH of the washing solution reached 7.0. Next, the rinsed shells were soaked in a 0.5M NaOH solution for 24 hours to remove proteins, and then rinsed with deionized water until the pH of the washing solution reached 7.0. This acid soaking + rinsing + alkali soaking + rinsing process was repeated five times to obtain chitin.

[0062] The extracted chitin was then immersed in a 40 wt% NaOH solution and heated at 80 °C for 12 h to deacetylate and convert it into chitosan. The degree of deacetylation was determined by acid-base titration, and the obtained chitosan had a degree of deacetylation of 78%. It was then dried in a vacuum oven at 105 °C for 24 h. Finally, it was ground using a high-speed centrifugal grinder and sieved to obtain shrimp shell chitosan powder with a particle size of 100-200 μm.

[0063] (3) Preparation of chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 (the preparation process diagram is shown in the figure below) Figure 1 (as shown in (c)) 2.2 g of shrimp shell chitosan, 0.17 g of fish scale hydroxyapatite, 10 mL of acetic acid solution (1 wt%), and 40 mL of ultrapure water were mixed and stirred at 60 °C for 3 h to obtain an aqueous phase solution. 2 mL of emulsifier Span 80 and 110 mL of paraffin oil were mixed and stirred at 60 °C for 40 min to obtain an oil phase solution.

[0064] The aqueous phase solution was added dropwise to the oil phase solution at 60℃. After the addition was complete, the mixture was stirred at 60℃ for 1 hour to form emulsion droplets. The mixture was then rapidly placed in an ice bath to cool to 5-6℃, allowing the emulsion droplets to transform into gel particles. Then, TPP at concentrations of 0 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, or 5 wt% (TPP as a crosslinking agent; concentration refers to the concentration of TPP added to the mixture) was added, and the mixture was stirred at 5-6℃ for 35 minutes to carry out the crosslinking reaction. After the crosslinking reaction was completed, the solid precipitate was collected by centrifugation at 2100 rpm for 20 minutes. The precipitate was washed five times with acetone to remove excess residual oil phase material, and then freeze-dried (pre-frozen at -80℃ for 9 hours, then dried at -50℃ under a vacuum of ≤20 Pa for 36 hours to constant weight) to obtain the final chitosan microspheres co-supported with hydroxyapatite and Mg(OH)₂ (CHM). A total of six different CHM concentrations with different crosslinking agent concentrations were obtained.

[0065] Test Example 1 Characterization data of CHM Figure 4 The surface morphology, composition, and elemental distribution of CHMs prepared in Example 1 at different crosslinking agent concentrations are presented using optical microscopy, SEM, EDS, and mapping. (a) and (b) are optical micrographs and SEM images of CHMs with crosslinking agent concentrations of 0 wt%, 1 wt%, 3 wt%, and 5 wt%, respectively, illustrating the surface morphology of the CHMs. (a) and (b) show that the CHM without crosslinking agent does not possess a spherical structure but exhibits an unevenly sized and dispersed blocky structure. This is due to structural collapse and degradation after drying, resulting in the inability to effectively encapsulate hydroxyapatite and Mg(OH)2. The CHM with 1 wt% crosslinking agent formed a rough spherical structure, but the size distribution of the spherical particles was uneven, and a large number of fragments were observed due to collapse and degradation, indicating that the degree of crosslinking was insufficient to fully support the integrity of the microsphere structure. When the crosslinking agent concentration increased to 3 wt% and 5 wt%, CHM exhibited a smooth and complete spherical structure, with a more stable microsphere structure, more uniform particle size distribution, and significantly enhanced encapsulation of hydroxyapatite and Mg(OH)2. Furthermore, there was no significant performance difference between the crosslinking agent concentrations of 3 wt% and 5 wt%, indicating that a TPP content of ≥3 wt% achieved sufficient crosslinking to effectively support the formation and stability of the spherical structure. (c) shows the elemental composition of the CHM surface prepared at different crosslinking agent concentrations. EDS analysis detected C, N, O, P, Ca, and Mg elements, confirming that hydroxyapatite (calcium and phosphorus) and Mg(OH)2 (magnesium) were successfully loaded onto the chitosan microspheres. Moreover, with increasing TPP content, the distribution of these elements within the microspheres became more uniform, and local enrichment significantly decreased, indicating that a higher TPP content helps improve the uniform dispersion of P, Ca, and Mg elements within the microspheres. To further understand the distribution of hydroxyapatite and Mg(OH)₂ on chitosan microspheres, the distribution of Ca, P, and Mg elements in CHM prepared with different crosslinking agent concentrations was analyzed using mapping technology. As shown in (d), without the addition of a crosslinking agent or with only 1 wt% TPP, the surface of CHM showed significant aggregation of Ca, P, and Mg elements. However, when 3 wt% or more TPP was added, a higher degree of crosslinking occurred, and these elements were more uniformly distributed throughout the microspheres.

[0066] Figure 5The XRD patterns of CHM (at a 3 wt% crosslinking agent concentration), chitosan, and chitosan-hydroxyapatite prepared in Example 1 are shown. In the figures, Chitosan represents chitosan, and Chitosan-HMP represents chitosan-hydroxyapatite (the preparation method is the same as in Example 1, except that the use of Mg(OH)2 is omitted). The XRD patterns show the crystal structures of chitosan, chitosan-hydroxyapatite, and CHM. The XRD pattern of chitosan shows two peaks at 12.1° and 21.8°. Seven new peaks were observed in the chitosan-hydroxyapatite pattern, located at 25.7°, 32.0°, 34.3°, 39.8°, 46.1°, 50.7°, and 53.6°, respectively, which are attributed to the bonding of hydroxyapatite. Three new peaks were observed in the CHM pattern, located at 8.5°, 38.3°, and 58.6°, corresponding to Mg(OH)2.

[0067] Test Example 2 CHM Performance Tests (1) Particle size distribution Particle size distribution analysis was performed based on SEM test results.

[0068] (2) Crosslinking degree test of CHM Take 5 mg of microsphere samples prepared at different crosslinking agent concentrations, add 2 wt% rhodamine reagent, and heat in boiling water for 20 min. After cooling to room temperature, measure the wavelength at 570 nm using an enzyme-linked immunosorbent assay (ELISA) to characterize the free amine content. Calculate the degree of crosslinking based on the free amine content. Repeat the experiment 5 times for each sample, and take the average value. The degree of crosslinking is calculated using the following formula: Degree of crosslinking (%) = (1 − C a / C s )×100% Among them, C s C represents the free amine content of the sample before crosslinking (i.e., the sample with a crosslinking agent concentration of 0 wt%). a This represents the free amine content of the cross-linked sample.

[0069] (3) Swelling ratio test of CHM Microsphere samples prepared at different crosslinking agent concentrations were immersed in deionized water to test the swelling ratio (q). 150 mg of each microsphere sample prepared at different crosslinking agent concentrations was added to 3 mL of ultrapure water and allowed to stand at 37°C. Samples were taken every 1 hour, excess water was gently removed with absorbent paper, and the mass of the microspheres was measured to calculate the swelling ratio. Each measurement was repeated 6 times, and the average swelling ratio (q) was calculated. The formula for calculating the swelling ratio is: q=(W s -W d ) / W d Among them, W d W is the dry weight of the microspheres before soaking. s This represents the wet weight of the microspheres after soaking.

[0070] (4) Degradability test of CHM To observe the degradation behavior of microspheres prepared at different cross-linking agent concentrations, 2 mL of phosphate-buffered saline (1×PBS) was used as the degradation solution, and 10 mg / mL of lysozyme was added to the phosphate-buffered saline. Microspheres (0.02 g) prepared at different cross-linking agent concentrations were added to the degradation solution, and their weight loss was monitored every 6 days until day 36. The weight loss rate was calculated using the following formula: Weight loss rate (%) = [(M b -M a ) / W b ]×100% Among them, M b M represents the weight of the microspheres before soaking. a This represents the weight of the microspheres after soaking.

[0071] (5) Analysis of the release behavior of magnesium, calcium and phosphorus elements Add 0.15 g of microsphere sample to 10 mL of ultrapure water and let stand at room temperature. Take samples every 6 days until day 36. After each sampling, centrifuge the sample at 3200 rpm for 10 min, collect the supernatant, and determine the concentrations of magnesium, calcium, and phosphorus in the supernatant using inductively coupled plasma mass spectrometry (ICP-MS).

[0072] Figure 6 The particle size distribution, degree of crosslinking, swelling ratio, degradability, and release behavior of magnesium, calcium, and phosphorus elements in CHM prepared at different crosslinking agent concentrations are shown in the figures. (a) shows the particle size distribution, (b) the degree of crosslinking test results, (c) the swelling ratio test results, (d) the degradation rate test results, (e) the release of Mg, and (f) the release of Ca. (a) shows that when the TPP content is between 1-5 wt%, the microsphere particle size is uniformly distributed in the range of 50-250 μm. When the TPP content is between 3-5 wt%, the microsphere particle size is concentrated in the range of 100-150 μm. Notably, at a TPP content of 3 wt%, the microsphere particle size reaches approximately 178 ± 17 μm. (b) shows that for all CHM samples, the degree of crosslinking increases with increasing crosslinking agent concentration, but the rate of increase slows down when the crosslinking agent concentration reaches 3 wt%. Furthermore, (b) also shows that when the crosslinking agent concentration is 3 wt%, the degree of crosslinking of CHM is approximately 67.0 ± 2.4%, which is consistent with... Figure 4The SEM images show that a cross-linking agent concentration ≥3 wt% is required to achieve the desired degree of cross-linking to form a complete microsphere structure. (c) shows that the swelling ratio of each CHM sample increases rapidly with time within 1 hour of immersion in water. After 1 hour, the rate of increase gradually approaches equilibrium, and the CHM samples reach water saturation within 1 hour, after which the swelling ratio does not change significantly. (d) shows that the weight loss rate increases with time, with a rapid increase before 24 days, attributed to glucosamine and N-acetylglucosamine. The uncross-linked chitosan component is exposed to lysozyme attack, leading to rapid degradation. After 24 days, the degradation rate slows down due to the more stable cross-linked structure and less degradation. (e) and (f) show that when CHM samples containing different concentrations of cross-linking agent are immersed in aqueous solution, the release of Mg and Ca increases with time. This is similar to the trend observed in (d). Before 24 days, Mg and Ca are released rapidly, but the release rate gradually slows down after 24 days. Before 24 days, the non-crosslinked components degraded, leading to a rapid release of Mg and Ca. Conversely, after 24 days, the crosslinked components degraded slowly, resulting in a slow release of Mg and Ca. It was also observed that the release rate of Mg and Ca decreased significantly with increasing crosslinking agent concentration. This is because a higher degree of crosslinking restricts ion diffusion. In summary, the study shows that a higher crosslinking agent concentration results in a better crosslinking effect. However, considering the issue of residual crosslinking agent and the needs of subsequent purification processes, 3 wt% crosslinking agent is considered the optimal condition for preparing CHM in this invention.

[0073] Example 2 Fabrication of 3D-printed composite materials for bone repair, 3D-printed filaments for bone repair, and bone scaffolds (process flow diagram shown in figure). Figure 1 (as shown in (d)) Table 1 lists the specific composition of nPHA / HMP and CPHA / CHM samples with different compositions. nPHA, nPHA-g-MA, and CHM or HMP were added to a mixer in proportion and mixed (at 85°C and 60 rpm for 10 min) to obtain a bone repair 3D printing composite material. The composite material was fed into an extruder (PHM30), and the filament specifications were controlled by adjusting the screw speed and traction speed to obtain a bone repair 3D printing filament with a diameter of approximately 1.75 ± 0.05 mm.

[0074] 3D printing filament is loaded into a fused deposition modeling (FDM) 3D printer. The filament is melted by a heater (130°C) and extruded through a nozzle. The printing height of each layer is 0.025 mm, the printing speed in the XY direction is 80 mm / s, and the Z-axis movement speed is 20 mm / s. The spatial structure of the scaffold is controlled by stacking layers one by one, so that the pore size of the resulting three-dimensional scaffold is about 250 μm, thus forming a three-dimensional porous scaffold for bone repair with a regular pore structure.

[0075] Table 1 In Table 1, nPHA represents polyhydroxyalkanoate; nPHA-g-MA represents compatibilizer (maleic anhydride (MA) grafted polyhydroxyalkanoate); HMP represents fish-scale hydroxyapatite (prepared in Example 1); CHM represents chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 (CHM prepared in Example 1 at a crosslinking agent concentration of 3 wt%); CPHA represents compatibilizer-modified nPHA (polyhydroxyalkanoate with added compatibilizer nPHA-g-MA, i.e., nPHA plus nPHA-g-MA). The units for nPHA and HMP (or CHM) in wt% represent that the sum of their masses is considered as 100 wt%. The unit of nPHA-g-MA is phr, representing the amount of additional nPHA-g-MA added to a base of nPHA + HMP (or CHM) = 100 wt%. (For example, 10 phr of nPHA-g-MA means that the mass of nPHA-g-MA is 10% of the sum of the masses of nPHA and HMP (or CHM). The percentages in nPHA / HMP 5, 10, 15, and 20 wt% refer to the mass percentage of HMP in nPHA + HMP, and the percentages in CPHA / CHM 5, 10, 15, and 20 wt% also refer to the mass percentage of CHM in nPHA + CHM. The specific raw material composition of the CPHA / CHM 10 wt% sample is: 90 parts by mass of nPHA + 10 parts by mass of nPHA-g-MA + 10 parts by mass of CHM (i.e., the amounts of each component given in Table 1 can also be considered as parts by mass).

[0076] Test Example 3 Structural characterization and mechanical property testing (1) XPS test and XRD test Figure 7XPS and XRD spectra of CPHA, nPHA / HPM 10wt%, CPHA / CHM 10wt%, and raw material nPHA prepared in Example 2 are shown. (a) shows the XPS spectra of CPHA, nPHA / HPM 10wt%, CPHA / CHM 10wt%, and raw material nPHA, and (b) shows the XRD spectra of nPHA / HPM 10wt%, CPHA / CHM 10wt%, and nPHA. The XPS spectra show that in the XPS spectrum of pure nPHA, the binding energies of carbon (C 1s) and oxygen (O 1s) are observed to be 281-292 eV and 533.5 eV, respectively. Compared to pure nPHA, a C 1s peak (CC=O) of 289.5 eV was detected in the spectrum of CPHA, which is attributed to the introduction of maleic anhydride in the compatibilizer. Furthermore, the spectrum of the 10wt% nPHA / HPM sample showed two new absorption peaks, at 348.3 eV (Ca 2p) and 102.9 eV (P 2p), indicating the binding of HPM to nPHA. The spectrum of the 10wt% CPHA / CHM sample, compared to the 10wt% nPHA / HPM sample, exhibited two new peaks, at 400.2 eV (N 1s) and 50.1 eV (Mg 2p). These peaks are attributed to the presence of the CHM component in the CPHA / CHM sample, provided by chitosan and Mg(OH)2 in CHM.

[0077] XRD patterns revealed the crystal structures of nPHA / HPM 10wt%, CPHA / CHM 10wt%, and nPHA. Pure nPHA exhibited 11 characteristic diffraction peaks at 13.2°, 16.6°, 19.8°, 21.6°, 22.7°, 25.5°, 27.3°, 30.7°, 35.2°, 37.7°, and 42.8°. Compared to pure nPHA, the nPHA / HPM sample showed six additional absorption peaks at 32.1°, 34.1°, 39.7°, 46.3°, 50.5°, and 53.5°. This phenomenon is attributed to the contribution of HMP to the peak positions in the XRD spectrum. Compared to the nPHA / HPM sample, three additional absorption peaks were observed at 18.1°, 38.2°, and 58.6° in the CPHA / CHM sample. The peaks at 38.2° and 58.6° are attributed to the presence of Mg(OH)2 in CPHA / CHM. The new absorption peak at 18.1° corresponds to the esterification caused by the reaction of maleic anhydride in the compatibilizer with the functional groups of CHM.

[0078] (2) Mechanical property testing The nPHA and the prepared composite material from Example 2 were fabricated into thin films (prepared on a hot press with the following parameters: temperature 85°C, pressure 8 MPa, heating time 4 min, cooling time 5 min) and tested under conditions of 20-30% humidity and 20-25°C. Tensile properties were tested using a general-purpose testing machine (Instron 3366, Instron Co.), with dumbbell-shaped samples tested according to ASTM D638. The test was performed at a speed of 3 mm / min until the sample fractured, generating stress-strain curves. Each value was based on the average of at least five repeated tests.

[0079] The fractured sample was fixed with carbon tape, plated with gold (at 25kV for 120s), and then placed in a scanning electron microscope to observe the morphology of the fractured surface. Energy dispersive X-ray spectroscopy (EDS) was used to analyze the material dispersion.

[0080] Figure 8 The results are shown in the tensile fracture test. (a)-(e) are scanning electron microscope (SEM) images of the tensile fracture surface of the nPHA(a), nPHA / HMP 10wt%(b), nPHA / HMP 20wt%(c), CPHA / CHM 10wt%(d), and CPHA / CHM 20wt%(e), respectively. (f)-(h) are the effects of HMP or CHM content on Young's modulus (E), tensile strength at break (δ), and elongation at break (ε), respectively.

[0081] As shown in (a)-(e), the fracture surface of the pure nPHA sample is relatively smooth. However, in the nPHA / HMP sample, HMP is not fully coated by nPHA, exhibiting poor interfacial adhesion, mainly due to the difference in hydrophilic / hydrophobic properties between HMP and the nPHA matrix. In contrast, the CPHA / CHM sample shows better adhesion, with CHM uniformly coated by CPHA. This improvement is attributed to the bonding between maleic anhydride in CPHA and polar groups in CHM, thereby enhancing interfacial adhesion.

[0082] The tensile property test results ((f)-(h)) show that the Young's modulus and tensile strength at break of the nPHA and CPHA samples (i.e., HMP or CHM content of 0) are 353±15 / 16.7±0.7 MPa and 349±16 / 16.5±0.8 MPa, respectively. The Young's modulus and tensile strength at break of CPHA are slightly lower than those of nPHA. This is because the addition of a compatibilizer to CPHA results in a more dispersed branched structure compared to nPHA. With increasing HMP content, the Young's modulus and tensile strength at break of the nPHA / HMP series samples show a significant decreasing trend. At an HMP content of 20 wt%, they decrease to 297±20 MPa and 10.19±1.6 MPa, respectively. This trend is similar to the SEM results, as poor interfacial adhesion between nPHA and HMP leads to reduced mechanical properties. Furthermore, the Young's modulus and tensile strength at break of CPHA / CHM increased with the addition of CHM, reaching their maximum values ​​of 393±18 MPa and 20.6±1.3 MPa, respectively, at a CHM content of 10 wt%. This is attributed to the bonding effect between CHM and CPHA, resulting in good adhesion within the composite material. However, when the CHM content exceeded 10 wt%, both the Young's modulus and tensile strength at break of the CPHA / CHM composite decreased slightly, which is attributed to the aggregation of CHM in the CPHA / CHM composite. In (h), the elongation at break of the nPHA / HMP and CPHA / CHM samples decreased with increasing HMP and CHM content. However, compared to the nPHA / HMP sample, the CPHA / CHM sample exhibited a slightly higher elongation at break. This is attributed to the better interfacial adhesion between CPHA and CHM, resulting in a higher elongation at break for the CPHA / CHM sample compared to the nPHA / HMP sample.

[0083] Test Example 4 1. Analysis of the water absorption, polysaccharide content, antioxidant activity and in vitro degradation behavior of the bone scaffold (1) Water absorption assessment Different series of bone scaffold samples prepared in Example 2 were dried to constant weight in a vacuum drying oven at 50±1℃. The bone scaffolds were then immersed in distilled water, and samples were taken every 6 days until day 36. The surface moisture of the samples was removed with absorbent paper, and the weight was recorded. The water absorption rate (W) was calculated using the following formula. A ): W A (%) = [(W w - W c ) / W c ]×100% In the formula, W w W represents the weight of the bone scaffold after soaking. c This represents the weight of the bone scaffold before soaking after drying.

[0084] (2) Polysaccharide content test Polysaccharide content was determined using the phenol-sulfuric acid method. 5g of sample was added to 50mL of dichloromethane and stirred at 350rpm until completely dissolved. Then, 0.3mL of the sample solution, 0.3mL of 5wt% phenol solution, and 1.5mL of concentrated sulfuric acid (98wt%) were mixed, allowed to stand for 30min, and the absorbance was measured at 490nm. The polysaccharide content in the sample was calculated using a glucose standard curve. Each sample was tested five times, and the average value was taken.

[0085] (3) Antioxidant activity analysis 1.5 mL of sample solution (0.015 g of the test sample was added to 1.5 mL of anhydrous ethanol, dispersed in an ultrasonic cleaner for 20 min, and then extracted at 150 rpm for 2 h at room temperature. After extraction, the solution was centrifuged at 10000 rpm for 10 min, the supernatant was collected, and filtered through a 0.22 μm microporous membrane; the resulting filtrate was the sample solution for antioxidant activity testing) was mixed with 0.3 mL of DPPH solution (0.1 mM, anhydrous ethanol). The mixture was placed in a dark room for 40 min, and the UV absorbance was measured at 515 nm. Each sample was tested five times, and the average value was taken. The free radical scavenging rate was calculated using the following formula: Clearance rate = (1-A) s / A c )×100% In the formula, A s Ac represents the absorbance of the sample solution mixed with DPPH solution at 515 nm; Ac represents the absorbance of the control group containing only DPPH solution and an equal volume of solvent (excluding sample) at 515 nm.

[0086] (4) Assessment of in vitro degradation behavior Different series of bone scaffold samples prepared in Example 2 were placed in 50 mL centrifuge tubes, 15 mL of 1×PBS solution was added, and the tubes were covered with aluminum foil and stored at room temperature. Samples were taken every 6 days until day 36, and weighed after being dried with paper towels to assess the degradation rate. The degradation rate (i.e., weight loss rate) was calculated according to the following formula: Degradation rate (%) = [(W f -W i ) / W i ] × 100% In the formula, W f W represents the weight of the sample after soaking in PBS. i This represents the initial weight of the sample before soaking in PBS.

[0087] Figure 9 Results of water absorption (a), in vitro degradation (b), polysaccharide content (c), and antioxidant activity (d) tests for nPHA, CPHA, nPHA / HMP (10wt% and 20wt%), and CPHA / CHM (10wt% and 20wt%) samples are presented. (a) shows that the water absorption rates of nPHA and CPHA samples are lower than those of nPHA / HMP and CPHA / CHM samples, because nPHA and CPHA are relatively hydrophobic materials. The water absorption rates of nPHA / HMP and CPHA / CHM samples increase with increasing HMP or CHM content, due to the porosity of HMP and CHM making the sample surfaces more absorbent. Furthermore, (a) also shows that at the same HMP or CHM content, nPHA / HMP has a slightly higher water absorption capacity than CPHA / CHM. This is because the binding between CPHA and CHM hinders the exposure of hydrophilic substances, leading to a decrease in water absorption.

[0088] (b) shows that the degradation rate of all samples increased with increasing immersion time in PBS. Furthermore, the degradation rates of the nPHA / HMP and CPHA / CHM samples were higher than those of nPHA or CPHA. This accelerated degradation is attributed to the hygroscopic properties of HMP and CHM, which promoted the in vitro degradation of the nPHA / HMP and CPHA / CHM samples. Additionally, (b) also shows that at the same HMP or CHM content, the degradation rate of CPHA / CHM was lower than that of nPHA / HMP. This is because interfacial bonding between CPHA and CHM enhanced the adhesion in the CPHA / CHM sample, resulting in a slightly lower degradation rate.

[0089] (c) shows that the polysaccharide content was mainly found in the CHM-containing composite system, and increased significantly with increasing CHM content from 10 wt% to 20 wt%. This result is attributed to the chitosan components (such as D-glucosamine and N-acetyl-D-glucosamine structural units) introduced into the CHM, while the nPHA, CPHA, and nPHA / HMP systems did not contain these components and only detected very low background signals. Furthermore, the presence of polysaccharides helps enhance the free radical scavenging capacity of the bone scaffold system and further promotes its antioxidant, anti-inflammatory, and antibacterial properties, such as… Figure 9 As shown in (d).

[0090] (d) shows that the nPHA / HMP sample exhibited almost no free radical scavenging activity. In the CPHA / CHM sample, the free radical scavenging rate increased with increasing CHM content. The free radical scavenging activity originates from the amino (-NH2) and hydroxyl (-OH) groups contained in the chitosan component of CHM. These functional groups can react with free radicals, thus demonstrating the observed free radical scavenging ability.

[0091] 2. Cell compatibility and mineralization assessment Both NIH3T3 and RAW-264.7 cells were commercially available, and cells from the same batch and passage number (e.g., P8) were used for testing. NIH3T3 cells were cultured in DMEM medium containing 10 wt% fetal bovine serum and 1 wt% penicillin-streptomycin at 37°C and 5 vol% CO2 until approximately 75% confluence. The medium was then replaced with osteogenic induction medium (basal medium supplemented with 50 μg / mL vitamin C, 10 mM β-glycerophosphate sodium, and 10 nM dexamethasone) for 21 consecutive days, with fresh medium replaced every 3 days. Osteogenic differentiation was assessed using ALP activity assays and alcinocyanine blue staining. RAW-264.7 cells were cultured in α-MEM medium containing 10 wt% fetal bovine serum and 1 wt% penicillin-streptomycin. After culturing under the same conditions to about 75% confluence, the osteoclast induction medium was replaced (75 ng / mL RANKL and 40 ng / mL LM-CSF were added to the basal medium). The cells were continuously induced and cultured for 7 days, with the medium being replaced every 2 days. The differentiation and function of osteoclasts were verified by TRAP staining and bone resorption pit experiments.

[0092] (1) Cell adhesion and cell cycle test Different samples were compared with 1×10 5 NIH3T3 cells / mL were co-cultured for 2 days in 6 cm diameter culture dishes. After removing the supernatant and washing with PBS, the cells were digested with trypsin to obtain a cell suspension, followed by centrifugation at 2300 rpm to obtain a cell pellet. The resulting cell pellet was grown in MEM-containing culture plates for 12 h. Cell growth was observed using an inverted microscope. Additionally, the same sample was cultured with 1.5 × 10⁶ cells / mL. 5 NIH3T3 cells / mL were cultured together for 2.5 days. The cells were then fixed with 3.5 wt% glutaraldehyde and dehydrated in different concentrations of ethanol (25 vol%, 35 vol%, 45 vol%, 75 vol%, 85 vol%, 95 vol%). Cell adhesion was observed by scanning electron microscopy.

[0093] (2) Cell viability test Different samples were immersed in 1×PBS solution for 24 days, then removed and co-cultured with differentiated osteoblasts and osteoclasts (immersed in 2×10⁻⁶ PBS solution). 4Cells were isolated with trypsin on days 1, 3, and 7, and then cultured for another day. Afterward, 150 µM MTT reagent was added and the cells were cultured for 3 hours to form soluble formazan. The formazan was dissolved in dimethyl sulfoxide (DMSO), and cell viability was measured using an enzyme-linked immunosorbent assay (ELISA) at 570 nm UV wavelength.

[0094] (3) Alkaline phosphatase (ALP) test ALP activity was determined using the pNPP method: After differentiation induction, the culture medium was discarded and the cells were washed with PBS. Then, 100 μL of cell lysis buffer and 100 μL of pNPP substrate solution were added, and the mixture was reacted at 37 °C for 30 min. After pNPP was converted to p-nitrophenol, NaOH was added to terminate the reaction. The absorbance was measured at 405 nm using a microplate reader to characterize ALP activity. A blank control group without cell lysis buffer was set up, and ALP activity was normalized according to the total protein content.

[0095] (4) Calcium deposition test Differentiated osteoblasts were fixed in 4 wt% formalin solution for 30 min, and then 0.5 wt% Alizarin Red S solution (all reagent concentrations refer to the concentration of the reagent in the mixture) was added and reacted for 30 min. Fluorescence changes in calcium deposition were observed using an inverted microscope. Subsequently, 10 wt% acetic acid was added to the sample, and after incubation for 30 min, the absorbance at 415 nm was measured to assess the level of calcium deposition.

[0096] Figure 10 The results of cell adhesion, cell cycle, and cell viability assays are shown below. (a) SEM images of NIH3T3 cell adhesion after 3 days of culture on control (blank control), nPHA, nPHA / HMP, and CPHA / CHM samples; (b)-(c) cell viability assay results of NIH3T3 cells (specifically osteoblasts differentiated from NIH3T3 cells) and RAW-264.7 cells (specifically osteoclasts differentiated from RAW-264.7 cells) on control, nPHA, nPHA / HMP, and CPHA / CHM samples, respectively; (d) alkaline phosphatase assay results; and (e) calcium deposition assay results. (a) shows no significant difference in cell adhesion morphology among the control, nPHA, nPHA / HMP, and CPHA / CHM samples. This indicates that none of the samples are toxic to mouse embryonic cells.

[0097] (b) showed that on day 1 of culture, there was no significant difference in the effect on osteoblast growth between the samples and the control group. However, on day 3, nPHA / HMP 15wt%, nPHA / HMP 20wt%, CPHA / CHM 15wt%, and CPHA / CHM 20wt% significantly enhanced osteoblast growth. On day 7, the relative growth rates of nPHA / HMP 20wt% and CPHA / CHM 20wt% reached 263.32±9.56% and 272.76±9.08%, respectively, which were higher than the 229.32±9.65% of the control group. This cell proliferation effect was mainly due to the release of calcium and phosphate ions from hydroxyapatite within HMP and CHM, which provided a favorable environment for osteoblast proliferation. In addition, CPHA / CHM showed slightly higher cell activity than the nPHA / HMP series, which was attributed to Mg 2+ The release of ions and the bioactivity of chitin in CHM materials. Both substances promote osteoblast activity in bone-related pathways and calcium-binding protein-related genes, thereby enhancing the proliferative effect.

[0098] (c) shows that osteoclast growth rate increased over time in the control group, nPHA group, and nPHA / HMP group. Compared with the control group, a more significant increase in osteoclasts was observed in the nPHA / HMP 15wt% and nPHA / HMP 20wt% groups on days 3 and 7, mainly due to the uptake of calcium / phosphate ions released from HMP in nPHA / HMP by osteoclasts, thereby promoting growth. In contrast, nPHA, which does not contain such additives, resulted in a less significant increase in osteoclast growth rate. However, in the CPHA / CHM group, a slight decrease in osteoclast activity was observed over time. This phenomenon may be attributed to the composition of CHM, which contains Mg(OH)2 and chitosan. These components act through various synergistic mechanisms, inhibiting osteoclast activity by regulating intracellular calcium ions and stimulating the secretion of calcitonin (an inhibitor of osteoclast activity).

[0099] (d) shows that purple fluorescent phosphorus aggregates were deposited on the composite samples after osteoblast culture. Both the control and nPHA samples showed less purple fluorescent phosphorus aggregate deposition. In contrast, the nPHA / HMP and CPHA / CHM series samples showed more significant purple fluorescence, i.e., phosphorus deposition. The intensity of purple fluorescence increased with increasing HMP or CHM content. This increase is mainly due to the enhanced osteoinduction effect of HMP or CHM interaction with osteoblasts, stimulating gene expression and activating multiple signaling pathways that promote alkaline phosphatase mineralization.

[0100] (e) shows the surface morphology of calcium aggregates and osteoblast activity in different samples after Alizarin Red S staining. Compared with the control group, the nPHA / HMP and CPHA / CHM samples showed more red fluorescence of calcium deposits. The amount of red fluorescence increased with increasing HMP or CHM content. This indicates that HMP or CHM components can chelate calcium ions into osteoblasts, promoting calcium deposition. Overall, these results confirm that osteoblast activity was enhanced by the PHA / HMP and CPHA / CHM samples, and show increased phosphate and calcium deposition on the cell surface.

[0101] 3. Simulated Body Fluid (SBF) Assessment 7.996 g NaCl, 0.350 g NaHCO3, 0.224 g KCl, 0.228 g K2HPO4·3H2O, 0.305 g MgCl2·6H2O, 0.278 g CaCl2, 0.071 g Na2SO4, and 6.057 g Tris (aminomethylpropanol) were sequentially added to 800 mL of deionized water and dissolved completely one by one. The pH was adjusted to 7.4 with 1 M HCl solution, and then deionized water was added to bring the total volume to 1000 mL. The solution was then filtered through a 0.2 μm filter to ensure sterility, yielding SBF. The prepared SBF solution was stored at 4 °C for later use. Samples were placed in SBF, and samples were taken every 6 days until day 36. The pH change of each sample was measured, and the samples were dried to constant weight in a vacuum drying oven at 50 ± 1 °C. The crystal morphology and calcium-to-phosphorus (Ca / P) ratio of the sample surface were analyzed (obtained by EDS analysis) to evaluate their properties.

[0102] Simulated body fluid assessment results as follows Figure 11As shown, (a)-(e) are EDS spectra and SEM images of crystal deposition in simulated body fluid after immersion in simulated body fluid for 30 days for nPHA, nPHA / HMP 10wt%, nPHA / HMP 20wt%, CPHA / CHM 10wt%, and CPHA / CHM 20wt% samples; (f) shows the Ca / P ratio of the crystals as shown by EDS spectral analysis; (g) and (h) show the changes in calcium and phosphate ion content in the simulated body fluid solution for each sample at different immersion times; (i) shows the pH changes of the simulated body fluid solution for each sample at different immersion times. (a)-(e) show that no obvious deposits were observed on the surface of nPHA, and its spectrum mainly showed C and O elements, as well as trace amounts of Ca and P, with a Ca / P ratio of approximately 0.65, which may be attributed to residual SBF solution. All surfaces of the nPHA / HMP and CPHA / CHM series samples showed obvious deposits, and EDS analysis indicated that these deposits were mainly composed of calcium, phosphorus, and oxygen. The calcium / phosphorus ratios of the nPHA / HMP (10wt% and 20wt%) and CPHA / CHM (10wt% and 20wt%) series were (1.70 and 1.73) and (1.66 and 1.68), respectively. The hydroxyapatite in the nPHA / HMP and CPHA / CHM samples underwent an ion exchange reaction with SBF solution to form spherical hydroxyapatite structures.

[0103] (f) shows that the Ca / P ratio of the mineralized crystal components in both nPHA and CPHA samples decreased slightly with increasing soaking time. The decrease in Ca / P ratio was more pronounced in the nPHA / HMP series samples with prolonged soaking time, primarily due to the reaction of HMP with the SBF solution. Conversely, the Ca / P ratio of the CPHA / CHM series samples increased with prolonged soaking time. This is because the HMP component encapsulated within the CPHA restricts its ion exchange reaction with the SBF solution, thus the Ca / P nucleation mineralization and crystallization behavior of CPHA / CHM differs from that of nPHA / HMP. After 30 days of soaking, the Ca / P ratio of CPHA / CHM approached the range of 1.6–1.8, similar to values ​​found in the human body. Furthermore, (g) and (h) show that the Ca and P ion contents in the SBF solution containing the nPHA / HMP sample decreased more rapidly than those in the CPHA / CHM sample. This further demonstrates that HMP coated with a chitosan outer layer in the CPHA / CHM sample does not readily undergo ion exchange with the SBF solution, thus leading to the difference in the crystallization rate behavior of the aforementioned Ca / P ratio.

[0104] (i) shows that the pH of the SBF solution containing nPHA remained relatively stable over time because almost no degradation occurred during the soaking period, resulting in minimal pH change. In contrast, the pH of the SBF solutions containing the nPHA / HMP and CPHA / CHM series samples gradually increased with soaking time. This pH increase is attributed to the ion exchange reaction between the SBF solution and the HMP or CHM present in the samples. Notably, the pH increase trend was more pronounced within 24 days, becoming milder after 24 days, which can be attributed to the fact that after 24 days, the HMP or CHM in the nPHA / HMP or CPHA / CHM samples could no longer effectively undergo ion exchange reactions with the SBF solution. The pH of the CPHA / CHM samples was lower than that of the nPHA / HMP samples because the CHM was encapsulated in the CPHA matrix, thereby reducing the release and cleavage rate.

[0105] 4. Antibacterial and anti-inflammatory performance test (1) Antibacterial performance test Immerse 1g of sample in 10mL of ultrapure water and allow it to degrade for 30 days before removing the sample. Then, add 1mL of Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) solution (1×10⁻⁶). 5 Add CFU / mL to the degradation solution and co-culture for 1.5 days, then spread on agar plates and incubate for 20 hours. Observe bacterial growth, take photos of colony growth, and record bacterial growth data.

[0106] (2) Anti-inflammatory performance test RAW 264.7 cells were cultured at a rate of 6 × 10⁻⁶. 5 Cells were seeded at a density in 24-well plates and cultured for 12 h. Then, each different test sample and LPS (10 μg / mL) were added to the wells containing the seeded cells, and the cells were cultured for another 12 h. The group with LPS served as the control group, and the group with DMSO served as the blank group. The anti-inflammatory activity of the cell culture supernatant of all samples was analyzed using an IL-6 ELISA kit at 450 nm.

[0107] The results of the antibacterial and anti-inflammatory performance tests are as follows: Figure 12As shown, (a) shows the IL-6 content in the cell culture supernatant, (b) and (c) show the growth rates of *Escherichia coli* and *Staphylococcus aureus*, respectively, and (d) shows the bacterial growth on agar medium. (a) shows that the IL-6 content in the nPHA, CPHA, and nPHA / HMP groups was slightly higher than that in the LPS group, indicating that the nPHA, CPHA, and nPHA / HMP materials had no or very weak anti-inflammatory effects. However, the IL-6 content in the CPHA / CHM (10wt% and 20wt%) samples gradually decreased, reaching approximately 81.26% and 73.96%, respectively. This is because the amino group (-NH2) of chitosan in CHM can react with inflammatory mediators (such as reactive oxygen species), inhibiting the expression of pro-inflammatory cytokines, thereby alleviating the inflammatory response and exerting a bioactive anti-inflammatory effect.

[0108] (b) and (c) show that *Escherichia coli* and *Staphylococcus aureus* exhibited stable growth trends in the control group, nPHA, or nPHA / HMP samples. However, the CPHA / CHM sample showed a significant inhibitory effect on the growth rate of both bacterial strains, and no bacterial survival was observed at 30 days in the CPHA / CHM (20 wt%) sample and 36 days in the CPHA / CHM (10 wt%) sample. Furthermore, (d) also shows that the CPHA / CHM sample significantly reduced bacterial growth at days 18 and 36 compared to the nPHA or nPHA / HMP samples. This antibacterial effect is attributed to the presence of Mg(OH)₂ and chitosan in the CHM of the CPHA / CHM sample. The released Mg... 2+ The synergistic effect of ions and the active groups of chitosan (such as -NH2 and -OH) effectively inhibits bacterial growth. Mg 2+ Active chitosan groups can oxidize microbial cell membranes and intracellular proteins, disrupt RNA and DNA metabolism, and lead to cell death.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A biodegradable polymer-based bone repair 3D printing composite material, characterized in that, By weight, the raw materials include: 80-95 parts of biodegradable polymer, 3-12 parts of compatibilizer, and 5-20 parts of chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2.

2. The biodegradable polymer-based bone repair 3D printing composite material as described in claim 1, characterized in that, The biodegradable polymers include one or more of polyhydroxyalkanoates, polycaprolactone, polylactic acid, polybutylene succinate, polylactic acid-glycolic acid copolymer, and polybutylene adipate-terephthalate copolymer. And / or, the compatibilizer is a maleic anhydride-grafted polyhydroxy fatty acid ester.

3. The biodegradable polymer-based bone repair 3D printing composite material as described in claim 2, characterized in that, The grafting rate of maleic anhydride in the maleic anhydride-grafted polyhydroxy fatty acid ester is 0.5-2 wt%.

4. The biodegradable polymer-based bone repair 3D printing composite material as described in claim 1, characterized in that, The preparation steps of the chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 include: mixing chitosan, natural hydroxyapatite, acetic acid solution and water to obtain an aqueous phase solution; mixing emulsifier and paraffin oil to obtain an oil phase solution; adding the aqueous phase solution dropwise to the oil phase solution, heating and stirring to react, then cooling to 5-6℃, and then adding a crosslinking agent to carry out a crosslinking reaction to obtain the chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2.

5. The biodegradable polymer-based bone repair 3D printing composite material as described in claim 4, characterized in that, The degree of deacetylation of the chitosan is 70-85%.

6. The biodegradable polymer-based bone repair 3D printing composite material as described in claim 4, characterized in that, The ratio of chitosan, natural hydroxyapatite, acetic acid solution, and water is 1.5-3.0g:0.1-0.3g:5-15mL:30-60mL; And / or, the concentration of the acetic acid solution is 0.5-2.0 wt%; And / or, the volume ratio of the emulsifier to the paraffin oil is 1:40-70; And / or, the crosslinking agent is a tripolyphosphate; And / or, the volume ratio of the aqueous phase solution to the oil phase solution is 1:30-60; And / or, the concentration of the crosslinking agent is 1-5 wt%.

7. The biodegradable polymer-based bone repair 3D printing composite material as described in claim 4, characterized in that, The heating and stirring reaction is carried out at a temperature of 50-70℃ for 2-5 hours. And / or, the crosslinking reaction takes 20-60 minutes.

8. A method for preparing a biodegradable polymer-based bone repair 3D printing composite material as described in any one of claims 1-7, characterized in that, Includes the following steps: The biodegradable polymer, compatibilizer, and chitosan microspheres co-loaded with hydroxyapatite and Mg(OH)2 are mixed to obtain the biodegradable polymer-based bone repair 3D printing composite material.

9. A bone repair 3D printing filament, characterized in that, The biodegradable polymer-based bone repair 3D printing composite material as described in any one of claims 1-7 is obtained by extrusion molding.

10. The application of the bone repair 3D printing filament as described in claim 9 in the 3D printing preparation of bone repair products or antibacterial coatings.

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