A cascade release stent for glenoid labrum injury repair and a preparation method and application thereof

Abstract

The application provides a novel cascade release stent for glenoid labrum injury repair and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The product comprises a decellularized matrix stent, at least three silicas and a connecting peptide, the silicas have different molar percentages of complex lipids, and different bioactive factors are respectively loaded in the silicas. The preparation method comprises the following steps: preparing at least three silicas loaded with different bioactive factors respectively; preparing a connecting peptide containing a maleimide group at one end and a DSPE-PEG group at the other end; generating a complex of the connecting peptide and the at least three silicas; and treating a decellularized matrix stent, mixing and incubating the complex, and obtaining the product. The application solves the technical problems that the tissue engineering stent in the prior art cannot realize the time-sequenced and ordered release of multiple bioactive factors, and solves the problem that the drug delivery system is difficult to be long-acting and enriched at a target tissue site in the body.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a novel cascade release stent for labrum injury repair and a preparation method and application thereof. BACKGROUND

[0002] The labrum of the hip joint is a fibrocartilage ring attached to the margin of the acetabulum, which is crucial for maintaining joint stability, load distribution and joint lubrication. Labral injury of the hip joint is a common cause of hip pain and limited activity, which seriously affects the quality of life of patients. Current treatment methods, such as traditional autologous tendon reconstruction, have many problems, including donor site complications, differences in biomechanics and biological functions between reconstructed tissues and natural labrum, and poor postoperative healing and high failure rate. Therefore, developing a new artificial labrum or repair stent that can simulate the structure and function of the natural labrum and actively promote tissue regeneration is a technical problem that needs to be solved in clinical practice.

[0003] Tissue engineering provides a new idea for labrum repair. An ideal tissue engineering stent not only needs to provide mechanical support and a three-dimensional space for cell attachment and growth, but also needs to create a microenvironment that can guide stem cells to differentiate into specific tissue types such as fibrocartilage. This usually requires the synergistic action of multiple bioactive factors. However, natural tissue repair is a highly dynamic and orderly process, and different growth factors play a dominant role at different stages. For example, in cartilage regeneration, stem cells first need to be induced to the chondrocyte precursor stage, then differentiate into mature chondrocytes, and finally the new tissue needs to establish a stable blood vessel network to obtain sustained nutritional support.

[0004] Traditional drug delivery methods, such as simply mixing multiple growth factors and implanting them into the body, cannot control their release sequence and time, leading to disordered drug action, low efficiency, and even possible antagonistic effects. How to construct an intelligent delivery system that can simulate the in vivo signal cascade and release multiple bioactive factors on demand and on time is a core challenge currently faced by the field of tissue engineering. In addition, delivery systems such as nanoparticles are easily cleared by the circulatory system in the body, making it difficult to achieve long-term and stable enrichment at the injury site, thereby limiting their therapeutic effect.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The present application aims to provide a novel cascade release stent for labrum injury repair and a preparation method and application thereof, which solves the technical problem that tissue engineering stents in the prior art cannot achieve the time-sequenced and ordered release of multiple bioactive factors, and also solves the problem that drug delivery systems are difficult to achieve long-term enrichment at the target tissue site in the body.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a novel cascaded release stent for labral injury repair, comprising:

[0009] A decellularized matrix (dECM) scaffold;

[0010] At least three types of silicate, wherein the at least three types of silicate have different molar percentages of complex lipids (CFLs) to achieve different release rates of the encapsulated drug; each silicate encapsulates a different bioactive factor;

[0011] A linker peptide connects the decellularized matrix scaffold and the at least three types of silica bodies together, wherein one end of the linker peptide is covalently linked to the decellularized matrix scaffold and the other end is linked to the outer surface of the at least three types of silica bodies.

[0012] Secondly, the present invention also provides a method for preparing the novel cascaded release stent for labral injury repair, comprising the following steps:

[0013] At least three types of silica bodies loaded with different bioactive factors were prepared, wherein the at least three types of silica bodies were composed of complex lipids (CFLs) with different molar percentages.

[0014] A linker peptide containing a maleimide group at one end and a DSPE-PEG group at the other end was prepared by chemical modification.

[0015] The linker peptide is mixed and incubated with at least three types of silica to allow the DSPE-PEG group of the linker peptide to insert into the silica membrane, forming a polypeptide-silica complex.

[0016] The decellularized matrix (dECM) scaffold is treated to generate free thiol groups, and then mixed and incubated with the peptide-silica complex to allow the maleimide groups of the linking peptide to react with the free thiol groups of the decellularized matrix scaffold, forming the novel cascade release scaffold.

[0017] Thirdly, the present invention also provides the application of the aforementioned scaffold in a medical product for repairing tissue damage.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The sequential delivery of cartilage tissue formation inducer (rapid release in 24h), transforming growth factor (continuous release in 3-7 days) and vascular endothelial growth factor (slow release in 7-14 days) was achieved by using silica-based drug delivery technology containing different proportions of CFL.

[0020] (2) By using the cross-linking technology of silica-peptide decellularized matrix, the circulation of nanoparticles in the body is avoided, and the drug is spatially specific to the labral injury site is enriched.

[0021] (3) The decellularized matrix provides a good local microenvironment and mechanical support for stem cells, and in combination with KLD-12 peptide, it specifically regulates the differentiation of stem cells into chondrocytes. This strategy breaks through the technical barriers of traditional combination drug administration, which is "uncontrollable in timing and imprecise in targeting", and provides a new idea for the regeneration and repair of labral tissue. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the structure of the drug cascade release stent of the present invention.

[0024] Figure 2 This is the chemical structural formula of the complex lipid (CFL) used in Example 1 of this invention.

[0025] Figure 3 These are transmission electron microscope (TEM) images of silicon bodies with different CFL ratios in Embodiment 1 of the present invention.

[0026] Figure 4 This is a particle size distribution diagram of silica with different CFL ratios in Example 1 of the present invention.

[0027] Figure 5 This is a ZETA potential diagram of silicon materials with different CFL ratios in Embodiment 1 of the present invention.

[0028] Figure 6 This is a diagram showing the drug encapsulation efficiency and drug loading of silica with different CFL ratios in Example 1 of the present invention.

[0029] Figure 7 This is the cumulative drug release curve of silica with different CFL ratios in Example 1 of the present invention over 21 days.

[0030] Figure 8 This is a characterization diagram of the preparation effect of decellularized matrix (dECM) in Example 1 of the present invention; wherein, Figure 8 In A, meniscus-1 / decellularized matrix-1 is observed macroscopically, while meniscus-2 / decellularized matrix-2 is observed in cross-section. Figure 8 B represents the assessment of DNA content. Figure 8C represents the assessment of GAG (glycosaminoglycan) content. Figure 8 D represents the collagen content assessment.

[0031] Figure 9 This is a schematic diagram of the linker peptide in Embodiment 1 of the present invention.

[0032] Figure 10 This is a graph showing the grafting rate of CFL and linker peptide under different feed ratios in Example 1 of the present invention.

[0033] Figure 11 These are Fourier transform infrared spectra of CFL at different molar concentrations before and after linkage with the linker peptide in Example 1 of the present invention.

[0034] Figure 12 This is a comparison chart of the free thiol content before and after the reaction of dECM with the linker peptide-silica complex in Example 1 of the present invention.

[0035] Figure 13 This is a scanning electron microscope of the decellularized matrix in Example 1 of the present invention; wherein, ECM: decellularized matrix; CFL: decellularized matrix + silica body; KLD: decellularized matrix + KLD polypeptide; ECK: decellularized matrix + silica body + KLD polypeptide.

[0036] Figure 14 The image is a laser confocal microscope fluorescence image of the composite scaffold successfully constructed according to Embodiment 1 of the present invention; wherein, ECM: decellularized matrix; CFL: decellularized matrix + silica body; KLD: decellularized matrix + KLD peptide; ECK: decellularized matrix + silica body + KLD peptide.

[0037] Figure 15 This is a verification diagram of the chondrogenic differentiation effect in Example 1 of the present invention.

[0038] Figure 16 This is a schematic diagram of a single CFL-scaled silica matrix encapsulating multiple growth factors in a comparative example. Detailed Implementation

[0039] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0040] As used in this article:

[0041] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0042] In these embodiments, unless otherwise specified, all parts and percentages are by mass. All undefined concentrations are arbitrary concentrations.

[0043] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0044] Unless otherwise specified, the PBS buffers used in the examples are all commonly used standard PBS buffers, namely 1X PBS pH 7.4 phosphate buffer.

[0045] A novel cascaded release stent for labral injury repair according to an embodiment of the present invention includes:

[0046] A decellularized matrix (dECM) scaffold;

[0047] At least three types of silicate, wherein the at least three types of silicate have different molar percentages of complex lipids (CFLs) to achieve different release rates of the encapsulated drug; each silicate encapsulates a different bioactive factor;

[0048] A linker peptide connects the decellularized matrix scaffold and the at least three types of silica bodies together, wherein one end of the linker peptide is covalently linked to the decellularized matrix scaffold and the other end is linked to the outer surface of the at least three types of silica bodies.

[0049] In some embodiments, the linker peptide is a modified KLD-12 polypeptide having a maleimide group at one end that can react with free thiol groups on the decellularized matrix scaffold, and a group at the other end that can link to the silica.

[0050] In a further embodiment, the group that can connect to the silica matrix is ​​a group obtained by modifying the KLD-12 polypeptide with DSPE-PEG2000-NHS.

[0051] In some embodiments, the at least three silica bodies include: a first silica body having a molar percentage of 20%-30% complex lipids (CFL); a second silica body having a molar percentage of 45%-55% complex lipids (CFL); and a third silica body having a molar percentage of 70%-80% complex lipids (CFL).

[0052] In some preferred embodiments, the at least three silica bodies include: a first silica body with a molar percentage of 25% complex lipids (CFL); a second silica body with a molar percentage of 50% complex lipids (CFL); and a third silica body with a molar percentage of 75% complex lipids (CFL).

[0053] In some embodiments, the structural formula of the complex lipid (CFL) is as follows:

[0054]

[0055] In some embodiments, the first silica body encapsulates the first bioactive factor, cartilage tissue formation inducer Kartogenin (KGN), enabling rapid release of KGN within 48 hours; the second silica body encapsulates the second bioactive factor, transforming growth factor (TGFβ3), enabling continuous release of TGFβ3 within 2-7 days; and the third silica body encapsulates the third bioactive factor, vascular endothelial growth factor (VEGF), enabling slow release of VEGF within 7-14 days.

[0056] A method for preparing a novel cascaded release stent for labral injury repair according to an embodiment of the present invention includes the following steps:

[0057] At least three types of silica bodies loaded with different bioactive factors were prepared, wherein the at least three types of silica bodies were composed of complex lipids (CFLs) with different molar percentages.

[0058] A linker peptide containing a maleimide group at one end and a DSPE-PEG group at the other end was prepared by chemical modification.

[0059] The linker peptide is mixed and incubated with at least three types of silica to allow the DSPE-PEG group of the linker peptide to insert into the silica membrane, forming a polypeptide-silica complex.

[0060] The decellularized matrix (dECM) scaffold is treated to generate free thiol groups, and then mixed and incubated with the peptide-silica complex to allow the maleimide groups of the linking peptide to react with the free thiol groups of the decellularized matrix scaffold, forming the novel cascade release scaffold.

[0061] In some embodiments, the silica body is formed by injecting an ethanol solution containing complex lipids (CFL), phospholipid organic compounds, and bioactive factors into an aqueous buffer solution, followed by ultrasonic treatment and self-assembly.

[0062] The phospholipid organic compound is preferably bisstearoylphosphatidylcholine (DSPC).

[0063] In some preferred embodiments, the linker peptide also has a lysine (K) side chain linked to FITC for tracking.

[0064] The structural formula of the linker peptide is as follows:

[0065]

[0066] In some embodiments, the treatment of the decellularized matrix (dECM) scaffold is performed using a tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) solution.

[0067] The application of the scaffold described in one embodiment of the present invention in a medical product for repairing tissue damage.

[0068] Example 1

[0069] (1) Preparation of silica

[0070] The complex lipid (CFL, with the structural formula shown below) will be used. Figure 2The lipids (as shown) were dissolved in anhydrous ethanol at a concentration of 10 mg / mL and hydrolyzed at room temperature for 8 hours. DSPC and DSPE-PEG-2000 were also dissolved in ethanol at a concentration of 10 mg / mL. Three EP tubes were used to prepare lipid mixtures with CFL molar percentages of 25%, 50%, and 75% respectively, based on a total lipid system of 2 mg. The remainder consisted of DSPC and DSPE-PEG-2000 (for the 75% complex lipids, the molar percentages of DSPC and DSPE-PEG-2000 were 15% and 10%, respectively; for the 50% complex lipids, the molar percentages were 40% and 10%, respectively; and for the 25% complex lipids, the molar percentages were 65% and 10%, respectively). Cy5.5 was added at a ratio of 3% of the system mass for subsequent tracking. KGN was added to a 25% CFL system at a CFL to KGN mass ratio of 10:1; TGFβ3 was added to a 50% CFL system at a CFL to TGFβ3 mass ratio of 10:1; and VEGF was added to a 75% CFL system at a CFL to VEGF mass ratio of 10:1. The three organic phases (ethanol solutions) were slowly injected into PBS buffer using the ethanol injection method under ultrasonic water bath conditions. After injection, the mixture was dialyzed to remove the ethanol, yielding three silica bodies encapsulated with KGN, TGFβ3, and VEGF, respectively designated as 25%CFL@KGN, 50%CFL@TGFβ3, and 75%CFL@VEGF.

[0071] The morphology of each group of silica (25% CFL, 50% CFL, 75% CFL) was observed using transmission electron microscopy, such as... Figure 3 As shown, all silicon bodies are spherical.

[0072] The particle size distribution of each group of silica (25% CFL, 50% CFL, 75% CFL) was determined using a Malvern particle size analyzer (ZETASIZERNANO ZSP analyzer, Malvern, UK). Figure 4 As shown, the particle sizes of 25%CFL, 50%CFL, and 75%CFL silica particles are 63.03 nm ± 2.59 nm, 76.10 nm ± 3.31 nm, and 66.51 nm ± 2.30 nm, respectively.

[0073] Figure 5 The ZETA potentials are shown to be -20.57mV±1.81mV, -21.10mV±1.63mV, and -22.00mV±1.06mV, respectively.

[0074] The encapsulation efficiency and drug loading of silica are as follows: Figure 6As shown, the encapsulation efficiencies of 25% CFL, 50% CFL, and 75% CFL silica were 80.973%±0.112%, 99.597%±0.036%, and 99.226%±0.061%, respectively; the drug loading of 25% CFL, 50% CFL, and 75% CFL silica were 8.097%±0.011%, 9.960%±0.004%, and 9.923%±0.006%, respectively.

[0075] Drug release behavior such as Figure 7 As shown, within 21 days, the cumulative release of 25% CFL@KGN reached 62.23%, exhibiting a rapid release pattern; the cumulative release of 50% CFL@TGFβ3 was 19.40%, exhibiting a medium-term sustained release pattern; while the cumulative release of 75% CFL@VEGF was only 12.74%, exhibiting a long-term slow release pattern. These results strongly demonstrate that precise control of drug release rate can be achieved by adjusting the CFL ratio.

[0076] (2) Preparation of decellularized matrix (dECM)

[0077] Fresh bovine meniscus lateral body section, approximately 2×0.5×0.5 cm in size, was cut, washed with PBS for 2 hours, incubated in 0.25% trypsin-EDTA solution at 4°C for 24 hours, then stirred in 1% sodium dodecyl sulfate (SDS) + 1% Triton X-100 solution for 48 hours; washed with ultrapure water for 3 days, then soaked in 75% ethanol + 0.1% peracetic acid solution for 5-8 hours for sterilization; soaked in ultrapure water for 24 hours, lyophilized for 72 hours, and stored at 4°C to obtain the dECM scaffold.

[0078] Characterization results as follows Figure 8 As shown, the cross-section of the acellular meniscus matrix (ECM) is triangular. DAPI staining of the cell nuclei reveals a large number of nuclei in the meniscus, while the ECM contains virtually no residual cells. Hematoxylin and eosin staining indicates a denser structure in the meniscus, while the ECM has a more porous structure. Safranin-Fix-Green and toluidine blue staining confirms the absence of proteoglycan deposition in the ECM. Compared to the meniscus, the ECM exhibits significantly lower levels of DNA, glycosaminoglycans (GAGs), and collagen, confirming the successful preparation of the acellular matrix.

[0079] (3) Preparation of KLD linker peptide

[0080] 1) Weigh 5g of 2-Chlorotrityl Chloride Resin resin with a degree of substitution of 0.84 mmol / g. Place the resin in a reaction tube, add DCM (15 ml / g), and shake for 30 min to allow the resin to swell. 2) Filter the solvent through a stencil to remove the solvent. Add 3 times the molar excess of Fmoc-L-LEU-OH amino acid, then add 10 times the molar excess of DIEA, and finally add DMF until dissolved. Shake for 1 h. Wash 6 times alternately with DMF and DCM to inoculate the first amino acid. 3) Add 15 ml of 20% piperidine DMF solution (15 ml / g). After 5 min, remove the solution and add another 15 ml of 20% piperidine DMF solution (15 ml / g). After 15 min, deprotect the resin. 4) Remove the piperidine solution. Take ten resin grains, wash three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105℃ for 5 min. A deep blue color indicates a positive reaction. 5) Wash twice with DMF (10 ml / g), twice with methanol (10 ml / g), and twice with DMF (10 ml / g). 6) Add a three-fold excess of the protecting amino acid (Fmoc-L-ASP(OTBU)-OH) and a three-fold excess of HBTU, both dissolved in as little DMF as possible. Add the mixture to the reaction tube, and immediately add a ten-fold excess of NMM (N-methylmorpholine (NMM) is an intermediate in the regulation of plant growth regulator amines). React for 30 minutes to complete the condensation. 7) Wash once with DMF (10 ml / g), twice with methanol (10 ml / g), and twice with DMF (10 ml / g). 8) Repeat steps 2)-7), linking the amino acids in the sequence from right to left. 9) After linking the last 3-MAL, remove the last K-side protecting group DDE with hydrazine hydrate, and react in DMF as the solvent for 45 minutes. Then add 3 times the amount of FITC / DIEA, react for 4 hours, wash twice with DMF (10 ml / g), twice with methanol (10 ml / g), twice with DMF (10 ml / g), and twice with DCM (10 ml / g). Dry under vacuum for 10 minutes to clean the resin. 10) Prepare the cutting solution (TFA 95%; water 1.5%; EDT 2.5%; TIS 1%). Place the resin in a flask or centrifuge tube, with a resin-to-cutting solution ratio of 10 ml / g, and shake at a constant temperature for 120 minutes. 11) precipitate with diethyl ether, wash six times with diethyl ether, and then evaporate to dryness at room temperature to obtain the crude peptide sequence. 12) Place 200 mg of crude peptide in a dish and dissolve it in 2-5 ml of 50% acetonitrile aqueous solution; filter the solution through a 0.45 μm filter membrane; for analysis: take 3 μL and analyze the crude product using analytical grade HPLC with water and acetonitrile as the mobile phase, for 30 min, using gradient elution. Equilibrate the HPLC with the initial gradient for 5 min before injection. The initial gradient is 95% water and 5% acetonitrile, and the final gradient is 5% water and 95% acetonitrile. For preparation: prepare the dissolved sample for injection.Prepare an HPLC equilibration for 10 min, with an initial gradient of 95% water and 5% acetonitrile, and an ending gradient of 25% water and 75% acetonitrile for 40 min. Collect the sample from the detector and perform purity and MS analysis. After passing the analysis, repeat the above steps to process all the remaining crude product. 13) Finally, freeze-dry the purified solution to obtain the final product. 14) Weigh 200 mg of DSPE-PEG2000-NHS and 3 equivalents of the linear peptide pure product, mix and dissolve in the organic solvent DMF, add DIEA dropwise, and stir for 4 h. 15) Cut 10 cm of a dialysis membrane with a specification of 2000MD, transfer the reaction solution into the dialysis membrane and seal it, dialyze in 2 L of PBS for 24 h, then dialyze again in 2 L of PBS for 24 h, and freeze-dry to obtain a white powdery linked peptide with the following structural formula. Figure 9 As shown.

[0081] (4) Linkage between the linker peptide KLD and the silicate CFL

[0082] The silica gel and the linker peptide were dissolved in PBS at a mass ratio of 2:1 and incubated at 60°C for 1-2 hours. Unintercalated peptides were then removed by dialysis. Figure 10 As shown, observing the FITC signal using a fluorescence spectrometer confirms the connection between the peptide and the silica body. Figure 11 As shown, Fourier transform infrared spectroscopy also confirmed the linker peptide and the three molar concentrations of silica.

[0083] (5) Preparation of the final composite scaffold

[0084] The decellularized matrix was immersed in 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) buffer and incubated at room temperature for 30 minutes to generate free thiol groups. The decellularized matrix was washed with PBS buffer to remove excess reducing agent. The linker peptide-silica complex was dissolved in PBS and mixed with the decellularized matrix at a 1:10 mass ratio, and incubated at room temperature for 2 hours to allow maleimide (Mal) to react with the thiol groups to form stable thioether bonds. The decellularized matrix was washed with PBS to remove unreacted linker peptide-silica complexes, finally obtaining the novel cascade release scaffold for labral injury repair, as described above. Figure 1 As shown.

[0085] like Figure 12 As shown, the free thiol content of the decellularized matrix before and after the reaction was determined by Ellman's reagent method, confirming that the free thiol content of the decellularized matrix was significantly reduced after the reaction.

[0086] like Figure 13As shown, SEM was used to evaluate the surface morphology of the decellularized meniscus. An ordered fibrous structure was observed on the surface of the decellularized matrix. After the decellularized matrix was modified with silica and linker peptides, a large number of particles were deposited on the surface, confirming the successful preparation of the composite scaffold.

[0087] like Figure 14 As shown, the fluorescence signal of the decellularized matrix was detected by laser confocal microscopy. The decellularized matrix showed Cy5.5 fluorescence, the linker peptide showed FITC fluorescence, and both fluorescences were present in the composite scaffold ECK, confirming the successful connection of the silica body, linker peptide and decellularized matrix.

[0088] Example 2

[0089] Bone marrow mesenchymal stem cells were co-cultured in vitro with different drug combinations prepared in Example 1. The expression levels of key genes for chondrogenic differentiation (such as the proteoglycan Aggrecan, ACAN) were detected using real-time quantitative PCR (RT-qPCR).

[0090] The results are as follows Figure 15 As shown, compared with the use of KGN or TGF-β3 alone, the combined application of 25% CFL@KGN and 50% CFL@TGFβ3 significantly increased the expression level of the ACAN gene, demonstrating the best effect in promoting chondrogenesis. This proves the superiority of the drug cascade release strategy designed in this invention in terms of biological function.

[0091] Examples 3-6

[0092] Examples 3-6 show the results of silicon matrix and linker peptides at mass ratios of 4:1, 1:1, 1:2, and 1:4, respectively. Figure 10 As shown, the connection between the peptide and the silica body can be confirmed by observing the FITC signal using a fluorescence spectrometer.

[0093] Comparative Example 1

[0094] Using the same materials as in Example 1, BMP2, TGF-β3, and FGF2F were encapsulated in a 75% CFL silica matrix. Figure 16 As shown, there may be disordered release and disordered modes of action among different growth factors.

[0095] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cascaded release stent for repairing labral injuries, characterized in that, include: A decellularized matrix scaffold; At least three types of silicate, said at least three types of silicate having different molar percentages of composite lipids; Each type of silica matrix encapsulates different bioactive factors; A linker peptide connects the decellularized matrix scaffold and the at least three types of silica bodies together, wherein one end of the linker peptide is connected to the decellularized matrix scaffold and the other end is connected to the outer surface of the at least three types of silica bodies; The linker peptide is a modified KLD-12 polypeptide, which has a maleimide group at one end that can react with the free thiol group of the decellularized matrix scaffold, and a group at the other end that can be linked to the silica body. The group that can connect to the silica is a group obtained by modifying the KLD-12 polypeptide with DSPE-PEG2000-NHS.

2. The cascaded release stent for labral injury repair according to claim 1, characterized in that, The at least three silicon-based materials include: The first silicate has a molar percentage of 20%-30% for the complex lipids; The composite lipids consist of a second silicate with a molar percentage of 45%-55%. The composite lipids consist of 70%-80% of a third silicate.

3. The cascaded release stent for labral injury repair according to claim 2, characterized in that, The first silica matrix encapsulates a first bioactive factor, a chondrocyte formation inducer. The second silica matrix encapsulates a second bioactive factor, transforming growth factor; The third silica matrix encapsulates the third bioactive factor, vascular endothelial growth factor.

4. A method for preparing a cascaded release stent for labral injury repair as described in any one of claims 1-3, characterized in that, Includes the following steps: At least three types of silica bodies loaded with different bioactive factors were prepared, wherein the at least three types of silica bodies were composed of composite lipids with different molar percentages. A linker peptide containing a maleimide group at one end and a DSPE-PEG group at the other end was prepared by chemical modification. The linker peptide is mixed and incubated with at least three types of silica to allow the DSPE-PEG group of the linker peptide to insert into the silica membrane, forming a polypeptide-silica complex. The decellularized matrix scaffold is treated to generate free thiol groups, and then mixed and incubated with the peptide-silica complex to allow the maleimide groups of the linking peptide to react with the free thiol groups of the decellularized matrix scaffold, forming the cascade release scaffold.

5. The preparation method according to claim 4, characterized in that, The silica body is formed by injecting an ethanol solution containing complex lipids, phospholipid organic compounds and bioactive factors into an aqueous buffer solution, followed by ultrasonic treatment and self-assembly.

6. The preparation method according to claim 4, characterized in that, The linker peptide also has a lysine side chain at one end linked to FITC for tracking.

7. The preparation method according to claim 4, characterized in that, The decellularized matrix scaffold was treated with tris(2-carboxyethyl)phosphonic acid hydrochloride solution.

8. The use of the scaffold according to any one of claims 1-3 in medical products for repairing tissue damage.

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