Preparation method of bone repair material

Abstract

The invention discloses a preparation method of a bone repair material, and belongs to the technical field of biomedical materials. According to the invention, Solidworks is adopted to construct a scaffold model, a 3D printing technology is utilized to prepare a sodium alginate / chitosan / mineralized collagen scaffold, and a bone repair material with good osteogenesis performance is formed; the mass ratio of the sodium alginate to the chitosan to the mineralized collagen in the material is 1: (0.2-0.6): 0.2. The stent is prepared by using a 3D printing technology, and meanwhile, different chitosan concentrations are selected, so that a series of stents with different properties are obtained; researches find that when the concentration of chitosan in the printing ink is 0.01-0.03 g / mL, the sodium alginate / chitosan / mineralized collagen printing ink forms a stable scaffold in an acidic crosslinking environment; the mechanical property of the stent is in a trend of increasing along with the increase of the concentration of the chitosan; the lower the chitosan concentration of the stent is, the faster the collagen is released in an environment with the pH value of 7.2-7.5, and the larger the release amount is.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of bone repair material, and belongs to the technical field of biomedical materials. BACKGROUND

[0002] Bone is an important and complex tissue structure of the human body, which provides mechanical support and biological microenvironment for the human body. In the human skeleton, organic substances such as collagen fibers endow the skeleton with flexibility, while inorganic substances such as apatite provide hardness. The combination of such organic and inorganic components makes the skeleton both strong and elastic. In the composition of the skeleton, the content of inorganic substances is the highest, usually between 60-70% (w / w), while the content of organic substances is 20-30% (w / w), and the remaining 10% (w / w) is water. Type I collagen is the main organic component in the skeleton, which is a force-sensitive protein with a fiber diameter of 80 to 100 nanometers. This collagen is composed of two α1 chains and one α2 chain, which form a triple helix structure through self-assembly and exhibit a characteristic 67-nanometer-spaced stripe pattern, which is called the D period. The inorganic component in the skeleton is mainly calcium phosphate mineral, the main components of which include hydroxyapatite, and octacalcium phosphate, tricalcium phosphate, amorphous calcium phosphate and carbonate-substituted hydroxyapatite.

[0003] Bone defects are mainly caused by traffic accidents, joint revision, pathological fractures, bone tumor resection and myelitis debridement, and the repair of bone defects still has great challenges in current clinical treatment. Although bone tissue has self-healing ability, it can only remodel defects of less than 6 mm. Bone grafting technology is commonly used in clinical practice to repair bone defects, so the scaffold material needs to have good osteogenic activity and meet specific biological and mechanical requirements to recruit and guide osteoblasts. SUMMARY

[0004] In order to construct a bone tissue engineering scaffold with bionic mechanical strength and promoting osteogenic differentiation, the present application provides a preparation method of bone repair material by 3D printing technology, which prepares a scaffold with good osteogenic performance.

[0005] The bone repair material provided by this invention involves cross-linking a 3D-printed scaffold in the presence of acetic acid. The addition of acetic acid protonates chitosan, giving it a positive charge and forming a polycation. Sodium alginate contains a large number of carboxyl groups, which become negatively charged after dissolving in water, forming a polyanion. Subsequently, chitosan and sodium alginate undergo electrostatic forces to form physical cross-links, making the scaffold less prone to collapse and deformation, and exhibiting excellent mechanical properties. Changing the concentration ratio of sodium alginate and chitosan in this invention also alters the mechanical properties of the scaffold. For example, cross-linking the scaffold in an acidic environment forms a dense hydrogel network, which may prevent the effective release of encapsulated mineralized collagen. However, the scaffold is used in a weakly alkaline environment (i.e., the application scenario of scaffold implantation in the human body). Therefore, this invention can regulate the release of mineralized collagen by changing the pH value of the microenvironment in which the scaffold is located, further improving the scaffold's bone repair capacity.

[0006] This invention provides a method for preparing a bone repair material. A scaffold model is constructed using Solidworks, and a sodium alginate (SA) / chitosan (CS) / mineralized collagen (MC) scaffold is prepared using 3D printing technology to form a bone repair material with good osteogenic properties. The mass ratio of sodium alginate, chitosan, and mineralized collagen in the material is 1:0.2 to 0.6:0.2.

[0007] The above preparation method specifically includes the following steps: The first step is the preparation of mineralized collagen. (1) At room temperature, 5.5g of bovine type I collagen gel (solid content of 1.36%) was dissolved in 0.5M acetic acid solution and stirred for 24h to form a collagen solution with a collagen concentration of 0.5mg / mL-1mg / mL, preferably 0.001g / mL; (2) Add 0.1 mol / L Ca to the collagen solution. 2+ Solution, stirred for 2 hours, Ca 2+ Add 100-150 mL of solution; (3) Add 0.1M phosphoric acid solution according to the calcium-to-phosphorus molar ratio of 1.67, and continue stirring for 2 hours; (4) Then add 1M sodium hydroxide solution to adjust the pH until pH=8±0.2; (5) Finally, pour out the supernatant and repeatedly centrifuge and wash until the pH of the supernatant is 7; (6) After freezing in a -20°C freezer for 2 hours, dry in a -60°C freeze dryer for 48 hours to remove excess moisture, and then grind and sieve to obtain mineralized collagen powder; The second step is the preparation of 3D printing ink: (1) Pour 0.2g of mineralized collagen powder into a beaker, add 20mL of deionized water, and sonicate for 30 minutes at room temperature using a magnetic ultrasonic cleaner to obtain a mineralized collagen suspension. (2) Add 1.0g of sodium alginate to the mineralized collagen suspension. At room temperature, set the stirring speed to 600r / min and stir continuously in a magnetic stirrer for 2 hours to obtain a viscous sodium alginate liquid. (3) Add chitosan to a mixed solution of sodium alginate and mineralized collagen. The concentration of chitosan in the mixed solution is 0.01~0.03 g / mL. Stir for 2 hours. (4) Centrifuge the stirred ink at 1000 r / min for 10 minutes to remove air bubbles; The third step involves fabricating the hydrogel scaffold using 3D printing: 3D printing ink was placed in a 5ml syringe, and a printing needle was connected. Under the following conditions, the printing time was 3-5 minutes: ambient temperature 25℃, humidity 40%, needle temperature 37℃, machine temperature 10℃, printing speed 6mm / s, extrusion speed 7mm / s, retraction 0.25mm, elevation 0.2mm, size 10*10 / 15*15mm, line spacing 1.2mm, layer height 0.2mm, and scaffold height 0.4mm. The hydrogel scaffold was obtained. After the scaffold was formed, it was immersed in an acetic acid solution for physical cross-linking.

[0008] Specifically, in the third step above, after the scaffold is printed, 1-3 mL of 1 mol / L acetic acid solution is added for cross-linking, and the collagen release performance will change with the increase of pH.

[0009] The lower the chitosan concentration of the scaffold prepared by this invention, and the faster and greater the collagen release in a weakly alkaline environment (pH=7.2-7.5), the better. This invention achieves the goal of regulating the release of mineralized collagen by changing the pH value of the microenvironment in which the scaffold is located, thereby further improving the bone repair capacity of the scaffold.

[0010] The beneficial effects of this invention are: (1) This invention uses 3D printing technology to prepare scaffolds and selects three different chitosan concentrations to obtain a series of scaffolds with different properties. Through research, it was found that when the chitosan concentration in the printing ink is 0.01~0.03g / mL, sodium alginate / chitosan / mineralized collagen printing ink forms a stable scaffold in an acidic cross-linking environment. (2) Polyanionic sodium alginate and polycationic chitosan interact to form a gel in an acidic environment. The scaffold printing performance is better with increasing chitosan concentration. The mechanical properties of the scaffold show an increasing trend with increasing chitosan concentration. Since the scaffold is cross-linked in an acidic environment after printing, the collagen release performance changes with increasing pH. The lower the chitosan concentration of the scaffold and the greater the collagen release in an environment with pH=7.2-7.5, the faster and the greater the release amount. (3) Regarding cell proliferation experiments, the presence of collagen in the mineralized collagen loaded on the scaffold gives it excellent cell proliferation-promoting ability, which promotes bone tissue regeneration. In the alizarin red staining experiment, the calcium nodules in the scaffold group were dark in color and had large nuclei. Due to the good osteogenic differentiation potential of hydroxyapatite, the mineralized collagen loaded on the scaffold effectively promoted osteogenic differentiation of cells by taking advantage of the excellent cell proliferation effect, showing good osteogenic differentiation ability. Attached Figure Description

[0011] Figure 1 SEM images of the bone repair materials prepared in Examples 1-3.

[0012] Figure 2 The images show the XRD patterns of the bone repair materials prepared in Examples 1-3.

[0013] Figure 3 The infrared spectra of Example 1 and sodium alginate, chitosan, and mineralized collagen are shown.

[0014] Figure 4 The tensile mechanical properties of the bone repair materials prepared in Examples 1-3 are shown in the diagram.

[0015] Figure 5 The diagram shows the collagen release performance of the bone repair materials prepared in Examples 1-3.

[0016] Figure 6 The images are CCK-8 images of the bone repair materials prepared in Examples 1-3.

[0017] Figure 7 These are live and dead staining images of the bone repair materials prepared in Examples 1-3.

[0018] Figure 8 The images show the ALP values ​​of the bone repair materials prepared in Examples 1-3.

[0019] Figure 9 Alizarin Red staining images of the bone repair materials prepared in Examples 1-3. Detailed Implementation

[0020] The present invention will be further illustrated by the following embodiments, but is not limited to the following embodiments. Example 1

[0021] 5.5 g of bovine type I collagen gel (solid content 1.36%) was dissolved in 0.5 M acetic acid solution and stirred for 24 h to form a collagen solution (concentration 0.001 g / mL). 100 mL of 0.1 mol / L Ca2+ was then added dropwise to the collagen solution. 2+ The solution was stirred for two hours, and 0.1M phosphoric acid solution was added according to a calcium-to-phosphorus molar ratio of 1.67. Then, 1M sodium hydroxide solution was added dropwise to adjust the pH until pH = 8 ± 0.2. Finally, the supernatant was poured off, and the solution was repeatedly centrifuged and washed until the pH of the supernatant was 7. After freezing at -20°C for 2 hours, it was dried in a freeze dryer at -60°C for 48 hours to remove excess moisture. The resulting mineralized collagen powder was obtained after grinding and sieving.

[0022] 0.2g of mineralized collagen powder was poured into a beaker, and 20ml of deionized water was added. The mixture was ultrasonically cleaned for 30 minutes at room temperature using a magnetic ultrasonic cleaner to obtain a mineralized collagen suspension. 1.0g of sodium alginate was added to the mineralized collagen suspension, and the mixture was stirred continuously for two hours at 600r / min at room temperature using a magnetic stirrer to obtain a viscous sodium alginate liquid. 0.2g of chitosan was added to the mixture of sodium alginate and mineralized collagen, and the mixture was stirred for 2 hours to obtain an ink of sodium alginate (0.05g / mL) / chitosan (0.01g / mL) / mineralized collagen (0.01g / mL). The stirred ink was centrifuged at 1000r / min for 10 minutes to remove air bubbles.

[0023] The obtained ink was placed in a syringe (BD-5mL), and a printing needle (0.25G) was connected. Under the following conditions, the printing time lasted for 5 minutes: ambient temperature 25℃, humidity 40%, needle temperature 37℃, machine temperature 10℃, printing speed 6mm / s, extrusion speed 7mm / s, pullback (SK) 0.25mm, lift (HP) 0.2mm, size 10*10mm, line spacing 1.2mm, layer height 0.2mm, and scaffold height 0.4mm. After printing, sodium alginate and chitosan were cross-linked in acetic acid solution to obtain a composite 3D printed hydrogel scaffold of sodium alginate (concentration 0.05g / mL) / chitosan (0.01g / mL) / mineralized collagen (0.01g / mL). Example 2

[0024] The preparation of mineralized collagen is the same as in Example 1.

[0025] 0.2g of mineralized collagen was poured into a beaker, and 20mL of deionized water was added. The mixture was ultrasonically cleaned for 30 minutes at room temperature using a magnetic ultrasonic cleaner to obtain a mineralized collagen suspension. 1.0g of sodium alginate was added to the mineralized collagen suspension, and the mixture was stirred continuously for two hours at 600r / min at room temperature using a magnetic stirrer to obtain a viscous sodium alginate liquid. 0.4g of chitosan was added to the mixed solution of sodium alginate and mineralized collagen, and the mixture was stirred for 2 hours to obtain an ink mixture of sodium alginate (0.05g / mL) / chitosan (0.01g / mL) / mineralized collagen (0.01g / mL). The stirred ink was centrifuged at 1000r / min for 10 minutes to remove air bubbles.

[0026] The obtained ink was placed in a syringe (BD-5mL), and a printing needle (0.25G) was connected. Under the following conditions, the printing time lasted for 5 minutes: ambient temperature 25℃, humidity 40%, needle temperature 37℃, machine temperature 10℃, printing speed 6mm / s, extrusion speed 7mm / s, pullback (SK) 0.25mm, lift (HP) 0.2mm, size 10*10mm, line spacing 1.2mm, layer height 0.2mm, and scaffold height 0.4mm. After printing, sodium alginate and chitosan were cross-linked with acetic acid solution to obtain a composite 3D printed hydrogel scaffold of sodium alginate (concentration 0.05g / mL) / chitosan (0.02g / mL) / mineralized collagen (concentration 0.01g / mL). Example 3

[0027] The preparation of mineralized collagen is the same as in Example 1.

[0028] 0.2g of mineralized collagen was poured into a beaker, and 20mL of deionized water was added. The mixture was ultrasonically cleaned for 30 minutes at room temperature using a magnetic ultrasonic cleaner to obtain a mineralized collagen suspension. 1.0g of sodium alginate was added to the mineralized collagen suspension, and the mixture was stirred continuously for two hours at 600r / min at room temperature using a magnetic stirrer to obtain a viscous sodium alginate liquid. 0.6g of chitosan was added to the mixture of sodium alginate and mineralized collagen, and the mixture was stirred for 2 hours to obtain an ink of sodium alginate (0.05g / mL) / chitosan (0.03g / mL) / mineralized collagen (0.01g / mL). The stirred ink was centrifuged at 1000r / min for 10 minutes to remove air bubbles.

[0029] The obtained ink was placed in a syringe (BD-5mL), and a printing needle (0.25G) was connected. Under the following conditions, the printing time lasted for 5 minutes: ambient temperature 25℃, humidity 40%, needle temperature 37℃, machine temperature 10℃, printing speed 6mm / s, extrusion speed 7mm / s, pullback (SK) 0.25mm, lift (HP) 0.2mm, size 10*10mm, line spacing 1.2mm, layer height 0.2mm, and scaffold height 0.4mm. After printing, sodium alginate and chitosan were cross-linked with acetic acid solution to obtain a composite 3D printed hydrogel scaffold of sodium alginate (concentration 0.05g / mL) / chitosan (0.03g / mL) / mineralized collagen (concentration 0.01g / mL).

[0030] The bone repair materials prepared using the above methods, employing a biomimetic approach and combined with 3D printing technology, create bone tissue engineering scaffolds that promote cell growth and differentiation, exhibiting excellent biocompatibility. They hold broad clinical application prospects in the field of bone defect repair.

[0031] Figure 1 shows SEM images of the bone repair materials prepared in Examples 1-3. The SA / CS / MC scaffolds prepared using 3D printing technology showed increasingly better printability with increasing chitosan concentration. Scanning electron microscopy images revealed a rough surface on the dried printed scaffolds. As the chitosan concentration increased, the outline of the printed grid scaffold became clearer, the degree of gel filament fusion gradually decreased, and the overall scaffold shape approximated the designed shape (i.e., printability gradually increased).

[0032] Figure 2 Examples 1-3 show the XRD images of the bone repair materials prepared. XRD is a phase analysis, structural analysis, and phase analysis of the sample crystals. (002), (211), and (004) are the crystal planes of hydroxyapatite. Based on the peak positions, the main inorganic component of the scaffold is hydroxyapatite.

[0033] Figure 3 Infrared spectral image of the bone repair material prepared in Example 1. Chitosan characteristic peaks include 1596 cm⁻¹. -1 The nearby amino (-NH2) peak, CH bending vibration at 1421 cm⁻¹ -1 Nearby. The main characteristic peaks of sodium alginate include those near 1620 cm⁻¹ in the molecular chain. -1 (-COO- asymmetry) and 1416 cm -1 Two carboxyl peaks (-COO- symmetrical). 1638 cm⁻¹ -1 and 1020cm -1 The amide I bond and PO4 bond, respectively, correspond to mineralized collagen. 3-Group. The amide II peak at 1600 cm⁻¹ in the infrared spectrum of the SA / CS / MC scaffold. -1 The significant enhancement, coupled with the absence of other individual amino or carboxyl peaks observed near this wavelength, indicates that sodium alginate exhibits a negatively charged carboxyl group (-COO). - ) and the positively charged amino groups (-NH3) of chitosan + The ionic interactions between ions. For example... Figure 3 1020 cm -1 The peak shown confirms PO4. 3- The presence of groups indicates that the scaffold was successfully loaded with mineralized collagen.

[0034] Figure 4 Images showing the tensile mechanical properties of the bone repair materials prepared in Examples 1-3. Mechanical properties are an important performance characteristic of 3D-printed tissue engineering scaffolds. To investigate the effect of chitosan concentration on the mechanical properties of SA / CS / MC scaffolds, tensile modulus of elasticity was tested on scaffolds from various experimental groups, and the modulus of elasticity extracted from the slope of the linear region of the stress-strain curve was plotted on... Figure 4 According to the results, the elastic moduli were 0.98, 1.16, and 1.42 MPa when the chitosan concentration was 0.01–0.03 g / mL, respectively. Analyzing the effect of chitosan concentration on the scaffold, we found that chitosan molecules contain a large number of amino and hydroxyl groups. These active groups give chitosan high chemical reactivity. The increased number of amino groups in the scaffold leads to the formation of more positively charged amino groups. The negatively charged carboxyl groups formed by the ionization of sodium alginate electrostatically crosslink with the positively charged amino groups, resulting in stronger intermolecular forces, tighter crosslinking of the scaffold, and improved overall scaffold strength. Therefore, we conclude that the scaffold has a greater elastic modulus with increasing chitosan concentration.

[0035] Figure 5 Images showing the collagen release properties of the bone repair materials prepared in Examples 1-3 are provided. The collagen release curves are shown below. Figure 5 As shown, where Figure 5 (a) is the standard curve for collagen concentration. Figure 5 (b) shows the collagen release curves of various scaffolds at different pH levels. Analysis at different pH environments revealed that the collagen release from scaffolds immersed at pH 7.4 was significantly greater than that from scaffolds immersed at pH 6.0. This is because the cross-linking environment of the scaffolds is acidic. When the immersion environment is weakly alkaline, the degree of cross-linking of the scaffolds decreases, the electrostatic interaction between sodium alginate and chitosan weakens, leading to easier degradation of the scaffolds and slower collagen release. Analysis of chitosan concentration changes showed that both the release rate and amount of collagen decreased with increasing chitosan concentration. This is mainly because higher chitosan concentrations result in tighter cross-linking of the scaffolds, making them less prone to degradation.

[0036] Figure 6 Examples 1-3 show CCK-8 images of the bone repair materials prepared. The absorbance of the cells increased with increasing culture time. On days 3 and 5, the relative number of cells in the chitosan scaffold extract groups with different concentrations was significantly higher than that in the blank control group (pure culture medium group). This may be because the mineralized collagen contained in the scaffold was released, and the hydroxyapatite in the mineralized collagen can continuously release Ca2+. 2+ and PO4 3- Plasma activates the calcium-sensitive receptor CaSR in osteoblasts, thereby initiating proliferation signaling pathways such as MAPK / ERK and PI3K / AKT. Simultaneously, collagen promotes the expression of cyclin proteins such as Cyclin D1 through integrin α2β1-mediated signal transduction, accelerating cell division. Therefore, scaffolds loaded with mineralized collagen exhibit superior cell proliferation-promoting capabilities.

[0037] Figure 7 Examples 1-3 show the live and dead cell staining images of the prepared bone repair materials. After 3 and 5 days of culture, the group containing mineralized collagen extract showed a large number of live cells and fewer dead cells compared to the blank control group. That is, the scaffold loaded with mineralized collagen had more live cells and fewer dead cells. The live and dead cell staining experiment and CCK-8 results jointly verified the cell proliferation-promoting ability of the scaffold.

[0038] Figure 8 Images of ALP levels in the bone repair materials prepared in Examples 1-3 are shown. The absorbance of the cells increased with prolonged culture time. On day 7, the ALP levels of chitosan scaffold extracts at different concentrations were significantly higher than those in the blank control group. This phenomenon is attributed to the activation of the focal adhesion kinase (FAK) signaling pathway by the RGD (arginine-glycine-aspartic acid) sequence in collagen, which promotes cell proliferation and osteogenic transformation through the Wnt / β-catenin pathway. The results indicate that the scaffold loaded with mineralized collagen has a better ability to promote cell differentiation.

[0039] Figure 9 Images of the bone repair materials prepared in Examples 1-3, stained with Alizarin Red. Alizarin Red was used to stain osteogenic-induced cells, where calcium nodules deposited on the cell surface appeared deep red after staining. Figure 9 It can be seen that, compared with the blank control group, the calcium nodules formed in the mineralized collagen-loaded group were darker in color and more numerous. This is because the hydroxyapatite in the mineralized collagen gradually and slowly releases calcium under physiological conditions. 2+ and PO4 3- This helps maintain stable local calcium and phosphorus concentrations, PO4 3- With Ca 2+These molecules combine to form crystal nuclei, accelerating the mineralization process of the extracellular matrix. Therefore, scaffolds loaded with mineralized collagen have superior osteogenic capacity.

Claims

1. A method for preparing a bone repair material, characterized in that: A scaffold model was constructed using Solidworks, and a sodium alginate / chitosan / mineralized collagen scaffold was prepared using 3D printing technology to form a bone repair material with good osteogenic properties. The mass ratio of sodium alginate, chitosan, and mineralized collagen in the material was 1:0.2~0.6:0.

2.

2. The method for preparing the bone repair material according to claim 1, characterized in that: Includes the following steps: The first step is the preparation of mineralized collagen. (1) At room temperature, 5.5g of bovine type I collagen gel was dissolved in 0.5M acetic acid solution and stirred for 24h to form a collagen solution; (2) Add 0.1 mol / L Ca to the collagen solution. 2+ Solution, stirred for 2 hours, Ca 2+ Add 100-150 mL of solution; (3) Add 0.1M phosphoric acid solution according to the calcium-to-phosphorus molar ratio of 1.67, and continue stirring for 2 hours; (4) Then add 1M sodium hydroxide solution to adjust the pH until pH=8±0.2; (5) Finally, pour out the supernatant and repeatedly centrifuge and wash until the pH of the supernatant is 7; (6) After freezing in a -20°C freezer for 2 hours, dry in a -60°C freeze dryer for 48 hours to remove excess moisture, and then grind and sieve to obtain mineralized collagen powder; The second step is the preparation of 3D printing ink: (1) Pour 0.2g of mineralized collagen powder into a beaker, add 20ml of deionized water, and sonicate for 30 minutes at room temperature using a magnetic ultrasonic cleaner to obtain a mineralized collagen suspension. (2) Add 1.0g of sodium alginate to the mineralized collagen suspension. At room temperature, set the stirring speed to 600r / min and stir continuously in a magnetic stirrer for 2 hours to obtain a viscous sodium alginate liquid. (3) Add chitosan to a mixed solution of sodium alginate and mineralized collagen. The concentration of chitosan in the mixed solution is 0.01~0.03 g / mL. Stir for 2 hours. (4) Centrifuge the stirred ink at 1000 r / min for 10 minutes to remove air bubbles; The third step involves fabricating the hydrogel scaffold using 3D printing: Place 3D printing ink into a 5mL syringe, connect the printing needle, and print for 3-5 minutes to obtain a hydrogel scaffold. After the scaffold is formed, immerse it in an acetic acid solution for physical cross-linking.

3. The method for preparing the bone repair material according to claim 2, characterized in that: In the first step, the solid content of bovine type I collagen gel was 1.36%, and the concentration of collagen solution was 0.5 mg / mL-1 mg / mL.

4. The method for preparing the bone repair material according to claim 2, characterized in that: In the third step, after the scaffold is printed, 1-3 mL of 1 mol / L acetic acid solution is added for cross-linking. The collagen release performance will change with the increase of pH.

5. The method for preparing the bone repair material according to claim 2, characterized in that: In the third step, the 3D printing conditions are as follows: ambient temperature 25℃, humidity 40%, needle temperature 37℃, machine temperature 10℃, printing speed 6mm / s, extrusion speed 7mm / s, retraction 0.25mm, lifting 0.2mm, size 10*10 or 15*15mm, line spacing 1.2mm, layer height 0.2mm, and support height 0.4mm.

6. A bone repair material prepared by the method according to any one of claims 1 to 5, characterized in that: The release of mineralized collagen can be regulated by changing the pH value of the microenvironment in which the scaffold is located: the lower the chitosan concentration and the stronger the collagen release in a weakly alkaline environment with pH=7.2-7.5, the faster and the greater the amount of collagen released.

Images