Nanofiber functional grading stent and preparation method thereof

Abstract

The invention discloses a nanofiber functional grading scaffold and a preparation method thereof. The preparation method comprises the following steps: step 1, respectively preparing a PLCL / silk fibroin spinning solution, a PLCL / silk fibroin / MDP1 spinning solution and a PLCL / silk fibroin / hydroxyapatite spinning solution; 2, processing the three spinning solutions into nanofiber yarns through electrostatic spinning; and step 3, by taking the PLCL / silk fibroin nanofiber yarns as warp yarns and taking the three kinds of nanofiber yarns as weft yarns in sequence, weaving on a machine to obtain the stent. In the nanofiber functional grading scaffold, polypeptide MDP1 is loaded in the nanofiber yarn for simulating a fibrous cartilage interface, so that the situation that the mechanical property of the whole structure is influenced by scar hyperplasia caused by excessive inflammation of a tendon-bone interface can be prevented; meanwhile, hydroxyapatite with an excellent osteogenesis promoting effect is loaded in the nanofiber yarn for simulating the bone interface, and the mechanical property of the tendon-bone interface is further enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a nanofiber functional graded scaffold and a preparation method thereof. Background Art

[0002] The rotator cuff refers to a group of four muscles and their attached tendon structures located around the shoulder joint, which are crucial for maintaining the stability of the shoulder joint and performing precise movement control. The rotator cuff is connected to the scapula and humerus through tendons, forming a dynamic support structure around the shoulder joint and is generally considered to be the core of shoulder stability. Under normal physiological conditions, the tendons of the rotator cuff ensure the stability of the humeral head in the glenoid fossa and prevent it from abnormal displacement. Because the rotator cuff tendons play an important role in stability and functional control in the shoulder joint, the integrity of its structure is crucial for the normal functioning of the shoulder joint. Rotator cuff injury or tear is the most common pathological condition in shoulder diseases, which is common in high-load, repetitive movements (such as throwing, weightlifting) or traumatic events. Rotator cuff injury often leads to shoulder pain, limited movement function and decreased muscle strength. In severe cases, it may affect daily life and work ability.

[0003] Physical bracing, nonsteroidal anti-inflammatory drugs, and steroid injections are commonly used to treat mild rotator cuff injuries. However, in clinical practice, severe rotator cuff injuries (such as complete tears or tears associated with shoulder instability) are typically treated with surgical procedures such as arthroscopic rotator cuff repair, open rotator cuff repair, rotator cuff reconstruction, and shoulder replacement. However, rotator cuff surgery merely reconnects the residual tendon to the footprint area, neglecting the tendon-bone connection area for stress transfer, resulting in poor postoperative repair outcomes. Furthermore, persistent aseptic inflammation in the rotator cuff prevents normal regeneration of the tendon-bone transition interface, which is replaced by scar tissue with even poorer mechanical properties. This, in turn, can lead to re-tear of the rotator cuff in a significant number of patients. Currently, there is a lack of an ideal artificial rotator cuff tendon-bone interface repair scaffold that provides mechanical support to the rotator cuff while slowly releasing loaded anti-inflammatory drugs, creating a suitable biological microenvironment for tendon-bone interface regeneration, and ensuring a simple and convenient surgical procedure. Therefore, the development of a functionally graded scaffold for tendon-bone injuries is of vital clinical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a nanofiber functional graded scaffold and a preparation method thereof in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect is to provide a method for preparing a nanofiber functional graded scaffold, comprising the following steps:

[0007] Step 1, preparing PLCL / silk fibroin spinning solution, PLCL / silk fibroin / MDP1 spinning solution and PLCL / silk fibroin / hydroxyapatite spinning solution respectively;

[0008] Step 2: Using silk fibroin yarn as the core layer, the three spinning solutions obtained in step 1 are processed into nanofiber yarns by electrospinning to obtain PLCL / silk fibroin nanofiber yarn, MDP1-loaded nanofiber yarn, and hydroxyapatite-loaded nanofiber yarn, respectively;

[0009] Step three, using the PLCL / silk fibroin nanofiber yarn as the warp, the PLCL / silk fibroin nanofiber yarn as the first weft, the nanofiber yarn loaded with MDP1 as the second weft, and the nanofiber yarn loaded with hydroxyapatite as the third weft, weaving them on a machine to obtain the nanofiber functional graded scaffold including a tendon interface simulation area, a fibrocartilage interface simulation area, and a bone interface simulation area in sequence.

[0010] Furthermore, the concentration of silk fibroin in the PLCL / silk fibroin spinning solution is 10-25 mg / mL, and the concentration of PLCL is 30-75 mg / mL;

[0011] The concentration of silk fibroin in the PLCL / silk fibroin / MDP1 spinning solution is 10-25 mg / mL, the concentration of PLCL is 30-75 mg / mL, and the concentration of MDP1 is 0.1-0.5 mg / mL;

[0012] The PLCL / silk fibroin / hydroxyapatite spinning solution has a silk fibroin concentration of 10-25 mg / mL, a PLCL concentration of 30-75 mg / mL, and a hydroxyapatite concentration of 1-10 mg / mL.

[0013] Furthermore, the MDP1 is a polypeptide derived from M2 macrophages, and its amino acid sequence is shown in SEQ ID NO.1.

[0014] Furthermore, the process parameters of the electrospinning are: yarn collection speed is 1-3 cm / s, spinning solution advancement rate is 1.5-2.0 mL / h, the distance between the two opposing syringe spinning heads and the rotating funnel is 8-20 cm, the voltage of the positive electric field is 8-14 KV, the voltage of the negative electric field is 8-14 KV, and the rotation speed of the rotating funnel is 400-800 rpm.

[0015] Furthermore, the electrospun nanofiber yarns were cross-linked in glutaraldehyde vapor for 24-36 hours to obtain nanofiber yarns with greater stability.

[0016] Furthermore, the silk yarn is a single thread with a diameter of 0.5-1.5 mm formed by rotating and stretching 10-20 single fibers with a diameter of 10-30 microns.

[0017] Furthermore, the total number of the warp threads is 12-36.

[0018] The second aspect is to provide a nanofiber functional graded scaffold prepared by the above preparation method, wherein the length of the scaffold is 1.5-2.0 cm and the width is 0.3-0.5 cm.

[0019] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0020] The nanofiber functional graded scaffold of the present invention adopts PLCL / silk fibroin nanofiber yarn, which makes the scaffold have good biocompatibility and mechanical properties; and the nanofiber yarn with PLCL and silk fibroin deposited on the surface is close to the morphology of the extracellular matrix, which can promote the attachment of tendon cells, fibrochondrocytes and mesenchymal stem cells to the surface of the scaffold.

[0021] In the nanofiber functional graded scaffold of the present invention, the nanofiber yarn used to simulate the fibrocartilage interface is loaded with the polypeptide MDP1, which can prevent scar hyperplasia caused by excessive inflammation at the tendon-bone interface and thus affect the overall structural mechanical properties; at the same time, the nanofiber yarn used to simulate the bone interface is loaded with hydroxyapatite with excellent osteogenesis effect, which further enhances the mechanical properties of the tendon-bone interface; in addition, the functional graded scaffold composed of tendon, fibrocartilage and bone interface has a large porosity, which is not only conducive to cell ingrowth and proliferation, but also can effectively promote the proliferation, maturation and matrix secretion of cells in the scaffold gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 (A) is the SEM image of PLCL / silk fibroin nanofiber yarn; (B) is the SEM image of nanofiber yarn loaded with MDP1; (C) is the SEM image of nanofiber yarn loaded with hydroxyapatite.

[0023] Figure 2 Schematic diagram of the functional graded nanofiber scaffold of the present invention.

[0024] Figure 3 The results of the performance evaluation of nanofiber functional graded scaffolds applied to the repair of rotator cuff tear injuries. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0026] The silk fibroin used in the following examples was purchased or prepared by the following method: Na2CO3 was added to 6L of double-distilled water at 100°C to prepare a 0.6mol / L solution, and then 60g of silk cocoons cut into about 2*2cm were added to the above solution and boiled at 100°C for 30 minutes. The cocoons were torn into pieces after each boiling, and this step was repeated twice to fully remove the silk fibroin and other impurities; the washed silk fibroin was placed in a vacuum drying oven for drying at a vacuum degree of -25kPa, a drying temperature of 35°C, and a drying time of 48h; 100mL of 9.1mol / L LiBr solution was prepared, 10g of the above-mentioned dried silk fibroin was added, and dissolved under stirring, and the obtained solution was filtered and dialyzed for 72h, and freeze-dried for use.

[0027] MDP1 (macrophage-derived peptide 1) is a polypeptide derived from M2 macrophages, with the sequence MCSVLLGIGLLEFGLLNTRLIKSQSFIRMRYLRGRGGR (SEQ ID NO. 1). MDP1 was selected from 231 M2 macrophage-derived polypeptides. It is a small peptide with strong biological activity, a suitable aliphatic amino acid index, and high stability. Analysis of the ORFfinder database revealed that MDP1 is derived from the lncRNA MM2P, suggesting that MDP1 may have similar functions in regulating M2 polarization as lncRNA MM2P. MDP1 used in the following examples was purchased or synthesized using the FMoc solid-phase method (and purity verified by HPLC).

[0028] Example 1

[0029] This embodiment provides a nanofiber functional graded scaffold for rotator cuff injury, and the preparation method thereof specifically comprises the following steps:

[0030] Step 1, spinning solution preparation:

[0031] 1) Preparation of PLCL (polylactic acid-polycaprolactone) / silk fibroin spinning solution: 0.25 g of silk fibroin and 0.75 g of PLCL were dissolved in 10 mL of hexafluoroisopropanol;

[0032] 2) Preparation of PLCL / silk fibroin / MDP1 spinning solution: 5 mg MDP1, 0.25 g silk fibroin, and 0.75 g PLCL were dissolved in 10 mL hexafluoroisopropanol;

[0033] 3) Preparation of PLCL / silk fibroin / hydroxyapatite spinning solution: 100 mg of hydroxyapatite, 0.25 g of silk fibroin and 0.75 g of PLCL were dissolved in 10 mL of hexafluoroisopropanol.

[0034] Step 2: Process the three spinning solutions into nanofiber yarns through electrospinning technology:

[0035] The silk yarn serves as the core layer, one end of which passes through the rotating funnel of the electrospinning machine and is fixed on the collector, and the other end is on the rotating yarn drum; the spinning solution is then transferred to the two syringes of the spinning machine, and the syringes are connected to the high-voltage electrostatic generator through a polytetrafluoroethylene tube. The two high-voltage electrostatic generator nozzles apply 14kV positive and negative high-voltage power supplies respectively, the syringe propulsion speed is 1.5-2.0mL / min, and the rotation speed of the rotating funnel is 400-800 rpm; finally, the collected yarns are placed in glutaraldehyde vapor for cross-linking for 24 hours to obtain PLCL / silk fibroin nanofiber yarn, MDP1-loaded nanofiber yarn and hydroxyapatite-loaded nanofiber yarn, respectively.

[0036] Scanning electron microscopy (SEM) was used to analyze the PLCL / silk fibroin nanofiber yarn ( Figure 1 Left), MDP1-loaded nanofiber yarn ( Figure 1 ) and hydroxyapatite-loaded nanofiber yarns ( Figure 1 Right), the results show that the nanofibers deposited on the surface of silk yarn are evenly distributed.

[0037] Step 3: Weaving nanofiber functional graded scaffolds:

[0038] With 24 PLCL / silk fibroin nanofiber yarns as warp, PLCL / silk fibroin nanofiber yarn as weft 1 (named as tendon interface simulation area, promoting tendon tissue growth and regeneration), nanofiber yarn loaded with MDP1 as weft 2 (named as fibrocartilage interface simulation area, local release of anti-inflammatory peptides to inhibit excessive inflammation), nanofiber yarn loaded with hydroxyapatite as weft 3 (named as bone interface simulation area, sustained release of hydroxyapatite to promote bone integration), a functional graded scaffold with continuous interface changes was prepared in sequence by a fully automatic multi-shuttle bifurcated pipeline loom. The size of each interface is 0.5 cm in length and 0.5 cm in width. The final scaffold size is 1.5 cm in length and 0.5 cm in width ( Figure 2 ).

[0039] Verification example (animal experiment)

[0040] To evaluate the in vivo repair ability of functionally graded scaffolds for tendon-bone interface repair in rabbits, a rotator cuff tear model was established. All animal experiments were conducted in accordance with the guidelines approved by the Animal Ethics Committee of Shanghai Tongren Hospital (approval number: A2024-031-01). Twenty-four 16-week-old male New Zealand White rabbits were randomly divided into three groups: a control group (suture repair), a PS scaffold group (PLCL / silk fibroin nanofiber yarn scaffold), and an MDP1 & HA scaffold group (functionally graded scaffold). Animals were anesthetized with intraperitoneal injections of 50 mg / kg ketamine and 5 mg / kg xylazine and then secured to the operating table. The rotator cuff area was shaved, and the skin was disinfected. A skin incision was made along the rotator cuff to expose the tendon, which was then sectioned at the fibrocartilage layer. The tendon interface of the scaffold was secured to the supraspinatus tendon using a Kessler suture technique with biodegradable 5-0 sutures. Simultaneously, holes (approximately 1 mm in diameter) were drilled in the bone attachment area, and surgical sutures were used through the bone channels to anchor the osteogenic interface of the scaffold to the bone tissue. After the surgery, the incisions were sutured, and the entire procedure was performed under sterile conditions. At 8, 16, and 24 weeks postoperatively, the animals were euthanized, and samples were collected for histological analysis.

[0041] The results are as follows Figure 3 As shown in the results, the nanofiber functional graded scaffold of the present invention has a good anti-inflammatory effect in vivo. Compared with the control group (suture repair) and the PS scaffold group (PLCL / silk fibroin nanofiber yarn scaffold), the in vivo anti-inflammatory effect is obvious, the collagen fibers are neatly arranged, the tissue interface at the tendon-bone integration site is clear, and the proliferation of scar tissue can be avoided.

[0042] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a nanofiber functional graded scaffold, characterized in that: The steps include: Step 1, preparing PLCL / silk fibroin spinning solution, PLCL / silk fibroin / MDP1 spinning solution and PLCL / silk fibroin / hydroxyapatite spinning solution respectively; Step 2: Using silk fibroin yarn as the core layer, the three spinning solutions obtained in step 1 are processed into nanofiber yarns by electrospinning to obtain PLCL / silk fibroin nanofiber yarn, MDP1-loaded nanofiber yarn, and hydroxyapatite-loaded nanofiber yarn, respectively; Step three, using the PLCL / silk fibroin nanofiber yarn as the warp, the PLCL / silk fibroin nanofiber yarn as the first weft, the nanofiber yarn loaded with MDP1 as the second weft, and the nanofiber yarn loaded with hydroxyapatite as the third weft, weaving them on a machine to obtain the nanofiber functional graded scaffold including a tendon interface simulation area, a fibrocartilage interface simulation area, and a bone interface simulation area in sequence.

2. The preparation method according to claim 1, characterized in that The concentration of silk fibroin in the PLCL / silk fibroin spinning solution is 10-25 mg / mL, and the concentration of PLCL is 30-75 mg / mL; The concentration of silk fibroin in the PLCL / silk fibroin / MDP1 spinning solution is 10-25 mg / mL, the concentration of PLCL is 30-75 mg / mL, and the concentration of MDP1 is 0.1-0.5 mg / mL; The PLCL / silk fibroin / hydroxyapatite spinning solution has a silk fibroin concentration of 10-25 mg / mL, a PLCL concentration of 30-75 mg / mL, and a hydroxyapatite concentration of 1-10 mg / mL.

3. The preparation method according to claim 1, characterized in that The MDP1 is a polypeptide derived from M2 macrophages, and its amino acid sequence is shown in SEQ ID NO.

1.

4. The preparation method according to claim 1, characterized in that The electrospinning process parameters are as follows: a yarn collection speed of 1-3 cm / s, a spinning solution advancement rate of 1.5-2.0 mL / h, a distance between the two opposing syringe spinning heads and the rotating funnel of 8-20 cm, a positive electric field voltage of 8-14 kV, a negative electric field voltage of 8-14 kV, and a rotating funnel speed of 400-800 rpm.

5. The preparation method according to claim 1, characterized in that The electrospun nanofiber yarns were cross-linked in glutaraldehyde vapor for 24-36 hours to obtain nanofiber yarns with strong stability.

6. The preparation method according to claim 1, characterized in that The silk yarn is a single thread with a diameter of 0.5-1.5 mm formed by rotating and stretching 10-20 single fibers with a diameter of 10-30 microns.

7. The preparation method according to claim 1, characterized in that The total number of the warp threads is 12-36.

8. A nanofiber functional graded scaffold prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The length of the stent is 1.5-2.0 cm, and the width is 0.3-0.5 cm.

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