A multifunctional nano material for rotator cuff injury combined with osteoporosis and a preparation method thereof

Abstract

This invention relates to the fields of improving the immune microenvironment, promoting angiogenesis and bone regeneration, and specifically to a multifunctional nanomaterial for rotator cuff injury complicated with osteoporosis and its preparation method. This invention develops a multifunctional nanomaterial, Nb2C@Mg-MOF (denoted as NM), for rotator cuff injury complicated with osteoporosis. This nanomaterial exhibits good biocompatibility, can scavenge reactive oxygen species, effectively inhibits inflammatory responses, adipogenic differentiation and osteoclastogenesis, promotes angiogenesis, osteogenic and myogenic differentiation, and can effectively inhibit the progression of osteoporosis and fatty infiltration, while promoting rotator cuff tendon-bone healing.

Description

Technical Field

[0001] This invention relates to the fields of improving the immune microenvironment, promoting angiogenesis and bone regeneration, and specifically to a multifunctional nanomaterial for rotator cuff injury with osteoporosis and its preparation method. Background Technology

[0002] Rotator cuff tears (RCTs) are one of the most common musculoskeletal disorders, typically causing shoulder pain and limited range of motion, accounting for approximately 50% of shoulder diseases. The incidence of RCTs increases with age; it is estimated that 25% of people over 60 and 50% of those over 80 are affected by large-area rotator cuff tears. With the deepening of population aging, this will inevitably place a significant burden on patients and health insurance systems. Although arthroscopic repair can alleviate shoulder pain and restore shoulder function by effectively suturing the torn rotator cuff, the re-tear rate remains high. Therefore, effectively promoting the regeneration of torn rotator cuffs and reducing postoperative re-tear rates are current top priorities.

[0003] During arthroscopic rotator cuff repair (RCT), irreversible degenerative changes such as fatty infiltration (FI), loss of angiogenesis, persistent and excessive oxidative stress and inflammation, and scarred tendon-bone healing significantly increase the difficulty of restoring the rotator cuff's anatomy and physiological function, thus greatly increasing the surgical failure rate. Furthermore, recent studies have shown that osteoporotic patients undergoing arthroscopic rotator cuff repair have a higher revision rate. Osteoporosis, a significant risk factor for re-tears after arthroscopic rotator cuff repair, is often overlooked in RCT treatment. Due to the negative impact of osteoporosis on bone quality, osteoporosis after arthroscopic rotator cuff repair significantly reduces the integrity of the tendon-bone interface, including tendon-bone healing efficiency and anchor loosening or even pull-out. Simultaneously, RCT reduces the range of motion of the affected limb, resulting in loss of mechanical stimulation and further leading to proximal humeral bone loss. Therefore, when combined with osteoporosis, simultaneously improving the quality of regenerated rotator cuff tissue and proximal humeral bone regeneration after arthroscopic rotator cuff repair is crucial.

[0004] Arthroscopic surgery is a commonly used treatment option in clinical practice. Although arthroscopic rotator cuff repair can effectively suture torn rotator cuffs, it still has several limitations. First, while current mainstream suturing techniques (such as single-row, double-row sutures, or suture bridge techniques) can restore the anatomical structure of the tendon-bone interface, they cannot completely replicate the biomechanical properties of a normal rotator cuff. Second, the rotator cuff tendons themselves have poor blood supply (especially the "junction zone" of the supraspinatus tendon), and postoperative healing relies on scar formation rather than the original fibrous structure. Patients with chronic tears or severe degeneration often have tendon fat infiltration and muscle atrophy, further reducing the healing potential. Studies have shown that even if the surgery is successful, the postoperative tendon healing rate is only 60%-80%. Third, the re-tear rate can reach 20%-70% in large or giant tears, especially in elderly patients or those with underlying diseases (such as osteoporosis). Re-tears are often associated with early postoperative overactivity, suture failure, or poor tendon-bone healing, and the effectiveness of secondary surgery is significantly reduced. Finally, while adjuvant techniques such as platelet-rich plasma (PRP), stem cells, or biological patches have been used to promote healing, their efficacy remains controversial. For example, there is no standardized approach to the timing and concentration of PRP application, and biological patches may trigger immune responses or mechanical failure. Summary of the Invention

[0005] In view of this, the present invention has developed a multifunctional nanomaterial Nb2C@Mg-MOF (denoted as NM) for rotator cuff injury complicated with osteoporosis. This nanomaterial has good biocompatibility, can scavenge reactive oxygen species, can effectively inhibit inflammatory response, adipogenic differentiation and osteoclastization, promote angiogenesis, osteogenic and myogenic differentiation, and can effectively inhibit the progression of osteoporosis and fat infiltration, and promote rotator cuff tendon-bone healing.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first technical objective of this invention is to provide a method for preparing a multifunctional nanomaterial for rotator cuff injury combined with osteoporosis, comprising the following steps: S1. Preparation of niobium carbide monolayer nanosheets using wet chemical etching and chemical exfoliation methods: Five grams of Nb₂AlC powder were immersed in 50 ml of hydrofluoric acid solution (HF, 50%) and stirred vigorously for 3 days to selectively etch aluminum. The resulting suspension was centrifuged at 11,000 rpm for 15 minutes to remove excess hydrofluoric acid. The collected multilayer Nb₂C was repeatedly washed with ultrapure water and anhydrous ethanol by centrifugation until the pH reached 6-7. The multilayer Nb₂C was stirred in 40 ml of TPAOH for 12 hours and then sonicated continuously for 12 hours. The supernatant was then collected and dialyzed for 3 days to remove residual TPAOH. The purified filtrate was freeze-dried to obtain monolayer Nb₂C nanosheets. S2. Preparation of magnesium-based metal-organic frameworks (Mg-MOF) using a hydrothermal method: Weigh 2.5 mmol Mg(NO3)2·6H2O and 0.8 mmol 2,5-dihydroxyterephthalic acid (H4DOBDC), and dissolve them in a pre-prepared mixed solution consisting of 67.5 mL N,N-dimethylformamide (DMF), 4.5 mL deionized water, and 4.5 mL anhydrous ethanol. Sonicate the mixture for 30 minutes until the solution is clear and transparent to obtain a Mg-MOF precursor solution. Transfer the precursor solution to a Teflon-lined autoclave at 125 °C, heat for 6 hours, and then allow it to cool naturally to room temperature. Collect the Mg-MOF and purify it by repeated centrifugation. Grind the purified Mg-MOF powder to obtain a pale yellow powder. S3. Preparation of niobium carbide-supported magnesium-based metal-organic frameworks (Nb2C@Mg-MOF) using a hydrothermal method. 4 mg Mg(NO3)2·6H2O, 5 mg 2,5-dihydroxyterephthalic acid (DOBDC) and 66 mg Nb2C were added to a 100 mL beaker, followed by 67.5 mL DMF, 4.5 mL deionized water and 4.5 mL ethanol. The resulting mixture was sonicated for 30 minutes and placed in a Teflon-lined autoclave at 125 °C. After reacting for 6 hours, the mixture was collected and purified by repeated centrifugation to obtain the multifunctional nanomaterial for rotator cuff injury combined with osteoporosis.

[0008] The second technical objective of this invention is to provide a multifunctional nanomaterial for rotator cuff injury combined with osteoporosis, prepared by the method described above.

[0009] Compared with the prior art, the beneficial effects of the present invention are: 1) This invention provides a method for preparing a multifunctional nanomaterial for rotator cuff injury combined with osteoporosis. This method yields a multifunctional nanomaterial (Nb2C@Mg-MOF, NM) assembled from niobium carbide (Nb2C) thin-layer nanosheets loaded with a magnesium-based metal-organic framework (Mg-MOF). Specifically, Nb2C can mimic multiple natural enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) to scavenge reactive oxygen species (ROS), thereby exerting anti-inflammatory properties; magnesium ions (Mg 2+ Magnesium is the fourth most abundant metal cation in the body and actively participates in bone, blood vessel, and muscle regeneration. Theoretically, delivering the magnesium organic framework (Mg-MOF) to the torn rotator cuff and gradually releasing Mg... 2+ It will promote the regeneration of related organizations.

[0010] Furthermore, Nb2C has a high drug-loading specific surface area, which enables Mg-MOF to be deposited on the Nb2C surface. 2+ Slow release enables effective repair and regeneration of osteoporotic RCTs after arthroscopic surgery. The Nb2C@Mg-MOF multifunctional nanomaterial (NM) prepared in this invention provides an ideal strategy for the treatment of rotator cuff injuries complicated with osteoporosis.

[0011] 2) Compared with traditional nanomaterials that promote tendon and bone healing, the multifunctional nanomaterials described in this invention combine the anti-inflammatory and antioxidant effects of Nb2C with those of Mg. 2+ It promotes tissue regeneration, and while promoting the healing of rotator cuff tendon and bone, it can also improve the local immune microenvironment, inhibit fat infiltration in the supraspinatus muscle and promote muscle regeneration, thereby achieving effective repair and regeneration of rotator cuff injury combined with osteoporosis after arthroscopic surgery, and providing a new treatment strategy for tendon and bone healing. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0013] Figure 1 This is a flowchart of the synthesis process of Nb2C@Mg-MOF of the present invention.

[0014] Figure 2 This is a characterization of the Nb2C@Mg-MOF of the present invention.

[0015] Figure 3 This invention evaluates the osteogenic differentiation effect of Nb2C@Mg-MOF in vitro.

[0016] Figure 4 This invention relates to the staining evaluation of Nb2C@Mg-MOF during the healing process in RCT.

[0017] Figure 5 This invention evaluates the in vivo inhibition of osteoporosis and promotion of osteogenic effects of Nb2C@Mg-MOF. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0020] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0021] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0022] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0023] This invention discloses a method for preparing multifunctional nanomaterials for rotator cuff injuries complicated with osteoporosis.

[0024] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0025] Example 1 A method for preparing a multifunctional nanomaterial for rotator cuff injury combined with osteoporosis includes the following steps: S1. Preparation of niobium carbide monolayer nanosheets using wet chemical etching and chemical exfoliation methods: Five grams of Nb₂AlC powder were immersed in 50 mL of hydrofluoric acid solution (HF, 50%) and stirred vigorously for 3 days to selectively etch aluminum. The resulting suspension was centrifuged at 11,000 rpm for 15 minutes to remove excess hydrofluoric acid. The collected multilayer Nb₂C was repeatedly washed with ultrapure water and anhydrous ethanol by centrifugation until the pH reached 6-7. The multilayer Nb₂C was stirred in 40 mL of TPAOH for 12 hours and then sonicated continuously for 12 hours. The supernatant was then collected and dialyzed for 3 days to remove residual TPAOH. The purified filtrate was freeze-dried to obtain monolayer Nb₂C nanosheets. S2. Preparation of magnesium-based metal-organic frameworks (Mg-MOF) using a hydrothermal method: Weigh 2.5 mmol Mg(NO3)2·6H2O and 0.8 mmol 2,5-dihydroxyterephthalic acid (H4DOBDC), and dissolve them in a pre-prepared mixed solution consisting of 67.5 mL N,N-dimethylformamide (DMF), 4.5 mL deionized water, and 4.5 mL anhydrous ethanol. Sonicate the mixture for 30 minutes until the solution is clear and transparent to obtain a Mg-MOF precursor solution. Transfer the precursor solution to a Teflon-lined autoclave at 125 °C, heat for 6 hours, and then allow it to cool naturally to room temperature. Collect the Mg-MOF and purify it by repeated centrifugation. After grinding, obtain a pale yellow Mg-MOF powder. S3. Preparation of niobium carbide-supported magnesium-based metal-organic frameworks (Nb2C@Mg-MOF) using a hydrothermal method. 4 mg Mg(NO3)2·6H2O, 5 mg 2,5-dihydroxyterephthalic acid (DOBDC) and 66 mg Nb2C were added to a 100 mL beaker, followed by 67.5 mL DMF, 4.5 mL deionized water and 4.5 mL ethanol. The resulting mixture was sonicated for 30 minutes and placed in a Teflon-lined autoclave at 125 °C. After reacting for 6 hours, the mixture was collected and purified by repeated centrifugation to obtain the multifunctional nanomaterial for rotator cuff injury combined with osteoporosis.

[0026] like Figure 1 The diagram shows the synthesis flow chart of Nb2C@Mg-MOF. Figure 2 (A) is a TEM image of the original Nb2AlC, Nb2C etched with HF, and Nb2C and NM layered with TPAOH. (B) is the elemental distribution map of NM (Nb, O, Mg, N). (C) is the thermogravimetric curve of Nb2C, Mg-MOF and NM at different temperatures. (D) is the X-ray diffraction (XRD) pattern of Nb2C, Mg-MOF and NM. (E) is the Fourier transform infrared (FTIR) spectrum of Nb2C, Mg-MOF and NM.

[0027] Specifically, this invention prepared bio-based Nb2C nanosheets (Nb2C@Mg-MOF) loaded with Mg-MOF using a wet chemical etching and hydrothermal method, and then characterized the gradually obtained products. Transmission electron microscopy (TEM) revealed that after high-frequency etching, the dense Nb2AlC layer expanded and separated, forming an accordion-like loose multilayer Nb2C structure. Then, the etched Nb2C was layered using TPAOH to obtain few-layer Nb2C with lateral and longitudinal nanoscale dimensions and thickness. To stably load Mg onto the Nb2C nanosheets... 2+ First, Mg is absorbed on the Nb2C surface.2+ Then, DHTA organic ligands were added to form a magnesium-based metal-organic framework. TEM images showed that Mg-MOF nanoparticles were uniformly distributed on the surface of few-layer Nb₂C. Elemental analysis showed that the major elements in NM were uniformly distributed, including carbon (C) and niobium (Nb) in Nb₂C, and magnesium (Mg) and nitrogen (N) in Mg-MOF. Thermogravimetric analysis also showed the loss of relative composition in each sample, proving the successful preparation of NM.

[0028] To further demonstrate the beneficial effects of the present invention and to better understand it, the following experimental examples further illustrate the technical features disclosed in the present invention, but should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above-described invention, without inventive effort, are also considered to fall within the protection scope of the present invention.

[0029] I. Cellular Experiments: 1. Grouping: Different concentrations of Nb2C@Mg-MOF were co-cultured with MC3T3-E1 osteoblasts, and a blank control group was set up.

[0030] Osteogenic activity assessment: MC3T3-E1 cells were seeded at an appropriate density in 6-well plates. When the cell density reached more than 90%, the cells were cultured for 14 days in osteogenic differentiation induction medium (10 mM ascorbic acid, 1 M β-glycerophosphate sodium and 1 mM dexamethasone) with different concentrations of Nb2C@Mg-MOF.

[0031] After co-culture, MC3T3-E1 cells were fixed with 4% paraformaldehyde for 30 minutes according to the manufacturer's ARS and ALP staining kit instructions, and washed three times with PBS. Images of the stained samples were captured under an inverted microscope, and the ARS and ALP positive areas were quantitatively analyzed using ImageJ software. Osteogenesis-promoting function in each group was determined based on the staining results. After co-culture, RNA was extracted, and qRT-PCR was used to verify changes in osteogenic gene expression. Results are shown below. Figure 3 .

[0032] II. Animal experiments: Animal experiments were conducted in accordance with relevant Chinese laws concerning animal experiments and were approved by the Ethics Committee of the Animal Center of Nanchang University. Forty 3-month-old female Sprague-Dawley rats (Beijing Sprague Biotechnology Co., Ltd., China) were used for in vivo studies. These animals were housed in a barrier environment and allowed free access to water and standard experimental feed. The rats were divided into six groups: normal, osteoporosis (OVX), osteoporosis + rotator cuff injury (OVX+RCT), osteoporosis + rotator cuff injury + Mg-MOF (OVX+RCT+MP), osteoporosis + rotator cuff injury + Nb2C (OVX+RCT+NP), and osteoporosis + rotator cuff injury + Nb2C@Mg-MOF (OVX+RCT+NMP).

[0033] A rat osteoporosis model was established by first undergoing bilateral ovariectomy to simulate postmenopausal osteoporosis in human women. The model was established after 3 months. Then, an acute rotator cuff injury repair model was developed, with thermosensitive hydrogels loaded with appropriate materials injected into the sutured tendon-bone interface according to group assignments. All rats were sacrificed 4 weeks post-surgery. Micro-CT scans were performed to analyze bone volume fraction (BV / TV, %), trabecular thickness (Tb.Th, pixel), and trabecular number (Tb.N, 1 / pixel). In addition, immunohistochemistry for inflammatory phenotypes (CD86 and CD163), immunofluorescence of the osteogenic gene (BMP2), Safranin-Fix-Green staining of the tendon-bone interface, and Oil Red staining of the supraspinatus muscle were performed, and quantitative analysis was conducted using ImageJ software.

[0034] Figure 4 , 5 The results showed that the nanomaterial Nb2C@Mg-MOF exhibited better anti-inflammatory, osteoporosis-inhibiting, fibrocartilage-promoting, and fat-infiltrating effects than other groups, demonstrating the material's promoting effect on tendon-bone healing.

[0035] Co-culturing Nb2C@Mg-MOF with osteoblasts demonstrated its osteogenic effect through alkaline phosphatase (ALP) staining and Alizarin Red (ARS) staining. In an osteoporosis-complicated acute rotator cuff injury repair model, the anti-inflammatory, osteogenic, osteoclast-inhibiting, and fat-infiltrating effects of the material were demonstrated through Micro-CT scanning and analysis, Safranin O / Fix Green (SO / FG) staining, CD86 / CD163 immunohistochemistry, BMP2 immunofluorescence, and supraspinatus oil red O staining.

[0036] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a multifunctional nanomaterial for rotator cuff injury combined with osteoporosis, characterized in that, Includes the following steps: S1. Preparation of niobium carbide monolayer nanosheets using wet chemical etching and chemical exfoliation methods: Nb2AlC powder was immersed in hydrofluoric acid solution and stirred vigorously for 3 days. The resulting suspension was centrifuged, and the collected multilayer Nb2C was repeatedly washed by centrifugation with ultrapure water and anhydrous ethanol until the pH value reached 6-7. The multilayer Nb2C was stirred in TPAOH for 12 hours, then continuously sonicated for 12 hours. The supernatant was collected and dialyzed. The purified filtrate was freeze-dried to obtain single-layer Nb2C nanosheets. S2. Preparation of magnesium-based metal-organic frameworks (Mg-MOF) using a hydrothermal method: Weigh Mg(NO3)2·6H2O and 2,5-dihydroxyterephthalic acid, dissolve them in a pre-prepared mixed solution consisting of N,N-dimethylformamide, deionized water, and anhydrous ethanol, and sonicate the mixture until the solution is clear and transparent to obtain a Mg-MOF precursor solution. Transfer the precursor solution to a Teflon-lined autoclave, heat it to react, and then allow it to cool naturally to room temperature. Collect the Mg-MOF and purify it by repeated centrifugation. After grinding, obtain a pale yellow Mg-MOF powder. S3. Preparation of niobium carbide-supported magnesium-based metal-organic frameworks (Nb2C@Mg-MOF) using a hydrothermal method. Mg(NO3)2·6H2O, 2,5-dihydroxyterephthalic acid, and the Nb2C nanosheets prepared in step S1 were added to a beaker, followed by the addition of DMF, deionized water, and ethanol. The resulting mixture was ultrasonically treated and then placed in a Teflon-lined autoclave. After heating and reaction, the mixture was collected and purified by repeated centrifugation to obtain the multifunctional nanomaterial Nb2C@Mg-MOF for rotator cuff injury combined with osteoporosis.

2. The preparation method according to claim 1, characterized in that, In S1, the ratio of Nb2AlC powder, hydrofluoric acid solution, and TPAOH is 5 g: 50 mL: 40 mL, and the concentration of the hydrofluoric acid solution is 50%.

3. The preparation method according to claim 1, characterized in that, In the mixed solution of S2, the molar ratio of N,N-dimethylformamide, deionized water and anhydrous ethanol is 67.5 mL: 4.5 mL: 4.5 mL, and the molar ratio of Mg(NO3)2·6H2O and 0.8 mmol 2,5-dihydroxyterephthalic acid is 2.5 mmol: 0.8 mmol.

4. The preparation method according to claim 1 or 3, characterized in that, In S2, the heating reaction temperature is 120-140℃, and the time is 6-8 hours.

5. The preparation method according to claim 1, characterized in that, In step S3, the ratio of N,N-dimethylformamide, deionized water and anhydrous ethanol is 67.5 mL: 4.5 mL: 4.5 mL, and the ratio of Mg(NO3)2·6H2O, 2,5-dihydroxyterephthalic acid and Nb2C nanosheets prepared in step S1 is 4 mg: 5 mg: 66 mg.

6. The preparation method according to claim 1 or 5, characterized in that, In S3, the heating reaction is carried out at a temperature of 120-140℃ for 6-8 hours.

7. A multifunctional nanomaterial for rotator cuff injury combined with osteoporosis, characterized in that, The material is prepared by the method described in claim 1.

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