Preparation method of a composite amniotic membrane lacrimal duct stent

Abstract

A preparation method of a composite amniotic membrane lacrimal duct stent disclosed by the present invention. The stent comprises three layers: an inner layer, a middle layer and an outer layer. The inner layer is a tubular composite inner core formed by melting and extruding PLGA or a mixture of PLGA / PCL into a hollow tube or braiding into a solid silk thread. The middle layer is an expanded sponge layer formed by a collagen solution containing one or more powders of amniotic membrane powder, mussel protein, hyaluronic acid, chitosan, etc. The expanded sponge layer is prepared into a columnar sponge layer by electrochemically depositing collagen, and one or more components of an anti-scarring and anti-adhesion component of amniotic membrane powder, a mussel protein adhesive material and a chitosan anti-inflammatory component material are sprayed; the outer layer is a biological amniotic membrane prepared by strengthening treatment. Compared with the existing lacrimal duct stent prepared from pure biological materials, it has good drainage effect, promotes healing and has an anti-inflammatory effect, and at the same time provides good support performance to maintain the smoothness of the channel.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a preparation method of a composite amniotic membrane lacrimal duct stent. Background Art

[0002] Lacrimal duct obstructive diseases are common ophthalmic diseases. They not only cause epiphora but also are prone to secondary bacterial infection, leading to acute and chronic dacryocystitis. Epiphora itself and the impregnation of the face by tears are detrimental to beauty, causing physical and psychological discomfort to patients. As an ocular infection focus, dacryocystitis can spread to the eyeball and orbit, and is often a contraindication for intraocular surgery.

[0003] At present, the main methods for treating lacrimal duct obstruction are as follows: lacrimal duct irrigation and probing, dacryocystorhinostomy, lacrimal duct silicone intubation nasolacrimal duct stent, dacryocystectomy, etc. Lacrimal duct irrigation and probing is simple and easy to perform, without the need for expensive equipment. However, the recurrence rate of treating adult lacrimal duct obstruction is relatively high, and it is easy to cause intimal damage, lacrimal punctum rupture, lacrimal canaliculus injury and false passage. Currently, it is mainly used for treating congenital nasolacrimal duct obstruction. Dacryocystorhinostomy is a classic surgical method for treating lacrimal duct obstruction, including transcutaneous, transnasal dacryorhinostomy, transnasal endoscopic laser dacryocystorhinostomy. No matter which anastomosis method is used, it destroys the integrity of the nasal mucosa and the normal anatomical and physiological structure of the lacrimal duct. The early postoperative effect is relatively ideal, but over time, the bone hole is easily blocked by scabs and proliferated granulation tissue, resulting in surgical failure. In China, it is more common to treat lacrimal duct obstructive diseases by implanting silicone tubes, other material artificial tubes or stents into the lacrimal duct. The silicone tube material has good support resilience, but it cannot be degraded in the body and needs to be removed by a second operation, causing secondary harm, and the effect is average. The above several methods have a relatively high short-term success rate, but a relatively low long-term indwelling success rate. Some materials need to be removed by a second operation, and there is a risk of secondary harm to the lacrimal duct. To avoid secondary harm, other biodegradable materials are often used to prepare lacrimal duct stents, such as stents prepared from polymers, which have good support effects, but lack the effect of promoting healing, and are prone to scarring and inflammation during the recovery period; lacrimal duct stents prepared from biomaterials have the effect of promoting healing, even anti-scarring and anti-inflammatory effects, but the support effect of the material is weak, and it is impossible to form a good dredging channel effect. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a composite amniotic membrane lacrimal duct stent, so as to solve the technical problems existing in the existing lacrimal duct stents, such as poor support effect of biomaterials, single function, poor biocompatibility and degradability.

[0005] To achieve the above object, the present invention provides the following technical solution: A composite amniotic membrane lacrimal duct stent, comprising an inner layer, a middle layer and an outer layer. The inner layer is a tubular composite inner core formed by melting and extruding a copolymer of polylactic acid and glycolic acid (PLGA) or a mixture of a copolymer of polylactic acid and glycolic acid and polycaprolactone into a hollow tube or braiding a solid silk thread; the middle layer is an expanded sponge layer formed by a collagen solution containing one or more powders of amniotic membrane powder, mussel protein, hyaluronic acid, chitosan, etc. The expanded sponge layer is prepared into a columnar sponge layer by electrochemically depositing collagen, and one or more components of an anti-scar and anti-adhesion component of amniotic membrane powder, a mussel protein adhesive material and a chitosan anti-inflammatory component material are sprayed; the outer layer is prepared by strengthening biological amniotic membrane.

[0006] The preparation method of the composite amniotic membrane lacrimal duct stent specifically comprises the following steps:

[0007] S1. The amniotic membrane is pretreated, chemically crosslinked and strengthened, and vacuum freeze-dried to obtain freeze-dried amniotic membrane. The freeze-dried amniotic membrane is cut into a fixed specification size according to the specification to obtain an amniotic membrane outer cover, and the remaining freeze-dried amniotic membrane is prepared by freeze-crushing to obtain freeze-dried amniotic membrane powder;

[0008] S2. A copolymer of polylactic acid and glycolic acid (PLGA) or a mixture of a copolymer of polylactic acid and glycolic acid and polycaprolactone (PLGA / PCL) is melted and extruded into a silk thread, wound around a high-purity titanium cylindrical core and braided into a tubular structure;

[0009] S3. The tubular structure prepared in step S2 and the high-purity titanium cylindrical core are used as the inner core to connect the negative electrode of the power supply as the cathode, immersed in a collagen solution, and a high-purity platinum is connected to the positive electrode of the power supply as the anode. Electric current is applied to electrochemically deposit collagen, so that collagen is deposited on the high-purity titanium cylindrical core to obtain a collagen gel. The collagen gel is crosslinked using an EDC / NHS solution to obtain a collagen stent;

[0010] S4. The collagen stent in step S3 is freeze-dried to obtain a stent collagen sponge layer;

[0011] S5. The freeze-dried amniotic membrane powder in step S1 is mixed evenly with a viscous protein powder containing mussel protein or fibrin and a powder containing hyaluronic acid or chitosan with anti-inflammatory and wound-healing effects, and evenly sprayed on the stent collagen sponge layer prepared in step S4 in the form of spraying to obtain an initial collagen sponge layer stent;

[0012] S6. Place the initially prepared collagen sponge layer scaffold in step S5 under a press to compress the sponge layer, then soak the compressed initially prepared collagen sponge layer scaffold in a mussel protein solution, wrap one or more layers of amniotic membrane outer coating, and bond and seal the edges with the mussel protein solution, place it in a drying oven for low-temperature drying, and remove the high-purity titanium cylindrical core to obtain a composite amniotic membrane lacrimal duct scaffold;

[0013] S7. Package the composite amniotic membrane lacrimal duct scaffold and then irradiate it for sterilization, and then perform secondary sealed packaging.

[0014] Preferably, the pretreatment, chemical cross-linking strengthening, and vacuum freeze-drying are specifically as follows: after washing with purified water, bluntly remove the amniotic membrane villous layer and residual blood stains, and then perform activation cross-linking treatment with a solution of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in 2-morpholinoethanesulfonic acid, wash and vacuum freeze-dry to prepare freeze-dried amniotic membrane, and then cut it into appropriate sizes to prepare the amniotic membrane outer coating.

[0015] Preferably, the ratio of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1 to 5:1, the concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is 0.1 mmol / L to 1 mmol / L; the concentration of 2-morpholinoethanesulfonic acid is 0.05 mol / L to 1 mol / L, the activation cross-linking temperature is 20°C to 37°C, and the activation time is 3 h to 20 h.

[0016] Preferably, the remaining freeze-dried amniotic membrane is placed in a low-temperature grinder for low-temperature grinding and screening, and the freeze-dried amniotic membrane powder with a particle size between 100 μm and 300 μm is selected.

[0017] Preferably, in step S2, the poly(lactic-co-glycolic acid) or a mixture of poly(lactic-co-glycolic acid) and polycaprolactone is placed in a melt extruder, start the equipment to extrude the poly(lactic-co-glycolic acid) or the mixture of poly(lactic-co-glycolic acid) and polycaprolactone into filaments. The ratio of the poly(lactic-co-glycolic acid) to the polycaprolactone in the mixture is 1 to 10:1, the melt extrusion temperature is controlled between 160°C and 220°C, the diameter of the nozzle is controlled between 0.1 mm and 0.5 mm, and filaments of a mixture of PLGA or PLGA / PCL with a diameter of 0.1 mm to 0.5 mm are prepared.

[0018] Preferably, take at least 3 strands or more of the mixture filaments of PLGA or PLGA / PCL, use a high-purity titanium cylindrical core with a diameter of 0.5 mm to 1 mm as the winding hard core, use a small amount of ethyl acetate solution of dichloromethane as the bonding solvent at the connection point, and braid the mixture filaments of PLGA or PLGA / PCL into a hollow cylindrical tube composite inner core with a diameter of 0.5 mm to 1 mm.

[0019] Preferably, in step S3, collagen is dissolved in acetic acid solution to prepare a 0.5% - 5% collagen acetic acid solution. The tubular structure and the high-purity titanium cylindrical core are immersed together in the collagen acetic acid solution. The high-purity titanium cylindrical core is placed directly above the center of the collagen acetic acid solution and connected to the negative electrode of the power supply. The silk thread of the tubular structure is kept immersed in the collagen acetic acid solution. Using high-purity platinum as the anode and connecting it to the positive electrode of the power supply, an electric current is applied at a voltage of 15V - 30V for 10min - 30min to deposit collagen onto the high-purity titanium cylindrical core to form the collagen gel. The prepared collagen gel is dried at 25°C - 37°C for 10min - 30min, and then transferred to an EDC / NHS solution for crosslinking. The crosslinking temperature is 20°C - 37°C, and the crosslinking time is 2h - 5h. The ratio of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the EDC / NHS solution is 1:1 - 5:1. The preparation method of the EDC / NHS solution is as follows: Prepare a phosphate buffer solution with a concentration of 0.1mol / L - 0.4mol / L, weigh EDC / NHS and dissolve it in the phosphate buffer solution to obtain an EDC / NHS solution with a concentration of 0.1mmol / L - 1mmol / L.

[0020] Preferably, in step S6, the press is a pneumatic press with a circular groove with a diameter of 1.2mm - 1.5mm after the upper and lower pressure plates are combined. Under a pressure of 0.1MPa - 0.5MPa, the initial collagen sponge layer scaffold is compressed to form a uniform cylindrical shape with a diameter of 1.2mm - 1.5mm. The concentration of the mussel protein solution is 0.1% - 1%. The amniotic membrane in step S1 is adhered to the initial collagen sponge layer scaffold for amniotic membrane fixation and dried at 30°C - 50°C for 30min - 120min.

[0021] Preferably, in step S7, the composite amniotic membrane lacrimal duct stent is subjected to blister packaging, irradiated and sterilized, and then subjected to secondary sealed packaging.

[0022] The present invention has the following beneficial effects compared with the prior art:

[0023] 1. A composite amniotic membrane lacrimal duct stent disclosed by the present invention comprises an inner layer, a middle layer and an outer layer. The inner layer is a tubular composite inner core formed by melting and extruding poly(lactic-co-glycolic acid) (PLGA) or a mixture of poly(lactic-co-glycolic acid) and polycaprolactone (PLGA / PCL) into a hollow tube or braiding solid silk threads into a tube. The middle layer is an expanded sponge layer formed by a collagen solution containing one or more powders of amniotic membrane powder, mussel protein, hyaluronic acid, chitosan, etc. The expanded sponge layer is prepared into a columnar sponge layer by electrochemically depositing collagen, and one or more components of amniotic membrane powder anti-scar and anti-adhesion components, mussel protein adhesive material and chitosan anti-inflammatory component material are sprayed; the outer layer is a biological amniotic membrane, which is prepared by strengthening treatment; a hollow reticular cylindrical structure is braided by melting and extruding PLGA or a PLGA / PCL mixture into silk threads. Compared with the existing lacrimal duct stent prepared from pure biological materials, it has good drainage effect and provides good support performance to maintain the smoothness of the channel;

[0024] 2. The composite amniotic membrane lacrimal duct stent disclosed by the method of the present invention has appropriate expansion support ability and drug loading ability. The sponge layer of the composite amniotic membrane lacrimal duct stent can expand automatically after absorbing liquid after being compressed, stably fix the material at the repair site to be repaired, and release materials such as healing-promoting, anti-inflammatory, adhesive protein, growth factor, etc. in the sponge layer to accelerate repair and reduce the sensitivity of the material to the body.

[0025] 3. The amniotic membrane outer layer of the composite amniotic membrane lacrimal duct stent prepared by the method of the present invention has good biological functions such as promoting mucosal repair and healing, reducing scar formation, reducing inflammation, preventing adhesion, etc.

[0026] 4. The composite amniotic membrane lacrimal duct stent prepared by the method of the present invention has appropriate anti-degradation performance and mechanical properties. After cross-linking the amniotic membrane to form a three-dimensional network structure, the mechanical properties of the amniotic membrane are improved, and at the same time, its anti-protease degradation performance is enhanced, thereby prolonging its in-vivo implantation time and effectively ensuring the completion of the repair of the lacrimal duct before the material degrades.

[0027] 5. Compared with silicone materials, the composite amniotic membrane lacrimal duct stent prepared by the method of the present invention has good biodegradability. The composite amniotic membrane lacrimal duct stent is wholly prepared from biodegradable materials, and no secondary removal is required for one operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a schematic structural diagram of the composite amniotic membrane lacrimal duct stent of the present invention;

[0029] Figure 2 is a schematic cross-sectional structural diagram of the composite amniotic membrane lacrimal duct stent of the present invention;

[0030] Figure 3Schematic structural diagram of the amniotic membrane outer coating of the composite amniotic membrane lacrimal duct stent of the present invention;

[0031] Figure 4 Schematic structural diagram of the composite inner core of PLGA or PLGA / PCL mixture of the composite amniotic membrane lacrimal duct stent of the present invention;

[0032] Figure 5 Schematic electrode structure diagram of the electro-deposition device in the preparation method of the composite amniotic membrane lacrimal duct stent of the present invention

[0033] Figure 6 Schematic structural diagram of the composite inner core and sponge layer of the composite amniotic membrane lacrimal duct stent of the present invention;

[0034] Figure 7 Schematic structural diagram of the composite amniotic membrane lacrimal duct stent after the composite inner core and the amniotic membrane outer coating are assembled and wrapped;

[0035] Figure 8 Physical diagram of the composite lacrimal duct amniotic membrane stent in Example 2 of the present invention;

[0036] Figure 9 Graph of dimensional changes of the composite lacrimal duct amniotic membrane stent in Example 2 of the present invention before and after rehydration and swelling;

[0037] Figure 10 Graph of CD31 expression in rabbit lacrimal ducts at three different time points in the animal experiment of Example 3 of the present invention;

[0038] Figure 11 Graph of α-SMA expression in rabbit lacrimal ducts at three different time points in the animal experiment of Example 3 of the present invention.

[0039] Reference numerals: 1 - outer layer; 2 - middle layer; 3 - inner layer. Detailed implementation manners

[0040] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with specific embodiments in conjunction with the accompanying drawings, but it is not limited to a specific example only, and this method can be applied to similar situations. In the embodiments, PLGA refers to poly(lactic-co-glycolic acid), and the PLGA / PCL mixture refers to a mixture of poly(lactic-co-glycolic acid) and polycaprolactone. In the following examples, the amniotic membrane was obtained by peeling from the placenta during cesarean section under sterile conditions after the parturient signed an informed consent form, and it is a discarded product separated from the living body. Example 1

[0041] Please refer to Figures 1 to 7, A composite amniotic membrane lacrimal duct stent, comprising three layers: an inner layer 3, a middle layer 2, and an outer layer 1. The inner layer 3 is a tubular structure formed by melt-extruding a poly(lactic-co-glycolic acid) (PLGA) or a mixture of poly(lactic-co-glycolic acid) and polycaprolactone into a hollow tube or braiding a solid silk thread. The middle layer 2 is an expanded sponge layer formed by a collagen solution containing one or more powders of amniotic membrane powder, mussel protein, hyaluronic acid, and chitosan. The expanded sponge layer is prepared by electrochemically depositing collagen into a columnar sponge layer and spraying one or more components of an anti-scar and anti-adhesion component of amniotic membrane powder, a mussel protein adhesive material, and a chitosan anti-inflammatory component material. The outer layer 1 is a biological amniotic membrane prepared by strengthening treatment. The specific preparation method includes the following steps:

[0042] Step 1. Preparation of the PLGA or PLGA / PCL mixture stent:

[0043] (1) Preparation of the PLGA or PLGA / PCL mixture silk thread:

[0044] Take a uniformly mixed mixture of PLGA or PLGA / PCL with a ratio of 1:1 and add it to a melt-extrusion device for melting and extrusion. Set the extrusion temperature at 180°C and the extrusion die aperture at 0.5 mm, and collect the extruded PLGA or PLGA / PCL mixture silk thread.

[0045] (2) Braiding of the PLGA or PLGA / PCL mixture stent:

[0046] Please refer to Figure 4 , Use a stainless high-purity titanium cylinder core with a diameter of 0.8 mm as the stent, wind multiple strands of silk thread around the stainless high-purity titanium cylinder core in a spiral form, and use an ethyl acetate solution containing dichloromethane as the bonding solvent at the silk thread connection point to prepare a spiral stent.

[0047] Step 2. Preparation of the outer layer wrapped with amniotic membrane:

[0048] (1) Cleaning:

[0049] The fresh amniotic membrane from a healthy cesarean section is bluntly cleaned with purified water 6 - 10 times, with each wash using no less than 500 ml of cleaning solution for 5 - 20 minutes to fully remove the chorionic layer and blood and dirt impurities;

[0050] (2) Crosslinking treatment:

[0051] The washed amniotic membrane was crosslinked by soaking it in an MES solution of EDC / HNS. The crosslinking buffer used was an MES solution with a concentration of 0.5 mol / l and a pH of 5.5, containing 0.5 mM of EDC and 0.1 mM of NHS solution. The crosslinking treatment was carried out at room temperature for 20 h. After crosslinking, the amniotic membrane was washed 5 - 10 times with purified water, with each wash using no less than 500 ml of washing solution and each wash lasting 10 min - 30 min. Ensure the safety of the material.

[0052] (3) Vacuum freeze-drying:

[0053] The crosslinked and washed amniotic membrane was laid flat in a vacuum freeze-dryer for vacuum freeze-drying.

[0054] (4) Cutting:

[0055] Please refer to Figure 3 , take the freeze-dried crosslinked amniotic membrane, cut it into a rectangular amniotic membrane according to the size requirements as the outer amniotic covering of the lacrimal duct stent. The remaining amniotic membrane material was placed in a ball mill pulverizer and intermittently pulverized at a speed of 1000 rpm / min, and the powder was sieved to retain the amniotic membrane powder with a particle size between 100 μm and 300 μm.

[0056] Step 3: Preparation of the middle sponge layer and assembly of the composite lacrimal duct amniotic membrane stent:

[0057] Please refer to Figure 5 , assemble the electrodeposition device. Take the stent prepared from the PLGA or PLGA / PCL mixture containing a high-purity titanium cylindrical core prepared in the above step 1 as the composite inner core, immerse it in a 1% collagen acetic acid solution until the woven material is completely submerged in the collagen solution, connect the negative electrode of the power supply as the cathode, use high-purity platinum as the anode and connect it to the positive electrode of the power supply, and also place it in the collagen solution, keep it parallel to the cathode high-purity titanium and at a distance of 1 cm, set the power supply voltage to 20 V, keep the power on for 30 min. After the electrodeposition is completed, transfer the material on the high-purity titanium to a blast drying oven and dry it at 37 °C for 15 min, then soak it in a 0.1 mol / L PB buffer solution with a ratio of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide of 1:1 and a concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride of 0.5 mmol / L, crosslink at 37 °C for 3 h. Then transfer it to 500 ml of purified water and gently rinse it 5 times, 5 min each time, and then transfer it to a vacuum freeze-dryer for freeze-drying to prepare the collagen sponge layer. For the structural schematic diagram of the composite inner core and the sponge layer, please refer to Figure 6 .

[0058] Mix amniotic membrane powder, mussel protein powder, and chitosan powder evenly in a ratio of 20:10:3. After grinding and sieving, use the spraying method to evenly spray the powder on the dried sponge layer above. Place the scaffold material after spraying the material in a pneumatic press with a circular groove of 1.5 mm in diameter after the upper and lower pressure plates are combined. Under a pressure of 0.4 MPa, compress it until the material forms a uniform cylindrical material with a diameter of 1.5 mm.

[0059] Then apply a 0.5% mussel protein solution to any longitudinal line on the sponge layer, take the biological amniotic membrane cut to the size specifications in Step 2 and wrap it in a winding form, and finally apply a 0.5% mussel protein solution to the amniotic membrane for bonding and fixing. Please refer to Figure 7 , and finally fix the prepared composite lacrimal duct amniotic membrane scaffold in the packaging box for packaging and sealing.

[0060] Step 4. Sterilization of the composite amniotic membrane lacrimal duct scaffold:

[0061] Sterilize the packaged composite lacrimal duct amniotic membrane scaffold by irradiation. Example 2

[0062] An experiment on the swelling performance of the composite lacrimal duct amniotic membrane scaffold prepared by the method for preparing a composite lacrimal duct amniotic membrane scaffold disclosed in the present invention. For the physical diagram of the composite lacrimal duct amniotic membrane scaffold, please refer to Figure 8 .

[0063] Experimental method: Take the composite lacrimal duct amniotic membrane scaffold and place it in physiological saline for testing to rehydrate, measure the size of the product before and after swelling, and observe the swelling performance of the product.

[0064] Experimental results: The composite lacrimal duct amniotic membrane scaffold in this example was tested for its size change before and after swelling in physiological saline for 5 minutes. For the statistical results, please refer to Figure 9 . The initial average size was about 1.7 mm, and the average size after water absorption and swelling was about 2.1 mm, proving that the composite lacrimal duct amniotic membrane scaffold prepared by the composite lacrimal duct amniotic membrane scaffold disclosed in the present invention has a certain liquid absorption and swelling performance. Example 3

[0065] Apply the composite lacrimal duct amniotic membrane scaffold prepared by the method for preparing a composite lacrimal duct amniotic membrane scaffold disclosed in the present invention to an animal experiment on rabbit lacrimal canaliculus injury.

[0066] Experimental method: Take the above composite lacrimal duct amniotic membrane scaffold for an animal experiment. Use the traditional silicone lacrimal duct scaffold as the control group and the modeling group as the blank group. Use the sclerosing agent lacrimal duct injection method to make a rabbit lacrimal canaliculus injury model. By analyzing the CD31 and a-SMA index data in the lacrimal canaliculus tissue after injury detected postoperatively, determine the effect of the composite lacrimal duct amniotic membrane scaffold.

[0067] Experimental results: The experimental data of rabbit models in the modeling group, silicone group and amniotic membrane group were summarized and statistically analyzed.

[0068] CD31, as a major adhesion molecule of vascular endothelial cells, is also expressed in the vascular endothelial cells of the lacrimal duct and is closely related to vascular proliferation activities. For the CD31 index in the animal experiment of this embodiment, the modeling group had the highest expression, the silicone group was the second, and the amniotic membrane group had the lowest expression. Please refer to Figure 10 .

[0069] α-smooth muscle actin (α-SMA) is expressed in the stromal myofibroblasts (MFB) of the lacrimal duct, reflects the degree of fibrosis of the lacrimal duct tissue, and participates in the central link of scar formation. In the normal rabbit lacrimal duct tissue, α-SMA is negatively expressed. For the α-SMA index in the animal experiment of this embodiment, the order of the average integrated optical density of α-SMA at each time point after surgery in the three groups was: modeling group > silicone group > amniotic membrane group. Please refer to Figure 11 .

[0070] The expressions of CD31 and α-SMA in the damaged rabbit lacrimal duct tissue were both significantly increased. The smaller the expression value, the closer it is to the expression value of the normal rabbit lacrimal duct tissue, and the smaller the damage. It can be seen from the experimental results that the composite lacrimal amniotic membrane stent has the effects of reducing inflammatory reactions, inhibiting scar formation and inflammatory neovascularization during the healing process of damaged rabbit lacrimal ducts.

[0071] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the scope of the essence of the present invention should also fall within the protection scope of the present invention. The parts not elaborated in detail in the present invention belong to the well-known technologies in the art.

Claims

1. A method for preparing a composite amniotic membrane lacrimal duct scaffold, characterized in that: The composite amniotic membrane tear duct stent comprises a three-layer structure of an inner layer (3), an intermediate layer (2) and an outer layer (1), wherein the inner layer (3) is a tubular composite inner core formed by melt extrusion of polylactic acid-glycolic acid copolymer PLGA or a mixture of polylactic acid-glycolic acid copolymer and polycaprolactone into a hollow tube or woven with solid silk threads; the intermediate layer (2) is an expanded sponge layer formed by a collagen solution containing one or more powders of amniotic membrane powder, mussel protein, hyaluronic acid, and chitosan, wherein the expanded sponge layer is prepared into a columnar sponge layer by electrochemical deposition of collagen, and one or more components of anti-scar and anti-adhesion components of amniotic membrane powder, mussel protein adhesive material, and chitosan anti-inflammatory component material are sprayed; the outer layer (1) is biological amniotic membrane, which is selected from healthy pregnant women undergoing cesarean section and prepared by enhanced treatment; The preparation method of the composite amniotic membrane lacrimal duct scaffold comprises the following steps: S1. The biological amniotic membrane is subjected to pretreatment, chemical cross-linking strengthening and vacuum freeze-drying to obtain freeze-dried amniotic membrane, the freeze-dried amniotic membrane is cut into fixed specifications to obtain an amniotic membrane coat, and the remaining freeze-dried amniotic membrane is frozen and crushed to obtain freeze-dried amniotic membrane powder; the biological amniotic membrane is selected from the amniotic membrane of healthy pregnant women undergoing cesarean section, the pretreatment, chemical cross-linking strengthening and vacuum freeze-drying specifically include washing with purified water, bluntly removing the chorionic villus layer and residual blood stains from the amniotic membrane, and then activating and cross-linking treatment with a solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 2-morpholineethanesulfonic acid of N-hydroxysuccinimide, washing and vacuum freeze-drying to prepare freeze-dried amniotic membrane, and then cutting to a suitable size to prepare an amniotic membrane coat; S2. A polylactic acid-glycolic acid copolymer PLGA or a polylactic acid-glycolic acid copolymer and a polycaprolactone PLGA / PCL mixture is melt-extruded and wound on a high-purity titanium cylindrical core to form a tubular structure; the polylactic acid-glycolic acid copolymer or the polylactic acid-glycolic acid copolymer and the polycaprolactone mixture is placed in a melt extruder, the equipment is started, and the polylactic acid-glycolic acid copolymer or the polylactic acid-glycolic acid copolymer and the polycaprolactone mixture is extruded into a thread, wherein the ratio of the polylactic acid-glycolic acid copolymer to the polycaprolactone in the mixture is 1 to 10:1, and the melt extruder is used to extrude the thread. The melt extrusion temperature is controlled between 160° C. and 220° C., and the diameter of the nozzle is controlled between 0.1 mm and 0.5 mm. A PLGA or PLGA / PCL mixture thread with a diameter of 0.1 mm to 0.5 mm is prepared. At least three strands of PLGA or PLGA / PCL mixture threads are taken and a high-purity titanium cylindrical core with a diameter of 0.5 mm to 1 mm is used as a winding hard core. A dichloromethane ethyl acetate solution is used as a bonding solvent at the connection point. The PLGA or PLGA / PCL mixture threads are woven into a hollow cylindrical tube composite inner core with a diameter of 0.5 mm to 1 mm. S3. The tubular structure prepared in step S2 and the high-purity titanium cylindrical core are used as the inner core and connected to the negative electrode of the power supply as the cathode, immersed in a collagen solution, connected to the positive electrode of the power supply with high-purity platinum as the anode, and powered on to electrochemically deposit collagen, so that the collagen is deposited on the high-purity titanium cylindrical core to obtain a collagen gel, and the collagen gel is cross-linked using an EDC / NHS solution to obtain a collagen scaffold; S4. freeze-drying the collagen scaffold in step S3 to obtain a scaffold collagen sponge layer; S5. The freeze-dried amniotic membrane powder in step S1 is mixed evenly with a viscous protein powder containing mussel protein or fibrin and an anti-inflammatory and healing-promoting powder containing hyaluronic acid or chitosan, and the mixture is evenly sprayed on the scaffold collagen sponge layer prepared in step S4 to obtain an initial collagen sponge layer scaffold; S6. placing the initial collagen sponge layer scaffold prepared in step S5 under a press to compress the sponge layer, then immersing the compressed initial collagen sponge layer scaffold in a mussel protein solution, wrapping one or more layers of amniotic membrane, and bonding and sealing the edges with a mussel protein solution, placing in a drying oven for low-temperature drying, and removing the high-purity titanium cylindrical core to obtain a composite amniotic membrane lacrimal duct scaffold; S7. After packaging, the composite amniotic membrane lacrimal duct stent is irradiated and sterilized, and then a secondary sealed package is performed.

2. The method for preparing the composite amniotic membrane lacrimal duct scaffold according to claim 1, characterized in that: The ratio of N (3-dimethylaminopropyl) N'ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1~5:1, the concentration of N (3-dimethylaminopropyl) N'ethylcarbodiimide hydrochloride is 0.1mmol / L~1mmol / L; the concentration of 2-morpholineethanesulfonic acid is 0.05mol / L~1mol / L, the activation cross-linking temperature is 20℃~37℃, and the activation time is 3h~20h.

3. The method for preparing the composite amniotic membrane lacrimal duct scaffold according to claim 1, characterized in that: The remaining freeze-dried amniotic membrane is placed in a low-temperature pulverizer for low-temperature pulverization and sieving, and freeze-dried amniotic membrane powder with a particle size between 100 μm and 300 μm is selected.

4. The method for preparing the composite amniotic membrane lacrimal duct scaffold according to claim 1, characterized in that: In the step S3, collagen is dissolved in acetic acid solution to prepare a 0.5% to 5% collagen acetic acid solution, the tubular structure and the high-purity titanium cylindrical core are immersed in the collagen acetic acid solution, the high-purity titanium cylindrical core is placed directly above the center of the collagen acetic acid solution, connected to the negative electrode of the power supply, and the silk thread of the tubular structure is kept immersed in the collagen acetic acid solution, high-purity platinum is used as an anode to connect the positive electrode of the power supply, and power is applied at a voltage of 15V to 30V for 10min to 30min to deposit collagen on the high-purity titanium cylindrical core to form the collagen gel; the prepared collagen gel is placed at 25°C to 37°C for 10min to 30min, and then transferred to EDC / NHS solution for cross-linking, the cross-linking temperature is 20°C to 37°C, and the cross-linking is 2h to 5h; N (3-dimethylaminopropyl) N'ethylcarbodiimide hydrochloride and N The ratio of hydroxysuccinimide is 1:1~5:1; the preparation method of the EDC / NHS solution is as follows: prepare a phosphate buffer with a concentration of 0.1mol / L~0.4mol / L, weigh EDC / NHS and dissolve it in the phosphate buffer to obtain an EDC / NHS solution with a concentration of 0.1mmol / L~1mmol / L.

5. The method for preparing the composite amniotic membrane lacrimal duct scaffold according to claim 1, characterized in that: In the step S6, the press is a pneumatic press with a circular groove with a diameter of 1.2mm to 1.5mm after the upper and lower pressing plates are combined. Under a pressure of 0.1MPa to 0.5MPa, the initial collagen sponge layer scaffold is compressed to form a uniform cylindrical shape with a diameter of 1.2mm to 1.5mm; the concentration of the mussel protein solution is 0.1% to 1%, and the amniotic membrane in the step S1 is adhered to the initial collagen sponge layer scaffold to fix the amniotic membrane, and is placed at 30℃ to 50℃ for drying for 30min to 120min.

6. The method for preparing the composite amniotic membrane lacrimal duct scaffold according to claim 1, characterized in that: In the step S7, the composite amniotic membrane lacrimal duct stent is packaged by blister packaging, irradiated and sterilized, and then sealed and packaged again.

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