WO2018219045A1 - 去细胞角膜基质透镜及其制备方法 - Google Patents

去细胞角膜基质透镜及其制备方法 Download PDF

Info

Publication number
WO2018219045A1
WO2018219045A1 PCT/CN2018/082470 CN2018082470W WO2018219045A1 WO 2018219045 A1 WO2018219045 A1 WO 2018219045A1 CN 2018082470 W CN2018082470 W CN 2018082470W WO 2018219045 A1 WO2018219045 A1 WO 2018219045A1
Authority
WO
WIPO (PCT)
Prior art keywords
lens
corneal stroma
stroma lens
preparation
physiological saline
Prior art date
Application number
PCT/CN2018/082470
Other languages
English (en)
French (fr)
Inventor
梁丽金
杨习锋
曾晨光
余克明
庄菁
Original Assignee
广州新诚生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州新诚生物科技有限公司 filed Critical 广州新诚生物科技有限公司
Publication of WO2018219045A1 publication Critical patent/WO2018219045A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/14—Eye parts, e.g. lenses, corneal implants; Implanting instruments specially adapted therefor; Artificial eyes
    • A61F2/16—Intraocular lenses
    • A61F2/1602—Corrective lenses for use in addition to the natural lenses of the eyes or for pseudo-phakic eyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • 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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • 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
    • A61L27/3641—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 characterised by the site of application in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • 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
    • 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
    • A61L27/3687—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 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • 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
    • 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
    • A61L27/3691—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 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2240/001—Designing or manufacturing processes
    • A61F2240/002—Designing or making customized prostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea

Definitions

  • the present disclosure relates to the field of tissue engineering technology, and more particularly to a decellularized corneal stroma lens and a method of preparing the same.
  • Hyperopia is a kind of refractive state that is imaged behind the retina after parallel rays enter the eye. When the refractive power of the eyeball is insufficient or the length of the axial length is insufficient, hyperopia occurs. Hyperopia, as a kind of eye disease, greatly affects people's quality of life, especially as the age increases, the incidence of hyperopia increases.
  • Hyperopia is a common clinical ametropia, and the correction of high hyperopia has always been a problem of refractive therapy.
  • the methods of hyperopia correction treatment mainly include frame glasses, contact lenses or refractive surgery.
  • Presbyopia is a physiological phenomenon that is not a pathological state nor a refractive error. It is a visual problem that people must inevitably appear after entering middle-aged and old age. As we age, patients with presbyopia must add a convex lens to get clear near vision.
  • implantable contact lenses are mostly made of synthetic materials, which can produce lenses of various degrees and sizes according to the refractive state of different patients.
  • Postoperative visual acuity recovery is fast, but its drawbacks are also obvious, such as: There may be an increase in astigmatism after surgery, and corneal haze may occur in the early stage; and its applicability is limited, and there is a risk of corneal flap, corneal damage may occur during long-term implantation, and visual quality may be degraded; in addition, its safety Lower, permanent foreign body, may cause immune rejection during long-term implantation. Therefore, for patients with high hyperopia, presbyopia, and anisometropia, it is necessary to find lens materials with wider indications, higher safety, better predictability, and better stability.
  • animal-derived corneas there are some problems in the use of animal-derived corneas, for example: (1) Although the animal-derived cornea has good histocompatibility, if its own cell removal is incomplete, there is a risk of generating an immune response; (2) In addition, although there are some methods for removing corneal stromal cells, the existing corneal acellular matrix preparation process is prone to cause swelling of the cornea, resulting in a change in the arrangement or conformation of the collagen fibers in the matrix, resulting in a decrease in the transparency of the corneal stroma. Morphology, curvature, etc.
  • the traditional corneal lens is usually made by special keratectomy, the process is relatively rough, may cause the cut surface to be not smooth, or the lens part is lost, the predictability and safety are poor. And the incidence of recent complications, such as astigmatism, is higher.
  • the purpose of the present disclosure includes providing a method for preparing a decellularized corneal stroma lens, and the object of the present disclosure further includes providing a decellularized corneal stroma lens to alleviate the low safety, poor transparency, and performance of a corneal lens existing in the prior art. Unstable technical problems.
  • the present disclosure provides a method for preparing a decellularized corneal stroma lens, comprising: sequentially subjecting a cell-bearing corneal stroma lens to cell lysis and cross-linking treatment, and then sterilizing to obtain the decellularized corneal stroma lens.
  • the present disclosure also provides the use of the above-described decellularized corneal stroma lens for the treatment of hyperopia, presbyopia, and anisometropia.
  • the method of cell lysis comprises: treating the corneal stroma lens with a cell lysate.
  • the cell lysate comprises at least one of SDS, NP40 and TritonX-100.
  • the concentration of the cell lysate is from 0.1% to 3%.
  • the method for cell lysis comprises: soaking with 0.9% physiological saline or phosphate buffer solution containing 0.1%-3% Triton X-100 for 12-48 hours, and then rinsing with 0.9% physiological saline or phosphate buffer solution 48 -96h.
  • the method of cross-linking treatment comprises: treating the corneal stroma lens after lysis of the cells with a cross-linking agent.
  • the crosslinking agent includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxythiosuccinimide, genipin and glutaraldehyde. kind.
  • the crosslinking method comprises: immersing in a 0.9% physiological saline or phosphate buffer solution containing a crosslinking agent, followed by rinsing with 0.9% physiological saline or a phosphate buffer solution for 24-96 hours, the crosslinking agent It is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxythiosuccinimide, genipin or glutaraldehyde.
  • the mass ratio of the crosslinking agent to the corneal stroma lens is 1:1 to 1:15.
  • the mass ratio of the crosslinking agent to the corneal stroma lens is 1:5-1:10.
  • the corneal stroma lens is further sterilized.
  • the corneal stroma lens is sterilized by treating the corneal stroma lens with a disinfectant comprising at least one of penicillin and streptomycin.
  • a disinfection treatment is further included, the method comprising: soaking with physiological saline containing a concentration of 0.01-0.1 mg/ml penicillin and a concentration of 0.05-0.5 mg/ml streptomycin. 1-5 h, then rinsed with 0.9% saline or phosphate buffer.
  • the corneal stroma lens after the cross-linking treatment is sterilized by irradiating the corneal stroma lens with radiation, and the radiation for radiation sterilization includes at least one of ultraviolet rays, X-rays and ⁇ rays. .
  • a sterilization treatment is further included, and the sterilization method includes: irradiating with ⁇ -rays, and the irradiation dose is 20 to 30 kGy.
  • the cell-bearing corneal stroma lens is obtained by full femtosecond laser technology.
  • the preparation method comprises:
  • Step (a) Disinfection: A corneal stroma lens with an accurate thickness of cells will be obtained by full femtosecond laser technology, with a concentration of 0.01-0.1 mg/ml penicillin and a concentration of 0.05-0.5 mg/ml streptomycin. Soak for 1-5 h in physiological saline, then rinse with 0.9% saline or phosphate buffer solution;
  • Step (b) cell lysis: soaking with 0.9% physiological saline or phosphate buffer solution containing 0.1%-3% TritonX-100 for 12-48h, then rinsing with 0.9% physiological saline for 48-96h;
  • Step (c) cross-linking: soaking in a 0.9% physiological saline or phosphate buffer solution containing a crosslinking agent, followed by rinsing with 0.9% physiological saline or a phosphate buffer solution for 24-96 hours, the crosslinking agent is 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide, or N-hydroxythiosuccinimide, or glutaraldehyde, or genipin; the amount and amount of the crosslinking agent used The mass ratio of the corneal stroma lens treated by cell lysis is 1:1 to 1:15;
  • Step (d) sterilization: irradiation with gamma rays, the irradiation dose is 20 to 30 kGy.
  • the present disclosure also provides a decellularized corneal stroma lens obtained according to the preparation method described.
  • the decellularized corneal stroma lens has a thickness of 10 to 90 ⁇ m.
  • the preparation method of the decellularized corneal stroma lens can not only effectively remove the cellular components in the matrix, reduce the immunogenicity of the matrix, improve the mechanical strength of the cornea, and effectively maintain the original morphology and transparency of the corneal stroma.
  • Decellularized corneal stroma lenses are characterized by greater safety, better transparency, and more stable performance.
  • the corneal stroma lens can be used as a scaffold for the growth of corneal stromal cells.
  • the corneal cells can migrate and grow in the lens, and finally integrate with the autologous cornea, which can be used as a permanent implant lens for clinical hyperopia. Correction of anisometropia provides new treatments.
  • the use of the lens as an implantable contact lens can avoid the occurrence of permanent foreign matter of the synthetic material type corneal lens and avoid the occurrence of immune rejection.
  • Figure 1 is a graph showing the results of HE staining of a corneal stroma lens before decellularization
  • FIG. 2 is a graph showing the results of HE staining of a corneal stroma lens after decellularization
  • Figure 3 is a graph showing the results of HE staining of a corneal stroma lens after decellularization and cross-linking
  • Figure 4 is a graph comparing light transmittance of a corneal stroma lens before and after decellularization.
  • the existing corneal stroma lens decellularization process is still not mature, and the obtained corneal stroma lens material has the problems of lower safety, poor transparency and unstable performance.
  • the present disclosure is directed to the improvements proposed in the prior art.
  • the present disclosure provides a method of preparing a decellularized corneal stroma lens.
  • the method comprises: sequentially performing cell lysis and cross-linking treatment with a corneal stroma lens with cells, and then sterilizing to obtain a decellularized corneal stroma lens.
  • the method of cell lysis includes: treating a corneal stroma lens with a cell lysate.
  • the cell lysate includes at least one of SDS, NP40 and TritonX-100, and the concentration of the cell lysate is 0.1% to 3%.
  • the 0.1%-3% Triton X-100 involved in the method of cell lysis may be, for example, but not limited to, 0.1% Triton X-100, 0.3% Triton X-100, 0.5% Triton X-100, 0.8% Triton X-100. , 1% Triton X-100, 1.5% Triton X-100, 2% Triton X-100, 2.5% Triton X-100 or 3% Triton X-100.
  • the method of cross-linking treatment comprises: treating the corneal stroma lens after cell lysis with a cross-linking agent.
  • the crosslinking agent involved in the crosslinking method may be, for example, but not limited to, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, or N-hydroxythiosuccinimide, or At least one of genipin, and glutaraldehyde.
  • the ratio of the crosslinking agent used in the cross-linking method to the corneal matrix lens is 1:1 to 1:15, and the mass ratio thereof can be, for example, but not limited to 1:1, 1:2, 1 :3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:10, 1:11, 1:12, 1:13, 1:14 Or 1:15.
  • the corneal stroma lens prior to cell lysis of the corneal stroma lens, the corneal stroma lens is also sterilized.
  • the corneal stroma lens is sterilized by treating the corneal stroma lens with a disinfectant comprising at least one of penicillin and streptomycin.
  • the disinfection method involves a concentration of 0.01-0.1 mg/ml penicillin and a physiological saline or phosphate buffer solution having a concentration of 0.05-0.5 mg/ml streptomycin, wherein the penicillin may be, for example, but not limited to 0.01.
  • the corneal stroma lens after the cross-linking treatment is sterilized by irradiating the corneal stroma lens with radiation, and the radiation for radiation sterilization includes at least one of ultraviolet rays, X-rays and ⁇ rays.
  • the radiation for radiation sterilization comprises gamma rays
  • the radiation dose involved in the sterilization method is 20-30 kGy, for example, but not limited to 20 kGy, 21 kGy, 22 kGy, 23 kGy, 24 kGy, 25 kGy, 26 kGy, 27kGy, 28kGy, 29kGy or 30kGy.
  • the corneal stroma lens with cells is derived from human or other animals (other animals such as, but not limited to, pigs, cows or sheep, etc.), preferably, the corneal stroma lens with cells is derived from humans.
  • SMILE Small Incision Lenticule Extraction
  • the cell-bearing corneal stroma lens referred to in the present disclosure refers to an untreated fresh corneal stroma lens that is directly removed from the eye of a human or other animal.
  • a plurality of lenses of precise thickness can be obtained, preferably having a thickness of 10-90 [mu]m.
  • the above-mentioned decellularized corneal stroma lens can be well applied to treat hyperopia, presbyopia and anisometropia, and there is no permanent foreign matter of synthetic material type corneal lens, and avoiding the phenomenon of immune rejection, and has a good application prospect. .
  • Step (a) Disinfection: A corneal stroma lens with an accurate thickness of cells obtained by full femtosecond laser technology is soaked in physiological saline containing a concentration of 0.01 mg/ml penicillin and a concentration of 0.1 mg/ml streptomycin. 3h, then rinsed with 0.9% saline;
  • Step (b) cell lysis: soaked in 0.9% physiological saline containing 0.5% TritonX-100 for 24h, then rinsed with 0.9% physiological saline for 96h;
  • Step (c) cross-linking: soaking in 0.9% physiological saline containing a crosslinking agent, followed by rinsing with 0.9% physiological saline for 24 hours, and the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethyl
  • the carbodiimide (EDC) the mass ratio of the cross-linking agent to the corneal matrix lens treated by cell lysis is 1:5;
  • Step (d) sterilization: the cross-linked corneal lens is irradiated with gamma rays, the irradiation dose is 25 kGy;
  • the corneal stroma lens with cells is derived from a human.
  • the inventors analyzed the properties of the decellularized corneal stroma lens prepared according to the method of Example 1, and the specific experiment is as follows.
  • HE staining of the decellularized corneal stroma lens prepared by the method of Example 1 was performed on the corneal stroma lens with cells, and the results were as shown in Fig. 1, Fig. 2 and Fig. 3, respectively, from the corneal stroma lens with cells.
  • Fig. 1 more cells were observed, and in the HE staining map of the decellularized corneal stroma lens (Fig. 2) prepared according to the method of Example 1, cells were substantially invisible.
  • the HE staining diagram of the decellularized corneal stroma lens (Fig. 3) obtained by cross-linking according to the method of Example 1 is substantially invisible, indicating that the method provided by the present disclosure can effectively remove Cells in the corneal stroma lens and maintain morphological stability of the corneal stroma lens.
  • the light transmittance of the decellularized corneal stroma lens prepared by the method of Example 1 was carried out by using a spectrophotometer in the visible light band (390 nm - 780 nm), and the result was as shown in FIG.
  • the visible light transmittance of the decellularized corneal stroma lens prepared by the method of Example 1 is substantially consistent with the fresh corneal stroma (ie, the corneal stroma lens with cells), indicating that the process has little effect on the light transmission performance of the corneal stroma lens. It can maintain a good light transmittance.
  • the decellularized corneal stroma is a decellularized corneal stroma lens
  • the fresh corneal stroma is a corneal stroma lens with cells.
  • Step (a) disinfection: a corneal stroma lens with an accurate thickness of cells obtained by full femtosecond laser technology, soaked in physiological saline containing a concentration of 0.05 mg/ml penicillin and a concentration of 0.2 mg/ml streptomycin 2h, and then rinsed with 0.9% saline;
  • Step (b) cell lysis: soaking with 0.9% physiological saline containing 0.3% TritonX-100 for 48h, then rinsing with 0.9% physiological saline for 96h;
  • Step (c) cross-linking: soaking in 0.9% physiological saline containing a crosslinking agent, followed by rinsing with 0.9% physiological saline for 24 hours, cross-linking agent N-hydroxythiosuccinimide (NHS); crosslinking agent
  • N-hydroxythiosuccinimide N-hydroxythiosuccinimide
  • Step (d) sterilization: the cross-linked corneal lens is irradiated with gamma rays, the irradiation dose is 25 kGy;
  • the corneal stroma lens with cells is derived from a human.
  • Step (a) Disinfection: Soak a fresh cell-bearing corneal stroma lens with physiological saline containing 0.1 mg/ml penicillin and a concentration of 0.5 mg/ml streptomycin for 1 h, and then use 0.9% physiological Rinse with salt water;
  • Step (b) cell lysis: soaking with 0.9% physiological saline containing 0.4% Triton X-100 for 36h, then rinsing with 0.9% physiological saline for 72h;
  • Step (c) cross-linking: soaking in 0.9% physiological saline containing a crosslinking agent, followed by rinsing with 0.9% physiological saline for 24 hours, and the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethyl
  • the ratio of the amount of the cross-linking agent to the corneal matrix lens treated by cell lysis is 1:10;
  • Step (d) sterilization: the cross-linked corneal lens is irradiated with gamma rays, the irradiation dose is 25 kGy;
  • the corneal stroma lens with cells is derived from a human.
  • Step (a) Disinfection: A corneal stroma lens with an accurate thickness of cells obtained by full femtosecond laser technology is soaked in physiological saline containing a concentration of 0.01 mg/ml penicillin and a concentration of 0.5 mg/ml streptomycin. 1h, then rinsed with 0.9% saline;
  • Step (b) cell lysis: soaked in 0.9% physiological saline containing 0.1% SDS for 24h, and then rinsed with 0.9% physiological saline for 96h;
  • Step (c) cross-linking: soaking in 0.9% physiological saline containing a crosslinking agent, followed by rinsing with 0.9% physiological saline for 24 hours, and the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethyl a carbodiimide and glutaraldehyde; the mass ratio of the cross-linking agent to the corneal matrix lens treated by cell lysis is 1:10;
  • Step (d) sterilization: the cross-linked corneal lens is irradiated with X-rays, the irradiation dose is 30kGy;
  • the corneal stroma lens with cells is derived from a human.
  • Step (a) disinfection: a corneal stroma lens with an accurate thickness of cells obtained by full femtosecond laser technology, soaked in physiological saline containing a concentration of 0.1 mg/ml penicillin and a concentration of 0.05 mg/ml streptomycin 3h, then rinsed with 0.9% saline;
  • Step (b) cell lysis: soaked in 0.9% physiological saline containing 3% NP40 for 24h, and then rinsed with 0.9% physiological saline for 96h;
  • Step (c) cross-linking: soaking in 0.9% physiological saline containing a crosslinking agent, followed by rinsing with 0.9% physiological saline for 24 hours, and the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethyl a carbodiimide and N-hydroxy sulfosuccinimide; a mass ratio of the cross-linking agent to the corneal matrix lens treated by cell lysis is 1:10;
  • Step (d) sterilization: the cross-linked corneal lens is irradiated with ultraviolet rays, the irradiation dose is 30 kGy;
  • the corneal stroma lens with cells is derived from a human.
  • Step (a) disinfection: a corneal stroma lens with an accurate thickness of cells obtained by full femtosecond laser technology, soaked in physiological saline containing a concentration of 0.05 mg/ml penicillin and a concentration of 0.05 mg/ml streptomycin 3h, then rinsed with 0.9% saline;
  • Step (b) cell lysis: soaked in 0.9% physiological saline containing 1% NP40 and 0.5% Triton X-100 for 12h, then rinsed with 0.9% physiological saline for 72h;
  • Step (c) cross-linking: soaking in 0.9% physiological saline containing a crosslinking agent, followed by rinsing with 0.9% physiological saline for 24 hours, and the crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethyl
  • the ratio of the amount of the cross-linking agent to the corneal matrix lens treated by cell lysis is 1:8;
  • Step (d) sterilization: the cross-linked corneal lens is irradiated with gamma rays, the irradiation dose is 30 kGy;
  • the corneal stroma lens with cells is derived from a human.
  • the obtained decellularized corneal stroma lens has the following advantages:
  • cell membrane lysis and cross-linking of the corneal stroma lens in a saline environment avoids the water-absorbent/dehydrated state of the corneal stroma lens and changes the conformation and arrangement of the collagen fibers, so that the corneal stroma lens can effectively maintain the original shape, thickness and curvature.
  • the cells in the corneal stroma lens are lysed and cross-linked, which can effectively remove the cellular components in the matrix, reduce the immunogenicity of the matrix, improve the mechanical strength of the cornea, and effectively maintain the original morphology of the corneal stroma lens. Transparency for a safer, more transparent and more stable corneal stroma lens.
  • the corneal stroma lens can be used as a 3-dimensional scaffold for the growth of corneal stromal cells.
  • the corneal cells can migrate and grow in the lens, and finally integrate with the autologous cornea. It can be used as a permanent implant lens for clinical height.
  • the correction of hyperopia and anisometropia provides new treatments.
  • the use of the lens as an implantable contact lens can avoid the occurrence of permanent foreign matter of the synthetic material type corneal lens and avoid the occurrence of immune rejection.
  • the full femtosecond laser technology can accurately cut, making the preparation of the cornea lens more precise, which can avoid the corneal lens cutting surface caused by the traditional cutting process is not smooth enough or the lens part is lost; and the application uses the full femtosecond laser technology for cutting The preparation process is more precise and predictive.
  • the decellularized corneal stroma lens provided by the present disclosure is characterized by higher safety, better transparency, and more stable performance.
  • the present disclosure provides a decellularized corneal stroma lens and a method of preparing the same, and relates to the technical field of tissue engineering.
  • the preparation method of the decellularized corneal stroma lens provided by the present disclosure not only can effectively remove the cellular components in the matrix, reduce the immunogenicity of the corneal stroma lens, improve the mechanical strength of the corneal lens, and effectively maintain the original shape and transparency of the corneal stroma.
  • the prepared decellularized corneal stroma lens has the characteristics of higher safety, better transparency and more stable performance; and can be used as a scaffold for proliferation and growth of corneal stromal cells, and finally integrated with the autologous cornea, which can be used as a permanent implant. Lens; in addition, the occurrence of phenomena such as immune rejection can be avoided. It has a good correction and treatment effect for hyperopia, presbyopia and anisometropia, and has broad application prospects.

Abstract

一种去细胞角膜基质透镜及其制备方法，涉及组织工程学的技术领域。该去细胞角膜基质透镜的制备方法不仅能够有效去除基质中细胞成分，降低角膜基质透镜的免疫原性，提高角膜透镜的力学强度，更能有效保持角膜基质的原始形态和透明度，所制得的去细胞角膜基质透镜具有安全性更高、透明度更佳和性能更稳定的特点；而且可以作为角膜基质细胞增殖生长的支架，最终与自体角膜融为一体，可作为永久性植入透镜；另外，还能够避免免疫排斥等现象的出现。

Description

去细胞角膜基质透镜及其制备方法
相关申请的交叉引用
本申请要求于2017年5月31日提交中国专利局的申请号为2017104030387、名称为“去细胞角膜基质透镜及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及组织工程学技术领域，尤其是涉及一种去细胞角膜基质透镜及其制备方法。
背景技术
远视是平行光线进入眼内后成像在视网膜之后的一种屈光状态，当眼球的屈光力不足或其眼轴长度不足时就产生远视。远视作为眼科疾病的一种，极大的影响了人们的生活质量，尤其是随着年龄的增大，远视的发生几率也会升高。
远视是临床常见的屈光不正眼病，高度远视的矫正一直是屈光治疗的难题。目前远视矫正治疗的方法主要包括框架眼镜、角膜接触镜或者屈光手术。
老视是一种生理现象不是病理状态也不属于屈光不正，是人们步入中老年后必然出现的视觉问题。随着年龄的增长，出现老视的患者必须增加凸透镜才能获得清晰的近视力。
目前，植入式的角膜接触镜多采用合成材料制成，可以根据不同患者的屈光状态生产出各种度数和型号大小的透镜，术后视力恢复快，但其弊端也比较明显，比如：术后可能出现散光增加，早期可能出现角膜雾状浑浊；而且其适用症有限，并存在角膜瓣相关风险，在长期植入过程中可能出现角膜损伤，视觉质量下降的情况；另外，其安全性较低，属于永久性异物，在长期植入过程中可能出现免疫排斥反应。因此，对于高度远视、老视及合并屈光参差的患者，需要找到适应症更广、安全性更高、预测性更强、稳定性更佳的透镜材料。
对于角膜基质移植所需的材料而言，不仅要求有良好的光学特性，还要求与人眼组织有良好的组织相容性，人工合成材料则在生物组织相容性上有很大的弊端，而利用动物源性的角膜，则能够在很大程度上缓解人工合成材料的组织相容性差的问题。
但使用动物源性角膜也存在着一些问题，例如：(1)虽然动物源性角膜的组织相容性较好，但如果其自身细胞去除不完全，则会存在产生免疫反应的风险；(2)此外，虽然目前已有一些去除角膜基质细胞的方法，但是现有的角膜去细胞基质制备工艺容易导致角膜 吸水肿胀，导致基质中的胶原纤维排列或构象发生改变，使角膜基质透明度下降，原始形态、曲率等发生改变；(3)而且，传统的角膜透镜通常是以特殊的角膜刀切削制得，工艺相对粗糙，可能造成削切面不够光滑，或者透镜部分丢失，预测性和安全性较差，且术后近期并发症，如散光等发生率较高。
因此，如何获得安全性更高、透明度更佳和性能更稳定的角膜基质透镜，仍需要进行不断的研究。安全、有效的人角膜基质的处理方法，在实际临床应用中，具有重大的意义。
有鉴于此，特提出本公开。
发明内容
本公开的目的包括，提供一种去细胞角膜基质透镜的制备方法，本公开的目的还包括，提供去细胞角膜基质透镜，以缓解现有技术中存在的角膜透镜安全性低、透明度差和性能不稳定的技术问题。
本公开提供了一种去细胞角膜基质透镜的制备方法，包括：将带有细胞的角膜基质透镜依次进行细胞裂解和交联处理，然后经过灭菌后得到所述去细胞角膜基质透镜。
本公开还提供了上述去细胞角膜基质透镜在治疗远视、老视和屈光参差中的应用。
进一步地，所述细胞裂解的方法包括：将所述角膜基质透镜用细胞裂解液处理。
进一步地，所述细胞裂解液包括SDS、NP40和TritonX-100中的至少一种。
进一步地，所述细胞裂解液的浓度为0.1％-3％。
进一步的，所述细胞裂解的方法包括：用含有0.1％-3％TritonX-100的0.9％的生理盐水或磷酸盐缓冲溶液浸泡12-48h，然后用0.9％生理盐水或磷酸盐缓冲溶液漂洗48-96h。
进一步的，所述交联处理的方法包括：将所述细胞裂解后的所述角膜基质透镜用交联剂处理。
进一步的，所述交联剂包括1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基硫代琥珀酰亚胺、京尼平和戊二醛中的至少一种。
进一步的，所述交联的方法包括：用含有交联剂的0.9％生理盐水或磷酸盐缓冲溶液中浸泡，然后用0.9％生理盐水或者磷酸盐缓冲溶液漂洗24-96h，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基硫代琥珀酰亚胺、京尼平或者戊二醛。
进一步的，所述交联剂的使用量与角膜基质透镜的质量比为1:1-1:15。
进一步的，所述交联剂的使用量与角膜基质透镜的质量比为1:5-1:10。
进一步的，在对所述角膜基质透镜进行细胞裂解之前，还包括对所述角膜基质透镜进行消毒处理。
进一步的，对所述角膜基质透镜进行消毒处理是将所述角膜基质透镜用消毒剂进行处 理，所述消毒剂包括青霉素和链霉素中的至少一种。
进一步的，在所述细胞裂解处理之前，还包括消毒处理，所述消毒的方法包括：用含有浓度为0.01～0.1mg/ml青霉素和浓度为0.05～0.5mg/ml链霉素的生理盐水浸泡1-5h，然后用0.9％生理盐水或磷酸盐缓冲溶液漂洗。
进一步的，对交联处理后的所述角膜基质透镜进行灭菌，是将所述角膜基质透镜进行辐射灭菌，用于辐射灭菌的射线包括紫外线、X射线和γ射线中的至少一种。
进一步的，在所述交联处理之后，还包括灭菌处理，所述灭菌的方法包括：用γ射线进行辐照，辐照剂量为20～30kGy。
进一步的，所述带有细胞的角膜基质透镜是通过全飞秒激光技术获得的。
进一步的，所述制备方法包括：
步骤(a)，消毒：将通过全飞秒激光技术获得带有细胞的准确厚度的角膜基质透镜，用含有浓度为0.01～0.1mg/ml青霉素和浓度为0.05～0.5mg/ml链霉素的生理盐水浸泡1-5h，然后用0.9％生理盐水或磷酸盐缓冲溶液漂洗；
步骤(b)，细胞裂解：用含有0.1％-3％TritonX-100的0.9％生理盐水或磷酸盐缓冲溶液浸泡12-48h，然后用0.9％的生理盐水漂洗48-96h；
步骤(c)，交联：用含有交联剂的0.9％生理盐水或磷酸盐缓冲溶液中浸泡，然后用0.9％生理盐水或者磷酸盐缓冲溶液漂洗24-96h，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺，或N-羟基硫代琥珀酰亚胺、或戊二醛、或京尼平；所述交联剂的使用量与所述经过细胞裂解处理的角膜基质透镜的质量比为1:1-1:15；
步骤(d)，灭菌：用γ射线进行辐照，辐照剂量为20～30kGy。
另外，本公开还提供了按照所述的制备方法得到的去细胞角膜基质透镜。
进一步地，所述去细胞角膜基质透镜的厚度为10-90μm。
一种上述去细胞角膜基质透镜在治疗远视、老视和屈光参差中的应用。
与现有技术相比，本公开的有益效果例如包括：
本公开提供的去细胞角膜基质透镜的制备方法，不仅能够有效去除基质中细胞成分降低基质的免疫原性，提高角膜的力学强度，更能有效保持角膜基质的原始形态和透明度，所制得的去细胞角膜基质透镜具有安全性更高、透明度更佳和性能更稳定的特点。而且，角膜基质透镜植入后可以作为角膜基质细胞生长增值的支架，角膜细胞可以在透镜中迁移和生长，最终与自体角膜融为一体，可作为永久性植入透镜，为临床上高度远视及屈光参差的矫正提供新的治疗方法。另外，以该透镜作为植入式角膜接触镜可以避免出现合成材料类角膜透镜的永久异物情况，避免免疫排斥等现象的出现。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为去细胞前的角膜基质透镜的HE染色结果图；
图2为去细胞处理后的角膜基质透镜的HE染色结果图；
图3为去细胞并进行交联后的角膜基质透镜的HE染色结果图；
图4为去细胞前后的角膜基质透镜的透光率比较图。
具体实施方式
下面将结合实施例对本公开的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
现有的角膜基质透镜去细胞工艺尚不成熟，制得的角膜基质透镜材料存在着安全性更低、透明度差和性能不稳定的问题。本公开就是针对现有技术中存在的这些问题，所提出的改进。
本公开提供了去细胞角膜基质透镜的制备方法。
包括：将带有细胞的角膜基质透镜依次进行细胞裂解和交联处理，然后经过灭菌后得到去细胞角膜基质透镜。
其中，细胞裂解的方法包括：将角膜基质透镜用细胞裂解液处理。细胞裂解液包括SDS、NP40和TritonX-100中的至少一种，细胞裂解液的浓度为0.1％-3％。
可选地，细胞裂解的方法中涉及的0.1％-3％TritonX-100，例如可以为，但不限于0.1％TritonX-100、0.3％TritonX-100、0.5％TritonX-100、0.8％TritonX-100、1％TritonX-100、1.5％TritonX-100、2％TritonX-100、2.5％TritonX-100或者3％TritonX-100。
其中，交联处理的方法包括：将细胞裂解后的角膜基质透镜用交联剂处理。交联的方法中涉及的交联剂例如可以为，但不限于1-(3-二甲氨基丙基)-3-乙基碳二亚胺，或者N-羟基硫代琥珀酰亚胺，或者京尼平，和戊二醛中的至少一种。
其中，交联的方法中涉及的交联剂的使用量与角膜基质透镜的质量比为1:1-1:15，其质量比例如可以为，但不限于1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:10、1:11、1:12、1:13、1:14或者1:15。
其中，在对角膜基质透镜进行细胞裂解之前，还包括对角膜基质透镜进行消毒处理。 对角膜基质透镜进行消毒处理是将角膜基质透镜用消毒剂进行处理，消毒剂包括青霉素和链霉素中的至少一种。
可选地，消毒的方法中涉及的浓度为0.01～0.1mg/ml青霉素和浓度为0.05～0.5mg/ml链霉素的生理盐水或磷酸盐缓冲溶液，其中青霉素例如可以为，但不限于0.01mg/ml、0.03mg/ml、0.05mg/ml、0.07mg/ml、0.1mg/ml；其中链霉素例如可以为，但不限于0.05mg/ml、0.06mg/ml、0.07mg/ml、0.08mg/ml、0.09mg/ml、0.1mg/ml、0.2mg/ml、0.3mg/ml、0.4mg/ml、0.5mg/ml。
其中，对交联处理后的角膜基质透镜进行灭菌，是将角膜基质透镜进行辐射灭菌，用于辐射灭菌的射线包括紫外线、X射线和γ射线中的至少一种。
可选地，用于辐射灭菌的射线包括γ射线，灭菌的方法中涉及的辐射剂量为20～30kGy，例如可以为，但不限于20kGy、21kGy、22kGy、23kGy、24kGy、25kGy、26kGy、27kGy、28kGy、29kGy或30kGy。
其中，带有细胞的角膜基质透镜来自于人或者其他动物(其他动物例如可以为，但不限于猪、牛或者羊等)，优选的，带有细胞的角膜基质透镜来自于人。
其中，涉及的全飞秒激光技术，是指利用全飞秒激光小切口角膜基质透镜取出术(Small Incision Lenticule Extraction,SMILE)进行切取的技术。SMILE是应用超短脉冲激光在角膜内完成两次脉冲扫描，制作角膜基质内镜片后将其取出，可以降低对角膜神经的损伤及生物与力学的影响。
另外，本公开中涉及的带有细胞的角膜基质透镜是指未经处理的、直接从人或者其他动物眼内取出的新鲜的角膜基质透镜。
需要注意的是，按照本公开提供的方法，能够获得多种厚度精确的透镜，优选的，厚度为10-90μm。
上述去细胞角膜基质透镜能够较好地应用于治疗远视、老视和屈光参差中，不会出现合成材料类角膜透镜的永久异物情况，避免免疫排斥等现象的出现，具有较好的应用前景。
为了有助于更清楚的理解本公开，现通过具体的实施例对本公开的内容进行详细的介绍。如未明确指出，以下实施例中涉及的实验操作方法为常用的分子生物学或医学操作方法，涉及的试剂、仪器为常规的市售试剂或者仪器。
实施例1 去细胞角膜基质透镜的制备方法
步骤(a)，消毒：将通过全飞秒激光技术获得的带有细胞的准确厚度的角膜基质透镜，用含有浓度为0.01mg/ml青霉素和浓度为0.1mg/ml链霉素的生理盐水浸泡3h，然后用0.9％的生理盐水漂洗；
步骤(b)，细胞裂解：用含有0.5％TritonX-100的0.9％的生理盐水浸泡24h，然后用0.9％的生理盐水漂洗96h；
步骤(c)，交联：用含有交联剂的0.9％的生理盐水中浸泡，然后用0.9％生理盐水漂洗24h，交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)；交联剂的使用量与经过细胞裂解处理的角膜基质透镜的质量比为1:5；
步骤(d)，灭菌：将交联后的角膜透镜用γ射线进行辐照，辐照剂量为25kGy；
需要说明的是，在本实施例1中，带有细胞的角膜基质透镜来自于人。
另外，发明人对按照实施例1的方法制备得到的去细胞角膜基质透镜的性能进行了分析，具体实验如下。
HE染色实验
分别对带有细胞的角膜基质透镜、按照实施例1的方法制备得到的去细胞角膜基质透镜进行HE染色，结果分别如图1、图2和图3所示，从带有细胞的角膜基质透镜的HE染色图(图1)中可以看到有较多的细胞存在，而从按照实施例1的方法制备得到的去细胞角膜基质透镜HE染色图(图2)中则基本看不到细胞，但基质纤维间存在一些空隙；而按照实施例1的方法进行交联后得到的去细胞角膜基质透镜HE染色图(图3)中则基本看不到空隙，说明本公开提供的方法可以有效除去角膜基质透镜中的细胞，并保持角膜基质透镜形态学稳定。
透光率检测实验
利用分光光度计在可见光波段(390nm-780nm)分别对带有细胞的角膜基质透镜、按照实施例1的方法制备得到的去细胞角膜基质透镜进行透光率分析，结果如图4所示，按照实施例1的方法制备得到的去细胞角膜基质透镜的可见光透光率基本与新鲜角膜基质(即带有细胞的角膜基质透镜)一致，说明该工艺对角膜基质透镜的透光性能影响较小，能使之保持较好的透光率。
其中，图4中，去细胞角膜基质即为去细胞角膜基质透镜；新鲜角膜基质即为带有细胞的角膜基质透镜。
实施例2 去细胞角膜基质透镜的制备方法
步骤(a)，消毒：将通过全飞秒激光技术获得的带有细胞的准确厚度的角膜基质透镜，用含有浓度为0.05mg/ml青霉素和浓度为0.2mg/ml链霉素的生理盐水浸泡2h，，然后用0.9％的生理盐水漂洗；
步骤(b)，细胞裂解：用含有0.3％TritonX-100的0.9％的生理盐水浸泡48h，然后用0.9％的生理盐水漂洗96h；
步骤(c)，交联：用含有交联剂的0.9％的生理盐水中浸泡，然后用0.9％生理盐水漂洗24h，交联剂N-羟基硫代琥珀酰亚胺(NHS)；交联剂的使用量与经过细胞裂解处理的角膜基质透镜的质量比为1:9；
步骤(d)，灭菌：将交联后的角膜透镜用γ射线进行辐照，辐照剂量为25kGy；
需要说明的是，在本实施例2中，带有细胞的角膜基质透镜来自于人。
实施例3 去细胞角膜基质透镜的制备方法
步骤(a)，消毒：将新鲜的带有细胞的角膜基质透镜，用含有浓度为0.1mg/ml青霉素和浓度为0.5mg/ml链霉素的生理盐水浸泡1h，，然后用0.9％的生理盐水漂洗；
步骤(b)，细胞裂解：用含有0.4％TritonX-100的0.9％的生理盐水浸泡36h，然后用0.9％的生理盐水漂洗72h；
步骤(c)，交联：用含有交联剂的0.9％的生理盐水中浸泡，然后用0.9％生理盐水漂洗24h，交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺；交联剂的使用量与经过细胞裂解处理的角膜基质透镜的质量比为1:10；
步骤(d)，灭菌：将交联后的角膜透镜用γ射线进行辐照，辐照剂量为25kGy；
需要说明的是，在本实施例3中，带有细胞的角膜基质透镜来自于人。
实施例4 去细胞角膜基质透镜的制备方法
步骤(a)，消毒：将通过全飞秒激光技术获得的带有细胞的准确厚度的角膜基质透镜，用含有浓度为0.01mg/ml青霉素和浓度为0.5mg/ml链霉素的生理盐水浸泡1h，然后用0.9％的生理盐水漂洗；
步骤(b)，细胞裂解：用含有0.1％SDS的0.9％的生理盐水浸泡24h，然后用0.9％的生理盐水漂洗96h；
步骤(c)，交联：用含有交联剂的0.9％的生理盐水中浸泡，然后用0.9％生理盐水漂洗24h，交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺和戊二醛；交联剂的使用量与经过细胞裂解处理的角膜基质透镜的质量比为1:10；
步骤(d)，灭菌：将交联后的角膜透镜用X射线进行辐照，辐照剂量为30kGy；
需要说明的是，在本实施例4中，带有细胞的角膜基质透镜来自于人。
实施例5 去细胞角膜基质透镜的制备方法
步骤(a)，消毒：将通过全飞秒激光技术获得的带有细胞的准确厚度的角膜基质透镜，用含有浓度为0.1mg/ml青霉素和浓度为0.05mg/ml链霉素的生理盐水浸泡3h，然后用0.9％ 的生理盐水漂洗；
步骤(b)，细胞裂解：用含有3％NP40的0.9％的生理盐水浸泡24h，然后用0.9％的生理盐水漂洗96h；
步骤(c)，交联：用含有交联剂的0.9％的生理盐水中浸泡，然后用0.9％生理盐水漂洗24h，交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺和N-羟基硫代琥珀酰亚胺；交联剂的使用量与经过细胞裂解处理的角膜基质透镜的质量比为1:10；
步骤(d)，灭菌：将交联后的角膜透镜用紫外线进行辐照，辐照剂量为30kGy；
需要说明的是，在本实施例5中，带有细胞的角膜基质透镜来自于人。
实施例6 去细胞角膜基质透镜的制备方法
步骤(a)，消毒：将通过全飞秒激光技术获得的带有细胞的准确厚度的角膜基质透镜，用含有浓度为0.05mg/ml青霉素和浓度为0.05mg/ml链霉素的生理盐水浸泡3h，然后用0.9％的生理盐水漂洗；
步骤(b)，细胞裂解：用含有1％NP40和0.5％TritonX-100的0.9％的生理盐水浸泡12h，然后用0.9％的生理盐水漂洗72h；
步骤(c)，交联：用含有交联剂的0.9％的生理盐水中浸泡，然后用0.9％生理盐水漂洗24h，交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺；交联剂的使用量与经过细胞裂解处理的角膜基质透镜的质量比为1:8；
步骤(d)，灭菌：将交联后的角膜透镜用γ射线进行辐照，辐照剂量为30kGy；
需要说明的是，在本实施例6中，带有细胞的角膜基质透镜来自于人。
按照本公开提供的去细胞角膜基质透镜的制备方法，制得的去细胞角膜基质透镜具有以下优点：
首先，在生理盐水环境对角膜基质透镜进行细胞裂解和交联，避免角膜基质透镜出现吸水/脱水状态，改变胶原纤维的构象和排列，这样能使角膜基质透镜有效地保持原始形态、厚度和曲率。在这种状态下对角膜基质透镜中的细胞进行裂解和交联，不仅能有效除去基质中细胞成分降低基质的免疫原性，提高角膜的力学强度，更能有效保持角膜基质透镜的原始形态和透明度，从而获得安全性更高、透明度更佳和性能更稳定的角膜基质透镜。
其次，角膜基质透镜植入后可以作为角膜基质细胞生长增值的3维支架，角膜细胞可以在透镜中迁移和生长，最终与自体角膜融为一体，可作为永久性植入透镜，为临床上高度远视及屈光参差的矫正提供新的治疗方法。另外，以该透镜作为植入式角膜接触镜可以避免出现合成材料类角膜透镜的永久异物情况，避免免疫排斥等现象的出现。
另外，全飞秒激光技术可以精确切削，使得角膜透镜的制备更加精密，可以避免传统切削工艺造成的角膜透镜切削面不够光滑或者透镜部分丢失；而本申请则利用了全飞秒激光技术进行切削，制备工艺更精密，预测性更强。
因此，本公开提供的去细胞角膜基质透镜具有安全性更高、透明度更佳和性能更稳定的特点。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对本公开的限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供了一种去细胞角膜基质透镜及其制备方法，涉及组织工程学的技术领域。本公开提供的去细胞角膜基质透镜的制备方法，不仅能够有效去除基质中细胞成分，降低角膜基质透镜的免疫原性，提高角膜透镜的力学强度，更能有效保持角膜基质的原始形态和透明度，所制得的去细胞角膜基质透镜具有安全性更高、透明度更佳和性能更稳定的特点；而且可以作为角膜基质细胞增殖生长的支架，最终与自体角膜融为一体，可作为永久性植入透镜；另外，还能够避免免疫排斥等现象的出现。对于远视、老视和屈光参差具有较好的矫正和治疗效果，具有广阔的应用前景。

Claims (20)

  1. 一种去细胞角膜基质透镜的制备方法，其特征在于，包括：将带有细胞的角膜基质透镜依次进行细胞裂解和交联处理，然后经过灭菌后得到所述去细胞角膜基质透镜。
  2. 根据权利要求1所述的制备方法，其特征在于，所述细胞裂解的方法包括：将所述角膜基质透镜用细胞裂解液处理。
  3. 根据权利要求2所述的制备方法，其特征在于，所述细胞裂解液包括SDS、NP40和TritonX-100中的至少一种。
  4. 根据权利要求3所述的制备方法，其特征在于，所述细胞裂解液的浓度为0.1％-3％。
  5. 根据权利要求1所述的制备方法，其特征在于，所述细胞裂解的方法包括：用含有0.1％-3％TritonX-100的0.9％的生理盐水或磷酸盐缓冲溶液浸泡12-48h，然后用0.9％生理盐水或磷酸盐缓冲溶液漂洗48-96h。
  6. 根据权利要求1所述的制备方法，其特征在于，所述交联处理的方法包括：将所述细胞裂解后的所述角膜基质透镜用交联剂处理。
  7. 根据权利要求6所述的制备方法，其特征在于，所述交联剂包括1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基硫代琥珀酰亚胺、京尼平和戊二醛中的至少一种。
  8. 根据权利要求5所述的制备方法，其特征在于，所述交联的方法包括：用含有交联剂的0.9％生理盐水或磷酸盐缓冲溶液中浸泡，然后用0.9％生理盐水或者磷酸盐缓冲溶液漂洗24-96h，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺、N-羟基硫代琥珀酰亚胺、京尼平或者戊二醛。
  9. 根据权利要求8所述的制备方法，其特征在于，所述交联剂的使用量与角膜基质透镜的质量比为1:1-1:15。
  10. 根据权利要求9所述的制备方法，其特征在于，所述交联剂的使用量与角膜基质透镜的质量比为1:5-1:10。
  11. 根据权利要求1-5任一项所述的制备方法，其特征在于，在对所述角膜基质透镜进行细胞裂解之前，还包括对所述角膜基质透镜进行消毒处理。
  12. 根据权利要求11所述的制备方法，其特征在于，对所述角膜基质透镜进行消毒处理是将所述角膜基质透镜用消毒剂进行处理，所述消毒剂包括青霉素和链霉素中的至少一种。
  13. 根据权利要求9所述的制备方法，其特征在于，在所述细胞裂解处理之前，还 包括消毒处理，所述消毒的方法包括：用含有浓度为0.01～0.1mg/ml青霉素和浓度为0.05～0.5mg/ml链霉素的生理盐水浸泡1-5h，然后用0.9％生理盐水或磷酸盐缓冲溶液漂洗。
  14. 根据权利要求1所述的制备方法，其特征在于，对交联处理后的所述角膜基质透镜进行灭菌，是将所述角膜基质透镜进行辐射灭菌，用于辐射灭菌的射线包括紫外线、X射线和γ射线中的至少一种。
  15. 根据权利要求13所述的制备方法，其特征在于，在所述交联处理之后，还包括灭菌处理，所述灭菌的方法包括：用γ射线进行辐照，辐照剂量为20～30kGy。
  16. 根据权利要求15所述的制备方法，其特征在于，所述带有细胞的角膜基质透镜是通过全飞秒激光技术获得的。
  17. 根据权利要求16所述的制备方法，其特征在于，所述制备方法包括：
    步骤(a)，消毒：将通过全飞秒激光技术获得带有细胞的准确厚度的角膜基质透镜，用含有浓度为0.01～0.1mg/ml青霉素和浓度为0.05～0.5mg/ml链霉素的生理盐水浸泡1-5h，然后用0.9％生理盐水或磷酸盐缓冲溶液漂洗；
    步骤(b)，细胞裂解：用含有0.1％-3％TritonX-100的0.9％生理盐水或磷酸盐缓冲溶液浸泡12-48h，然后用0.9％的生理盐水漂洗48-96h；
    步骤(c)，交联：用含有交联剂的0.9％生理盐水或磷酸盐缓冲溶液中浸泡，然后用0.9％生理盐水或者磷酸盐缓冲溶液漂洗24-96h，所述交联剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺，或N-羟基硫代琥珀酰亚胺、或戊二醛、或京尼平；所述交联剂的使用量与所述经过细胞裂解处理的角膜基质透镜的质量比为1:1-1:15；
    步骤(d)，灭菌：用γ射线进行辐照，辐照剂量为20～30kGy。
  18. 按照权利要求1-17任一项所述的制备方法得到的去细胞角膜基质透镜。
  19. 根据权利要求18所述的去细胞角膜基质透镜，其特征在于，所述去细胞角膜基质透镜的厚度为10-90μm。
  20. 一种如权利要求18-19任一项所述的去细胞角膜基质透镜在治疗远视、老视和屈光参差中的应用。
PCT/CN2018/082470 2017-05-31 2018-04-10 去细胞角膜基质透镜及其制备方法 WO2018219045A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710403038.7A CN107233143A (zh) 2017-05-31 2017-05-31 去细胞角膜基质透镜及其制备方法
CN201710403038.7 2017-05-31

Publications (1)

Publication Number Publication Date
WO2018219045A1 true WO2018219045A1 (zh) 2018-12-06

Family

ID=59986004

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/082470 WO2018219045A1 (zh) 2017-05-31 2018-04-10 去细胞角膜基质透镜及其制备方法

Country Status (2)

Country Link
CN (1) CN107233143A (zh)
WO (1) WO2018219045A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021130276A1 (en) * 2019-12-27 2021-07-01 Gebauer-Klopotek Patent Verwaltungs-UG (haftungsbeschränkt) Stabilization of collagen scaffolds

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107233143A (zh) * 2017-05-31 2017-10-10 广州新诚生物科技有限公司 去细胞角膜基质透镜及其制备方法
CN109568663B (zh) * 2018-11-27 2021-01-15 中山大学中山眼科中心 角膜支架材料的制备方法及角膜支架材料
CN111803437A (zh) * 2020-06-03 2020-10-23 广州新诚生物科技有限公司 一种药物缓释动物角膜基质透镜及制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1985778A (zh) * 2005-12-20 2007-06-27 广州知光生物科技有限公司 生物型人工角膜
CN101066471A (zh) * 2007-04-25 2007-11-07 中山大学中山眼科中心 一种脱细胞角膜基质及其制备方法
US20100215717A1 (en) * 2009-02-26 2010-08-26 Shay Soker Endothelial scaffolds
CN103908700A (zh) * 2013-01-06 2014-07-09 陕西佰傲再生医学有限公司 一种脱细胞角膜的制备方法
CN104001215A (zh) * 2014-06-13 2014-08-27 深圳艾尼尔角膜工程有限公司 一种脱细胞角膜基质及其制备方法
CN107233143A (zh) * 2017-05-31 2017-10-10 广州新诚生物科技有限公司 去细胞角膜基质透镜及其制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102793593B (zh) * 2012-09-03 2015-06-10 于好勇 用于后巩膜加固术的异种巩膜片及其制备方法
CN103550826B (zh) * 2013-10-31 2015-07-08 广州优得清生物科技有限公司 异种角膜材料的制备方法
CN103892938A (zh) * 2014-04-01 2014-07-02 薛春燕 一种角膜板层片及其制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1985778A (zh) * 2005-12-20 2007-06-27 广州知光生物科技有限公司 生物型人工角膜
CN101066471A (zh) * 2007-04-25 2007-11-07 中山大学中山眼科中心 一种脱细胞角膜基质及其制备方法
US20100215717A1 (en) * 2009-02-26 2010-08-26 Shay Soker Endothelial scaffolds
CN103908700A (zh) * 2013-01-06 2014-07-09 陕西佰傲再生医学有限公司 一种脱细胞角膜的制备方法
CN104001215A (zh) * 2014-06-13 2014-08-27 深圳艾尼尔角膜工程有限公司 一种脱细胞角膜基质及其制备方法
CN107233143A (zh) * 2017-05-31 2017-10-10 广州新诚生物科技有限公司 去细胞角膜基质透镜及其制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021130276A1 (en) * 2019-12-27 2021-07-01 Gebauer-Klopotek Patent Verwaltungs-UG (haftungsbeschränkt) Stabilization of collagen scaffolds
CN115103695A (zh) * 2019-12-27 2022-09-23 盖鲍厄-克洛波泰克佩坦特弗瓦尔屯格斯-尤格公司(有限责任) 胶原蛋白支架的稳定

Also Published As

Publication number Publication date
CN107233143A (zh) 2017-10-10

Similar Documents

Publication Publication Date Title
WO2018219045A1 (zh) 去细胞角膜基质透镜及其制备方法
Sasaki et al. In vivo evaluation of a novel scaffold for artificial corneas prepared by using ultrahigh hydrostatic pressure to decellularize porcine corneas
Snibson Collagen cross‐linking: a new treatment paradigm in corneal disease–a review
US6544286B1 (en) Pre-fabricated corneal tissue lens method of corneal overlay to correct vision
US10072244B2 (en) Method for preparing heterogenetic corneal material
CN103908700B (zh) 一种脱细胞角膜的制备方法
AU2001278947A1 (en) Pre-fabricated corneal tissue lens and method of corneal overlay to correct vision (ii)
WO2014089548A1 (en) Oculoplasty grafts
CN104971381B (zh) 一种异种角膜移植物的无菌加工制备方法
CN112426566A (zh) 一种再生性角膜材料与其制备方法及用途
CN107233144A (zh) 去细胞角膜基质透镜在治疗眼科疾病中的应用
RU2676434C1 (ru) Комбинированный способ лечения заболеваний роговицы с применением кератопластики и кросслинкинга
CN111686301B (zh) 一种高度透明的脱细胞角膜基质及其制备方法
CN110420352A (zh) 一种生物型眼组织修复材料及其制备方法
Wand et al. Riboflavin–UV-A Crosslinking for Fixation of Biosynthetic Corneal Collagen Implants
Zhang et al. Update of research progress on small incision lenticule extraction (SMILE) lenticule reuse
CN112295018B (zh) 一种高性能再生性角膜材料与其制备方法及用途
Thirunavukarasu et al. Electron beam-irradiated donor cornea for on-demand lenticule implantation to treat corneal diseases and refractive error
CN114191613A (zh) 一种用鱼鳔制备的生物角膜及其制备方法和应用
CN109069696B (zh) 一种脱细胞猪板层干燥角膜及其使用方法和用途
JP4463124B2 (ja) 生体組織に含まれる細胞量を減少させる方法及び無細胞化された生体組織
US20230404805A1 (en) Tissue-Augmented Corneal Inlay Surgery Technique
US20220409361A1 (en) Ablatable Corneal Inlay For Correction Of Refractive Errors And/Or Presbyopia
CN115006595B (zh) 一种复合型高性能脱细胞角膜与其制备方法及用途
Almodin et al. Intrastromal implantation of chicken corneal grafts into the cornea of rabbits for corneal thickening: an experimental study

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18808739

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 02.04.2020)

122 Ep: pct application non-entry in european phase

Ref document number: 18808739

Country of ref document: EP

Kind code of ref document: A1