3D printing bio-ink, mineralized scaffold, preparation method of 3D printing bio-ink and application of 3D printing bio-ink in graded regeneration of mineralized tissues

Abstract

The invention provides 3D printing bio-ink, a mineralized scaffold, a preparation method and application in mineralized tissue grading regeneration, and belongs to the technical field of bone tissue engineering materials. The preparation method comprises the following steps: firstly, preparing an organic matter microcapsule with a bionic shape and a porous network, rapidly generating mineralization deposition in the microcapsule by introducing a polyphenol nucleation site to form a bone-like hydroxyapatite nanosheet aggregate, then taking the aggregate as a main functional unit of the bio-ink, and stably assembling the microcapsule through an organic matter continuous phase to obtain the biological ink. The personalized accurate control of the number of mineralization nucleation sites and the growth domain in the implant is realized. The mineralized scaffold is prepared through 3D printing, the mineralized scaffold provided by the invention can form rich and homogeneous mineralized growth domains in microcapsules, can effectively promote the growth of mesenchymal stem cells and form mineralized nodules to become artificial ossification centers, and accelerates the in-situ mineralization and bone regeneration processes of new tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of bone tissue engineering materials, and particularly relates to a 3D printing bioink, a mineralized scaffold, a preparation method thereof, and an application thereof in hierarchical regeneration of mineralized tissues. Background Art

[0002] There are different mineralized structures formed by a molecular bottom-up self-assembly mechanism in human tissues, which have specific excellent functions. For example, bone tissue is formed by mineralized crystals mainly composed of nano-hydroxyapatite arranged orderly in a collagen matrix to form a hard composite structure for bearing force. Bone and tendon are tightly combined through a gradient mineralized integration region to effectively transmit loads. The formation, growth, and integration of ossification centers are key steps in the bone development process, determining the final morphology and motor function quality of tissues such as bone and joint. However, when tissue defects occur, it is still challenging to reconstruct fine mineralized structures through regenerative implants.

[0003] When large-area tissue defects occur, it is difficult to reconstruct the natural mineralized matrix structure. Developing 3D printed tissue repair implants to promote high-quality tissue reconstruction is an ideal design for clinical applications. However, the existing printed implants guide the formation process of bone matrix mainly by mediating the random distribution, growth, integration, and on-demand remodeling of small pieces of newly formed mineralized organic matter, and it is difficult to regionally regulate the mineralization growth process. In areas such as the skull, spine, and joints, inappropriate regional matrix mineralization and bone regeneration processes are likely to cause serious adverse reactions such as nerve compression and soft tissue degeneration, which are the key factors leading to the decline of patients' motor function and disability. During development, the positioning, growth, and fusion of ossification centers play a key regulatory role in the distribution of regenerated mineralized matrix. Therefore, the importance of developing 3D printed regenerative implants with controllable assembled artificial ossification centers to guide the mineralized tissue reconstruction process has been gradually recognized.

[0004] Studies have shown that during the development of both intramembranous ossification and endochondral ossification, a key process of mineralized matrix growth and integration mediated by osteoblasts is included in the spheroidal ossification center. Compared with endochondral ossification involving multi-stage fine regulation, intramembranous ossification is relatively simple. The formation of the ossification center is initiated by the differentiation of condensed mesenchymal stem cells into osteoblasts. In a microenvironment saturated with mineralization precursors, the collagen network secreted by cells provides a growth space as a matrix skeleton, and molecules such as bone sialoprotein act as nucleation sites to regulate mineralization deposition and growth. With the continuous replacement of surface mesenchymal stem cells, the newly formed mineralized bone matrix expands from the ossification center to the periphery.

[0005] In recent years, bio-mineralization engineering strategies have shown excellent potential in simulating developmental ossification centers to construct bioactive mineralization initiation units that guide bone matrix formation. By using molecules such as citric acid and RNA to replace proteins that are difficult to purify and expensive as artificial mineral nucleation sites, polymer-induced calcium phosphate precursors can be effectively combined with three-dimensional organic matter to construct active composite mineralization structures, thus becoming bone induction growth domains to accelerate autologous tissue mineralization and bone regeneration processes. However, as aggregate units, polymer-induced calcium phosphate precursors enter the organic matter through diffusion to induce mineralization deposition. This introduction method makes the mineralization effect vulnerable to diffusion characteristics such as the density and volume of the organic matter, and it is difficult to simulate the morphology of the ossification center and achieve precise control of mineralization growth.

[0006] Based on the above problems, some studies have designed a printed implant that incorporates polyaspartic acid and alkaline phosphatase into a bioink to construct distributed mineral nucleation sites and calcium ion and phosphate ion growth domains. It is clear that the increase in nucleation sites can improve the in-situ mineralization saturation in the implant, and the subsequently grown mineralized products also have the biological activity to accelerate the bone regeneration process. However, although controllable and uniform assembly can be achieved through printing, the physically mixed functional molecules in the matrix are prone to rapid loss and it is difficult to maintain stable regulation of the growth in the mineralized area. Therefore, it is necessary to further optimize the design of the bioink in order to achieve controllable and stable assembly of artificial ossification centers in regenerative implants, thereby promoting the bionic reconstruction of natural mineralized tissues.

[0007] Therefore, there is an urgent need to provide a 3D printing bioink and its scaffold to achieve controllable assembly of artificial ossification centers for hierarchical regeneration of mineralized tissues. Summary of the Invention

[0008] The present invention is to solve the above technical problems, and thus provides a 3D printing bioink, a mineralized scaffold, a preparation method thereof, and an application in achieving controllable assembly of artificial ossification centers in hierarchical regeneration of mineralized tissues. The technical objective of the present invention is to provide a bioink and a 3D printed mineralized scaffold to achieve the construction of controllable assembly of artificial ossification centers, so as to solve the problems that existing bone regeneration scaffold materials cannot achieve bionic mineralization inside the scaffold and cannot form a homogeneous regional selective mineralization structure inside the scaffold, thereby providing a method for efficiently achieving bone regeneration and hierarchical regeneration of mineralized tissues.

[0009] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0010] The present invention first provides a 3D printing bioink, which includes 8 - 30 wt% GelMA solution, 5 - 30 wt% gelatin, 0.5 wt% LAP (photoinitiator), and 5 - 50 wt% mineralized microcapsules. Among them, the preparation method of the mineralized microcapsules is:

[0011] (1) Prepare a porous organic microcapsule from a GelMA solution;

[0012] (2) Immerse the porous organic microcapsule obtained in step (1) in a tannic acid solution to form a microcapsule with nucleation sites;

[0013] (3) Mix the microcapsule with nucleation sites obtained in step (2) with a CaCl 2 solution and a K 2 HPO 4 solution for biomineralization to obtain the mineralized microcapsule.

[0014] The present invention first successfully prepared gelatin-based spherical bionic organic microcapsules with uniform and adjustable sizes and a connected network structure through microfluidic technology and ice-template pore-making technology. Research shows that the microcapsules have excellent permeability and modifiability, and are suitable for the uniform infiltration and combination of nucleation site molecules and mineralization precursors; as a growth matrix for cells, they can well provide a micro-growth domain conducive to tissue regeneration. The aggregated structure formed by assembling as functional units has rich body interaction sites and spaces.

[0015] Furthermore, the present invention prepares the above microcapsules into a bioink and combines it with 3D printing technology to successfully design a printable artificial ossification center capable of self-mineralizing growth, and selectively assembles it into a customizable implant to guide the hierarchical reconstruction of mineralized tissues. Experiments prove that as the main functional unit of the bioink, the mineralized microcapsules are stably assembled through an organic continuous phase, realizing the personalized and precise control of the number of mineralization nucleation sites and growth domains in the implant, and facilitating the preparation of spinal fusion devices with a reinforced shell layer and selectively mineralized implantable patches for tendon-bone healing. In a body fluid environment, the microcapsules that effectively retain the mineralized structure can not only continue to self-mineralize and grow, but the gradually released polyphenol / precursor system can stably combine with the surrounding continuous organic matter to form more secondary mineralization nucleation and growth domains, thereby promoting the controllable regional bionic mineralization integration in the implant. In terms of regeneration and repair, the surface and interior of the printed microcapsule assembly structure have a large amount of host cell growth space, which effectively promotes the growth of bone marrow mesenchymal stem cells and the formation of mineralized nodules, and accelerates the in-situ mineralization of new tissues and the bone regeneration process.

[0016] In the construction of mineralized scaffolds, the present inventors have found that when other polyphenol materials (such as dopamine, EGCG) are used to replace tannic acid, physical cross-linking can be formed by the binding of polyphenols to microcapsules, but it cannot serve as a nucleation site to form a homogeneous mineralized structure; while when the polyphenol substance tannic acid is selected, it can quickly and efficiently bind to microcapsules as the primary mineralization nucleation site. After the microcapsules are assembled into the scaffold, they can gradually be released from it and combine with the surrounding organic matter to form secondary nucleation sites, thereby forming homogeneous mineralization inside the scaffold. At the same time, when the scaffold is directly modified with tannic acid, a too dense organic matter will be formed, which also prevents the mineralization from growing continuously inward and cannot form homogeneous mineralization inside the scaffold. In addition, mineralized microcapsules of different sizes also affect the mineralization growth inside the scaffold.

[0017] Furthermore, the preparation method of the porous organic matter microcapsules in step (1) is a combined microfluidic-ice template technology.

[0018] Furthermore, the concentration of the tannic acid solution in step (2) is 4-10 wt%.

[0019] Furthermore, the soaking time in step (2) is 4-24 h.

[0020] Furthermore, in step (3), the dosage ratio of the microcapsules with nucleation sites, CaCl 2 solution and K 2 HPO 4 solution is 20-200 μg microcapsules / mL calcium phosphate incubation solution, and the concentration range of Ca 2+ ,HPO 4 2 in the calcium phosphate incubation solution is 1-10 mM.

[0021] The second object of the present invention is to provide a preparation method of the 3D printing bioink as described above, including the following steps:

[0022] S1: Prepare mineralized microcapsules, and remove the excess liquid in the mineralized microcapsule dispersion by centrifugation;

[0023] S2: Add a polymer continuous phase solution composed of 10 wt% GelMA, 5 wt% gelatin and 0.5 wt% LAP to the mineralized microcapsules obtained in step (1) to prepare a printable bioink.

[0024] Furthermore, the centrifugation process in step S1 is a rotation speed of 2000-5000 rpm and a centrifugation time of 5-15 minutes.

[0025] Furthermore, the preparation process in step S2 is extrusion-based bioprinting.

[0026] A third object of the present invention is to provide a 3D printed mineralized scaffold, which is prepared by crosslinking the above-mentioned 3D printed bioink after visible light irradiation.

[0027] A fourth object of the present invention is to provide the use of the above-mentioned 3D printed mineralized scaffold in the preparation of bone repair and regeneration materials.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention proposes a developmental artificial ossification center design and corresponding regional selective printing technology for customizing bioactive regenerative implants that can guide the hierarchical regeneration of locatable mineralized tissues. First, organic microcapsules with a biomimetic shape and porous network are prepared by combining ice templating and microfluidics technology. By introducing polyphenol nucleation sites, mineralization deposition rapidly occurs in the microcapsules, forming aggregates of bone-like hydroxyapatite nanosheets. As the main functional unit of the bioink, the microcapsules are stably assembled through an organic continuous phase, achieving precise control of the number of mineralization nucleation sites and growth domains in the implant. In a body fluid environment, the microcapsules that effectively retain the mineralized structure can not only continue to grow by self-mineralization, but the gradually released polyphenol / precursor system can stably combine with the surrounding continuous organic matter to form more secondary mineralization nucleation and growth domains, thereby promoting controllable regional biomimetic mineralization integration in the implant. The rich mineralization growth domains formed inside the microcapsules can effectively promote the ingrowth of mesenchymal stem cells in the bone marrow and form mineralized nodules to become artificial ossification centers, accelerating the in-situ mineralization and bone regeneration process of new tissues. Description of the Drawings

[0030] Figure 1 Characterization of organic matter micro-units: (a) optical microscope image and (b) SEM image; (c) optical microscope image of TA-treated micro-units; (d) SEM image, (e) EDS image, (f) XRD image (* marks the characteristic peaks of hydroxyapatite), and (g) TEM image of microcapsules after 1-day mineralization incubation growth.

[0031] Figure 2 For microcapsules after 5-day mineralization incubation growth: (a) optical microscope image (the dotted circle indicates the boundary of the mineralized shell layer); (b) alizarin red staining image; (c) statistical chart of particle size changes; (d) SEM image, and (e) EDS element distribution surface scan image.

[0032] Figure 3 For the bioink: (a) temperature-sensitive viscosity change characteristics; (b) temperature-sensitive storage modulus / loss modulus change characteristics, and (c) shear thinning characteristics.

[0033] Figure 4 For (a) digital photos of printed and assembled structures of mineralized microcapsules with different loading amounts; (b) optical microscope image of the assembled structure.

[0034] Figure 5 Optical microscope images of the mineralization growth in the assembled structure at different time points (the dotted circles and arrows indicate the mineralization products); alizarin red staining images of the assembled structure after 7 days of incubation; SEM images; and XRD patterns (* indicates the characteristic peaks of hydroxyapatite).

[0035] Figure 6 Digital photos of the spinal fusion cage model and micro-CT reconstructions of the mineralized growth structure; digital photos and optical microscope images of the tendon-bone healing biphasic tissue repair patch model before and after mineralization growth.

[0036] Figure 7 Proliferation, adhesion, and secretion of mineralized nodules of bone marrow mesenchymal stem cells on the Min-Mcap assembled structure; (a) confocal microscope images of cell viability and (b) related statistical results after 3 and 7 days of co-culture; (c) cell adhesion in the assembled structure after 10 days of co-culture; (d) alizarin red staining images of the secretion of mineralized nodules by cells in the microcapsule assembled structure after 14 days of co-culture (the dotted circles indicate the mineralized nodules around the microcapsules).

[0037] Figure 8 Function of the microcapsule assembled implant in the cranial defect repair model; (a) gross observation of the surgical site 3 weeks after implantation, with the implant marked by a black dotted line; (b) HE and Goldner staining results showing the mineralization growth around the microcapsules in the newly formed tissue (the mineralized area and the microcapsule area are indicated by blue dotted lines and red dotted circles respectively, and the blue arrows indicate the regenerated mineralized matrix around the microcapsules); (c) statistical analysis of the Goldner staining results (***P < 0.001 vs. blank control group; ##P < 0.01 vs. B-Mcaps group).

[0038] Figure 9 Mineralization growth of the printed assembled structure of mineralized microcapsules of different sizes in the incubation solution.

[0039] Figure 10 Mineralization growth in the GelMA-based continuous phase printed structure directly treated with TA; (a) direct optical microscope observation images; (b) calcein staining and (c) alizarin red staining results. Specific Embodiments

[0040] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be specifically described below in conjunction with embodiments. It should be noted that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the protection scope of the present invention.

[0041] Example 1

[0042] I. Experimental method

[0043] 1. Preparation and characterization of mineralized microcapsules

[0044] (1) Preparation of mineralized microcapsules

[0045] GelMA was synthesized by the microfluidic method and combined with the ice-templating treatment method to prepare porous organic microcapsules with diameters of 100, 150, and 200 μm (denoted as B-Mcaps).

[0046] Tannic acid (TA) was dissolved in water to prepare solutions with concentrations of 4%, 8%, and 10% (wt%). Then, the porous organic microcapsules were dispersed in different concentration solutions and soaked overnight to prepare microcapsules with nucleation sites (denoted as NS-Mcaps). After washing the NS-Mcaps in each group several times with deionized water, they were observed using an optical microscope to analyze the effect of TA treatment on their morphology.

[0047] To prepare mineralized microcapsules (denoted as Min-Mcaps), the NS-Mcaps were first added to a calcium chloride solution and dispersed evenly; subsequently, the dispersion was mixed with an equal volume of K 2 HPO 4 solution and incubated at 37 °C. After a specific period of time, the Min-Mcaps were collected and washed with deionized water for subsequent research.

[0048] (2) Characterization of microcapsules

[0049] ① Alizarin red staining

[0050] Alizarin red staining was used to analyze the distribution of mineralized deposition products on the Min-Mcaps. After adding the Min-Mcaps dispersion to a 24-well plate, it was stained with the alizarin red working solution for 30 minutes, washed with deionized water, and observed under a microscope.

[0051] ② SEM-EDS

[0052] After the microcapsules were freeze-dried and sputter-coated with gold, a scanning electron microscope was used to observe the microscopic morphology of the mineralized deposition products, and an energy dispersive spectrometer was used in combination to analyze the microscopic distribution of the mineralized substances.

[0053] ③ XRD

[0054] Wide-angle XRD was used to further study the phase transformation of the Min-Mcaps.

[0055] ④ TEM

[0056] After cryosection treatment, TEM was used to characterize the micro-nano morphology of the mineralized deposition products in Min-Mcaps.

[0057] 2. Preparation and Rheological Characteristics of Bioink

[0058] Excess liquid in the microcapsule dispersion was removed by centrifugation, and a printable bioink was prepared by adding a polymer continuous phase solution composed of 10 wt% GelMA / 5 wt% gelatin and 0.5 wt% LAP.

[0059] A rheometer was used to measure the rheological properties of the bioink loaded with Min-Mcaps and the pure GelMA / gelatin bioink. The thermosensitive characteristics of the bioink were studied under the condition of a temperature range of 4 - 37 °C (strain: 1%, frequency: 1 Hz). At room temperature, the rheological characteristics of the ink were studied at a shear rate of 0.1 - 100 rad / s.

[0060] 3. Printability of Bioink

[0061] B-Mcaps and Min-Mcaps were introduced into the GelMA / gelatin solution at low and high loading doses respectively to prepare bioinks for printability studies. The printed structures were crosslinked by visible light irradiation and soaked in deionized water at 37 °C overnight to remove gelatin. The morphology and distribution of microcapsules in the structure were observed by optical microscopy.

[0062] 4. Alizarin Red Staining of Printed Structures

[0063] The printed structures were placed in 24-well plates and stained with alizarin red solution respectively to analyze the structural stability and distribution of Min-Mcaps under the shear force during printing.

[0064] 5. Mineralization Growth in Min-Mcaps Assembled Structures

[0065] To clearly observe the distribution of mineralized deposition growth in the printed structures, we used Min-Mcaps pre-mineralized for 1 day to prepare bioink. B-Mcaps were introduced into the GelMA / gelatin solution and printed in the same way to prepare structures loaded with B-Mcaps. The GelMA / gelatin solution was printed to prepare blank control structures. The printed structures of each group were immersed in 10-fold simulated body fluid for incubation according to a certain solid-liquid ratio; after a specific time, they were collected and washed with deionized water to remove unstably bound deposits, and the morphology and distribution of the mineralized deposits inside the structures were analyzed by optical microscopy and alizarin red staining.

[0066] (1) XRD

[0067] Wide-angle XRD was used to study the mineralized growth phases in the structures.

[0068] (2) SEM images of the mineralized structures

[0069] After being rinsed with deionized water, freeze-dried, and sputter-coated with gold, the surface and cross-section of the mineralized structures were observed using SEM.

[0070] (3) Micro-CT detection of the mineralized structures

[0071] After being rinsed with deionized water and freeze-dried, the samples were scanned using Micro-CT. The data results were analyzed by three-dimensional reconstruction and by selecting the distribution pattern of the gray values of the mineralized substances.

[0072] 6. Proliferation, adhesion, and osteogenic differentiation of bone marrow mesenchymal stem cells on the Min-Mcaps assembled structures

[0073] (1) Cell culture, proliferation, and adhesion

[0074] Bone marrow mesenchymal stem cells were isolated from SD-Dawley rats and cultured using the method described previously. The printed GelMA structures, B-Mcap assembled structures, and Min-Mcap assembled structures were placed in 24-well culture plates respectively. Bone marrow mesenchymal stem cells (2×10 4 ) were seeded onto each construct. After co-culturing for 3 days and 7 days, they were stained with 5-chloromethylfluorescein diacetate and propidium iodide at 37 °C for 30 min and observed under a confocal fluorescence microscope. To detect the cell adhesion on the structures, bone marrow mesenchymal stem cells (1×10 4 ) were evenly seeded onto each construct, fixed with paraformaldehyde fixative after co-culturing for 10 days, stained with rhodamine-phalloidin and DAPI, and then observed under a confocal fluorescence microscope.

[0075] (2) Alizarin red staining

[0076] Bone marrow mesenchymal stem cells were seeded onto the printed structures at a density of 2×10 4 cells / well, fixed after co-culturing in osteogenic induction medium for 14 days, stained with alizarin red solution, and observed and analyzed for the secretion of mineralized nodules by cells under an optical microscope.

[0077] 7. In vivo mineralization and tissue regeneration

[0078] (1) In situ defect repair model

[0079] A skull defect repair model was established to analyze the in-vivo mineralization promotion and tissue regeneration characteristics of the implant. Thirty healthy male Sprague-Dawley rats (8 weeks old) were divided into 3 groups: (1) untreated group, (2) B-Mcap assembled implant, and (3) Min-Mcap assembled implant. A defect with a diameter of 4 mm was created in each rat. After implanting the material (φ4×1.5 mm 3 ), the incision was closed. Three weeks after implantation, the animals were sacrificed and the skulls were collected, fixed with fixative for further analysis (n = 5).

[0080] (2) Histological evaluation

[0081] The samples were pretreated, embedded in polymethyl methacrylate, and then sectioned. The sections were stained with hematoxylin / eosin (HE) and Goldner's trichrome for histological analysis.

[0082] 8. Statistical analysis

[0083] The data were expressed as mean ± standard deviation, and one-way analysis of variance (ANOVA) was performed to compare the statistically significant differences between groups.

[0084] II. Experimental results

[0085] 1. Controllable preparation and characterization of microcapsules

[0086] Using an aqueous solution of GelMA as the raw material, spherical microorganic matter units with controllable sizes and similar morphology to the ossification center were prepared through a microfluidic device. Then, the ice templating method was used to process the microorganic matter units to form a connected porous structure, providing more binding areas for subsequent mineralization deposition. First, micro-units with a size of about 150 μm were prepared as a preliminary research model. From Figure 1 the optical microscope observation results, it can be seen that the prepared micro-units have uniform sizes and are consistent with the expected size. Due to the formation of internal pores, its light transmittance is relatively low. From Figure 1 the scanning electron microscope results in b, it can be seen that pore structures with a size of 10 - 20 μm are distributed inside the microspheres, and the surface of the organic matrix material inside the pores is smooth.

[0087] Furthermore, tannic acid (TA) molecules rich in polyphenols were designed to modify the microorganic matter units to introduce mineralization nucleation sites. First, the polyphenol groups in tannic acid can stably bind to the amino groups in the organic matter units through hydrogen bonding. Subsequently, the benzene ring in the tannic acid molecule can conjugate with calcium ions to initiate the nucleation process; phosphate ions and calcium ions in the environment are successively assembled and grown at the conjugate binding sites, enabling the gradual formation of amorphous calcium phosphate and then crystals.

[0088] In the experiment, GelMA micro-units were first incubated with tannic acid (TA) solutions of different concentrations. After overnight incubation at 37 °C, it could be visually observed that the particles gradually changed from white to light yellow. Observation under an optical microscope revealed that treatment with TA molecules led to a decrease in the light transmittance of the microparticles and a certain reduction in particle size ( Figure 1 as shown in c) of []. This is mainly due to TA acting as a binding point, which further cross-links the gelatin molecular chain network. Due to the uniform binding of TA in the microspheres, the re-cross-linked particles still maintain their regular spherical appearance. The effects of different concentrations of TA treatment on the appearance of the micro-units are not significantly different. As can be seen above, TA treatment makes the pore size inside the microspheres smaller and the pore structure denser, which is consistent with the analysis by optical microscopy. In addition, the degree of pore densification shows a concentration dependence on TA. The pore size inside the microspheres treated with 10% TA is significantly smaller than that of the microcapsules treated with 4% TA.

[0089] After removing the free TA, the treated micro-units were immersed in a calcium-phosphorus source mineralization solution for incubation to observe the formation of mineralized microcapsules. According to the SEM results ( Figure 1 as shown in d) of []. After 1 day of incubation, mineral particles were tightly bound to the porous organic matter surface of the micro-units treated with different concentrations of TA. The single particles were nanoneedle-shaped, and after aggregative growth, they presented as mineralized lamellar regions, all closely adhering to the organic matter. Through EDS elemental mapping analysis ( Figure 1 as shown in e) of []. It could be seen that calcium and phosphorus elements were evenly dispersed, which was consistent with the morphology of the microcapsules, demonstrating the uniformity of the mineralized deposition on the microcapsule surface.

[0090] XRD detection was used to analyze the phase of the formed mineralized products ( Figure 1 as shown in f) of []. It was found that the peak positions of the minerals deposited in the micro-units treated with different concentrations of TA were the same, and the characteristic peaks of hydroxyapatite appeared. Analysis of the peak intensities showed that compared with the micro-units treated with 4% TA concentration, the spectra of the micro-units treated with 8% and 10% TA solutions had stronger hydroxyapatite characteristic peaks. Through the processing and calculation of the characteristic peak areas, it was shown that the sizes of the mineralized crystal grains deposited in the micro-units treated with 8% and 10% TA concentrations were relatively close, being 28.9 nm and 29.7 nm respectively. Based on this, 10% TA concentration was selected for subsequent experiments to modify the organic matter. TEM was used to observe the cryosection profile of the mineralized micro-units to analyze the crystal unit morphology and arrangement of the mineralized products inside the organic matter. As Figure 1 shown in g) of []. The mineralized units deposited on the gray organic matter were needle-shaped, with lengths between 30 - 50 nm, which was close to the XRD calculation results.

[0091] 2. Growth of Microcapsules

[0092] After it was confirmed that the micro-organic matter units treated with TA could rapidly form mineralized microcapsules and the phase of their deposition products after incubation in a calcium phosphate mineralization solution for 24 h, the growth of Min-Mcaps was further observed as the incubation time increased. After 5 days of incubation, the Min-Mcaps were collected and washed several times with deionized water for subsequent studies. First, observation was carried out by optical microscope ( Figure 2 as shown in a) of Figure 2 . It could be clearly seen that the particle size of Min-Mcaps became larger than that after 24 h of incubation, and a relatively uniform and transparent sediment shell layer was formed on the surface, with a thickness of 14.08 ± 1.30 μm. By performing alizarin red staining analysis on Min-Mcaps ( as shown in b) of

[0093] ), it could be seen that the sediment reacted with the staining solution to show red and was evenly distributed, indicating that the sediment shell layer was a calcium-based substance. Figure 2

[0094] Furthermore, according to the observation results of the optical microscope, statistical analysis was carried out on the particle size changes of the microspheres at each time point before and after mineralization ( Figure 2 as shown in c) of Figure 2 ). The results showed that the particle sizes of the unmodified micro-organic matter units, TA-modified micro-organic matter units, and microcapsules formed after 1 day and 5 days of mineralization were 172.41 ± 14.63 μm, 129.31 ± 9.44 μm, 155.17 ± 6.90 μm, and 190.80 ± 7.76 μm, respectively. Treatment with TA caused physical re-crosslinking inside the microcapsules, resulting in a smaller particle size. As the mineralization incubation time extended, the particle size of the microcapsules gradually increased, which was mainly caused by two factors: 1) During the incubation process, TA was gradually released from the microcapsules, making the molecular chain network of the basal organic matter become loose, thus increasing the volume of the micro-units; 2) The gradual formation of the mineralized shell layer further increased the particle size. Combining the above results, it could be found that the gradual release of TA did not hinder mineralization growth. This was mainly because a large amount of TA was bound to the micro-organic matter units. Although part of it was released during the incubation process, there was still a sufficient amount stably bound to provide mineralization initiation sites.

[0095] 3. Preparation and Rheological Properties of Bioink

[0096] Use GelMA / gelatin mixed ink as the continuous phase for loading Min-Mcaps. GelMA is a temperature-sensitive material that exhibits good printability in the range of 15 - 20 °C. Adding gelatin, on the one hand, acts as a mechanical reinforcement phase to improve the structural stability of the printed structure during the printing process; on the other hand, as a pore-forming aid, it can be easily removed after the formation of the printed structure, forming voids conducive to ion penetration.

[0097] For temperature-sensitive bioinks, selecting an appropriate printing temperature is crucial for regulating rheological properties and the formation of the structure. Therefore, after the ink is formulated, its rheological characteristics are first explored. As can be seen from Figure 3 the viscosity-temperature change curve in a), all groups of inks show temperature sensitivity. As the temperature increases, the viscosity of the ink gradually decreases. As expected, below the phase transition temperature, the viscosity of the GelMA / gelatin composite ink is significantly higher than that of the GelMA ink. In addition, the addition of Min-Mcaps acts as a reinforcement phase, which can further increase the viscosity of the ink.

[0098] Combined with the analysis of the results of the temperature-dependent curves of the storage modulus and loss modulus of the three inks ( Figure 3 in b), the phase transition temperature of the GelMA ink is around 20 °C, and the phase transition temperature of the GelMA / gelatin composite ink is around 25 °C. The addition of gelatin increases the phase transition temperature of the ink. The addition of Min-Mcaps does not have a significant effect on the phase transition temperature, and the phase transition temperature of the composite ink remains around 25 °C. Based on this result, the rheological characteristics of each group of inks at 15 °C and 25 °C are investigated. As can be seen from Figure 3 c, all groups of bioinks exhibit shear thinning characteristics. In the angular frequency range of 0 - 40°, the addition of gelatin makes the viscosity of the GelMA / gelatin composite ink significantly higher than that of the GelMA group, while the addition of Min-Mcaps does not affect the reinforcement effect of gelatin.

[0099] 4. Printability of Bioink

[0100] In terms of printing performance, Min-Mcaps with a particle size of 150 - 200 μm are used to prepare low-loading and high-loading bioinks for experiments. As can be seen from Figure 4It can be seen that bioinks containing different doses of Min-Mcaps all have excellent printability. At low loading levels, the printed structures exhibit the transparent color of the continuous phase GelMA, and Min-Mcaps are evenly distributed among the continuous phases, mainly covered inside the continuous phase. While the structures formed by printing high-loading bioinks exhibit the yellow color of Min-Mcaps, with significantly reduced transparency, and prominent Min-Mcaps can be clearly seen on the surface of the printed filaments. In terms of the formation of three-dimensional structures, the ink maintains excellent structural stability and pore connectivity after being assembled layer by layer along the planned regular paths. In addition, the ink can be assembled along irregular curved paths without structural deformation and collapse.

[0101] 5. Role of Min-Mcaps in promoting the mineralized growth of printed structures

[0102] After clarifying that Min-Mcaps have mineralized micro-nano structures and growth trends similar to those of ossification centers, we then immersed the printed structures of each group in simulated body fluid to investigate the mineralized growth of Min-Mcaps in the continuous phase system. Min-caps with a diameter of 150 - 200 μm and pre-mineralized for 1 day were selected to prepare bioinks to construct a research model for printed structures. GelMA structures and B-Mcaps assembled structures were used as control groups. After immersing the structures of each group in simulated body fluid for 7 days, they were rinsed several times with deionized water to remove the unstable mineralized substances for subsequent research. From Figure 5 the optical microscope observation results in a), it can be seen that at 3 days of incubation, granular dispersed deposits can be clearly seen in the structures assembled with Min-Mcaps, and the quantity is significantly higher than that in the GelMA structure group and the B-Mcaps structure group. As the incubation time extends, mineral growth is gradually observed in the structures of each group. Among them, the structures assembled with Min-Mcaps have the most deposits, presenting sheet-like aggregates, which are widely distributed on the surface and around the mineralized microcapsules.

[0103] By tracking the distribution of calcium deposition through alizarin red staining ( Figure 5 in b)), it was observed that: compared with the difficult-to-observe stable deposits on the GelMA structures, the mineralized substances in the structures assembled with B-Mcaps are mainly stably deposited on the surface of B-Mcaps and within the assembly gaps; the mineralized substances are evenly deposited in the structures assembled with Min-Mcaps. The microscopic morphology results of the deposited mineralized substances on the surface and inside the structures of each group observed by SEM show ( Figure 5In c), some network-like degradation products are deposited on the surface of the GelMA structure, and some inorganic substances are distributed therein; there are few infiltrating growth deposits inside. The deposits on the B-Mcaps assembled structure are all aggregates assembled by nano-needle-shaped calcium phosphate mineralized particles, which are closely attached to the GelMA organic matter. The surface and internal deposit aggregates of the Min-Mcaps structure are significantly more than those grown on the B-Mcaps structure.

[0104] Furthermore, after 7 days of mineralization growth, the XRD detection was carried out on the printed structures of each group to analyze the phase of the mineralization products. From Figure 5 As can be seen from d), due to the small amount of deposited mineralization products, only weak characteristic peaks of mineralized substances were detected in the GelMA structure and the B-Mcaps structure. The characteristic peaks of hydroxyapatite can be seen in the spectra of the GelMA and Min-Mcaps structures. The characteristic peaks in the Min-Mcaps structure are significantly stronger than those in the GelMA structure.

[0105] Based on the above results, it can be speculated that Min-Mcaps promotes the overall mineralization deposition of the printed structure as an artificial ossification center mainly through two ways: 1) The TA bound therein provides nucleation sites for calcium phosphate mineralization deposition, promoting its gradual growth; 2) The TA therein is gradually released and combined with the continuous phase, so that the surrounding organic matrix has secondary nucleation sites. In addition, more importantly and worthy of note is that the topological structure of the organic matrix has a significant regulatory effect on the deposition of mineralized substances, which is mainly manifested in the following aspects: 1) The voids formed by the Gelatin sacrificial material in the continuous phase gelatin matrix enable the gradually released TA to better infiltrate the continuous phase to provide nucleation sites, and the mineralized substances have growth space. 2) The introduction of Mcaps makes a certain void in the contact space between the continuous phase and Mcaps, which is conducive to the accumulation growth of mineralization.

[0106] Based on the action law of the Min-Mcaps structure mineralization growth, a spinal fusion device model with an irregular shape and a multi-material integrated device model were designed and constructed to further investigate its application potential. First, a bioink loaded with Min-Mcaps was prepared. Referring to the commonly used clinical implants, a spinal fusion device model with a thickness of 3 mm and a maximum width of 10 mm was designed and printed ( Figure 6 as shown in a)). To enhance the structural stability, an outer edge shell layer with a width of 0.33 mm was added. After the model was soaked in simulated body fluid for 7 days, it was rinsed with deionized water to remove the unstably bound calcium deposits and freeze-dried for detection. As can be seen from the Micro-CT detection results, in the printed structure with a large volume, the mineralization deposition promoted by Min-Mcaps is also evenly distributed throughout the device model.

[0107] Next, GelMA ink and Min-Mcaps-based bioink were respectively selected to construct a biphasic tissue patch device model for tendon-bone repair with a thickness of 1.5 mm by multi-nozzle printing technology and observe the mineralization growth therein( Figure 6 as shown in b) of. The number of printing layers of Min-Mcaps-based ink and GelMA ink are 2 layers and 3 layers respectively. The thickness of the GelMA structure is 0.9 mm, and the thickness of the Min-Mcaps structure is 0.6 mm. After incubating the printed structure in simulated body fluid for 7 days, the unstable conjugates were washed with deionized water and then observed. From the appearance, the shape of the patch model did not change before and after mineralization. Due to the release of TA, TA in the Min-Mcaps assembly part was gradually released, making the color turn yellow. It was found by optical microscopy that abundant mineralized particles in the upper Min-Mecaps structure aggregated and deposited around the Min-Mcaps, while the surface of the bottom GelMA structure was smooth and there were few mineralized deposits

[0108] The above results indicate that Min-Mcaps formulated as a functional unit in bioink, combined with the printing structure design, can not only complete the construction of large-volume personalized devices and promote their uniform mineralization growth in body fluid, but also be used for selective mineralization in multi-material personalized structures. It has the potential application in the bionic repair of gradient mineralized structure tissues such as articular bone-cartilage and tendon-bone, which are difficult to achieve with conventional methods

[0109] 6. Proliferation, adhesion and osteogenic differentiation of mesenchymal stem cells in the Min-Mcaps assembly structure

[0110] A biological ink was prepared using Min-Mcaps pre-mineralized for 1 day and a printed structure research model was fabricated. It was co-cultured with bone marrow mesenchymal stem cells to investigate the biosecurity of the mineralized microcapsule assembly device and its effect on osteogenic differentiation. As can be seen from Figure 7 a) and b) of, during the co-culture from 3 days to 7 days, the stem cells had good viability on each group of structures, with few dead cells, and the cell viability was above 85%. As the co-culture time of the cells with each group of structures increased, the cells gradually spread well and proliferated on the printed filaments. At 10 days of co-culture, it was observed that the cells gradually connected and adhered to the printed filaments in a sheet-like manner on each group of printed structures( Figure 7 as shown in c) of. Especially on the Mcaps assembly structure, not only can the cells be seen growing and spreading densely on the microcapsules, but also their growth and connection can be observed between the pores formed by printing. Thus, it can be seen that the Mcaps assembly structure has good cell compatibility

[0111] The secretion of mineralized nodules by osteoblasts is an important natural process in bone regeneration. Therefore, the generation of mineralized nodules by cells on the Min-Mcaps assembled structure was then observed. After 14 days of co-culture, alizarin red staining was performed and it was found that ( Figure 7 as shown in d) of Figure 7 , there were few cells secreting mineralized nodules on the structure assembled by B-Mcaps, while cells secreting mineralized nodules and stained red were evenly dispersed in the Min-Mcaps assembled structure. In addition, the mineralization products were mainly distributed around Min-Mcaps. Thus, it can be seen that Min-Mcaps, as a bioactive device, has the ability to promote osteogenic differentiation and the formation of mineralized nodules.

[0112] 7. In vivo mineralization and tissue repair promotion ability of the Min-Mcaps assembled structure

[0113] An in-situ cranial defect model was constructed, and the printed structure was implanted into the defect site to investigate the in vivo mineralization growth of Min-Mcaps and its effect on tissue regeneration ( Figure 8 as shown in a) of Figure 8 . After the structure was implanted, no animals died, preliminarily indicating good tissue compatibility. Three weeks after implantation, the animals were sacrificed and the surgical site was exposed. It was found that a thin layer of soft tissue grew in the defect of the blank control group. The microcapsule assembled structure group was surrounded by new tissue and no adverse reactions were seen. Combining the results of HE and Goldner trichrome histological staining and analysis, it was found that ( Figure 8 as shown in b) of Figure 8 , mainly loose organic soft tissue was formed in the control group. A layer of mineralized substances was deposited on its surface. The presence of microcapsules could be observed at the defect sites of other groups. Consistent with the in vitro mineralization experiment results, the new tissue between the pores of the B-Mcaps assembled structure was similar to that growing in the blank group, mainly loose soft tissue, and the mineralization deposition was mainly around B-Mcaps. Abundant new osteoid could be observed between the pores of the Min-Mcaps assembled structure, and abundant mineralized areas could be seen around Min-Mcaps. Statistical analysis of the results showed that the proportions of newly formed mineralized tissue at the defect sites in the blank control group, the B-Mcaps tissue structure group, and the Min-Mcaps assembled structure group were 10.59%, 15.27%, and 28.06% respectively ( Figure 8 as shown in c) of Figure 8 . Compared with the control group, as an artificial ossification center, Min-Mcaps assembled in the structure could effectively promote the mineralization of surrounding tissues (P < 0.001).

[0114] Comparative Example 1

[0115] Study the mineralization growth in mineralized microcapsule assembled structures of different sizes:

[0116] Prepare porous organic matter microspheres with an average particle size of about 100 m and about 250 μm respectively, and use the same method as in Example 1 to prepare small-sized mineralized microcapsules (Min-SMcaps) and large-sized mineralized microcapsules (Min-BMcaps) in the incubation solution, prepare the corresponding bioink and print and assemble the structure.

[0117] The microstructural changes after 3 days and 7 days of incubation were observed by optical microscopy, and the results are as Figure 9 shown. It can be found that (a) mineralized deposits gradually aggregate and grow around the mineralized microcapsules (marked by blue dotted circles and arrows); (b) the results of calcein staining and (c) alizarin red staining show that the degree of mineralization and homogeneity in the printed structure gradually decrease as the particle size of the microcapsules increases. This may be because under the same mass and the same volume of suspended dispersion matrix, the number of small-sized microcapsules is the largest, so that there can be more abundant nucleation sites distributed in the organic matter continuous phase.

[0118] Comparative Example 2

[0119] Study the growth and mineralization of the structure directly treated with TA:

[0120] The GelMA-based continuous phase printed structure was directly treated with tannic acid (TA), and the mineralization growth in the structure was observed. The results are as Figure 10 shown. From Figure 10 it can be seen that after calcein staining and alizarin red staining, it was observed that only a small amount of mineralized deposits grew on the surface of the network structure. This may be because the organic matter is too dense after being modified with TA, and it is difficult for the mineralization precursor to enter its interior for mineralization growth. However, in the continuous organic matter designed in the present invention, there are mineralization growth sites as TA is gradually released from the microcapsules; it does not affect the entry of the mineralization precursor into the interior of the organic matter, and through the regulation of the loading quantity of the microcapsules, homogeneous mineralization can be rapidly formed in the presence of nucleation sites.

[0121] Comparative Example 3

[0122] Replace tannic acid with other polyphenol materials (such as EGCG), prepare microcapsules and mineralize them according to the method of Example 1. It was found that the above-mentioned polyphenol substances would form a cross-linked network with the scaffold, and it was impossible to form a homogeneous region-controllable mineralized structure by providing fixed-point multi-level nucleation sites like mineralized microspheres.

Claims

1. A 3D printing bio-ink, characterized in that: The invention comprises 8-30wt% GelMA solution, 5-30wt% gelatin, 0.5wt% LAP and 5-50wt% mineralized microcapsules, wherein the preparation method of the mineralized microcapsules is as follows: (1) GelMA solution was prepared into porous organic microcapsules by microfluidics and ice template method; (2) soaking the porous organic microcapsules obtained in step (1) in a tannic acid solution to form microcapsules with nucleation sites; (3) The microcapsules with nucleation sites obtained in step (2) are mixed with a CaCl2 solution and a K2HPO4 solution and then mineralized and grown to obtain the mineralized microcapsules.

2. The 3D printing bio-ink according to claim 1, characterized in that: The porous organic microcapsules described in step (1) are prepared by using a microfluidics-ice template combination technology.

3. The 3D printing bio-ink according to claim 1, characterized in that: The concentration of the tannic acid solution in step (2) is 4 to 10 wt %.

4. The 3D printing bio-ink according to claim 1, characterized in that: The soaking time in step (2) is 4-24 hours.

5. The 3D printing bio-ink according to claim 1, characterized in that: The amount ratio of the microcapsules with nucleation sites, the CaCl2 solution and the K2HPO4 solution in step (3) is 20-200 μg microcapsules / mL calcium-phosphate incubation solution, and the Ca in the calcium-phosphate incubation solution is 1. 2+ 、HPO4 2 The concentration range was 1-10 mM.

6. The method for preparing the 3D printing bio-ink according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: preparing mineralized microcapsules, and removing excess liquid from the mineralized microcapsule dispersion by centrifugation; S2: Add a polymer continuous phase solution consisting of 10 wt% GelMA, 5 wt% gelatin and 0.5 wt% LAP to the mineralized microcapsules obtained in step (1) to prepare a printable bio-ink.

7. The preparation method according to claim 6, characterized in that: The centrifugal treatment process in step S1 is a rotation speed of 2000-5000 rpm and a centrifugation time of 5-15 minutes.

8. The preparation method according to claim 6, characterized in that: The preparation process in step S2 uses extrusion bioprinting.

9. A 3D printed mineralized scaffold, characterized in that: The 3D printing bio-ink according to any one of claims 1 to 5 is prepared by cross-linking after visible light irradiation.

10. Use of the 3D printed mineralized scaffold according to claim 9 in the preparation of bone repair and regeneration materials.

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