CN111330081B - Preparation method of medicine-carrying amniotic membrane and influence of medicine-carrying amniotic membrane on corneal epithelium repair - Google Patents
Preparation method of medicine-carrying amniotic membrane and influence of medicine-carrying amniotic membrane on corneal epithelium repair Download PDFInfo
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- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
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Abstract
The preparation of the drug-loaded amnion comprises the following steps: and selecting the human amniotic membrane preserved by the glycerol to prepare the amniotic membrane. Soaking in recombinant human nerve growth factor (rhNGF) at 4 deg.C for certain concentration, taking out amnion at different time periods, transferring to small culture dish containing Phosphate Buffered Saline (PBS) at room temperature, and screening for appropriate soaking time and concentration. The drug-loaded amniotic membrane prepared by the method is transplanted at a corneal epithelial defect part, and can promote the regeneration of corneal epithelium, the recovery of corneal sensitivity and the regeneration of nerve fibers of a nerve plexus under the corneal epithelium. The invention has the beneficial effects that: not only can reduce the frequency of drug administration after transplantation, reduce the influence of eye drops on the amnion and prevent the amnion from falling off in advance, but also can slowly release rhNGF to play the role of promoting the growth of epithelium and nerve. Therefore, the prepared amnion drug-carrying system with double functions has important clinical application value.
Description
Technical Field
The invention belongs to the field of medical application or transplantation biomaterials, and relates to preparation of a drug-loaded amniotic membrane for promoting regeneration and repair of corneal epithelium and sub-epithelial nerves, in particular to preparation of an amniotic membrane loaded with recombinant human nerve growth factor (rhNGF).
Background
Corneal epithelial defects and difficulties in healing are common conditions in ophthalmology. Delayed healing of the corneal epithelium is also a troublesome problem for ophthalmologists, under the influence of ocular local corneal lesions or systemic diseases such as diabetes. Clinically, amnion transplantation is usually combined with medicaments for promoting epithelial and nerve healing to treat the diseases. Among them, amnion transplantation was reported as early as 1940 for ocular surface reconstruction, and can restore the cornea surface seriously damaged by chemical burn and thermal burn, etc., and amnion promotes the healing of the persistent corneal epithelial defect with or without the incorporated ulcer. However, the treatment of corneal epithelial defects by amniotic membrane transplantation has certain limitations, and for some patients with severe corneal epithelial lesions or systemic diseases such as diabetes and the like, double-layer amniotic membrane transplantation or multiple amniotic membrane transplantations are required to improve symptoms, so that the risk of multiple operations and the economic burden of the patients are increased. In addition to the treatment of the amnion transplantation operation, the drug therapy is a good treatment scheme, and experimental studies at home and abroad find that Nerve Growth Factor (NGF) can promote the repair of corneal epithelium and nerves under the corneal epithelium. Currently, there is no clinically applied NGF eye drops, but recombinant human nerve growth factor (rhNGF) eye drops are on the market abroad, but the price is expensive. The rhNGF has been developed in phase I clinical trials of healthy people and phase I/II clinical trials of patients with neurotrophic keratitis. At present, there is no research on the combined application of amnion transplantation and rhNGF eye drops.
Disclosure of Invention
Based on the background technology, the invention aims to provide a medicine-carrying amniotic membrane and a method for treating corneal epithelial defect, and particularly relates to the method for carrying out medicine carrying on the amniotic membrane in vitro, detecting a medicine release curve of the amniotic membrane, screening optimal concentration and medicine carrying time, and using the medicine-carrying amniotic membrane for treating corneal epithelial and nerve injury.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a drug-loaded amnion comprises the following steps:
(1) selecting the human amniotic membrane preserved by glycerol, and washing the glycerol on the surface of the amniotic membrane to be clean by 0.9% sodium chloride injection.
(2) Placing the amnion obtained in the step (1) on sterilized infusion film paper, and sucking the residual sodium chloride solution by using special laboratory absorbent paper.
(3) And (3) adding a layer of infusion film paper on the dried amnion obtained in the step (2), flattening and compacting. And measuring, drawing and cutting by using a ruler to manufacture the amniotic membrane.
(4) Soaking the amniotic membrane prepared in the step (3) in rhNGF (recombinant human nerve growth factor) with a certain concentration at 4 ℃, taking out the amniotic membrane after a period of time, and transferring the amniotic membrane to a small culture dish containing PBS at room temperature.
Further, the size of the goat film sheet prepared in the step (3) is 15mmx15mm square.
Further, the rhNGF concentration in the step (4) is 100ug/ml and 500 ug/ml.
Further, the amnion was removed after 30min in step (4) and transferred to a small dish containing PBS at room temperature.
A method for treating corneal epithelial defect by using a medicament-carrying amniotic membrane specifically comprises the following steps:
(1) selecting the human amniotic membrane preserved by glycerol, and washing the glycerol on the surface of the amniotic membrane to be clean by 0.9% sodium chloride injection.
(2) Placing the amnion obtained in the step (1) on sterilized infusion film paper, and sucking the residual sodium chloride solution by using special laboratory absorbent paper.
(3) And (3) adding a layer of infusion film paper on the dried amnion obtained in the step (2), flattening and compacting. And measuring, drawing and cutting by using a ruler to manufacture the amniotic membrane.
(4) And (4) soaking the goat diaphragm prepared in the step (3) in rhNGF with a certain concentration at 4 ℃, taking out the goat diaphragm after a period of time, and transferring the goat diaphragm to a small culture dish containing PBS at room temperature.
(5) Transplanting the amniotic membrane prepared in the step (4) to the corneal epithelial defect.
Further, the size of the goat film sheet prepared in the step (3) is 15mmx15mm square.
Further, in step (4), after 30min, the goat membranes were removed and transferred to a small petri dish containing PBS at room temperature.
Further, the rhNGF concentration in the step (4) is 100ug/m and 500 ug/ml.
An effect of a drug-loaded amniotic membrane on corneal epithelium repair, comprising: the medicine-carrying amniotic membrane prepared by the method is transplanted at a corneal epithelial defect part, and can promote the regeneration of corneal epithelium, the recovery of corneal sensitivity and the regeneration of nerve fibers of nerve plexuses under the corneal epithelium.
Further, the defective corneal epithelium is rabbit corneal epithelium.
The invention has the beneficial effects that: the amniotic membrane is soaked in the rhNGF solution to play a role in adsorbing the medicament and then slowly releasing the medicament, and is transplanted to an animal model with corneal epithelium defect, so that the application frequency after transplantation can be reduced, the influence of eye drops on the amniotic membrane can be reduced, the amniotic membrane can be prevented from falling off in advance, and the rhNGF can be slowly released to play a role in promoting the growth of epithelium and nerve. Therefore, the prepared amnion drug-carrying system with double functions has important clinical application value.
Drawings
FIG. 1 is a graph of the in vitro release profile of amniotic membrane after soaking rhNGF therein;
FIG. 2 is a graph showing the concentration of the rhNGF-loaded amniotic membrane after transplantation in experiment 1;
FIG. 3 shows changes in corneal sensitivity at various time points after corneal epithelial injury;
FIG. 4 shows the change in the rate of corneal epithelial repair at various times post-operatively;
figure 5 shows the change in the density of subepithelial nerve regeneration 3 weeks after corneal epithelial injury.
Detailed Description
For the convenience of understanding, the technical scheme of the invention is further described in detail by embodiments with reference to the attached drawings:
a preparation method of a drug-loaded amnion comprises the following steps:
(1) selecting the human amniotic membrane preserved by glycerol, and washing the glycerol on the surface of the amniotic membrane to be clean by 0.9% sodium chloride injection.
(2) Placing the amnion obtained in the step (1) on sterilized infusion film paper, and sucking the residual sodium chloride solution by using special laboratory absorbent paper.
(3) And (3) adding a layer of infusion film paper on the dried amnion obtained in the step (2), flattening and pressing, measuring by a ruler, drawing, and cutting to prepare a square amnion sheet with the size of 15mmx15 mm.
(4) Soaking the amniotic membrane prepared in the step (3) in rhNGF with the concentration of 100ug/ml and 500ug/ml at 4 ℃, taking out the amniotic membrane after 30min, and transferring the amniotic membrane to a small culture dish containing PBS at room temperature.
Experiment 1: the release kinetics of the rhNGF are researched by detecting the content of the rhNGF by an ELISA method so as to verify the relationship between the release amount of the rhNGF and the concentration and the soaking time of the soaking medicament, and the specific experimental steps are as follows:
(1) the goat membranes soaked in rhNGF with the concentration of 100ug/ml and 500ug/ml are taken out after 24h, 3h, 1h, 30min and 15min respectively and transferred to a small culture dish containing PBS at room temperature.
(2) The PBS solution was collected continuously over 2 weeks.
The experimental results show that: the amnion is soaked in the rhNGF solution with the same concentration, the amount of the amnion loaded drugs is different at different soaking times, the amnion can continuously release the rhNGF within 2 weeks after soaking the drugs for 24h, 3h, 1h, 30min and 15min, and the release amount is stable and has no obvious burst release phenomenon after soaking for 30 min. Under the same soaking time, the medicine carrying capacity of the soaked sheep membrane with the rhNGF concentration of 500ug/ml is more than that of the soaked sheep membrane with the rhNGF concentration of 100ug/ml, and the medicine release capacity is larger. See figure 1.
It follows therefore that: the rhNGF release amount is related to the concentration of the soaked medicament, and the higher the soaking concentration is, the more the amnion medicament-carrying amount is, and the larger the medicament release amount is.
A method for treating corneal epithelial defect by using a medicament-carrying amniotic membrane specifically comprises the following steps:
(1) selecting the human amniotic membrane preserved by glycerol, and washing the glycerol on the surface of the amniotic membrane to be clean by 0.9% sodium chloride injection.
(2) Placing the amnion obtained in the step (1) on sterilized infusion film paper, and sucking the residual sodium chloride solution by using special laboratory absorbent paper.
(3) And (3) adding a layer of infusion film paper on the dried amnion obtained in the step (2), flattening and compacting. And measuring, drawing and cutting by using a ruler to prepare a square sheepskin sheet with the size of 15mmx15 mm.
(4) Soaking the goat diaphragm prepared in the step (3) in rhNGF with the concentration of 100ug/ml and 500ug/ml at 4 ℃, taking out the goat diaphragm after 30min, and transferring the goat diaphragm to a small culture dish containing PBS at room temperature.
(5) Transplanting the amniotic membrane prepared in the step (4) to the corneal epithelial defect.
Experiment 2: animal experiments verify that the prepared medicament-carrying amnion has the effect of repairing corneal epithelium:
the specific experimental steps are as follows:
1) the animal experiment is carried out by adopting a sheep membrane with the soaking temperature of 4 ℃, the soaking rhNGF concentration of 500ug/ml and the soaking time of 30 min.
2) Selecting 30 New Zealand rabbits, anesthetizing ear vein, opening eyelid with an eyelid opener, and cutting off the third palpebral fissure. After surface anesthesia, a corneal trephine with a diameter of 7.5mm is gently rotated on the corneal surface to make corneal impressions. And scraping all corneal epithelium in the indentation along the corneal indentation by using an Algerbrush II electric corneal epithelium knife, trimming the edge neatly, and establishing a corneal epithelium defect model with the monocular diameter of 7.5 mm.
3) The animal model is randomly divided into a blank control group and a medicament-carrying amniotic membrane transplantation group by adopting a random digital table method, and each group is provided with 10 animals.
4) Except for the blank control group, the medicament-carrying amniotic membrane transplantation group is subjected to medicament-carrying amniotic membrane transplantation after modeling.
5) Two groups of 48h corneal epithelium regeneration were observed by sodium corneal fluorescein staining. The Cochet-Bonnet tactile meter measures the change in corneal sensitivity 3 weeks after surgery. After 3 weeks of operation, corneal material was taken and nerve staining was performed, and changes in the density of corneal epithelial plexus nerve fibers were calculated using an Image J1.45 s Image analysis system. The amnion transplantation component is used for obtaining amnion materials after 2, 4, 6 and 8 days of operation, and the rhNGF content in the amnion transplantation piece is detected by an ELISA method.
The experimental results show that: the rhNGF can be slowly and continuously released within 8 days after the drug-loaded amniotic membrane transplantation, and is shown in figure 2. The transplantation of the medicament-loaded amniotic membrane can promote the regeneration of rabbit corneal epithelium, and is shown in figure 4. The transplantation of the loaded amniotic membrane can promote the recovery of corneal sensitivity, as shown in figure 3. The transplantation of the drug-loaded amniotic membrane can promote the regeneration of nerve fibers of nerve plexus under corneal epithelium, as shown in figure 5.
Claims (3)
1. A preparation method of a medicine-carrying amnion is characterized by comprising the following steps:
(1) selecting human amniotic membrane preserved by glycerol, and washing the glycerol on the surface of the amniotic membrane to be clean by 0.9% sodium chloride injection;
(2) placing the amnion obtained in the step (1) on sterilized infusion film paper, and sucking the residual sodium chloride solution by using special laboratory absorbent paper;
(3) adding a layer of infusion film paper on the dried amnion obtained in the step (2), flattening and pressing, measuring and drawing by a ruler, and cutting to manufacture an amnion sheet;
(4) and (4) soaking the amniotic membrane prepared in the step (3) in rhNGF with a certain concentration at 4 ℃, taking out the amniotic membrane after 30min, and transferring the amniotic membrane to a small culture dish containing PBS at room temperature.
2. The method for preparing a drug-loaded amniotic membrane according to claim 1, wherein the method comprises the following steps: the size of the goat film sheet prepared in the step (3) is 15mmx15mm square.
3. The method for preparing a drug-loaded amniotic membrane according to claim 1, wherein the method comprises the following steps: the rhNGF concentration in the step (4) is 100ug/ml and 500 ug/ml.
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| EP1926746A1 (en) * | 2005-09-23 | 2008-06-04 | Associazione Levi-Montalcini A.P.S. | Method for the production of biologically active rhngf |
| CN101040616B (en) * | 2006-03-24 | 2011-08-17 | 珠海亿胜生物制药有限公司 | Pharmaceutical amniotic membrane and processes for their preparation |
| EP2077900A2 (en) * | 2006-10-30 | 2009-07-15 | SHIUEY, Yichieh | Methods and systems for immobilizing corneal prostheses |
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