A biodegradable tympanic membrane repair material and preparation method thereof

Abstract

The invention discloses a degradable biological tympanic membrane repair material and a preparation method thereof, and relates to the field of ear tympanic membrane repair technology. The method comprises the following steps: providing a gelatin spinning solution and a cross-linking solution, and forming a regenerated loose fiber membrane with a disordered loose fiber structure by an electrostatic spinning process; wherein the thickness of the regenerated loose fiber membrane is 50-270 μm, the cross-linking degree is 50-90%, and the spinning fiber diameter is 400-600 nm; wherein the gelatin can be a drug-modified gelatin, and the method can further include forming a shielding dense layer by an electrostatic spinning process, and forming a double-layer structure with the regenerated loose fiber membrane layer. The biological tympanic membrane repair material of the present invention can replace autologous tissues such as temporalis fascia to realize tympanic membrane perforation repair, is suitable for a variety of surgical procedures such as butterfly cartilage tympanoplasty, and can provide continuous drug sustained release during tympanic membrane repair to ensure the effect of tympanic membrane regeneration and healing.

Description

Technical Field

[0001] The invention relates to an eardrum repair material, and in particular to a degradable biological eardrum repair material and a preparation method thereof. Background Art

[0002] The tympanic membrane is located between the external auditory canal and the tympanic cavity. It is the outer wall of the tympanic cavity, making the tympanic cavity a closed middle ear cavity. The tympanic membrane is composed of the epithelium, fibrous layer, and mucosa layer from the outside to the inside. The tympanic membrane of an adult is about 9 mm high, 8 mm wide, and 0.1 mm thick. Because the tympanic membrane itself is thin and close to the outside world, tympanic membrane perforation is relatively common in clinical practice. Its causes are mostly acute and chronic otitis media, iatrogenic injury, pressure injury, tympanic tube placement, etc. After the tympanic membrane is perforated, external pollutants can enter the middle ear through the perforation and cause infection and pus discharge. Ear pain, tinnitus, and hearing loss are the main clinical manifestations of tympanic membrane perforation.

[0003] Acute tympanic membrane perforation caused by various reasons or smaller tympanic membrane perforation has a high self-healing rate, while larger tympanic membrane perforation or tympanic membrane perforation caused by chronic otitis media with repeated infection and pus discharge usually cannot heal on its own and requires intervention through tympanic membrane repair surgery.

[0004] The commonly used materials for tympanic membrane repair surgery in clinical practice are temporalis fascia, cartilage-perichondrium, and fat. The use of autologous repair materials not only requires the creation of a second surgical area during the operation, resulting in secondary trauma, but also increases the risk of bleeding infection, hematoma, pain, and scar formation at the incision. Moreover, after tympanic membrane repair surgery, it takes a certain amount of time for the Eustachian tube function to recover. Before the Eustachian tube function is restored, the middle ear is always in a hypoxic environment and negative pressure state, which can easily lead to complications such as displacement, depression, adhesion, infection, and re-perforation of the graft. In addition, the current autologous repair materials have the problem of repair failure due to infection, and the long-term anti-inflammatory effect after surgery is not good. It is easy to breed bacteria, scarring, and cause otitis media. At the same time, if autologous tissues and non-degradable tissues represented by cartilage are used, when the tympanic membrane repair heals, the tympanic membrane position cannot be restored to its original transparent state, which is not conducive to the later ear endoscopy diagnosis.

[0005] At the same time, current artificial tympanic membrane repair materials are mostly homogeneous structures, are not removable, cannot meet the needs of various clinical procedures, and have poor versatility.

[0006] In summary, there is an urgent need for an artificial tympanic membrane repair material that can replace autologous tissue, is suitable for a variety of clinical procedures, is degradable and absorbable, has good support and repair properties, releases drugs over a long period of time, and can prevent postoperative tympanic cavity adhesions. Summary of the invention

[0007] In view of the above problems existing in the tympanic membrane repair materials in the prior art, the present invention provides a degradable biological tympanic membrane repair material and a preparation method thereof.

[0008] In one aspect, the present invention provides a method for preparing a biodegradable biological tympanic membrane repair material, the method comprising the following steps:

[0009] providing a spinning solution and a cross-linking solution, wherein the spinning solution comprises gelatin;

[0010] The spinning solution and the cross-linking solution are formed into a regenerated loose fiber membrane through an electrostatic spinning process;

[0011] The thickness of the regenerated loose fiber membrane is 50-270 μm, the cross-linking degree is 50-90%, and the diameter of the spun fiber is 400-600 nm.

[0012] In some embodiments of the present invention, the electrospinning process conditions for forming the regenerated loose fiber membrane are: humidity of 40-50%, rotation speed of 60-120 rpm, and voltage of 10-20 kV.

[0013] In some embodiments of the present invention, the gelatin is drug-modified gelatin, and the preparation of the drug-modified gelatin includes: chlorination of the drug to form a chlorinated drug, and then mixing the chlorinated drug with gelatin to form the drug-modified gelatin.

[0014] In some embodiments of the present invention, the chlorination treatment is to react the drug with thionyl chloride.

[0015] In some embodiments of the present invention, the chloracylated drug is grafted onto gelatin via an ester bond to form drug-modified gelatin.

[0016] In some embodiments of the present invention, the drug is selected from one or more of the following drugs: levofloxacin, ofloxacin, norfloxacin, pefloxacin, enoxacin and ciprofloxacin.

[0017] In some embodiments of the present invention, the drug is ciprofloxacin, the drug-modified gelatin is gelatin-ciprofloxacin GEL-CiFX, and in the spinning solution formed by the drug-modified gelatin, the content of gelatin-ciprofloxacin (GEL-CiFX) is 10% w / v-35% w / v.

[0018] In some embodiments of the present invention, the preparation of the drug-modified gelatin includes: forming a solution from gelatin GEL, chloroacylated ciprofloxacin CiFX-COCl and a solvent thereof, wherein the content of GEL is 10% w / v-35% w / v, and the content of chloroacylated ciprofloxacin is 3% w / v-10.5% w / v.

[0019] In some embodiments of the present invention, in the drug-modified gelatin, the grafting rate of the drug ciprofloxacin is 80%-95%.

[0020] In some embodiments of the present invention, the spinning solution comprises 2.5% w / v-5% w / v sodium alginate.

[0021] In some embodiments of the present invention, the preparation method further comprises the following step: forming a shielding dense layer by an electrostatic spinning process, wherein the spinning solution of the shielding dense layer comprises a degradable hydrophobic polymer.

[0022] In some embodiments of the present invention, the electrospinning process conditions of the shielding dense layer are: humidity of 40%-50%, rotation speed of 2500-3500 rpm, and voltage of 10-20 kV.

[0023] In some embodiments of the present invention, the thickness of the shielding dense layer is 30-100 μm, and the fiber diameter is 600-800 nm.

[0024] In some embodiments of the present invention, the shielding dense layer is formed before the regenerated loose fiber membrane is formed, and the regenerated loose fiber membrane is formed with the shielding dense layer as a base layer.

[0025] In some embodiments of the present invention, the shielding dense layer is a hydrophobic film with a water contact angle of 120-133°, and the regenerated loose fiber membrane is a hydrophilic fiber membrane.

[0026] In some embodiments of the present invention, the fibers of the shielding dense layer are arranged in a directional manner, and the fibers of the regenerated loose fiber membrane are arranged in a disordered manner.

[0027] In some embodiments of the present invention, the shielding dense layer and the regenerated loose fiber membrane can be peeled off from each other, and the peeling force is 0.71-1.15N.

[0028] In some embodiments of the present invention, the degradable hydrophobic polymer is selected from one or more of the following materials: polylactic acid PLA, polylactic acid-glycolic acid copolymer PLGA, polycaprolactone PCL and derivatives thereof.

[0029] In some embodiments of the present invention, the degradable hydrophobic polymer is polylactic acid (PLA), and the spinning solution of the shielding dense layer further contains polylysine (EPL).

[0030] In some embodiments of the present invention, in the spinning solution of the shielding dense layer, the content of polylactic acid PLA is 10% w / v-25% w / v, and the content of polylysine EPL is 2.5% w / v-5% w / v.

[0031] On the other hand, the present invention provides a degradable biological tympanic membrane repair material prepared according to the above preparation method.

[0032] Compared with the prior art, the present invention has the following beneficial technical effects:

[0033] (1) The biodegradable biological tympanic membrane repair material prepared according to the present invention can replace autologous tissues such as temporalis fascia, cartilage-perichondrium and fat to repair tympanic membrane perforation, effectively improving the efficiency of repair surgery and avoiding secondary trauma to patients.

[0034] (2) When the biodegradable biological tympanic membrane repair material prepared by the present invention has a double-layer structure consisting of a shielding dense layer and a regenerated loose fiber membrane, by adjusting the electrospinning process parameter design, a shielding dense layer with a dense and highly oriented fiber structure and a regenerated loose fiber membrane with a disordered loose fiber structure are obtained. The shielding dense layer can provide the tensile strength required for tympanic membrane repair, and is not prone to deformation and contracture during the implantation process. At the same time, the hydrophobic design can prevent postoperative adhesion of the tympanic cavity; the regenerated loose fiber membrane can provide adhesion sites for cells, and can effectively induce the proliferation and repair of the epithelial layer and the mucosal layer. At the same time, the drugs carried by the regenerated loose fiber membrane can provide continuous drug release during the tympanic membrane repair process to ensure the effect of tympanic membrane regeneration and healing.

[0035] (3) The degradable biological tympanic membrane repair material with a double-layer structure prepared by the present invention has a structural difference and a surface wettability difference between the shielding dense layer and the regenerated loose fiber membrane, which makes it removable. In addition to the traditional explantation method, the endoplantation method and the sandwich method, it is also suitable for butterfly cartilage myringoplasty. It is easy to operate clinically, has good versatility, and is suitable for a variety of surgical procedures. When polylysine is added to the shielding dense layer of the degradable biological tympanic membrane repair material of the present invention, the connection firmness between the double-layer structure can be enhanced, and the mechanical properties of the double-layer structure composite membrane can be further improved.

[0036] (4) In the biodegradable biological tympanic membrane repair material prepared by the present invention, the ester bond connection of the regenerated loose fiber membrane allows gelatin to retain sufficient RGD sequences even when the drug is grafted onto gelatin, which can specifically bind to a variety of cell integrins, provide cell adhesion sites, and promote the adhesion and aggregation of epithelial cells and mucosal cells at the perforation site, thereby facilitating the repair of perforated tympanic membranes. At the same time, when sodium alginate SA is added, epithelialization can be further promoted during the tympanic membrane repair process, shortening the repair time at the perforation site and achieving a better repair effect.

[0037] (5) The biodegradable biological tympanic membrane repair material prepared by the present invention realizes drug grafting, so that the biological tympanic membrane has a continuous drug release function during the tympanic membrane repair process. In addition, drug grafting is achieved by grafting with the hydroxyl group of hydroxyproline in gelatin, which not only avoids the cell adhesion site of the RGD sequence that does not affect the perforation repair process, but also provides continuous drug release during the repair process, plays an antibacterial and anti-infection role, and solves the problem of recurrence of inflammation at the perforation site and repair failure caused by infection of the autologous repair material. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1a This is a scanning electron microscope image of the surface of the shielding dense layer of the degradable biological tympanic membrane repair material of Example 7 of the present invention.

[0039] Figure 1b This is a scanning electron microscope image of the surface of the regenerated loose fiber membrane of the degradable biological eardrum repair material of Example 7 of the present invention.

[0040] Figure 2 The live and dead cell fluorescence adhesion staining images of the biodegradable tympanic membrane repair materials of Examples 1-7 of the present invention and the repair materials of Comparative Examples 1-3 after fibroblast proliferation.

[0041] Figure 3 It is a graph showing the in vivo drug release curve of the degradable biological tympanic membrane repair material of Examples 2-4 of the present invention. DETAILED DESCRIPTION

[0042] Various aspects of the present invention will be described in detail below in conjunction with specific embodiments. These specific embodiments are only used to illustrate the present invention and do not constitute any limitation on the protection scope of the present invention.

[0043] Interpretation

[0044] The "oriented fibers" described herein refer to fibers formed by electrospinning that are regularly arranged, for example, substantially parallel along the length direction of the fibers, thereby forming a dense structure, providing scaffold support, and preventing adhesion on the tympanic side.

[0045] The "disordered fibers" described herein refer to fibers formed by electrospinning that are irregularly arranged and randomly disordered, thereby forming a loose fiber structure suitable for cell adhesion.

[0046] In a specific embodiment of the present invention, the regenerated loose fiber membrane has a disordered loose fiber structure and a continuous drug sustained release effect, which can effectively guide the repair of tympanic membrane defects and ensure the regeneration and healing effect of the tympanic membrane. The gelatin spinning solution for forming the regenerated loose fiber membrane uses gelatin or drug-modified gelatin, and the cross-linking agent uses glyoxal. The cross-linking agent can also use other cross-linking agents, such as: one or a combination of glyoxal, glutaraldehyde, and formaldehyde.

[0047] In a specific embodiment of the present invention, the drug in the drug-modified gelatin is ciprofloxacin, and other drugs may also be selected, such as levofloxacin, ofloxacin, norfloxacin, pefloxacin, or enoxacin. The drug is continuously released during the tympanic membrane repair process to play an antibacterial and anti-infection role, thereby solving the problem of recurrence of inflammation at the perforation site and repair failure caused by infection of the autologous repair material.

[0048] In a specific embodiment of the present invention, the spinning solution for forming the regenerated loose fiber membrane contains 2.5% w / v-5% w / v sodium alginate to promote cell climbing.

[0049] In a specific embodiment of the present invention, the shielding dense layer has a directional dense fiber structure and a hydrophobic design. In a specific embodiment, the spinning solution forming the shielding dense layer includes polylactic acid PLA, and other degradable hydrophobic polymers can also be used, for example: one or more of the following degradable hydrophobic polymers can be selected: polylactic acid-glycolic acid copolymer PLGA, polycaprolactone PCL and their derivatives.

[0050] In a specific embodiment of the present invention, the spinning solution for forming the shielding dense layer further comprises polylysine to enhance the connection firmness between the regenerated loose fiber membrane and the shielding dense layer.

[0051] In a specific embodiment of the present invention, the electrospinning conditions of the regenerated loose fiber membrane and the shielding dense layer can be adjusted according to the design requirements of the regenerated loose fiber membrane and the shielding dense layer of the present invention, for example, according to the fiber disordered structure and drug sustained release requirements of the regenerated loose fiber membrane and the fiber oriented structure and density requirements of the shielding dense layer.

[0052] Example 1

[0053] The biological tympanic membrane repair material of this embodiment is prepared by an electrospinning process, which includes the following steps:

[0054] (1) Forming a gelatin spinning solution: Weigh gelatin, add a water-acetic acid-ethyl acetate mixed solvent, stir to dissolve, and prepare a gelatin spinning solution with a mass volume ratio of 20% w / v.

[0055] (2) Weigh glyoxal and dissolve it in water to form a glyoxal solution with a concentration of 15%wt.

[0056] (3) During the spinning process, the gelatin spinning solution and the glyoxal solution are cross-linked in real time; wherein, the electrospinning conditions are: humidity of 40%, rotation speed of 100 rpm, and voltage of 12 kV; spinning forms a regenerated loose fiber membrane, and the spinning fibers are arranged in a disordered manner. The structural parameters of the formed regenerated loose fiber membrane are: layer thickness of 200 μm, and fiber diameter of 400-600 nm.

[0057] (4) The spun fiber membrane obtained in step 3 was cross-linked at 80°C and vacuum treated for 24 hours, with a cross-linking degree of 85%.

[0058] Example 2

[0059] The difference between the biological eardrum repair material of this embodiment and the embodiment 1 is that: drug-modified gelatin is used as the spinning raw material, and the preparation of the biological eardrum repair material includes the following steps:

[0060] (1) Chloroacylation of the drug: Weigh 1 g of ciprofloxacin (CiFX), add 400 mL of a mixture of SOCl2 and THF, heat to 80 °C and reflux for 4-6 h, then perform rotary evaporation, sedimentation, and drying to obtain chloroacylated ciprofloxacin (CiFX-COCl).

[0061] (2) Preparation of drug-modified gelatin: Weigh gelatin (GEL) and CiFX-COCl obtained in step (1), dissolve in acetic acid solution, adjust pH to <5, continue stirring, dialyze, freeze-dry and pulverize to obtain drug-modified gelatin GEL-CiFX. In the resulting solution, the mass volume ratio of gelatin is 10% w / v, the mass volume ratio of CiFX-COCl is 3% w / v, and the drug grafting rate of GEL-CiFX is 80%.

[0062] (3) Preparation of GEL-CiFX spinning solution: GEL-CiFX and SA were weighed, and a water-acetic acid-ethyl acetate mixed solvent was added, and the mixture was stirred to dissolve, thereby obtaining a regenerated loose fiber membrane spinning solution; wherein the mass volume ratio of GEL-CiFX was 10% w / v, and the mass volume ratio of SA was 5% w / v.

[0063] (4) Weigh glyoxal and dissolve it in water to form a glyoxal solution with a concentration of 2%wt.

[0064] (5) During the spinning process, the drug-modified gelatin spinning solution GEL-CiFX and the glyoxal solution were cross-linked in real time; the electrospinning conditions were: humidity 50%, rotation speed 120 rpm, and voltage 10 kV; spinning formed a regenerated loose fiber membrane, and the spinning fibers were arranged in a disordered manner. The structural parameters of the regenerated loose fiber membrane formed were: layer thickness 50 μm, and fiber diameter 400-600 nm.

[0065] (6) The spun fiber membrane obtained in step (5) was cross-linked at 80°C and vacuum treated for 24 hours, with a cross-linking degree of 50%.

[0066] Example 3

[0067] The preparation of the biological tympanic membrane repair material of this embodiment includes the following steps:

[0068] (1) Chloroacylation of the drug: Weigh 1 g of ciprofloxacin (CiFX), add 400 mL of a mixture of SOCl2 and THF, heat to 80 °C and reflux for 4-6 h, then perform rotary evaporation, sedimentation, and drying to obtain chloroacylated ciprofloxacin (CiFX-COCl).

[0069] (2) Preparation of drug-modified gelatin: Weigh gelatin (GEL) and CiFX-COCl and dissolve them in acetic acid solution. Adjust the pH to <5. After continuous stirring, dialyze, freeze-dry and pulverize to obtain drug-modified gelatin (GEL-CiFX). In the resulting solution, the mass volume ratio of gelatin is 35% w / v, the mass volume ratio of CiFX-COCl is 10.5% w / v, and the drug grafting rate of GEL-CiFX is 95%.

[0070] (3) Preparation of GEL-CiFX spinning solution: GEL-CiFX and SA were weighed, and a water-acetic acid-ethyl acetate mixed solvent was added, and the mixture was stirred to dissolve, thereby obtaining a regenerated loose fiber membrane spinning solution; wherein the mass volume ratio of GEL-CiFX was 35% w / v, and the mass volume ratio of SA was 2.5% w / v.

[0071] (4) Weigh glyoxal and dissolve it in water to form a glyoxal solution with a concentration of 20%wt.

[0072] (5) During the spinning process, the GEL-CiFX spinning solution and the glyoxal solution were cross-linked in real time; the electrospinning conditions were: humidity 40%, rotation speed 60 rpm, and voltage 20 kV; the spinning formed a regenerated loose fiber membrane, and the spinning fibers were arranged in a disordered manner. The structural parameters of the regenerated loose fiber membrane formed were: layer thickness 270 μm, and fiber diameter 400-600 nm.

[0073] (6) The spun fiber membrane obtained in step (5) was cross-linked at 80°C and vacuum treated for 24 hours, with a cross-linking degree of 90%.

[0074] Example 4

[0075] The preparation of the biological tympanic membrane repair material of this embodiment includes the following steps:

[0076] (1) Chloroacylation of the drug: Weigh 1 g of CiFX, add 400 mL of a mixture of SOCl2 and THF, heat to 80 °C and reflux for 4-6 h, then perform rotary evaporation, sedimentation, and drying to obtain CiFX-COCl.

[0077] (2) Preparation of drug-modified gelatin: GEL and CiFX-COCl were weighed and dissolved in acetic acid solution, the pH was adjusted to <5 and stirred continuously, followed by dialysis, freeze-drying and pulverization to obtain GEL-CiFX; in the resulting solution, the mass volume ratio of GEL was 25% w / v, and the mass volume ratio of CiFX-COCl was 6.9% w / v; the drug grafting rate of GEL-CiFX was 90%.

[0078] (3) Preparation of GEL-CiFX spinning solution: GEL-CiFX and SA were weighed, and a water-acetic acid-ethyl acetate mixed solvent was added, and the mixture was stirred to dissolve, thereby obtaining a regenerated loose fiber membrane spinning solution; wherein the mass volume ratio of GEL-CiFX was 20% w / v, and the mass volume ratio of SA was 3% w / v.

[0079] (4) Weigh glyoxal and dissolve it in water to form a glyoxal solution with a concentration of 15%wt.

[0080] (5) During the spinning process, the GEL-CiFX spinning solution and the glyoxal solution were cross-linked in real time; the electrospinning conditions were: humidity of 40%, rotation speed of 100 rpm, and voltage of 12 kV; the spinning formed a regenerated loose fiber membrane, and the spinning fibers were arranged in a disordered manner. The structural parameters of the regenerated loose fiber membrane formed were: layer thickness of 200 μm, and fiber diameter of 400-600 nm.

[0081] (6) The spun fiber membrane obtained in step (5) was cross-linked at 80°C and vacuum treated for 24 hours, with a cross-linking degree of 85%.

[0082] Example 5

[0083] The preparation of the biological tympanic membrane repair material of this embodiment includes the following steps:

[0084] (1) Preparing a spinning solution for a shielding dense layer: weigh polylactic acid (PLA) and polylysine (EPL), dissolve them in acetone, and mix them evenly to obtain a spinning solution for a shielding dense layer, wherein the mass volume ratio of PLA is 10% w / v, and the mass volume ratio of EPL is 2.5% w / v.

[0085] (2) The spinning solution prepared in step (1) is used to spin a shielding dense layer having a directional fiber structure through an electrospinning process; the electrospinning conditions are: humidity 40%, rotation speed 2500 rpm, and voltage 10 kV; the structural parameters of the formed shielding dense layer are: layer thickness 50 μm, and fiber diameter 600-800 nm.

[0086] (3) The shielding dense layer formed in step (2) is used as the base layer, and a regenerated loose fiber membrane is further formed on the shielding dense layer by electrospinning. The preparation steps of the regenerated loose fiber membrane are according to the steps described in Example 4, and finally a biological eardrum with a double-layer structure of a shielding dense layer and a regenerated loose fiber membrane is obtained.

[0087] Example 6

[0088] The difference between the biological eardrum repair material of this embodiment and that of embodiment 5 is that the dense layer is shielded. The preparation of the biological eardrum repair material includes the following steps:

[0089] (1) Preparing a spinning solution for a shielding dense layer: weighing PLA and EPL, dissolving them in acetone, and mixing them evenly to obtain a spinning solution for a shielding dense layer, wherein the mass volume ratio of PLA is 25% w / v, and the mass volume ratio of EPL is 5% w / v.

[0090] (2) The spinning solution prepared in step (1) is used to form a shielding dense layer having an oriented fiber structure through an electrospinning process; the electrospinning conditions are: humidity 50%, rotation speed 3500 rpm, voltage 20 kV; the structural parameters of the formed shielding dense layer are: layer thickness 100 μm, oriented fiber diameter 600-800 nm.

[0091] (3) The shielding dense layer formed in step (2) is used as the base layer, and a regenerated loose fiber membrane is further formed on the shielding dense layer by electrospinning. The preparation steps of the regenerated loose fiber membrane are according to the steps described in Example 4, and finally a biological eardrum with a double-layer structure of a shielding dense layer and a regenerated loose fiber membrane is obtained.

[0092] Example 7

[0093] The difference between the biological eardrum repair material of this embodiment and that of embodiment 5 is that the dense layer is shielded. The preparation of the biological eardrum repair material includes the following steps:

[0094] (1) Preparing a spinning solution for a shielding dense layer: PLA and EPL were weighed, dissolved in acetone, and mixed evenly to obtain a spinning solution for a shielding dense layer, wherein the mass volume ratio of PLA was 12% w / v, and the mass volume ratio of EPL was 3% w / v.

[0095] (2) The spinning solution prepared in step (1) is used to form a shielding dense layer having an oriented fiber structure through an electrospinning process; the electrospinning conditions are: humidity 50%, rotation speed 3000 rpm, and voltage 12 kV; the structural parameters of the formed shielding dense layer are: layer thickness 30 μm, and oriented fiber diameter 600-800 nm.

[0096] (3) The shielding dense layer formed in step (2) is used as the base layer, and a regenerated loose fiber membrane is further formed on the shielding dense layer by electrospinning. The preparation steps of the regenerated loose fiber membrane are according to the steps described in Example 4, and finally a biological eardrum with a double-layer structure of a shielding dense layer and a regenerated loose fiber membrane is obtained.

[0097] Comparative Example 1

[0098] The only difference between the repair material of Comparative Example 1 and Example 7 is that the regenerated loose fiber membrane is not prepared, and the preparation steps of the shielding dense layer are the same as those of Example 7.

[0099] Comparative Example 2

[0100] The repair material of Comparative Example 2 is different from that of Example 7 only in that polylysine EPL is not added to the spinning solution of the shielding dense layer, and the remaining preparation steps are the same as those of Example 7.

[0101] Comparative Example 3

[0102] The only difference between the repair material of Comparative Example 3 and Example 7 is that sodium alginate is not added to the spinning solution of the regenerated loose fiber membrane, and the remaining preparation steps are the same as Example 7.

[0103] In order to further illustrate the beneficial technical effects of the degradable biological tympanic membrane repair material prepared by the present invention, the relevant properties of the degradable tympanic membrane repair material prepared according to the specific embodiment of the present invention will be tested below.

[0104] 1. Scanning electron microscopy observation

[0105] The degradable tympanic membrane repair material prepared in Example 7 was observed using a scanning electron microscope to observe the surface structures of the shielding dense layer and the regenerated loose fiber membrane of the repair material. The corresponding SEM images are shown in FIG. Figure 1a and Figure 1b As shown by Figure 1a It can be seen from the shielding dense layer shown that the shielding dense layer is composed of a closely arranged directional fiber structure, in which the directional fibers are basically oriented in the same way, have certain mechanical properties, and play a supporting role; Figure 1b It can be seen from the regenerated loose fibrous membrane shown that the regenerated loose fibrous membrane is composed of disordered fibers and has a three-dimensional porous structure, which can effectively guide the growth of epithelial and mucosal layer cells.

[0106] 2. Mechanical properties test

[0107] An electronic universal testing machine was used with a speed of 50 mm / min and a sample size of 15 mm×50 mm to test the tensile strength and peeling force of the tympanic membrane repair materials prepared in Examples 1-7 and the film materials prepared in Comparative Examples 1-3. The test results are shown in Table 1 below.

[0108] Table 1 Tensile strength and peel force test results

[0109]

[0110] From the tensile strength test data shown in Table 1, it can be seen that the tympanic membrane repair materials of Examples 5-7 have a shielding dense layer and have strong tensile resistance, that is, the mechanical properties of the double-layer structure composite membrane with a shielding dense layer and a regenerated loose fiber membrane are better. From the peeling force test data shown in Table 1, it can be seen that the tympanic membrane repair materials of Examples 5-7 add polylysine to enhance the connection firmness between the double-layer structures. In addition, the sodium alginate in the regenerated loose fiber membrane can be electrostatically adsorbed with polylysine, which can further improve the interface firmness.

[0111] It can be seen that the tympanic membrane repair materials of Examples 5-7 of the present invention are removable and can be applied to various procedures such as explantation, endoplantation, sandwich, butterfly cartilage myringoplasty, etc., and are easy to operate clinically and have strong versatility. Specifically, they are mainly reflected in the following aspects:

[0112] ① Structural composition: The double-layer structure of this biological eardrum has obvious differences in fiber orientation and surface wettability. The dual differences in structure and composition give it a clear double-layer boundary and a peelable property.

[0113] ② Interface connection: The EPL in the shielding dense layer can not only chemically cross-link with the unreacted residual glyoxal in the regenerated loose fiber membrane, providing interface strength while reducing solvent residues to ensure safety, but also the cation-rich EPL can also electrostatically attract the anions of the regenerated loose fiber membrane SA. The synergistic effect of chemical cross-linking and electrostatic attraction ensures the firmness of the interface connection between the double-layer structure.

[0114] ③ Clinical operation: This biological tympanic membrane can be used without peeling. Chemical cross-linking and electrostatic attraction ensure the close connection between the two layers of membrane, so that it will not fall off and delaminate without external force. It is suitable for traditional explantation, endoplantation and sandwich methods. It is convenient for the surgeon to perform peeling operation under external force conditions. The electrospinning process is used to construct fiber orientation differences and surface wettability differences to achieve the peelability of the biological tympanic membrane, which effectively solves the problem that a single-layer homogeneous tissue repair material cannot meet the requirements of the butterfly cartilage myringoplasty procedure.

[0115] 3. Cell Adhesion Performance

[0116] In vitro cell culture was used to investigate the cell adhesion of the regenerated loose fiber membrane surfaces of Examples 1-7, Comparative Examples 2-3, and the shielding dense layer surface of Comparative Example 1. The samples were prepared into discs with a diameter of 1.1 mm. After treatment, they were placed in a 48-well cell culture plate, washed with PBS to remove surface debris, and then added with an appropriate amount of culture medium and placed in a cell culture incubator for incubation for 12 h. L929 fibroblasts were inoculated onto the disc material, placed in a cell culture incubator for incubation for 30 min, the culture plate was taken out, an appropriate amount of complete culture medium was added, and then placed in a cell culture incubator for culture. The culture conditions were 37°C, 5% CO2, and 95% humidity. The complete culture medium was replaced every 48 h. After culturing to the 7th day, cell adhesion was observed by staining. The corresponding live and dead cell fluorescent adhesion staining images of the regenerated loose fiber membrane surfaces of Examples 1-7, Comparative Examples 2-3, and the shielding dense layer surface of Comparative Example 1 are shown in the following figure. Figure 2 As shown in the figure, it can be seen that the repair material with a regenerated loose fibrous membrane structure significantly promotes cell adhesion and proliferation, and therefore can effectively promote the growth of epithelial cells and mucosal cells; in addition, by comparing the repair materials of Examples 1-7 with those of Comparative Example 3, it can be seen that the addition of sodium alginate can further facilitate cell climbing, promote epithelialization, shorten the repair time of the perforation position, and ensure the regeneration and healing effect of the tympanic membrane; the cell adhesion of the shielding dense layer is lower than that of the regenerated loose fibrous membrane, which can prevent postoperative tympanic cavity adhesion.

[0117] 4. Water contact angle test

[0118] A contact angle tester was used to drop 4 μl of liquid onto the surface of the membrane material, and the hydrophilic and hydrophobic properties of the tympanic membrane repair materials of Examples 1-7 and the membrane materials of Comparative Examples 1-3 were tested, and the test results are shown in Table 2. The contact angle test results shown in Table 2 show that the shielding dense layer is hydrophobic and the regenerated loose fiber membrane is highly hydrophilic, so the degradable tympanic membrane biorepair material having the shielding dense layer and the regenerated loose fiber membrane can prevent tympanic adhesion and promote cell adhesion, which is beneficial to the repair of the perforation position.

[0119] Table 2 Contact angle test results

[0120]

[0121] 5. Drug release performance in vivo

[0122] The biological tympanic membrane repair materials prepared in Examples 2-4 were implanted subcutaneously in rats to test the drug release performance of the biological tympanic membrane in vivo. The animals were killed at 1 week, 4 weeks, 8 weeks, 10 weeks and 12 weeks after implantation to obtain samples for analysis. The incompletely degraded biological tympanic membrane was placed in 30 ml acetonitrile solution for 20 minutes of ultrasound and the drug release behavior was tested by liquid phase. The test results are as follows: Figure 3 As shown. Figure 3The in vivo drug release test results shown indicate that, in the biological tympanic membrane repair material of the present invention, chemically linking the drug molecules to gelatin can effectively prolong the drug release cycle and achieve sustained antibacterial and anti-inflammatory effects at the tympanic membrane perforation site.

[0123] 6. Test on the change of the number of ε-amino groups in the side chain of gelatin protein peptide chain

[0124] The change in the number of side chain ε-amino groups in the gelatin protein peptide chain was determined by the TNBS (2,4,6-trinitrobenzenesulfonic acid) method for GEL-CiFX prepared by steps (1)-(2) of Examples 2, 3, and 4, respectively.

[0125] The ε-amino group content is calculated as follows, in mol / g:

[0126]

[0127] Where, A - absorption value; 1.46×10 4 - molar absorption coefficient of trinitrobenzene derivatives; b - optical path, unit: cm; X - sample weight, unit: g;

[0128] The test results are shown in Table 3.

[0129] Table 3. Number of amino groups on the surface of drug-modified gelatin

[0130]

[0131] From the results in Table 3, it can be seen that there is no significant change in the number of amino groups on the gelatin surface after drug grafting. Drug grafting avoids the cell adhesion site of the RGD sequence and does not affect the perforation repair process, providing a growth scaffold for epithelial cells and mucosal cells. At the same time, drug grafting can be achieved to achieve long-term anti-inflammatory effects after surgery.

[0132] Combined with Table 3 Figure 2 and Figure 3 It can be seen that the repair material of the embodiment of the present invention realizes drug grafting while protecting the RGD sequence, ensuring that cell adhesion and long-term drug release are promoted simultaneously during the repair process of tympanic membrane perforation. Specifically, the GEL in the regenerated loose fiber membrane has an RGD sequence, which can promote the adhesion and aggregation of epithelial cells and mucosal cells at the site of tympanic membrane perforation, and the loose porous structure is also conducive to the orderly climbing of cells; drug grafting is realized by ester bond connection between the drug and gelatin in the regenerated loose fiber membrane, which not only avoids the RGD sequence and thus does not affect the cell adhesion site in the perforation repair process, but also provides drug sustained release in the tympanic membrane repair process, so that the biological tympanic membrane has the function of drug sustained release in the repair process, and then plays a continuous antibacterial and anti-infection role, solving the problem of recurrence of inflammation at the perforation site and repair failure caused by infection of autologous repair materials.

[0133] 7. Animal Tympanic Membrane Repair Experiment

[0134] A left tympanic membrane perforation model was made in 50 guinea pigs, and a CO2 laser was used to create a hole in the tympanic membrane at the opening of the external auditory canal. The experimental groups are shown in Table 4. After surgery, 1% lidocaine was injected locally into the ears of the mice for analgesia, and the repair rate (healing area of ​​the perforation site> 99%) was calculated 12 weeks after surgery.

[0135] 12 weeks after the tympanic membrane perforation surgery, 20 ears were repaired with the biological tympanic membrane prepared in Example 7, and the healing rate of the repair under different surgical procedures was 100%, which was better than that of the temporalis fascia group; there were 10 ears in the blank control group, 8 ears with unhealed tympanic membrane perforations, and the healing rate was 20%. The healing rate of each group was tested by chi-square test, indicating that the healing rate of different groups was significantly different (P < 0.01), and the results are shown in Table 4.

[0136] Table 4. Comparison of healing rates of tympanic membrane perforations

[0137]

[0138] From the animal tympanic membrane repair experiment, it can be seen that the repair material of the embodiment of the present invention can successfully replace the temporalis muscle fascia, cartilage-perichondrium, and fat commonly used in current clinical repair. Compared with these autologous tissue repairs, the advantages of the degradable biological tympanic membrane repair material prepared by the present invention in achieving the replacement of autologous tissue repair are reflected in: the drug load can have an anti-infection effect, can improve the success rate of surgery, and has a good tympanic membrane repair effect. At the same time, it can be applied to a variety of surgical procedures and has strong universal performance.

[0139] The present invention has been specifically described above in conjunction with specific embodiments. These specific embodiments are only used for illustration and cannot be used to limit the scope of protection of the present invention. Those skilled in the art may make various modifications, changes or substitutions to the present invention without departing from the essence and scope of the present invention. Therefore, various equivalent changes made according to the present invention still belong to the scope covered by the present invention.

Claims

1. A method for preparing a biodegradable biological tympanic membrane repair material, It is characterized in that The method comprises the following steps: Providing a spinning solution and a cross-linking solution, wherein the spinning solution comprises gelatin and 2.5% w / v-5% w / v sodium alginate; The spinning solution and the cross-linking solution are formed into a regenerated loose fiber membrane through an electrostatic spinning process; The thickness of the regenerated loose fiber membrane is 50-270 μm, the cross-linking degree is 50-90%, and the diameter of the spun fiber is 400-600 nm; Forming a shielding dense layer by an electrostatic spinning process, wherein the spinning solution of the shielding dense layer includes a degradable hydrophobic polymer; the spinning solution of the shielding dense layer also contains polylysine EPL; The shielding dense layer is formed before the regenerated loose fiber membrane is formed, and the regenerated loose fiber membrane is formed with the shielding dense layer as a base layer; The shielding dense layer is a hydrophobic film with a water contact angle of 120-133°, and the regenerated loose fiber membrane is a hydrophilic fiber membrane; The fibers of the shielding dense layer are arranged in a directional manner, and the fibers of the regenerated loose fiber membrane are arranged in a disordered manner; The shielding dense layer and the regenerated loose fiber membrane can be peeled off from each other.

2. The preparation method according to claim 1, It is characterized in that The electrospinning process conditions of the regenerated loose fiber membrane are: humidity of 40-50%, rotation speed of 60-120rpm, and voltage of 10-20kV.

3. The preparation method according to claim 1, It is characterized in that The gelatin is drug-modified gelatin, and the preparation of the drug-modified gelatin includes: chlorination treatment of the drug to form a chlorinated drug, and then mixing the chlorinated drug with gelatin to form the drug-modified gelatin.

4. The preparation method according to claim 3, It is characterized in that The chlorinated drugs are grafted onto gelatin via ester bonds to form drug-modified gelatin.

5. The preparation method according to claim 3, It is characterized in that The drug is selected from one or more of the following drugs: levofloxacin, ofloxacin, norfloxacin, pefloxacin, enoxacin and ciprofloxacin.

6. The preparation method according to claim 3, It is characterized in that The drug-modified gelatin is gelatin-ciprofloxacin GEL-CiFX. In the spinning solution formed by the drug-modified gelatin, the mass volume ratio of GEL-CiFX is 10% w / v-35% w / v.

7. The preparation method according to claim 6, It is characterized in that The preparation of the drug-modified gelatin comprises: forming a solution from gelatin GEL, chloroacylated ciprofloxacin CiFX-COCl and a solvent thereof, wherein the content of GEL is 10% w / v-35% w / v, and the content of CiFX-COCl is 3% w / v-10.5% w / v.

8. The preparation method according to claim 6, It is characterized in that In drug-modified gelatin, the grafting rate of drug ciprofloxacin is 80%-95%.

9. The preparation method according to claim 1, It is characterized in that The electrospinning process conditions of the shielding dense layer are: humidity of 40-50%, rotation speed of 2500-3500rpm, and voltage of 10-20kV.

10. The preparation method according to claim 1, It is characterized in that The thickness of the shielding dense layer is 30-100 μm, and the fiber diameter is 600-800 nm.

11. The preparation method according to claim 1, It is characterized in that The peeling force between the shielding dense layer and the regenerated loose fiber membrane is 0.71-1.15N.

12. The preparation method according to claim 1, It is characterized in that The degradable hydrophobic polymer is selected from one or more of the following materials: polylactic acid PLA, polylactic acid-glycolic acid copolymer PLGA, polycaprolactone PCL and derivatives thereof.

13. The preparation method according to claim 12, It is characterized in that The degradable hydrophobic polymer is polylactic acid PLA.

14. The preparation method according to claim 13, It is characterized in that In the spinning solution of the shielding dense layer, the content of polylactic acid PLA is 10% w / v-25% w / v, and the content of polylysine EPL is 2.5% w / v-5% w / v.

15. A biodegradable biological tympanic membrane repair material, made by the preparation method according to any one of claims 1 to 14.

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