3D bio-printing radial porous scaffold for staged bone regeneration and preparation method of 3D bio-printing radial porous scaffold

Abstract

The invention discloses a 3D bio-printing radial porous scaffold for staged bone regeneration and a preparation method thereof, the 3D bio-printing radial porous scaffold can provide a bionic cell microenvironment to support permeation of host repair cells to a defect part and promote osseointegration after implantation, and in addition, the 3D bio-printing radial porous scaffold can be used for repairing the defect part. The three key steps of staged bone regeneration can be completed through sequential release of double growth factors contained in the 3D biological printing radial porous scaffold and continuous release of PO4 < 3-> and Ca < 2 + > in the later period, efficient bone regeneration can be achieved, and a bionic staged regeneration strategy with prospects is represented.

Description

Technical Field

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

[0002] Bone defects may be caused by trauma, fractures, infection, deformity or tumors. For defects exceeding a critical size, artificial intervention is usually required. Currently, autologous transplantation is considered the gold standard. However, autologous transplantation has many limitations, such as the need for secondary surgery, pain, risk of infection and poor wound healing. In addition, allogeneic transplantation also faces the potential risk of immune rejection and disease transmission. With the rapid development of tissue engineering and regenerative medicine, biomaterial scaffolds have been considered a promising alternative strategy that can effectively overcome the limitations of autologous and allogeneic transplantation.

[0003] Biomaterial scaffolds suitable for bone regeneration should provide a biomimetic cell microenvironment to support the infiltration of host repair cells into the defect site and promote bone integration after implantation. Radially arranged scaffolds can guide host repair cells to migrate from the host tissue to the defect center in the radial direction. Currently, 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 has high requirements for temperature control and requires precise control of the cooling rate and direction. In addition, since the microstructure of the scaffold depends on the growth process of ice crystals, its structural fineness 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 facing this field. Summary of the Invention

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

[0005] In the present invention, the inventors introduced BMP-2 loaded black phosphorus nanosheets (BP@BMP-2) into a precursor solution containing PDGF-BB, GelMA and TCP for the first time, and used 3D bioprinting technology to construct a hydrogel scaffold with a radial porous structure. Under the stimulation of dual growth factors (BMP-2 and PDGF-BB), BMSCs were recruited and differentiated into mature osteoblasts. Subsequently, 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 scaffolds composited with black phosphorus nanosheets can play a good role in bone regeneration applications and represent a promising biomimetic staged regeneration strategy.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] A first aspect of the present invention provides a method for preparing a 3D bioprinted radial porous scaffold for staged bone regeneration.

[0008] Furthermore, the method includes the following steps: incubating black phosphorus (BP) nanosheets with a BMP-2 solution and centrifuging to obtain BMP-2-loaded black phosphorus nanosheets (BP@BMP-2); dissolving methacrylated gelatin (GelMA) in a mixed solution of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) and lemon yellow to obtain a GelMA solution; adding β-tricalcium phosphate (TCP) nanoparticles, BP@BMP-2 nanosheets, and platelet-derived growth factor-BB (PDGF-BB) solution to the GelMA solution to obtain bio-ink, and loading 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.

[0009] In the present invention, the 3D bioprinted radial porous scaffold contains the sequential release of dual growth factors and the late PO4 3- and Ca 2+ The sustained release of 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: inflammation and hematoma formation, callus formation and endochondral ossification, and bone remodeling and mature bone formation. These three stages are a continuous and interconnected process, each with its own specific cellular and molecular events that collectively promote bone regeneration and repair. When using the 3D bioprinted radial porous scaffold for bone repair, the scaffold needs to provide appropriate support and microenvironment for bone regeneration at different stages to promote smooth bone tissue regeneration.

[0011] Inflammation and hematoma formation period: After bone injury occurs, the body first initiates an inflammatory response. Blood vessels at the injured site rupture and bleed, forming a hematoma, which provides a place for the subsequent aggregation of repair cells and the release of growth factors. At the same time, inflammatory cells such as macrophages and neutrophils will rapidly migrate to the injury site, clear necrotic tissue and bacteria, and release a variety of cytokines and growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β). These factors can recruit repair cells such as mesenchymal stem cells, laying 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 the initial callus. Simultaneously, chondrocytes also form cartilage tissue, which gradually transforms into bone tissue through endochondral ossification. During this stage, blood vessels gradually grow into the callus tissue, providing nutrients and oxygen for bone regeneration and 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 function, absorbing and remodeling excess callus tissue. Simultaneously, osteoblasts continuously secrete bone matrix, continuously remodeling and optimizing bone tissue, ultimately forming mature bone tissue and restoring its normal structure and function. During this stage, the mechanical properties of bone tissue gradually return to near-normal levels.

[0014] To establish a programmed dual delivery system, a 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 used 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 a folded surface. They have excellent biocompatibility, adjustable band gap and degradability. Due to their unique structure, BP nanosheets can effectively load drugs or bioactive factors. During their degradation process, BP nanosheets release phosphate ions (PO4 3- ) can react with calcium ions (Ca 2+ ) combine to form calcium phosphate, thereby promoting bone 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, and the bone injury includes but is not limited to: skull defect, maxillofacial bone defect, humerus defect, ulna and radius defect, femur defect, tibia and fibula defect, vertebral body defect, lamina defect, pelvic bone defect, metacarpal bone defect, phalangeal defect, metatarsal bone defect, toe bone defect, etc.

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

[0018] (1) Synthesis of GelMA: Pigskin gelatin type A was dissolved to obtain a completely transparent gelatin solution, methacrylic anhydride was slowly added dropwise to the gelatin solution, stirred, and the solution was transferred to a dialysis membrane for dialysis. The solution was centrifuged, and the supernatant was collected and freeze-dried to prepare GelMA.

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

[0020] (3) Dissolving the GelMA in a mixed solution of LAP and lemon yellow to prepare a GelMA solution, adding TCP nanoparticles, the BP@BMP-2 nanosheets, and PDGF-BB solution to the GelMA solution to obtain 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.

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

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

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

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

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

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

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

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

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

[0030] Optionally, the molecular weight cutoff of the dialysis in step (1) is 3500 Da;

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

[0032] Optionally, the centrifugation conditions in step (1) are 5000 rpm, 10 min;

[0033] Optionally, the freeze-drying conditions in step (1) are -80°C and freeze-drying for 48 hours.

[0034] Furthermore, the amount of the BP nanosheet dispersion 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] Furthermore, the amount of the BP nanosheet dispersion 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 conditions for centrifuging the BP nanosheet dispersion in step (2) are 10,000 rpm for 10 min;

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

[0040] Optionally, the centrifugation conditions after incubation in step (2) are 10,000 rpm and 5 min.

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

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

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

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

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

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

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

[0048] Optionally, the amount of BP@BMP-2 nanosheets used 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] Furthermore, the dosage of 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 used 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 used in step (3) is 10 mg;

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

[0057] Optionally, the amount of the 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 invention, the 3D bioprinter is a digital light processing 3D bioprinter, which is a 3D printing device that uses a digital micromirror device (DMD) to achieve photocuring.

[0060] Digital light processing 3D bioprinters utilize digital micromirror technology. Computer-controlled micromirror arrays on a DMD chip project ultraviolet light according to a pre-designed pattern into a resin tank containing bio-ink. The bio-ink contains a photoinitiator, which rapidly solidifies under UV light, forming layers upon layers, ultimately forming a three-dimensional biological structure.

[0061] The digital light processing 3D bioprinter has the following advantages in the field of biomaterial printing: it can achieve high printing resolution and precisely control the solidification position and shape of the bioink, which is conducive to the construction of complex biological structures, such as tissue models with fine vascular networks; a layer of bioink can be solidified with a single exposure, and the printing speed is relatively fast, which can complete the printing of larger-sized biological structures in a shorter time, improving efficiency; a variety of types of bioinks can be used, including bioinks based on materials such as hydrogels and proteins, providing more options for printing different tissues and organs.

[0062] A second aspect of the present invention provides any of the following products:

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

[0064] (2) A bio-ink for 3D bioprinting, the bio-ink 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, PDGF-BB solution and their amounts are as described in the first aspect of the present invention;

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

[0067] In the present invention, the bio-ink is a specialized material used in 3D bioprinting. It is typically composed of biomaterials, bioactive molecules, and other components. It can precisely control the distribution and arrangement of cells during the printing process, mimicking the structure and function of natural tissues, providing a suitable environment for cell survival and growth, and ultimately forming biologically active tissue or organ analogs. In specific embodiments of the present invention, the bio-ink refers to the material used in 3D bioprinting to produce the aforementioned 3D bioprinted radially porous scaffold.

[0068] A third aspect of the present invention provides the application of any of the following aspects:

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

[0070] (2) Application of the bio-ink described in the second aspect of the present invention in the preparation of 3D bio-printed radial porous scaffolds for staged bone regeneration.

[0071] In addition, the present invention also provides a method for bone repair or bone regeneration, which comprises the following steps: precisely placing the printed 3D bioprinted radial porous scaffold described in the second aspect of the present invention at the bone defect of a subject in need, ensuring that the scaffold fits tightly with the surrounding healthy bone tissue.

[0072] In some embodiments, the subject includes mammals and non-mammals. The mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The primates are preferably monkeys, apes, or humans. In preferred embodiments of the present invention, the subject is a human. In specific embodiments of the present invention, the subject is a patient with a bone defect.

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

[0074] In some embodiments, during the implantation of the scaffold, if necessary, a fixing device such as screws or steel plates 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 entire bone repair or regeneration process, the 3D-printed radially porous scaffold provided by the present invention can provide three-dimensional spatial support and guidance for the growth of bone tissue. Its radially porous structure is conducive to the exchange of nutrients, cell migration and blood vessel growth, thereby promoting the staged regeneration of bone tissue and ultimately achieving effective repair of bone defects.

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

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

[0078] Figure 1 : Characterization of the physical properties of BP nanosheets, including: Figure A: zeta potential of BP and BP@BMP-2 nanosheets; Figure B: particle size distribution and average diameter of BP nanosheets; Figure C: particle size distribution and average diameter of BP@BMP-2 nanosheets; Figure D: transmission electron microscopy image of BP nanosheets; Figure E: transmission electron microscopy image of BP@BMP-2 nanosheets; Figure F: Raman spectra of BP and BP@BMP-2 nanosheets; Figure G: X-ray photoelectron spectroscopy of BP and BP@BMP-2 nanosheets; Figure H: UV-visible absorption spectrum recording the degradation of BP and BP@BMP-2 nanosheets at 0, 2, 4, 6, 8 and 10 days; Figure I: degradation image of BP and BP@BMP-2 nanosheets; Figure J: in vitro PO4 3- Release curve;

[0079] Figure 2Figure 3: Characterization of 3D bioprinted hydrogel scaffolds, including: A: Fourier transform infrared spectra of gelatin and GelMA; B: Nuclear magnetic resonance spectra of gelatin and GelMA; C: X-ray diffraction of TCP nanoparticles; D: Computer simulation model of 3D bioprinted radial pore scaffold; E: Cross-sectional and longitudinal section views of freeze-dried 3D bioprinted scaffold; F: Macro- and microstructures of 3D bioprinted radial pore scaffold;

[0080] Figure 3 : Mechanical properties and sustained release capacity of hydrogel scaffolds, including: Figure A: frequency dependence curve; Figure B: time dependence curve; Figure C: loss factor tandelta; Figure D: shear rate-viscosity curve; Figure E: stress-strain curve; Figure F: compression strength results obtained by compression test; Figure G: expansion rate of hydrogel scaffold; Figure H: degradation rate of hydrogel scaffold in PBS buffer; Figure I: degradation rate of hydrogel scaffold in collagenase solution; Figure J: cumulative release curve of BMP-2 from hydrogel scaffold; Figure K: cumulative release curve of PDGF-BB from hydrogel scaffold; Figure L: continuous release of BMP-2 and PDGF-BB from hydrogel scaffold, *P<0.05, **P<0.01, ***P<0.001;

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

[0082] Figure 5: Scaffolds enhance bone regeneration in critical-sized calvarial defects in rats. (A) Representative images of the in vivo experimental timeline and surgical procedure; (B) Representative microCT images of bone defects at 4 and 8 weeks after scaffold treatment, as well as quantitative analysis: (C) BMD, (D) BV / TV; (E) Representative images of HE staining demonstrating the effects of different scaffold types on bone defect regeneration; (F) Representative images of Masson's trichrome staining demonstrating the effects of different scaffold types on bone defect regeneration, *P<0.05, **P<0.01, ***P<0.001;

[0083] Figure 6 : Immunohistochemical staining was used to evaluate the effects of the blank group, G, GT, GTP, GTB, and GTPB scaffolds on bone regeneration after 4 and 8 weeks. A: Immunohistochemical images of OPN after 4 and 8 weeks; B: Immunohistochemical images of OCN after 4 and 8 weeks; C: Immunohistochemical images of Runx2 after 4 and 8 weeks; D: Immunohistochemical images of COL I after 4 and 8 weeks; E: Immunohistochemical images of VEGF after 4 and 8 weeks, and the corresponding quantitative analysis: F: OPN, G: OCN, H: Runx2, I: COL I, J: VEGF, *P<0.05, **P<0.01, ***P<0.001; Figure 7 Live / Dead staining images of BMSCs cultured with hydrogel scaffold extract on days 1, 3, 5, and 7. In the Live / Dead staining, almost no cell death was observed in all groups, and the cells showed high viability. DETAILED DESCRIPTION

[0084] The present invention will be further described below with reference to specific embodiments. The following specific embodiments are intended only to illustrate the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0085] The reagents, raw materials, and experimental consumables used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0086] Example A 3D bioprinted radial porous scaffold for staged bone regeneration and its effect verification

[0087] 1. Experimental Materials

[0088] Gelatin type A was purchased from Sigma-Aldrich (St. Louis, MO). β-tricalcium phosphate (TCP) nanoparticles were purchased from Beijing Inno Innovation Technology Co., Ltd. (Beijing). Black phosphorus (BP) nanosheet dispersion was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai). Bone marrow mesenchymal stem cells (BMSCs) were purchased from ScienCell Research Laboratories (Carlsbad, MD, USA). Total RNA extraction kits, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Madison, MD, USA). Antibodies against osteopontin (OPN), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), type I collagen (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 Beyotime Biotechnology Co., Ltd. (Shanghai, China). BMP-2 and PDGF-BB ELISA kits were purchased from Sangon Biotech Biotechnology Co., Ltd. (Shanghai).

[0089] 2. Experimental methods

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

[0091] The synthesis method of GelMA is as follows: First, 5g of pig skin gelatin type A was dissolved in 50mL of PBS buffer and magnetically stirred in a 50℃ water bath until a completely transparent solution was obtained. Then, 3mL of methacrylic anhydride was slowly added dropwise to the gelatin solution and stirred continuously at 50℃ for 1 hour to complete the methacrylation reaction. Subsequently, the solution was transferred to a dialysis membrane (molecular weight cutoff of 3500Da) and dialyzed with deionized water at 40℃ for 3 days to remove impurities. The dialyzed solution was centrifuged at 5000rpm for 10 minutes, the precipitate was removed, the supernatant was collected, and lyophilized at -80℃ for 48 hours to obtain GelMA. GelMA was stored at -20℃ for subsequent use.

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

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

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

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

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

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

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

[0099] The particle size and zeta potential of BP and BP@BMP-2 nanosheets were measured using a Zetasizer Nano ZS-90 (Malvern, UK). The particle size was measured using dynamic light scattering at a 90° scattering angle, and the zeta potential was measured using laser Doppler microelectrophoresis. The microstructure of BP and BP@BMP-2 nanosheets was recorded using a transmission electron microscope (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 is based on the light scattering effect and provides information on molecular vibration modes by measuring the frequency changes of scattered light. 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 the degradation performance was monitored by UV-visible spectrophotometry every two days until the 10th day. 3- Release curves. 3D bioprinted hydrogel scaffolds were prepared using BP nanosheets (with or without BMP-2 loading) as described above. The scaffolds were immersed in 1 mL of deionized water and incubated at 37°C with shaking at 100 rpm. 200 μL of the incubation solution was collected on days 1, 2, 5, 7, 14, 21, and 28 and replaced with an equal volume of fresh deionized water. PO4 in the incubation solution was quantified using a detection kit and a microplate reader. 3- concentration.

[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 slides and pressed into a sheet. The infrared spectrum of the sample was measured in a dry environment from 4000 to 400 cm -1 The wavelength range is collected with a resolution of 4cm -1 The chemical environment of hydrogen atoms in the hydrogel scaffold was analyzed using nuclear magnetic resonance (NMR). Different chemical shifts reflect the electronic environment around hydrogen atoms and provide clues to the material composition and functional group distribution. The structure and phase content of TCP were analyzed by X-ray diffraction (XRD). Cu K Diffraction was performed with a 2θ angle range of 10° to 80°. In order to observe the microstructure of the hydrogel scaffold, the scaffold was cut into squares, 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 base and sputtered with gold for 60 seconds. The morphology was subsequently photographed using a scanning electron microscope (SEM, Sigma 500, Zeiss, Jena, Germany) at 5kV.

[0102] (6) Rheological properties

[0103] Rheological tests were performed to characterize the hydrogels using a Haake Mars 60 rheometer (ThermoFisher Scientific, Waltham, USA). The hydrogel prepolymer solution was placed in a mold to prepare hydrogel samples with a diameter of 20 mm and a height of 2 mm, and the samples were stored at 4 °C. Silicone oil was added to the edges of the hydrogel samples to prevent water loss. In order to determine the frequency dependence of the storage modulus (G') and loss modulus (G"), all hydrogel samples were measured in the range of 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. The shear rate was varied from 1 to 100 s -1 ), keeping the temperature and strain constant, shear rate-viscosity curves were obtained to evaluate the printing performance.

[0104] (7) Mechanical strength

[0105] Hydrogel samples were prepared into uniform shapes (10 mm × 10 mm × 3 mm). Compression tests were then performed at a speed of 1 mm / min using a biomaterial universal mechanical testing machine (WDW-10, Jinan Chuanbai Instrument Co., Ltd., Shandong, China). Stress-strain curves 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 PBS buffer. After the preset time interval, the hydrogel was removed from 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 reaches swelling equilibrium. The swelling ratio is calculated according to the following formula: Swelling ratio (%) = W t / W0×100%. The degradation rate was measured in a similar manner. The initial mass of the freeze-dried hydrogel was W0. The hydrogel was then immersed in PBS buffer or 5U / mL collagenase solution. The degradation solution was replaced every 3 days. At predetermined time intervals, the hydrogel was removed from the PBS buffer or collagenase solution, freeze-dried, and weighed (W tThe degradation rate of the hydrogel was calculated according to the following formula: Degradation rate (%) = (W0-W t ) / W0×100%.

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

[0109] To quantify the release rates of BMP-2 and PDGF-BB, the scaffolds were immersed in PBS buffer and maintained at a constant temperature of 37°C. At each pre-determined 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 from the scaffolds was measured using ELISA kits (Wuhan Cusabio Technology Co., Ltd., China) according to the manufacturer's protocol, and cumulative release curves were then plotted.

[0110] (10) Cytocompatibility evaluation

[0111] The pre-prepared hydrogel samples were sterilized by ethylene oxide for use in all subsequent biological experiments. The hydrogel samples were incubated in DMEM medium for 24 hours to obtain the extract, which was performed according to ISO 10993-12. BMSCs (5×10 4 cells / well) to assess cell proliferation and viability. To determine cell proliferation, a CCK-8 assay (ThermoFisher Scientific, Madison, USA) was performed to measure the changes in optical density (OD value) at 450 nm after cells were incubated with hydrogels for 1, 3, 5, and 7 days. Cell viability was assessed by Live / Dead staining and recorded using a confocal laser scanning microscope (LSM800, Zeiss, Jena, Germany). The cells were fixed with 4% paraformaldehyde and treated with Triton X-100 (Sigma-Aldrich, St. Louis, USA). Then, FITC-phalloidin (Solarbio Technology Co., Ltd., Beijing, China) was used for staining for 20 minutes, and the cytoskeleton and cell extension were observed under a confocal laser scanning microscope. A cell scratch assay was performed to study the effect of the scaffold on cell migration. BMSCs (1×10 5 Cells / well) were seeded into culture plates and incubated at 37°C, 5% CO2 for 24 hours. Use the tip of a pipette to scratch the cell layer, making sure the scratch is perpendicular to the cell layer. Then, wash with PBS buffer to remove the detached cells. The hydrogel extract was added to the wells and co-cultured with the cells. Cell migration was monitored using a fluorescent inverted microscope at 0, 12, and 24 hours. The migration rate was calculated according to the following formula: Migration rate (%) = (R0-R t ) / R0×100%, where R0 is the initial scratch area, R t The remaining scratch areas at different time points.

[0112] (11) In vitro osteogenic properties

[0113] The scaffold-induced BMSCs (5×10 4 The cells were incubated with the scaffold extract for 7, 14, and 21 days before being fixed with formalin and stained with either 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, WI, USA).

[0114] (12) qRT-PCR experiments

[0115] After 7 days of 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). Subsequently, gene expression was analyzed using the TB Green qRT-PCR Kit (Takara, Shiga, Japan) in a StepOne Plus real-time fluorescence quantitative PCR instrument (ThermoFisher Scientific, Madison, USA). Using GAPDH as an internal reference gene, the mRNA expression levels of ALP, Runx2, OCN, OPN, and Col I were analyzed.

[0116] (13) Skull defect model

[0117] The in vivo osteogenic properties of the scaffold were evaluated using a critical-size skull defect model in 6-week-old male Sprague-Dawley rats. Animal experimental procedures and housing conditions were in accordance with the Guidelines for the Use of Laboratory Animals of the National Institutes of Health (NIH) and were supervised by the Ethics Committee of Peking Union Medical College Hospital. During the modeling process, a critical-size bone defect (4 mm) was created on the skull of each rat, and the hydrogel sample was implanted into the defect site. Tissue samples were collected 4 and 8 weeks after surgery for subsequent histological and imaging evaluations.

[0118] (14) MicroCT analysis

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

[0120] (15) Histological staining and immunohistochemistry

[0121] Skull tissue samples were collected and prepared for tissue sections at 4 and 8 weeks postoperatively. The bone tissue was formalin-fixed, decalcified, dehydrated, cleared, and paraffin-infiltrated. Routine staining included hematoxylin and eosin (HE) and Masson's trichrome staining.

[0122] Immunohistochemical staining was performed using four monoclonal antibodies closely associated with osteogenesis and angiogenesis: osteopontin (OPN), osteocalcin (OCN), Runx2, collagen type I (COL I), and vascular endothelial growth factor (VEGF). After staining, pathological sections were imaged using an Olympus BX53 light microscope (Shinjuku, Tokyo, Japan). Quantitative analysis of immunohistochemical staining 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 composed of phosphorus atoms arranged in an accordion pattern, forming a unique honeycomb two-dimensional structure. BP nanosheets have a large surface area and can be used to load small-molecule drugs, proteins, and peptides for drug delivery systems. Unlike other two-dimensional materials such as graphene, BP nanosheets degrade into non-toxic phosphate ions in the body, demonstrating excellent biocompatibility and biosafety.

[0126] The zeta potential of BP and BP@BMP-2 nanosheets is shown in Figure 2. Figure 1 As shown in A. The zeta potential of BP nanosheets was measured to be -12.5±1.3 mV. After loading with BMP-2, the zeta potential of BP@BMP-2 nanosheets increased to 8.9±2.3 mV. The change in zeta potential indicated the presence of electrostatic interaction between BMP-2 and BP nanosheets, confirming that BMP-2 was successfully loaded onto BP nanosheets. Zeta potential values ​​of -30 or 30 mV have been used as criteria for nanoparticle dispersion. The electrostatic repulsion between nanoparticles that meet these criteria can offset the attractive force, thereby increasing the distance and spacing between nanoparticles, ultimately reducing the risk of particle aggregation and precipitation. The particle size distribution of BP and BP@BMP-2 nanosheets was analyzed by a nanoparticle size analyzer ( Figure 1 B and 1C). The results showed that the average particle size of BP nanosheets was 635.4 ± 1.5 nm. After loading with BMP-2, the average particle size of BP@BMP-2 nanosheets slightly increased to 653.5 ± 0.1 nm.

[0127] TEM images showed that both BP and BP@BMP-2 nanosheets exhibited a characteristic two-dimensional sheet morphology ( Figure 1 The measured lattice spacing of the BP and BP@BMP-2 nanosheets was 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 measurement results.

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

[0129] The XPS results of BP nanosheets showed characteristic peaks at 133eV and 197eV, corresponding to P2p and P2s, respectively, which are consistent with the peaks of BP nanosheets reported previously. After loading BMP-2, the XPS results of BP@BMP-2 nanosheets showed an increase in the concentrations of carbon, oxygen, and nitrogen, while retaining the P2p and P2s peaks similar to those of BP nanosheets ( Figure 1 G). These results confirm that BMP-2 was successfully loaded onto BP nanosheets.

[0130] A digital camera was used to observe the degradation appearance of BP and BP@BMP-2 nanosheets on days 0, 2, 4, 6, 8, and 10. It was observed that the BP@BMP-2 nanosheets faded more slowly than BP. On day 10, the brown color of the BP nanosheets had almost completely faded, indicating that the BP nanosheets had been largely degraded into phosphate and other oxidation products. In contrast, the BP@BMP-2 nanosheets still retained some brown color, but the color was slightly weakened ( Figure 1 I). UV-vis absorption spectroscopy 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 be: (1) BMP-2 binds to the BP nanosheets, possibly partially covering their surface and forming a protective film that reduces their direct exposure to water and oxygen, thereby slowing down the oxidation reaction; (2) at a neutral pH, BMP-2 carries a positive charge, while BP carries a negative charge, which, through electrostatic interactions, affects the electron transfer process after photoexcitation, thereby slowing down BP degradation. In summary, these results indicate that BMP-2 successfully modifies BP, enhancing its stability and making it less susceptible to degradation.

[0131] The degradation of BP will release PO4 3- into the solution, thus measuring PO4 3- The concentration of can be used to evaluate the degree of BP degradation. Figure 1 As shown in J, PO4 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 the 28th day, the accumulated PO4 3- The release rates were 60.3% and 46.5%, respectively. In conclusion, BMP-2 enhanced the stability of BP nanosheets and made their PO4 3- The release is slower and more sustained.

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

[0133] GelMA forms a stable three-dimensional structure through photopolymerization, and its physical properties can be adjusted by factors such as the degree of methacrylate, crosslinking agent and concentration. These characteristics make GelMA widely used in 3D printing. In the FTIR spectra of gelatin and GelMA, 1637.3 cm -1 The characteristic absorption peak at 1540.8 cm corresponds to the stretching vibration of the C=O bond and represents the amide I conformation. -1 The peak at 1240.5 cm is attributed to the bending vibration of the NH bond coupled with the stretching vibration of the CH bond, indicating the presence of amide II conformation. -1 The peaks at are due to the bending vibrations of the NH and CN bonds, corresponding to the amide III conformation, respectively. 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 resulted in 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 showed the presence of new acrylic acid proton peaks (=CH2) at 5.3 and 5.5 ppm, confirming the successful grafting of methacrylate groups onto gelatin.

[0134] TCP is the main inorganic component of bone tissue and has excellent biocompatibility, without triggering immune rejection or inflammatory responses. In the body, TCP degrades and is gradually replaced by newly formed bone tissue, making it a widely used material for bone repair. Figure 2 Figure C shows the XRD spectrum of TCP. Obvious diffraction peaks were observed at 2θ angles of 17.08°, 25.88°, 28.02°, 31.12°, 34.54°, 47.06°, and 53.16°, confirming the crystalline nature of TCP.

[0135] An ideal scaffold is not only able to recruit the host's endogenous cells to the bone defect site, but also promote osteogenic differentiation and bone matrix deposition. Previous studies have shown that good nanotopology can regulate cell behavior, such as guiding cell migration and promoting directional cell differentiation. Mac et al. designed a radiation-aligned mineralized collagen fiber scaffold containing nanosilicon. Experiments showed that the 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 accuracy of this collagen fiber scaffold may be insufficient. In contrast, 3D printing technology can accurately print advanced structures and complex properties based on digital models, which is suitable for personalized medical needs. Figure 2 D shows the top and front views of the 3D bioprinted model, showing a double-layer radial structure. Figure 2 E shows cross-sectional and longitudinal views of the freeze-dried 3D bioprinted scaffold, clearly demonstrating the interconnected radiating channels that are expected to facilitate cell migration from the surrounding bone tissue into the defect site. Figure 2 Figure F shows the macro- and microscopic morphology of the 3D bioprinted scaffold. The introduction of TCP nanoparticles and BP nanosheets slightly reduced the printability of the bioink, resulting in slightly blurred edges of the bilayered radial structures in the GelMA / TCP / BP scaffold. Inverted microscopy images further confirmed the presence of a bilayered porous structure within the scaffold. SEM images clearly revealed a well-defined channel structure and a microscopic porous network within the scaffold. These porous structures exhibited excellent integrity and permeability, providing ample space for cell attachment and proliferation.

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

[0137] The shear-thinning properties of the 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 increases the viscosity of the hydrogel, and the viscosity is highest when TCP and BP are added at the same time. This may be attributed to the large specific surface area and interfacial effect of BP nanosheets. When BP nanosheets are introduced into GelMA hydrogel, strong interactions such as van der Waals forces and hydrogen bonds may 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 limits the movement of GelMA molecular chains due to the interaction between TCP and the polymer chains, thereby increasing the viscosity. When BP and TCP are added to GelMA hydrogel at the same time, they may produce a synergistic effect, further enhancing the viscosity. In short, TCP and BP can enhance the cohesion and viscosity of the hydrogel scaffold.

[0138] According to the literature, biomaterials that promote tissue regeneration need to match the initial mechanical properties of the host tissue. Excessive mechanical strength can hinder bone regeneration and the stability of the bone-implant interface. Conversely, materials with insufficient strength fail to provide the necessary mechanical environment for bone regeneration. Figure 3 E shows the stress-strain curves of the hydrogel scaffold. It can be observed that the addition of BP and TCP enhances the compressive strength of the scaffold. The compressive strength of the GelMA / TCP / BP hydrogel scaffold at 40% strain is 70.2±6.9 ​​kPa, while the compressive strength of the GelMA hydrogel scaffold decreases significantly to 10.8±0.1 kPa ( Figure 3 F). This may be due to the varying degrees of polymer chain aggregation in different hydrogel samples. Another possible reason is that BP and TCP may enhance the physical interactions within the hydrogel (such as hydrogen bonds, van der Waals forces, and electrostatic interactions) and the covalent cross-linked network, making the scaffold more resistant to deformation. These results suggest that the addition of BP and TCP influences the mechanical properties, forming a relatively dense and robust network structure.

[0139] Too high an expansion ratio may cause the material to expand in volume after absorbing water, thereby compressing the surrounding tissues or organs and affecting the stability and biocompatibility of the material. In addition, if the expansion ratio is too high, the material may become soft, reduce mechanical strength, and impair its supporting function in bone regeneration. Figure 3 As shown in Figure 2, the swelling ratio of GelMA hydrogel in PBS buffer can reach 52%, but after adding TCP or BP, the swelling ratio of the hydrogel is significantly reduced. This is because TCP releases Ca in the hydrogel. 2+ , interacting 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 and makes the network structure more compact. However, the degradation of BP produces acidic products, which may change the pH value of the hydrogel and cause swelling. The simultaneous addition of TCP and BP makes the hydrogel exhibit higher structural stability and lower swelling when absorbing water, which helps the scaffold maintain its shape and structure in the long term. In general, all expansion curves have a significant increase in the initial stage because the hydrogel contains a large number of hydrophilic groups and a porous network structure. After 8 hours, the swelling force gradually decreases and eventually reaches equilibrium. In summary, the introduction of TCP and BP can regulate the network structure of the hydrogel, thereby affecting the swelling performance 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. Considering that hydrogels usually have a fast degradation rate, the designed new hydrogel needs to degrade slowly to provide initial mechanical support for bone tissue regeneration. Figure 3 H and 3I show the degradation performance of the hydrogel in PBS buffer and collagenase solution. It can be observed that with the addition of TCP or BP, the degradation rate of the hydrogel decreased in both PBS buffer and collagenase solution. When both materials were added at the same time, the degradation rate was the slowest. The introduction of TCP or BP increased the cross-linking density and stability of the hydrogel. A higher cross-linking density is generally associated with a slower degradation rate because a denser molecular network limits the penetration of water molecules or enzymes, thereby delaying degradation. In addition, the phosphoric acid produced during the degradation of BP may trigger the 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 is able to match the regeneration rate of new bone tissue.

[0141] Literature reports that the main challenges of BMP-2 delivery are the use of supraphysiological doses and the burst release phenomenon in the early stages. High concentrations of BMP-2 are known to be associated with a variety of 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 controlled and sustained release of BMP-2. We measured the release kinetics of BMP-2 in hydrogels. Compared with G / TCP / BP / BMP-2 hydrogels, the release rate of G / TCP / BP@BMP-2 hydrogels was slower and more sustained ( 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 for the gradual degradation of BP and the sustained release of BMP-2. This sustained-release property helps continuously stimulate the host stem cells to undergo osteogenic differentiation, promoting long-term bone regeneration.

[0142] Supplementation of PDGF-BB stimulated the gradual migration of BMSCs in the hydrogel. Figure 3 As shown in Figure K, the gradual degradation of the hydrogel resulted in the sustained release of PDGF-BB, with a cumulative release of approximately 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 validated the sequential release pattern of two growth factors. Figure 3 As shown in Figure 1, both BMP-2 and PDGF-BB were continuously released 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 goals of cell recruitment prior to cell differentiation and angiogenesis prior to new bone formation.

[0144] (3) In vitro cytocompatibility 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 a complex three-dimensional structure, closely resembling the extracellular matrix, making them an ideal carrier for cell adhesion and proliferation, and also a reservoir 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 showed cell proliferation over a period of time ( Figure 4 D). The OD values ​​of the GTPB scaffold on days 1, 3, 5, and 7 were higher than those of the other groups, indicating that the GTPB scaffold had a strong effect on promoting cell growth. In Live / Dead staining, since almost no cell death was observed in all groups, the cells showed high activity ( Figure 7 Cytoskeleton staining showed that cells exhibited better expansion after co-culture on GTPB scaffolds ( Figure 4 A). Cell scratch test showed that the scratch area of ​​GTPB scaffold was the smallest ( Figure 4 B). Quantitative analysis showed that after 24 hours, the migration rates of the Control, G, GT, GTP, GTB, and GTPB groups were 33.6%, 37.0%, 39.3%, 62.3%, 57.3%, and 70.6%, respectively. The migration rates of the GTP and GTPB scaffolds were higher than those of the other groups ( Figure 4 E) These results suggest a role for PDGF-BB in promoting cell recruitment.

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

[0148] qRT-PCR was used to detect the expression levels of osteogenesis and angiogenesis marker genes (OPN, OCN, Runx2, COL I and VEGF genes). qRT-PCR experiments showed that GTPB scaffolds showed the highest gene expression levels ( Figure 4 HL). These results indicate that the GTPB scaffold has the best osteogenic ability.

[0149] (4) In vivo osteogenic potential

[0150] Animal models play a crucial role in the validation process of medical devices before clinical trials. In the field of bone regeneration, the critical-size skull defect model is widely used. This model is considered an ideal tool for studying the effects of biomaterials and growth factors on bone regeneration because it effectively simulates the clinical bone defect scenario ( Figure 5 A).

[0151] We first used microCT to evaluate the in vivo osteogenesis of the hydrogel. MicroCT is widely used to evaluate and quantify new bone formation and the structure-function relationship between bone tissue and biomaterials. Figure 5 As shown in Figure 2, new bone formation in all groups increased significantly over time, with bone tissue growing from the edge of the defect toward the center. Among all 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, compared with the blank group, the BMD of the GTPB scaffolds increased by 4.1 times, and the BV / TV ratio increased by 2.9 times.

[0152] In addition, histological staining, including HE staining and Masson trichrome staining, was performed. From 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 appeared more mature. In contrast, the blank group showed only fibrous connective tissue in the defect area, while the other scaffolds had a small amount of new bone tissue. Masson trichrome staining showed similar results, with the GTPB scaffold showing more mature purple bone tissue, while the other groups showed immature bone or fibrous connective tissue in blue ( Figure 5 F).

[0153] Immunohistochemical staining was used to evaluate the expression levels of osteogenic and angiogenic proteins. The results showed that the immunostaining of osteogenic and angiogenic proteins was strongest in GTPB scaffolds at 4 and 8 weeks ( Figure 6 AE). Quantitative results showed that the proportion of positive cells in the GTPB scaffolds was significantly higher than that in the other groups, which was consistent with the trend observed in the staining images ( Figure 6 These results confirmed that the GTPB scaffold (i.e., the GelMA / TCP / PDGF-BB / BP@BMP-2 scaffold prepared by the present invention) has excellent osteogenesis and angiogenesis abilities.

Claims

1. A method for preparing a 3D bioprinted radial porous scaffold for staged bone regeneration, characterized in that: The method comprises the following steps: Black phosphorus (BP) nanosheets were incubated with a BMP-2 solution and centrifuged to prepare BMP-2-loaded black phosphorus nanosheets (BP@BMP-2). Methacryloylated gelatin (GelMA) was dissolved in a mixed solution of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) and tartrazine to prepare a GelMA solution. 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 a 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.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) Synthesis of GelMA: Pigskin gelatin type A was dissolved to obtain a completely transparent gelatin solution, methacrylic anhydride was slowly added dropwise to the gelatin solution, stirred, and the solution was transferred to a dialysis membrane for dialysis. The solution was 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, and the precipitate was collected to prepare BP nanosheets. The BP nanosheets were incubated with a BMP-2 solution and centrifuged to prepare BP@BMP-2 nanosheets. (3) Dissolving the GelMA in a mixed solution of LAP and lemon yellow to prepare a GelMA solution, adding TCP nanoparticles, the BP@BMP-2 nanosheets, and PDGF-BB solution to the GelMA solution to obtain 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.

3. The method according to claim 2, characterized in that The amount of pigskin gelatin type A in step (1) is 1-10 g; Optionally, the solvent for dissolving the pigskin gelatin type A in step (1) is PBS buffer, deionized water, acetic acid solution, hydrochloric acid solution or sodium hydroxide solution; Optionally, the amount of the PBS buffer is 10-100 mL; Optionally, the amount of methacrylic anhydride used in step (1) is 0.5-6 mL.

4. The method according to claim 3, characterized in that The amount of pigskin gelatin type A in step (1) is 5 g; Optionally, the solvent for dissolving the pigskin gelatin type A in step (1) is PBS buffer; Optionally, the amount of the PBS buffer is 50 mL; Optionally, the amount of methacrylic anhydride used in step (1) is 3 mL; Optionally, the stirring condition in step (1) is 50° C., 1 h; Optionally, the molecular weight cutoff of the dialysis in step (1) is 3500 Da; Optionally, the dialysis condition in step (1) is 40° C. and dialysis for 3 days; Optionally, the centrifugation conditions in step (1) are 5000 rpm, 10 min; Optionally, the freeze-drying conditions in step (1) are -80°C and freeze-drying for 48 hours.

5. The method according to claim 2, characterized in that The amount of the BP nanosheet dispersion in step (2) is 0.1-5 mL, 0.05-2 mg / mL; Optionally, the amount of the BMP-2 solution in step (2) is 0.1-5 mL, 50-150 ng / mL.

6. The method according to claim 5, characterized in that The amount of the BP nanosheet dispersion in step (2) is 1 mL, 0.2 mg / mL; Optionally, the amount of the BMP-2 solution in step (2) is 1 mL, 100 ng / mL; Optionally, the centrifugation conditions for centrifuging the BP nanosheet dispersion in step (2) are 10,000 rpm for 10 min; Optionally, the incubation condition in step (2) is room temperature and overnight incubation; Optionally, the centrifugation conditions after incubation in step (2) are 10,000 rpm and 5 min.

7. The method according to claim 2, characterized in that The LAP and tartrazine mixed solution in step (3) is prepared by dissolving LAP and tartrazine in deionized water; Optionally, the dosage of LAP is 0.2-5 mg; Optionally, the amount of tartrazine is 0.1-3 mg; Optionally, the amount of deionized water is 0.2-5 mL; Optionally, the amount of GelMA used in step (3) is 0.05-1 g; Optionally, the mass volume ratio of GelMA in step (3) is 5-15%; Optionally, the amount of TCP nanoparticles used in step (3) is 5-15 mg; Optionally, the amount of BP@BMP-2 nanosheets used in step (3) is 0.05-1 mg; Optionally, the amount of the PDGF-BB solution in step (3) is 5-15 μL.

8. The method according to claim 7, characterized in that The dosage of LAP is 1 mg; Optionally, the amount of tartrazine is 0.6 mg; Optionally, the amount of deionized water is 1 mL; Optionally, the amount of GelMA used in step (3) is 0.1 g; Optionally, the mass volume ratio of GelMA in step (3) is 10%; Optionally, the amount of TCP nanoparticles used in step (3) is 10 mg; Optionally, the amount of BP@BMP-2 nanosheets used in step (3) is 0.1 mg; Optionally, the amount of the PDGF-BB solution in step (3) is 10 μL; Optionally, the 3D bioprinter in step (3) is a digital light processing 3D bioprinter.

9. Any of the following products: (1) A 3D bioprinted radial porous scaffold for staged bone regeneration prepared by the method according to any one of claims 1 to 8; (2) A bio-ink for 3D bioprinting, the bio-ink comprising a GelMA solution, TCP nanoparticles, BP@BMP-2 nanosheets, and a PDGF-BB solution; in, The GelMA solution, TCP nanoparticles, BP@BMP-2 nanosheets, PDGF-BB solution and their amounts are as described in any one of claims 1 to 8; Optionally, the GelMA solution, TCP nanoparticles, BP@BMP-2 nanosheets, and PDGF-BB solution are mixed to obtain bio-ink.

10. Any of the following applications: (1) Use of the 3D bioprinted radial porous scaffold described in claim 9 in the preparation of bone repair and regeneration materials; (2) Use of the bio-ink described in claim 9 in the preparation of 3D bio-printed radial porous scaffolds for staged bone regeneration.

Images