3D printing nHA / PCL-CSMA-Exos jaw bone defect repair material and preparation method thereof

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to a 3D printing nHA / PCL-CSMA-Exos jaw bone defect repair material and a preparation method thereof. The present application prepares a biomimetic porous 20%nHA / PCL support through high-temperature melting deposition, constructs a biological active interface through ultraviolet light cross-linking CSMA, and loads bone marrow mesenchymal stem cell exosomes BMSC-Exos based on electrostatic action to obtain a composite support nHA / PCL-CSMA-Exos. The support has good hydrophilicity and biocompatibility, can significantly promote cell proliferation and cell migration, and can promote the generation of bone tissue. The composite support combining 3D printing precise forming, hydrogel biological active coating and exosome regulation function provides a new solution with structural adaptability, mechanical adaptability and biological adaptability for the regeneration and repair of oral and maxillofacial bone defects, and has important clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a 3D-printed nHA / PCL-CSMA-Exos jawbone defect repair material and its preparation method. Background Technology

[0002] Facial bone defects caused by congenital defects (such as alveolar clefts), acquired trauma, and tumor resection severely affect patients' physiological functions, including chewing, speech, and appearance, as well as their mental and physical health. Currently, autologous bone grafting is the "gold standard" for bone defect repair, but it has problems such as donor site complications, limited bone volume, and bone resorption. Therefore, the development of tissue-engineered scaffolds with good repair effects has become a research hotspot.

[0003] In recent years, the combination of 3D printing technology and tissue engineering has provided a new strategy for personalized and precise jawbone defect repair. Chinese invention patent CN115998960A discloses a 3D-printed customized multi-functional jawbone defect repair material and its preparation method. It uses coaxial 3D printing technology to prepare an inner and outer double-layer scaffold. The outer layer, HA / SA gel, is loaded with minocycline for early antibacterial and anti-inflammatory effects, while the inner layer, a HA / SA complex, is loaded with deer antler powder for later osteogenic effects. A layered drug release strategy has initially solved the problem of temporal regulation of antibacterial and osteogenic functions. However, this technology still has the following limitations: 1) The scaffold based on hydroxyapatite / sodium alginate (HA / SA) has weak mechanical properties, making it difficult to meet the high mechanical support requirements of jawbone defects; 2) Its functionalization mainly relies on the simple loading of antibacterial drugs and traditional Chinese medicine powders and the physical release controlled by the double-layer structure. The precision and sustainability of bioactivity regulation need to be improved, especially lacking the ability to actively and finely regulate cell behavior (such as migration and differentiation).

[0004] To address the aforementioned issues, there is an urgent need in this field to develop a jawbone defect repair material that combines excellent mechanical properties, precise structural adaptability, and high bioactivity regulation. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention constructs an nHA / PCL scaffold with excellent mechanical properties and customizable structure using 3D printing, and builds a CSMA hydrogel bioactive interface on its surface and within its pores. Then, bone marrow mesenchymal stem cell exosomes (BMSC-Exos) are loaded through electrostatic interaction to obtain a composite repair material with excellent mechanical, structural, and biocompatibility.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A method for preparing a 3D-printed nHA / PCL-CSMA-Exos jawbone defect repair material is provided, comprising the following steps: S1: A porous nHA / PCL composite scaffold was fabricated using 3D printing technology; S2: Preparation of methacrylamide chitosan hydrogel precursor solution; S3: Mix bone marrow mesenchymal stem cell exosomes with the hydrogel precursor solution prepared in step S2 to form an Exos-CSMA mixed solution; S4: The Exos-CSMA mixed solution obtained in step S3 is injected into the pores of the nHA / PCL scaffold obtained in step S1, and ultraviolet light crosslinking is performed to solidify the CSMA hydrogel in situ on the surface of the scaffold and inside the pores, and exosomes are loaded to obtain the 3D printed nHA / PCL-CSMA-Exos jaw defect repair material.

[0007] Preferably, in step S1, the mass ratio of nano-hydroxyapatite to polycaprolactone in the nHA / PCL scaffold is 1:4.

[0008] Preferably, in step S1, the 3D printing technology is fused deposition modeling; the printing parameters are: layer thickness 0.1 mm, speed 200 mm / s.

[0009] Preferably, in step S3, after the bone marrow mesenchymal stem cell exosomes are mixed with the hydrogel precursor solution, the concentration of the exosomes is 20-50 μg / mL.

[0010] Preferably, in step S4, the ultraviolet crosslinking process is as follows: first, pre-crosslinking for 1 second to allow the Exos-CSMA mixed solution to initially adhere, then injecting it into the pores of the scaffold, and then photocuring for another 10 seconds to achieve complete crosslinking.

[0011] The invention also provides a 3D-printed nHA / PCL-CSMA-Exos jawbone defect repair material prepared by any of the above methods, characterized in that it comprises a porous nHA / PCL composite scaffold matrix and a methacrylamide chitosan hydrogel coating loaded with bone marrow mesenchymal stem cell exosomes, the coating being attached to the surface and pores of the scaffold matrix.

[0012] The beneficial effects of this invention are as follows: This invention utilizes high-temperature fused deposition modeling (FTDM) to fabricate a biomimetic porous 20% nHA / PCL scaffold. A bioactive interface is constructed by cross-linking CSMA with ultraviolet light, and bone marrow mesenchymal stem cell exosomes (BMSCs) are loaded based on electrostatic interactions to obtain a composite scaffold, nHA / PCL-CSMA-Exos. This scaffold exhibits excellent hydrophilicity and biocompatibility, significantly promoting cell proliferation and migration, while also promoting bone tissue regeneration. This composite scaffold, combining precise 3D printing, a hydrogel bioactive coating, and exosome-mediated regulation, provides a novel solution for the regenerative repair of maxillofacial bone defects, offering structural, mechanical, and biocompatible advantages, and possesses significant clinical translational value. Attached Figure Description

[0013] Figure 1 Macroscopic images of a 3D-printed 20% nHA / PCL scaffold; where A is the top / bottom view of the scaffold, and B is the side view of the scaffold; Figure 2 Scanning electron microscope images of a 20% nHA / PCL stent; where A and B are frontal views of the stent, and C is a cross-sectional view of the stent; Figure 3 EDS energy spectrum of 20% nHA / PCL scaffold; Figure 4 The stress-strain curve for a 20% nHA / PCL stent; Figure 5 For the identification of exosomes and zeta potential; where A is the microscopic morphology of exosomes as shown by TEM, B is the particle size distribution of exosomes as shown by NTA analysis, C is the expression of exosome surface marker proteins as shown by WB, and D is the exosome zeta potential. Figure 6 The image shows the general layout of the nHA / PCL-CSMA and nHA / PCL-CSMA-Exos composite stents; where A is the nHA / PCL-CSMA stent and B is the nHA / PCL-CSMA-Exos stent. Figure 7 Scanning electron microscope (SEM) images of the nHA / PCL-CSMA and nHA / PCL-CSMA-Exos composite scaffolds; where A is nHA / PCL-CSMA and B is nHA / PCL-CSMA-Exos. Figure 8 The contact angles and contact angle bar charts for the three composite scaffolds at 1s, 30s, and 60s are shown. Figure 9 Images and bar charts showing the hemolysis rates of the three composite stents; Figure 10 Bar chart showing the OD values ​​of the three CCK8 support structures; Figure 11Images showing cell viability and mortality staining on three sets of scaffolds; Figure 12 Photographs of cell scratches and bar charts of cell migration rates at 0h, 12h, and 24h for three groups of scaffolds; Figure 13 Images of ALP and ARS staining and bar charts of ALP and ARS quantitative data are shown for three sets of scaffolds; where A is an ALP staining image; B is an ARS staining image; C is an ALP quantitative bar chart; and D is an ARS quantitative bar chart. Detailed Implementation

[0014] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0015] Example 1: Preparation of 3D-printed nHA / PCL-CSMA-Exos jawbone defect repair material Step 1: Fabrication and characterization of 3D-printed 20% nHA / PCL scaffolds: Weigh 4 g of PCL and 1 g of hydroxyapatite with a particle size of 200 nm. Measure 17.1 mL of dichloromethane using a graduated cylinder to prepare a polymer solution with a concentration of 15 wt%. In a fume hood, pour the measured dichloromethane from the graduated cylinder into a plastic bottle, then add the PCL to a beaker. Stir at room temperature and 500 r / min until completely dissolved. Add 1 g of hydroxyapatite and stir for 4 hours. After stirring, pour the solution evenly onto the surface of a clean tray, ensuring a uniform distribution. Secure the tray in the fume hood and maintain it for 24 hours to allow the solvent to fully evaporate, obtaining the printing ink material. Add the prepared printing ink material to the printer head cartridge. Turn on the printer and import the slicing path planning file. Set the corresponding printing parameters according to the material requirements: layer thickness 0.1 mm, speed 200 mm / s, to print a 20% nHA / PCL scaffold.

[0016] like Figure 1 The scaffold has a regular shape, measuring 10 mm × 10 mm × 5 mm, and is a white porous structure with uniform and regular internal pores. After drying the 20% nHA / PCL scaffold, it was fixed to the operating stage using conductive adhesive, and the surface was sprayed with gold. The microstructure of the scaffold was observed using a scanning electron microscope under an accelerating voltage of 10 kV. Figure 2The scaffold surface was observed to be smooth and flat, with a microporous structure of varying sizes. White granular nHA material was visible inside the scaffold through the cut surface. The microstructure of the scaffold was observed using a scanning electron microscope at an accelerating voltage of 10 kV. The elemental types, distribution, and content of the scaffold surface were analyzed using an energy dispersive spectroscopy (EDS) instrument at an accelerating voltage of 20 kV. Figure 3 Four elements were detected on the surface of the scaffold: O, C, Ca, and P.

[0017] The scaffold was printed to a size of 5×5×5mm, and a compression test was performed using an electronic universal testing machine at a compression speed of 2 mm / min. The results showed that the compressive strength of the 20% nHA / PCL scaffold was (13.76±1.33) MPa, which is consistent with the compressive strength of human cancellous bone. Figure 4 The stress-strain curves show that as stress increases, the deformation is rapid at first and then slow.

[0018] Step 2: Extraction and identification of bone marrow mesenchymal stem cell exosomes (BMSC-Exos): Take healthy 3rd-5th generation BMSCs and, when the cells reach 70-80% confluence, replace the medium with DMEM complete medium containing 10% exosome-free serum and continue culturing for 48 h. Collect the cell supernatant, centrifuge at 2,000 xg for 10 min to remove cell debris. Collect the supernatant again, centrifuge at 2,000 xg at 4°C for 30 min. Collect the supernatant again, centrifuge at 10,000 xg at 4°C for 45 min. Collect the supernatant, filter through a 0.45 μm filter membrane, and collect the filtrate. Centrifuge the filtrate at 4°C at 100,000 xg for 70 min. Remove the supernatant, resuspend the pellet in PBS, filter through a 0.22 μm filter membrane, and ultracentrifuge again at 4°C at 100,000 xg for 70 min. Remove the supernatant and resuspend the pellet in PBS.

[0019] Exosome morphology was observed using transmission electron microscopy (TEM), exosome size distribution was analyzed using NTA, and the expression of exosome surface marker proteins was detected using Western blotting (WB). The charge on exosomes was also detected using zeta potential. Figure 5TEM analysis revealed that the extracted exosomes exhibited a typical saucer-like shape and a complete bilayer membrane structure. NTA particle size analysis showed that the extracted exosomes ranged in size from 30 to 150 nm, with an average particle size of 82.9 nm. Western blotting analysis of exosome surface marker proteins showed positive expression of CD9, CD81, and TSG101 proteins, while Calnexin protein was negatively expressed. These results are consistent with the characteristics of exosome detection, confirming the successful isolation, purification, and extraction of exosomes from healthy BMSCs. Zeta potential analysis revealed that the extracted exosomes carried a negative charge, ranging from -13 to -92 mV with an average potential of -35.87 mV.

[0020] Step 3: Preparation of CSMA precursor solution: The CSMA precursor solution was purchased from Engineering For Life, model number EFL-S-CSMA-100K. The solution had been sterilized with a 0.22μm filter and was a sterile product.

[0021] Step 4: Preparation and characterization of the composite scaffold: After pre-crosslinking the CSMA precursor solution for 1 second, it was injected into the pores of the nHA / PCL scaffold. After the CSMA was fully attached to the nHA / PCL scaffold, it was photocured again for 10 seconds to obtain the nHA / PCL-CSMA composite scaffold.

[0022] After the exosomes and CSMA hydrogel precursor solution were mixed evenly at a concentration of 30 μg / mL, they were pre-crosslinked for 1 s using a curing lamp and then injected into the pores of the nHA / PCL scaffold. After the hydrogel and exosomes were fully attached to the nHA / PCL scaffold, they were photocured again for 10 s to obtain the nHA / PCL-CSMA-Exos composite scaffold.

[0023] like Figure 6 In both the nHA / PCL-CSMA scaffold and the nHA / PCL-CSMA-Exos scaffold, transparent CSMA hydrogel was observed filling the porous structure. The microstructure of the nHA / PCL-CSMA and nHA / PCL-CSMA-Exos composite scaffolds was observed using scanning electron microscopy. Figure 7 Both sets of scaffolds can adhere tightly to the scaffold surface.

[0024] The surface contact angle of tissue-engineered artificial bone fabricated using 3D printing technology was measured using a water contact angle meter via the static droplet method. After drying, nHA / PCL, nHA / PCL-CSMA, and nHA / PCL-CSMA-Exos scaffolds were placed on the operating table. The experimental liquid was 5 μL of deionized water. Images were taken and the contact angle was calculated. Figure 8 The results showed that the contact angles of all three material groups were less than 90°, indicating good hydrophilicity. Among them, the contact angles of the nHA / PCL-CSMA scaffold and the nHA / PCL-CSMA-Exos group were smaller than those of the nHA / PCL group. This demonstrates that the addition of CSMA can improve the hydrophilicity of the material surface.

[0025] Example 2: Performance Testing of Composite Scaffold 1. Blood compatibility of composite stents Rabbit blood was used to prepare a 4% erythrocyte suspension, which was then co-incubated with three groups of scaffolds at 37°C for 4 hours. PBS was used as a negative control, and distilled water as a positive control. After removing the scaffold material, centrifugation was performed, and the color of the solution after co-incubation was captured using a camera. The hemolysis rate was calculated based on the absorbance of the supernatant at 540 nm using a microplate reader. The formula is as follows: Hemolysis rate = (OD sample - OD negative control group) / (OD positive control group - OD negative control group) × 100%. Figure 9 The hemolysis rate of all three stents was less than 5%, indicating that all three materials have good blood cell compatibility.

[0026] 2. Cell proliferation and cell viability staining of composite scaffolds BMSCs were co-cultured with three groups of sterile scaffolds, and the OD values ​​of each group at 450 nm were detected using a microplate reader at 12, 24, 48, and 72 h using a CCK-8 assay kit.

[0027] BMSCs were co-cultured with three sets of sterile scaffolds and stained using a cell live / dead staining kit on day 3. The staining was observed using an inverted fluorescence microscope, and the entire process was conducted in the dark.

[0028] like Figure 10 As time progressed, the OD values ​​of all groups increased, indicating that the number of cells in each scaffold increased over time, demonstrating that all composite scaffolds could promote BMSC proliferation. The nHA / PCL-CSMA-Exos scaffold group showed the most significant cell proliferation ability. Figure 11 Cell viability staining showed that most BMSCs stained green, indicating they were live cells, while only a few dead cells stained red. This indicates that all three scaffold groups had good cell compatibility.

[0029] 3. Cell migration of composite scaffolds BMSCs were evenly seeded into wells of a plate. Once the cells adhered and reached a density of 90%, a scratching technique was performed. Three sets of sterile scaffolds were then added to the wells, and the culture medium was replaced with serum-free culture medium for further incubation. Cell migration was observed under a microscope at 12 and 24 hours. Figure 12 The three composite materials all promoted cell migration, with the nHA / PCL-CSMA-Exos group showing the most significant migration ability, followed by the nHA / HA-CSMA group. This indicates that CSMA hydrogel can promote cell migration of the nHA / PCL scaffold, and the addition of exosomes can synergistically improve the cell migration ability of the scaffold with CSMA hydrogel. Furthermore, the sustained-release effect of CSMA hydrogel prolongs the duration of exosome action on cells.

[0030] 4. Osteogenic induction of composite scaffolds When BMSCs reached 60%-70% confluence, scaffolds from the nHA / PCL, nHA / PCL-CSMA, and nHA / PCL-CSMA-Exos groups were added, and the medium was replaced with osteogenic induction medium. After 7 days of osteogenic induction, alkaline phosphatase staining was performed; after 21 days of osteogenic induction, alizarin red staining was performed. Quantitative analysis was then conducted. Figure 13 Blue-purple ALP and red calcium nodules were observed in all groups. The nHA / PCL-CSMA-Exos group showed the most significant osteogenic effect, followed by the nHA / PCL-CSMA group. Quantitative analysis confirmed this result. CSMA hydrogel can promote early and late osteogenic formation of the nHA / PCL scaffold in vitro, and the addition of exosomes further improved the early and late osteogenic capacity of the nHA / PCL-CSMA group.

[0031] In summary, the present invention combines 3D-printed nHA / PCL scaffolds with CSMA hydrogels and BMSC-Exos to prepare an nHA / PCL-CSMA-Exos composite scaffold that exhibits synergistic enhancements in mechanical support, structural adaptation, hydrophilicity, and biocompatibility, especially in promoting cell migration and osteogenic differentiation. It is a promising material for jawbone defect repair.

[0032] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0033] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a 3D-printed nHA / PCL-CSMA-Exos jawbone defect repair material, characterized in that, Includes the following steps: S1: A porous nHA / PCL composite scaffold was fabricated using 3D printing technology; S2: Preparation of methacrylamide chitosan hydrogel precursor solution; S3: Mix bone marrow mesenchymal stem cell exosomes with the hydrogel precursor solution prepared in step S2 to form an Exos-CSMA mixed solution; S4: The Exos-CSMA mixed solution obtained in step S3 is injected into the pores of the nHA / PCL scaffold obtained in step S1, and ultraviolet light crosslinking is performed to solidify the CSMA hydrogel in situ on the surface of the scaffold and inside the pores, and exosomes are loaded to obtain the 3D printed nHA / PCL-CSMA-Exos jaw defect repair material.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of nano-hydroxyapatite to polycaprolactone in the nHA / PCL scaffold is 1:

4.

3. The preparation method according to claim 1, characterized in that, In step S1, the 3D printing technology is fused deposition modeling; the printing parameters are: layer thickness 0.1 mm, speed 200 mm / s.

4. The preparation method according to claim 1, characterized in that, In step S3, after the bone marrow mesenchymal stem cell exosomes are mixed with the hydrogel precursor solution, the concentration of the exosomes is 20-50 μg / mL.

5. The preparation method according to claim 1, characterized in that, In step S4, the ultraviolet crosslinking process is as follows: first, pre-crosslinking for 1 second allows the Exos-CSMA mixed solution to initially adhere, then inject it into the pores of the scaffold, and then photocuring for another 10 seconds to achieve complete crosslinking.

6. The 3D-printed nHA / PCL-CSMA-Exos jawbone defect repair material prepared by the method according to any one of claims 1-5, characterized in that, The scaffold includes a porous nHA / PCL composite matrix and a methacrylamide chitosan hydrogel coating loaded with bone marrow mesenchymal stem cell exosomes, the coating being attached to the surface and pores of the scaffold matrix.

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