Method for preparing calcium carbonate microcrystal by carbonic anhydrase regulating microalgae mineralization, calcium carbonate microcrystal and application thereof

Abstract

The application discloses a method for preparing calcium carbonate microcrystals by carbonic anhydrase-regulated microalgae mineralization, the calcium carbonate microcrystals and application thereof. The method comprises the following steps: S1. microalgae culture and oil accumulation: proliferating and culturing chlorella to make algal cells reach the late logarithmic phase or the stationary phase; then, nitrogen limitation or nitrogen starvation induction culture is carried out to make the algal body accumulate oil; S2. mineralization induction culture: the algal liquid is transferred into a mineralization culture medium, and carbonic anhydrase is added to culture for 5-48 h; the mineralization culture medium contains Ca 2+ and HCO3 ‑ ; S3. microcrystal collection: the algal body subjected to the mineralization induction culture is collected, and microcrystals are separated to obtain calcium carbonate microcrystals. The calcium carbonate microcrystals obtained by the above method can be quickly converted into carbonic acid-doped hydroxyapatite, and further applied to 3D printing medical materials. The method can realize the co-production of biomaterials and high-oil algal powder in combination with a low-nitrogen induction process of microalgae.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and relates to biomineralization. Specifically, it relates to a method for preparing calcium carbonate microcrystals by regulating microalgal mineralization with carbonic anhydrase, calcium carbonate microcrystals and their applications; more specifically, it relates to the application of calcium carbonate microcrystals prepared by regulating microalgal mineralization with carbonic anhydrase in the preparation of carbonic acid-doped hydroxyapatite and 3D printing materials. Background Technology

[0002] Calcium carbonate microcrystals hold great promise in medical materials and 3D printing. Aragonite, in particular, is widely used in drug delivery and bone repair materials due to its porous structure and controllable recrystallization properties. Recent studies have shown that aragonite can be rapidly converted into carbonate-doped hydroxyapatite (Ca-P) within hours, achieving highly biomimetic inorganic coatings or particle structures. Simultaneously, composite systems of hydrogels such as Alginate and GelMA with Ca-P or silicate particles have been widely used in the fabrication of extruded 3D-printed bone repair scaffolds, indicating that calcium carbonate microcrystals are expected to become an important raw material for 3D-printed medical materials.

[0003] Currently, the most commonly used microbial induced calcium carbonate deposition (MICP) technology is the urease-bacterial method. For example, Li Jianshan et al. selected the urease-producing bacteria Bacillus NS-6 and soybean urease to induce CaCO3 precipitation, and used it as biocement for ecological restoration, leakage sealing and other fields (Li Jianshan, Yang Li'an, Yan Changhao, et al. Study on CaCO3 precipitation induced by urease-producing bacteria NS-6 and soybean urease [J]. Bioprocessing, 2025, 23(5): 530-537). The biological urease method mainly relies on chemical supersaturation drive and lacks the biological regulation characteristics of "enzyme-microenvironment" synergistic effect. In contrast, the microalgal mineralization process has more biomimetic potential. Carbonic anhydrase (CA) is a key catalytic enzyme in the algal mineralization process, which can accelerate the CO2 hydration reaction and regulate the local pH and inorganic carbon supply. During the growth of microalgae, inhibiting carbonic anhydrase will significantly reduce HCO3. - The dependent photosynthesis and carbon concentration capacity indicate that CA plays a decisive role in carbon supply to algae and the balance of external carbonates. In vertebrates, CA also participates in bone mineralization and bone resorption, achieving bone remodeling by regulating the acidification environment of osteoclasts, indicating that this enzyme has a common regulatory role in mineralization processes across species.

[0004] Therefore, if we can achieve the preparation of calcium carbonate microcrystals by regulating microalgal mineralization with carbonic anhydrase, realize controllable biomineralization, and apply the obtained calcium carbonate microcrystals to 3D printing medical materials, it will open up new preparation routes and new applications for bio-derived calcium carbonate microcrystals. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a method for preparing calcium carbonate microcrystals by regulating microalgal mineralization using carbonic anhydrase, thus achieving the goal of preparing calcium carbonate microcrystals by regulating microalgal mineralization using carbonic anhydrase. This method, combined with a low-nitrogen induction process for microalgae, enables the co-production of biomineralized materials and high-oil algal powder, thereby increasing the overall value of microalgae products.

[0006] The specific technical solution is as follows:

[0007] One objective of this invention is to provide a method for preparing calcium carbonate microcrystals by regulating microalgal mineralization using carbonic anhydrase, comprising the following steps:

[0008] S1. Microalgae culture and lipid accumulation: Coccoyxa subellipsoidea is proliferated to bring the algal cells to the end-log phase or stationary phase, during which a large amount of biomass is accumulated; then, nitrogen restriction or nitrogen starvation induction culture is carried out to induce lipid accumulation in the algae; a higher lipid yield can be obtained in this way.

[0009] S2. Mineralization induction culture: The algal solution is transferred to a mineralization medium and carbonic anhydrase is added for 5–48 h of culture; the mineralization medium contains Ca. 2+ and HCO3 - ;

[0010] S3. Microcrystal collection: The algae that have undergone mineralization-induced culture are harvested and microcrystals are separated to obtain calcium carbonate microcrystals.

[0011] The mechanism of this invention is as follows: Microalgae increase the extracellular pH value through photosynthesis, and accelerate the reaction of CO2 and HCO3 under the action of extracellular carbonic anhydrase. - / CO3 2- The interconversion between these elements induces supersaturation and nucleation of calcium carbonate in a Ca²⁺-containing environment. This invention utilizes *Chlorella vulgaris* to obtain calcium carbonate microcrystals rich in vaterite under the action of exogenous carbonic anhydrase. The crystal phase can be controlled by regulating carbonic anhydrase activity, solution pH, and mineralization time. Simultaneously, at the microalgal metabolic level, this invention significantly improves the lipid yield of *Chlorella vulgaris* through a two-stage "proliferation-induction" culture strategy, increasing the lipid content of *Chlorella vulgaris* cells from the initial 10%–15% (dry weight percentage) to 35%–41%. Coupling this process with the mineralization stage achieves the co-production of lipids and calcium carbonate microcrystals.

[0012] Further, in step S1: CO2 is used as the carbon source. Specifically, propagation culture is carried out in a photobioreactor, and CO2 is introduced into it. The CO2 volume fraction is preferably 0.5%~3%, and the aeration rate is preferably 0.5~1 vvm. The photobioreactor is preferably made of glass or transparent plastic.

[0013] Furthermore, in step S1: the light intensity is preferably 150~300 μmol·m -2 ·s -1 .

[0014] During the proliferation culture process, the optimal light intensity is 150–250 μmol·m⁻¹. -2 ·s -1 .

[0015] During nitrogen restriction or nitrogen starvation induction culture, the light intensity is preferably 200–300 μmol·m⁻¹. -2 ·s -1 .

[0016] Furthermore, in step S1, the preferred culture temperature is 22~28 ℃.

[0017] Furthermore, in step S1: the algal culture is increased until the optical density (OD) of the algal solution reaches a certain level. 680 The concentration is 1.5~3.0, at which point the dry weight of the algae can generally reach 0.6~1.0 g·L. -1 This completes the microalgae proliferation stage.

[0018] Furthermore, in step S1: it is preferable to use BASAL or modified BASAL basal medium for proliferation culture.

[0019] Further, in step S1: nitrogen restriction or nitrogen starvation induction culture is performed using a nitrogen-restricted or nitrogen-starved medium. Specifically, based on the medium used for proliferation culture, the concentration of nitrate or other nitrogen sources in the medium is reduced by 70% to 90%. Through nitrogen restriction or nitrogen starvation induction culture, the lipid content of *Chlorella vulgaris* cells can be increased from the initial 10% to 15% (dry weight percentage) to 35% to 41%, completing lipid accumulation and providing a basis for lipid production.

[0020] Furthermore, in step S1, the nitrogen restriction or nitrogen starvation induction culture time is preferably 24~48 h.

[0021] Furthermore, in step S2, the preferred amount of carbonic anhydrase added is 4-8 U·mL. -1 .

[0022] Furthermore, in step S2: the mineralization medium preferably contains Ca. 2+ 5~20 mM and HCO3 - 10~50 mM.

[0023] Furthermore, in step S2, the preferred culture temperature is 22~28℃.

[0024] Furthermore, in step S2: it is preferable to maintain the culture medium at pH 8.4~9.0. This can be achieved by using a pH-stat control system or by manually adding NaHCO3 or NaOH to stably maintain the system at pH 8.4~9.0, more preferably pH 8.6±0.2.

[0025] Furthermore, in step S2: no CO2 is supplied. Carbon restriction induces high expression of the algal extracellular carbonic anhydrase eCA.

[0026] Furthermore, in step S2, the volume ratio of algal solution to mineralization culture medium is preferably controlled at 1:(1~3). Specifically, the mineralization culture medium can be added to the same photobioreactor or another independent mineralization reactor.

[0027] Furthermore, in step S2, the mineralization culture time is preferably 5~24 h, more preferably 5~8 h.

[0028] Furthermore, in step S3: microcrystals are separated by centrifugation or membrane filtration. Since the diameter of *Chlorella vulgaris* is greater than 5 μm, effective separation can be achieved. The particle size is mainly distributed in the range of 0.1–5 μm. SEM observation shows that under carbonic anhydrase-regulated conditions, a large number of spherical or near-spherical microcrystals formed after 6 h of mineralization-induced culture have diameters mainly concentrated in the range of 0.5–2 μm, and their surfaces exhibit porous or rough structures, making them suitable as precursors for subsequent transformations and composite materials.

[0029] Furthermore, in step S3: after separating the calcium carbonate microcrystals, the algal cells are dried and pulverized to obtain high-oil algal powder; alternatively, the oil in the algal cells can be collected. Specifically, organic solvents can be used to extract the oil from the algal cells.

[0030] A second objective of this invention is to provide a microcrystalline calcium carbonate prepared by the above method. The calcium carbonate microcrystals obtained by the above preparation method are rich in aragonite.

[0031] A third objective of this invention is to provide the application of the aforementioned calcium carbonate microcrystals in the preparation of carbonate-doped hydroxyapatite. These calcium carbonate microcrystals are suitable as precursors for subsequent transformations and composite materials. In particular, after 6 hours of mineralization-induced culture, a large number of spherical or near-spherical microcrystalline particles are formed, with amorphous calcium carbonate and aragonite as the main crystalline phases. These particles have porous or rough surfaces, and their diameters are mainly concentrated between 0.5 and 2 μm.

[0032] Furthermore, the method for preparing carbonate-doped hydroxyapatite from the aforementioned calcium carbonate microcrystals includes: placing the calcium carbonate microcrystals in a phosphate buffer solution containing bicarbonate ions to obtain carbonate-doped hydroxyapatite (CAp). The obtained carbonate-doped hydroxyapatite is in the form of nanosheets, needles, or particles.

[0033] Specifically, the concentration of the phosphate buffer solution is preferably 0.05~0.5 M, and the pH value is preferably 7.4~8.0.

[0034] Specifically, the calcium carbonate microcrystals are placed in a phosphate buffer solution containing bicarbonate and allowed to stand at 25-45°C for 4-32 h, preferably 10-12 h.

[0035] The fourth objective of this invention is to provide the application of the above-mentioned calcium carbonate microcrystals in the preparation of 3D printing materials.

[0036] Specifically, carbonate-doped hydroxyapatite (CAp), obtained by phase inversion of the aforementioned calcium carbonate microcrystals, is mixed with an aqueous solution of biomaterials to prepare a composite ink, thereby obtaining an extrusion-type 3D printing material. The biomaterial is one or more of alginate, methacrylamide gelatin (GelMA), and polylactic-co-glycolic acid copolymer (PLGA).

[0037] The preferred content of carbonate-doped hydroxyapatite (CAp) in the system is 8wt% to 20wt%.

[0038] The total concentration of the aqueous solution of the biological material is 4wt% to 15wt%; for example, the aqueous solution contains 1wt% to 5wt% alginate and 3wt% to 10wt% methacrylamide gelatin.

[0039] Photoinitiators, such as LAP, are also added to the composite ink system.

[0040] The aforementioned materials can be used as medical materials, especially bone repair materials. The printing path and structure can be designed as a multi-layered mesh, columnar, or anatomically shaped bone repair scaffold. After extrusion printing, the material is ionically crosslinked and / or photocrosslinked to obtain the bone repair scaffold. The resulting printed scaffold exhibits good shape retention and high forming accuracy in a wet state, with a uniform pore structure and adjustable pore size through printing parameters.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] This invention uses carbonic anhydrase to regulate the mineralization of Chlorella vulgaris, and the obtained microcrystalline calcium carbonate crystals are rich in aragonite, with particle sizes mainly concentrated in the range of 0.1~5 μm; in particular, a large number of spherical or near-spherical microcrystalline particles formed after 6 h of mineralization culture have diameters mainly concentrated in the range of 0.5~2 μm and have porous or rough structures on their surfaces, making them suitable as precursors for subsequent transformation and composite materials.

[0043] The algal-derived calcium carbonate microcrystals of this invention can be rapidly converted into carbonate-doped hydroxyapatite under mild conditions, possessing a chemical composition, microscale, and biocompatibility that match the structure of natural bone minerals. The mineralization products of this invention are highly biomimetic and can be rapidly converted into medical caps, meeting the processing requirements of 3D-printed medical materials.

[0044] The microcrystalline calcium carbonate exhibits high controllability in crystal form and particle size. The formation of ACC, aragonite, or calcite can be programmably controlled through the adjustment of cultivation conditions, enabling the preparation of high-purity calcium carbonate microcrystals with narrow particle size distribution. The microcrystals (CaCO3 or CAp) obtained by this invention have excellent compatibility with alginate, GelMA, and other matrices, enabling the formation of composite inks with suitable rheological properties and high printing precision.

[0045] This invention not only achieves efficient CO2 fixation, but also forms an integrated system of "co-production of oil and calcium carbonate microcrystals". Through a two-stage cultivation strategy, microalgal oil production and calcium carbonate deposition are coupled, realizing bidirectional high-value CO2 production within the same system. The economic benefits are far greater than those of a single-product system.

[0046] This invention features low pollution, high carbon efficiency, and suitability for large-scale production. Unlike urease-dependent MIP technology, this invention produces no ammonia emissions, making the system green and environmentally friendly, and suitable for continuous operation of photobioreactors. Attached Figure Description

[0047] Figure 1 This is an electron microscopy image of calcium carbonate microcrystals after 6 hours of mineralization culture in Example 1;

[0048] Figure 2 Electron microscopy images of calcium carbonate microcrystals after 24 h of mineralization culture in Comparative Example 2;

[0049] Figure 3 This is an electron microscope image of hydroxyapatite after 12 hours of phase inversion in Example 2. Detailed Implementation

[0050] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] In the specific implementation, the carbonic anhydrase used is a conventional commercially available biochemical preparation (EC 4.2.1.1; 9001-03-0, Aladdin).

[0052] Example 1

[0053] The method for preparing calcium carbonate microcrystals by regulating microalgal mineralization using carbonic anhydrase is as follows:

[0054] S1. Microalgae cultivation and lipid accumulation:

[0055] Coccoyxa subellipsoidea was cultured for proliferation. Coccoyxa subellipsoidea was selected as the main mineralizing agent and inoculated into BASAL basal medium. Its basic composition is shown in Table 1.

[0056] Table 1. Basic Composition of BASAL Basal Culture Medium

[0057]

[0058] The culture was carried out in a glass-based photobioreactor. The culture conditions were: temperature 25±2℃, light intensity 150 μmol·m⁻¹. -2 ·s -1 A continuous aeration of 1% CO2 (v / v) was introduced at a flow rate of 0.5 vvm. The algae were cultured under these conditions for 72 hours. At this point, the optical density (OD) of the algal solution was... 680 Reaching 2.5 indicates the completion of the microalgae proliferation stage.

[0059] Nitrogen-limited culture was performed: the culture medium was replaced with a nitrogen-limited medium (based on the basic composition shown in Table 1, but with the potassium nitrate concentration changed to 250 mg / L), and the light intensity was increased to 300 μmol·m⁻¹. -2 ·s -1 Under the same conditions, continue culturing for another 48 hours to allow the algae to accumulate lipids, thus providing a basis for co-production of lipid products.

[0060] S2. Mineralization Induction Culture: After nitrogen-limited culture, the algal solution was transferred to a mineralization medium for mineralization induction culture. The mineralization medium was an aqueous solution containing 10 mM CaCl2·2H2O and 20 mM NaHCO3, with the initial pH adjusted to 8.6 using NaOH or HCl. The volume ratio of algal solution to mineralization medium was controlled at 1:3, and both were added to the same photobioreactor. Carbonic anhydrase was added to the reactor to achieve a final concentration of 8 U·mL. -1 While maintaining constant light and temperature conditions, the continuous CO2 supply is stopped, and the system relies entirely on HCO3. - Provide inorganic carbon. Add NaHCO3 or NaOH dropwise to stabilize the pH of the system at 8.6 ± 0.2. Take samples at 1 h, 6 h, 24 h, and 48 h of incubation.

[0061] S3. The obtained culture medium was subjected to gradient centrifugation. First, algal cells were collected by centrifugation at 4000 rpm for 10 min. Then, the speed was increased to 12000 rpm for 10 min to obtain microcrystalline calcium carbonate precipitate. After gently washing twice with deionized water, the precipitate was dried under vacuum at 40℃ for 10 h to obtain microcrystalline calcium carbonate. The lipids in the wet algal cells were extracted using a methanol / chloroform solution (volume ratio 1:2), and the lipid content was determined by differential gravimetric analysis. The microcrystalline calcium carbonate particles were analyzed by sieve and scanning electron microscopy, confirming that the particle size was mainly distributed in the range of 0.1–5 μm.

[0062] Comparative Example 1

[0063] Referring to Example 1, the difference from Example 1 is that in step S2, carbonic anhydrase is not added, and the continuous CO2 introduction is not stopped. The amount and method of CO2 introduction are the same as in step S1, and the pH value of the solution is not controlled during this period.

[0064] Comparative Example 2

[0065] Referring to Example 1, the difference from Example 1 is that in step S2, the continuous CO2 introduction is not stopped, and the amount and method of CO2 introduction are the same as in step S1.

[0066] Test 1

[0067] The solid products obtained in Example 1 and the comparative example were characterized by scanning electron microscopy (SEM) to monitor the Ca content during the culture process. 2+ Concentration changes and oil accumulation.

[0068] The crystal form comparisons of Example 1 and Comparative Examples 1 and 2 are shown in Table 2.

[0069] Table 2. Crystalline comparison of examples with different mineralization cultivation times and comparative examples.

[0070]

[0071] The results showed that, in the presence of carbonic anhydrase, the sample from Example 1, after 6 hours of mineralization, was mainly composed of amorphous calcium carbonate (ACC) and vaterite. The crystal forms were relatively dispersed, but the vaterite content was high. The electron micrograph is shown below. Figure 1As shown in the figure, after 24 h of reaction, aragonite remained the dominant crystalline phase, and a small amount of aragonite or calcite characteristic peaks began to appear. By 48 h, some aragonite transformed into aragonite or calcite, but studies showed that the overall crystal form could still be controlled within the expected range by adjusting the pH. Comparative Example 1 samples were taken at 1 h and 6 h of mineralization reaction and found almost no visible flocculent precipitate in the system, and very little solid was obtained after centrifugation. After 24 h and 48 h of reaction, the solid product increased, but effective separation was difficult to achieve. This was because the pH of the culture medium decreased due to the introduction of CO2, which affected the precipitation of calcium carbonate. Comparative Example 2 obtained a higher yield of calcium carbonate precipitate within 24 h. XRD analysis showed that it mainly existed in the stable calcite crystal form, with almost no precursor phases such as ACC or aragonite observed, indicating a single crystal phase. The electron micrograph of Comparative Example 2 after 24 h of mineralization culture is shown in the figure. Figure 2 As shown.

[0072] SEM observations showed that in Example 1, under carbonic anhydrase-regulated conditions, the numerous spherical or near-spherical microcrystalline particles formed after 6 hours of mineralization culture had diameters mainly concentrated between 0.5 and 2 μm, and exhibited porous or rough surface structures, making them suitable as precursors for subsequent transformation and composite materials. SEM results from Comparative Example 2 showed that the obtained particles were mostly large-sized rhombohedral particles or aggregates with a wide particle size distribution, which is insufficient to meet the requirements of uniform particle size and controllable surface structure for medical powders.

[0073] Monitoring and recording changes in oil content and Ca in Example 1 2+ Consumption rate was used to assess the co-production effect. Analysis showed that the oil content remained at a high level during the mineralization stage, while the Ca content... 2+ The concentration decreased rapidly within the first 6-24 hours, indicating that the CaCO3 nucleation and growth process induced by *Chlorella vulgaris* was significantly accelerated under the participation of exogenous carbonic anhydrase.

[0074] During the mineralization culture process, since Example 1 and Comparative Examples 1 and 2 were in a nitrogen-deficient state, the lipids in the microalgal cells were further accumulated; the highest lipid content in the cells reached 38.31%, 37.9%, and 40.9%, respectively. The lipid content of the microalgal cells obtained in Example 1 compared with Comparative Examples 1 and 2 is shown in Table 3.

[0075] Comparative Example 1: Ca during mineralization 2+ Monitoring of concentration changes revealed that, within the same time period, Ca... 2+ The consumption rate was significantly lower than in Example 1. There was no significant decrease in the first 6 hours, and a slow decreasing trend only appeared after 24 hours, indicating that the mineralization process was significantly slower under the condition of no carbonic anhydrase regulation.

[0076] Table 3. Comparison of oil content between examples and comparative examples with different mineralization cultivation times.

[0077]

[0078] Example 2

[0079] Using the aragonite-enriched calcium carbonate microcrystals obtained in Example 1 as raw materials, a phase transformation was performed on them. The method steps are as follows:

[0080] The aragonite-enriched microcrystalline calcium carbonate sample obtained from the 6-hour mineralization culture in Example 1 was sieved to obtain powder with a particle size range of 0.1–5 μm. This powder was added to a pre-prepared 50 mM phosphate buffer solution, and the pH was adjusted to 7.6 with dilute hydrochloric acid or NaOH. The reaction temperature was controlled at 37°C, and the solution was soaked under magnetic stirring or gentle shaking. During this process, bicarbonate ions (NaHCO3) were added to stabilize the pH at 7.6 and enhance the degree of carbonic acid doping. Carbonic acid-doped hydroxyapatite (CAp) was obtained. Samples of the solid were taken at 4 h, 8 h, and 12 h of soaking treatment.

[0081] Test 2

[0082] XRD analysis was performed on the solids obtained from immersion treatment for different times in Example 2. During immersion of 4–8 h, the original characteristic peaks of aragonite gradually weakened, and obvious characteristic peaks of carbonate-doped hydroxyapatite (CAp) appeared, indicating that phase transformation began to occur on the microcrystal surface. After immersion for 12 h, the aragonite peaks in the XRD pattern basically disappeared, and the highly crystalline peaks of CAp were clearly visible, indicating that most of the calcium carbonate microcrystals had been transformed into CAp.

[0083] The solid obtained after phase transformation in Example 2 was analyzed by scanning electron microscopy (SEM). The SEM observations after 12 h of immersion treatment are as follows: Figure 3 The results show that the transformed particles form a large number of nanosheet-like or needle-like crystals on their surface, creating a highly rough surface structure with a large specific surface area, which further facilitates subsequent interactions with cells or matrix materials. Since this transformation process is carried out under neutral or weakly alkaline conditions, without the need for high temperatures, high alkalinity, or strong acids and bases, it has minimal impact on the biocompatibility of the materials, making it suitable for the preparation of medical materials.

[0084] Example 3

[0085] The carbonate-doped hydroxyapatite (CAp) obtained by soaking for 12 h in Example 2 was used to prepare 3D printed medical composite materials. The method steps are as follows:

[0086] A mixed aqueous solution of sodium alginate (2 wt%) and methacrylamide gelatin (5 wt%) was prepared, and 0.05 wt% photoinitiator LAP was added. CAp powder was added under stirring to make its mass fraction in the system reach 10 wt%, and degassing treatment was performed to obtain an extrudable composite ink.

[0087] The composite ink was loaded into a syringe and extruded through a nozzle with a diameter of 2 mm at room temperature. After printing, the sodium alginate was ionically crosslinked with 100 mM CaCl2 solution and then photocured under 405 nm wavelength light for 40 s to complete the photocrosslinking of methacrylamide gelatin and obtain a 3D printed bone repair scaffold.

[0088] The resulting 3D-printed bone repair scaffold exhibits good shape retention and high forming accuracy in a wet state, with a uniform pore structure and adjustable pore size through printing parameters.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing calcium carbonate microcrystals by regulating microalgal mineralization using carbonic anhydrase, characterized in that, Includes the following steps: S1. Microalgae culture and lipid accumulation: Proliferate and culture *Gnaphalium arachnoideum* until the algal cells reach the late logarithmic phase or stationary phase; then, induce nitrogen restriction or nitrogen starvation culture to accumulate lipids in the algae. S2. Mineralization induction culture: The algal solution is transferred to a mineralization medium and carbonic anhydrase is added for 5–48 h of culture; the mineralization medium contains Ca. 2+ and HCO3 - ; S3. Microcrystal collection: The algae that have undergone mineralization-induced culture are harvested and microcrystals are separated to obtain calcium carbonate microcrystals.

2. The method according to claim 1, characterized in that, In step S1: Using CO2 as a carbon source; Light intensity 150~300 μmol·m -2 ·s -1 ; The incubation temperature is 22~28℃; The nitrogen restriction or nitrogen starvation induction culture time is 24-48 h.

3. The method according to claim 1, characterized in that, In step S2: The amount of carbonic anhydrase added is 4-8 U / mL. -1 ; The mineralization medium contains Ca. 2+ 5~20 mM and HCO3 - 10~50 mM.

4. The method according to claim 1, characterized in that, In step S2: The incubation temperature is 22~28℃; Maintain the culture medium at pH 8.4-9.

0.

5. The method according to claim 1, characterized in that, In step S2: No CO2 is supplied.

6. According to the method of claim 1, in step S2: the volume ratio of algal solution to mineralized culture medium is controlled at 1:(1~3).

7. Calcium carbonate microcrystals, characterized in that, Prepared by the method described in any one of claims 1 to 6.

8. The application of the calcium carbonate microcrystals as described in claim 7 in the preparation of carbonate-doped hydroxyapatite.

9. The application according to claim 1, characterized in that, Calcium carbonate microcrystals were placed in a phosphate buffer solution containing bicarbonate ions to obtain carbonate-doped hydroxyapatite.

10. The application of calcium carbonate microcrystals as described in claim 7 in the preparation of 3D printing materials.

Images