CN111437441A

CN111437441A - Drug-loaded KGN (KGN) nano-fiber scaffold and preparation method and application thereof

Info

Publication number: CN111437441A
Application number: CN202010423013.5A
Authority: CN
Inventors: 杨力; 陈培兴
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2020-07-24

Abstract

The invention relates to a drug loaded KGN nano-fiber scaffold as well as a preparation method and application thereof, belonging to the field of preparation of biological scaffolds. The invention discloses a medicine-carrying KGN nano-fiber scaffold, which is characterized in that the surface of Silk Fibroin (SF) nano-fiber is modified with Polydopamine (PD), amino groups of PD and carboxyl groups of active medicine KGN are utilized, and KGN is loaded on the surface of the nano-fiber through EDC/NHS amide condensation reaction, so that oxidative stress can be eliminated in the early stage, inflammatory reaction can be reduced, and the transformation from an inflammation stage to a tissue regeneration stage and a remodeling stage can be promoted; meanwhile, KGN is continuously slowly released in the middle and later stages to induce the formation of fibrocartilage tissues, and finally the regeneration of interface tissues is realized; in addition, in vitro experiment results prove that the drug-loaded KGN nano-fiber scaffold has an inflammation regulating function, can promote the formation of fibrocartilage of a bone-tendon interface tissue, and has great application potential in interface tissue engineering.

Description

Drug-loaded KGN (KGN) nano-fiber scaffold and preparation method and application thereof

Technical Field

The invention belongs to the field of biological scaffold preparation, and particularly relates to a drug loaded KGN nano-fiber scaffold as well as a preparation method and application thereof.

Background

The interface tissue of the musculoskeletal system can be divided into three major regions: (1) a bone; (2) fibrocartilage; (3) the tendon/ligament, in which fibrocartilage attenuates stress concentration to the maximum extent because it can buffer impact, plays a crucial role in the efficient transmission of mechanical loads. The injury of interface tissues such as bone-tendon/ligament is easily caused in daily physical activities, and when the bone and tendon/ligament are directly combined again through an operation, the original tissue structure, biological function and microenvironment in the interface region are lost, so that the two cannot be effectively integrated with each other, the stability is poor, the secondary injury rate is high, and the clinical important difficulty is caused.

The repair process following tissue injury can go through three important stages, respectively: (1) the inflammatory phase; (2) the tissue regeneration period; (3) a tissue remodeling stage. In the early inflammatory stage, neutrophils, including lymphocytes, monocytes, and macrophages, reach the site of injury to inhibit foreign infection and eliminate necrotic tissue cells. However, the proteases and active oxygen released by these inflammatory cells into the surrounding tissue environment can cause serious damage to fibroblast-like cells, so that the secretion and deposition of collagen and glycosaminoglycans (GAGs) are hindered, and the healing recovery of the tissue is not facilitated. In addition, the timely transition from the initial inflammatory phase to the subsequent tissue neogenesis and remodeling phase following injury is also a key regulatory point for tissue repair^[170](ii) a At the same time, during the tissue regeneration and remodeling stages, many chemical, mechanical and cellular signals are involved in the repair process, including migration, proliferation and directed differentiation in specific directions. In particular, due to the characteristics of interface tissue structure and function, the self-repair guiding ability after injury is insufficient, and the scar-forming vascular tissue cannot meet the biological function of mechanical load transmission. Based on the process, the early inflammatory reaction after tissue injury is supposed to be reduced, and the transformation from the inflammatory phase to the tissue regeneration and remodeling phases is quickened; meanwhile, a local microenvironment which is favorable for cell adhesion and proliferation and can guide cells to directionally differentiate towards the fibrocartilage is provided in the middle and later stages, so that the regeneration of an interface tissue is promoted, and the biological function of the interface tissue is recovered.

The application of the biological material as an ideal tissue engineering strategy has the potential of regulating and controlling the repair and regeneration of damaged tissues. The SF nanofiber scaffold (fibroin nanofiber scaffold) is used as a biomaterial of a bionic ECM (extracellular matrix) structure, has good biocompatibility and mechanical properties, can have different functional characteristics through different surface modifications, and can realize a desired regulation and control effect in tissue engineering. The PD coating (polydopamine coating) on the surface of the nanofiber has good stability and oxidation resistance, and can inhibit inflammatory reaction caused by Reactive Oxygen Species (ROS). In addition, groups abundant in PD (polydopamine), such as catechol, amino groups, imino groups, etc., can provide binding sites for further surface modification, and PD has also been widely studied as a bioactive compound. Kartogenin (kgn), a hydrophobic small molecule drug discovered in recent years, can promote differentiation of Mesenchymal Stem Cells (MSCs) into cartilage-like cells and induce regeneration of cartilage tissue through early research. Subsequent researches find that KGN also has a promoting effect on the repair of interface tissues and can induce the formation of interface fibrocartilage. As a non-toxic and stable synthetic small molecule, KGN itself has a carboxyl group, can be covalently bound to an amino group, and can be simply and conveniently loaded on a biological material. The strategy of drug slow release not only can improve the stability and the availability of the drug, but also has the advantages of orientation, long duration and the like.

In summary, based on the amino structure rich in the reductive PD coating, the small molecule drug KGN with the cartilage induction function is loaded on the SF nanofiber to design and construct a drug-loaded KGN nanofiber scaffold.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a drug-loaded KGN nanofiber scaffold; the second purpose of the invention is to provide a preparation method of the drug-loaded KGN nano-fiber scaffold; the invention also aims to provide application of the medicine-carrying KGN nano-fiber scaffold in interface tissue engineering.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a drug-loaded KGN nanofiber scaffold comprising a fibroin (SF) nanofiber scaffold, a Polydopamine (PD) coating layer modified on the surface of the scaffold, and 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid (KGN) crosslinked with the polydopamine coating layer.

Preferably, the crosslinking is specifically: through amide condensation reaction, covalent crosslinking is carried out on KGN and Polydopamine (PD), and then KGN is introduced onto a nanofiber material to form a drug-loaded KGN nanofiber scaffold.

2. The preparation method of the drug loaded KGN nano-fiber scaffold comprises the following steps:

(1) preparing a fibroin nanofiber scaffold with a polydopamine-modified surface;

(2) preparing a drug-loaded KGN nano-fiber scaffold: and (3) immersing the fibroin nanofiber scaffold with the surface modified with polydopamine into the activated KGN solution, stirring for reaction for at least 24h, taking out, and washing to obtain the drug-loaded KGN nanofiber scaffold.

Preferably, the silk fibroin nanofiber scaffold with the surface modified with polydopamine in the step (1) is prepared by the following method:

a. placing sheet-shaped silkworm cocoon in 0.5% NaHCO₃Boiling the water solution and washing with double distilled water;

b. repeating the operation in the step a to obtain degummed silk, and then placing the degummed silk in a drying oven for drying;

c. placing dried silk in CaCl₂、CH₃CH₂OH and H₂Boiling O in a ternary system composed of O according to a molar ratio of 1:2:8 until the O is completely dissolved, filtering with a membrane with the aperture of 4.5um, dialyzing with double distilled water to remove other ions to obtain a solution, freeze-drying the solution to obtain pure silk fibroin in a sponge state, and storing the pure silk fibroin at the temperature of minus 20 ℃ for later use;

d. c, putting the pure silk fibroin obtained in the step c into hexafluoroisopropanol, and stirring until the solution is clear and transparent to obtain a silk fibroin electrostatic spinning solution;

e. and (3) preparing the fibroin electrostatic spinning solution into a fibroin nano-fiber scaffold by using an electrostatic spinning instrument, and storing the fibroin nano-fiber scaffold at room temperature for later use after airing.

Further preferably, the method specifically comprises the following steps:

a. placing 0.5cm × 0.5cm sheet-shaped Bombyx Bombycis in 0.5% NaHCO solution₃Aqueous solutionBoiling for 30min, and washing with double distilled water;

b. repeating the operation in the step a to obtain degummed silk, and placing the degummed silk in a drying oven for drying;

c. placing dried silk in 55g CaCl₂Heating silk to 6 hours at 70 ℃ in a ternary system formed by mixing 58.2ml of absolute ethyl alcohol and 72ml of triple distilled water to completely dissolve the silk to obtain a 10% silk fibroin solution, filtering the silk fibroin solution by a membrane with the pore diameter of 4.5 microns, dialyzing the silk fibroin solution for 3 days by double distilled water, replacing the double distilled water every 6 hours to remove other ions to obtain a solution, freeze-drying the solution to obtain pure silk fibroin in a sponge state, and storing the pure silk fibroin at-20 ℃ for later use;

d. dissolving the pure silk fibroin prepared in the step c into hexafluoroisopropanol according to the proportion of 1:10, g: ml, and stirring for 3d on a magnetic stirrer to obtain a fibroin electrostatic spinning solution in hexafluoroisopropanol with the zero degree of 10%;

e. sucking the fibroin electrostatic spinning solution by using an injector, preparing SF (sulfur hexafluoride) nano-fibers on an electrostatic spinning instrument by using a 22G dispensing needle, setting the parameters of the electrostatic spinning instrument as the injection speed of 1m L/h, the rotating speed of a receiver of 200rpm, the voltage of 15kV and the receiving distance of 8cm, obtaining a fibroin nano-fiber bracket, and storing the fibroin nano-fiber bracket at room temperature for later use after air drying.

Preferably, the activated KGN solution in step (2) is prepared as follows: adding KGN storage solution into 2-morpholine ethanesulfonic acid (MES) buffer solution, stirring uniformly, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (Sulfo-NHS), and stirring and activating for at least 30min to obtain the activated KGN solution.

Preferably, the mass ratio of the 2-morpholine ethanesulfonic acid, KGN, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxy thiosuccinimide is 310:1:180: 66.

Further preferably, the concentration of 2-morpholinoethanesulfonic acid in the 2-morpholinoethanesulfonic acid (MES) buffer is 0.1 mol/L.

More preferably, the 2-morpholine ethanesulfonic acid (MES) buffer solution is prepared by adding MES (2-morpholine ethanesulfonic acid) and 29.22g NaCl into deionized water according to a molar ratio of 1:5, stirring until the MES and the NaCl are completely dissolved, adjusting the pH to 5.5-6.0 by using a NaOH solution, and keeping the volume to 1L by using the deionized water.

Preferably, the KGN concentration in the KGN stock solution is 20 g/L.

Further preferably, the KGN stock solution is prepared by completely dissolving KGN (Kartogenin) in DMSO in a mass-to-volume ratio of 20:1 to g: L and storing at 4 ℃ until use.

3. The drug loaded KGN nano-fiber scaffold is applied to interface tissue engineering.

The invention has the beneficial effects that:

the invention discloses a drug-loaded KGN nano-fiber scaffold, which is based on an amino structure rich in a reductive PD coating, loads a micromolecule drug KGN with a cartilage induction function on SF nano-fibers, namely, the KGN and the PD are covalently crosslinked through EDC/NHS amide condensation reaction, and then the KGN is introduced onto a nano-fiber material so as to enhance the fiber cartilage inductivity of the material. The drug-loaded KGN nano fiber scaffold can eliminate oxidative stress and reduce inflammatory reaction in early stage, and promote the transformation from the inflammatory stage to the tissue regeneration and remodeling stage; meanwhile, KGN is continuously slowly released in the middle and later stages to induce the formation of fibrocartilage tissues, and finally the regeneration of interface tissues is realized; in addition, in vitro experiment results prove that the drug-loaded KGN nano-fiber scaffold has an inflammation regulating function, can promote the formation of fibrocartilage of a bone-tendon interface tissue, and has great application potential in interface tissue engineering.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.

Drawings

For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 is a process for preparing a drug-loaded KGN nanofiber scaffold according to the present invention;

FIG. 2 is FTIR (A) and KGN-PD-SF nanofiber scaffolds¹H NMR (B) spectrum;

FIG. 3 is a diagram showing the morphology of SF, PD-SF and the drug loaded KGN nanofiber scaffolds prepared in example 1;

fig. 4 is a comparison graph of the oxidation resistance and hydrophilicity of the drug-loaded KGN nanofiber scaffolds, SF, and PD-SF prepared in example 1, wherein a is a contact angle of different materials, B is a color change graph of different materials after reaction with DPPH solution, C is a statistical graph of water contact angles of different materials, and D is the oxidation resistance activity of different materials;

FIG. 5 is the KGN release profile of the drug-loaded KGN nanofiber scaffolds prepared in example 1;

fig. 6 shows the cell activities of different materials (drug loaded KGN nanofiber scaffold (KGN-PD-SF), silk fibroin nanofiber (SF), and polydopamine modified silk fibroin nanofiber (PD-SF)), where a is an immunofluorescence staining pattern of BMSCs cultured on the material for 1, 3, and 7 days, B is a quantitative analysis of cell adhesion of the different materials, and C is the proliferation of BMSCs cultured on the different materials for 1, 3, and 7 days;

FIG. 7 is the intracellular ROS inhibiting ability of a drug loaded KGN nanofiber scaffold (KGN-PD-SF);

fig. 8 shows the in vitro induction capacity of different materials (drug loaded KGN nanofiber scaffold (KGN-PD-SF), silk fibroin nanofiber (SF), and polydopamine modified silk fibroin nanofiber (PD-SF)) for BMSCs differentiation into fibrocartilage;

fig. 9 is an immunofluorescence image of different materials (drug loaded KGN nanofiber scaffold (KGN-PD-SF), silk fibroin nanofiber (SF), and polydopamine modified silk fibroin nanofiber (PD-SF));

FIG. 10 is an immunofluorescent staining of inflammatory markers in the SF, PD-SF and KGN-PD-SF groups 1 week after surgery, wherein A is an immunofluorescent staining pattern for marker COX-2, B is quantitative statistics for marker COX-2, C is an immunofluorescent staining pattern for marker I L-1 β, and D is quantitative statistics for marker I L-1 β;

FIG. 11 is the result of histological staining analysis of the interface region, wherein A is a comparison graph after 4 weeks and 8 weeks, B is an analysis comparison graph of sirius red staining on collagen tissue, C is a comparison graph of interface width, and D is a comparison graph of osteo-tenodesis;

fig. 12 is a schematic diagram of in vivo biomechanical properties for assessing bone-tendon integration effect, wherein a is application of nanofiber material in ligament reconstruction; b is the macroscopic form of the surface of the femoral cartilage; c is the biomechanical profile of the SF, PD-SF and KGN-PD-SF groups after 4 and 8 weeks of surgery.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It should be noted that, in the following embodiments, features in the embodiments may be combined with each other without conflict.

Example 1

The preparation process of the drug loaded KGN nano-fiber scaffold is shown in figure 1, and the specific method comprises the following steps:

(1) preparing an SF nanofiber scaffold (PD-SF) with PD modification on the surface:

a. placing 0.5cm × 0.5cm sheet-shaped Bombyx Bombycis in 0.5% NaHCO solution₃Boiling in water solution for 30min, and washing with double distilled water;

c. placing dried silk in 55g CaCl₂Heating silk to 6 hours at 70 ℃ in a ternary system formed by mixing 58.2ml of absolute ethyl alcohol and 72ml of triple distilled water to completely dissolve the silk to obtain a 10% silk fibroin solution, filtering the silk fibroin solution by a membrane with the pore diameter of 4.5 microns, dialyzing the silk fibroin solution for 3 days by double distilled water, replacing the double distilled water every 6 hours to remove other ions to obtain a solution, freeze-drying the solution to obtain pure silk fibroin in a sponge state, and storing the pure silk fibroin at-20 ℃ for later use; (ii) a

e. sucking the fibroin electrostatic spinning solution by using an injector, preparing SF (sulfur hexafluoride) nano-fibers on an electrostatic spinning instrument by using a 22G dispensing needle, setting the parameters of the electrostatic spinning instrument as the injection speed of 1m L/h, the rotating speed of a receiver of 200rpm, the voltage of 15kV and the receiving distance of 8cm, obtaining a fibroin nano-fiber bracket, and storing the fibroin nano-fiber bracket at room temperature for later use after air drying;

(2) preparing MES activation buffer solution, namely weighing 19.52g of MES (2-morpholine ethanesulfonic acid) and 29.22g of NaCl, adding deionized water, stirring until the MES and the NaCl are completely dissolved, adjusting the pH value to be between 5.5 and 6.0 by using NaOH solution, and continuously adding deionized water until the volume is constant to 1L to obtain MES activation buffer solution;

(3) KGN stock solution, weighing 20mg KGN (Kartogenin), adding 1m L DMSO, stirring to dissolve completely, and storing in a refrigerator at 4 deg.C for use;

(5) activation of KGN carboxyl, namely adding 150u L KGN storage solution into 50m L MES buffer solution, stirring to mix uniformly, then adding 200mg EDC and 550mg N-hydroxy thiosuccinimide (Sulfo-NHS), stirring slightly at room temperature, and carrying out activation reaction for 30 minutes to obtain an activated KGN solution;

(6) preparing a drug-loaded KGN nano-fiber scaffold: and (2) putting the PD modified SF nano-fiber scaffold prepared in the step (1) into the activated KGN solution prepared in the step (5), stirring for reacting for at least 24h, taking out, washing with deionized water, and removing residual EDC, NHS and unreacted KGN on the surface to obtain the drug loaded KGN nano-fiber scaffold (KGN-PD-SF).

Example 2

A drug-loaded KGN nanofiber scaffold (KGN-PD-SF) prepared in example 1 was characterized and the results are shown in FIG. 2, wherein A in FIG. 2 is an FTIR spectrum showing that KGN-PD-SF has a stronger amide absorption peak than PD-SF (PD-modified SF nanofiber scaffold), while KGN-PD-SF has no absorption peak of ammonium salt, and B in FIG. 2 is¹H NMR spectrum, also known as KGN andcompared with PD-SF, KGN-PD-SF has a characteristic absorption peak (7.3-7.9 ppm) of KGN benzene ring protons. The above results show that KGN and PD-SF are covalently cross-linked through an amide bond in a drug-loaded KGN nanofiber scaffold (KGN-PD-SF).

Fig. 3 is a diagram showing a morphology of SF, PD-SF and a drug loaded KGN nanofiber scaffold (KGN-PD-SF) prepared in example 1, from which the drug loaded KGN nanofiber scaffold of the present invention is randomly arranged to form a porous network structure, which is beneficial for intercellular interaction, including transmission of nutrients and gases, and meets the basic requirements of an ideal ECM biomaterial.

Fig. 4 is a comparison graph of the oxidation resistance and hydrophilicity of the drug-loaded KGN nanofiber scaffolds, SF, and PD-SF of the present invention, wherein a is a contact angle of different materials, B is a color change graph of different materials after reaction with DPPH solution, C is a statistical graph of water contact angles of different materials, and D is the oxidation resistance activity of different materials. As can be seen from a and C in fig. 4, after the coating poly-dopamine is added on the SF surface, the contact angle is obviously reduced, and the contact angle of the nanofiber material is increased after KGN is continuously crosslinked, but there is no significant difference, which indicates that the hydrophilicity of the drug-loaded KGN nanofiber scaffold is weakened, the main reason is that the number of amino groups on the surface of the PD functional coating is reduced after KGN crosslinking, but the hydrophilic property of PD has various groups (including catechol, imino group, etc.), so the hydrophilic effect of the amino groups is weakened after KGN crosslinking, but the influence on the overall hydrophilicity of the nanofiber is small; as can be seen from B and D in FIG. 4, the antioxidant capacity of PD-SF is not significantly affected after KGN crosslinking.

Fig. 5 shows the property of the drug-loaded KGN nanofiber scaffold prepared in example 1 to release drug KGN, from which it can be seen that KGN in the drug-loaded KGN nanofiber scaffold can be sustained and released for up to 7 weeks, and is 81.90% during cumulative release. Compared with the prior art, the medicament wrapped in the nano-fiber can be released from the fiber rapidly, and can generate a strong bursting effect within 24 hours, and show a lasting medicament release characteristic, which is mainly attributed to that an amide covalent bond formed between an amino group on PD and KGN carboxyl is stronger than the interaction of physical adsorption and an ionic bond, so that the lasting slow-release characteristic is more consistent with the characteristic of long middle and later periods of interfacial tissue regeneration repair, and a long-term stable inductivity microenvironment can be provided for tissue repair.

In order to evaluate the cell activities of the drug-loaded KGN nanofiber scaffold (KGN-PD-SF), the fibroin nanofiber Scaffold (SF), and the polydopamine-modified fibroin nanofiber scaffold (PD-SF) prepared in example 1, BMSCs on the scaffold material were detected using L IVE/DEAD and MTS kits, respectively, and the results are shown in fig. 6, where a is an immunofluorescence staining pattern of BMSCs cultured on the material for 1, 3, and 7 days, B is a quantitative analysis of cell adhesion of different materials, and C is a proliferation condition of BMSCs cultured on different materials for 1, 3, and 7 days.

To explore the intracellular ROS inhibiting ability of KGN crosslinked drug-loaded KGN nanofiber scaffolds (KGN-PD-SF), BMSCs were also seeded onto the nanofiber scaffolds, and H was added₂O₂To induce intracellular ROS production, final assay using DCFH-DA was performed, the results of which are shown in FIG. 7 (scale 100 μm). From FIG. 7, it was found that KGN-PD-SF has similar fluorescence intensity to PD-SF, both weaker than that of the TCP group, indicating that KGN has no significant effect on ROS scavenging effect of PD after crosslinking.

Next, the induction ability of the KGN-loaded nanofiber scaffolds (KGN-PD-SF) to differentiate BMSCs into fibrocartilage in vitro was continuously studied, BMSCs were inoculated with the material for 7 days, normalization was performed with SF group as control, and qRT-PCR results are shown in fig. 8, where P <0.05, indicating significant difference. From the results of fig. 8, it can be seen that the gene expression level of cartilage and tendon markers was significantly increased in the KGN-PD-SF group compared to the SF group; meanwhile, the gene expression of KGN-PD-SF group Col II and Sox9 is higher than that of other groups; TNMD and SCX expression level trends are similar, and the SF group and the PD-SF group have no significant difference; in addition, the expression level of Col III in KGN-PD-SF group is also obviously higher than that of SF and PD-SF groups.

Immunofluorescence images of fibrocartilage marker proteins were analyzed in comparison, as shown in fig. 9 (scale 50 μm). As can be seen from the results in FIG. 9, after BMSCs were inoculated into the material and cultured for 7 days, the markers related to the development of cartilage and tendon, including TNC, TNMD, Col II and Col III, showed stronger fluorescence intensity on KGN-PD-SF material than the SF and PD-SF groups. This result is consistent with the results of qRT-PCR, indicating that the protein expression level trend remains the same as the mRNA level.

Based on the performance of the drug-loaded KGN nanofiber scaffold (KGN-PD-SF) in the analysis, the regulation and control effect of the drug-loaded KGN nanofiber scaffold on the in vivo oxidative stress inflammatory response is continuously researched, immunofluorescent staining is carried out on two inflammation-related markers, namely COX-2 and I L-1 β, of a tissue sample after 1 week of surgery, and the result is shown in fig. 10 (a ruler is 200 microns), wherein A is an immunofluorescent staining graph of the marker COX-2, B is quantitative statistics of the marker COX-2, C is an immunofluorescent staining graph of the marker I L-1 β, and D is quantitative statistics of the marker I L-1 β.

Histological staining analysis of the interface area was performed 4 and 8 weeks after ligament reconstruction surgery and the results are shown in fig. 11. The hematoxylin-eosin staining results of the tissue sections can show that after 4 weeks, the groups have no significant difference; after 8 weeks, there was significant fibrocartilage-like transitional tissue in the bone-tendon interface region of KGN-PD-SF group, as shown in a in fig. 11. Based on the histological scoring results, it was found that the KGN-PD-SF drug loaded KGN nanofiber scaffold group had higher bone-tendon integration than the SF group (as shown in D in fig. 11). The collagen tissue can be analyzed by sirius red staining, and the result shows that the collagen tissue of the KGN-PD-SF group is significantly more than that of other experimental groups (as shown in B in figure 11); in addition, the collagen fiber orientation consistency of the KGN-PD-SF group interface area is good 8 weeks after the operation, and the anchoring between the fiber and the bone is tighter. The interface width is also an important reference index for evaluating the integration of bone-tendon, and the smaller the interface width is, the better the integration effect can be shown to some extent. As shown in fig. 11, C, there was no significant difference in the width of the interface between the three groups at 4 weeks post-surgery; after 8 weeks, however, the interface width of the KGN-PD-SF group was significantly smaller than that of the other groups (223.5. + -. 5.90. mu.m [ SF group ], 198.1. + -. 28.77. mu.m [ PD-SF group ], 148.5. + -. 17.71. mu.m [ KGN-PD-SF group ]).

Since biomechanical properties are closely related to integration of bone-tendon and the degree of interfacial healing, in vivo biomechanical properties were continuously studied to evaluate the integration effect of bone-tendon. The mechanical tensile results are shown in fig. 12 (wherein a is a schematic diagram of the application of the nanofiber material in ligament reconstruction; B is a macroscopic morphology of the femoral cartilage surface; and C is a biomechanical statistical diagram of the SF, PD-SF and KGN-PD-SF groups after 4 and 8 weeks of operation), and it can be found that the ultimate mechanical load of each group is enhanced from 4 weeks to 8 weeks after operation: wherein there was no significant difference in limit loading between the three groups of samples at 4 weeks post-surgery; after 8 weeks, the ultimate load of KGN-PD-SF and PD-SF material groups is obviously higher than that of SF material groups, and the functional modification is shown to improve the integration effect of the nanofiber material on bone-tendon.

In conclusion, the drug-loaded KGN nano-fiber scaffold (KGN-PD-SF) disclosed by the invention is characterized in physicochemical properties, the functional effects of the drug-loaded KGN nano-fiber scaffold on the cellular level and the in vivo histological level are discussed, and the effects of the multifunctional nano-fiber scaffold on inducing the regeneration of the bone-tendon interface tissue and promoting the integration of the bone-tendon interface tissue and the tendon interface tissue are evaluated. The main results are as follows: (1)¹h NMR and Fourier transform infrared spectrum results show that KGN is successfully crosslinked with the PD coating on the surface of the nanofiber through amide condensation reaction; (2) the contact angle experiment and DPPH method result show that the original physicochemical properties of the nanofiber, including hydrophilicity, inoxidizability and the like, are not obviously changed after KGN functional modification; (3) the drug release characteristics prove that the multifunctional nanofiber scaffold can realize the continuous slow release of the active drug KGN, the release duration can last for 8 weeks, and the period of tissue repair and regeneration can be covered; (4) the results of cell activity and active oxygen detection show that the multifunctional nanofiber scaffold has good cell compatibility and the ability of eliminating intracellular ROS; (5) PCR and immunofluorescence staining experimentsThe results show that the nano-fiber scaffold after KGN functional modification can induce BMSCs to differentiate towards cartilage and tendon directions, can promote the expression of major components of fibrocartilage tissues and has fibrocartilage induction function; (6) in vivo experiment results show that the multifunctional nanofiber scaffold can effectively reduce oxidative stress and inflammatory reaction of an injury area in the early stage of tissue repair based on the synergistic function of PD and KGN, quicken the transition from an inflammation stage to a tissue regeneration and remodeling stage, and can continuously and slowly release inductive molecular drugs in the middle and later stages to promote the repair and regeneration of interface tissues; (7) the result of a biomechanical tensile experiment proves that the multifunctional nanofiber scaffold can promote the functional integration of bone and tendon and improve the bearing capacity of ultimate load.

The invention discloses a multifunctional nano-fiber stent capable of continuously releasing active drugs, namely a drug loaded KGN nano-fiber stent (KGN-PD-SF). Based on the SF nanofiber with the modified PD surface, the KGN is loaded on the surface of the nanofiber through EDC/NHS amide condensation reaction by utilizing amino groups of the PD and carboxyl groups of an active drug KGN, and the multifunctional nanofiber scaffold prepared by the method has the following advantages: (1) the PD coating modified on the surface of the nanofiber has rich amino functional groups, and the nanofiber has higher specific surface area, and a fiber surface modification mode is adopted on the basis of the characteristic, so that the loading capacity of active molecules KGN can be increased, and the interaction between KGN and the periphery is facilitated; (2) the active drug molecule KGN is combined with the nanofiber carrier through an amido bond, and the combination mode of chemical crosslinking is more stable than physical wrapping or adsorption, so that long-term slow release of the active drug molecule KGN can be realized; (3) KGN is used as a hydrophobic micromolecule, the bioavailability of the KGN in a body is low, and the nano-fiber scaffold with good biocompatibility is used as a drug carrier, so that the defect of hydrophobicity is overcome, the utilization rate of the KGN can be improved through continuous slow release, and the side effect is reduced; (4) the PD surface modified SF nano-fiber has ROS removing capacity, can effectively eliminate inflammatory reaction in the early stage of tissue injury, can provide a good local environment for the function of active drug molecules KGN, and the synergistic interaction of PD and KGN is also beneficial to the repair and regeneration of interface fibrocartilage. The nano-fiber scaffold material prepared by the method has great application potential in interface tissue engineering.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Claims

1. The drug-loaded KGN nanofiber scaffold is characterized by comprising a fibroin nanofiber scaffold, a polydopamine coating modified on the surface of the scaffold, and 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid crosslinked with the polydopamine coating.

2. The drug-loaded KGN nanofiber scaffold of claim 1, wherein the cross-linking is specifically: the 2- ([1, 1-biphenyl ] -4-yl carbamoyl) benzoic acid and polydopamine are subjected to covalent crosslinking through an amide condensation reaction, and then the 2- ([1, 1-biphenyl ] -4-yl carbamoyl) benzoic acid is introduced onto a nanofiber material to form a drug loaded KGN nanofiber scaffold.

3. The preparation method of the drug-loaded KGN nano-fiber scaffold, according to any one of claims 1-2, is characterized in that the method comprises the following steps:

(2) preparing a drug-loaded KGN nano-fiber scaffold: and (2) soaking the fibroin nanofiber scaffold with the surface modified with polydopamine into an activated 2- ([1, 1-biphenyl ] -4-yl carbamoyl) benzoic acid solution, stirring for reaction for at least 24h, taking out, and washing to obtain the drug loaded KGN nanofiber scaffold.

4. The preparation method according to claim 3, wherein the silk fibroin nanofiber scaffold with the surface modified with polydopamine in the step (1) is prepared by the following method:

c. placing dried silk in CaCl₂、CH₃CH₂OH and H₂Cooking O in a ternary system according to the molar ratio of 1:2:8 until the O is completely dissolved, performing membrane filtration and double distilled water dialysis to remove other ions, performing freeze drying on the obtained solution to obtain pure silk fibroin in a sponge state, and storing at-20 ℃ for later use;

5. The preparation method according to claim 4, wherein the method is specifically as follows:

6. The method according to claim 3, wherein the activated 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid solution in the step (2) is prepared as follows: adding 2- ([1, 1-biphenyl ] -4-yl carbamoyl) benzoic acid storage solution into 2-morpholine ethanesulfonic acid buffer solution, stirring uniformly, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide, stirring and activating for at least 30min to obtain the activated 2- ([1, 1-biphenyl ] -4-yl carbamoyl) benzoic acid solution.

7. The method according to claim 6, wherein the mass ratio of 2-morpholinoethanesulfonic acid, 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxythiosuccinimide is 310:1:180: 66.

8. The preparation method of claim 6, wherein the concentration of 2-morpholinoethanesulfonic acid in the 2-morpholinoethanesulfonic acid buffer solution is 0.1 mol/L, and the 2-morpholinoethanesulfonic acid buffer solution is prepared by adding 2-morpholinoethanesulfonic acid and NaCl into deionized water according to a molar ratio of 1:5, stirring until complete dissolution, adjusting the pH to 5.5-6.0 with NaOH solution, and fixing the volume to 1L with deionized water.

9. The method according to claim 6, wherein the concentration of 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid in the stock solution of 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid is 20 g/L, and the stock solution of KGN is prepared by completely dissolving 2- ([1, 1-biphenyl ] -4-ylcarbamoyl) benzoic acid in DMSO in a mass-to-volume ratio of 20:1, g: L, and storing the resulting solution at 4 ℃ for use.

10. The use of the drug-loaded KGN nanofiber scaffold of any one of claims 1-2 in interface tissue engineering.