Shape memory silk protein / magnesium oxide bone repair scaffold and preparation method thereof

Abstract

The application discloses a shape memory silk fibroin / magnesium oxide bone repair stent and a preparation method thereof, which comprises silk fibroin SF and magnesium oxide MgO, and the mass ratio of MgO and silk fibroin is (0.1-3):10. The application introduces MgO nanoparticles into the SF material system, constructs a shape memory silk fibroin / magnesium oxide (SF / MgO) bone repair stent which has excellent mechanical properties, is customizable and has water / blood response, and realizes critical bone defect regeneration.

Description

Technical Field

[0001] This invention relates to the field of bone tissue engineering technology, specifically to a shape memory silk / magnesium oxide bone repair scaffold and its preparation method. Background Technology

[0002] Regeneration of critical-sized bone defects caused by trauma, tumors, or infections is a major challenge for orthopedic surgeons. To date, autologous bone grafting remains the clinical gold standard for treating bone defects; however, autologous grafts have limitations such as postoperative pain, large-segment bone loss, and potential donor complications. Clinical alternatives to bone prostheses, such as titanium alloys and polyetherketones, while possessing excellent mechanical properties, suffer from poor integration between the graft and host bone tissue due to their inherent bioinertness and poor biodegradability, leading to loosening, detachment, and ineffective bone regeneration. Most importantly, most existing bone substitutes lack shape adaptability, resulting in inadequate matching to defects; expanding bone defects into normal bone defects through open surgery increases surgical difficulty and cost, and causes severe trauma, pain, and significant functional impairment for patients.

[0003] Shape memory polymers (SMPs), as an emerging intelligent biomaterial, offer hope for patients with bone defects. SMPs possess the ability to switch between their initial shape and programmed state in response to various stimuli, such as light, heat, electric fields, magnetic fields, or water. They can be implanted into bone tissue via minimally invasive methods and "adapt" to the shape of the bone defect, enabling the scaffold material to achieve close contact with surrounding tissues. However, for biomedical devices, the most physiologically readily available shape-restoring stimuli are temperature and water. The need for external stimulation for SMPs inevitably increases the complexity, cost, and time required for clinical procedures. Therefore, inducing rapid restoration of the material's original shape during patient surgery using water / blood would be highly appealing. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a shape memory silk protein / magnesium oxide bone repair scaffold with excellent mechanical properties, customizability, and water / blood responsiveness, as well as its preparation method. Through simple trimming, it can quickly achieve matching with regular / irregular bone defects and promote in-situ bone regeneration of bone defects.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A shape memory silk fibroin / magnesium oxide bone repair scaffold comprises silk fibroin (SF) and magnesium oxide (MgO), wherein the mass ratio of MgO to silk fibroin is (0.1-3):10, preferably 1:10.

[0007] The method for preparing the shape memory magnesium oxide bone repair scaffold includes the following steps:

[0008] (1) Mix the suspension of MgO nanoparticles and the silk protein solution and stir; then react at -15 to -10℃ for 12 to 24 h to obtain SF / MgO composite cryogel;

[0009] (2) Take out the SF / MgO composite cryogel and thaw it at room temperature to obtain the SF / MgO composite scaffold;

[0010] (3) The thawed SF / MgO composite scaffold is then placed at -80℃ to -20℃ for 12 to 24 hours and freeze-dried to obtain a shape memory silk protein / magnesium oxide bone tissue repair scaffold.

[0011] In step (1), the suspension of MgO nanoparticles is obtained by dissolving MgO nanoparticles in phosphate buffer (PBS).

[0012] In step (1), the concentration of the suspension of MgO nanoparticles is 45-120 mg / mL; preferably 90 mg / mL.

[0013] In step (1), the mass ratio of the MgO nanoparticles to the solute in the silk protein solution is (0.1-3):10, preferably 1:10.

[0014] In step (1), the concentration of the silk protein solution is 50-100 mg / mL.

[0015] In step (1), the silk fibroin solution is a mixed solution of silk fibroin, ethylene glycol diglycidyl ether (EGDE), and tetramethylethylenediamine (TEMED). Preferably, the amount of EGDE added is 1-3 mmol / g per gram of silk fibroin solute; the amount of TEMED added is 0-0.5 v / v% per milliliter of silk fibroin solution, more preferably 0.25 v / v%.

[0016] In step (1), the stirring is a thorough vortex stirring, and the stirring time is 2 to 15 minutes, preferably 5 minutes.

[0017] In step (2), the thawing time is 2 to 12 hours, preferably 6 hours.

[0018] The principle of this invention is as follows: (1) After mixing MgO nanoparticles and silk protein solution, the mixture reacts at a specific temperature for a certain time, resulting in a gel transformation of the silk protein and forming a double-network cryogel of the silk protein. At the same time, the MgO nanoparticles and the double-network cryogel of the silk protein form a composite system with chemical cross-linking, physical cross-linking and electrostatic interaction, thereby obtaining an SF / MgO composite cryogel; (2) The SF / MgO composite cryogel is thawed at room temperature to obtain a solid porous material, namely an SF / MgO composite scaffold; (3) The silk protein used in this invention comes from silkworm cocoons. The SF / MgO composite cryogel has excellent mechanical properties, fatigue resistance and water-induced shape memory properties, and also has excellent bone marrow mesenchymal stem cell (compatibility) and tissue compatibility; (4) Magnesium is an essential element for bone health. It participates in the physiological processes of bone tissue formation, bone metabolism and bone mineral crystallization. Therefore, the magnesium oxide (MgO) used in this invention will not degrade rapidly in the body nor produce hydrogen. It can be used as Mg in bone tissue materials. 2+ Source; (5) This invention introduces MgO nanoparticles into the SF material system to construct a shape memory silk protein / magnesium oxide (SF / MgO) bone repair scaffold with excellent mechanical properties, customizability, and water / blood response, thereby achieving critical bone defect regeneration.

[0019] Compared with the prior art, the present invention has the following advantages and effects:

[0020] (1) The bone repair scaffold of the present invention has excellent mechanical properties: due to the hierarchical structure of silk protein itself, its mechanical properties are excellent. At the same time, the SF / MgO scaffold is a composite system of chemical cross-linking, physical cross-linking and electrostatic interaction, so the SF / MgO scaffold has excellent mechanical properties.

[0021] (2) The bone repair scaffold of the present invention has water / blood responsiveness: the molecular chain of silk protein is mainly composed of heavy chain and light chain; wherein, the heavy chain part is composed of alternating hydrophilic region and hydrophobic region, and the hydrophilic region realizes the water / blood responsiveness of SF / MgO scaffold.

[0022] (3) The bone repair scaffold of the present invention is trimmable: mulberry silk has been used as an FDA-certified clinical suture for many years and has the ability to be trimmed. The SF / MgO scaffold is an organic composite of regenerated silk protein material from silk and nano magnesium oxide particles, which has similar performance characteristics to porous sponges and also has the ability to be trimmed.

[0023] (4) The bone repair scaffold of the present invention exhibits good bioactivity and biocompatibility: the silk fibroin is composed of natural components, such as silk, which has been used clinically as surgical sutures for many years; magnesium is an essential element for bone health, participating in the physiological processes of bone tissue formation, bone metabolism, and bone mineral crystallization; magnesium oxide (MgO) is an oxide of magnesium that neither rapidly degrades nor produces hydrogen in the body. It can serve as a source of magnesium in bone tissue materials. 2+ The source of.

[0024] (5) The bone repair scaffold of the present invention is safe and non-toxic, and the materials used are all naturally derived, and the degradation products are safe and non-toxic.

[0025] (6) The manufacturing process of the present invention is simple and the cost is low. The silk protein used for the scaffold is widely available and the cost is low. Attached Figure Description

[0026] Figure 1 This is the stress-strain curve of the compressive mechanics of the stent of the present invention.

[0027] Figure 2 The figures show the compressive stress-strain curves of the scaffold of the present invention and the pure SF scaffold under cyclic loading-unloading.

[0028] Figure 3 The bracket of this invention can withstand more than 1000 cycles of load-unload compression testing.

[0029] Figure 4 This is a diagram illustrating how the stent of the present invention recovers its original shape in water / blood after being fully compressed.

[0030] Figure 5 This is a diagram illustrating the ability of the stent of the present invention to recover its original shape after being fully compressed in a wet state, compared to a pure SF stent.

[0031] Figure 6 This is a schematic diagram of the bone defect implanted through a small-diameter defect after the scaffold of the present invention has been trimmed.

[0032] Figure 7 The image shows the cell morphology after co-culturing the extract of the scaffold of the present invention with mouse embryonic osteoblasts (MC3T3-E1).

[0033] Figure 8 This is a CT image of the rat skull defect repair using the scaffold of the present invention.

[0034] Figure 9 This is a statistical analysis of bone volume fraction and bone mineral density in rats using the bone scaffold of the present invention for repairing skull defects.

[0035] Figure 10 This is a histological evaluation of bone repair in the scaffold of the present invention.

[0036] Figure 11 This is an immunological evaluation of bone repair after in vivo using the scaffold of the present invention. Detailed Implementation

[0037] To facilitate understanding of the present invention, specific embodiments will be described in detail below. These embodiments will help those skilled in the art to further understand the present invention; however, they are not intended to limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements to the present invention without departing from its conceptual framework, and these modifications and improvements all fall within the scope of protection of the present invention.

[0038] In this invention, the preparation method of the silk protein solution preferably refers to the relevant steps of patent application number 201911257846.2, "A method for preparing a tissue-engineered cartilage scaffold".

[0039] Example 1

[0040] A shape memory silk protein / magnesium oxide bone repair scaffold was prepared using the following steps:

[0041] 1) Dissolve 10g of NaHCO3 in 2L of ultrapure water to prepare a 0.5% NaHCO3 solution, and heat to boiling;

[0042] 2) Add 10g of chopped silkworm cocoons to a NaHCO3 solution (boiling) and boil for 1 hour;

[0043] 3) Remove the silk that has been boiled for 1 hour and rinse it 5 times in ultrapure water;

[0044] 4) Remove the silkworm silk, spread it out on a clean tray, and place it in a 45℃ oven to dry;

[0045] 5) Dissolve 8g of dried silk in 100mL of LiBr (9.3mol / L) and boil in a water bath at 40℃ for 3h until dissolved;

[0046] 6) Filter the dissolved silk protein solution through double-layered gauze into an Erlenmeyer flask;

[0047] 7) The filtered silk protein solution is placed into a dialysis bag with a molecular weight cutoff of 8,000 to 14,000 and dialyzed in ultrapure water for 3 days.

[0048] 8) Pour the dialyzed silk protein solution into centrifuge tubes and place them in a centrifuge. Centrifuge at 12,000 r / min for 15 minutes.

[0049] 9) The centrifuged silk fibroin solution is placed into a dialysis bag with a molecular weight cutoff of 4500. The dialysis bag is then placed in a polyethylene glycol (PEG) solution with a weight / volume percentage concentration of 10% w / v for reverse dialysis to concentrate the silk fibroin concentration to 5-6% w / v, thus obtaining the desired silk fibroin solution.

[0050] 10) Further, a silk protein solution with a weight / volume percentage concentration of 5.5% w / v was stirred with 2 mmol / g ethylene glycol diglycidyl ether (EGDE) and 0.25 v / v % tetramethylethylenediamine (TEMED) and vortexed for 5 min to obtain a mixed solution.

[0051] 11) Dissolve MgO nanoparticles in PBS buffer to obtain a suspension of MgO nanoparticles with a concentration of 90 mg / mL;

[0052] 12) Subsequently, the suspension from 11) was added to the mixed solution from 10) in a ratio of 1:10 between the content of MgO nanoparticles and the content of solute in the silk protein solution, and named SF-1nMgO.

[0053] 13) Pour the mixed solution into 3 mL centrifuge tubes and place the centrifuge tubes in a constant temperature reactor at -10℃ for 24 h;

[0054] 14) Then remove the centrifuge tubes and thaw them in room temperature water for 12 hours;

[0055] 15) Remove the water-containing scaffold from the centrifuge tube, place it in a -40℃ freezer for 24 hours, and then freeze-dry it in a freeze dryer for 24 hours to finally obtain the shape memory silk protein / magnesium oxide bone tissue repair scaffold.

[0056] Examples 2-4

[0057]

[0058] Comparative Example

[0059] A pure silk protein scaffold (i.e., a pure SF scaffold) is prepared using the following steps:

[0060] 1) Dissolve 10g of NaHCO3 in 2L of ultrapure water to prepare a 0.5% NaHCO3 solution, and heat to boiling;

[0061] 2) Add 10g of chopped silkworm cocoons to a NaHCO3 solution (boiling) and boil for 1 hour;

[0062] 3) Remove the silk that has been boiled for 1 hour and rinse it 5 times in ultrapure water;

[0063] 4) Remove the silkworm silk, spread it out on a clean tray, and place it in a 45℃ oven to dry;

[0064] 5) Dissolve 8g of dried silk in 100mL of LiBr (9.3mol / L) and boil in a water bath at 40℃ for 3h until dissolved;

[0065] 6) Filter the dissolved silk protein solution through double-layered gauze into an Erlenmeyer flask;

[0066] 7) The filtered silk protein solution is placed into a dialysis bag with a molecular weight cutoff of 8,000 to 14,000 and dialyzed in ultrapure water for 3 days.

[0067] 8) Pour the dialyzed silk protein solution into centrifuge tubes and place them in a centrifuge. Centrifuge at 12,000 r / min for 15 minutes.

[0068] 9) The centrifuged silk fibroin solution is placed into a dialysis bag with a molecular weight cutoff of 4500. The dialysis bag is then placed in a polyethylene glycol (PEG) solution with a weight / volume percentage concentration of 10% w / v for reverse dialysis to concentrate the silk fibroin concentration to 5-6% w / v, thus obtaining the desired silk fibroin solution.

[0069] 10) Pour the silk protein solution into a 3 mL centrifuge tube and place the centrifuge tube in a constant temperature reactor at -10℃ for 24 h;

[0070] 11) Then remove the centrifuge tubes and thaw them in room temperature water for 12 hours;

[0071] 12) Remove the water-containing scaffold from the centrifuge tube, place it in a -40℃ freezer for 24 hours, and then freeze-dry it in a freeze dryer for 24 hours to finally obtain a pure silk protein scaffold.

[0072] Test Example 1: Mechanical Property Test

[0073] The compressive mechanical properties of the SF / MgO scaffold of this invention were measured using a dynamic mechanical analyzer. Quasi-static and cyclic compression test specimens were cut to 4 mm × 5 mm (diameter × height). Quasi-static compression tests were conducted at a strain rate of -30% / min. Young's compression modulus was calculated based on the initial linear strain range (5%-10%). In low-cycle cyclic compression tests, pure SF scaffolds and SF-1nMgO were fully immersed in phosphate-buffered saline (PBS) solution (pH = 7.4, 37°C) and then subjected to a strain rate of -30% / min from 0% to -30% strain. In high-cycle cyclic compression tests, SF-1nMgO was fully immersed in phosphate-buffered saline (PBS) solution (pH = 7.4, 37°C) and then subjected to a strain rate of -100% / min from -2% to -22% strain.

[0074] Figure 1This is the compression stress-strain curve of the SF / MgO scaffold of the present invention after being fully immersed in phosphate-buffered saline (PBS) solution (pH = 7.4, 37°C), where SF is the comparative example, SF-1nMgO is the scaffold prepared in Example 1, and SF-3nMgO is the scaffold prepared in Example 5. Figure 1 It can be seen that the SF / MgO scaffold of the present invention can withstand more than 70% compressive strain.

[0075] Figure 2 A comparison of the load-unload cyclic compression test between the pure SF scaffold and the SF / MgO scaffold of this invention shows that the SF / MgO scaffold material of this invention has better shape memory properties. The scaffold of this invention is a multi-crosslinked network structure material integrating covalent crosslinking, physical crosslinking, and electrostatic interaction. After compression, the SF amorphous structure deforms, but the structure is not destroyed. During this process, internal stress is generated in the scaffold, promoting its recovery to its original state. More importantly, the silk fibroin consists of relatively disordered hydrophilic regions (amorphous structural domains) and crystalline (β-crystalline structural domains) hydrophobic regions. The hydrophilic blocks provide solubility in water and are responsible for the elasticity and toughness of the silk fibroin. These two factors give the scaffold excellent shape memory properties in water and blood.

[0076] Figure 3 The SF / MgO stent of this invention withstood more than 1,000 load-unload cyclic compression tests. The curves remained intact and changed regularly during the test, indicating that the SF / MgO stent did not suffer structural damage during the test and has excellent shape memory effect and fatigue resistance.

[0077] Test Example 2: Performance Test of Water / Blood Response Recovery

[0078] The ability of the SF / MgO stent of the present invention to recover its shape after being fully compressed was tested using room temperature water and room temperature blood. Multiple stents were flattened together and placed in water and blood, and the recovery time was recorded respectively.

[0079] Figure 4 This is a diagram illustrating how the SF / MgO stent of the present invention recovers its original shape in water / blood after being fully compressed. As can be seen from the diagram, the SF / MgO stent has an extremely fast recovery speed in water, while the rebound speed is relatively slower in relatively viscous blood, but it can still recover its original state within 10 seconds.

[0080] Figure 5 The ability of a pure SF scaffold and the SF / MgO scaffold of this invention to recover their original shape after compression is compared. It can be seen that the SF / MgO scaffold of this invention has better shape memory capability than the pure SF scaffold.

[0081] Test Example 3: Bone defect implantation achieved after material trimming.

[0082] Figure 6 This is a schematic diagram of the SF / MgO scaffold material of the present invention, which is implanted into a bone defect through a small-diameter defect after being trimmed. The diagram shows that the SF / MgO scaffold material of the present invention has the potential for personalized implantation through trimming during clinical surgery.

[0083] Test Example 4: Evaluation of Material Cell Compatibility

[0084] The extract of SF-1nMgO scaffold was prepared according to ISO 10993-12 standard. Simply put, the fixed volume to medium volume ratio (1.25 cm⁻¹) was... 2 mL -1 Sterilized scaffolds were immersed in Dulbecco modified Eagle medium (DMEM, high glucose, Invitrogen, USA) containing 10% (v / v) fetal bovine serum (FBS, Gibco, USA) and 1% (v / v) penicillin / streptomycin (Gibco, USA) (total 30 mL) for 24 hours in a 5% CO2 chamber at 37°C. Cell spreading morphology was assessed using the CLSM method. After fixation with 4% paraformaldehyde, cells were treated with 0.2% Triton X-100 (Sigma, USA) in PBS. Cells were stained with phalloidin-FITC (1:200 dilution, Solarbio, China) for 30 min, and observed using CLSM on slides covered with DAPI-containing medium (Solarbio, China).

[0085] Figure 7 The image shows the cell morphology after co-culturing the extract of the scaffold of the present invention with mouse embryonic osteoblasts (MC3T3-E1). It can be seen that the cells maintained good cell morphology, which indicates that the scaffold material of the present invention has good cell compatibility.

[0086] Test Example 5: Experiment on Bone Defect Repair Using Materials

[0087] A rat skull defect model was used to evaluate the regenerative capacity of scaffolds (Approval No.: 202110-02). The rats were divided into a blank group (defect only), a control group (defect received SF scaffold), and an experimental group (defect received SF / 1nMgO scaffold). After anesthesia and routine preparation, two critical-sized defects of 4 mm in diameter were created in each rat, and scaffolds of approximately 5 mm in diameter were implanted to support the defect tissue. Physical examinations were performed daily throughout the postoperative period. At 4 and 8 weeks postoperatively, rats were sacrificed by injection of an overdose of sodium pentobarbital, and the implanted scaffolds and surrounding tissues were removed. Samples were fixed and processed for further research. All samples were scanned using a micro-CT scanner. After scanning, three-dimensional reconstruction of the bone was achieved using a CT analyzer, and the percentage of bone volume to total bone volume (BV / TV) and local volumetric bone mineral density (BMD) were measured. Histological analysis of the skull reconstruction was performed using H&E and Masson trichrome staining. In addition, to further assess bone formation and angiogenesis, immunofluorescence or immunohistochemical staining was used to detect OCN, BMP-2 and Runx2, VEGF, and CD31 antibodies. Images of stained sections were obtained using a digital slide scanner, and image analysis was performed using IPP 6.0 to quantify expression.

[0088] Figure 8 The images show CT scans of rat skull defects repaired using the bone repair scaffold of the present invention. It can be seen that the addition of MgO particles to the SF material in the bone repair scaffold of the present invention enhances the repair of bone defects.

[0089] Depend on Figure 9 Quantitative analysis showed that the SF-1nMgO group exhibited enhanced bone regeneration, with the highest bone mineral density (BMD) and bone volume / total tissue volume (BV / TV) values. At 8 weeks, the BMD and BV / TV of the SF-1nMgO scaffold group were 0.25 g / cm³. 3 The concentration was 27.2%, and in the SF stent group it was 0.19 g / cm³. 3 The concentration was 24%, while the blank group had only 0.08 g / cm³. 3 and 13%.

[0090] Depend on Figure 10 The H&E staining results showed that the SF-1nMgO group had the highest amount of newly formed bone tissue at each time point. This newly formed bone tissue grew within the scaffold pores and integrated with the scaffold. Conversely, the control group was mostly covered by abundant blue fibrous connective tissue, while the SF group showed a small amount of fibrous tissue and a small amount of new bone tissue. Masson's trichrome staining was used to assess bone regeneration; the SF-1nMgO group showed more purple staining, indicating mature bone, while other groups showed more blue-stained immature woven bone or fibrous tissue.

[0091] Depend on Figure 11It was found that at 8 weeks post-surgery, the SF-1nMgO group had a higher number of CD31-positive cells in the defect site, and the number of blood vessels was 4.1 times and 3.4 times that of the control group, respectively. Furthermore, immunohistochemical staining images of osteogenic-associated proteins BMP-2, Runx-2, OCN, and VEGF showed similar results. The results indicated that osteogenic-associated proteins were expressed at the highest level in the SF-1nMgO group, with weaker expression in the SF group, and limited expression in the control group.

[0092] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a shape memory silk protein / magnesium oxide bone repair scaffold, characterized in that... Includes the following steps: (1) Mix and stir the suspension of MgO nanoparticles and the silk protein solution; then react at -15 to -10℃ for 12 to 24 h to obtain SF / MgO composite cryogel; the mass ratio of MgO nanoparticles to solute in the silk protein solution is (0.1 to 3): 10; (2) Take out the SF / MgO composite cryogel and thaw it at room temperature to obtain the SF / MgO composite scaffold; (3) The thawed SF / MgO composite scaffold is then placed at -80℃ to -20℃ for 12 to 24 hours and freeze-dried to obtain a shape memory silk protein / magnesium oxide bone tissue repair scaffold.

2. The method for preparing the shape memory silk / magnesium oxide bone repair scaffold according to claim 1, characterized in that: In step (1), the suspension of MgO nanoparticles is obtained by dissolving MgO nanoparticles in phosphate buffer.

3. The method for preparing the shape memory silk / magnesium oxide bone repair scaffold according to claim 1, characterized in that: In step (1), the concentration of the suspension of MgO nanoparticles is 45–120 mg / mL.

4. The method for preparing the shape memory silk / magnesium oxide bone repair scaffold according to claim 1, characterized in that: In step (1), the concentration of the silk protein solution is 50~100 mg / mL.

5. The method for preparing the shape memory silk / magnesium oxide bone repair scaffold according to claim 1, characterized in that: In step (1), the silk protein solution is a mixed solution of silk protein, ethylene glycol diglycidyl ether, and tetramethylethylenediamine; 1-3 mmol / g of ethylene glycol diglycidyl ether is added per gram of silk protein solute, and 0-0.5 v / v% of tetramethylethylenediamine is added per milliliter of silk protein solution.

6. The method for preparing the shape memory silk / magnesium oxide bone repair scaffold according to claim 1, characterized in that: In step (1), the stirring is a thorough vortex stirring, and the stirring time is 2 to 15 minutes.

7. The method for preparing the shape memory silk / magnesium oxide bone repair scaffold according to claim 1, characterized in that: In step (2), the thawing time is 2 to 12 hours.

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