A three-dimensional porous bioactive scaffold, vascularized bone organoids, their preparation methods and applications

Abstract

This invention relates to a three-dimensional porous bioactive scaffold, a vascularized bone organoid, its preparation method, and its applications, belonging to the field of tissue engineering technology. The bioactive scaffold includes a scaffold body, a Piezo1 channel agonist loaded on the scaffold body, and an Nrf2 agonist and / or Nrf2 expression vector loaded on the scaffold body; the scaffold body is a 3D-printed PMBG / β-TCP composite porous scaffold. The vascularized bone organoid uses the three-dimensional porous bioactive scaffold as a supporting framework and also includes cellular components, including co-cultured bone marrow mesenchymal stem cells and endothelial cells. Compared with existing technologies, this invention successfully constructs a novel vascularized bone organoid capable of targeting the Piezo1-Nrf2 signaling axis and possessing anti-endothelial aging and mechanical enhancement functions.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, and in particular to a three-dimensional porous bioactive scaffold, a vascularized bone organoid, its preparation method and application. Background Technology

[0002] Osteoporosis is an age-related degenerative bone disease characterized by decreased bone mass, deterioration of bone microstructure, and increased bone fragility, leading to a significantly increased risk of fractures. Recent studies have found that H-type blood vessels in bone play a crucial role in osteogenic coupling, and their density is positively correlated with bone quality. During aging, H-type blood vessels are significantly lost, accompanied by senescence and functional decline of bone endothelial cells, which is one of the important reasons for impaired bone regeneration capacity in the context of osteoporosis.

[0003] Piezo1 is a mechanosensitive ion channel that can be activated by mechanical forces (such as fluid shear stress) on the cell membrane, mediating the ion exchange of calcium ions (Ca). 2+ Piezo1 influx plays a crucial role in maintaining vascular homeostasis and bone metabolism. Studies have shown that Piezo1 activation can downstream regulate calcium... 2+ The calmodulin (CaM)-AMPK / Nrf2 signaling pathway. Nrf2 is a core transcription factor for cellular anti-oxidative stress, and its activation is closely related to delaying cellular senescence. However, how to apply the Piezo1-Nrf2 mechanotransmission axis to tissue engineering to construct functional constructs that can resist endothelial senescence and efficiently promote vascularized bone regeneration remains an unsolved technical challenge in this field.

[0004] In addition, existing bone tissue engineering strategies often focus on inducing osteoblast differentiation or simply introducing pro-angiogenic factors, neglecting the regulation of cell fate by the biomechanical microenvironment, and in particular failing to effectively address the bottleneck problem of dysfunction in senescent endothelial cells.

[0005] Therefore, developing a bone organoid that can mimic mechanical stimulation, actively reverse endothelial cell aging, and synergistically promote osteogenic and angiogenesis is of great significance for the treatment of osteoporotic bone defects in the elderly. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects of the prior art and provide a three-dimensional porous bioactive scaffold, a vascularized bone organoid, its preparation method and application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] The present invention first provides a three-dimensional porous bioactive scaffold, the bioactive scaffold comprising a scaffold body, a Piezo1 channel agonist loaded on the scaffold body, and an Nrf2 agonist and / or an Nrf2 expression vector loaded on the scaffold body;

[0009] The main body of the scaffold is a PMBG / β-TCP composite porous scaffold obtained by 3D printing.

[0010] Furthermore, the pore size of the support body is 100-200 μm, and the porosity is 60-70%.

[0011] Furthermore, the support body has a mesoporous structure with an average pore size of 8-10 nm.

[0012] This invention utilizes ultrafast photocurable PMBG as a binder for TCP (Cellular Tissue Injection), followed by photocuring 3D printing to prepare a PMBG / TCP scaffold. The 3D-printed PMBG / TCP scaffold has a large porosity, which can serve as a three-dimensional porous bioactive scaffold to support cellular components and provide them with a mechanical microenvironment. Furthermore, the PMBG / TCP scaffold also contains a mesoporous structure, making it suitable as a carrier for sustained drug release. It can effectively achieve the sequential, slow, and continuous release of Yoda1 and oe-Nrf2, thus ensuring long-lasting bioactivity.

[0013] Furthermore, the Piezo1 channel agonist is any one or more of Yoda1, Yoda2, Jedi2, Yaddle1, and Piezo1agonist 1-d2.

[0014] Furthermore, the Piezo1 channel agonist is preferably Yoda1.

[0015] Furthermore, the Nrf2 agonist is any one or more of sulforaphane and dimethyl fumarate.

[0016] Furthermore, the Nrf2 expression vector is any one or more of an adeno-associated virus vector, lentiviral vector, adenovirus vector, or plasmid DNA carrying the Nrf2 gene.

[0017] Furthermore, the Nrf2 expression vector is an adeno-associated virus vector (AAV9-Nrf2 viral vector) carrying the Nrf2 gene.

[0018] This invention also provides a method for preparing a three-dimensional porous bioactive scaffold, the method specifically comprising the following steps:

[0019] S1: Preparation of PMBG precursor sol containing SiO2, CaO, P2O5 and photosensitizing modifier;

[0020] S2: Mix PMBG precursor sol with β-TCP and photoinitiator to obtain PMBG / TCP composite photosensitive paste;

[0021] S3: Photopolymerization 3D printing was performed using PMBG / TCP composite photosensitive paste as raw material to obtain a PMBG / β-TCP composite porous scaffold.

[0022] S4: Prepare a working solution containing a Piezo1 channel agonist, an Nrf2 agonist, and / or an Nrf2 expression vector, immerse the PMBG / β-TCP composite porous scaffold in the working solution, and obtain the three-dimensional porous bioactive scaffold by critical volume saturation adsorption-freeze drying.

[0023] Further, in step S1, the molar ratio of SiO2, CaO and P2O5 in the PMBG precursor sol is (40-50):(20-30):(2-10), preferably 45:25:5.

[0024] Further, in step S1, the molar ratio of the photosensitive modifier to SiO2 is 1:(5-10).

[0025] Further, in step S1, the total solid content (based on the total mass of oxides) of SiO2, CaO, P2O5 and photosensitive modifier in the sol ranges from 5 wt% to 30 wt%, preferably from 10 wt% to 20 wt%.

[0026] Further, in step S1, the photosensitive modifier is preferably 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) to impart photocuring properties to the material.

[0027] Further, in step S2, the mass fraction of β-TCP in the PMBG / TCP composite photosensitive slurry is 5~20wt%, preferably 10wt%.

[0028] Furthermore, in step S2, the amount of photoinitiator added is 1 to 2 wt% of the total mass of the slurry.

[0029] Further, in step S2, the photoinitiator is preferably phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide.

[0030] Further, in step S2, the PMBG / TCP composite photosensitive slurry is ball-milled and degassed before use.

[0031] Furthermore, in step S3, the wavelength of the light source for the photopolymerization 3D printing is 365-420 nm, preferably 400-420 nm. The 365-460 nm band covers the ultraviolet to blue light region, which can effectively activate commonly used photoinitiators (such as BAPO), and also covers the mainstream wavelengths of SLA and DLP printers on the market.

[0032] Furthermore, in step S3, the light intensity for the photopolymerization 3D printing is 5-50 mW / cm². 2 The preferred value is 10-20 mW / cm 2 Low light intensity is beneficial for control precision, while high intensity is beneficial for increasing speed and expanding adaptability.

[0033] Furthermore, in step S3, the photocured 3D printed material is subjected to a secondary curing process for 1-30 minutes under a 365-420 nm ultraviolet light source to ensure complete cross-linking of the material and improve its mechanical strength.

[0034] Further, in step S4, the final concentration of the Piezo1 channel agonist in the working solution is 5-20 mg / mL.

[0035] Further, in step S4, the final concentration of the Nrf2 agonist is 0-20 mg / mL.

[0036] Further, in step S4, the final titer of the Nrf2 expression vector in the working solution is 1 × 10⁻⁶. 12 vg / mL ~1×10 13 vg / mL.

[0037] Further, in step S4, the specific steps of the critical volume saturated adsorption-freeze-drying are as follows:

[0038] Ensure that the working fluid completely submerges the support to achieve critical saturation of the pores;

[0039] Air is expelled from the pores through vacuum treatment, which assists in filling with the working fluid.

[0040] After sealing, incubate at 0-4 ℃ and 50-70 rpm for 24-72 h until adsorption equilibrium is reached;

[0041] After the adsorption process is complete, the scaffold is freeze-dried to obtain the three-dimensional porous bioactive scaffold.

[0042] This invention has significant technical advantages in the preparation of three-dimensional porous bioactive scaffolds.

[0043] First, this invention employs a low-temperature photocuring-freeze-drying process, avoiding high-temperature sintering throughout. It relies solely on ultraviolet light to initiate double-bond cross-linking and curing, supplemented by freeze-drying. This mild process environment (<40 ℃) not only perfectly preserves the grafted photosensitive active groups, but more importantly, it allows for the loading of thermosensitive biomolecules and small molecule drugs via physical adsorption after scaffold formation—something existing high-temperature processes cannot achieve. While traditional high-temperature calcination processes can improve ceramic strength, they lead to two fatal problems: first, all organic photosensitive groups (methacrylates) are completely decomposed, rendering the material unusable for subsequent photocuring and reprocessing; second, the high-temperature environment cannot load any bioactive drugs (such as Yoda1) or thermosensitive gene vectors (such as viruses), thus only pure inorganic scaffolds can be prepared, preventing in-situ drug delivery.

[0044] Furthermore, the synthesis and preparation of this invention does not require a pore-forming template agent. It can directly utilize the self-assembly of inorganic precursors during the sol-gel process and the pores left by ice crystal sublimation during freeze-drying to construct a hierarchical porous structure. This method simplifies the process and avoids the cytotoxicity risks associated with incomplete template agent removal.

[0045] The present invention also provides a vascularized bone organoid, which is supported by a three-dimensional porous bioactive scaffold and also includes cellular components.

[0046] Furthermore, the cellular components include co-cultured bone marrow mesenchymal stem cells and endothelial cells.

[0047] Furthermore, the endothelial cells are vascular endothelial cells, selected from one or more of human umbilical vein endothelial cells (HUVECs), human microvascular endothelial cells, human aortic endothelial cells, human peripheral blood-derived endothelial progenitor cells, or endothelial cells differentiated from induced pluripotent stem cells, preferably human umbilical vein endothelial cells (HUVECs).

[0048] Furthermore, the ratio of bone marrow mesenchymal stem cells (BMSCs) to endothelial cells is (1-2):1, preferably 2:1. This ratio is to balance the supporting role of osteoblasts in vascular cells with the competition between the two for space / nutrients. BMSCs, as stromal cells, participate in mineralization and support vascular endothelial cells. If the proportion of BMSCs is too high, it will consume too much nutrition, leading to inhibited endothelial cell growth and difficulty in forming vascularized organoids; if the proportion of HUVECs is too high, the osteogenic mineralization capacity of the organoids will be significantly reduced, failing to meet the mechanical requirements of bone repair.

[0049] This invention also provides a method for preparing vascularized bone organoids, comprising the following steps:

[0050] T1: Collect expanded bone marrow mesenchymal stem cells and endothelial cells and seed them in liquid matrix gel to obtain a cell-gel mixture;

[0051] T2: The cell-gel mixture is dropped onto a three-dimensional porous bioactive scaffold, allowing the cell-gel mixture to naturally penetrate and fill the porous network inside the scaffold;

[0052] T3: The matrix gel is thermally crosslinked and cured in situ on a three-dimensional porous bioactive scaffold, and the vascularized bone organoid is obtained by culturing it in a composite induction medium.

[0053] Furthermore, in step T1, the concentration of the bone marrow mesenchymal stem cells is (1-2)×10⁻⁶. 6 cells / mL.

[0054] Furthermore, in step T1, the concentration of the liquid matrix adhesive is 6-10 mg / mL.

[0055] Furthermore, the matrix adhesive is Matrigel.

[0056] Furthermore, in step T3, the thermal crosslinking curing time is 30-45 minutes.

[0057] Furthermore, in step T3, the composite induction medium is composed of osteogenic induction medium and endothelial cell medium mixed in a 1:1 volume ratio.

[0058] Furthermore, in step T3, the fresh composite culture medium is replaced every 24-48 hours, and the culture period can be up to 6 weeks. During precursor formation, BMSCs differentiate into osteogenic lineages under the induction of osteogenic factors, while HUVECs self-assemble to form microvascular networks under the influence of EGM-2 factor and the microenvironment.

[0059] The present invention also provides the use of a three-dimensional porous bioactive scaffold and / or vascularized bone organoid in the preparation of drugs, biological products or devices for repairing bone defects caused by osteoporosis in the elderly.

[0060] Compared with the prior art, the present invention has the following technical advantages:

[0061] (1) This invention innovatively applies the biological mechanism of targeting the Piezo1-Nrf2 mechanical signal axis to alleviate endothelial cell aging to the construction of vascularized bone organoids, thereby fundamentally improving the microenvironment for bone regeneration in aging organisms.

[0062] (2) The three-dimensional porous bioactive scaffold of the present invention innovatively loads Piezo1 channel agonist and Nrf2 expression vector on the scaffold, and through the synergistic effect of the two, achieves the organic unity of three major functions: anti-endothelial aging, angiogenesis and enhanced osteogenic differentiation, thus solving the problem of single function of traditional bone tissue engineering.

[0063] (3) The present invention innovatively uses 3D printed PMBG / TCP scaffold as the main body of drug-loaded scaffold and the main support body of vascularized bone organoids. It can not only provide suitable mechanical support and degradation performance, but its mesoporous structure can also serve as a drug sustained-release carrier, successfully realizing the sequential, slow and continuous release of Piezo1 channel agonist and Nrf2 expression carrier, ensuring long-term biological activity.

[0064] (4) Based on the preparation of PMBG / TCP scaffolds and the loading of bone marrow mesenchymal stem cells and endothelial cells, this invention successfully constructed a novel vascularized bone organoid capable of simulating mechanical stimulation, actively reversing endothelial cell senescence, and synergistically promoting osteogenic and angiogenesis. In vitro experiments confirmed that this construct can significantly prolong organoid survival time, enhance osteogenic gene expression, and promote endothelial tube formation. In vivo animal experiments further demonstrated that this vascularized bone organoid can effectively promote H-type angiogenesis and new bone formation in an aged rat skull defect model, with a repair effect significantly better than that of the drug-free scaffold. It can be used to prepare regenerative medicine products such as drugs or biological products for repairing bone defects caused by senile osteoporosis, which is of great significance for the clinical treatment of osteoporotic bone defects in the elderly. Attached Figure Description

[0065] Figure 1 This is a graph showing the results of the angiogenesis capacity test in this invention.

[0066] Figure 2 This is a diagram showing the expression results of the P53 protein in this invention.

[0067] Figure 3 This is a diagram showing the expression results of the CAM protein in this invention.

[0068] Figure 4 This is a diagram showing the expression results of the CAM protein in this invention.

[0069] Figure 5 This image shows the in vitro survival time of the vascularized bone organoid precursor of this invention. A and B are microscopic photographs taken before and after staining, respectively.

[0070] Figure 6 The results show the expression of Col IA and Runx2 osteogenic genes and the localization and expression level of P53 protein in the vascularized bone organoid precursor of this invention.

[0071] Figure 7Macroscopic morphology images of the PMBG stent (left) prepared in Comparative Example 1 and the drug-loaded PMBG@Yoda1+oeNrf2 composite stent (right) prepared in Example 1.

[0072] Figure 8 The results of energy dispersive X-ray spectroscopy (EDS) analysis and Fourier transform infrared spectroscopy (FTIR) analysis of the PMBG / TCP composite scaffold are shown.

[0073] Figure 9 The mechanical properties of the PMBG / TCP stent and the drug-eluting composite stent (PMBG@Yoda1+oeNrf2) are shown in the figure.

[0074] Figure 10 The in vitro degradation curves of the PMBG / TCP scaffold and the drug-eluting composite scaffold (PMBG@Yoda1+oeNrf2) in simulated body fluids are shown.

[0075] Figure 11 For PMBG / TCP stents and drug-eluting composite stents (PMBG@Yoda1+oeNrf2), calcium ions (Ca) 2+ ) and silicate ions (SiO4) 4- Release curve.

[0076] Figure 12 The in vitro release curves of Yoda1 (A) and oe-Nrf2 (B) are shown.

[0077] Figure 13 Cell viability diagram of the PMBG@Yoda1+oeNrf2 vascularized organoid constructed in this invention.

[0078] Figure 14 The results show the ALP activity of the PMBG@Yoda1+oeNrf2 vascularized organoids constructed in this invention, as well as the Control and PMBG groups.

[0079] Figure 15 The expression levels of Runx2 and Col IA genes in the PMBG@Yoda1+oeNrf2 vascularized organoid constructed in this invention.

[0080] Figure 16 The figure shows the angiogenesis test results of the PMBG@Yoda1+oeNrf2 vascularized organoid constructed in this invention.

[0081] Figure 17 Micro-CT images of different scaffolds / organoids implanted in animal testing.

[0082] Figure 18Histological staining (H&E and Masson) results after implantation of different scaffolds / organoids in animal testing.

[0083] Figure 19 The results are shown for immunofluorescence in animal testing. Detailed Implementation

[0084] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0085] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0086] The Piezo1 channel agonist used in the following embodiments of the present invention is Yoda1, with a purity of 99.97% and MCE.

[0087] The Nrf2 expression vector used in the following embodiments of the present invention is AAV9-Nrf2 adeno-associated virus solution with a titer of 1×10⁻⁶. 13 vg / mL, purchased from Shanghai Jikai Gene.

[0088] I. Verification of the role of targeting the Piezo1-Nrf2 axis in delaying endothelial cell senescence

[0089] This invention innovatively applies the biological mechanism of "targeting the Piezo1-Nrf2 biomechanical signaling axis to alleviate endothelial cell senescence" to the construction of vascularized bone organoids, fundamentally improving the microenvironment for bone regeneration in aging organisms.

[0090] To verify the role of targeting the Piezo1-Nrf2 axis in delaying endothelial cell senescence, this invention first used human umbilical vein endothelial cells (HUVECs, purchased from the Chinese Academy of Sciences Cell Bank, P2-P4 generations) for experiments, and set up a control group, a laminar shear stress (LSS) treatment group, an LSS + Piezo1 inhibitor GsMTx4 group, and a Piezo1 agonist Yoda1 treatment group.

[0091] In the control group, HUVECs were cultured in T25 cell culture flasks using complete endothelial cell culture medium (EGM-2) in a conventional static culture incubator at 37 ℃ and 5% CO2. No fluid shear force or exogenous drug stimulation was applied during the culture process; only routine medium changes were performed.

[0092] In the laminar flow shear stress treatment group, HUVECs were seeded on fluidized bed slides coated with fibronectin (10 μg / mL). Once the cell confluence reached over 90% and a dense monolayer was formed, the cells were connected to a parallel plate flow chamber system (purchased from Leimeng Biotechnology). The peristaltic pump flow rate was adjusted to apply a constant-direction, stable-magnitude laminar shear stress to the cells. The shear stress intensity was set to 12 dyne / cm². 2 The action time was set to 4 hours, and the system was run in a 37 ℃ constant temperature chamber.

[0093] The LSS+Piezo1 inhibitor GsMTx4 group was largely the same as group 2, but the cells were pretreated with the drug before LSS loading. The cell culture medium was replaced with fresh EGM-2 medium containing the Piezo1-specific inhibitor GsMTx4 (5 μM, purchased from Abcam) for 30 minutes.

[0094] After pretreatment, maintain the drug-containing culture medium environment at this concentration, and follow the same parameters as described in Group 2 (12 dyne / cm). 2 Laminar shear stress was applied to the cells. This group aimed to verify the changes in the response of endothelial cells to mechanical signals after blocking the Piezo1 channel.

[0095] The Piezo1 agonist Yoda1 treatment group used HUVECs cultured in T25 cell culture flasks. The culture medium was replaced with fresh EGM-2 medium containing the Piezo1 chemoagonist Yoda1 (10 μM, purchased from Sigma-Aldrich) for 24 hours. This group was used to verify whether specific chemical activation could mimic the biological effects induced by LSS.

[0096] Subsequently, angiogenesis capacity was tested using a Tube Formation assay, and Ca 2+ Intracellular calcium ion concentration was detected using fluorescent probes, and the protein expression levels of CaM, p-AMPK, Nrf2, and P53 were detected by Western blotting.

[0097] The specific method for detecting angiogenesis ability using the Tube Formation assay is as follows:

[0098] (1) Corning gel plating: Pre-cool the 96-well cell culture plate on ice. Add 50 μL of thawed Corning gel to each well and gently shake to spread the gel evenly at the bottom of the well. Then incubate the culture plate in a 37 ℃ cell culture incubator for 30 minutes.

[0099] (2) Cell seeding: HUVECs treated under different conditions (LSS shear force, Yoda1 drug, etc.) were digested and counted. The cell density was adjusted by adding 1×10⁶ cells to each well. 5 Each cell.

[0100] (3) Tube culture and observation: The inoculated culture plates were placed in an incubator at 37 ℃ and 5% CO2. After 6 hours of culture, the cell morphology was observed and images were taken using an inverted phase contrast microscope.

[0101] like Figure 1-4 As shown, compared with the control group, both LSS and Yoda1 treatments significantly enhanced the tube-forming ability of HUVECs and promoted Ca2+ formation. 2+ Influx of Piezo1 upregulated the expression of CaM, p-AMPK, and Nrf2 proteins, while downregulating the expression of the key aging protein P53; and GsMTx4 reversed the effects of LSS. These results successfully demonstrate that activation of the Piezo1-Nrf2 axis can effectively delay endothelial cell senescence.

[0102] II. Construction and Functional Assessment of Vascularized Bone Organoid Precursors

[0103] To preliminarily assess whether the construction targeting the Piezo1-Nrf2 axis has high osteogenic potential, this invention further constructed a vascularized bone organoid precursor using a microfluidic chip and tested its osteogenic properties.

[0104] The method for constructing the vascularized bone organoid precursor of the present invention specifically includes the following steps:

[0105] 1. Cell preparation and mixing with matrix gel:

[0106] (1) Human bone marrow mesenchymal stem cells (hBMSCs, purchased from Shangen Biotechnology, P2-P4 generation) and human umbilical vein endothelial cells (HUVECs, purchased from the Cell Bank of the Chinese Academy of Sciences, P2-P4 generation) were resuscitated and expanded. They were counted after digestion.

[0107] (2) Prepare Matrigel (Corning, catalog number 356230) for basement membrane under ice bath conditions (0-4 ℃) and adjust Matrigel to a working concentration of 8 mg / mL.

[0108] (3) Resuspend hBMSCs and HUVECs in the above-mentioned cold Matrigel at a ratio of 2:1. The final density of hBMSCs was 2 × 10⁻⁶. 6 cells / mL, final density of HUVECs was 1×10⁻⁶ 6cells / mL. The operation must be rapid and always performed on ice to prevent Matrigel from solidifying prematurely, resulting in a cell-gel mixture.

[0109] 2. Microfluidic chip seeding and cross-linking:

[0110] (1) Selection and parameters of microfluidic chip: A commercial microfluidic cell culture chip based on polydimethylsiloxane (PDMS) material (purchased from AIM BIOTECH) was selected. The chip design parameters are as follows: the main channel width is 1000-1500 μm (for accommodating gel); the gap between micropillars and microchannels is 10-20 μm (for confining the gel but allowing nutrients and factors to diffuse instantly); and the height is 100-250 μm.

[0111] (2) Inoculation: Using a pre-cooled micropipette, aspirate the cell-gel mixture prepared in step 1 and carefully inject it into the central gel channel of the microfluidic chip. The injection volume per chip channel is approximately 10-20 μL (depending on the chip model).

[0112] (3) Polymerization and solidification: After injection, the chip is immediately placed in a 37 ℃, 5% CO2 cell culture incubator and incubated for 30 minutes. Under body temperature conditions, Matrigel will undergo thermosensitive cross-linking, changing from a liquid state to a solid hydrogel scaffold, thereby encapsulating and fixing cells to form vascularized bone organoid precursors.

[0113] 3. Co-culture system and culture medium formulation:

[0114] After the gel has completely solidified, add a special composite induction medium for vascularized bone precursors to the media channels on both sides of the chip. This medium is a 1:1 volume mixture of osteogenic induction medium and endothelial cell culture medium.

[0115] (1) Component A (osteogenic inducing agent): α-MEM basal medium + 10% fetal bovine serum (FBS) + 100 nM dexamethasone + 10 mM sodium β-glycerophosphate + 50 μM ascorbic acid.

[0116] (2) Component B (endothelial maintenance medium): EGM-2 Endothelial Growth Medium (purchased from Lonza), containing growth factor additives such as VEGF, hFGF-B, and R3-IGF-1.

[0117] (3) Medium replacement protocol: Fresh composite medium should be replaced every 24-48 hours, and the culture period can be up to 6 weeks. During precursor formation, BMSCs differentiate into osteogenic lineages under the induction of osteogenic factors, and HUVECs self-assemble to form microvascular networks under the action of EGM-2 factor and microenvironment.

[0118] Performance testing of the vascularized bone organoid precursors was conducted in three groups: a control group, a Yoda1-treated group, and a Yoda1 + Nrf2 agonist group. Following the aforementioned method, vascularized bone organoid precursors consisting of a mixture of BMSCs and HUVECs were prepared, wherein:

[0119] The control group received no additional treatment;

[0120] The Yoda1 treatment group had an additional 2.5 μM of Yoda1 (dissolved in DMSO, final DMSO concentration <0.1%) added to the aforementioned composite culture medium (osteogenic inducing medium + endothelial maintenance medium) to continuously act on the culture process;

[0121] In the Yoda1+Nrf2 agonist group, on day 2 after cells were mixed with Matrigel, lentiviral particles carrying the human Nrf2 gene (purchased from Shanghai Jikai Gene) were added to the composite medium. The multiplicity of infection was set to 20, and 5 μg / mL Polybrene was added to improve infection efficiency. The medium was switched back to standard composite medium 24 hours after transfection.

[0122] The survival time of organoid precursors was observed by live / dead staining, the expression of osteogenic genes (Col IA, Runx2) was detected by RT-PCR, and the localization and expression level of P53 protein were detected by immunofluorescence.

[0123] like Figure 5 As shown, in the Yoda1+Nrf2 agonist group, the in vitro survival time of bone organoid precursors can exceed 42 days.

[0124] like Figure 6 As shown, the mRNA expression levels of Col IA and Runx2 were significantly upregulated, while the protein expression of P53 was significantly downregulated. This indicates that targeting the Piezo1-Nrf2 axis successfully constructed a vascularized bone organoid precursor with long-term survival capability and high osteogenic potential.

[0125] III. Construction and characterization of three-dimensional porous bioactive scaffolds.

[0126] In order to achieve effective support for cellular components and provide them with a mechanical microenvironment in vascularized bone organoids, this invention innovatively designs a three-dimensional porous bioactive scaffold.

[0127] Example 1:

[0128] This embodiment provides a three-dimensional porous bioactive scaffold, denoted as the PMBG@Yoda1+oeNrf2 composite scaffold. The specific preparation method is as follows:

[0129] (1) Fabrication of PMBG / TCP scaffolds using a photopolymerization 3D printer:

[0130] A. Raw material preparation and synthesis of PMBG precursor sol:

[0131] First, raw materials were prepared according to a molar ratio of SiO2: CaO: P2O5 = 45: 25: 5. The silicon source was tetraethyl orthosilicate (TEOS, 98.0% purity, Sigma-Aldrich), the calcium source was calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, 99.0% purity, Sigma-Aldrich), and the phosphorus source was triethyl phosphate (TEP, 99.0% purity, Aladdin reagent). The photosensitizing agent was propyl 3-(trimethoxysilyl)methacrylate (TMSPMA, 97% purity, Sigma-Aldrich), used to impart photocuring properties to the material.

[0132] 0.045 mol of TEOS and 0.010 mol of TEP were dissolved in 60 mL of anhydrous ethanol and magnetically stirred for 30 minutes at room temperature. Then, deionized water containing 0.5 M HCl (catalyst) was added, and hydrolysis was continued with stirring for 1 hour. Next, 0.025 mol of calcium nitrate tetrahydrate was added and stirred until completely dissolved. To introduce photocurable groups, 0.005 mol of TMSPMA was slowly added dropwise to the above mixed sol, and the mixture was refluxed and stirred at 60 °C in the dark for 6 hours to obtain a photocurable mesoporous bioactive glass (PMBG) precursor sol.

[0133] B. Formulation of PMBG / TCP composite photosensitive paste:

[0134] Weigh out β-TCP sintered powder (Ca3(PO4)2 purity ≥ 98%, Sigma-Aldrich). Mix 90 wt% of the above PMBG precursor sol with 10 wt% of β-TCP powder by mass percentage.

[0135] To initiate the photopolymerization reaction, a photoinitiator, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO / Irgacure 819), was added to the mixture at an amount of approximately 1.5 wt% of the total slurry mass.

[0136] The above mixture was placed in a planetary ball mill and ball-milled at 300 rpm for 2 hours. Then, it was placed in a vacuum drying oven in the dark for 30 minutes to remove bubbles, resulting in a uniform and stable PMBG / TCP composite photosensitive slurry.

[0137] C.DLP 3D printing molding:

[0138] The prepared composite photosensitive slurry was poured into the resin tank of a 3D printer (model: Pico2, Asiga, Australia) based on Digital Light Processing (DLP) technology. The designed cylindrical porous support model (e.g., 5 mm diameter, 2 mm height, pore size 100-200 μm, porosity 60-70%) was then imported, and the printing parameters were set as follows:

[0139] Light source wavelength: 405 nm UV LED;

[0140] Light intensity: 10-20 mW / cm 2 ;

[0141] Single layer thickness: 50 μm;

[0142] Single-layer exposure time: The bottom layer exposure time is 15 s, and the exposure time for the remaining layers is 2.5 - 4.0 s;

[0143] Z-axis lifting speed: 5 mm / min.

[0144] D. Post-processing:

[0145] After printing, the scaffold was removed from the printing platform and ultrasonically cleaned three times, each time for 5 minutes, with anhydrous ethanol and deionized water to remove any uncured residual paste from the pores. Subsequently, the scaffold was placed in a full-function UV curing chamber (wavelength 400 nm, power 50 W, Grayhill (Shenzhen) Technology Co., Ltd.) for secondary curing for 10 minutes to ensure complete cross-linking of the material and improve its mechanical strength. Finally, the scaffold was freeze-dried for 24 hours to obtain the final PMBG / TCP composite porous scaffold.

[0146] (2) Yoda1 and oe-Nrf2 were loaded into the scaffold by critical saturation adsorption-freeze drying method to obtain PMBG@Yoda1+oeNrf2 scaffold:

[0147] A. Preparation of the compound drug working solution:

[0148] First, prepare a stock solution of Piezo1 channel agonist (Yoda1): Weigh an appropriate amount of Yoda1 powder (purity: 99.97%, MCE) and dissolve it in dimethyl sulfoxide to prepare a high-concentration stock solution. Then, in a sterile laminar flow hood, use sterile phosphate-buffered saline (PBS, pH=7.4) as a diluent. Mix the above Yoda1 stock solution with Nrf2 expression vector (AAV9-Nrf2 adeno-associated virus solution, titer 1×10⁻⁶). 13 (vg / mL, purchased from Shanghai Jikai Gene) was mixed and diluted proportionally. The final concentrations of each component in the mixed solution are as follows: the final concentration of Yoda1 is 10 mg / mL, and the final titer of AAV9-Nrf2 viral vector is 1×10⁻⁶.12 vg / mL. After mixing, gently pipette to mix thoroughly, and store temporarily in a 4 ℃ refrigerator until use.

[0149] B. Critical saturation adsorption process:

[0150] Place the PMBG / TCP scaffold prepared in step (1) and sterilized by UV into a sterile 12-well cell culture plate (one scaffold per well). Add sufficient amount of the above-mentioned composite drug working solution to each well, and the amount added should completely submerge the scaffold (for example, add 1 mL to each scaffold, the specific volume is calculated according to the porosity of the scaffold, to reach the critical saturation state of the pores).

[0151] To ensure that the Yoda1 and Nrf2 expression vectors could penetrate deeply into the mesoporous channels of the PMBG scaffold and the macroscopic pores of the scaffold, the culture plate after sample loading was placed in a vacuum drying oven and subjected to three short-term vacuum treatments (30 seconds each, vacuum degree -0.08 MPa) to expel air from the pores and assist in drug filling. Subsequently, the culture plate was sealed (to prevent solvent evaporation) and placed on a temperature-controlled horizontal shaker.

[0152] Adsorption conditions: Temperature controlled at 4 ℃, shaker speed at 60 rpm, and continuous incubation for 48 hours. During this process, Yoda1 molecules and AAV9-Nrf2 viral vector are enriched on the surface of the scaffold material and inside the mesopores through physical adsorption and diffusion, reaching adsorption equilibrium.

[0153] C. Freeze-drying:

[0154] After adsorption, the scaffold was removed from the drug solution and gently held with sterile forceps to remove excess liquid from the surface. The drug-loaded scaffold was immediately placed in an ultra-low temperature freezer at -80 ℃ for 4 hours to rapidly freeze the drug solution within the pores into ice crystals, thus fixing the drug distribution. The pre-frozen scaffold was then transferred to a vacuum freeze dryer (model: SCIENTZ-10N / A, Ningbo). The freeze-drying parameters were set as follows: cold trap temperature -50 ℃ to -60 ℃, vacuum degree <10 Pa, drying time 48 hours. The final product was a PMBG@Yoda1+oeNrf2 composite scaffold loaded with Piezo1 agonist and Nrf2 expression vector. This drying process maintained the biological activity of the drug and virus and ensured their stable attachment within the scaffold's micro / nano structure.

[0155] Comparative Example 1:

[0156] This comparative example provides a drug-free three-dimensional porous scaffold, which differs from Example 1 in that it is fabricated solely by 3D printing and does not load Yoda1 and oe-Nrf2, denoted as PMBG / TCP.

[0157] Figure 7The images show the macroscopic morphology of the PMBG scaffold (left) prepared in Comparative Example 1 and the drug-loaded PMBG@Yoda1+oeNrf2 composite scaffold (right) prepared in Example 1. As can be seen from the images, both scaffolds exhibit a regular cylindrical shape with a diameter of approximately 5 mm, maintaining structural integrity without significant shrinkage, deformation, or collapse. This indicates that photopolymerization 3D printing technology possesses good structural integrity and printing precision, accurately reproducing the design model, and that the material has good molding ability. The scaffold surface formed by 3D printing exhibits a clear and uniform mesh structure, with regular large square pores formed through orthogonal stacking (0° / 90°). From a macroscopic visual perspective, the pores show good connectivity; this interconnected porous network facilitates subsequent cell migration, nutrient delivery, and blood vessel ingrowth. Furthermore, comparing the blank PMBG stent on the left with the PMBG@Yoda1+oeNrf2 composite stent on the right, it can be seen that after loading Yoda1 and Nrf2 viral vectors and undergoing freeze-drying, the macroscopic framework structure of the stent remained unchanged, and the pores remained open, without any pore blockage caused by excessive drug coating. This indicates that the critical saturation adsorption-freeze-drying process is mild and controllable, and does not damage the original physical structure of the stent.

[0158] Figure 8 The chemical composition and structure of the PMBG / TCP composite scaffold are characterized. Figure A shows the results of energy-dispersive X-ray spectroscopy (EDS) analysis, and Figure B shows the Fourier transform infrared spectroscopy (FTIR) analysis.

[0159] like Figure 8 As shown in Figure A, the elemental composition analysis of the scaffold material revealed that the sample mainly contained four elements: oxygen (O), silicon (Si), calcium (Ca), and phosphorus (P). The red area in the figure represents oxygen, which constitutes the largest proportion, consistent with the characteristics of oxide ceramic materials (SiO2, CaO, P2O5). The blue, yellow, and cyan areas represent silicon, calcium, and phosphorus, respectively. The detection and abundance distribution of the three key elements, Si, Ca, and P, directly confirm that bioactive glass (mainly contributing Si, Ca, and P) and β-tricalcium phosphate (mainly contributing Ca and P) were successfully composited in the scaffold matrix using the sol-gel method and physical mixing process. The "Others" shown in the figure are mainly attributed to residual carbon (C) and other organic components in the photocurable resin component. The EDS results strongly demonstrate the consistency between the scaffold's bulk chemical composition and the designed formulation.

[0160] like Figure 8 As shown in Figure B, the infrared spectrum of the PMBG / TCP scaffold exhibits characteristic absorption peaks in the fingerprint region, further verifying the chemical bonding structure of the material. (1000-1100 cm⁻¹) -1A broad and strong absorption band is observed in the vicinity: the most significant absorption peak is observed within this wavenumber range, primarily attributable to the asymmetric stretching vibrations of the Si-O-Si bonds in the silicate network, superimposed with the stretching vibrations of the PO bonds. This is a hallmark feature of the formation of bioactive glass framework structures. (500-600 cm⁻¹) -1 Nearby absorption peaks: Characteristic absorption peaks appearing in the low wavenumber region correspond to the bending vibrations of the PO bonds in the phosphate groups and the bending vibrations of Si-O-Si. This confirms the stable presence of phosphorus in the β-TCP crystal phase and glass network. Furthermore, no obvious impurity peaks appeared in the spectrum, indicating that the composite material reacted fully during preparation and photocuring, forming the expected inorganic-organic composite network structure.

[0161] Figure 9 The mechanical properties of the PMBG / TCP stent and the drug-eluting composite stent (PMBG@Yoda1+oeNrf2) are shown in the figure. Figure 9 China A and Figure 9 The compressive modulus and ultimate compressive strength of the blank PMBG / TCP scaffold and the drug-loaded PMBG@Yoda1+oeNrf2 composite scaffold were compared. The compressive modulus of the blank PMBG / TCP scaffold was approximately 13.5 ± 1.2 MPa, while that of the drug-loaded PMBG@Yoda1+oeNrf2 scaffold increased to approximately 14.8 ± 1.5 MPa, indicating a slight improvement in the resistance to elastic deformation of the drug-loaded composite scaffold. The ultimate compressive strength of the blank PMBG / TCP scaffold was approximately 7.2 ± 0.8 MPa, while that of the drug-loaded scaffold was approximately 8.2 ± 0.6 MPa. This data indicates that the drug-loaded composite scaffold has an enhanced ability to withstand maximum destructive loads while still meeting the mechanical requirements for cancellous bone repair. During the photocuring and subsequent solvent removal processes, the organic-inorganic hybrid network of the original PMBG / TCP scaffold may contain microscopic defects such as fine pores or uneven cross-linking density. During the "critical saturation adsorption-freeze-drying" process, the solution containing the Yoda1 small molecule and AAV9 viral vector protein penetrates deeply into these microscopic defects. After freeze-drying, the remaining solid drug molecules and biomacromolecules partially fill the microcracks at the microscale, reducing stress concentration points under stress.

[0162] Figure 10 The in vitro degradation curves of the PMBG / TCP scaffold and the drug-eluting composite scaffold (PMBG@Yoda1+oeNrf2) in simulated body fluid are shown. The weight loss of both scaffolds showed a continuous increasing trend with prolonged immersion time. During the 28-day test period, the degradation curves showed a roughly linear increase, without any instantaneous material disintegration or degradation stagnation. This indicates that the scaffold material has good chemical stability in the body fluid environment, and the degradation process is controllable.

[0163] Figure 11 In Figure A, calcium ions (Ca) in the PMBG / TCP stent and drug-eluting composite stent (PMBG@Yoda1+oeNrf2) are present. 2+ Release curve, Figure 11 B represents the silicate ions (SiO4) in the PMBG / TCP stent and drug-eluting composite stent (PMBG@Yoda1+oeNrf2). 4- Release curve.

[0164] Both sets of scaffolds exhibited a trend of rapid initial release followed by a decrease and eventual stabilization. During the initial immersion phase, the non-bridging oxygen bonds on the scaffold surface rapidly hydrolyzed, leading to the release of calcium ions. 2+ Rapidly released into the solution. On day 1, the Ca of both sets of scaffolds... 2+ The concentrations all reached their peak values, approximately 60 μg / mL. Subsequently, the Ca in the solution... 2+ The concentration of [agent / material] showed a significant decreasing trend. This does not mean that release has stopped, but rather marks the beginning of the biomineralization process. With the formation of the silica layer, the Ca in the solution [deteriorates / increases / increases]. 2+ Reacting with phosphate ions, the hydroxyapatite layer is redeposited on the scaffold surface, consuming calcium ions in the solution. After day 7, the release and consumption reach a dynamic equilibrium, with the concentration stabilizing between 15-25 μg / mL. The curve trends of the drug-loaded composite scaffold and the blank scaffold are basically consistent, indicating that the loading of drugs and viral vectors did not hinder the normal release of calcium ions and subsequent surface mineralization deposition.

[0165] The silicon ion release from both scaffolds exhibited a sustained, slow increase and long-lasting release characteristic. As the PMBG glass network framework gradually degraded, silicon was released primarily as SiO4. 4- The silicon ion concentration was continuously released into the medium. It gradually increased from approximately 10 μg / mL on day 1. By day 14, the silicon ion concentration reached a plateau, remaining at around 20-25 μg / mL. The silicon release rate of the drug-loaded composite scaffold was slightly lower than that of the blank scaffold. This may be because the Yoda1 molecules and viral proteins loaded on the pore surface formed a physical barrier, which to some extent slowed down the contact between the glass matrix and water and the hydrolysis rate. This sustained-release effect is beneficial in application, as it can avoid the potential cytotoxicity caused by excessively high local silicon ion concentrations.

[0166] This invention further employs an in vitro extraction method to determine the in vitro drug release performance of Yoda1 and oeNrf2 in the drug-loaded composite scaffold. The specific operational steps of the in vitro extraction release experiment are as follows:

[0167] (1) Take the prepared PMBG@Yoda1+oeNrf2 cylindrical support sample (n=3) and weigh it accurately.

[0168] (2) Immerse the scaffold into 2 mL centrifuge tubes and add 1.0 mL of phosphate buffer (PBS, pH 7.4) to each tube as the release medium.

[0169] (3) Place the centrifuge tube with the support in a 37 ℃ constant temperature shaker and shake it continuously at 100 rpm to simulate the body fluid circulation environment.

[0170] (4) At the preset time points (days 1, 2, 3, 5, 7, 14, 21, 28, and 42), aspirate all 1.0 mL of the release solution for post-testing, and immediately add 1.0 mL of fresh preheated PBS buffer to continue incubation.

[0171] Detection methods and concentration conversion:

[0172] A. Detection of Piezo1 agonist (Yoda1) release - UV spectrophotometry / HPLC:

[0173] The concentration of Yoda1 in the release solution was detected using high performance liquid chromatography (HPLC) or ultraviolet-visible spectrophotometer.

[0174] Standard curve plotting: Prepare a series of Yoda1 standard solutions with known concentrations (e.g., 0.5~50 μg / mL), measure the absorbance at characteristic absorption wavelengths (e.g., 210 nm or 280 nm), and plot the concentration-absorbance standard curve.

[0175] Sample Measurement and Conversion: Measure the absorbance of the released liquid at each time point, and substitute it into the standard curve equation to calculate the concentration (C). n (mg / mL).

[0176] Cumulative release rate calculation: Cumulative release rate (%) = ×100%.

[0177] in, c i Let i be the concentration measured in the i-th sample. v 0 The release medium volume is 1 mL. M total Yoda1 represents the total mass of the actual load in the support.

[0178] B. Detection of Nrf2 expression vector release - Micro-BCA protein quantification method:

[0179] Viral capsid protein was used as a detection marker, and the concentration was measured using a Micro-BCA protein assay kit. 150 μL of the release buffer was added to a 96-well plate, followed by an equal volume of BCA working solution. After incubation at 37 °C for 2 hours, the absorbance was measured at 562 nm using a microplate reader. The viral protein concentration was calculated based on the BSA standard curve, and the cumulative release percentage was further calculated using the formula described above.

[0180] Figure 12 China A and Figure 12 Figure B shows the in vitro release curves of Yoda1 and oe-Nrf2, respectively. As shown in the figure, the release curves of the two active ingredients both exhibit typical "biphasic release" characteristics: in the first 7 days, both Yoda1 and oe-Nrf2 show relatively fast release rates. Figure 12 According to data from China A, the cumulative release of Yoda1 reached approximately 55% by day 7. Figure 12 The results from the B-mode imaging showed that the cumulative release of oe-Nrf2 reached approximately 60% by day 7. This rapid release was primarily attributed to drug molecules adsorbed on the outer surface of the scaffold and the superficial layers of the macropore walls. These molecules could quickly contact and diffuse with the medium, facilitating the rapid achievement of effective therapeutic concentrations in the early stages of implantation and initiating osteogenic and antioxidant signaling pathways. After day 7, the slopes of the two curves noticeably flattened, entering a sustained and stable plateau release phase. By day 42, the cumulative release rate of Yoda1 was approximately 80%; by day 28, the cumulative release rate of oe-Nrf2 was approximately 85%. The drug in this phase mainly originated from the portion deeply adsorbed within the mesopores of the scaffold. The confinement effect of the mesopores increased molecular diffusion resistance, achieving long-lasting sustained release. The release cycle covered the critical window of bone repair (>4 weeks), without any explosive releases that could lead to cytotoxicity, meeting the design requirements of a bone tissue engineering scaffold drug delivery system.

[0181] The above verification demonstrates that this invention successfully fabricated a three-dimensional porous bioactive scaffold (PMBG@Yoda1+oeNrf2 scaffold) with a PMBG / TCP composite scaffold as the main body and loaded with Yoda1 and oe-Nrf2. This three-dimensional porous bioactive scaffold not only provides suitable mechanical support and degradation performance, but its mesoporous structure also serves as a drug sustained-release carrier, enabling the sequential, slow, and continuous release of Piezo1 channel agonists (such as Yoda1) and Nrf2 expression carriers (oe-Nrf2), ensuring long-term bioactivity and meeting the design requirements of bone tissue engineering scaffold drug delivery systems.

[0182] IV. Construction and Functional Assessment of Vascularized Bone Organoids

[0183] Based on the successful construction of the Piezo1-Nrf2 axis and the successful preparation of the PMBG / TCP three-dimensional porous bioactive scaffold, this invention further constructs a vascularized bone organoid.

[0184] This vascularized osteoid organoid specifically includes:

[0185] Cellular components: Contains co-cultured bone marrow mesenchymal stem cells (BMSCs) and endothelial cells, preferably human umbilical vein endothelial cells (HUVECs), which form a vascular network structure in a three-dimensional environment.

[0186] Scaffold components: PMBG / TCP three-dimensional porous bioactive scaffold, used to support cellular components and provide them with a mechanical microenvironment.

[0187] Active factors: The PMBG / TCP three-dimensional porous bioactive scaffold is loaded with a Piezo1 channel agonist and an Nrf2 expression vector. The Piezo1 channel agonist is preferably Yoda1; the Nrf2 expression vector is a plasmid or viral vector that overexpresses Nrf2, preferably an adeno-associated virus vector (oe-Nrf2) carrying the Nrf2 gene.

[0188] Example 2:

[0189] This embodiment provides a vascularized bone organoid, the specific preparation method of which is as follows:

[0190] 1. Pretreatment of drug-eluting stents:

[0191] The PMBG@Yoda1+oeNrf2 composite scaffold (cylindrical, 5 mm in diameter, 1 mm in height) prepared in Example 1 and freeze-dried was sterilized with ultraviolet light in a clean bench for 48 hours.

[0192] Before co-culture begins, place the scaffold in a sterile culture dish and pre-wet it for 30 minutes with a small amount of basal medium (α-MEM, serum-free) to remove any residual air bubbles in the scaffold micropores and prepare for cell adhesion.

[0193] 2. Co-seeding of co-cultured cells and scaffolds:

[0194] (1) Preparation of cell-gel mixture: Human bone marrow mesenchymal stem cells (hBMSCs, purchased from Shangen Biotechnology, P2-P4 generation) and human umbilical vein endothelial cells (HUVECs, purchased from the Cell Bank of Chinese Academy of Sciences, P2-P4 generation) were resuscitated and expanded. They were counted separately after digestion.

[0195] (2) Inoculation procedure: Prepare Matrigel (Corning, catalog number 356230) under ice bath conditions (0-4 ℃), and adjust the Matrigel to a working concentration of 8 mg / mL. Resuspend hBMSCs and HUVECs in the above-mentioned ice-cold Matrigel at a quantitative ratio of 2:1. The final density of hBMSCs was 2 × 10⁻⁶. 6 cells / mL, final density of HUVECs was 1×10⁻⁶ 6 cells / mL. The procedure must be performed quickly and always on ice, ultimately yielding a cell-gel mixture.

[0196] (3) In situ perfusion / colonization: Using a micropipette, the above-mentioned gel mixture containing two types of cells was slowly dripped onto the top and side pores of the pre-moistened PMBG@Yoda1+oeNrf2 scaffold. The capillary force and gravity generated by the interconnected macroporous structure of the scaffold allowed the cell-gel mixture to naturally penetrate and fill the porous network inside the scaffold.

[0197] (4) Gel curing: After the composite is added, place it in a 37 ℃ incubator for 30-45 minutes to allow the Matrigel that has penetrated into the pores of the scaffold to undergo in-situ thermal cross-linking and curing.

[0198] 3. Establishment and condition control of the co-cultivation system:

[0199] (1) Culture medium addition: After the gel solidifies, add sufficient composite induction culture medium (formulation is the same as in Example 2, i.e. osteogenic induction medium and endothelial culture medium are mixed in a 1:1 ratio) to the culture well.

[0200] (2) Validation of in-situ drug delivery and sustained-release effects:

[0201] In this co-culture system, the PMBG@Yoda1+oeNrf2 scaffold served as the sole drug source. The Yoda1 small molecule and AAV9-Nrf2 viral vector loaded in the scaffold were continuously and slowly released into the microenvironment within the scaffold pores over time.

[0202] (3) Long-term dynamic culture: The constructed organoids are placed in a 37 ℃, 5% CO2 incubator. The conventional compound induction medium is replaced every 2 days. The culture cycle is set to 14-21 days to culture vascularized bone organoids containing Yoda1+oeNrf2.

[0203] Comparative Example 2:

[0204] The difference between this comparative example and Example 2 is that an unloaded PMBG / TCP stent was used; all other operations are the same as in Example 2.

[0205] Based on the successful construction of the aforementioned vascularized bone organoids, this invention further evaluated their in vivo and in vitro biological functions, as detailed below:

[0206] I. In vitro biological function evaluation

[0207] In the in vitro biological function evaluation, cell proliferation was detected by CCK-8 assay, early osteogenic differentiation was assessed by ALP staining, osteogenic genes were detected by RT-qPCR, and angiogenesis capacity was assessed by Tube Formation assay.

[0208] CCK-8 test:

[0209] (1) BMSCs were loaded with 2×10 3 Inoculate at a density of 100 cells / well in 96-well plates and incubate for 24 hours.

[0210] (2) Discard the old culture medium and replace it with a complete culture medium containing different concentrations of scaffold extract. Set up a blank control group and continue culturing for 1, 3 and 5 days.

[0211] (3) At each time point, add 10 μL of CCK-8 solution to each well and incubate in a 37℃ incubator for 1-2 hours in the dark. Measure the absorbance at 450 nm using a microplate reader.

[0212] ALP staining:

[0213] (1) BMSCs were seeded in 24-well plates. After the cell confluence reached 80%, the medium was replaced with osteogenic induction medium containing scaffold extract and cultured continuously for 7 days.

[0214] (2) Discard the culture medium, wash twice with PBS, add 4% paraformaldehyde fixative, and fix at room temperature for 15-20 minutes.

[0215] (3) Use the BCIP / NBT staining kit (Beyotime ALP staining kit) and stain for 30-60 minutes in the dark.

[0216] (4) Under a microscope, the depth and distribution area of ​​the blue-purple precipitate represent the activity of ALP and the degree of osteogenic differentiation.

[0217] Tube Formation Experiment:

[0218] (1) Corning gel plating: Pre-cool the 96-well cell culture plate on ice. Add 50 μL of thawed Corning gel to each well and gently shake to spread the gel evenly at the bottom of the well. Then incubate the culture plate in a 37 ℃ cell culture incubator for 30 minutes.

[0219] (2) Cell seeding: HUVECs treated under different conditions (untreated, PMBG extract treated, and PMBG@Yoda1+oeNrf2 extract treated) were digested and counted. Cell density was adjusted by adding 1×10⁻⁶ cells to each well. 5 Each cell.

[0220] (3) Tube culture and observation: The inoculated culture plates were placed in an incubator at 37 ℃ and 5% CO2. After 6 hours of culture, the cell morphology was observed and images were taken using an inverted phase contrast microscope.

[0221] like Figure 13 As shown, the vascularized organoids constructed in Example 2 exhibited good cell viability. Live / dead cell staining and a steadily increasing proliferation curve over time strongly confirmed that the PMBG@Yoda1+oeNrf2 composite scaffold material and its sustained-release Yoda1 and Nrf2 have excellent biocompatibility, did not produce significant cytotoxicity to seed cells (BMSCs), and can support long-term cell survival and expansion in a complex three-dimensional environment, providing the cell biology basis for constructing vascularized bone organoids. In contrast, the PMBG group organoids in Comparative Example 2 lacked the biological stimulation effect of the Yoda1 and oeNrf2 carriers. Although their cell viability remained at a high level on days 3 and 5 of culture, it showed slight fluctuations or a decreasing trend.

[0222] like Figure 14 As shown, the vascularized organoids constructed in Example 2 (PMBG@Yoda1+oeNrf2 group) exhibited the deepest and densest blue-purple precipitate, indicating that the cells not only proliferated extensively but also that most had entered the early stage of active osteogenic differentiation, with the strongest ALP activity. In comparison, the ALP staining intensity in Comparative Example 2 group was somewhat higher than that in the Control group, demonstrating the inherent basic osteogenic induction ability of the bioactive glass material, but its osteogenic promotion effect was significantly weaker than that of Example 2.

[0223] like Figure 15 As shown, the Runx2 and ColIA genes were most highly expressed in the vascularized organoids constructed in Example 2 (PMBG@Yoda1+oeNrf2 group). In contrast, the Runx2 and ColIA gene expression in Comparative Example 2 group was increased to some extent compared with the Control group, demonstrating the inherent basic osteogenic induction ability of bioactive glass materials, but its osteogenic promotion effect was significantly weaker than that of Example 2.

[0224] like Figure 16As shown, in the vascularized organoids constructed in Example 2 using the PMBG@Yoda1+oeNrf2 group, HUVECs formed the most complete and dense tubular structures. Numerous clear, robust, and closed vascular loops were visible, constructing a highly complex biomimetic vascular network. Quantitative analysis further confirmed that the total number of loops in this group was approximately 20, and the number of standardized nodes exceeded 200% of the control group, both being the highest among all groups. In contrast, although Comparative Example 2 induced a certain number of vascular-like structures in HUVECs, and the number of vascular loops and nodes was significantly better than the blank control group, demonstrating that the active silicon ions released by the PMBG material have a certain pro-angiogenic effect, the vascular network it formed had poor connectivity, a thinner diameter, and more breakpoints and unclosed areas. Quantitative data showed that the number of vascular loops and nodes in Comparative Example 2 was only about half that of Example 2.

[0225] The above results demonstrate that the PMBG@Yoda1+oeNrf2 scaffold loaded with hBMSCs and HUVECs (i.e., the vascularized bone organoid constructed in Example 2) has good biocompatibility, excellent osteogenic induction and angiogenesis capabilities, and can serve as a potential vascularized bone organoid with mechanical responsiveness and anti-cellular aging function.

[0226] II. Evaluation of in vivo bone repair effect

[0227] To verify the in vivo application effect of the vascularized bone organoid constructed in this invention on the efficient repair of bone defects in senile osteoporosis, this invention selected aged SD rats and established a critical size skull defect model.

[0228] In the experiment, participants were randomly divided into three groups: a blank control group, a PMBG scaffold group, and a PMBG@Yoda1+oeNrf2 scaffold group. The specific procedures are as follows:

[0229] 1. Selection of experimental animals:

[0230] Healthy male Sprague-Dawley (SD) rats aged 18-20 months (corresponding to old age in humans) weighing 500-600 g were selected and purchased from Shanghai Shengchang Biotechnology. All animals were acclimatized in an SPF-grade animal facility for one week with free access to food and water.

[0231] 2. Surgical mold creation steps:

[0232] (1) Anesthesia: Isoflurane gas anesthesia was used. The induction concentration was 4-5%, and the maintenance concentration was 1-3%. After the rats lost their righting reflex and had reduced pain sensation, they were fixed in a prone position on the operating table. The respiratory rate, mucosal color and other anesthetic indicators were closely monitored.

[0233] (2) Skin preparation and disinfection: Shave the hair on the top of the rat’s skull and disinfect with povidone-iodine three times from the center of the surgical incision outward.

[0234] (3) Incision and exposure: Make a longitudinal incision of about 1.5 cm along the midline of the skull to cut the skin, subcutaneous tissue and periosteum. Use a periosteal elevator to bluntly separate the periosteum to both sides to fully expose the flat bilateral parietal bones.

[0235] (4) Drilling of bone defects: Using a medical electric bone drill equipped with a 5 mm diameter slow-speed sterilization trephine, circular defects were drilled symmetrically on both sides of the midline suture of the rat skull.

[0236] (5) Material implantation: Rats were randomly divided into three groups (n=6) and treated as follows:

[0237] Control Group: No material was implanted at the defect site, and only the blood clot was retained as a negative control.

[0238] PMBG scaffold assembly: A UV-sterilized PMBG 3D-printed scaffold loaded with hBMSCs and HUVECs (5 mm in diameter, 1 mm in thickness, matching the size of the defect) is tightly implanted at the defect site.

[0239] PMBG@Yoda1+oeNrf2 scaffold assembly: A PMBG@Yoda1+oeNrf2 scaffold (5 mm in diameter, 1 mm in thickness, matching the size of the defect) loaded with hBMSCs and HUVECs from Example 2 was tightly implanted at the defect site.

[0240] (6) Suturing: After confirming that the material is firmly implanted and there is no active bleeding, use 4-0 absorbable sutures to suture the periosteum and skin incision in layers.

[0241] (7) Postoperative care: For three consecutive days after the operation, administer penicillin (40,000 U / rat) intramuscularly to prevent infection, and closely observe the rats' recovery status and wound healing.

[0242] Six weeks post-surgery, new bone volume (BV / TV) was analyzed by Micro-CT scanning, histological observation was performed by HE staining and Masson trichrome staining, and H-type angiogenesis was assessed by CD31 / Emcn immunofluorescence double labeling.

[0243] Depend on Figure 17 and Figure 18 The results showed that the PMBG@Yoda1+oeNrf2 group had significantly more new bone formation than the other two groups.

[0244] Depend on Figure 19The results showed that the PMBG@Yoda1+oeNrf2 group had the highest number of CD31+ / Emcn+ H-type vessels, confirming that the PMBG@Yoda1+oeNrf2 scaffold loaded with hBMSCs and HUVECs can effectively repair bone defects in the context of senile osteoporosis by promoting H-type angiogenesis and bone regeneration.

[0245] In summary, this invention has successfully developed a bone organoid that can target the Piezo1-Nrf2 signaling axis, simulate mechanical stimulation, actively reverse endothelial cell senescence, and synergistically promote osteogenic and angiogenesis, which is of great significance for the treatment of osteoporotic bone defects in the elderly.

[0246] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A three-dimensional porous bioactive scaffold, characterized in that, The bioactive scaffold includes a scaffold body, a Piezo1 channel agonist loaded on the scaffold body, and an Nrf2 agonist and / or an Nrf2 expression vector loaded on the scaffold body. The main body of the scaffold is a photocurable mesoporous bioactive glass / β-TCP composite porous scaffold obtained by 3D printing.

2. The three-dimensional porous bioactive scaffold according to claim 1, characterized in that, The pore size of the main body of the support is 100-200 μm, and the porosity is 60-70%. The main body of the support has a mesoporous structure with an average pore size of 8-10 nm.

3. The three-dimensional porous bioactive scaffold according to claim 1, characterized in that, The Piezo1 channel agonist is any one or more of Yoda1, Yoda2, Jedi2, Yaddle1, and Piezo1 agonist 1-d2; The Nrf2 agonist is any one or more of sulforaphane and dimethyl fumarate; The Nrf2 expression vector is any one or more of an adeno-associated virus vector, lentiviral vector, adenovirus vector, or plasmid DNA carrying the Nrf2 gene.

4. A method for preparing a three-dimensional porous bioactive scaffold according to any one of claims 1-3, characterized in that, The preparation method specifically includes the following steps: S1: Preparation of photocurable mesoporous bioactive glass precursor sol containing SiO2, CaO, P2O5 and photosensitive modifier; S2: The photocurable mesoporous bioactive glass precursor sol is mixed with β-TCP and a photoinitiator to obtain a photocurable mesoporous bioactive glass / TCP composite photosensitive slurry; S3: Photocurable 3D printing was performed using photocurable mesoporous bioactive glass / TCP composite photosensitive slurry as raw material to obtain a photocurable mesoporous bioactive glass / β-TCP composite porous scaffold. S4: Prepare a working solution containing Piezo1 channel agonist, Nrf2 agonist and / or Nrf2 expression vector, immerse the working solution in the photocurable mesoporous bioactive glass / β-TCP composite porous scaffold, and obtain the three-dimensional porous bioactive scaffold by critical volume saturation adsorption-freeze drying.

5. The method for preparing a three-dimensional porous bioactive scaffold according to claim 4, characterized in that, In step S1, the molar ratio of SiO2, CaO and P2O5 in the photocurable mesoporous bioactive glass precursor sol is (40-50): (20-30): (2-10), and the molar ratio of the photosensitive modifier to SiO2 is 1: (5-10). In step S2, the mass fraction of β-TCP in the photocurable mesoporous bioactive glass / TCP composite photosensitive slurry is 5~20wt%, and the amount of photoinitiator added is 1~2wt% of the total mass of the slurry; In step S3, the wavelength of the light source for the photopolymerization 3D printing is 400-420 nm, and the light intensity for the photopolymerization 3D printing is 10-20 mW / cm². 2 .

6. The method for preparing a three-dimensional porous bioactive scaffold according to claim 4, characterized in that, In step S4, the final concentration of the Piezo1 channel agonist in the working solution is 5-20 mg / mL; The final concentration of the Nrf2 agonist is 0-20 mg / mL; The final titer of the Nrf2 expression vector in the working solution is 1×10⁻⁶. 12 vg / mL ~ 1×10 13 vg / mL.

7. A vascularized osteoid organoid, characterized in that, The three-dimensional porous bioactive scaffold according to any one of claims 1-3 is used as a supporting framework and further includes cellular components; The cell components include co-cultured bone marrow mesenchymal stem cells and endothelial cells, with a ratio of (1-2):

1.

8. A method for preparing a vascularized bone organoid according to claim 7, characterized in that, Includes the following steps: T1: Collect expanded bone marrow mesenchymal stem cells and endothelial cells and seed them in liquid matrix gel to obtain a cell-gel mixture; T2: The cell-gel mixture is dropped onto a three-dimensional porous bioactive scaffold, allowing the cell-gel mixture to naturally penetrate and fill the porous network inside the scaffold; T3: The matrix gel is thermally crosslinked and cured in situ on a three-dimensional porous bioactive scaffold, and the vascularized bone organoid is obtained by culturing it in a composite induction medium.

9. The method for preparing vascularized bone organoids according to claim 8, characterized in that, In step T1, the concentration of the bone marrow mesenchymal stem cells is (1-2)×10⁻⁶. 6 cells / mL, the concentration of liquid matrix gel is 6-10 mg / mL; In step T3, the thermal crosslinking curing time is 30-45 minutes; In step T3, the composite induction medium is composed of osteogenic induction medium and endothelial cell medium mixed in a 1:1 volume ratio.

10. The use of the vascularized bone organoid of claim 7 in the preparation of a drug, biological product or device for repairing bone defects caused by osteoporosis in the elderly.

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