Bone cement as well as preparation method and application thereof in alveolar bone repair

Abstract

The invention discloses bone cement as well as a preparation method and application thereof in alveolar bone repair, and belongs to the field of biomedical materials. The bone cement comprises a solid phase and a liquid phase, and the liquid-solid ratio is 0.4-0.6 mL / g; the solid phase is composed of alpha-calcium sulfate hemihydrate powder, alpha-tricalcium phosphate powder and mineralized collagen / fibroin powder according to the mass ratio of (55-77): (14-36): 9; mineralized collagen and fibroin in the mineralized collagen / fibroin powder are combined for use, and the mass ratio of the mineralized collagen to the fibroin is 2: (1-4); the liquid phase is a carboxymethyl chitosan solution; firstly, a CMCS solution with the concentration of 1-5 wt% is prepared, and then the solid phase is added according to the liquid-solid ratio to obtain the bone cement. The prepared bone cement composite material not only has excellent collapse resistance and injectability, but also can promote adhesion and proliferation of osteoblasts, and can induce osteogenic differentiation in vitro and promote osseointegration.

Description

Technical Field

[0001] This invention relates to a bone cement, its preparation method, and its application in alveolar bone repair, belonging to the field of biomedical materials. Background Technology

[0002] Alveolar bone repair is an important topic in oral and maxillofacial surgery, commonly seen in cases of bone defects after tooth extraction, bone resorption due to periodontal disease, and congenital bone deformities. Ideal bone repair materials must meet three core requirements: injectability (adapting to complex defect morphology and simplifying surgery), collapse resistance (maintaining structural stability in the body fluid environment after implantation), and osseointegration capacity (promoting new bone formation and functional recovery). However, existing bone cement materials still have significant limitations in these aspects (Kang J, Lian X, He Z, et al. Journal of Biomaterials Applications, 2025, 40(4): 547-559.).

[0003] Traditional injectable bone cements, represented by calcium phosphate bone cement (CPC), achieve minimally invasive injection by mixing liquid and solid to form a paste. However, to improve fluidity, the liquid-to-solid ratio often needs to be increased, leading to prolonged curing time (from several minutes to tens of minutes). Furthermore, ions and organic matter in blood plasma further delay in vivo curing, increasing the difficulty of the surgical procedure. More importantly, uncured paste is easily dispersed by blood (poor anti-collapse properties), not only failing to form properly, but the micro- and nano-powders may also cause serious complications such as vascular blockage through blood circulation. In addition, traditional CPC is relatively brittle and has limited biocompatibility, with a weak effect on promoting osteoblast attachment and proliferation. Therefore, developing a bone cement that combines rapid curing, anti-collapse properties, degradation compatibility, and high osteogenic activity is an urgent need in the field of alveolar bone repair.

[0004] While autologous bone grafting is considered the gold standard, its clinical application is limited by issues such as donor site complications (e.g., 45.7% of patients experience acute gait disorder after iliac bone harvesting), limited bone volume, and secondary surgical trauma. Other alternative materials, such as deproteinized beef bone (DBB), while possessing some osteoconductive properties, degrade extremely slowly (residues may remain even 10 years post-surgery) and lack osteogenic activity, resulting in low new bone formation efficiency. Hydroxyapatite (HA) materials, on the other hand, present challenges in shaping and stable retention within the defect area, requiring the use of a barrier membrane, which increases surgical complexity and patient burden.

[0005] To address these issues, researchers have attempted to optimize performance through material modification: for example, adding additives such as hydroxymethyl cellulose improves injectability, but prolongs curing time (Takechi M, Miyamoto Y, Ishikawa K, et al. Biomaterials, 1998, 19(22): 2057-2063.); introducing sodium alginate to form a gel network using calcium ions to enhance anti-collapse properties, but with limited improvement in mechanical properties (Momota Y, Miyamoto Y, Ishikawa K, et al. Journal of Biomedical Materials Research, 2002, 63(5): 542-547.); although 3D printing technology can customize porous structures (such as 60° oriented scaffolds), the printability and in vivo degradation matching of printed materials still need to be optimized (Korn P, Ahlfeld T, Lahmeyer F, et al. Frontiers in bioengineering and biotechnology, 2020, 8: 217.). In addition, although complex bioactive ingredients (such as BMP-2) can promote osteogenic formation, they may trigger excessive bone resorption or inflammatory response (Shen H, Zhi Y, Zhu F, et al. Dental Materials Journal, 2021, 40(1): 191-201.).

[0006] In summary, existing materials struggle to balance injectability, collapse resistance, and bone repair efficiency, especially in the unique environment of alveolar bone subjected to chewing forces and fluid erosion. The mechanical stability and long-term osseointegration capacity of these materials present significant challenges. Developing novel bone cements that combine high injectability (e.g., achieving over 90% extrusion rate through viscosity control), strong collapse resistance (no disintegration in body fluids within 30 minutes), and excellent biocompatibility is crucial for simplifying alveolar bone repair surgery and improving clinical efficacy. Summary of the Invention

[0007] To address the problem that existing materials struggle to balance injectability, collapse resistance, and bone repair efficiency, this invention provides a bone cement for alveolar bone repair and its preparation method. This bone cement composite material not only possesses excellent collapse resistance and injectability but also promotes osteoblast attachment and proliferation. Furthermore, it can induce osteogenic differentiation in vitro and promote bone integration.

[0008] This invention is based on calcium phosphate / calcium sulfate bone cement composite material, and improves the slump resistance and injectability of bone cement by combining mineralized collagen / silk fibroin powder and carboxymethyl chitosan curing liquid. The mineralized collagen / silk fibroin powder optimizes material properties through multiple effects in the modification of bone cement. From an interfacial perspective, the mineralized collagen / silk fibroin contains polar groups such as carboxyl and hydroxyl groups, which can interact with the α-TCP surface Ca... 2+ PO4 3- and α-CSH's Ca 2+ Hydrogen bonds and electrostatic interactions are formed, enhancing the interfacial bonding between the organic phase (mineralized collagen / silk fibroin) and the inorganic phase, reducing the risk of separation. Regarding structural stability, mineralized collagen / silk fibroin and carboxymethyl chitosan can construct a three-dimensional network through cross-linking reactions to synergistically encapsulate α-TCP and α-CSH particles, reducing disintegration in body fluids and improving compressive strength and anti-collapse properties. In terms of bioactivity, mineralized collagen / silk fibroin provides attachment sites for osteoblasts, synergistically releasing Ca2+ from α-TCP and α-CSH. 2+ PO4 3- This process promotes cell proliferation and osteogenic differentiation; simultaneously, it regulates the degradation rate to match new bone formation, avoiding insufficient support or hindering bone integration. Furthermore, mineralized collagen / silk fibroin mimics natural bone components, enhancing material biocompatibility, aiding osteoconduction and bone integration, and optimizing the clinical applicability of bone cement in alveolar bone repair. The bone cement composite material prepared in this invention exhibits excellent biocompatibility; the gel network formed by collagen / silk fibroin and carboxymethyl chitosan significantly improves the compressive strength of the bone cement, demonstrating excellent anti-collapse and injectability properties. The mechanism by which mineralized collagen / silk fibroin and carboxymethyl chitosan bind to α-TCP and α-CSH mainly involves the following aspects: (a) Hydrogen bonding and electrostatic interactions enhance interfacial adhesion. ① Interactions of mineralized collagen / silk fibroin with α-TCP and α-CSH: Both mineralized collagen and silk fibroin contain polar functional groups such as carboxyl and hydroxyl groups, which can undergo hydrogen bonding and electrostatic attraction with calcium ions and phosphate ions on the surface of α-TCP and calcium ions on the surface of α-CSH. For example, the carboxyl group interacts with the calcium ions on the surface of α-TCP. 2+ Electrostatic interactions are formed, and hydroxyl groups form hydrogen bonds with the water of crystallization of α-CSH, which enhances the interfacial bonding between the organic and inorganic phases and improves the overall stability of the material.

[0009] ② Interaction between carboxymethyl chitosan and α-TCP, α-CSH: Carboxymethyl chitosan (CMCS) interacts with α-TCP and α-CSH via its carboxyl and hydroxyl groups. 2+ It forms coordination bonds or hydrogen bonds, achieving organic-inorganic integration. Interaction with α-TCP enhances interfacial bonding, slows degradation, and improves toughness; interaction with α-CSH inhibits crystal growth, reduces hydration shrinkage, and extends workability.

[0010] (ii) Crosslinking reaction to build a stable network structure ① Synergistic effect of mineralized collagen / silk fibroin on cross-linking with α-TCP and α-CSH: Silk fibroin in Ca 2+ In their presence, ionic cross-linking can occur, forming a three-dimensional gel network. α-TCP and α-CSH continuously release Ca during hydration. 2+ This provides a sufficient ion source for the cross-linking of silk fibroin, making the gel network more stable. At the same time, collagen fibers in mineralized collagen may also participate in the construction of this cross-linked network, enhancing the overall stability and mechanical properties of the material.

[0011] ② Carboxymethyl chitosan can crosslink with other components: Carboxymethyl chitosan has the ability to form crosslinked structures. When combined with α-TCP and α-CSH, it may crosslink with other components (such as MC, SF, and Ca) through intermolecular interactions or through interactions with other components (such as MC, SF, and Ca). 2+ Cross-linking reactions occur in bridges (such as α-TCP and α-CSH). This cross-linking can form a more stable three-dimensional structure, which not only helps maintain the shape and structural integrity of the material, but also improves the material's ability to bind α-TCP and α-CSH, further improving the material's anti-collapse performance and overall mechanical properties.

[0012] (III) Mineralization promotes structural stability and osseointegration Organic-inorganic synergistic mineralization: Collagen fibers in mineralized collagen provide a template for mineralization, guiding Ca... 2+ and PO4 3- Orderly deposition. If silk fibroin is involved, its molecular structure and properties may also influence the ion deposition process, synergistically interacting with mineralized collagen and α-TCP / α-CSH to form a more stable composite structure. This organic-inorganic synergistic mineralization not only enhances the material's mechanical properties but also improves its integration with surrounding bone tissue, promoting the repair and healing of bone defects.

[0013] (iv) Bioactivity regulation promotes cellular response and bone regeneration ① Promoting effects on cell adhesion and proliferation: Mineralized collagen, silk fibroin, and carboxymethyl chitosan all possess good biocompatibility and can provide attachment sites for osteoblasts. α-TCP and α-CSH release Ca... 2+ Silk fibroin can activate osteoblast proliferation signaling pathways and may also have the ability to load and slowly release bioactive substances, further promoting cell proliferation. Components in mineralized collagen may also interact with receptors on the cell surface, regulating cell behavior and jointly promoting bone tissue regeneration and repair.

[0014] ② Induction of osteogenic differentiation: The combination of mineralized collagen, silk fibroin, and α-TCP and α-CSH can provide a suitable microenvironment for osteoblasts. α-TCP releases PO4... 3- and Ca released by α-CSH 2+ It can participate in the formation of bone matrix, synergistically induce osteoblast differentiation, increase alkaline phosphatase activity, and promote the formation of mineralized nodules. Some components of silk fibroin and mineralized collagen may interact with intracellular signaling pathways to regulate the expression of osteoblast-related genes, further promoting osteoblast differentiation and maturation, and accelerating bone tissue repair and reconstruction.

[0015] In summary, mineralized collagen / silk fibroin and carboxymethyl chitosan, along with α-TCP and α-CSH, form a stable organic-inorganic composite structure through hydrogen bonding, electrostatic interactions, and cross-linking reactions. Combined with the synergistic effects of mineralization and bioactivity, this ultimately achieves comprehensive optimization of the material's anti-collapse properties, injectability, and bone regeneration and integration capabilities, providing an ideal biomaterial basis for bone defect repair.

[0016] This invention provides a collapse-resistant, injectable bone cement composite material, comprising a solid phase and a liquid phase, with a liquid-to-solid ratio of 0.4~0.6 mL / g; the solid phase is composed of α-calcium sulfate hemihydrate (α-CSH) powder, α-tricalcium phosphate (α-TCP) powder, and mineralized collagen / silk fibroin (MC / SF) powder in a mass ratio of (55~77):(14~36):9; wherein collagen and silk fibroin are used in combination in the MC / SF, with a mass ratio of 2:(1~4); the liquid phase is a carboxymethyl chitosan (CMCS) solution with a concentration of 1 wt%~5 wt%; a CMCS solution with a concentration of 1 wt%~5 wt% is first prepared, and then added to the solid phase according to the above liquid-to-solid ratio to obtain bone cement.

[0017] This invention provides a method for preparing the above-mentioned bone cement, the preparation steps of which are as follows: Step 1: Synthesis of α-CSH powder; α-CSH is synthesized hydrothermally using calcium sulfate dihydrate as the raw material; Step 2: Synthesis of α-TCP powder; α-TCP is synthesized by high-temperature sintering of calcium carbonate, phosphoric acid, and calcium hydroxide as raw materials; Step 3: Synthesis of MC / SF powder; MC / SF is synthesized using bovine collagen and silk fibroin as raw materials through biomimetic mineralization. Step 4: Prepare carboxymethyl chitosan (CMCS) solution. The preparation method is as follows: Dissolve CMCS powder in water to prepare a CMCS solution with a concentration of 1 wt% to 5 wt%. Step 5: Mix the three powders synthesized in steps 1 to 3 in a certain proportion to form a solid phase, with a liquid-to-solid ratio of 0.4 to 0.6 mL / g, and mix with CMCS solution to form a homogeneous slurry; Step 6: At room temperature, the solidification time is 10-16 minutes; the compressive strength after 24 hours of solidification is ≥10MPa.

[0018] Furthermore, the preparation method of α-CSH powder in step one is as follows: (1) Weigh an appropriate amount of calcium sulfate dihydrate (CSD) as raw material; (2) Using the hydrothermal synthesis method, CSD is placed in a vertical pressure steam sterilizer; (3) The temperature was set at 127℃, and the reaction was carried out under saturated vapor pressure for 9 h; (4) After taking it out, place it in an oven to dry to obtain blocky solid α-CSH; (5) Then the blocky solid α-CSH is ground and sieved to obtain white α-CSH powder.

[0019] The specific process for preparing α-TCP powder in step two is as follows: (1) At room temperature, add 29.98 g of phosphoric acid (H3PO4) to 1 L of deionized water to make a 0.26 mol / L phosphoric acid solution, and stir it evenly in a magnetic stirrer; (2) Add 13.13 g of calcium carbonate (CaCO3) and stir magnetically for 90 min to allow it to react fully; (3) Add 10.13 g of calcium hydroxide (Ca(OH)2) to 1 L of deionized water to make a 0.13 mol / L calcium hydroxide solution, and stir it evenly in a magnetic stirrer; (4) Add the solution obtained in steps (1) and (2) above into a separatory funnel, drop it into the solution in step (3), and stir continuously for 4 h; (5) After the solution has been added dropwise, add 13.13 g of calcium carbonate (CaCO3), stir continuously for 12 h, and then let stand for 4-5 h; (6) After settling, the precipitate is evenly loaded into centrifuge tubes and centrifuged at 3000 r / min for 15 min. (7) After centrifugation, the precipitate is taken out and ball-milled for 2 hours; (8) After repeating step (6), perform vacuum drying at 80℃ for 24 h; (9) Grind the tricalcium phosphate (Ca3(PO4)2) prepared above into powder, sinter it in a high-temperature furnace at 1400 °C for 5.5 h, and then quickly take it out and cool it down. (10) Grind and sieve to obtain α-TCP powder with a particle size distribution mainly in the range of 0.13~0.56 μm and an average particle size of 0.21 μm.

[0020] The method for synthesizing MC / SF powder in step three is as follows: (1) Weigh 75 mg of bovine collagen, add 75 mL of deionized water and 2.14 mL of glacial acetic acid, and stir to dissolve for 24 h; (2) Add a CaCl2 solution made of 18 mL of deionized water and 1.04 g of CaCl2 powder, and stir for 2 h; (3) Add another 360 μL of H3PO4 solution and stir for 2 h; (4) Then, use NaOH to adjust the pH of the above solution to around 8, add 75 mg of silk fibroin to dissolve, and stir overnight for 12 h; (5) Let the sediment stand for 3-24 hours, and centrifuge 3-5 times until the pH of the supernatant is 7; (6) Take the precipitate, freeze dry it, grind it and sieve it to obtain MC / SF powder.

[0021] This invention provides the application of the above-mentioned bone cement in alveolar bone repair.

[0022] The beneficial effects of this invention are: (1) Significantly improved mechanical properties: The gel network of mineralized collagen / silk fibroin and carboxymethyl chitosan can fill the gaps between α-TCP and α-CSH particles, enhance the interfacial bonding force, and significantly improve the compressive strength of bone cement.

[0023] (2) Excellent biocompatibility and osteogenic activity: Mineralized collagen / silk fibroin and carboxymethyl chitosan provide attachment sites for osteoblasts, and together with the Ca released by α-TCP and α-CSH, they provide attachment sites for osteoblasts. 2+ PO4 3- It promotes cell proliferation and differentiation, providing suitable conditions for bone regeneration.

[0024] (3) Optimization of degradation and bone regeneration matching: The biodegradability of mineralized collagen / silk fibroin and carboxymethyl chitosan is synergistic with the degradation of α-TCP and α-CSH, avoiding insufficient support due to excessively rapid degradation or hindering new bone ingrowth due to excessively slow degradation, which can promote bone integration.

[0025] (4) Material handling performance is adapted to clinical needs: Organic phase adjusts the viscosity of the slurry and improves injectability, such as by adjusting the concentration or liquid-solid ratio of carboxymethyl chitosan solution to optimize the surgical operation window.

[0026] (5) The bone cement composite material prepared by this invention has good biocompatibility; the gel network formed by collagen / silk fibroin and carboxymethyl chitosan significantly improves the compressive strength of the bone cement, and exhibits excellent anti-collapse performance and injectability; at the same time, the bone cement composite material promotes the attachment and proliferation of osteoblasts, and it can induce osteogenic differentiation in vitro. In summary, the comprehensive advantages of this composite bone cement in terms of mechanical strength, bioactivity, degradation regulation and handling performance make it a promising candidate for alveolar bone repair. Attached Figure Description

[0027] Figure 1 This is a diagram illustrating the preparation process and mechanism of the bone cement composite material of the present invention; Figure 2 Electron micrographs of the bone cement composite materials prepared in Comparative Examples 1-6 and Examples 1-3 are shown below; In the figures, (A): α-TCP / α-CSH; (B): α-TCP / α-CSH-MC; (C): α-TCP / α-CSH-MC / SF; (D): α-TCP / α-CSH-MC / CMCS1; (E): α-TCP / α-CSH-MC / CMCS3; (F): α-TCP / α-CSH-MC / CMCS5; (G): α-TCP / α-CSH-MC / SF / CMCS1; (H): α-TCP / α-CSH-MC / SF / CMCS3; (I): α-TCP / α-CSH-MC / SF / CMCS5; Figure 3 The graph shows the setting time results of the bone cement composite materials prepared in Comparative Examples 1-6 and Examples 1-3; Figure 4 The graph shows the compressive strength results of the bone cement composite materials prepared in Comparative Examples 1-6 and Examples 1-3; Figure 5 The graph shows the injection test results of the bone cement composite materials prepared in Comparative Examples 1-6 and Examples 1-3; Figure 6 The results of the anti-collapse test of the bone cement composite materials prepared by Comparative Examples 1-3, Comparative Examples 5-6, and Examples 1-3 are shown in Figure (A): Static anti-collapse result diagram; (B): Dynamic anti-collapse result diagram. Figure 7 The results of CCK8 testing are for the bone cement composite materials prepared in Comparative Examples 1-3, Comparative Example 6, and Example 3. Figure 8 The relative cell proliferation rate of the bone cement composite materials prepared in Comparative Examples 1-3, Comparative Example 6, and Example 3; Figure 9 The results of ALP testing are for the bone cement composite materials prepared in Comparative Examples 1-3, Comparative Example 6, and Example 3. Figure 10 The results of ARS testing of bone cement composite materials prepared in Comparative Examples 1-3, Comparative Example 6, and Example 3 are shown in the figure. (A): Blank; (B): α-TCP / α-CSH; (C): α-TCP / α-CSH-MC; (D): α-TCP / α-CSH-MC / SF; (E): α-TCP / α-CSH-MC / CMCS5; (F): α-TCP / α-CSH-MC / SF / CMCS5. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below, but the implementation of the present invention is not limited thereto.

[0029] The sources of the relevant materials involved in the embodiments of this invention are as follows: The phosphoric acid, calcium carbonate, calcium hydroxide, calcium sulfate dihydrate, collagen, and silk used in the examples were all commercially available chemically pure raw materials.

[0030] α-Tricalcium phosphate (α-TCP) is prepared by the following method: (1) At room temperature, add 29.98 g of phosphoric acid (H3PO4) to 1 L of deionized water to make a 0.26 mol / L phosphoric acid solution, and stir it evenly in a magnetic stirrer; (2) Add 13.13 g of calcium carbonate (CaCO3) and stir magnetically for 90 min to allow it to react fully; (3) Add 10.13 g of calcium hydroxide (Ca(OH)2) to 1 L of deionized water to make a 0.13 mol / L calcium hydroxide solution, and stir it evenly in a magnetic stirrer; (4) Add the solution obtained in steps (1) and (2) above into a separatory funnel, drop it into the solution in step (3), and stir continuously for 4 h; (5) After the solution has been added dropwise, add 13.13 g of calcium carbonate (CaCO3), stir continuously for 12 h, and then let stand for 4~5 h; (6) After settling, the precipitate is evenly loaded into centrifuge tubes and centrifuged at 3000 r / min for 15 min. (7) After centrifugation, the precipitate is taken out and ball-milled for 2 hours; (8) After repeating step (6), perform vacuum drying at 80℃ for 24 h; (9) Grind the tricalcium phosphate (Ca3(PO4)2) prepared above into powder, sinter it in a high-temperature furnace at 1400 °C for 5.5 h, and then quickly take it out and cool it down. (10) Grind and sieve to obtain α-TCP powder with a particle size distribution mainly in the range of 0.13~0.56 μm.

[0031] α-Calcium sulfate hemihydrate (α-CSH) is prepared by the following method: (1) Weigh an appropriate amount of calcium sulfate dihydrate (CSD) as raw material; (2) Using the hydrothermal synthesis method, CSD is placed in a vertical pressure steam sterilizer; (3) The temperature was set at 127℃, and the reaction was carried out under saturated vapor pressure for 9 h; (4) After taking it out, place it in an oven to dry to obtain blocky solid α-CSH; (5) Then the blocky solid α-CSH is ground and sieved to obtain white α-CSH powder.

[0032] Silk fibroin (SF) is prepared by the following method: (1) Weigh out 50 g of silk as raw material; (2) Boil 2000 mL of deionized water, add 5 g of Na2CO3, dissolve, then add silk and stir for 30 min; (3) Rinse three times with deionized water and rub vigorously; (4) Repeat step (3) 3 times, put it into the dryer to dry, and complete the SF degumming; (5) Weigh the dried silk and add the corresponding mass of LiBr powder and deionized water to prepare a LiBr solution; (6) The dried silk was gradually dissolved in a small amount of the prepared LiBr solution in a 65°C water bath. (7) After complete dissolution, perform water dialysis for 2 days, and then freeze dry to obtain SF powder.

[0033] Mineralized collagen / silk fibroin (MC / SF) is prepared by the following method: (1) At room temperature, add 2.14 mL of glacial acetic acid to 75 mL of deionized water to make a 0.5 mol / L glacial acetic acid solution, and stir it evenly in a magnetic stirrer; (2) Add 75 mg of collagen and stir evenly for 24 h; (3) Add 1.04 g of CaCl2 powder to 18 mL of deionized water to make a 0.5 mol / L CaCl2 solution, add it to the above solution, and stir for 2 h; (4) Add another 360 μL of H3PO4 solution and stir for 2 h; (5) After adjusting the pH of the above solution to around 8 using NaOH solution, add 75 mg of silk fibroin powder and stir overnight for 12 h; (6) After standing for 3-12 hours to settle, take the precipitate and centrifuge it 3-5 times until pH=7; (7) Freeze-dry for 24 h, grind and sieve to obtain MC / SF powder.

[0034] Specific embodiments and comparative examples are given below: Comparative Example 1 (Curing solution is deionized water) Using deionized water as the curing liquid, 2.55 g of α-CSH dry powder and 0.45 g of α-TCP dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this example was labeled α-CSH / α-TCP.

[0035] Comparative Example 2 Using deionized water as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this example was labeled α-CSH / α-TCP / MC.

[0036] Comparative Example 3 Using deionized water as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC / SF dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this example was labeled α-CSH / α-TCP / MC / SF.

[0037] Comparative Example 4 Using a 1 wt% carboxymethyl chitosan solution as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this example was labeled α-CSH / α-TCP / MC / CMCS1.

[0038] Comparative Example 5 Using a 3wt% carboxymethyl chitosan solution as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this example was labeled α-CSH / α-TCP / MC / CMCS3.

[0039] Comparative Example 6 Using a 5wt% carboxymethyl chitosan solution as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this example was labeled α-CSH / α-TCP / MC / CMCS5. Example 1

[0040] Using a 1 wt% carboxymethyl chitosan solution as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC / SF dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the mold was removed to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this example was labeled α-CSH / α-TCP / MC / SF / CMCS1. Example 2

[0041] Using a 3wt% carboxymethyl chitosan solution as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC / SF dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The sample prepared in this embodiment was labeled α-CSH / α-TCP / MC / SF / CMCS3. Example 3

[0042] Using a 5wt% carboxymethyl chitosan solution as the curing liquid, 2.55 g of α-CSH dry powder, 0.45 g of α-TCP dry powder, and 0.3 g of MC / SF dry powder were placed in a mixing container and mixed thoroughly. Deionized water was added to the mixed powder at a liquid-to-solid ratio (liquid volume to total solid mass) of 0.6 mL / g, and the mixture was rapidly stirred until homogeneous to obtain bone cement slurry. A portion of the bone cement slurry was quickly injected into a mold, avoiding the introduction of air bubbles during the process. After standing until completely solidified, the slurry was demolded to obtain bone cement columns and bone cement particles, which were used for mechanical property testing and cell proliferation and osteogenic induction experiments, respectively. Another portion of the bone cement slurry was loaded into a 5 mL syringe with an outlet diameter of 2.0 mm using a stirring spoon for injectability testing and anti-collapse performance experiments. The samples prepared in this example were labeled α-CSH / α-TCP / MC / SF / CMCS.

[0043] Embodiments 1-3 of the present invention employ Figure 1 The process shown is used for preparation.

[0044] Figure 2 Electron micrographs of the bone cement composite materials prepared in Comparative Examples 1-6 and Examples 1-3 are shown; Figure 2 It can be observed that the surface of the bone cement composite material is mainly composed of rod-shaped α-CSH, granular α-TCP, and plate-shaped crystals that generate CSD. They all have rod-like structures, but after the addition of mineralized collagen, a small number of aggregated particles appear, and this phenomenon becomes more pronounced after the addition of silk fibroin. Furthermore, as the CMCS increases, the gaps between materials decrease, and the structure becomes more compact.

[0045] Figure 3The results show the setting times of the bone cement composites prepared in Examples 1-6 and Examples 1-3. The setting time of α-CSH / α-TCP was 33.14 ± 1.35 min. With increasing CMCS concentration, the curing time of the composite material decreased, ranging from 10 to 30 min, all reaching the optimal curing time for bone cement. The shortest curing time was 16.17 ± 1.47 min for a CMCS concentration of 5% mixed with MC / SF. When CMCS and MC / SF are added together, their synergistic effect optimizes solution viscosity, promotes interfacial reactions, enhances calcium ion release and complexation, and forms a more stable network structure, thereby accelerating the hydration reaction and setting process, and shortening the setting time.

[0046] Figure 4 The results show the compressive strength of the bone cement composites prepared in Examples 1-6 and Examples 1-3. Overall, the addition of CMCS improves the mechanical properties of the bone cement, and the mechanical properties gradually increase with increasing CMCS concentration. Furthermore, the composite exhibits the highest compressive strength (11.59 ± 1.03 MPa) when the CMCS concentration is 5%. This is because MC / SF and CMCS cross-link with dissolved calcium ions to form a hydrogel, significantly improving the compressive strength of the composite. Additionally, CMCS, composed of parallel H-linked chains, is structurally similar to collagen in natural bone, thus enhancing the mechanical properties of the composite. This indicates that the gel network formed by carboxymethyl chitosan (CMCS) and mineralized collagen / silk fibroin (MC / SF) can fill the gaps between α-TCP and α-CSH particles, enhancing interfacial bonding and significantly improving the compressive strength of the bone cement.

[0047] Figure 5 The results show the injectability of the bone cement composite materials prepared in Comparative Examples 1-6 and Examples 1-3. The injectability of the unadded bone cement is around 50%, while the injectability of the composite bone cement in Comparative Example 4 is only about 20%. The injectability of the remaining composite bone cements is all above 40%. Among them, the injectability of α-CSH / α-TCP / MC / SF / CMCS5 is 97.01% ± 0.37%. This indicates that appropriate proportions of CMCS and MC / SF can utilize the thickening properties of CMCS and the gel network of MC / SF to adjust the viscosity of the bone cement paste and improve injectability.

[0048] Figure 6 Figure A shows the static anti-collapse results of the bone cement composite materials prepared in Examples 1-3, 5-6, and Examples 1-3, while Figure B shows the dynamic anti-collapse results of the bone cement composite materials prepared in Examples 1-3, 5-6, and Examples 1-3. Figure 6As shown, unadded bone cement exhibits poor collapse resistance. In bone cements with different concentrations of CMCS, the collapse resistance improves with increasing CMCS concentration. Furthermore, dynamic collapse resistance testing reveals that bone cement with 5% CMCS and MC / SF composite exhibits only minimal collapse, demonstrating superior collapse resistance. This is because as the mass proportion of carboxymethyl chitosan increases, it chelates with dissolved calcium ions to form a water-soluble colloidal solution with good viscosity. This solution effectively prevents water penetration into the material during the paste-like stage of the bone cement, thus avoiding collapse. This indicates that the gel network formed by MC / SF and CMCS increases the cohesion and viscosity of the bone cement composite, making the mixture more stable, less prone to collapse, and improving collapse resistance.

[0049] Figure 7 The results of CCK8 testing on the bone cement composite materials prepared in Comparative Examples 1-3 and 6, and Example 3, show the cell proliferation of the blank group and control group (α-CSH / α-TCP group, α-CSH / α-TCP / MC, α-CSH / α-TCP / MC / SF, α-CSH / α-TCP / MC / CMCS5, and α-CSH / α-TCP / MC / SF / CMCS5) after 1, 3, and 5 days of culture. The results show that the cell survival rate was similar in all groups on the first day, all groups showed good cell proliferation characteristics on the third day, and the OD values ​​of all groups increased on the fifth day, indicating that the cell survival rate increased over time. The cell survival rate of the α-CSH / α-TCP / MC / SF / CMCS5 group increased by 31.74% compared to the control group (Comparative Example 1), showing better biocompatibility. This also indicates that the addition of MC / SF and CMCS is more conducive to cell growth and proliferation.

[0050] Figure 8 The relative growth ratio (RGR) of each group was calculated on day 5, and its cytotoxicity was analyzed. All groups showed values ​​greater than 80%, with a cytotoxicity grade of 1. This indicates that the bone cement composite material has good cell compatibility and its cytotoxicity to MC3T3 cells is negligible. Furthermore, the RGR values ​​of the group with added α-CSH / α-TCP / MC were generally higher than those of the control group. Moreover, the addition of α-CSH / α-TCP to CMCS and MC / SF did not cause cytotoxicity and promoted cell proliferation and growth. This is because CMCS and MC / SF are natural polymer materials with good biocompatibility, which can promote cell proliferation and differentiation.

[0051] Figure 9The results of ten-day ALP tests on the bone cement composite materials prepared in Examples 1-3 and 6, and Example 3, are as follows: The absorbance of the Blank group was 0.43, which, as a blank group, was higher than that of the control group (0.43), but the difference was not statistically significant. Components with added MC showed significantly higher absorbance than the Blank group, indicating that MC has a certain promoting effect on ALP activity, which is beneficial to osteoblast differentiation. Among them, the α-CSH / α-TCP / MC / SF / CMCS group had the highest absorbance value, indicating that this group had the strongest promoting effect on ALP activity. Compared with the Blank group and the Control group, the α-CSH / α-TCP / MC, α-CSH / α-TCP / MC / SF, α-CSH / α-TCP / MC / CMCS5, and α-CSH / α-TCP / MC / SF / CMCS5 groups all significantly increased ALP activity, indicating that the composite material is conducive to osteoblast differentiation. MC and SF have good biocompatibility and bioactivity, and can promote osteoblast adhesion and differentiation. CMCS can further improve the material microenvironment, making cells easier to grow, while providing a favorable ion exchange environment and enhancing osteogenic activity. In summary, the synergistic effect of MC / SF and CMCS significantly increases ALP activity.

[0052] Figure 10 The results of the alizarin red test on the bone cement composite materials prepared in Examples 1-3 and 6 (Examples 3) after ten days show that orange-red mineralized nodules were visible within the cells under a microscope. The Blank group showed the lightest staining, with sparse and small nodules and the worst calcium nodule formation. The Control group showed relatively light staining and average nodule density. The α-CSH / α-TCP / MC group exhibited multiple dense clusters of red nodules with good area and staining depth. The α-CSH / α-TCP / MC / SF group showed dense and deeply stained red areas and good overall performance. The α-CSH / α-TCP / MC / CMCS5 group showed large, blocky red areas with high staining depth and excellent performance. The α-CSH / α-TCP / MC / SF / CMCS5 group showed large-area fusion of red areas with extremely deep staining and optimal calcium nodule formation. Therefore, in this experiment, the α-CSH / α-TCP / MC / SF / CMCS group of the bone cement composite material showed a certain promoting effect on osteogenic induction and had a positive impact on osteogenic differentiation of cells.

[0053] Table 1. Composition ratio of composite materials

[0054] Relative cell proliferation rate (%) = OD value of experimental group / OD value of blank control group × 100 Note: In the calculation of relative growth ratio, the blank control group is a group of normally cultured cells without material intervention, and its growth rate is set to 100%.

[0055] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the protection scope of the present invention.

Claims

1. A bone cement, characterized in that, The solid phase and the liquid phase are included, and the liquid-solid ratio is 0.4-0.6 mL / g; the solid phase is composed of α-calcium sulfate hemihydrate powder, α-tricalcium phosphate powder and mineralized collagen / silk powder in a mass ratio of (55-77):(14-36):9; the mineralized collagen / silk powder is composed of mineralized collagen and silk, and the mass ratio of the two is 2:(1-4); and the liquid phase is a carboxymethyl chitosan solution.

2. Bone cement according to claim 1, characterized in that The concentration of the carboxymethyl chitosan solution is 1 wt%-5 wt%.

3. A method of preparing the bone cement according to claim 1 or 2, characterized in that The method comprises the following steps: Step one, synthesis of α-CSH powder; α-CSH is hydrothermally synthesized by taking calcium sulfate dehydrate as a raw material; Step two, synthesis of α-TCP powder; α-TCP is synthesized by high-temperature sintering by taking calcium carbonate, phosphoric acid and calcium hydroxide as raw materials; Step three, synthesis of MC / SF powder; MC / SF is synthesized by biomimetic mineralization by taking bovine collagen and silk as raw materials; Step four, preparation of a carboxymethyl chitosan solution; the preparation method is as follows: the CMCS powder is dissolved in water to prepare a CMCS solution with a concentration of 1 wt%-5 wt%; Step five, the three powders synthesized in steps one to three are mixed in a proportion as the solid phase, and are mixed with the CMCS solution in a liquid-solid ratio of 0.4-0.6 mL / g to form a uniform slurry by stirring; Step six, the setting time is 10-16 minutes at room temperature; and the compressive strength after 24 hours of solidification is greater than or equal to 10 MPa.

4. The method of preparing bone cement according to claim 3, characterized in that, The preparation method of the α-CSH powder in step one is as follows: (1) calcium sulfate dehydrate is weighed as a raw material; (2) the CSD is placed in a vertical pressure steam sterilizer by using a hydrothermal synthesis method; (3) the temperature is set to 127 DEG C, and the reaction is carried out under saturated steam pressure for 9 h; (4) after taking out, drying in an oven can obtain blocky solid α-CSH; (5) then the blocky solid α-CSH is ground, sieved, and α-CSH white powder is obtained.

5. The method of preparing bone cement according to claim 3, characterized in that, The preparation process of the α-TCP powder in step two is as follows: (1) at room temperature, 29.98 g of phosphoric acid is added to 1 L of deionized water to prepare a 0.26 mol / L phosphoric acid solution, which is stirred uniformly in a magnetic stirrer; (2) 13.13 g of calcium carbonate is added, and magnetic stirring is carried out for 90 min to make it fully react; (3) 10.13 g of calcium hydroxide is added to 1 L of deionized water to prepare a 0.13 mol / L calcium hydroxide solution, which is stirred uniformly in a magnetic stirrer; (4) the solutions prepared in steps (1) and (2) are added to a separatory funnel, and the solution in step (3) is added dropwise, and stirring is continued for 4 h; (5) after the solution is added dropwise, 13.13 g of calcium carbonate is added, and stirring is continued for 12 h, and then it is left to stand for 4-5 h; (6) after standing is completed, the precipitate is evenly loaded into a centrifuge tube for centrifugal separation at 3000 r / min for 15 min; (7) after the precipitate separated by centrifugation is taken out, ball milling is carried out for 2 h; (8) after repeating step (6), vacuum drying is carried out at 80 DEG C for 24 h; (9) the tricalcium phosphate prepared above is ground into powder, and after sintering in a high-temperature furnace at 1400 DEG C for 5.5 h, it is quickly taken out for quenching; (10) The α-TCP powder with a particle size distribution mainly ranging from 0.13 to 0.56 μm and an average particle size of 0.21 μm was obtained by grinding and sieving.

6. The method of preparing bone cement according to claim 3, characterized in that, The method for synthesizing the MC / SF powder in Step three is as follows: (1) 75 mg of bovine collagen was weighed and added into 75 mL of deionized water and 2.14 mL of glacial acetic acid, and stirred and dissolved for 24 h; (2) A CaCl2 solution prepared by mixing 18 mL of deionized water and 1.04 g of CaCl2 powder was further added, and stirred for 2 h; (3) 360 μL of H3PO4 solution was further added, and stirred for 2 h; (4) Then, the pH of the above solution was adjusted to about 8 by using NaOH, and 75 mg of silk fibroin was further added to dissolve, and stirred overnight for 12 h; (5) The precipitate was allowed to stand for 3-24 h, and centrifuged for 3-5 times until the pH of the supernatant was 7; (6) The precipitate was subjected to freeze-drying, grinding and sieving to obtain the MC / SF powder.

7. Use of the bone cement according to claim 1 or 2 in alveolar bone repair.

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