Application of bone cell specific Wnt signal in critical bone defect repair

Abstract

The invention discloses application of a bone cell specific Wnt signal in critical bone defect repair, and relates to the field of medicine. A 3D printed composite scaffold for use in the treatment of critical bone defects, bone nonunion or osteoporosis, which is applied in the form of a 3D printed composite scaffold comprising bio-ink and polycaprolactone PCL, the bio-ink comprising a bone-derived acellular extracellular matrix (dECM) in a drug-activated bone cell Wnt signal. The dECM bio-ink based on drug activated bone cell Wnt signals and the PCL are used for constructing the composite scaffold through 3D printing, the composite scaffold is used for repairing critical bone defects and retaining a microstructure and bioactive components of a natural bone matrix, and osteogenic differentiation, osteoclast generation, angiogenesis and neurogenesis of bone marrow stromal cells BMSCs can be remarkably promoted.

Description

Technical Field

[0001] This invention relates to the medical field, and more particularly to the application of osteocyte-specific Wnt signaling in the repair of critical bone defects. Background Technology

[0002] Repairing critical bone defects is a major challenge in the field of orthopedics. Existing methods such as autologous bone and allogeneic bone transplantation have problems such as limited donors and secondary surgical trauma, while synthetic materials often result in poor repair effects due to insufficient bioactivity.

[0003] In recent years, tissue-engineered scaffolds have been used to promote bone regeneration by mimicking the natural bone microenvironment. However, traditional scaffolds often lack versatility and struggle to simultaneously regulate osteogenic, osteoclast, vascular, and neural regeneration processes. The osteocyte Wnt signaling pathway plays a crucial role in bone metabolism, but its application in bone defect repair has not been fully explored.

[0004] Existing technologies largely rely on transgenic animal models, such as Wnt signaling-activated mice, which limits their clinical feasibility.

[0005] Therefore, developing a Wnt signaling regulation strategy based on non-GMO methods (such as drug activation) combined with bio-ink technology is an urgent problem to be solved.

[0006] Therefore, this invention proposes the application of osteocyte-specific Wnt signaling in the repair of critical bone defects. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of osteocyte-specific Wnt signals in the repair of critical bone defects.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The application of osteocyte-specific Wnt signaling in the repair of critical bone defects is used to treat critical bone defects, nonunion, or osteoporosis. It is applied in the form of a 3D printed composite scaffold, which includes bio-ink and polycaprolactone PCL. The bio-ink includes bone-derived decellularized extracellular matrix (dECM) that activates osteocyte Wnt signaling in osteocytes.

[0009] Preferably, the preparation method of the bio-ink is as follows: bone-derived decellularized extracellular matrix (dECM) is mixed with methacrylamide gelatin (GelMA) to prepare a bio-ink with a concentration of 10 mg / mL. The ink has a uniform porous structure, and after swelling in PBS for 24 hours, the swelling rate is 80% ± 2%, and the degradation rate is 65% ± 1% within 30 days.

[0010] Preferably, the method for preparing bone-derived decellularized extracellular matrix (dECM) based on drug-activated osteoblast Wnt signaling includes the following steps: Bone tissue was obtained from wild-type animals, and the Wnt signaling pathway was activated in vitro using a drug activator. Then, it was demineralized, defatted, and enzymatically digested to remove cells, resulting in dECM that retains collagen and glycosaminoglycans. The drug activator is selected from lithium chloride or other Wnt signaling agonists, and the residual DNA content is reduced by more than 95% after decellularization, while the retention rate of collagen and glycosaminoglycans is not less than 80%.

[0011] A method for repairing critical bone defects involves implanting a 3D-printed composite scaffold into the bone defect area.

[0012] A bone tissue engineering kit, characterized by comprising: dECM for the application of osteocyte-specific Wnt signaling in critical bone defect repair, bio-ink for the application of osteocyte-specific Wnt signaling in critical bone defect repair, as well as 3D printing equipment and cell culture reagents, for rapidly constructing personalized bone repair scaffolds.

[0013] The beneficial effects of this invention are as follows: 1. This invention utilizes a bone-derived decellularized extracellular matrix (dECM) bio-ink based on drug-activated Wnt signaling in osteocytes, and constructs a composite scaffold with polycaprolactone (PCL) via 3D printing for the repair of critical bone defects. It preserves the microstructure and bioactive components of the natural bone matrix and can significantly promote osteogenic differentiation, osteoclast formation, angiogenesis, and neurogenesis of bone marrow stromal cells (BMSCs). Attached Figure Description

[0014] Figure 1 Preparation and characterization of bone tissue-derived bioink: (A) Flowchart of decellularized matrix bioink preparation. (B) Printability of dECM-Bioink. (C-E) Quantitative analysis of DNA, collagen, and GAGs on cellular and decellularized scaffolds. *P<0.05 compared to the DBM group; (F) Live / dead staining to assess cell viability of BMSCs on dECM scaffolds after 1, 4, and 7 days of culture, showing live cells (green) and dead cells (red). (G) CCK-8 analysis of cell proliferation on days 1, 4, and 7 of the three scaffolds. Images and data represent n = 3 separate experiments. *P<0.05 compared to the GelMA group; #P<0.05 compared to the GelMA / WT-dECM group. Differences between groups were analyzed using one-way ANOVA.

[0015] Figure 2Characterization of bio-ink performance: (A) Scanning electron microscope image of the bio-ink. Scale bar = 100 μm. (B) Swelling behavior of the bio-ink in PBS. (C) Degradation characteristics of the bio-ink in PBS at different times within 37°C. n = 3. * p <0.05, compared to the GelMA group; # p <0.05, compared with the WT-BdECM group. One-way ANOVA.

[0016] Figure 3 Determination of the osteogenic and osteoclast-promoting differentiation performance of Wnt-dECM (A, B) AP staining and AP activity detection of bone marrow mesenchymal stem cells after 7 days of culture under 2D and 3D conditions, respectively. (C) Expression of osteogenic genes in bone marrow mesenchymal stem cells after 7 days of culture under 2D and 3D conditions, respectively. (D) Alizarin Red S staining of mineralized nodules deposited by bone marrow mesenchymal stem cells after 21 days of culture in osteogenic induction medium. (E) Quantitative mineralization test on day 21. (F) TRAP staining. Images and data represent n = 3 individual experiments. *P < 0.05 compared with GelMA group; #P < 0.05 compared with GelMA / WT-dECM group. Differences between groups were analyzed by one-way ANOVA, n = 3.

[0017] Figure 4 Effects of Wnt-dECM on angiogenesis and neurogenesis: (A) HUVEC migration assay. (B) Expression of angiogenesis marker genes. (C) HUVEC tubule formation. (D) Quantitative analysis of total tubule length and branching. (E) SHY5Y migration assay. (F) Expression of neurotrophic factors. (G) SHY5Y immunofluorescence of β3-tubulin (green). Images and data represent n = 3 separate experiments. *P < 0.05 compared with the GelMA group; #P < 0.05 compared with the GelMA / WT-dECM group. Differences between groups were analyzed using one-way ANOVA. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0019] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0020] Example 1: Drug-activated bone cell Wnt signaling and preparation of dECM First, bone tissue was extracted from the femur of 8-week-old wild-type mice, and Wnt signaling in osteocytes was activated in vitro using drugs (such as lithium chloride or other Wnt signaling agonists). After washing with ddH2O and mincing, the bone tissue was demineralized in 0.5N HCl at room temperature for 24 h, followed by defatting with a 1:1 chloroform / methanol solution for 1 h. The tissue was then washed alternately with methanol and ddH2O and lyophilized. Cells were decellularized in a solution containing 0.05% trypsin and 0.02% EDTA at 37°C with stirring for 24 h, followed by stirring in 1xPBS containing antibiotics for another 24 h. The tissue was then lyophilized and stored at -80°C. The decellularization effect was verified by HE staining and DNA quantification; the residual DNA content was reduced by more than 95%, and the retention rates of collagen and glycosaminoglycans (GAG) reached 83% and 88%, respectively.

[0021] Example 2: Formulation and Characterization of Bio-Ink Drug-activated dECM was dissolved and mixed with methacrylamide gelatin (GelMA) to prepare a bio-ink with a concentration of 10 mg / mL. Scanning electron microscopy showed that the ink had a uniform porous structure. Degradation experiments showed that the degradation rate was approximately 65% ​​after 30 days.

[0022] Example 3: Construction and Biocompatibility Evaluation of 3D Printed Composite Scaffolds Based on the STL file, a scaffold model was designed. A 3D bioprinter was used to first melt PCL at 95℃ to print the supporting framework, followed by printing bio-ink containing BMSCs. The resulting composite scaffold was then cross-linked using 405 nm blue light. Live / dead cell staining showed a cell viability exceeding 90%, and CCK-8 assays indicated that the proliferation activity of the drug-activated dECM group was significantly higher than that of the control group.

[0023] Example 4: In vitro biological function analysis After co-printing BMSCs with bio-ink, ALP staining and qPCR showed upregulated expression of bone genes in drug-activated dECM components and an increase in mineralized nodules. Osteoclast induction experiments showed an increase in TRAP-positive cells, and angiogenesis and neural differentiation experiments both confirmed the promoting effect.

[0024] Example 5: In vitro osteoclast differentiation experiment To evaluate the effect of drug-activated dECM (determined extracellular matrix) of osteocytes, a source of Wnt signaling, on osteoclast differentiation, bone marrow mononuclear cells (BMMs) were extracted from wild-type mouse bone marrow and seeded into bio-ink-coated 48-well plates. Induction medium containing RANKL (50 ng / mL) and M-CSF (25 ng / mL) was added, and the plates were cultured for 5 days. TRAP staining was used to observe multinucleated osteoclast formation. The results showed that the number of TRAP-positive cells in the drug-activated dECM group was significantly higher than that in the control group (GelMA and WT-dECM). This experiment confirms that drug-activated dECM can promote osteoclast generation during bone remodeling, providing a basis for the bone metabolic homeostasis function of the scaffold.

[0025] Example 6: In vitro angiogenesis experiment To investigate the pro-angiogenic effect of drug-activated dECM, human umbilical vein endothelial cells (HUVECs) were seeded in bio-ink-coated 24-well plates and cultured for 3 days. Cell migration was assessed using a Transwell assay. HUVECs were seeded in the upper chamber, and culture medium containing drug-activated dECM was added to the lower chamber. After 8 hours of culture, crystal violet staining was used to count migrating cells. The number of migrating cells in the drug-activated dECM group was 1.5 times that of the WT-dECM group and 4.2 times that of the GelMA group. Simultaneously, tubule formation assays were performed in Matrigel-coated plates. ImageJ analysis after 6 hours of culture showed a significant increase in total tubule length and branch number in the drug-activated dECM group. qPCR detection of upregulated expression of angiogenesis genes (VEGF, HIF1a) confirmed its pro-angiogenic potential.

[0026] Example 7: In vitro neural differentiation experiment To assess the effect of drug-activated dECM on neurogenesis, human neuroblastoma cells (SH-SY5Y) were seeded on bio-ink coated plates and cultured for 7 days. Transwell assays showed that the number of migrating cells in the drug-activated dECM group was 2.7 times that of the WT-dECM group and 5.4 times that of the GelMA group. Immunofluorescence staining for β3-tubulin expression showed a strong positive signal and the appearance of synaptic structures in the drug-activated dECM group. qPCR confirmed the upregulation of neurotrophic factors (GDNF, NTF3, FGF).

[0027] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Use of bone cell-specific Wnt signaling in the repair of critical bone defects, characterized in that, The application is applied to the treatment of critical bone defects, nonunion or osteoporosis, and is applied in the form of a 3D-printed composite scaffold, which comprises a biological ink and polycaprolactone (PCL), and the biological ink comprises dECM which activates Wnt signals of bone cells by drugs.

2. Use of bone cell-specific Wnt signaling in the repair of critical bone defects according to claim 1, characterized in that, The configuration method of the biological ink is to mix bone-derived decellularized extracellular matrix (dECM) and methacrylated gelatin (GelMA) to prepare a biological ink with a concentration of 10 mg / mL, the ink has a uniform porous structure, and the swelling rate is 80%±2% after swelling in PBS for 24 hours, and the degradation rate is 65%±1% within 30 days.

3. The use of bone cell-specific Wnt signaling in the repair of critical bone defects according to claim 1, characterized in that, The preparation method of the bone-derived decellularized extracellular matrix (dECM) based on the drug-activated Wnt signals of bone cells comprises the following steps: Bone tissue is obtained from wild-type animals, the Wnt signal pathway is activated in vitro by a drug activator, and then decalcification, delipidation and enzymatic decellularization are performed to obtain dECM which retains collagen and glycosaminoglycan; The drug activator is selected from lithium chloride or other Wnt signal agonists, the DNA residual amount after decellularization is reduced by more than 95%, and the retention rate of collagen and glycosaminoglycan is not less than 80%.

4. A method of repairing a critical bone defect, characterized by: The repair method is to implant the 3D-printed composite scaffold into the bone defect area.

5. A bone tissue engineering kit, characterized by: The dECM in the application of the bone cell-specific Wnt signal in claim 1 in the repair of critical bone defects, the biological ink in the application of the bone cell-specific Wnt signal in claim 2 in the repair of critical bone defects, and 3D printing equipment and cell culture reagents are used to quickly construct personalized bone repair scaffolds.

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