A method for preparing a hydroxyapatite bone tissue engineering scaffold

Abstract

This invention relates to the field of biomedical materials technology, and more particularly to a method for preparing a hydroxyapatite bone tissue engineering scaffold, comprising the following steps: preparing acetylated or alkylated modified cellulose nanocrystals; using the modified cellulose nanocrystals as a biodispersant to synthesize hydroxyapatite; and using the synthesized hydroxyapatite to prepare a bone tissue engineering scaffold. The preparation method of this invention is simple, requires minimal experimental conditions, and does not require expensive equipment; it also does not introduce organic surfactants, and the dispersant used is essentially harmless to the environment and organisms; the prepared HA exhibits good dispersibility; and the hydroxyapatite bone tissue engineering scaffold has good biocompatibility, which is beneficial for osteoblast proliferation and differentiation, and for the structural remodeling and construction of bone defect tissue.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a method for preparing a hydroxyapatite bone tissue engineering scaffold. Background Technology

[0002] In recent years, bone tissue engineering scaffolds have become a research hotspot in the field of bone injury and repair due to their high cost-effectiveness and simple manufacturing process. Scaffolds serve as temporary matrices supporting tissue and cell growth, providing essential support for cell proliferation and differentiation. The success of bone tissue engineering scaffolds in aiding cell and tissue growth depends primarily on porosity (cell proliferation, cell differentiation, waste and nutrient exchange), sufficient mechanical properties (depending on applied biomechanical stress), superior biocompatibility (within the time frame required to provide effective cell regeneration), and the economics of the entire process (from research to implementation). The main criteria required for bone tissue engineering scaffolds can be customized according to the application, through targeted selection of polymer type, scaffold raw materials, and preparation methods. Traditional scaffold designs include hydrogels and fibers. Natural bone is a highly dynamic connective tissue with a porous, coaxial, ring-like structure; the excellent properties of natural bone tissue are closely related to its internal cells, material composition, and microstructure. During transplantation, defects in the original natural bone tissue may induce immune rejection. Bone tissue engineering scaffolds are required to be absorbed or degraded by the environment within a certain timeframe.

[0003] Hydroxyapatite (HA) is a common bioceramic material used for bone repair and regeneration due to its biocompatibility, osteoconductivity, and ability to accelerate the reconstruction of damaged bone tissue. Hydroxyapatite is a significant component of natural bone, accounting for up to 50%. Its excellent biocompatibility and strong affinity with human tissues make it ideal for applications combining biomedicine and human tissues. Hydroxyapatite is naturally produced through biomineralization processes in living cells and can also be chemically synthesized in the laboratory. Synthetic HA exhibits different morphologies and a unique Ca / P ratio, with a stoichiometric ratio of 1.67, similar to natural HA in human bone. Therefore, HA nanoparticles have been widely used in biomedical applications as bioactive compounds to promote bone and tooth repair in composite materials with biopolymers.

[0004] Currently, there are many methods for synthesizing hydroxyapatite (HA) in polymer matrices, including in-situ mineralization and wet chemical precipitation or pyrolysis before mixing and dispersion. However, these methods often produce HA with poor crystallinity, or in aggregated and agglomerated form, resulting in low strength and affecting the dispersibility and mechanical properties of the composite material, thus limiting its application range. Therefore, we can use biodispersants to improve the stability and surface activity of hydroxyapatite. Among naturally occurring dispersants, cellulose nanocrystals (CNCs) are a promising biodispersant due to their good biocompatibility. Cellulose nanocrystals (CNCs) can be acid-hydrolyzed to eliminate amorphous structural regions and retain crystalline regions, and are widely used as reinforcing agents in the materials and food industries as well as tissue engineering scaffolds in biomedical applications. In addition to providing mechanical properties, the extensive colloidal distribution of CNCs in PVA matrices has been well-documented, exhibiting biocompatibility, high mechanical properties, and crystallinity. Using CNCs as dispersants is a promising method for synthesizing hydroxyapatite with excellent properties. Therefore, composite materials combining HA nanoparticles with organic matter have been extensively studied in order to prepare high-performance artificial bone tissue engineering scaffolds.

[0005] To date, although there have been studies on the synthesis of HA using organic surfactants or some inorganic dispersants, these methods have the following drawbacks: (1) Hydroxyapatite nanoparticle sols synthesized at high temperatures have low crystallinity and uneven particle morphology; (2) The surfactants or stabilizers used are highly toxic and cannot be used in the biomedical field; (3) After drying, HA is prone to agglomeration, and the particle size is inconsistent, resulting in poor uniformity; (4) The reaction conditions are high (high temperature, high pressure), the equipment requirements are high, the cost is high, the safety is poor, the yield is low, and it is not conducive to industrial production; (5) It consumes a lot of energy, pollutes the environment, and cannot achieve sustainable development.

[0006] Therefore, it is crucial to invent a stable method for synthesizing high-performance hydroxyapatite bone tissue engineering scaffolds. Summary of the Invention

[0007] This invention provides a method for preparing a hydroxyapatite bone tissue engineering scaffold to solve the aforementioned technical problems existing in the prior art.

[0008] According to a first aspect of the present invention, the present invention provides a method for preparing a hydroxyapatite bone tissue engineering scaffold, comprising the following steps:

[0009] Preparation of acetylated or alkylated modified cellulose nanocrystals;

[0010] The modified cellulose nanocrystals were used as a biodispersant to synthesize hydroxyapatite;

[0011] Bone tissue engineering scaffolds were prepared using the synthesized hydroxyapatite.

[0012] This invention first modifies CNCs by alkylation or acetylation to prepare modified CNCs with high thermal stability and hydrophobic properties. The modified and unmodified CNCs are used as biodispersants to synthesize hydroxyapatite. The synthesized hydroxyapatite is then incorporated into a colloidal polymer matrix (such as polyvinyl alcohol) to prepare a bone tissue engineering scaffold, further improving the mechanical stability and pore uniformity of the bone tissue engineering scaffold. This invention provides a simple method for preparing a hydroxyapatite bone tissue engineering scaffold, requiring minimal experimental conditions and no expensive equipment. Furthermore, it does not introduce organic surfactants, and the dispersant used is essentially harmless to the environment and organisms. The prepared hydroxyapatite (HA) exhibits excellent dispersibility. The hydroxyapatite bone tissue engineering scaffold demonstrates good biocompatibility, promoting osteoblast proliferation and differentiation, and facilitating the structural remodeling and reconstruction of bone defect tissue. The hydroxyapatite in the bone tissue engineering scaffold more closely resembles the organic-inorganic composition of human bone, exhibiting high plasticity, high porosity, and uniform pore distribution during scaffold molding, resulting in stronger compressive strength. The phases are uniformly dispersed and possess good homogeneity. This method expands the application scope of HA in biomedical clinical settings and has significant application value.

[0013] Furthermore, the preparation of acetylated modified cellulose nanocrystals includes the following steps:

[0014] Step A1: Disperse CNCs in toluene solution using ultrasonication under ice-water bath conditions, and then use an ultrasonic cleaner to help disperse them evenly to obtain a CNCs suspension.

[0015] Step A2: Add the CNCs suspension obtained in step A1 into the reaction vessel, and add glacial acetic acid, acetic anhydride and perchloric acid in sequence. After ultrasonic-assisted reaction at room temperature for 3-5 hours, add anhydrous ethanol to terminate the reaction and obtain the reaction product.

[0016] Step A3: Dialyze the reaction product obtained in step A2; collect the dialyzed mixture and freeze-dry it to obtain A-CNCs.

[0017] In the above scheme, during the preparation of acetylated modified cellulose nanocrystals, CNCs are first ultrasonically dispersed in toluene solution under ice-water bath conditions to obtain a uniformly dispersed CNCs suspension. Then, acetic anhydride is used as an acetylation agent to prepare modified A-CNCs with high thermal stability and hydrophobic properties.

[0018] Further, in step A1, the weight-to-volume ratio of the CNCs to the toluene solution is (0.2-0.5):10.

[0019] In the above scheme, by limiting the weight-to-volume ratio of CNCs to toluene solution in step A1 to a reasonable range, it is beneficial to form a stable and uniform CNCs suspension.

[0020] Further, in step A2, the volume ratio of the glacial acetic acid, the acetic anhydride, the perchloric acid, and the anhydrous ethanol is 8:5:0.02:20.

[0021] In the above scheme, limiting the volume ratio of glacial acetic acid, acetic anhydride, perchloric acid and anhydrous ethanol in step A2 to a reasonable range is beneficial to the full and effective progress of the acetylation reaction.

[0022] Further, in step A3, the specific steps of dialysis are as follows: the obtained reaction product is dialyzed with anhydrous ethanol for 2-3 days in a dialysis bag with a molecular weight of 7000Da-14000Da, and then transferred to deionized water for dialysis for one week.

[0023] In the above scheme, by adopting a specific dialysis process, the reaction products can be better separated and purified, thereby improving the purification rate.

[0024] Furthermore, the preparation of alkylated modified cellulose nanocrystals includes the following steps:

[0025] Step K1: CNCs are ultrasonically dispersed in an ethanol solution under ice-water bath conditions to obtain a CNCs suspension;

[0026] Step K2: Prepare a KH-550 working solution with a weight percentage of 10%-15%, add it to the CNCs suspension obtained in step K1, adjust the pH of the mixture to between 4 and 5, and perform an ultrasonic-assisted reaction at room temperature; add anhydrous ethanol to terminate the reaction and obtain the reaction product.

[0027] Step K3: Dialyze the reaction product obtained in step K2; freeze-dry the dialyzed suspension to obtain K-CNCs.

[0028] In the above scheme, during the preparation of alkylated modified cellulose nanocrystals, CNCs are first ultrasonically dispersed in an ethanol solution under ice-water bath conditions to obtain a uniformly dispersed CNCs suspension. Then, KH-550 working solution is used as an alkylating agent to prepare modified K-CNCs with high thermal stability and hydrophobic properties.

[0029] Further, in step K1, the weight-to-volume ratio of the CNCs to the ethanol solution is (0.2-0.5):20.

[0030] In the above scheme, by limiting the weight-to-volume ratio of CNCs to ethanol solution in step K1 to a reasonable range, it is beneficial to form a stable and uniform CNCs suspension.

[0031] Furthermore, in step K1, the ultrasound time is 5 min to 15 min.

[0032] In the above scheme, limiting the ultrasonic time in step K1 to a reasonable range is beneficial to forming a stable and uniform CNCs suspension.

[0033] Furthermore, in step K2, the ultrasound-assisted reaction time is 8-10 hours.

[0034] In the above scheme, limiting the time of the ultrasonic-assisted reaction in step K2 to a reasonable range is beneficial to the alkylation reaction and improves the reaction efficiency.

[0035] Further, in step K3, the specific steps of dialysis are as follows: the reaction product obtained in step K2 is dialyzed with deionized water for one week in a dialysis bag with a molecular weight of 7000Da-14000Da until the pH value is stable and neutral.

[0036] In the above scheme, by adopting a specific dialysis process, the reaction products can be better separated and purified, thereby improving the purification rate.

[0037] Furthermore, the modified cellulose nanocrystals are used as a biodispersant to synthesize hydroxyapatite via an in-situ precipitation method, specifically including the following steps:

[0038] A modified cellulose nanocrystal suspension was prepared and reacted with calcium hydroxide to obtain a mixture; phosphoric acid was added dropwise to the mixture, and the reaction continued to obtain a hydroxyapatite suspension; the hydroxyapatite suspension was washed until the pH value was neutral, and hydroxyapatite particles were obtained.

[0039] In the above scheme, modified cellulose nanocrystals are mixed and reacted with calcium hydroxide. Phosphoric acid is added to continue the reaction to obtain a hydroxyapatite suspension. The pH value of the hydroxyapatite suspension is neutral after washing, which can obtain hydroxyapatite particles with high crystallinity. The obtained hydroxyapatite particles have good dispersibility, have a calcium-phosphorus ratio similar to that of natural HA, and have excellent performance.

[0040] Furthermore, the modified cellulose nanocrystal suspension has a weight percentage of 3%-5%.

[0041] In the above scheme, limiting the weight percentage of the modified cellulose nanocrystal suspension within a reasonable range is beneficial to obtaining hydroxyapatite particles with better performance.

[0042] Furthermore, the reaction time of the modified cellulose nanocrystal suspension with calcium hydroxide is 1h-3h, and the temperature is 55℃-65℃.

[0043] In the above scheme, by limiting the reaction time and temperature of the modified cellulose nanocrystal suspension with calcium hydroxide within a reasonable range, the mixing reaction is facilitated and the reaction efficiency is improved.

[0044] Furthermore, the concentration of the calcium hydroxide is 0.5M-1.5M, preferably 1M.

[0045] In the above scheme, limiting the concentration of calcium hydroxide within a reasonable range is beneficial to the mixed reaction and improves the reaction efficiency.

[0046] Further, phosphoric acid is added dropwise to the resulting mixture, and the reaction continues for 4-6 hours.

[0047] In the above scheme, limiting the reaction time to a reasonable range is beneficial for the alkylation reaction to proceed fully and improves the reaction efficiency.

[0048] Furthermore, the preparation of a bone tissue engineering scaffold using the synthesized hydroxyapatite specifically includes the following steps:

[0049] The synthesized hydroxyapatite was formulated into a hydroxyapatite suspension; the hydroxyapatite suspension was added to a PVA solution and mixed; air bubbles in the suspension after the reaction were removed by ultrasonic treatment; and the suspension was cast onto a 6-well plate and freeze-dried under vacuum to obtain a bone tissue engineering scaffold.

[0050] Furthermore, the weight percentage of the hydroxyapatite suspension is 3%-12%.

[0051] In the above scheme, limiting the weight percentage of hydroxyapatite suspension within a reasonable range is beneficial for the formation of bone tissue engineering scaffolds.

[0052] Furthermore, the PVA solution has a weight percentage of 5%-10%.

[0053] In the above scheme, limiting the weight percentage of PVA solution within a reasonable range is beneficial for the formation of bone tissue engineering scaffolds.

[0054] Furthermore, the hydroxyapatite suspension is added to the PVA solution and mixed for 3-5 hours.

[0055] In the above scheme, by limiting the mixing time of the hydroxyapatite suspension into the PVA solution to a reasonable range, it is beneficial to the formation of the bone tissue engineering scaffold, so that the prepared bone tissue engineering scaffold has a uniform pore distribution, high compressive strength, high mechanical strength, and a highly dense porous interconnected structure at the nanoscale, while also having good uniformity.

[0056] Furthermore, the ultrasonic treatment time is 15-25 minutes.

[0057] In the above scheme, limiting the ultrasonic treatment time to a reasonable range is beneficial to the uniform distribution of pores in the bone tissue scaffold, and the formation of a dense interconnected pore channel structure after freeze-drying.

[0058] The beneficial effects of this invention are:

[0059] (1) The method for preparing a hydroxyapatite bone tissue engineering scaffold of the present invention does not introduce chemical reagents. The dispersant used is green and natural, with good dispersibility and biocompatibility, and is harmless to the environment and organisms.

[0060] (2) The HA prepared by the nano-biodispersant used in this invention has high crystallinity, good dispersibility, and a calcium-to-phosphorus ratio similar to that of natural HA. It has excellent performance and broad application prospects.

[0061] (3) The bone tissue engineering scaffold prepared by the present invention has good biocompatibility, which can promote the proliferation, differentiation and mineralization of osteoblasts, and is beneficial to repairing bone defect tissue and reshaping bone tissue structure.

[0062] (4) The hydroxyapatite in the bone tissue engineering scaffold of the present invention is closer to the ratio of organic and inorganic components in human bone, and has high plasticity and high porosity during scaffold molding.

[0063] (5) The bone tissue engineering scaffold prepared by the present invention has uniform pore distribution, high compressive strength, high mechanical strength, and a highly dense porous interconnected structure at the nanoscale. It also has good uniformity and meets the principle of personalized customization of scaffolds.

[0064] (6) The reaction conditions of this invention are mild, the process is simple and easy to implement, the experimental conditions are simple, the equipment requirements are low, and no expensive equipment is required. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0066] Figure 1 This is a process flow diagram of a method for preparing a hydroxyapatite bone tissue engineering scaffold according to the present invention;

[0067] Figure 2 EDS analysis diagrams of different hydroxyapatite particles of the present invention;

[0068] Figure 3 These are TEM images of different hydroxyapatite particles from the present invention.

[0069] Figure 4 This is a SEM image of the PHC-K bone tissue engineering scaffold in an embodiment of the present invention;

[0070] Figure 5 This is a SEM image of the PHC-A bone tissue engineering scaffold in an embodiment of the present invention;

[0071] Figure 6 This is a SEM image of the PHC-C bone tissue engineering scaffold in the comparative example of this invention;

[0072] Figure 7 This is a SEM image of the PHC-P bone tissue engineering scaffold in the comparative example of this invention;

[0073] Figure 8 This is a SEM image of the PHC-HA bone tissue engineering scaffold in the comparative example of this invention;

[0074] Figure 9 The image shown is an FTIR image of the PHC-K stent in an embodiment of the present invention.

[0075] Figure 10 The image shown is an FTIR image of the PHC-A stent in an embodiment of the present invention.

[0076] Figure 11 This is the FTIR image of the PHC-P stent in the comparative example of this invention;

[0077] Figure 12 This is the FTIR image of the PHC-C stent in the comparative example of this invention;

[0078] Figure 13 This is the FTIR image of the PHC-HA stent in the comparative example of this invention;

[0079] Figure 14 The XRD pattern of the PHC-K stent in this embodiment of the invention;

[0080] Figure 15 The image shows the XRD pattern of the PHC-A stent in this embodiment of the invention.

[0081] Figure 16 The XRD pattern of the PHC-P stent in the comparative example of this invention;

[0082] Figure 17 The image shows the XRD pattern of the PHC-C stent in the comparative example of this invention.

[0083] Figure 18 The XRD pattern of the PHC-HA stent in the comparative example of this invention;

[0084] Figure 19 This is a graph showing the cell viability of different bone tissue engineering scaffolds of the present invention;

[0085] Figure 20 This is a diagram showing the cell proliferation capacity of different bone tissue engineering scaffolds of the present invention.

[0086] Figure 21 These are cell live / dead fluorescence staining images of different bone tissue engineering scaffolds of the present invention;

[0087] Figure 22 This is an ALP activity diagram of different bone tissue engineering scaffolds of the present invention. Detailed Implementation

[0088] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0089] Example 1

[0090] This embodiment provides a method for preparing a hydroxyapatite bone tissue engineering scaffold, the process flow diagram of which is shown below. Figure 1 As shown, the specific steps include the following:

[0091] (1) Preparation of alkylated modified cellulose nanocrystals: 0.4 g of CNCs were ultrasonically dispersed in 20.0 mL of ethanol solution under ice-water bath conditions to obtain a CNCs suspension. The ultrasonic time was 10 min. Then, 10% (w / w) KH-550 working solution was prepared and added to the CNCs suspension. The pH of the mixture was adjusted to 4-5 using glacial acetic acid. The reaction was ultrasonically assisted at room temperature for 8-10 h. 20 mL of anhydrous ethanol was added to terminate the reaction. The reaction product was dialyzed with deionized water in a dialysis bag with a molecular weight of 7000 Da-14000 Da for one week until the pH value was stable and neutral. Finally, the lyophilized suspension was obtained as K-CNCs.

[0092] (2) Synthesis of hydroxyapatite using modified cellulose nanocrystals as a biodispersant: A 3% (w / w) aqueous suspension of K-CNCs was prepared and mixed with 1M calcium hydroxide (pH=10) at 60℃ for 3 h. Then, 0.6M phosphoric acid was immediately added dropwise to the mixture for 6 h. The hydroxyapatite suspension was washed with distilled water until the pH value was 7, yielding hydroxyapatite particles, i.e., HC-K.

[0093] (3) Bone tissue engineering scaffolds were prepared using synthetic hydroxyapatite: 3%, 6%, 9%, and 12% (w / w) hydroxyapatite suspensions prepared with K-CNCs as dispersant were prepared using water as solvent. The suspensions were mixed in 5% (w / w) PVA solution for 3 hours. After the reaction was completed, the air bubbles in the suspensions were removed by ultrasonic treatment for 20 minutes. The suspensions were then cast onto 6-well plates and freeze-dried under vacuum for 3 days to obtain bone tissue engineering scaffolds, namely PHC-K scaffolds.

[0094] Example 2

[0095] This embodiment provides a method for preparing a hydroxyapatite bone tissue engineering scaffold, the process flow diagram of which is shown below. Figure 1 As shown, the specific steps include the following:

[0096] (1) Preparation of acetylated modified cellulose nanocrystals: 0.4 g of CNCs were ultrasonically dispersed in 10.0 mL of toluene solution under ice-water bath conditions, and the dispersion was assisted in an ultrasonic cleaner for 20 min. Then, the uniformly mixed CNCs suspension was added to a reaction vessel, followed by 8.0 mL of glacial acetic acid, 5 mL of acetic anhydride, and 0.02 mL of perchloric acid. After ultrasonic-assisted reaction for 3 h at room temperature, 20 mL of anhydrous ethanol was added to terminate the reaction. Subsequently, the reaction product was dialyzed with anhydrous ethanol in a dialysis bag with a molecular weight (7000-14000 Da) for 2-3 days, and then transferred to deionized water for one week. Finally, the dialyzed mixture was collected and freeze-dried to obtain A-CNCs.

[0097] (2) Synthesis of hydroxyapatite using modified cellulose nanocrystals as a biodispersant: A 3% (w / w) suspension of A-CNCs was prepared and mixed with 1M calcium hydroxide (pH=10) at 60℃ for 3 h. Then, 0.6M phosphoric acid was immediately added dropwise to the mixture for 6 h. The hydroxyapatite suspension was washed with distilled water until the pH value was 7, yielding hydroxyapatite particles, namely HC-A.

[0098] (3) Bone tissue engineering scaffolds were prepared using synthetic hydroxyapatite: 3%, 6%, 9%, and 12% (w / w) hydroxyapatite suspensions prepared with A-CNCs as dispersant were prepared and mixed in 5% (w / w) PVA solution for 3 hours. After the reaction was completed, the air bubbles in the suspension were removed by ultrasonic treatment for 20 minutes and then cast onto a 6-well plate. The bone tissue engineering scaffolds, namely PHC-A scaffolds, were obtained by vacuum freeze-drying for 3 days.

[0099] Comparative Example 1

[0100] This comparative example provides a method for preparing a hydroxyapatite bone tissue engineering scaffold, specifically including the following steps:

[0101] A 3% (w / w) suspension of commercially available cellulose nanocrystals (CCNCs, from ScienceK) was prepared and mixed with 1M calcium hydroxide (pH=10) at 60°C for 3 hours. Immediately afterwards, 0.6M phosphoric acid was added dropwise to the mixture over 6 hours. The hydroxyapatite suspension was washed with distilled water until the pH reached 7, yielding hydroxyapatite particles, i.e., HC-C.

[0102] Hydroxyapatite suspensions prepared with CCNCs as dispersant at concentrations of 3%, 6%, 9%, and 12% (w / w) were prepared and mixed in a 5% (w / w) PVA solution for 3 hours. After the reaction was completed, the suspensions were ultrasonically treated for 20 minutes to remove air bubbles. The suspensions were then cast onto 6-well plates and freeze-dried under vacuum for 3 days to obtain bone tissue engineering scaffolds, namely PHC-C scaffolds.

[0103] Comparative Example 2

[0104] This comparative example provides a method for preparing a hydroxyapatite bone tissue engineering scaffold, specifically including the following steps:

[0105] Prepare a 3% (w / w) CNCs suspension and mix it with 1M calcium hydroxide (pH=10) at 60°C for 3 hours. Then immediately add 0.6M phosphoric acid dropwise to the mixture and continue for 6 hours. Wash the hydroxyapatite suspension with distilled water until the pH value is 7 to obtain hydroxyapatite particles, i.e., HC-P.

[0106] Hydroxyapatite suspensions prepared with CNCs as dispersant at concentrations of 3%, 6%, 9%, and 12% (w / w) were prepared and mixed in a 5% (w / w) PVA solution for 3 hours. After the reaction was completed, the suspensions were ultrasonically treated for 20 minutes to remove air bubbles. The suspensions were then cast onto 6-well plates and freeze-dried under vacuum for 3 days to obtain a bone tissue engineering scaffold, namely the PHC-P scaffold.

[0107] Comparative Example 3

[0108] This comparative example provides a method for preparing a hydroxyapatite bone tissue engineering scaffold, specifically including the following steps:

[0109] 1M calcium hydroxide (pH=10) and 0.6M phosphoric acid were mixed and reacted at 60℃ for 6 hours. The hydroxyapatite suspension was washed with distilled water until the pH value reached 7, yielding hydroxyapatite particles, which is HA.

[0110] 3%, 6%, 9%, and 12% (w / w) hydroxyapatite suspensions were prepared and mixed in 5% (w / w) PVA solution for 3 hours. After the reaction was completed, the suspensions were ultrasonically treated for 20 minutes to remove air bubbles. The suspensions were then cast onto 6-well plates and freeze-dried under vacuum for 3 days to obtain bone tissue engineering scaffolds, namely PHC-HA scaffolds.

[0111] Comparative Example 4

[0112] A 5% (w / w) PVA solution was prepared, and air bubbles in the suspension were removed by ultrasonic treatment for 20 minutes. The solution was then cast onto a 6-well plate and freeze-dried under vacuum for 3 days to obtain a scaffold, which is the PVA scaffold.

[0113] Test Example 1

[0114] Energy dispersive spectroscopy (EDS) analysis: Energy dispersive spectroscopy (EDS) combined with SEM is used to detect the elemental composition of the sample surface and estimate the calcium-to-phosphorus ratio representing HA.

[0115] Transmission electron microscopy (TEM) analysis: A drop of hydroxyapatite suspension prepared with modified cellulose nanocrystals as a dispersant was cast onto a carbon-coated copper grid and rinsed with deionized water. The sample was dried at room temperature and imaged using a transmission electron microscope.

[0116] The test results are as follows:

[0117] As shown in Table 1, the calcium-to-phosphorus ratio closest to that of natural hydroxyapatite (1.67) is 1.60, which is the hydroxyapatite synthesized using alkylated modified cellulose nanocrystals as a dispersant, as in Example 2.

[0118] Table 1. Elemental content of hydroxyapatite particles

[0119]

[0120]

[0121] EDS results are as follows Figure 2 As shown, Figure 2 In the diagram, A represents the EDS analysis chart of HA in Comparative Example 3, B represents the EDS analysis chart of HC-C in Comparative Example 1, C represents the EDS analysis chart of HC-P in Comparative Example 2, D represents the EDS analysis chart of HC-A in Example 2, and E represents the EDS analysis chart of HC-K in Example 1. Figure 2 The results show that the elemental composition of the hydroxyapatite synthesized by the four dispersants (Comparative Example 2-CNCs, Comparative Example 1-CCNCs, Example 2-A-CNCs, and Example 1-K-CNCs) is consistent, consisting of carbon, oxygen, calcium, and phosphorus.

[0122] TEM results as follows Figure 3 As shown, Figure 3 In the diagram, A represents the TEM image of HC-K in Example 1, B represents the TEM image of HC-A in Example 2, C represents the TEM image of HC-C in Comparative Example 1, D represents the TEM image of HC-P in Comparative Example 2, and E represents the TEM image of HA in Comparative Example 3. The interaction between hydroxyapatite and the surface of CNCs was observed. Hydroxyapatite was synthesized using A-CNCs, K-CNCs, CNCs, and CCNCs as dispersants. In the presence of calcium hydrolysis and phosphoric acid, hydroxyapatite formed spherical particles decorated along the rods of CNCs.

[0123] Test Example 2

[0124] Scanning electron microscopy (SEM) analysis: The microstructure of the bone tissue engineering scaffold was investigated using SEM. Samples were mounted on gold-coated carbon ribbons and imaged at accelerating voltages of 15–20 kV. The average pore size of the bone tissue engineering scaffold was calculated using SEM images and ImageJ software.

[0125] Porosity analysis: Ethanol was used as the solvent to determine the porosity of the bone tissue engineering scaffold.

[0126] Soak the sample for 48 hours or until saturated. The porosity percentage is calculated using the following formula:

[0127]

[0128] Where V1 is the volume of ethanol, V2 is the volume of ethanol and sample, and V3 is the volume of ethanol remaining after removing the sample.

[0129] Equilibrium swelling rate analysis: The equilibrium swelling rate is calculated as follows: after 24 hours or until the weight is constant, at a constant weight (W)... dry The difference between the swollen weight in PBS and the weight in phosphate-buffered saline (PBS) divided by the constant weight of the sample (W). dry ):

[0130]

[0131] Compression test analysis: A universal testing machine was used at a strain rate of 1 mm / min. -1 A 1 kN load cell was applied to a cylindrical (5 × 10 mm) specimen until the specimen height reached 70% deformation. The mechanical modulus was estimated based on the initial linear area of ​​the compression curve (1-5% strain).

[0132] Fourier transform infrared (FTIR) spectroscopy analysis: The potassium bromide pelleting method was used. 1 mg of each sample was weighed and mixed with 50-60 mg of dry potassium bromide (KBr). The powder mixture was pressed under 10 MPa pressure using a hydraulic press for 1 min until a uniform film was obtained. The spectral recording range was 4000-650 cm⁻¹. -1 The spectra of the prepared thin films were recorded on FTIR.

[0133] X-ray diffraction (XRD) analysis: The sample was scanned using Cu-Kα rays (λ = 0.15418 nm). At room temperature, with a 2θ range of 3-50°, a scanning speed of 2° / min, a voltage of 40 kV, and a current of 40 mA, a 20 mg powder sample was scanned on an X-ray diffractometer. The crystallinity index (CrI) was calculated from the ratio of the height of the 002 peak to the height of the amorphous peak.

[0134] Cytotoxicity and cell viability analysis: For cell culture, PVA, PHC-P9, and PHC-K9 scaffolds (2 mm thick) were sterilized with UV light and immersed in ascorbic acid-free osteoblast α-MEM for at least 1 h before cell seeding. MC3T3-E1 osteoblasts were seeded on the scaffolds for cell attachment and mineralization studies to evaluate the cytotoxicity of the bone tissue engineering scaffolds. MC3T3-E1 osteoblasts were seeded at 30,000 cells / cm². 2 Cells were seeded at a density of 30,000 cells / cm² on cell culture plates and incubated at 37°C / 5% CO₂ for 24 h. PVA, PHC-P9, and PHC-K9 bone tissue engineering scaffolds were added to the culture medium, and the cells were cultured again for 24 h. A scaffold-free cell group served as a negative control. After culturing osteoblasts, a cell proliferation assay (MTS) was performed. In all experiments, 100 mL of MTS reagent was added at a ratio of 10:1, and the cells were cultured at 37°C / 5% CO₂ for 2 h. The optical density (OD) was measured on the microplates using absorbance at 490 nm. A scaffold-free cell group served as a blank control. Cell proliferation capacity on the scaffolds was assessed using MTS at 1, 4, 7, and 10 days according to the kit instructions. MC3T3-E1 osteoblasts were seeded on PVA, PHC-P9, and PHC-K9 scaffolds and cultured at 37°C / 5% CO₂ for a specified time. 2After incubation, MTS measurements were performed. The scaffold-free cell group served as a blank control. Corrected absorbance was obtained by subtracting the absorbance value at 490 nm from the corresponding blank reading. Each experiment was performed in triplicate. Cell viability on the scaffold was compared with data recorded on the PVA. Simultaneously, cell adhesion and viability were confirmed using a mammalian cytotoxicity kit containing calcein acetoxymethyl ester (AM) (2 mm) and homodimer etidium (4 mm), followed by cell staining and observation.

[0135] Alkaline phosphatase analysis: MC3T3-E1 osteoblasts were analyzed at a concentration of 30,000 cells / cm³. 2 Cells were seeded at high densities on PVA, PHC-P9, and PHC-K9 bone tissue engineering scaffolds and cultured for 14 days in osteogenic medium containing 10 mM β-glycerophosphate and 50 mg / mL L-ascorbic acid. Alkaline phosphatase (ALP) activity was determined using a colorimetric assay kit. Cell culture medium was collected at 4, 7, 10, and 14 days. The medium was changed every 2 days. The collected medium samples were then incubated with p-nitrophenyl phosphate at 25°C for 60 min. After the addition of solution was stopped, absorbance was measured at 405 nm using a microplate reader to determine the total protein content of the cells. ALP activity and protein content were calculated using a standard curve, and ALP activity was further normalized to the total protein concentration of the cells.

[0136] The test results are as follows:

[0137] like Figure 4 The image shown is a SEM image (×400; ×1.20k) of the PHC-K bone tissue engineering scaffold. Figure 5 The image shown is a SEM image (×400; ×1.20k) of the PHC-A bone tissue engineering scaffold. Figure 6 The image shown is a SEM image (×400; ×1.20k) of the PHC-C bone tissue engineering scaffold. Figure 7 The image shown is a SEM image (×400; ×1.20k) of the PHC-P bone tissue engineering scaffold. Figure 8 The image shown is a SEM image (×400; ×1.20k) of the PHC-HA bone tissue engineering scaffold. Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 In the diagram, A, B, C, D, a, b, c, and d represent: A: PHC-3, B: PHC-6, C: PHC-9, D: PHC-12, a: magnified structural diagram of PHC-3, b: magnified structural diagram of PHC-6, c: magnified structural diagram of PHC-9, and d: magnified structural diagram of PHC-12, respectively; 3, 6, 9, and 12 represent the concentrations of hydroxyapatite used in the scaffold.

[0138] Depend on Figures 4-8 As shown, the microstructural changes observed by SEM reveal the influence of hydroxyapatite content on the morphology of bone tissue engineering scaffolds. The macroporous structure of the bone tissue engineering scaffolds was preserved through freeze-drying and ice sublimation. Figure 4 This study demonstrates that all bone tissue engineering scaffolds successfully prepared using ice template technology possess highly dense porous structures with interconnected pore matrices and pore channels. SEM experiments revealed that the porosity and pore uniformity of the bone tissue engineering scaffold PHC-HA, prepared from hydroxyapatite synthesized without CNCs as a dispersant, were significantly lower than those prepared from hydroxyapatite synthesized with modified CNCs as a dispersant.

[0139] The porosity, equilibrium swelling ratio, and pore size results are shown in Table 2. Among the bone tissue engineering scaffolds prepared using hydroxyapatite synthesized with four dispersants—K-CNCs, A-CNCs, CNCs, and CCNCs—the bone tissue engineering scaffold prepared with hydroxyapatite synthesized using K-CNCs as a dispersant in Example 1 of this invention exhibits relatively high porosity and equilibrium swelling ratio, and the pores are relatively uniform. Among the bone tissue engineering scaffolds prepared with different hydroxyapatite addition amounts of 3%, 6%, 9%, and 12%, the highest porosity and equilibrium swelling ratio were observed at an addition amount of 9%.

[0140] Table 2 Porosity, average diameter, and equilibrium swelling ratio of bone tissue engineering scaffolds

[0141]

[0142]

[0143] Note: Different letters (ah) indicate that there are significant differences between the values ​​(p<0.05).

[0144] FTIR analysis such as Figures 9-13 As shown, where, Figure 9 The image shown is the FTIR plot of the PHC-K stent. Figure 10 The image shown is the FTIR plot of the PHC-A stent. Figure 11 The image shown is the FTIR plot of the PHC-P stent. Figure 12 The image shown is the FTIR plot of the PHC-C stent. Figure 13 The image displayed is the FTIR spectrum of the PHC-HA stent, from... Figure 9-13 It can be seen that the combination of hydroxyapatite and PVA forms hydrogen interactions, altering the chemical functional groups of individual components and leading to changes in peak intensity in the FTIR spectrum. The FTIR curves of PVA and bone tissue engineering scaffolds (3-12%) are at 3430 cm⁻¹. -1(-OH group), 2900cm -1 (CH2 asymmetric stretching), 1430cm -1 (-CH2 and CH vibrations in cellulose), 918cm -1 (CO stretch) and 845cm -1 The presence of characteristic peaks at (CC stretching) indicates the presence of cellulose and PVA. Absorption bands at 1035, 682, 601, and 536 cm⁻¹ indicate the presence of cellulose and PVA. -1 Phosphate groups (PO4) can be seen in hydroxyapatite. 3- Different vibration modes of 1032cm -1 The peak values ​​at these points were designated as the asymmetric stretching mode of phosphate, while the bending modes of OPO corresponded to 682, 601, and 536 cm⁻¹. -1 The peak value at which the phosphate group (PO4) is observed. As the amount of hydroxyapatite added increases, the phosphate group (PO4)... 3- The intensity of the peak increases.

[0145] Figures 14-18 The XRD patterns of the bone tissue engineering scaffold are shown, in which... Figure 14 The image shown is the XRD pattern of the PHC-K stent. Figure 15 The image shown is the XRD pattern of the PHC-A stent. Figure 16 The image shown is the XRD pattern of the PHC-P stent. Figure 17 The image shown is the XRD pattern of the PHC-C stent. Figure 18 The XRD pattern of the PHC-HA scaffold is shown. The characteristic peaks at 2θ = 19.4 and 2θ = 40.1 represent the crystallization peaks of PVA. The pattern shows peaks around 2θ = 22.5 (200) and 25.7 (002), 31.6 (210), 32.6 (211), 34.0 and 40.1 (310), and 46.5 (222), corresponding to CNCs and hydroxyapatite, respectively, due to the formation of hydroxyapatite along the CNCs surface. The main peak at 2θ = 19.4 belongs to PVA, while the shoulder peak at 2θ = 22.5 and the broad peak at 2θ = 25.5-32.6 are from CNCs and hydroxyapatite, respectively. With the increase of hydroxyapatite addition, the intensity of the main peak at 2θ = 19.4 in all bone tissue engineering scaffold curves gradually decreases, resulting in a decrease in crystallinity, indicating the formation of [HO-]-Ca through the interaction of -OH groups. 2+ The -[-OH] bond disrupts the natural crystal structure of PVA.

[0146] The relationship between the type and amount of hydroxyapatite and the compressive modulus is shown in Table 3. With increasing hydroxyapatite content, the compressive modulus gradually increases, indicating that hydroxyapatite helps improve the mechanical properties of bone tissue engineering scaffolds. When the hydroxyapatite content is 9%, the mechanical properties of the hydroxyapatite bone tissue engineering scaffold PHC-K9, prepared using alkylated modified cellulose nanocrystals as a dispersant, reach their maximum (0.085±0.004 MPa). The compressive modulus of PHC-K9 is approximately 286.4% higher than that of PVA. The mechanical strength of the hydroxyapatite bone tissue engineering scaffold prepared using modified cellulose nanocrystals as a dispersant is significantly higher than that prepared using unmodified cellulose nanocrystals, demonstrating the importance of modified cellulose nanocrystals as a biodispersant in bone tissue engineering. The physical and chemical properties and morphological analysis of the PHC-K9 bone tissue engineering scaffold indicate that the PHC-K9 bone tissue engineering scaffold has good biocompatibility.

[0147] Table 3 Compression Modulus of Bone Tissue Engineering Scaffolds

[0148]

[0149]

[0150] Note: Different letters (ah) indicate that there are significant differences between the values ​​(p<0.05).

[0151] The cell viability of osteoblast MC3T3-E1 cell lines cultured for 24 hours is as follows: Figure 19 As shown, the bone tissue-engineered scaffolds were non-toxic to osteoblasts. Cell viability was approximately 90% higher in all samples compared to the control group. Cell viability was similar for PHC-K9 (98.02±0.84%) and PHC-P9 (93.02±6.47%) compared to the control group (101.85±2.05%). Cell viability was slightly lower with the PVA scaffold, at 90.25±2.62%. Both PVA and HC-prepared scaffolds contained non-cytotoxic materials.

[0152] Figure 20 The results are shown in the figure, which evaluated the survival of MC3T3-E1 cells cultured on PHC-K9, PHC-P9, and PVA scaffolds. Figure 20MTS assay results showed that MC3T3-E1 metabolized MTS to a brown methylamine product in PHC-K9, with the metabolic value changing over time. The results indicated that osteoblasts cultured in PHC-K9 had higher proliferation capacity than those cultured in PVA on days 4 and 7, reaching a peak of 282.19 ± 10.63% on day 10. Osteoblasts cultured in PHC-P9 also showed a similar trend, reaching 229.15 ± 15.14% on day 10, while osteoblasts cultured in PVA reached 163.62 ± 10.86% on day 10. The proliferation capacity of bone tissue engineering scaffolds prepared with modified and unmodified nanobiodispersants was higher than that of bone tissue engineering scaffolds without biodispersants.

[0153] Figure 21 The live / dead cell staining further confirmed cell viability. During the 10-day culture period, MC3T3-E1 osteoblasts grown on PHC-K9 and PHC-P9 scaffolds exhibited high survival rates and viable cell growth. With increasing culture time, cells showed colony growth morphology and uniform distribution. In contrast, cells on the PVA surface showed green spots in the cytoplasm and a round, less elongated cell morphology. Cell density on the PVA scaffold did not increase over time. Regarding cell structure, MC3T3-E1 cells on the PHC-K9 scaffold exhibited a spreading spindle shape at days 7 and 10, indicating ample cell growth and proliferation. Cells were long and extended into adjacent areas. The proliferation morphology of MC3T3-E1 cells grown on PHC-K9 and PHC-P9 scaffolds at days 4, 7, and 10 showed good cell growth. The fluorescence pattern of MC3T3-E1 was consistent with the cell proliferation report results, indicating that PHC-K9 supports osteoblast growth during extended culture periods. Improved cell-cell interactions were observed on PHC-K9 and PHC-P9 scaffold samples on day 10 of culture. In cell viability studies, modified biodispersant / nanohydroxyapatite particles loaded in bone tissue engineering scaffolds significantly improved osteoblast growth, proliferation, and differentiation compared to PVA.

[0154] Figure 22This indicates the mineralization status of osteoblasts. Mineralized deposition of osteoblasts on the scaffold was detected at 4-14 days in differentiation medium. The ALP activity released by osteoblasts grown on each scaffold material differed, with the PHC-K9 scaffold exhibiting stronger proliferation and differentiation capabilities. Compared to PHC-K9, the PVA scaffold material exhibited lower mineralization within 14 days of culture. The ALP activity of PHC-K9 was 0.59±0.09 (ALP activity / mg protein) on day 4, peaking at 1.71±0.25 (ALP activity / mg protein) on day 14; the ALP activity of PHC-P9 was 0.44±0.18 (ALP activity / mg protein) on day 4, peaking at 1.36±0.17 (ALP-activity / mg protein) on day 14, while the ALP activity of PVA was 0.22±0.08 (ALP-activity / mg protein) on day 4 and 0.33±0.07 (ALP / mg protein) on day 14. The ALP activity of PHC-K9 gradually increased in the early stages of culture, rising further after 4 days. The presence of ALP activity in osteoblasts enhanced the osteogenic potential of the scaffold. Significantly greater cellular mineralization was observed on the PHC-K9 scaffold compared to PVA.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a hydroxyapatite bone tissue engineering scaffold, characterized in that, Includes the following steps: Preparation of acetylated or alkylated modified cellulose nanocrystals; The modified cellulose nanocrystals were used as a biodispersant to synthesize hydroxyapatite; Bone tissue engineering scaffolds were prepared using the synthesized hydroxyapatite; The preparation of acetylated modified cellulose nanocrystals includes the following steps: Step A1: Disperse CNCs in toluene solution using ultrasonication under ice-water bath conditions, and then use an ultrasonic cleaner to help disperse them evenly to obtain a CNCs suspension. Step A2: Add the CNCs suspension obtained in step A1 into the reaction vessel, and add glacial acetic acid, acetic anhydride and perchloric acid in sequence. After ultrasonic-assisted reaction at room temperature for 3-5 hours, add anhydrous ethanol to terminate the reaction and obtain the reaction product. Step A3: Dialyze the reaction product obtained in step A2; collect the dialyzed mixture and freeze-dry it to obtain A-CNCs; The preparation of alkylated modified cellulose nanocrystals includes the following steps: Step K1: CNCs are ultrasonically dispersed in an ethanol solution under ice-water bath conditions to obtain a CNCs suspension; Step K2: Prepare a KH-550 working solution with a weight percentage of 10%-15%, add it to the CNCs suspension obtained in step K1, adjust the pH of the mixture to between 4 and 5, and perform an ultrasonic-assisted reaction at room temperature; The reaction was terminated by adding anhydrous ethanol, yielding the reaction product; Step K3: Dialyze the reaction product obtained in step K2; freeze-dry the dialyzed suspension to obtain K-CNCs; The preparation of bone tissue engineering scaffolds using the synthesized hydroxyapatite specifically includes the following steps: The synthesized hydroxyapatite was formulated into a hydroxyapatite suspension; the hydroxyapatite suspension was added to a PVA solution and mixed; air bubbles in the suspension after the reaction were removed by ultrasonic treatment; and the suspension was cast onto a 6-well plate and freeze-dried under vacuum to obtain a bone tissue engineering scaffold.

2. The preparation method according to claim 1, characterized in that, In step A1, the weight-to-volume ratio of the CNCs to the toluene solution is (0.2-0.5):10; And / or, in step A2, the volume ratio of the glacial acetic acid, the acetic anhydride, the perchloric acid, and the anhydrous ethanol is 8:5:0.02:

20.

3. The preparation method according to claim 1, characterized in that, In step A3, the specific steps of dialysis are as follows: the obtained reaction product is dialyzed with anhydrous ethanol for 2-3 days in a dialysis bag with a molecular weight of 7000Da-14000Da, and then transferred to deionized water for dialysis for one week.

4. The preparation method according to claim 1, characterized in that, In step K1, the weight-to-volume ratio of the CNCs to the ethanol solution is (0.2-0.5):

20. And / or, in step K1, the ultrasound time is 5 min-15 min; And / or, in step K2, the duration of the ultrasound-assisted reaction is 8-10 hours; And / or, in step K3, the specific steps of dialysis are as follows: the reaction product obtained in step K2 is dialyzed with deionized water for one week in a dialysis bag with a molecular weight of 7000Da-14000Da until the pH value is stable and neutral.

5. The preparation method according to claim 1, characterized in that, The modified cellulose nanocrystals were used as a biodispersant to synthesize hydroxyapatite via an in-situ precipitation method, specifically including the following steps: A modified cellulose nanocrystal suspension was prepared and reacted with calcium hydroxide to obtain a mixture; phosphoric acid was added dropwise to the mixture, and the reaction continued to obtain a hydroxyapatite suspension; the hydroxyapatite suspension was washed until the pH value was neutral, and hydroxyapatite particles were obtained.

6. The preparation method according to claim 5, characterized in that, The modified cellulose nanocrystal suspension has a weight percentage of 3%-5%; And / or, the reaction time of the modified cellulose nanocrystal suspension with calcium hydroxide is 1h-3h, and the temperature is 55℃-65℃; And / or, the concentration of the calcium hydroxide is 0.5M-1.5M; And / or, phosphoric acid is added dropwise to the resulting mixture, and the reaction continues for 4-6 hours.

7. The preparation method according to claim 6, characterized in that, The concentration of the calcium hydroxide is 1M.

8. The preparation method according to claim 1, characterized in that, The hydroxyapatite suspension has a weight percentage of 3%-12%; And / or, the PVA solution contains 5%-10% by weight; And / or, the hydroxyapatite suspension is added to the PVA solution and mixed for 3-5 hours; And / or, the duration of the ultrasonic treatment is 15 min to 25 min.

Images