A 3d bioprinted radial porous scaffold for staged bone regeneration and method of making the same

Abstract

The application discloses a 3D bioprinted radial porous scaffold for staged bone regeneration and a preparation method thereof, the 3D bioprinted radial porous scaffold can provide a biomimetic cell microenvironment to support the infiltration of host repair cells to a defect site, and promote bone integration after implantation, in addition, the sequential release of double growth factors and the sustained release of PO4 3‑ and Ca 2+ in the 3D bioprinted radial porous scaffold can complete the three key steps of staged bone regeneration, can realize efficient bone regeneration, and represents a promising biomimetic staged regeneration strategy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bone repair materials, and particularly relates to a 3D bioprinted radial porous scaffold for staged bone regeneration and a preparation method thereof. BACKGROUND

[0002] Bone defects can be caused by trauma, fracture, infection, deformity or tumor. For defects beyond the critical size, artificial intervention is usually required. At present, autograft is considered as the gold standard. However, autograft has many limitations, such as the need for secondary surgery, pain, infection risk and poor wound healing. In addition, allograft also faces potential risks of immune rejection and disease transmission. With the rapid development of tissue engineering and regenerative medicine, biomaterial scaffolds have been considered as a promising alternative strategy to effectively overcome the limitations of autograft and allograft.

[0003] Biomaterial scaffolds suitable for bone regeneration should provide a biomimetic cellular microenvironment to support the infiltration of host repair cells to the defect site and promote bone integration after implantation. Radially arranged scaffolds can guide host repair cells to migrate from host tissue to the center of the defect along the radial direction. At present, the main methods for constructing radially arranged scaffolds include directional freeze casting technology and 3D printing technology. Directional freeze casting technology is widely used and has the advantages of low cost and environmental protection, but the technology requires high temperature control and needs to accurately control the cooling rate and direction. In addition, since the microstructure of the scaffold depends on the growth process of ice crystals, the structural precision is lower compared to 3D printing technology. How to use 3D printing technology to prepare a 3D bioprinted scaffold that can be effectively used for staged bone regeneration is still one of the main technical problems faced by the field. SUMMARY

[0004] In order to overcome the technical problems currently faced by the field, the purpose of the present application is to provide a 3D bioprinted radial porous scaffold for staged bone regeneration and a preparation method thereof.

[0005] In the present application, the inventors of the present application first introduced BMP-2 loaded black phosphorus nanosheets (BP@BMP-2) into a precursor solution containing PDGF-BB, GelMA and TCP, and constructed a hydrogel scaffold with radial porous structure using 3D bioprinting technology. Under the stimulation of double growth factors (BMP-2, PDGF-BB), BMSCs were recruited and differentiated into mature osteoblasts, and then BP nanosheets and TCP nanoparticles continuously released PO4 3- and Ca 2+ , promoting the formation and deposition of calcium phosphate on the hydrogel scaffold. Therefore, the 3D bioprinted radial porous scaffold with black phosphorus nanosheet composite can play a good effect in bone regeneration applications and represents a promising biomimetic staged regeneration strategy.

[0006] The application achieves the above-mentioned application purposes by adopting the following technical solutions.

[0007] The first aspect of the application provides a preparation method of a 3D bioprinted radial porous scaffold for staged bone regeneration.

[0008] Further, the method comprises the following steps: incubating black phosphorus (BP) nanosheets with a BMP-2 solution to prepare BMP-2-loaded black phosphorus nanosheets (BP@BMP-2) after centrifugation; dissolving methacrylated gelatin (GelMA) in a mixed solution of lithium phenyl-2,4,6-trimethylbenzoyl phosphonate (LAP) and tartrazine to prepare a GelMA solution; adding beta-tricalcium phosphate (TCP) nanoparticles, BP@BMP-2 nanosheets and platelet-derived growth factor-BB (PDGF-BB) solution to the GelMA solution to obtain a bio-ink, and loading the bio-ink into a 3D bioprinter to prepare a 3D bioprinted radial porous scaffold GelMA / TCP / PDGF-BB / BP@BMP-2 for staged bone regeneration.

[0009] In the application, the 3D bioprinted radial porous scaffold contains sequential release of double growth factors and late PO4 3- and sustained release of Ca 2+ , which can complete the three key steps of staged bone regeneration and achieve efficient bone regeneration.

[0010] In some embodiments, the three key steps of bone regeneration include the inflammation and hematoma formation period, the callus formation and endochondral bone formation period, and the bone remodeling and mature bone formation period. These three stages are a continuous and interrelated process, each stage has its specific cellular and molecular events, which together promote the regeneration and repair of bone. When using the 3D bioprinted radial porous scaffold for bone repair, the scaffold needs to provide corresponding support and microenvironment for bone regeneration at different stages to promote the smooth regeneration of bone tissue.

[0011] Inflammation and hematoma formation period: after bone injury occurs, the body first initiates an inflammatory response. The blood vessels at the injury site rupture and bleed, forming a hematoma, which provides a site for the aggregation and growth of subsequent repair cells and the release of growth factors. At the same time, inflammatory cells such as macrophages and neutrophils rapidly migrate to the injury site, clearing necrotic tissue and bacteria, and releasing various cytokines and growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), which can recruit mesenchymal stem cells and other repair cells to lay the foundation for bone regeneration.

[0012] Callus formation and endochondral ossification: Under the stimulation of growth factors, mesenchymal stem cells differentiate into osteoblasts and chondrocytes. Osteoblasts begin to secrete bone matrix, forming initial callus. At the same time, chondrocytes also form cartilage tissue, which gradually transforms into bone tissue through endochondral ossification. In this stage, blood vessels gradually grow into the callus tissue, providing nutrients and oxygen for bone regeneration, promoting further growth and mineralization of bone tissue.

[0013] Bone remodeling and mature bone formation: As the callus continues to mineralize and mature, osteoclasts begin to play a role in absorbing and remodeling excess callus tissue. At the same time, osteoblasts continue to secrete bone matrix, allowing bone tissue to be continuously remodeled and optimized, ultimately forming mature bone tissue and restoring normal structure and function of the bone. In this stage, the mechanical properties of bone tissue gradually recover to near normal levels.

[0014] To establish a programmed dual delivery system, the common method disclosed in the prior art is to load growth factors into multi-scale carriers to achieve differentiated pharmacokinetic properties. In the present invention, the inventors use black phosphorus (BP) nanosheets as a sustainable release platform for growth factors.

[0015] BP nanosheets are a two-dimensional crystalline nanomaterial with a layered structure and folded surface, with excellent biocompatibility, adjustable band gap, and degradability. Due to its unique structure, BP nanosheets can effectively load drugs or bioactive factors. During its degradation process, the phosphate ions (PO4 3- ) released by BP nanosheets can combine with calcium ions (Ca 2+ ) to form calcium phosphate, thereby promoting bone tissue mineralization and maintaining the mechanical strength of bone tissue.

[0016] In some embodiments, the 3D bioprinted radial porous scaffold is used for bone injury repair, including but not limited to: skull defects, maxillofacial bone defects, humeral defects, ulnar and radial defects, femoral defects, tibial and fibular defects, vertebral defects, lamina defects, pelvic bone defects, metacarpal defects, phalangeal defects, metatarsal defects, phalangeal defects, etc.

[0017] Further, the method comprises the following steps:

[0018] (1) Synthesis of GelMA: Dissolve porcine skin gelatin type A to obtain a completely transparent gelatin solution, slowly add methacrylic anhydride to the gelatin solution, stir, transfer the solution to a dialysis membrane, centrifuge, collect the supernatant, and freeze-dry to prepare GelMA;

[0019] (2) Synthesis of BP@BMP-2: the dispersion of BP nanosheets was centrifuged to remove N-methyl-2-pyrrolidone, the precipitate was collected to prepare BP nanosheets, and the BP nanosheets were incubated with a BMP-2 solution, and then centrifuged to prepare BP@BMP-2 nanosheets;

[0020] (3) The GelMA was dissolved in a mixed solution of LAP and tartrazine to prepare a GelMA solution, TCP nanoparticles, the BP@BMP-2 nanosheets, and a PDGF-BB solution were added to the GelMA solution to obtain a bio-ink, and the bio-ink was loaded into a 3D bioprinter to prepare a 3D bioprinted radial porous scaffold GelMA / TCP / PDGF-BB / BP@BMP-2 for staged bone regeneration.

[0021] Further, the amount of the pigskin gelatin type A in step (1) is 1-10 g;

[0022] Alternatively, the solvent for dissolving the pigskin gelatin type A in step (1) is a PBS buffer, deionized water, an acetic acid solution, a hydrochloric acid solution, or a sodium hydroxide solution;

[0023] Alternatively, the amount of the PBS buffer is 10-100 mL;

[0024] Alternatively, the amount of the methacrylic anhydride in step (1) is 0.5-6 mL.

[0025] Further, the amount of the pigskin gelatin type A in step (1) is 5 g;

[0026] Alternatively, the solvent for dissolving the pigskin gelatin type A in step (1) is a PBS buffer;

[0027] Alternatively, the amount of the PBS buffer is 50 mL;

[0028] Alternatively, the amount of the methacrylic anhydride in step (1) is 3 mL;

[0029] Alternatively, the stirring condition in step (1) is 50°C for 1 h;

[0030] Alternatively, the molecular weight cut-off for dialysis in step (1) is 3500 Da;

[0031] Alternatively, the dialysis condition in step (1) is 40°C for 3 days;

[0032] Alternatively, the centrifugation condition in step (1) is 5000 rpm for 10 min;

[0033] Alternatively, the freeze-drying condition in step (1) is -80°C for 48 h.

[0034] Further, the amount of the BP nanosheet dispersion solution in step (2) is 0.1-5 mL, 0.05-2 mg / mL;

[0035] Optionally, the amount of the BMP-2 solution in step (2) is 0.1-5 mL, 50-150 ng / mL.

[0036] Further, the amount of the BP nanosheet dispersion solution in step (2) is 1 mL, 0.2 mg / mL;

[0037] Optionally, the amount of the BMP-2 solution in step (2) is 1 mL, 100 ng / mL.

[0038] Optionally, the centrifugation condition of the BP nanosheet dispersion solution in step (2) is 10000 rpm, 10 min;

[0039] Optionally, the incubation condition in step (2) is room temperature, incubation overnight.

[0040] Optionally, the centrifugation condition after incubation in step (2) is 10000 rpm, 5 min.

[0041] Further, the LAP and lemon yellow mixed solution in step (3) is prepared by dissolving LAP and lemon yellow in deionized water;

[0042] Optionally, the amount of the LAP is 0.2-5 mg;

[0043] Optionally, the amount of the lemon yellow is 0.1-3 mg;

[0044] Optionally, the amount of the deionized water is 0.2-5 mL;

[0045] Optionally, the amount of the GelMA in step (3) is 0.05-1 g;

[0046] Optionally, the mass-volume ratio of the GelMA in step (3) is 5-15%;

[0047] Optionally, the amount of the TCP nanoparticle in step (3) is 5-15 mg;

[0048] Optionally, the amount of the BP@BMP-2 nanosheet in step (3) is 0.05-1 mg;

[0049] Optionally, the amount of the PDGF-BB solution in step (3) is 5-15 μL.

[0050] Further, the amount of the LAP is 1 mg;

[0051] Optionally, the amount of tartrazine is 0.6 mg;

[0052] Optionally, the amount of deionized water is 1 mL;

[0053] Optionally, the amount of GelMA in step (3) is 0.1 g;

[0054] Optionally, the mass-volume ratio of GelMA in step (3) is 10%;

[0055] Optionally, the amount of TCP nanoparticles in step (3) is 10 mg;

[0056] Optionally, the amount of BP@BMP-2 nanosheet in step (3) is 0.1 mg;

[0057] Optionally, the amount of PDGF-BB solution in step (3) is 10 μL;

[0058] Optionally, the 3D bioprinter in step (3) is a digital light processing 3D bioprinter.

[0059] In a specific embodiment of the present application, the 3D bioprinter is a digital light processing 3D bioprinter, which is a 3D printing device that uses a digital micromirror device (DMD) to realize light curing forming.

[0060] The digital light processing 3D bioprinter uses digital micromirror technology to project ultraviolet light according to a pre-designed pattern onto a resin tank containing biological ink by controlling the micromirror array on the DMD chip through a computer. The biological ink contains a photoinitiator, which rapidly solidifies the biological ink under the irradiation of ultraviolet light, and the layers are stacked to form a three-dimensional biological structure.

[0061] The digital light processing 3D bioprinter has the following advantages in the field of biological material printing: it can achieve high printing resolution, accurately control the solidification position and shape of biological ink, and help build complex biological structures such as tissue models with fine blood vessel networks; one exposure can solidify a layer of biological ink, the printing speed is relatively fast, and a large size of biological structure can be printed in a short time, improving the efficiency; a variety of types of biological ink can be used, including biological ink based on hydrogel, protein and other materials, providing more choices for the printing of different tissues and organs.

[0062] The second aspect of the present application provides any one of the following products:

[0063] (1) a 3D bioprinted radial porous scaffold for staged bone regeneration prepared by the method according to the first aspect of the present application;

[0064] (2) A bio-ink for 3D bioprinting, comprising a GelMA solution, TCP nanoparticles, BP@BMP-2 nanosheets, and a PDGF-BB solution;

[0065] wherein the GelMA solution, TCP nanoparticles, BP@BMP-2 nanosheets, and PDGF-BB solution and the amounts thereof are as described in the first aspect of the present application.

[0066] Optionally, the GelMA solution, TCP nanoparticles, BP@BMP-2 nanosheets, and PDGF-BB solution are mixed to obtain the bio-ink.

[0067] In the present application, the bio-ink is a special material for 3D bioprinting, which is usually composed of biological materials, bioactive molecules and other components, and can accurately control the distribution and arrangement of cells during the printing process, simulate the structure and function of natural tissues, provide a suitable environment for cell survival and growth, and ultimately form a tissue or organ analog with biological activity. In a specific embodiment of the present application, the bio-ink refers to a material for 3D bioprinting to obtain a 3D bioprinted radial porous scaffold as described above.

[0068] The third aspect of the present application provides the use of any one of the following aspects:

[0069] (1) The use of the 3D bioprinted radial porous scaffold described in the second aspect of the present application in the preparation of bone repair and regeneration materials;

[0070] (2) The use of the bio-ink described in the second aspect of the present application in the preparation of a 3D bioprinted radial porous scaffold for staged bone regeneration.

[0071] In addition, the present application also provides a method for bone repair or bone regeneration, which comprises the following steps: accurately placing the printed 3D bioprinted radial porous scaffold described in the second aspect of the present application at the bone defect site of a subject in need thereof, and ensuring that the scaffold is closely attached to the surrounding healthy bone tissue.

[0072] In some embodiments, the subject includes a mammal, a non-mammal. The mammal is preferably a rodent, an even-toed ungulate, an odd-toed ungulate, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a human. In a preferred embodiment of the present application, the subject is a human, and in a specific embodiment of the present application, the subject is a patient with a bone defect.

[0073] In some embodiments, the method further comprises anesthesia and incision, that is, selecting a suitable anesthesia method according to the specific condition of the patient, and then making a proper incision at the bone defect site of the patient to fully expose the bone defect area to facilitate subsequent implantation of the scaffold.

[0074] In some embodiments, during the implantation of the scaffold, if necessary, a fixing device such as a screw or a steel plate can be used to fix the scaffold in a suitable position to ensure its stability and provide a stable mechanical environment for bone regeneration.

[0075] During the whole bone repair or bone regeneration process, the 3D printed radial porous scaffold provided by the application can provide three-dimensional space support and guidance for the growth of bone tissue, and the radial porous structure is beneficial to the exchange of nutrients, the migration of cells and the ingrowth of blood vessels, thereby promoting the staged regeneration of bone tissue and finally achieving effective repair of bone defects.

[0076] Compared with the prior art, the application has the following advantages and beneficial effects:

[0077] The application provides a novel 3D bioprinted radial porous scaffold for staged bone regeneration, which can provide a biomimetic cell microenvironment to support the infiltration of host repair cells into the defect site and promote bone integration after implantation. In addition, the sequential release of double growth factors and the sustained release of PO4 3- and Ca 2+ in the 3D bioprinted radial porous scaffold can complete the three key steps of staged bone regeneration, enabling efficient bone regeneration and representing a promising biomimetic staged regeneration strategy. BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1 : Physical property characterization of BP nanosheets, wherein, A figure: zeta potential of BP and BP@BMP-2 nanosheets; B figure: particle size distribution and average diameter of BP nanosheets; C figure: particle size distribution and average diameter of BP@BMP-2 nanosheets; D figure: transmission electron microscope image of BP nanosheets; E figure: transmission electron microscope image of BP@BMP-2 nanosheets; F figure: Raman spectrum of BP and BP@BMP-2 nanosheets; G figure: X-ray photoelectron spectroscopy of BP and BP@BMP-2 nanosheets; H figure: degradation of BP and BP@BMP-2 nanosheets recorded by ultraviolet-visible absorption spectrum at 0, 2, 4, 6, 8 and 10 days; I figure: degradation image of BP and BP@BMP-2 nanosheets; J figure: in vitro PO4 3- release curve of BP and BP@BMP-2 nanosheets;

[0079] Figure 2: Characterization of 3D bioprinted hydrogel scaffolds, where A. FTIR spectra of gelatin and GelMA; B. NMR spectra of gelatin and GelMA; C. X-ray diffraction of TCP nanoparticles; D. Computer simulation model of 3D bioprinted radial pore scaffolds; E. Cross-sectional and longitudinal views of freeze-dried 3D bioprinted scaffolds; F. Macro- and micro-structure of 3D bioprinted radial pore scaffolds;

[0080] Figure 3 : Mechanical properties and sustained release capacity of hydrogel scaffolds, where A. Frequency-dependent curves; B. Time-dependent curves; C. Loss factor tandelta; D. Shear rate-viscosity curves; E. Stress-strain curves; F. Compression strength results from compression tests; G. Swelling ratio of hydrogel scaffolds; H. Degradation rate of hydrogel scaffolds in PBS buffer; I. Degradation rate of hydrogel scaffolds in collagenase solution; J. Cumulative release curves of BMP-2 from hydrogel scaffolds; K. Cumulative release curves of PDGF-BB from hydrogel scaffolds; L. Continuous release of BMP-2 and PDGF-BB from hydrogel scaffolds, *P < 0.05, **P < 0.01, ***P < 0.001;

[0081] Figure 4 : Cell compatibility and in vitro osteogenic performance of scaffolds, where A. Cytoskeleton staining with FITC-phalloidin; B. Cell scratch test of BMSCs; C. ALP and Alizarin Red S staining of BMSCs cultured with hydrogel scaffold extract at days 7, 14, and 21; D. CCK-8 cell viability test of BMSCs cultured with hydrogel scaffold extract at days 1, 3, 5, and 7; E. Quantitative cell migration rate of BMSCs; F. Quantitative analysis of ALP staining of BMSCs; G. Quantitative analysis of Alizarin Red S staining of BMSCs; qRT-PCR analysis quantified the effect of five scaffolds on osteogenic and angiogenic genes in BMSCs cultured with hydrogel extract: H. OPN, I. OCN, J. Runx2, K. COL I, L. VEGF, *P < 0.05, **P < 0.01, ***P < 0.001;

[0082] Figure 5: The effect of the scaffold on the bone regeneration of the critical size skull defect of the rat is enhanced, wherein, A figure: the time schedule of the in vivo experiment and the representative image of the surgical procedure; B figure: the representative micro-CT image of the bone defect after the scaffold treatment for 4 weeks and 8 weeks, and the quantitative analysis: C figure: BMD, D figure: BV / TV; E figure: the representative image of HE staining shows the influence of different types of scaffolds on the bone defect regeneration; F figure: the representative image of Masson trichrome staining shows the influence of different types of scaffolds on the bone defect regeneration, *P<0.05, **P<0.01, ***P<0.001;

[0083] Figure 6 : The effect of the blank group, the G, GT, GTP, GTB and GTPB scaffolds on the bone regeneration after 4 weeks and 8 weeks is evaluated by immunohistochemical staining, wherein, A figure: the immunohistochemical image of OPN after 4 weeks and 8 weeks; B figure: the immunohistochemical image of OCN after 4 weeks and 8 weeks; C figure: the immunohistochemical image of Runx2 after 4 weeks and 8 weeks; D figure: the immunohistochemical image of COL I after 4 weeks and 8 weeks; E figure: the immunohistochemical image of VEGF after 4 weeks and 8 weeks, and the corresponding quantitative analysis: F figure: OPN, G figure: OCN, H figure: Runx2, I figure: COL I, J figure: VEGF, *P<0.05, **P<0.01, ***P<0.001; Figure 7 : The Live / Dead staining images of BMSCs cultured with the hydrogel scaffold extract liquid at 1st, 3rd, 5th and 7th days. In the Live / Dead staining, almost no cell death is observed in all groups, and the cells show high activity. DETAILED DESCRIPTION

[0084] The present application will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present application, and should not be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

[0085] The reagents, raw materials and experimental consumables used in the present application are easily obtained by those skilled in the art, and can be obtained from commercial channels if not otherwise specified, and the experimental methods not specified in the present application are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. In particular, the following examples are only used to illustrate the present application, and should not limit the scope of the present application in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present application, and should not and will not limit the present application described in detail in the claims.

[0086] Example 3D bioprinted radial porous scaffolds for staged bone regeneration and validation of their effects

[0087] 1. Experimental materials

[0088] Gelatin type A was purchased from Sigma-Aldrich (St. Louis, MO, USA). β-tricalcium phosphate (TCP) nanoparticles were purchased from Beijing Innoway Technology Co., Ltd. (Beijing, China). Black phosphorus (BP) nanoplatelet dispersion was purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Bone marrow mesenchymal stem cells (BMSCs) were purchased from ScienCell Research Laboratories (Carlsbad, USA). Total RNA extraction kit, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Madison, USA). Antibodies against osteopontin (OPN), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), collagen type I (COL I), and vascular endothelial growth factor (VEGF) were purchased from Proteintech (Wuhan, China). Alkaline phosphatase (ALP) staining reagent and alizarin red S staining solution were purchased from Biyun Tian Biotechnology Co., Ltd. (Shanghai, China). BMP-2 and PDGF-BB ELISA kits were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

[0089] 2. Experimental methods

[0090] (1) Synthesis of methacrylated gelatin (GelMA)

[0091] The synthesis of GelMA was as follows: first, 5 g of porcine skin gelatin type A was dissolved in 50 mL of PBS buffer, and magnetic stirring was performed under a 50 °C water bath until a completely transparent solution was obtained. Then, 3 mL of methacrylic anhydride was slowly added to the gelatin solution, and stirring was continued at 50 °C for 1 hour to complete the methacrylation reaction. Subsequently, the solution was transferred to a dialysis membrane (molecular weight cut-off of 3500 Da) and dialyzed against deionized water at 40 °C for 3 days to remove impurities. After dialysis, the solution was centrifuged at 5000 rpm for 10 minutes, the precipitate was removed, the supernatant was collected, and it was freeze-dried at -80 °C for 48 hours to obtain GelMA. GelMA was stored at -20 °C for subsequent use.

[0092] (2) Preparation of black phosphorus nanoplatelets loaded with BMP-2 (BP@BMP-2)

[0093] The preparation method of the BP nanosheets is as follows: 1 mL of a BP nanosheet dispersion solution with a concentration of 0.2 mg / mL is centrifuged at 10,000 rpm for 10 minutes to remove N-methyl-2-pyrrolidone (NMP), the precipitate is collected and resuspended in deionized water, and this process is repeated 3 times to ensure complete removal of residual NMP, thereby obtaining BP nanosheets.

[0094] Subsequently, the BP nanosheets are incubated with 1 mL of a BMP-2 solution (100 ng / mL) at room temperature overnight. After incubation, the solution is centrifuged at 10,000 rpm for 5 minutes to obtain BP@BMP-2 nanosheets formed by π-π stacking and electrostatic interactions.

[0095] (3) Manufacture of 3D bioprinted scaffolds

[0096] To prepare the photoinitiator solution, 1 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and 0.6 mg of tartrazine are dissolved in 1 mL of deionized water. Subsequently, 0.1 g of GelMA is dissolved in the solution to obtain a 10% GelMA solution. To prepare the bio-ink, 10 mg of TCP nanoparticles, 0.1 mg of BP@BMP-2 nanosheets, and 10 μL of a platelet-derived growth factor-BB (PDGF-BB) solution are added to the GelMA solution. A digital light processing (DLP) 3D bioprinter (EFL-BP-6601, Yongquan Intelligent Equipment Co., Ltd., Suzhou, China) is used to directly program the control of the bio-ink delivery system and the motion frame. The prepared bio-ink is loaded into the bioprinter, and the bioprinting parameters are selected for manufacturing. Finally, the 3D bioprinted radial scaffold is washed with sterile PBS buffer for 3 times for subsequent application.

[0097] Specifically, the process of 3D bioprinting is as follows: the bio-ink is loaded into the print cartridge, and 3D bioprinting is performed through a nozzle with a diameter of 200 μm to obtain a 3D printed scaffold. In the process of 3D bioprinting, the printing parameters are set as follows: layer thickness is 0.5 mm, layer number is 2 layers, printing speed is 5 mm / s, exposure time is 20 s, light intensity is 17 mW / cm 2 , and the scanning mode is cross-grid scanning; the printing temperature is 25°C, and the cooling plate is pre-cooled to 4°C.

[0098] (4) Characterization of BP@BMP-2 nanosheets

[0099] The particle size and zeta potential of BP and BP@BMP-2 nanosheets were measured by Zetasizer Nano ZS-90 (Malvern, UK). The particle size was measured using dynamic light scattering method at 90° scattering angle, and the zeta potential was measured using laser Doppler microelectrophoresis method. The microstructure of BP and BP@BMP-2 nanosheets was recorded using transmission electron microscopy (TEM, Tecnai G2 F20S-TWIN, FEI, Hillsboro, USA) at 5 kV. The molecular structure and chemical composition of BP@BMP-2 nanosheets were characterized by Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). Raman spectroscopy provides information on molecular vibration patterns by measuring the frequency change of scattered light based on light scattering effects. XPS analyzes the type, chemical state, and distribution of elements by measuring the energy distribution of photoelectrons in the material. BP and BP@BMP-2 nanosheets were dispersed in deionized water and placed under light. The degradation rate was observed using a camera, and its degradation performance was monitored by ultraviolet-visible spectrophotometry every two days until the 10th day. To determine the in vitro PO4 3- release profile, 3D bioprinted hydrogel scaffolds were prepared using BP nanosheets (with and without BMP-2 loading) as described above. The scaffolds were immersed in 1 mL of deionized water and incubated at 37°C with 100 rpm shaking. 200 μL of incubation solution was collected on days 1, 2, 5, 7, 14, 21, and 28, and replaced with an equal volume of fresh deionized water. The PO4 3- concentration in the incubation solution was quantified using a detection kit and a microplate reader.

[0100] (5) Characterization of 3D bioprinted scaffolds

[0101] The chemical structure of the hydrogel was characterized by Fourier transform infrared spectroscopy (FTIR, Nicolet iS10, Thermo Fisher, Madison, USA). 8 mg of dry sample was placed between two glass sheets and pressed into a sheet. The infrared spectrum of the sample was collected in a dry environment from a wavelength range of 4000 to 400 cm -1 with a resolution of 4 cm -1 . Nuclear magnetic resonance (NMR) was used to analyze the chemical environment of hydrogen atoms in the hydrogel scaffold. Different chemical shifts reflect the electronic environment around hydrogen atoms, providing clues about the composition and functional group distribution of the material. The structure and phase content of TCP were analyzed by X-ray diffraction (XRD) using Cu K Diffraction was performed in the 2theta angle range from 10° to 80°. To observe the microstructure of the hydrogel scaffold, the scaffold was cut into cubes, frozen in liquid nitrogen for 30 minutes, and then immediately placed in a freeze dryer for 24 hours. The freeze-dried sample was mounted on a copper holder and sputtered with gold for 60 seconds. Subsequently, the morphology was photographed using a scanning electron microscope (SEM, Sigma 500, Zeiss, Jena, Germany) at a voltage of 5 kV.

[0102] (6) Rheological properties

[0103] Rheological tests of the hydrogels were characterized using a Haake Mars 60 rheometer (ThermoFisher Scientific, Waltham, USA). The hydrogel prepolymer solution was placed in a mold to prepare a hydrogel sample with a diameter of 20 mm and a height of 2 mm, and it was stored at 4°C. Silicon oil was added to the edge of the hydrogel sample to prevent moisture loss. To determine the frequency dependence of the storage modulus (G’) and the loss modulus (G”), the measurement range of all hydrogel samples was 1 to 100 Hz. The time sweep method was used to evaluate the viscoelastic changes of the hydrogels at different time scales. The loss factor tandelta was measured to further characterize the viscoelastic properties of the hydrogels. By changing the shear rate (1 to 100 s -1 ), the temperature and strain were kept constant, and the shear rate-viscosity curve was obtained to evaluate the printing performance.

[0104] (7) Mechanical strength

[0105] The hydrogel samples were prepared into uniform size shapes (10 mm x 10 mm x 3 mm). Then, using a universal mechanical testing machine for biomaterials (WDW-10, Jinan Chuangbai Instrument Co., Ltd., Shandong, China) at a speed of 1 mm / min, compression tests were performed. The stress-strain curve and compression modulus were automatically recorded by the machine.

[0106] (8) Swelling and degradation behavior

[0107] The freeze-dried hydrogel was weighed, and the initial mass was W0. Then, the hydrogel was placed in a PBS buffer. After reaching the preset time interval, the hydrogel was taken out of the PBS, and excess water was removed using filter paper. The mass of the hydrogel at different time points was recorded as W t , until the hydrogel reached swelling equilibrium. The swelling rate was calculated according to the following formula: Swelling rate (%) = W t / W0 x 100%. In a similar manner, the degradation rate was measured. The initial mass of the freeze-dried hydrogel was W0. Then, the hydrogel was immersed in a PBS buffer or a 5 U / mL collagenase solution. The degradation solution was replaced every 3 days. At the predetermined time interval, the hydrogel was taken out of the PBS buffer or the collagenase solution, freeze-dried, and weighed (W t). The degradation rate of hydrogels was calculated according to the following formula: Degradation rate (%) = (W0-W t ) / W0x 100%.

[0108] (9) Release of BMP-2 and PDGF-BB

[0109] To quantify the release rate of BMP-2 and PDGF-BB, the scaffolds were immersed in PBS buffer and kept at a constant temperature of 37°C. At each preset time point, 1 mL of PBS buffer was collected and replenished with the same volume of fresh buffer. The amount of BMP-2 and PDGF-BB released by the scaffolds was measured using an ELISA kit (Cusabio Technology Co., Ltd., Wuhan, China) according to the manufacturer's protocol, and then the cumulative release curve was plotted.

[0110] (10) Evaluation of cell compatibility

[0111] Pre-prepared hydrogel samples were sterilized by ethylene oxide for subsequent use in all biological experiments. Hydrogel samples were incubated in DMEM medium for 24 hours to obtain the extract, according to ISO 10993-12. BMSCs (5x10 4 cells / well) were used to evaluate cell proliferation and viability. To determine cell proliferation, a CCK-8 experiment (ThermoFisher Scientific, Madison, USA) was performed to measure the change in optical density (OD value) at 450 nm after 1, 3, 5, and 7 days of co-incubation of cells with hydrogels. Cell viability was evaluated by Live / Dead staining and recorded using a confocal laser scanning microscope (LSM800, Zeiss, Jena, Germany). By fixing with 4% paraformaldehyde, cells were treated with Triton X-100 (Sigma-Aldrich, St. Louis, USA). Then, FITC-phalloidin (Solarbio Technology Co., Ltd., Beijing, China) was used to stain for 20 minutes, and the cytoskeleton and cell expansion were observed under a confocal laser scanning microscope. A cell scratch experiment was performed to study the effect of scaffolds on cell migration. BMSCs (1x10 5 cells / well) were seeded into culture plates and incubated at 37°C, 5% CO2 for 24 hours. A pipette tip was used to scratch the cell layer, ensuring that the scratch was perpendicular to the cell layer. Then, it was washed with PBS buffer to remove the detached cells. Hydrogel extract was added to the wells and co-cultured with the cells. Cell migration was monitored using a fluorescence inverted microscope at 0, 12, and 24 hours. The migration rate was calculated according to the following formula: Migration rate (%) = (R0-R t ) / R0x 100%, where R0 is the initial scratch area and R t is the remaining scratch area at different time points.

[0112] (11) In vitro osteogenic properties

[0113] Scaffold-induced BMSCs (5 × 10⁶) were assessed by ALP and Alixin S staining. 4 The ability of cells / well to differentiate osteogenic genes in vitro was assessed. Cells were co-incubated with scaffold extract for 7, 14, and 21 days, then fixed with formalin and stained with ALP or Alcian Red S for 30 minutes. The concentrations of ALP and Alcian Red S were then measured using a microplate reader (Thermo Fisher Scientific, Madison, USA).

[0114] (12) qRT-PCR experiment

[0115] After 7 days of co-incubation with the scaffold, total RNA was extracted from the cells using Trizol (Invitrogen, Carlsbad, USA). Reverse transcription was performed using the Revert Aid first-strand cDNA synthesis kit (Takara, Shiga, Japan). Gene expression was then analyzed using the TB Green qRT-PCR kit (Takara, Shiga, Japan) in a StepOne Plus real-time quantitative PCR system (ThermoFisher Scientific, Madison, USA). GAPDH was used as an internal control gene to analyze the mRNA expression levels of ALP, Runx2, OCN, OPN, and Col I.

[0116] (13) Skull defect model

[0117] The in vivo osteogenic properties of the scaffold were assessed using a critical-sized skull defect model in 6-week-old male Sprague-Dawley rats. Animal handling and housing conditions complied with the National Institutes of Health (NIH) guidelines for laboratory animal use and were supervised by the Ethics Committee of Peking Union Medical College Hospital. During modeling, a critical-sized bone defect (4 mm) was created on the skull of each rat, and a hydrogel sample was implanted into the defect site. Tissue samples were collected at 4 and 8 weeks post-surgery for subsequent histological and imaging evaluation.

[0118] (14) MicroCT Analysis

[0119] Skull tissue samples were collected at 4 and 8 weeks post-surgery and fixed in 4% paraformaldehyde solution for preservation. After appropriate trimming, the bone defect sites were scanned using a micro-CT scanner (Somatom Definition Flash, Siemens Healthineers, Volzheim, Germany). Based on the micro-CT data, bone mineral density (BMD) and bone volume / total defect volume (BV / TV) of the newly formed bone tissue were quantitatively calculated.

[0120] (15) Histological staining and immunohistochemistry

[0121] Calvaria tissue samples were collected at 4 and 8 weeks postoperatively and tissue sections were prepared. Bone tissues were fixed with formalin, decalcified, dehydrated, transparentized and paraffin infiltrated. Routine procedures included hematoxylin-eosin (HE) staining and Masson’s trichrome staining.

[0122] Immunohistochemical staining was performed with four monoclonal antibodies closely related to osteogenesis and angiogenesis: osteopontin (OPN), osteocalcin (OCN), Runx2, collagen type I (COL I) and vascular endothelial growth factor (VEGF). After staining, images of pathological sections were taken using an Olympus BX53 optical microscope (Shinjuku, Tokyo, Japan). Quantitative analysis of immunohistochemistry was performed by counting positive cells.

[0123] 3. Experimental results

[0124] (1) Characteristics of BP@BMP-2 nanosheets

[0125] BP is a phosphorus allotrope, each layer of which is composed of phosphorus atoms arranged in an accordion-like manner, forming a unique honeycomb-like two-dimensional structure. BP nanosheets have a large specific surface area and can be used to load small molecule drugs, proteins and peptides, and are applied to drug delivery systems. Unlike other two-dimensional materials such as graphene, BP nanosheets can be degraded into non-toxic phosphate ions in vivo, and have superior biocompatibility and biosafety.

[0126] The zeta potentials of BP and BP@BMP-2 nanosheets are shown in Figure 1 The zeta potential of BP nanosheets was measured to be -12.5 ± 1.3 mV. After loading BMP-2, the zeta potential of BP@BMP-2 nanosheets increased to 8.9 ± 2.3 mV. The change in zeta potential indicates that there is an electrostatic interaction between BMP-2 and BP nanosheets, confirming the successful loading of BMP-2 onto BP nanosheets. Zeta potential values of -30 or 30 mV have been used as a standard for nanoparticle dispersion. The electrostatic repulsion between nanoparticles that meet these standards can counteract the attractive forces, thereby increasing the distance and spacing between nanoparticles, ultimately reducing the risk of particle aggregation and sedimentation. The particle size distribution of BP and BP@BMP-2 nanosheets was analyzed by nanoparticle size analyzer Figure 1 B and 1C). The results show that the average particle size of BP nanosheets is 635.4 ± 1.5 nm. After loading BMP-2, the average particle size of BP@BMP-2 nanosheets slightly increased to 653.5 ± 0.1 nm.

[0127] TEM images show that both BP and BP@BMP-2 nanosheets exhibit a characteristic two-dimensional sheet-like morphologyFigure 1 D and 1E). The lattice spacing of BP and BP@BMP-2 nanosheets were measured to be 0.22 nm and 0.25 nm, respectively. The increase in lattice spacing after BMP-2 loading indicates an increase in the size of the nanosheets, which is consistent with the trend of the particle size measurements.

[0128] The Raman scattering spectrum of BP nanosheets shows three characteristic peaks at 365.1 cm -1 , 443.1 cm -1 , and 468.2 cm -1 , corresponding to A1g, B2g, and A2g vibration modes, confirming the two-dimensional sheet-like structure of BP nanosheets Figure 1 After BMP-2 loading, the Raman scattering spectrum of BP@BMP-2 nanosheets retains these three characteristic peaks, but with slight redshift. The redshift can be attributed to the electrostatic interaction between BP nanosheets and BMP-2, which weakens the bond strength, thus lowering the vibration frequency and shifting the characteristic peaks to lower wavenumbers (i.e., redshift). Overall, these results confirm the successful preparation of BP@BMP-2 nanosheets.

[0129] XPS of BP nanosheets shows characteristic peaks at 133 eV and 197 eV, corresponding to P2p and P2s, respectively, which are consistent with previously reported peaks for BP nanosheets. After BMP-2 loading, the XPS results of BP@BMP-2 nanosheets show increased carbon, oxygen, and nitrogen concentrations, while retaining similar P2p and P2s peaks as BP nanosheets Figure 1 G). These results confirm the successful loading of BMP-2 onto BP nanosheets.

[0130] The degradation appearance of BP and BP@BMP-2 nanosheets was observed using a digital camera at days 0, 2, 4, 6, 8, and 10. It was observed that the fading speed of BP@BMP-2 nanosheets was slower compared to BP. At day 10, the brown color of BP nanosheets almost completely faded, indicating that BP nanosheets have mostly degraded into phosphate and other oxidation products. In contrast, BP@BMP-2 nanosheets still retained some brown color, but slightly faded Figure 1 I). UV-vis absorption spectra further confirmed this trend, showing that the absorbance of BP and BP@BMP-2 nanosheets gradually decreased over time. However, the absorbance of BP@BMP-2 nanosheets was still higher than that of BP nanosheets, which is consistent with the observed color fading pattern Figure 1H). Potential reasons for this enhanced stability may include: (1) BMP-2 binding to BP nanosheets may partially cover their surface, forming a protective film that reduces direct exposure to water and oxygen, thereby slowing down oxidation; (2) Under neutral pH conditions, BMP-2 carries a positive charge while BP carries a negative charge, influencing the electron transfer process after photoexcitation through electrostatic interactions, thus slowing down BP degradation. In summary, these results indicate that BMP-2 successfully modifies BP, enhancing its stability and making it less prone to degradation.

[0131] Because the degradation of BP releases PO4 3- It is added to the solution, thus measuring PO4. 3- The concentration can be used to assess the extent of BP degradation. For example... Figure 1 As shown in J, PO4 in BP@BMP-2 nanosheets 3- The release rate was slower than that of BP nanosheets, indicating that the interaction between BP and BMP-2 effectively delayed the degradation of BP. On day 28, the cumulative PO4 content of BP and BP@BMP-2 nanosheets was [data missing]. 3- The release rates were 60.3% and 46.5%, respectively. In summary, BMP-2 enhances the stability of BP nanosheets, making their PO4 content higher. 3- The release is slower and more continuous.

[0132] (2) Characteristics of 3D bioprinted scaffolds

[0133] GelMA forms stable three-dimensional structures through photopolymerization, and its physical properties can be tuned by factors such as the degree of methacrylate, crosslinking agent, and concentration. These properties make GelMA widely applicable in 3D printing. In the FTIR spectra of gelatin and GelMA, 1637.3 cm⁻¹... -1 The characteristic absorption peak at 1540.8 cm⁻¹ corresponds to the stretching vibration of the C=O bond, representing the amide I conformation. -1 The peak at 1240.5 cm⁻¹ is attributed to the coupling of the bending vibration of the NH bond and the stretching vibration of the CH bond, indicating the presence of the amide II conformation. Furthermore, the peak at 1240.5 cm⁻¹... -1 The peak at that point is due to the bending vibrations of the NH and CN bonds, corresponding to the amide III conformation, respectively. For example... Figure 2 As shown in Figure A, the characteristic peaks of amides I, II, and III in GelMA are significantly stronger than those in gelatin, indicating that methacrylate groups were successfully introduced into the gelatin molecular chain. This modification led to the formation of new amide bonds, confirming the successful synthesis of GelMA. Figure 2 B shows the NMR spectra of gelatin and GelMA. The NMR results indicate the presence of novel acrylic proton peaks (=CH2) at 5.3 and 5.5 ppm, confirming the successful grafting of methacrylate groups onto the gelatin.

[0134] TCP is the main inorganic component of bone tissue, with excellent biocompatibility, and does not cause immune rejection or inflammatory response. In vivo, TCP can be degraded and gradually replaced by newly formed bone tissue, making it a widely used material for bone repair. Figure 2 C shows the XRD spectrum of TCP. The presence of clear diffraction peaks at 2 theta angles of 17.08°, 25.88°, 28.02°, 31.12°, 34.54°, 47.06°, and 53.16° confirms the crystalline nature of TCP.

[0135] An ideal scaffold not only recruits host endogenous cells to the bone defect site, but also promotes osteogenic differentiation and bone matrix deposition. Previous studies have shown that a good nanotopology can regulate cell behavior, such as guiding cell migration and promoting cell directional differentiation. Mac et al. designed a radiation-aligned mineralized collagen fiber scaffold containing nanosilicon. Experiments showed that cells on the scaffold were elongated and aligned with the direction of the fibers, effectively promoting the directional migration of cells from the edge to the center. However, the precision of this collagen fiber scaffold may not be sufficient. In contrast, 3D printing technology can accurately print advanced structures and complex characteristics according to digital models, suitable for personalized medical needs. Figure 2 D shows the top and front views of the 3D bioprinted model, presenting a double-layered radiation structure. Figure 2 E shows the cross-sectional and longitudinal views of the freeze-dried 3D bioprinted scaffold, clearly showing the interconnected radiation channels. These radiation channels are expected to promote cell migration from the surrounding bone tissue to the defect site. Figure 2 F demonstrates the macro- and micro-morphology of the 3D bioprinted scaffold. After introducing TCP nanoparticles and BP nanosheets, the printability of the bio-ink slightly decreased, resulting in a slight blurring of the double-layered radiation structure edges in the GelMA / TCP / BP scaffold. Inverted microscope images further confirm the presence of a double-layered porous structure in the scaffold. SEM images clearly show that the scaffold has a clear channel structure and a micro-porous network. These porous structures exhibit excellent integrity and permeability, providing sufficient space for cell attachment and proliferation.

[0136] We further determined the rheological properties of the hydrogel. Figure 3 A and 3B respectively demonstrate the changes of G' and G" with frequency and time. When G' is greater than G", the hydrogel remains in a gel state. As shown in the figures, G' is always greater than G" with the increase of frequency and time, indicating that a stable three-dimensional network structure is formed within the hydrogel. In addition, the viscoelasticity of the hydrogel is evaluated by the loss factor tandelta. As shown in Figure 3The loss factor tandelta of GelMA / TCP / BP hydrogels was lower than other groups, as shown in C, indicating that the addition of TCP nanoparticles and BP nanosheets enhanced the viscoelasticity of the hydrogels. These results show that TCP nanoparticles and BP nanosheets have a synergistic effect in improving the mechanical properties of hydrogel scaffolds.

[0137] The shear-thinning properties of hydrogels can verify their injectability and printability. By analyzing the relationship between viscosity and shear rate, we observed that the viscosity of all hydrogels decreased with increasing shear rate, verifying their printability Figure 3 D). In addition, the addition of TCP or BP increased the viscosity of the hydrogels, and the highest viscosity was observed when TCP and BP were added simultaneously. This can be attributed to the large specific surface area and interfacial effects of BP nanosheets. When BP nanosheets are introduced into GelMA hydrogels, strong interactions such as van der Waals forces and hydrogen bonds can occur between BP and GelMA molecular chains. These interactions increase the internal friction within the GelMA matrix, thereby increasing the viscosity. The addition of TCP nanoparticles increases the viscosity due to the interactions between TCP and polymer chains, which restrict the movement of GelMA molecular chains. When BP and TCP are added to GelMA hydrogels simultaneously, they can produce a synergistic effect, further enhancing the viscosity. In summary, TCP and BP can enhance the cohesiveness and viscosity of hydrogel scaffolds.

[0138] According to the literature, biomaterials that promote tissue regeneration need to match the initial mechanical properties of host tissues. If the mechanical strength is too high, it can hinder bone tissue regeneration and the stability of the bone-implant interface. Conversely, materials with insufficient strength cannot provide the required mechanical environment for bone regeneration. Figure 3 E shows the stress-strain curves of the hydrogel scaffolds. It can be observed that the addition of BP and TCP enhances the compressive strength of the scaffolds. The compressive strength of GelMA / TCP / BP hydrogel scaffolds at 40% strain was 70.2 ± 6.9 kPa, while the compressive strength of GelMA hydrogel scaffolds decreased significantly to 10.8 ± 0.1 kPa Figure 3 F). This can be due to the different degrees of polymer chain aggregation in different hydrogel samples. Another possible reason is that BP and TCP can enhance the physical interactions (such as hydrogen bonds, van der Waals forces, and electrostatic interactions) within the hydrogel and the covalent crosslinking network, making the scaffold more resistant to deformation. These results show that the addition of BP and TCP affects the mechanical properties, forming a relatively dense and robust network structure.

[0139] An excessively high expansion ratio can cause the material to swell upon absorbing water, compressing surrounding tissues or organs and affecting its stability and biocompatibility. Furthermore, a high expansion ratio may soften the material, reducing its mechanical strength and impairing its supporting function in bone regeneration. Figure 3 As shown in Figure G, the swelling ratio of GelMA hydrogel in PBS buffer can reach 52%, but the swelling ratio decreases significantly after the addition of TCP or BP. This is because TCP releases CaO into the hydrogel. 2+ BP interacts with negatively charged groups (such as carboxyl or phosphate groups) to form ionic crosslinks. Ionic crosslinking increases the crosslinking density between polymer chains, making the hydrogel more compact when absorbing water. The introduction of BP increases the crosslinking density of the hydrogel, making the network structure more compact. However, the degradation of BP produces acidic products, which may change the pH of the hydrogel and cause swelling. The simultaneous addition of TCP and BP results in higher structural stability and lower swelling of the hydrogel when absorbing water, which helps the scaffold maintain its shape and structure over the long term. Overall, all swelling curves show a significant increase in the initial stage due to the presence of numerous hydrophilic groups and a porous network structure in the hydrogel. After 8 hours, the swelling force gradually decreases and eventually reaches equilibrium. In conclusion, the introduction of TCP and BP can modulate the network structure of the hydrogel, thereby affecting its swelling properties to meet the needs of bone repair.

[0140] The degradation rate of a hydrogel is a key indicator because it determines whether the hydrogel can match the rate of new bone regeneration. Given that hydrogels typically have a relatively fast degradation rate, novel hydrogels need to be designed to degrade slowly, providing initial mechanical support for bone tissue regeneration. Figure 3 H and 3I showed the degradation performance of the hydrogel in PBS buffer and collagenase solution. It was observed that the degradation rate of the hydrogel decreased in both PBS buffer and collagenase solution with the addition of TCP or BP. The degradation rate was slowest when both materials were added simultaneously. The introduction of TCP or BP increased the crosslinking density and stability of the hydrogel. Higher crosslinking density is generally associated with a slower degradation rate because the denser molecular network restricts the penetration of water molecules or enzymes, thus delaying degradation. Furthermore, the phosphate generated during BP degradation may trigger acidic degradation of the hydrogel matrix, resulting in an overall degradation rate faster than that of the GelMA / TCP hydrogel. These results indicate that the GelMA / TCP / BP hydrogel can match the regeneration rate of new bone tissue.

[0141] Literature reports that the main challenges for BMP-2 delivery are the use of supraphysiological doses and burst release at early stages. High concentrations of BMP-2 are known to be associated with various adverse clinical outcomes, such as wound infection, delayed healing, bone resorption, tumor formation, and ectopic bone. These risks highlight the importance of optimizing the release pattern and delivery platform to achieve a controlled and sustained release of BMP-2. We measured the release kinetics of BMP-2 in the hydrogels. The release rate of G / TCP / BP@BMP-2 hydrogels was slower and more sustained than that of G / TCP / BP / BMP-2 hydrogels Figure 3 J). This is due to the electrostatic interaction between BP and BMP-2, which effectively controls the release rate of BMP-2. By incorporating BMP-2 into BP nanosheets, a sustained release system is formed, allowing the gradual degradation of BP and the sustained release of BMP-2. The sustained release property helps to continuously stimulate host stem cells for osteogenic differentiation, promoting long-term bone regeneration.

[0142] Supplemental PDGF-BB stimulated the gradual migration of BMSCs in the hydrogel. As shown in Figure 3 K, the gradual degradation of the hydrogel led to the sustained release of PDGF-BB, with an accumulative release of about 61.9% after 21 days.

[0143] According to the different stages of bone regeneration, a time-dependent release pattern of growth factors is necessary to ensure that each factor is released at the appropriate time. We verified the sequential release pattern of two growth factors. As shown in Figure 4 L, both BMP-2 and PDGF-BB were able to be released continuously from the hydrogel scaffold throughout the release curve. The early release rate of PDGF-BB was higher than that of BMP-2, which is consistent with the biological goal of cell recruitment preceding cell differentiation, and vascular regeneration preceding new bone formation.

[0144] (3) In vitro cell compatibility and osteogenic potential

[0145] According to the composition of the scaffold, the GelMA scaffold is abbreviated as G, the GelMA / TCP scaffold is abbreviated as GT, the GelMA / TCP / PDGF-BB scaffold is abbreviated as GTP, the GelMA / TCP / BP@BMP-2 scaffold is abbreviated as GTB, and the GelMA / TCP / PDGF-BB / BP@BMP-2 scaffold is abbreviated as GTPB.

[0146] Hydrogels have complex three-dimensional structures, closely resembling extracellular matrix, making them ideal carriers for cell adhesion and proliferation, as well as reservoirs for growth factors. Therefore, verifying the cell compatibility of hydrogel scaffolds is an important step in evaluating their potential biological applications. CCK-8 experiments showed that cells in all groups exhibited cell proliferation Figure 7 D). The OD values of GTPB scaffolds at 1, 3, 5, and 7 days were higher than those of other groups, indicating that GTPB scaffolds had a strong effect on promoting cell growth. In Live / Dead staining, cells exhibited high activity because almost no cell death was observed in all groups Figure 4 ). Cytoskeleton staining showed that cells exhibited better expansion after co-culture with GTPB scaffolds Figure 4 A). Cell scratch experiments showed that the scratch area of GTPB scaffolds was the smallest Figure 4 B). Quantitative analysis showed that the migration rates of Control, G, GT, GTP, GTB, and GTPB groups were 33.6%, 37.0%, 39.3%, 62.3%, 57.3%, and 70.6% after 24 hours. The migration rates of GTP and GTPB scaffolds were higher than those of other groups Figure 4 E). These results indicated the role of PDGF-BB in promoting cell recruitment.

[0147] ALP staining was used to evaluate the effect of scaffolds on promoting osteogenic differentiation of BMSCs. At 7 and 14 days, GTPB scaffolds showed the deepest purple staining, followed by GTB and GTP scaffolds Figure 4 C). Quantitative analysis showed that the ALP activity of GTPB scaffolds was higher than that of other groups Figure 4 F). Subsequently, Alizarin Red S staining was performed to evaluate the mineralization ability of BMSCs. At 14 and 21 days, GTPB scaffolds exhibited the most significant calcium nodules, followed by GTB and GTP scaffolds Figure 4 C). Quantitative analysis showed that GTPB scaffolds had the highest number of calcium nodules Figure 4 G). These results indicated that GTPB scaffolds had the ability to promote osteogenic differentiation and mineralization of BMSCs.

[0148] qRT-PCR was used to detect the expression levels of osteogenic and angiogenic marker genes (OPN, OCN, Runx2, COL I, and VEGF genes). qRT-PCR experiments showed that GTPB scaffolds exhibited the highest gene expression levels Figure 5 H-L). These results indicated that GTPB scaffolds had the most excellent osteogenic ability.

[0149] (4) In vivo osteogenic potential

[0150] Animal models play a crucial role in the validation process of medical devices prior to clinical trials. In the field of bone regeneration, the critical-sized calvarial defect model is widely used. This model is considered as an ideal tool to study the influence of biomaterials and growth factors on bone regeneration as it effectively mimics the clinical bone defect scenario Figure 5 A).

[0151] We first evaluated the in vivo osteogenic effect of the hydrogels using micro-CT. Micro-CT is widely used to assess and quantify the newly formed bone tissue and the structure-function relationship between the bone tissue and the biomaterials. As shown in Figure 5 B, the new bone formation in all groups significantly increased over time, with the bone tissue growing from the edge of the defect towards the center. Among the groups, the GTPB scaffold showed the most bone tissue and calcified tissue formation. Further quantitative analysis showed that the BMD and BV / TV of the GTPB scaffold were significantly higher than those of the other groups Figure 5 C and 5D). At week 8, the BMD of the GTPB scaffold increased by 4.1-fold and the BV / TV increased by 2.9-fold compared to the blank group.

[0152] In addition, histological staining, including HE staining and Masson’s trichrome staining, was performed. From the HE staining, it can be observed that the GTPB scaffold showed the most bone tissue at both 4 and 8 weeks Figure 5 E). The newly formed bone tissue was tightly integrated with the surrounding tissue and looked more mature. In contrast, the blank group only showed fibrous connective tissue in the defect area, while the other scaffolds had a small amount of new bone tissue. Masson’s trichrome staining showed similar results, with the GTPB scaffold showing more mature purple bone tissue, while the other groups showed blue immature bone or fibrous connective tissue Figure 6 F).

[0153] Immunohistochemical staining was performed to evaluate the expression level of osteogenic and angiogenic proteins. The results showed that the GTPB scaffold had the strongest immunostaining of osteogenic and angiogenic proteins at 4 and 8 weeks Figure 6 A-E). The quantitative results showed that the proportion of positive cells in the GTPB scaffold was significantly higher than that in the other groups, which was consistent with the trend observed in the staining images ​ F-J). These results confirmed that the GTPB scaffold, i.e., the GelMA / TCP / PDGF-BB / BP@BMP-2 scaffold prepared by the present application, has excellent osteogenic and angiogenic capacity.

Claims

1. A method for fabricating a 3D bioprinted radial porous scaffold for staged bone regeneration, characterized in that, The method includes the following steps: Black phosphorus nanosheets BP@BMP-2 loaded with BMP-2 were prepared by incubating and centrifuging black phosphorus BP nanosheets with BMP-2 solution; GelMA solution was prepared by dissolving methacryloyl gelatin in a mixed solution of lithium phenyl-2,4,6-trimethylbenzoylphosphonate LAP and lemon yellow; β-tricalcium phosphate TCP nanoparticles, BP@BMP-2 nanosheets, and platelet-derived growth factor-BB PDGF-BB solution were added to the GelMA solution to obtain bio-ink; the bio-ink was loaded into a 3D bioprinter to prepare a 3D bioprinted radial porous scaffold GelMA / TCP / PDGF-BB / BP@BMP-2 for staged bone regeneration; The 3D bioprinted radial porous scaffold contains a dual growth factor sequential release and late PO4 phase. 3- and Ca 2+ The sustained release of [something] can complete the three key steps of staged bone regeneration.

2. The method according to claim 1, characterized in that, The method includes the following steps: (1) Synthesis of GelMA: Pig skin gelatin type A was dissolved to obtain a completely transparent gelatin solution. Methacrylic anhydride was slowly added to the gelatin solution and stirred. The solution was transferred to a dialysis membrane for dialysis, centrifuged, and the supernatant was collected and freeze-dried to prepare GelMA. (2) Synthesis of BP@BMP-2: The BP nanosheet dispersion was centrifuged to remove N-methyl-2-pyrrolidone, the precipitate was collected, and BP nanosheets were prepared. The BP nanosheets were incubated with BMP-2 solution and centrifuged to prepare BP@BMP-2 nanosheets. (3) Dissolve the GelMA in a mixed solution of LAP and lemon yellow to obtain a GelMA solution. Add TCP nanoparticles, the BP@BMP-2 nanosheets, and PDGF-BB solution to the GelMA solution to obtain a bio-ink. Load the bio-ink into a 3D bioprinter to obtain a 3D bioprinted radial porous scaffold GelMA / TCP / PDGF-BB / BP@BMP-2 for staged bone regeneration.

3. The method according to claim 2, characterized in that, The amount of pigskin gelatin type A mentioned in step (1) is 1-10g.

4. The method according to claim 2, characterized in that, In step (1), the solvent used to dissolve the porcine gelatin type A is PBS buffer, deionized water, acetic acid solution, hydrochloric acid solution or sodium hydroxide solution.

5. The method according to claim 4, characterized in that, The amount of PBS buffer used is 10-100 mL.

6. The method according to claim 2, characterized in that, The amount of methacrylic anhydride used in step (1) is 0.5-6 mL.

7. The method according to claim 3, characterized in that, The amount of pigskin gelatin type A used in step (1) is 5 g.

8. The method according to claim 4, characterized in that, The solvent used to dissolve the porcine gelatin type A in step (1) is PBS buffer.

9. The method according to claim 5, characterized in that, The amount of PBS buffer used is 50 mL.

10. The method according to claim 6, characterized in that, The amount of methacrylic anhydride used in step (1) is 3 mL.

11. The method according to claim 2, characterized in that, The stirring conditions described in step (1) are 50°C and 1 h.

12. The method according to claim 2, characterized in that, The molecular weight cutoff for dialysis described in step (1) is 3500 Da.

13. The method according to claim 2, characterized in that, The dialysis conditions described in step (1) are 40°C and dialysis for 3 days.

14. The method according to claim 2, characterized in that, The centrifugation conditions described in step (1) are 5000 rpm and 10 min.

15. The method according to claim 2, characterized in that, The freeze-drying conditions described in step (1) are -80°C and freeze-drying for 48 h.

16. The method according to claim 2, characterized in that, The amount of BP nanosheet dispersion used in step (2) is 0.1-5 mL, 0.05-2 mg / mL.

17. The method according to claim 2, characterized in that, The amount of BMP-2 solution used in step (2) is 0.1-5 mL, 50-150 ng / mL.

18. The method according to claim 16, characterized in that, The amount of BP nanosheet dispersion used in step (2) is 1 mL, 0.2 mg / mL.

19. The method according to claim 17, characterized in that, The amount of BMP-2 solution used in step (2) is 1 mL, 100 ng / mL.

20. The method according to claim 2, characterized in that, In step (2), the centrifugation conditions for the BP nanosheet dispersion are 10,000 rpm and 10 min.

21. The method according to claim 2, characterized in that, The incubation conditions described in step (2) are room temperature and overnight incubation.

22. The method according to claim 2, characterized in that, In step (2), the centrifugation conditions after incubation are 10,000 rpm and 5 min.

23. The method according to claim 2, characterized in that, The LAP and lemon yellow mixed solution mentioned in step (3) is prepared by dissolving LAP and lemon yellow in deionized water.

24. The method according to claim 23, characterized in that, The dosage of LAP is 0.2-5 mg.

25. The method according to claim 23, characterized in that, The dosage of the tartrazine is 0.1-3 mg.

26. The method according to claim 23, characterized in that, The amount of deionized water used is 0.2-5 mL.

27. The method according to claim 2, characterized in that, The amount of GelMA used in step (3) is 0.05-1 g.

28. The method according to claim 2, characterized in that, The mass-to-volume ratio of the GelMA solution in step (3) is 5-15%.

29. The method according to claim 2, characterized in that, The amount of TCP nanoparticles used in step (3) is 5-15 mg.

30. The method according to claim 2, characterized in that, The amount of BP@BMP-2 nanosheets used in step (3) is 0.05-1 mg.

31. The method according to claim 2, characterized in that, The amount of PDGF-BB solution used in step (3) is 5-15 μL.

32. The method according to claim 24, characterized in that, The dosage of LAP is 1 mg.

33. The method according to claim 25, characterized in that, The dosage of lemon yellow is 0.6 mg.

34. The method according to claim 26, characterized in that, The amount of deionized water used is 1 mL.

35. The method according to claim 27, characterized in that, The amount of GelMA used in step (3) is 0.1 g.

36. The method according to claim 28, characterized in that, The mass-volume ratio of the GelMA solution in step (3) is 10%.

37. The method according to claim 29, characterized in that, The amount of TCP nanoparticles used in step (3) is 10 mg.

38. The method according to claim 30, characterized in that, The amount of BP@BMP-2 nanosheets used in step (3) is 0.1 mg.

39. The method according to claim 31, characterized in that, The amount of PDGF-BB solution used in step (3) is 10 μL.

40. The method according to claim 2, characterized in that, The 3D bioprinter mentioned in step (3) is a digital light processing 3D bioprinter.

41. A 3D bioprinted radial porous scaffold for staged bone regeneration prepared by the method according to any one of claims 1-40.

42. A bio-ink for 3D bioprinting prepared by the method according to any one of claims 1-40, wherein the bio-ink comprises GelMA solution, TCP nanoparticles, BP@BMP-2 nanosheets, and PDGF-BB solution.

43. The application of the 3D bioprinted radial porous scaffold of claim 41 in the preparation of bone repair and regeneration materials.

44. The use of the bio-ink of claim 42 in the preparation of a 3D bioprinted radially porous scaffold for staged bone regeneration.

Images