Multi-dimensional bionic scaffold with space-time synergistic osteogenesis function and preparation method

Abstract

The invention discloses a multi-dimensional bionic scaffold with a space-time synergistic osteogenesis function and a preparation method. The bionic scaffold comprises loofah, a hydroxyapatite coating and polyvinyl alcohol hydrogel, the preparation method comprises the following steps: immersing natural towel gourds in a NaClOacetic acid solution, boiling to obtain white towel gourds, and then alternately immersing the white towel gourds in a calcium solution and a phosphorus solution to obtain an intermediate product WLH; adding Mg-MOFs synthesized by a hydrothermal method into a polyvinyl alcohol solution to obtain a hydrogel precursor solution; and immersing WLH into the hydrogel precursor solution, and carrying out vacuum, freezing and unfreezing circular treatment to obtain the loofah-shaped multi-dimensional bionic scaffold. The invention has the following beneficial effects: the scaffold has a cortical-cancellous structure and mechanical characteristics simulating a natural bone, and has good biocompatibility and biological functions of immunoregulation-angiogenesis-osteogenesis induction coupling.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, in particular to a multidimensional bionic scaffold with spatiotemporal coordinated osteogenesis function and a preparation method thereof. Background Art

[0002] Reconstructing critical-sized bone defects resulting from severe trauma or congenital deformity is a long-standing clinical challenge in orthopedics. While autologous bone grafting remains the gold standard, donor site morbidity and supply limitations necessitate the development of advanced alternatives. Conventional bioscaffold strategies struggle to replicate the triaxially anisotropic structure of native bone—comprising the outer cortical bone and inner cancellous bone. Cortical bone, with its highly dense, biaxially anisotropic lamellar structure composed of longitudinally aligned Haversian grooves, provides the primary mechanical support for the bone. In contrast, cancellous bone consists of a heterogeneous porous structure with trabecular arrangement. Their synergistic combination forms a unique triaxially anisotropic hierarchical architecture. During bone healing, this architecture synergistically regulates mechanotransduction, immunomodulation, and vascularized osteogenesis, and collectively influences cell proliferation, migration, aggregation, and secretion, thereby orchestrating bone regeneration. Therefore, this biological complexity requires scaffolds that simultaneously mimic the anatomical structure and recapitulate regenerative microenvironmental cues.

[0003] Existing technologies still have significant limitations: 3D printing, renowned for its ability to customize complex geometries, represents a paradigm shift in tissue regeneration. It offers significant advantages over traditional bioscaffolds by faithfully replicating large, complex tissue structures. However, 3D printing is limited by resolution and cannot simultaneously construct anisotropic Haversian tubular structures and three-dimensionally interconnected cancellous bone pores. Gradient scaffolds fabricated by layer-by-layer stacking lack a well-defined cortical-cancellous bone interface, cannot replicate the triaxial anisotropic characteristics of natural bone, and can only display pore gradients. More importantly, current designs prioritize structural mimicry (e.g., Haversian channels) or biochemical functions, neglecting their coordinated regulation of multifunctional coupling. These deficiencies highlight the unmet need for a single scaffold system that can both holistically replicate the multiscale architecture of bone and dynamically coordinate cellular responses. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art. The scaffold has the cortical-cancellous structure and mechanical characteristics that simulate natural bone, has good biocompatibility and the biological function of immune regulation-angiogenesis-osteoblast induction coupling, and is a multidimensional bionic scaffold with spatiotemporal synergistic osteogenesis function and a preparation method.

[0005] The purpose of the present invention is achieved through the following technical solutions: a multidimensional bionic scaffold with spatiotemporal synergistic osteogenesis function, comprising loofah, hydroxyapatite coating and polyvinyl alcohol hydrogel; the loofah is a white loofah obtained by boiling natural loofah; The hydroxyapatite coating is formed by calcium-phosphorus alternately mineralizing on the surface of the white luffa; The polyvinyl alcohol hydrogel contains a magnesium-based metal organic framework material.

[0006] Further, the calcium-phosphorus alternately mineralizing refers to alternately immersing the white luffa in a calcium solution and a phosphorus solution.

[0007] Further, the calcium solution is a 200 mM CaCl2 / Tris-HCl solution (pH 7.4), and the phosphorus solution is a 120 mM Na2HPO4 solution (pH 7.4).

[0008] Preferably, the magnesium-based metal organic framework material is synthesized by a hydrothermal method.

[0009] Preferably, the concentration of the magnesium-based metal organic framework material in the polyvinyl alcohol hydrogel is 0.4 wt%, The concentration of the polyvinyl alcohol is 5 wt%.

[0010] In another aspect, a preparation method of a luffa-shaped multi-dimensional biomimetic scaffold with space-time synergistic osteogenesis function is provided, which comprises the following steps: (1) immersing a natural luffa (NL) in a NaClO2 acetic acid solution and boiling to obtain a white luffa (WL); (2) alternately immersing the WL in a calcium solution and a phosphorus solution to obtain an intermediate product WLH; (3) synthesizing a magnesium-based metal organic framework material (Mg-MOFs) by a hydrothermal method; (4) dissolving polyvinyl alcohol (PVA) powder in deionized water, and then adding the Mg-MOFs to obtain a hydrogel precursor solution; (5) immersing the WLH in the hydrogel precursor solution, and performing vacuum, freezing and thawing cycle treatment. Finally, immersing the sample in ultrapure water for 3 hours, and freeze-drying to obtain a final scaffold product WLHPM.

[0011] The present application has the following advantages: 1. The boiling purpose in the boiling to obtain the white luffa is to remove lignin, so as to effectively loosen the close connection and interaction between cellulose microfibers, and soften the material to a cell-friendly modulus, so that the porosity can be greatly improved.

[0012] 2. The white luffa (WL) is immersed in 50 mL of a calcium solution at 37 °C for 1 hour, and then the excess water is absorbed by absorbing paper, and then the WL is washed with deionized water and wiped dry; then, the WL is immersed in a phosphorus solution at 37 °C for 1 hour, and the excess water is also removed, and this alternately immersing process is repeated five times.

[0013] 3. In step (3) of the present invention, Mg-MOFs are synthesized by hydrothermal method. The operation of the preferred embodiment is as follows: MgCl2 is added to 5 mL of ddH2O and gently stirred to dissolve it; gallic acid is then added and the mixture is vigorously stirred for 30 minutes to produce a milky white precipitate; 10 M KOH aqueous solution is added dropwise and the pH value is adjusted to 10.0, at which time the precipitate gradually dissolves and the color changes to brownish yellow; then, the mixture is heat-treated at 120°C for 24 hours; post-synthesis purification includes continuous centrifugation cycles and washing with ddH2O to remove residues; and the resulting material is then freeze-dried for 24 hours to obtain Mg-MOFs.

[0014] The polyvinyl alcohol powder in step (4) of the present invention has poor solubility in cold water and needs to be stirred at 90°C, preferably with magnetic stirring.

[0015] In step (5) of the present invention, after the WLH is immersed in the hydrogel precursor solution, it is preferably placed in a vacuum environment for 10 minutes to promote the hydrogel solution to enter the WLH channel.

[0016] In step (5) of the present invention, after the WLH is immersed in the hydrogel precursor solution, it is preferably subjected to vacuum, freezing, and thawing cycles three times; application of the scaffold in the treatment of critical bone defects.

[0017] 4. The scaffold of the present invention has a triaxial anisotropic structure: the lignified loofah used in the present invention has an inherent cortical-cancellous bone structure, which can provide the main mechanical support for the skeleton. The addition of hydrogel greatly improves the mechanical properties of the scaffold.

[0018] Good effect in treating critical bone defects: The scaffold of the present invention is not only structurally compatible with natural bone, but also has the mechanical conduction of natural bone. At the same time, it synergizes bone regeneration through multimodal immunoregulation-angiogenesis-osteoinduction, and has a good therapeutic effect on critical bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison diagram of the morphological structure of loofah before and after delignification in Example 1; Figure 2 The SEM image and energy dispersive spectrometer (EDS) image of Mg-MOFs in Example 1; Figure 3 Cross-sectional micro-CT and SEM images of WL, WLH, WLHP, and WLHPM in Example 1; Figure 4 Figure 1 is a mechanical test diagram of the multi-dimensional bionic scaffold at each stage in Example 1; Figure 5 The cell staining images after 1 day and 3 days of co-culture of cells and scaffolds in Example 2; Figure 6This is a graph showing cell viability after 1, 3, and 7 days of co-culture of cells and scaffolds in Example 2; Figure 7 Images of immunofluorescence staining using CD206 (green) and INOS (red) after RAW264.7 cells were co-cultured with different scaffolds for 24 hours in Example 3; Figure 8 Graph showing the experimental results of promoting angiogenesis of the multidimensional bionic scaffold in Example 4; Figure 9 ALP and ARS staining images after co-culture of cells with each group of scaffolds in Example 5; Figure 10 Micro-CT and three-dimensional reconstruction images of the stent in Example 6 after 4 and 12 weeks of transplantation. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings, and the protection scope of the present invention is not limited to the following: Example

[0021] A preparation method of a loofah-shaped multidimensional bionic scaffold with spatiotemporal synergistic osteogenesis function: (1) Natural loofah (NL) was immersed in 3 wt% NaClO2 acetic acid solution (pH ≈ 4.6) and boiled for 1 h to remove lignin to obtain white loofah (WL); (2) Alternately immerse the WL in two solutions: 200 mM CaCl2 / Tris-HCl solution (Ca solution, pH 7.4) and 120 mM Na2HPO4 solution (P solution, pH 7.4). After soaking the WL in 50 mL of Ca solution at 37 °C for 1 hour, remove excess water with absorbent paper, then rinse the WL with deionized water and wipe dry. Then, immerse the sample in P solution at 37 °C for 1 hour, and also remove excess water. This alternating immersion process was repeated five times. The sample was thoroughly washed, freeze-dried, and labeled as WLH; (3) Synthesis of magnesium-based MOFs by hydrothermal method. Gently stir 5 mL of ddH2O to dissolve MgCl2. Then add GA and stir the mixture vigorously for 30 minutes to produce a milky white precipitate. Add 10 M KOH aqueous solution dropwise and adjust the pH to 10.0. The precipitate gradually dissolves and the color changes to brownish yellow. After stirring for 30 minutes, the mixture is hydrothermally treated at 120 °C for 24 hours. Centrifuge at 8000 rpm for 10 minutes, wash with ddH2O to remove the residue, and centrifuge at 8000 rpm for another 10 minutes. The resulting material is then freeze-dried for 24 hours to obtain Mg-MOFs. (4) At 90 °C, PVA powder was dissolved in deionized water with magnetic stirring. After the mixture was cooled to room temperature, Mg-MOFs were added to make the final Mg-MOFs concentration of 0.4 wt% and PVA concentration of 5 wt%. The WLH was immersed in the hydrogel precursor solution at the bottom of a beaker. The beaker was placed under vacuum for 10 min to facilitate the solution entering the WLH channels. After 12 h of freezing at -40 °C and thawing for 6 h to room temperature, this freeze-thaw cycle was repeated three times for the preparation of Mg-MOFs-loaded multi-dimensional biomimetic scaffold (WLHPM). Finally, the sample was soaked in ddH2O for 3 h to remove any residual contaminants; (5) WLHP (without Mg-MOFs) was prepared in the same way.

[0022] Figure 1 For the comparison of the morphological structure of the luffa before and after delignification, a) photographs of NL and WL. b) scanning electron microscope (SEM) images of NL and WL. From Figure 1 It can be seen that the native luffa (NL) was subjected to delignification treatment to obtain a luffa template (WL) rich in white cellulose. After delignification, the fiber microstructure became obviously different, the lignin content decreased to 1.31 ± 0.13%, the hemicellulose decreased to 11.22 ± 0.44%, and the cellulose ratio increased to 83.72 ± 0.72%. This treatment method effectively loosens the tight connection and interaction between cellulose microfibers, greatly improving the porosity.

[0023] Figure 2 For the SEM and EDS images of Mg-MOFs. The synthesized Mg-MOFs have a polyhedral morphology (diameter 5 ~7 μm) with uniform distribution of carbon (C), magnesium (Mg) and oxygen (O) elements. It is proved that Mg-MOFs are successfully synthesized and can be further used.

[0024] Figure 3 For the cross-sectional micro-CT and SEM images of WL, WLH, WLHP and WLHPM. The three-dimensional structure reconstruction by micro-CT scanning shows that WL exhibits anisotropic characteristics similar to native bone in three orthogonal directions. In the X-axis direction (cross-sectional view), the cross-section of individual fibers is composed of porous tubular structures. In the Y-axis direction (longitudinal view), axially layered fiber bundles are observed, and their tubular structures are very similar to the Haversian canals in cortical bone. This oriented fiber tube system provides a physical migration path for cells, which is crucial for guiding cell directional migration.

[0025] In order to further characterize the properties of the scaffold, mechanical tests were performed on the scaffolds prepared at each stage, and the results are as follows Figure 4As shown. (A) and (C) are representative compressive stress-strain curves of the scaffold along the Z and Y axes, respectively. (B) and (D) are the compressive strength of the scaffold along the Z and Y axes, respectively. The compressive strength along the Z axis (parallel to the cancellous bone) and Y axis (parallel to the cortical bone) was evaluated through compression testing. As shown in the figure, due to the heterogeneity of the cortical-cancellous bone structure, the mechanical properties exhibited distinct directionality, with the compressive strength in the Y axis significantly higher than that in the Z axis. Notably, the WLH scaffold exhibited improved compressive strength in both directions compared to the WL scaffold, which may be related to the mineralized coating. Furthermore, the addition of PVA hydrogel significantly enhanced the mechanical properties of the WLH scaffold. These results demonstrate that integrating a structurally oriented scaffold template with a hydrogel not only retains the advantages of mechanical anisotropy but also achieves superior overall mechanical properties.

[0026] Example 2: In vitro biocompatibility of multidimensional biomimetic scaffolds The specific preparation method of the scaffold was the same as that in Example 1. After the scaffold was prepared, rat bone marrow mesenchymal stem cells (BMSCs) and mouse embryonic osteoblast precursor cells (MC3T3-E1) were co-cultured with the scaffold for 1 day and 3 days, and then the cells were stained. The results were as follows: Figure 5 A and Figure 5 As shown in B, the more green dots, the more living cells. The results showed that BMSCs and MC3T3-E1 cells co-cultured with all four groups of scaffolds had very few apoptosis and necrosis. Furthermore, the cell survival rates after 1, 3 and 7 days of co-culture with the scaffolds were as follows: Figure 6 A and Figure 6 As shown in Figure B, after 1, 3, and 7 days of co-culture with the four groups of scaffolds, the cell survival rates were very high and were no different from those in the control group. This indicates that the scaffolds have optimal biocompatibility.

[0027] Example 3: Immunomodulatory properties of multidimensional biomimetic scaffolds The specific preparation method of the scaffold is consistent with that of Example 1. To elucidate the immune regulation cascade, mouse mononuclear macrophage leukemia cells (RAW264.7) activated by lipopolysaccharide were co-cultured with different scaffolds for 24 hours in a simulated inflammatory microenvironment. After co-culture, immunofluorescence staining was performed using CD206 (M2 marker, green) and INOS (M1 marker, red). The results are shown in Figure 2. Figure 7 As shown, the CD206 expression in the WLHPM group was significant, and the INOS signal was very low, indicating that the multidimensional bionic scaffold had an immunomodulatory effect.

[0028] Example 4: Angiogenic activity of multidimensional biomimetic scaffolds The specific preparation method of the scaffolds was the same as that in Example 1. After culturing human umbilical vein endothelial cells (HUVECs) using four sets of scaffolds, A) wound healing assay; B) Transwell migration assay after 24 hours of culture; and C) HUVEC angiogenesis assay were performed. The results are shown in Figure 2. Figure 8 As shown in the figure, it can be clearly seen that the multidimensional bionic scaffold can promote the migration and tube formation of human umbilical vein endothelial cells, and has the effect of promoting blood vessel growth.

[0029] Example 5: Osteogenic Induction Performance of Multidimensional Bionic Scaffolds The specific preparation method of the scaffold was the same as that in Example 1. Four groups of scaffolds were used to co-culture BMSCs for 7 and 14 days. Alkaline phosphatase (ALP) staining was performed after 7 days of culture; Alizarin red S (ARS) staining was performed after 14 days of culture. The results are shown in Figure 2. Figure 9 Figure 1 shows the results of ALP staining after 7 days of co-culture of BMSCs with the four scaffolds. Alkaline phosphatase is a hallmark enzyme of mature osteoblasts, and the intensity of the color indicates the number of mature osteoblasts. As shown in the figure, all groups cultured in osteogenic induction medium showed positive ALP staining at all time points. Notably, WLHPM exhibited the strongest osteogenic potential on day 7, while WLH and WLHP showed relatively weak ALP activity. Figure 2 shows the results of ARS staining after 14 days of co-culture of BMSCs with the four scaffolds. ARS is an anthraquinone dye that binds to calcium ions to form a red complex. The degree of staining can be used to assess the calcium ion content and, therefore, the degree of calcification. The figure shows that BMSCs in each group exhibited progressive mineralization. Both macroscopically and microscopically, WLHPM exhibited the most intense red color and densely distributed calcium nodules. This demonstrates that the multidimensional biomimetic scaffold can induce osteogenic differentiation of stem cells and possess osteogenic induction properties.

[0030] Example 6: Bone defect reconstruction effect of multidimensional bionic scaffold The specific preparation method of the scaffold is consistent with that of Example 1. In order to evaluate the bone defect reconstruction effect after scaffold transplantation, micro-CT images and three-dimensional reconstruction images of the four groups of scaffolds were analyzed at 4 weeks and 12 weeks after transplantation. Figure 10As shown in the figure, cross-sectional CT images of the femur at 4 weeks revealed dense cortical bone destruction in all groups. At 12 weeks postoperatively, significant cortical bone regeneration was observed in the scaffold groups, particularly in the WLHPM group, where the newly formed bone bridge completely covered the defect area. Three-dimensional reconstructed images of new bone formation in each group are shown in the figure. The quantity and quality of regenerated bone varied among the groups, with the WLHPM group showing the highest bone repair efficiency and the greatest amount of newly formed bone tissue. Both the WLH and WLHP groups demonstrated superior osteoinductive properties compared to the WL group, but bone development in the WLHP group was slightly slower than that in the WLH group. Notably, in the WLH, WLHP, and WLHPM groups, newly formed bone grew and deposited along the oriented structure of the luffa fibers, forming a bone density gradient consistent with the cortical-cancellous structure of the scaffold, indicating that the scaffold structure not only supports but also guides osteogenesis.

[0031] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A multidimensional bionic scaffold with spatiotemporal synergistic osteogenesis, characterized by: including loofah, hydroxyapatite coating, and polyvinyl alcohol hydrogel; The sponge gourd is a white sponge gourd obtained by boiling natural sponge gourd; The hydroxyapatite coating is formed by alternating mineralization of calcium and phosphorus on the surface of the white loofah; The polyvinyl alcohol hydrogel contains a magnesium-based metal organic framework material.

2. The multidimensional bionic scaffold with spatiotemporal coordinated osteogenesis according to claim 1, characterized in that: The calcium-phosphorus alternating mineralization refers to the white loofah being alternately soaked in a calcium solution and a phosphorus solution.

3. The multidimensional bionic scaffold with spatiotemporal coordinated osteogenesis according to claim 2, characterized in that: The calcium solution was a 200 mM CaCl2 / Tris-HCl solution with a pH value of 7.4, and the phosphorus solution was a 120 mM Na2HPO4 solution with a pH value of 7.

4.

4. The multidimensional bionic scaffold with spatiotemporal coordinated osteogenesis according to claim 1, characterized in that: The magnesium-based metal organic framework material is synthesized by a hydrothermal method.

5. The multi-dimensional bionic scaffold with spatiotemporal coordinated osteogenesis according to claim 2, characterized in that: The concentration of the magnesium-based metal organic framework material in the polyvinyl alcohol hydrogel is 0.4 wt %, and the concentration of the polyvinyl alcohol is 5 wt %.

6. The multidimensional bionic scaffold with spatiotemporal coordinated osteogenesis and preparation method according to claim 5, characterized in that: The following steps are involved: S1, immersing natural loofah NL in NaClO2 acetic acid solution and boiling to obtain white loofah WL; S2, alternately soaking the white loofah WL in a calcium solution and a phosphorus solution to obtain an intermediate product WLH; S3. Hydrothermal synthesis of magnesium-based metal organic framework materials Mg-MOFs; S4, dissolving polyvinyl alcohol (PVA) powder in deionized water, and then adding Mg-MOFs to obtain a hydrogel precursor solution; S5. Immerse WLH in a hydrogel precursor solution and undergo a vacuum, freeze, and thaw cycle. Finally, soak the sample in ultrapure water for 3 hours and freeze-dry to obtain the final scaffold product WLHPM.

7. A multidimensional bionic scaffold with spatiotemporal coordinated osteogenesis according to any one of claims 1 to 6, characterized in that: Also included is the use of the scaffold in treating critical bone defects.

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