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WO2014038866A1 - Composition pour traitement des maladies angiogéniques à l'aide d'une membrane de matrice extracellulaire de cellules dérivées de cartilage, et matériel pour greffe de cornée ou de conjonctive - Google Patents

Composition pour traitement des maladies angiogéniques à l'aide d'une membrane de matrice extracellulaire de cellules dérivées de cartilage, et matériel pour greffe de cornée ou de conjonctive Download PDF

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Publication number
WO2014038866A1
WO2014038866A1 PCT/KR2013/008018 KR2013008018W WO2014038866A1 WO 2014038866 A1 WO2014038866 A1 WO 2014038866A1 KR 2013008018 W KR2013008018 W KR 2013008018W WO 2014038866 A1 WO2014038866 A1 WO 2014038866A1
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Prior art keywords
extracellular matrix
corneal
chondrocyte
composition
neovascular disease
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PCT/KR2013/008018
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English (en)
Korean (ko)
Inventor
민병현
박소라
최병현
양재욱
윤정호
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Ajou University Industry Academic Cooperation Foundation
REGENPRIME CO Ltd
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Ajou University Industry Academic Cooperation Foundation
REGENPRIME CO Ltd
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Priority claimed from KR1020130106092A external-priority patent/KR101539700B1/ko
Application filed by Ajou University Industry Academic Cooperation Foundation, REGENPRIME CO Ltd filed Critical Ajou University Industry Academic Cooperation Foundation
Priority to CN201380057870.6A priority Critical patent/CN104837494A/zh
Publication of WO2014038866A1 publication Critical patent/WO2014038866A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a composition for treating neovascular disease using a chondrocyte-derived extracellular matrix membrane and to a corneal or conjunctival implant, and more particularly to corneal neoplasia such as pterygium by using the inhibitory effect of angiogenesis of the chondrocyte-derived extracellular matrix membrane.
  • a composition for treating vascular diseases and a corneal or conjunctival implant is a composition for treating neovascular diseases using a chondrocyte-derived extracellular matrix membrane and to a corneal or conjunctival implant.
  • the present invention utilizes an angiogenesis-inhibiting effect of a support prepared in the form of a membrane using chondrocyte-derived extracellular matrix formed by secretion from cartilage tissue and / or cartilage-derived chondrocytes of an animal, and thus, corneal neoplasia such as pterygium.
  • a composition for treating vascular diseases and a corneal or conjunctival implant is provided.
  • the present invention inhibits vascular endothelial cell adhesion and proliferation and inhibits vascular endothelial cell migration by inhibiting vascular endothelial cell adhesion and proliferation of animal cartilage tissue and / or chondrocyte-derived extracellular matrix membranes, thereby ultimately inhibiting neovascularization. It relates to novel uses related to efficacy.
  • Angiogenesis means the creation of new blood vessels. Normal neovascularization should be induced or regulated in relation to developmental or wound healing, but it is not a desirable phenomenon when neovascularization induces tissue degeneration. In particular, neovascularization is closely related to diseases such as cancer, cartilage neovascularization, corneal neovascularization, diabetic retinopathy, choroidal neovascular disease, and macular degeneration. Can increase.
  • neovascular inducers such as VEGF (vascular endothelial growth factor), FGF, and PIGF are expressed from cancer cells or tissues to form microvascular vessels from surrounding blood vessels. Expression of the factor is known to induce ischemic damage and inflammatory responses.
  • VEGF vascular endothelial growth factor
  • FGF vascular endothelial growth factor
  • PIGF vascular endothelial growth factor
  • the cornea is avascular tissue and should always be transparent to preserve vision.
  • neovascularization is known to occur in the eye and cause neovascular related diseases of the eye.
  • neovascularization in the cornea inhibits eye transparency and leads to loss of vision
  • neovascularization in the retina leads to abnormal blood vessels resulting in blood exudation, leading to blindness through degeneration of the retinal cells.
  • Related diseases include corneal neovascularization, diabetic retinopathy, choroidal neovascular disease, macular degeneration, and the like. Therefore, neovascularization in the eye is not a desirable phenomenon and is preferably suppressed as much as possible.
  • neovascular disease of the cornea medication is performed, but in severe cases, surgical procedures such as removal and autoconjunctival transplantation are performed.
  • high recurrence rate (10-45%) and complications are problematic.
  • surgery or reoperation for a wide range of pterygium there is a limitation that the above-described surgical procedure is difficult to apply.
  • the corneal therapeutic materials released to date include collagen porous sponges mainly used for glaucoma surgery, and amnion, a medical material derived from living tissue, used to treat burns, corneal damage and pterygium.
  • domestic products include AmniSite-Cornea of Bioland Inc., and most of them are US products.
  • Ambio-dry2 TM of IOP Company is imported into Korea and used for pterygium surgery and corneal damage treatment. .
  • these products are all non-medical insurance items, and the burden on the patient for surgery costs is very large.
  • Korean Patent Publication No. 10-0947553 discloses a method for preparing amnion tissue graft by treating amniotic membrane isolated from an animal or human body with alcohol, trypsin, alkaline solution and acid solution. The treatment of amniotic tissue implants in the eye of induced dogs has been shown to have a corneal epithelial regeneration effect.
  • Korean Patent Publication No. 100996846 discloses corneal or conjunctival implants that contribute to stabilizing the ocular surface, including mucin-secreting cells, which are abundant as corneal or conjunctival implants containing amnion and non-mucosal epithelial cells, and which have high rates of engraftment and persistence after transplantation. have.
  • the present inventors have conducted intensive studies on new medical materials that can prevent neovascularization by paying attention to the complications of corneal haze, decreased vision and blindness due to neovascularization as described above. It has been confirmed that the cell-derived extracellular matrix membrane has excellent efficacy in inhibiting angiogenesis, and thus, it has been used to develop a composition for treating neovascular disease and a corneal or conjunctival implant.
  • an object of the present invention is to treat corneal neovascular disease such as pterygium by using the inhibitory effect of the angiogenesis of chondrocyte-derived extracellular matrix membrane secreted from cartilage-derived chondrocytes of cultured animals and formed as a membrane-like support.
  • corneal neovascular disease such as pterygium
  • angiogenesis of chondrocyte-derived extracellular matrix membrane secreted from cartilage-derived chondrocytes of cultured animals and formed as a membrane-like support.
  • the present invention provides a novel composition for treating neovascular disease and a corneal or conjunctival implant.
  • composition for treatment of neovascular disease of the present invention comprises chondrocyte-derived extracellular matrix formed by secretion from animal cartilage as an active ingredient.
  • the chondrocyte-derived extracellular matrix is characterized in that consisting of collagen and protein (glycosaminoglycan).
  • the chondrocyte-derived extracellular matrix is obtained by decellularizing chondrocytes and neutralizing after treatment with proteolytic enzymes, or cells formed by secreting chondrocytes in monolayer culture of chondrocytes isolated from chondrocytes. It is characterized in that the outer stromal membrane produced by decellularization.
  • composition for treating neovascular disease of an embodiment of the present invention is characterized by inhibiting vascular endothelial cell adhesion and proliferation.
  • composition for treating neovascular disease of an embodiment of the present invention is characterized by inhibiting neovascularization by inhibiting angiogenesis and vascular infiltration.
  • the composition for treating neovascular disease of one embodiment of the present invention can be used to treat corneal neovascular disease, and in particular can be used to treat pterygium or traumatic corneal ulcer.
  • the composition for treating neovascular disease of one embodiment of the present invention may be used to treat diabetic retinopathy, choroidal neovascular disease or macular degeneration.
  • composition for treating neovascular disease of one embodiment of the present invention may be provided as an eye drop or injection in the form of eye drops by mixing the chondroblast-derived extracellular matrix membrane with a pharmaceutically acceptable carrier.
  • composition for treating neovascular disease of one embodiment of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings and the like can be used in combination as pharmaceutically acceptable carriers.
  • buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers and the like can be used in combination.
  • injectables may be prepared in unit dosage ampoules or in multiple dosage forms.
  • bases, excipients, lubricants, preservatives and the like can be used during topical administration.
  • the present invention is not limited thereto, and those skilled in the art will understand that the composition for treating the neovascular disease of the present invention may be formulated in various forms, and may include various carriers or additives. .
  • the route of administration of the composition for treatment of neovascular disease of the present invention can be administered through any general route as long as the chondrocyte-derived extracellular matrix membrane component can reach the eye.
  • Eye periphery, cornea, conjunctiva, eye periphery, anterior, subretinal, or subconjunctival administration may be, but is not limited thereto.
  • a therapeutically effective amount of the composition for treating neovascular disease of the present invention means an amount required for administration in order to expect the neovascular disease therapeutic effect.
  • the disease type of the patient, the severity of the disease, the type of chondrocyte-derived extracellular matrix membrane formulation to be administered, the age, sex, weight, health condition, diet, administration time and method of administration of the therapeutic composition, route of administration and It can be adjusted according to the discharge rate.
  • the composition for treating neovascular disease of the present invention when administering the composition for treating neovascular disease of the present invention to an adult, it may be administered at a dose of 0.1 ⁇ g / kg to 100 mg / kg once daily.
  • the corneal or conjunctival graft material of the present invention comprises: 1) chondrocyte-derived extracellular matrix obtained by decellularizing cartilage tissue and treating and neutralizing it with protease; Or 2) chondrocyte-derived extracellular matrix prepared by decellularizing the extracellular matrix membrane formed by secretion of chondrocytes in monolayer culture of chondrocytes isolated from chondrocytes.
  • Corneal or conjunctival graft material of an embodiment of the present invention may be cultured by implanting eye cells on the chondrocyte-derived extracellular matrix membrane.
  • the eye cells are characterized in that the corneal stem cells that are cells that grow cells by cell division at the edge of the eye surface surrounding the cornea.
  • epithelial stem cells, oral mucosa cells, or non-mucosa cells of the corneal limbus may be transplanted and cultured on the chondrocyte-derived extracellular stromal membrane.
  • Corneal or conjunctival implants of one embodiment of the present invention are characterized by inhibiting vascular endothelial cell adhesion and proliferation.
  • the corneal or conjunctival implant of an embodiment of the present invention is characterized by inhibiting angiogenesis by inhibiting blood vessel formation and blood vessel penetration.
  • the present invention can provide novel compositions and corneal or conjunctival implants for the treatment of corneal neovascular diseases such as pterygium using the angiogenesis inhibitory efficacy of chondrocyte derived extracellular matrix membranes.
  • corneal neovascular diseases such as pterygium
  • the treatment of corneal neovascular diseases such as pterygium is performed by using the inhibitory effect of the angiogenesis of chondrocyte-derived extracellular matrix membranes secreted from cartilage-derived chondrocytes of cultured animals and formed as membrane support.
  • Novel compositions and corneal or conjunctival implants can be provided.
  • novel compositions and corneal or conjunctival implants for treating the neovascular disease of the present invention are secreted from cartilage-derived chondrocytes of cultured animals, and the chondrocyte-derived extracellular matrix membrane formed as a support in the form of membrane is attached and proliferated vascular endothelial cells. Inhibition of blood vessels and inhibition of vascular endothelial cell migration can inhibit angiogenesis and blood vessel penetration and ultimately inhibit angiogenesis.
  • corneal haze caused by neovascularization after ulceration or lesion of cornea by preventing neovascularization of cornea, resulting visual acuity Lowering and complications of blindness can be prevented.
  • CD-ECM chondrocyte-derived extracellular matrix membrane
  • Figure 1 (a) is a graph showing the chondrocyte adhesion rate (%) and vascular endothelial cell adhesion rate (%) for the culture plate, CD-ECM experimental group and amnion experimental group transplanted with chondrocytes, respectively
  • b) is a graph showing the measured fluorescence intensity indicating the adherent cell number of the culture plate, CD-ECM test group and amnion test group transplanted with chondrocytes, respectively.
  • Figure 2 is a graph showing chondrocyte proliferation rate (%) and vascular endothelial cell proliferation rate (%) for the culture plate, CD-ECM experimental group and amnion experimental group transplanted with chondrocytes, respectively.
  • Figure 3 is a photograph showing the degree of angiogenesis after performing the implantation and culture of vascular endothelial cells for the geltrex-coated control group, geltrex / CD-ECM powder experimental group and geltrex / amnion powder experimental group, respectively.
  • FIG. 4 shows the number and length of blood vessels formed per visual field after transplantation and culture of vascular endothelial cells for the geltrex-coated control group, the geltrex / CD-ECM powder test group, and the geltrex / amniotic powder test group, respectively. It is a graph measured and shown.
  • FIG. 5 is a photograph of each sample collected from the spine at 1 week after the injection of Matrigel alone, Matrigel / CD-ECM powder, and Matrigel / amniotic powder into the nude mouse spine. Three-dimensional image photograph.
  • FIG. 6 shows chemical analysis of individual sample tissue sections collected from the spine at one week after injection of Matrigel alone, Matrigel / CD-ECM powder, and Matrigel / amniotic powder into the nude mouse spine, respectively.
  • FIG. 7 is a graph of the number of blood vessels per area and the diameter of blood vessels measured and quantified using the IMAGE J program in the micrograph image stained in FIG. 6.
  • FIG. 8 shows immunization of each sample tissue section collected from the spine at one week after injection of Matrigel alone, Matrigel / CD-ECM powder, and Matrigel / amniotic powder into the nude mouse spine, respectively. After chemical staining (VEGF, HIF-1a, endostatin), the immunostaining state was taken under a microscope.
  • FIG. 9 shows that CD-ECM was implanted into the left eye (experimental group) and nothing was implanted into the right eye (control) after the experimental development of corneal neovascularization in both rabbits, compared to the right eye control group without CD-ECM. It is a photograph showing that much blood vessel formation was suppressed in the left eye experimental group treated with -ECM.
  • FIG. 10 shows experimentally developing corneal neovascularization, implanted CD-ECM in the left eye (experimental group), and extracting the eyeballs of rabbits with nothing implanted in the right eye (control), followed by immunochemical staining (CD31). A picture of the immunostaining state under a microscope.
  • Figure 11 is an experimental development of corneal neovascularization and implantation of CD-ECM in the left eye (experimental group) and the eyes of rabbits with nothing implanted in the right eye (control) after analysis of the expression level of VEGF protein in eye tissues Western blot results are photographs.
  • FIG. 12 is a microscopic picture of a cell sheet that is engraftd after implantation on the CD-ECM of the present invention.
  • Figure 13 is transplanted epithelial cell sheet cultured by epithelial cells transplanted on the CD-ECM of the present invention to the epithelial region of the corneal tissue of the rabbit and cultured for 3 weeks after transplanting it to the dorsal subcutaneous tissue of the mouse after transplantation of the transplanted tissue Micrograph showing the results of histological analysis.
  • Figure 14 is transplanted epithelial cell sheet cultured by epithelial cells transplanted on the CD-ECM of the present invention to the epithelial region of the corneal tissue of the rabbit and cultured for 3 weeks after transplanting it to the dorsal subcutaneous tissue of the mouse after transplantation of the transplanted tissue Is a photomicrograph showing the results of immunochemical analysis.
  • Figure 15 is an electron micrograph observed after transplanting the epithelial cell sheet cultured by the epithelial cells transplanted on the CD-ECM of the present invention in the epithelial region of the rabbit corneal tissue and cultured for 3 weeks after implantation in the dorsal subcutaneous tissue of the mouse As a result, the epithelial cells form a tight junction with the lower corneal stroma.
  • the present invention provides a novel composition for treating neovascular disease and a corneal or conjunctival implant.
  • composition for treatment of neovascular disease of the present invention comprises chondrocyte-derived extracellular matrix formed by secretion from animal cartilage as an active ingredient.
  • the chondrocyte-derived extracellular matrix is characterized in that consisting of collagen and protein (glycosaminoglycan).
  • the chondrocyte-derived extracellular matrix is obtained by decellularizing chondrocytes and neutralizing after treatment with proteolytic enzymes, or cells formed by secreting chondrocytes in monolayer culture of chondrocytes isolated from chondrocytes. It is characterized in that the outer stromal membrane produced by decellularization.
  • composition for treating neovascular disease of an embodiment of the present invention is characterized by inhibiting vascular endothelial cell adhesion and proliferation.
  • composition for treating neovascular disease of an embodiment of the present invention is characterized by inhibiting neovascularization by inhibiting angiogenesis and vascular infiltration.
  • Corneal or conjunctival graft material of the present invention comprises: 1) chondrocyte-derived extracellular matrix obtained by decellularizing chondrocytes and neutralizing after treatment with proteolytic enzymes; Or 2) chondrocyte-derived extracellular matrix prepared by decellularizing the extracellular matrix membrane formed by secretion of chondrocytes in monolayer culture of chondrocytes isolated from chondrocytes.
  • Corneal or conjunctival graft material of an embodiment of the present invention may be cultured by implanting eye cells on the chondrocyte-derived extracellular matrix membrane.
  • the eye cells are characterized in that the corneal stem cells that are cells that grow cells by cell division at the edge of the eye surface surrounding the cornea.
  • epithelial stem cells, oral mucosa cells, or non-mucosa cells of the corneal limbus may be transplanted and cultured on the chondrocyte-derived extracellular stromal membrane.
  • Corneal or conjunctival implants of one embodiment of the present invention are characterized by inhibiting vascular endothelial cell adhesion and proliferation.
  • the corneal or conjunctival implant of an embodiment of the present invention is characterized by inhibiting angiogenesis by inhibiting blood vessel formation and blood vessel penetration.
  • Chondrocyte-derived extracellular matrix membrane used as a composition for treating corneal neovascular disease of the present invention and as a base material for corneal or conjunctival implants It is a support in the form of a film prepared as follows.
  • extracellular matrix membrane in the form of a membrane formed by collecting and culturing chondrocytes of sterile pig knee cartilage tissue. These extracellular matrix membranes derived from chondrocytes are currently used clinically only for the purpose of regenerating cartilage in orthopedics.
  • the extracellular matrix secreted by culturing chondrocytes isolated from sterile pig knee cartilage in a monolayer is in the form of a membrane or film. After drying, decellularized with an enzyme such as DNase I and obtained by washing with PBS buffer.
  • the extracellular matrix membrane is a biofilm having a thickness of about 10 to 20 ⁇ m, and has collagen and protein sugar as main components, and has a tensile strength of about 25 N / mm 2 and an elongation of 10% (Korea Patent Publication No. 10- 0816395).
  • chondrocytes obtained by pulverizing sterile pig knee cartilage tissue in powder form and decellularized with enzymes such as hypotonic lysis buffer and DNase I and washed with distilled water or PBS (Korea Patent Publication No. 10-1056069) After treatment with acidic aqueous solution added with protease, it is neutralized and modified to prepare a membrane-like support.
  • the amnion used as a control for the treatment of neovascular disease using the chondrocyte-derived extracellular matrix membrane and corneal or conjunctival implant of the present invention uses a product sold by Bioland Co., Ltd. (Cheonan, Chungcheongnam-do, Korea).
  • the powder of the CD-ECM and the amniotic membrane used in the examples to be described later were freeze-dried at each of these films at -70 ° C and then using a cryogenic sample crusher (JFC-300, JAI, Japan). Prepare and sterilize with ionic gas at 27 ° C. for 24 hours before use.
  • a cryogenic sample crusher JFC-300, JAI, Japan
  • vascular endothelial cells and chondrocytes attached to the chondrocyte-derived extracellular matrix membrane (CD-ECM) of the present invention prepared in Example 1 and the control amnion, respectively, used in the in vitro and in vivo tests described below are as follows. It was prepared by incubation beforehand.
  • HUVEC vascular endothelial cell
  • endothelial growth factors Promega, Wisconsin, USA
  • 10 ⁇ g / ml bFGF 10 ⁇ g / ml bFGF
  • FBS 2% FBS
  • Dog cells were seeded and incubated at 37 ° C. in a 5% CO 2 incubator.
  • Two days after seeding of vascular endothelial cells (HUVEC) unattached cells were removed and the cells attached to the plate were further cultured.
  • vascular endothelial cells (HUVECs) were removed by trypsin treatment and centrifuged at 180 g for 7 minutes. Cell pellets obtained after centrifugation were resuspended in cell culture and 1 ⁇ 10 6 cells per plate were seeded again for cell proliferation and cultured to 70-80% density.
  • cartilage pieces were taken from the knee cartilage of two-week-old female New Zealand white rabbits, and then treated with 1 mg / ml collagenase (Worthington, NJ, USA) in DMEM to separate chondrocytes. .
  • the chondrocyte suspension obtained after collagenase treatment was filtered only with chondrocytes by a nylon mesh cell strainer (BD, MA, USA), and centrifuged (1700 rpm, 10 min) to separate only chondrocytes.
  • the isolated chondrocyte pellets were resuspended in DMEM with 10% FBS and 1% antibiotic-antimycotic, seeded 1.5 ⁇ 10 6 per plate in the culture plate, and then 37 ° C. in a 5% CO 2 incubator. Incubated at. The culture medium was changed every three days.
  • the adhesion rate of chondrocytes and vascular endothelial cells (HUVEC) according to the difference between the chondrocyte-derived extracellular matrix membrane (CD-ECM) of the present invention prepared in Example 1 and the control amnion It was confirmed as follows.
  • CD-ECM and amnion were attached to a separate 24-well dish and dried, followed by ion gas sterilization.
  • 1 ⁇ 10 3 chondrocytes and vascular endothelial cells were transplanted separately into a dish coated with CD-ECM and amnion, and 40 ⁇ l Calcein AM (2 ⁇ g / 1 mL) (Invitrogen) was used for fluorescence staining of cells. , Wisconsin, USA) were added and then incubated at 37 ° C. in a 5% CO 2 incubator for 24 hours.
  • a medium containing DMEM and 10% FBS was used for culturing chondrocytes, and an endothelial cell growth medium, 10 ⁇ g / ml bFGF and 2% FBS were used for culturing vascular endothelial cells. Used.
  • an endothelial cell growth medium 10 ⁇ g / ml bFGF and 2% FBS were used for culturing vascular endothelial cells.
  • the transplantation and culture of chondrocytes and vascular endothelial cells were performed under the same conditions as described above using a conventional cell culture plate without the films attached thereto.
  • the culture medium was removed to check the number of unattached cells, and based on this, the adhesion rate was calculated and quantified.
  • the amount of attached cells was quantified by measuring the intensity of fluorescence stained by fluorescence staining using a microplate reader. Quantitative Results Data analysis was performed by Student t-test using GraphPad software and each experiment was repeated five times and expressed as mean value and standard deviation (statistical significance: * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
  • the proliferation rate difference between chondrocytes and vascular endothelial cells (HUVEC) according to the difference between the chondrocyte-derived extracellular matrix membrane (CD-ECM) of the present invention prepared in Example 1 and the control amniotic membrane is as follows. It was confirmed as follows.
  • CD-ECM and amnion were attached to a separate 24-well dish and dried, followed by ion gas sterilization.
  • 1 ⁇ 10 3 chondrocytes and vascular endothelial cells were separately transplanted into a plate coated with CD-ECM and amnion, respectively, and cultured at 37 ° C. in a 5% CO 2 incubator for a total of 7 days.
  • DMEM and 10% FBS medium were used for culturing chondrocytes, and endothelial cell growth media, 10 ⁇ g / ml bFGF and 2% FBS were used for vascular endothelial cell culture. Used.
  • the percentage of vascular endothelial cell proliferation was higher in the amnion experimental group in the proliferation rate of the vascular endothelial cells on the 4th and 7th day after the culture. I could confirm it.
  • chondrocyte-derived extracellular matrix membrane (CD-ECM) material of the present invention prepared in Example 1 can inhibit angiogenesis of vascular endothelial cells as follows.
  • Geltrex base membrane base membrane
  • Geltrex base membrane base membrane
  • CD-ECM powder and amnion powder prepared in Example 1 were placed in each well coated with Geltrex polymerized, and then 1.9 ⁇ 10 4 vascular endothelial cells were transplanted and incubated for 24 hours.
  • staining was performed by adding calcein (Calcein) AM (2 ⁇ g / 1 mL) (Invitrogen, Wisconsin, USA) to stain for angiogenesis after culture.
  • the endothelial cell growth media (endothelial cell growth media), 100 ng / ml bFGF and 10% FBS was added to the culture of vascular endothelial cells.
  • the control group was transplanted and cultured vascular endothelial cells under the same conditions as described above using a 24-well dish coated with only CD-ECM powder and amnion powder without geltrex coating.
  • the CD-ECM powder of the present invention strongly inhibited the vascular endothelial cell production, whereas the conventional amnion powder did not significantly interfere with the vascular endothelial cell production. .
  • the CDECM material of the present invention can suppress vascular endothelial cell adhesion and proliferation and inhibit vascular endothelial cell vascular infiltration by inhibiting angiogenesis of vascular endothelial cells. And ultimately inhibit angiogenesis.
  • chondrocyte-derived extracellular matrix membrane (CD-ECM) material of the present invention prepared in Example 1 can inhibit vascular invasion of the matrigel (BDBioscience, Cat. 354234 and 356234) penetration
  • the assay was performed and evaluated visually.
  • the CD-ECM powder or amnion powder prepared in Example 1 was mixed with Matrigel, and then injected subcutaneously under the nude mouse, and the control group was injected with Matrigel subcutaneously under the nude mouse.
  • Matrigel "Matrigel”, “Matrigel CD-ECM Powder” and “Matrigel + Amniotic Powder” were injected along each of 10 nude mouse vertebrae each side to be implanted in the longitudinal direction and at 1 week after transplantation Samples were collected from the spine, and visual observation was performed to confirm the shape, size, and vascular invasion of the sample.
  • the CD-ECM material of the present invention effectively inhibits blood vessel penetration as compared with the conventional amniotic membrane material.
  • the chondrocyte-derived extracellular matrix membrane (CD-ECM) material of the present invention prepared in Example 1 was able to inhibit the formation and penetration of blood vessels after performing a matrigel (matrigel) penetration assay. Histochemical analysis and evaluation.
  • the CD-ECM powder or amnion powder prepared in Example 1 was mixed with Matrigel, and then injected subcutaneously under the nude mouse, and the control group was injected with Matrigel subcutaneously under the nude mouse.
  • Matrigel "Matrigel”, “Matrigel + CD-ECM Powder” and “Matrigel + Amniotic Powder” were injected along each side of 10 nude mouse vertebrae to be transplanted in the longitudinal direction, and 1 week after transplantation Samples were collected from the spine at.
  • H & E staining hematoxylin / eosin staining
  • angiogenesis was confirmed by microscope (Nikon E600, Japan) through chemical staining (H & E) and immunochemical staining (staining for a-SMA and CD31) for each sample (see FIG. 6).
  • the number of blood vessels per area and the diameter of the blood vessels in the micrograph image was measured and quantified using an IMAGE J program (Wayne Rasband, National Institutes of Health, USA) (see FIG. 7).
  • 6 is a photograph taken with a microscope (Nikon E600, Japan) at 400 times magnification after chemical staining (H & E) and immunochemical staining (staining for a-SMA and CD31) for each sample tissue section.
  • H & E chemical staining
  • immunochemical staining staining for a-SMA and CD31
  • CD-ECM powder was measured and quantified by measuring the number of capillary structure (mm2) and diameter (diameter of capillary structure ⁇ m) formed per area through a slide of stained tissue. There was no blood vessel measured in this mixed Matrigel experimental group. On the other hand, in the matrigel experimental group mixed with the amniotic membrane powder, the length of the formed blood vessels was longer and the number of blood vessels was also observed when compared with the materigel experimental group in which the CD-ECM powder was mixed as well as the experimental group using the matrigel alone.
  • the CD-ECM material of the present invention effectively inhibits angiogenesis and vascular penetration as compared to the conventional amniotic membrane material.
  • Example 7 the experiments were carried out in the same or similar manner as in Example 7, and each sample was collected to determine whether the angiogenic factor and the anti-angiogenic factor were expressed in each experimental group. Immunochemical analysis and evaluation were performed for the purpose of identification.
  • angiogenesis factors VEGF, HIF-1a
  • angiogenesis inhibitors Endostatin
  • Immunochemical staining was performed according to known conventional methods. Then, the presence or absence of angiogenesis was confirmed by microscope (Nikon E600, Japan) through immunochemical staining (staining for VEGF, HIF-1a and Endostatin) for each sample (see FIG. 8).
  • FIG. 8 is a photograph taken with a microscope (Nikon E600, Japan) at 400 times magnification after immunochemical staining (staining for VEGF, HIF-1a and Endostatin) for each sample tissue section.
  • immunochemical staining staining for VEGF, HIF-1a and Endostatin
  • the presence of angiogenesis was confirmed by immunochemical staining of VEGF and HIF-1a, angiogenesis factors. It was confirmed that the immunochemical staining, but in the matrigel experimental group mixed with the CD-ECM powder was not expressed because VEGF and HIF-1a was not expressed and the vascular structure could not be observed.
  • the CD-ECM material of the present invention was confirmed to effectively inhibit angiogenesis and vascular penetration compared to the conventional amniotic membrane material, it was possible to observe the expression of angiogenesis inhibitory factor in the blood vessel formed tissues.
  • the CD-ECM material of the present invention can inhibit vascular endothelial cell adhesion and proliferation, inhibit vascular endothelial cell migration, as well as angiogenesis and vascular infiltration. By effectively inhibiting the ultimate angiogenesis was confirmed that can be suppressed. Therefore, the CD-ECM material of the present invention can be usefully used as a composition for treating neovascular disease and as a corneal or conjunctival implant.
  • CD-ECM material of the present invention can be utilized as a composition for treating neovascular disease and as a corneal or conjunctival implant.
  • corneal neovascularization was experimentally generated in both eyes of 8-week-old New Zealand white rabbits, and then CD-ECM was implanted in one eye (left eye: experimental group) and none in the other eye (right eye: control). Without comparing the inhibitory effect of corneal neovascularization of CD-ECM of the present invention.
  • the experimental process and the experimental results are described as follows.
  • a 4 point suture is applied to the mid stromal level using 7-0 mersilk in the limbal portion of the New Zealand white rabbit.
  • CD-ECM was prepared from Example 1.
  • the amniotic membrane was separated from the placenta immediately after the caesarean section of the mother, which was negative on serum tests such as hepatitis B, hepatitis C, HIV, syphilis and the like.
  • the separated amniotic membranes were washed with PBS (phosphphate buffered saline), and then spread thinly on nitrocellulose paper so that the epithelial side of the amniotic membrane faced oppositely. 1 was added to the medium mixed with -70 °C stored in the refrigerator.
  • the CD-ECM and amnion used were sutured with 10-0 nylon to fix around the neovascularization.
  • Levofloxacin (Cravit®, Santen Pharmaceutical Company, Osaka, Japan) eye drops were administered three times a day for one week.
  • CD-ECM was performed after removal of the suture on day 4 of neovascular induction. After 7 days, anesthesia was visually observed to examine the degree of angiogenesis. In the visual observation as shown in FIG. 9, it was confirmed that the formation of blood vessels was much suppressed in the left eye experimental group treated with CD-ECM compared to the right eye control group not treated with CD-ECM.
  • anesthetized rabbits were sacrificed in a CO 2 chamber and oculars were removed for histological analysis.
  • immunochemical staining of CD31 a marker of vascular endothelial cells
  • FIG. 10 after performing immunochemical staining for CD31, a marker of vascular endothelial cells, the eye tissue sections were analyzed.
  • CD-ECM was transplanted compared to the right eye control group without CD-ECM. In the left eye experimental group, it was confirmed that blood vessel formation and CD31 expression were significantly low.
  • VEGF protein which plays an important role in angiogenesis
  • Western blot As shown in FIG. 11, VEGF expression was detected in the left eye experimental group transplanted with CD-ECM compared to the right eye control group without CD-ECM transplantation as a result of extracting protein from eye tissue and examining the expression level of VEGF protein by Western blot. It was confirmed that the amount was remarkably low.
  • the CD-ECM material of the present invention when used as a composition for treating neovascular disease and a corneal or conjunctival implant, corneal haze caused by neovascularization after ulceration or lesion of the cornea by preventing corneal neovascularization, This can prevent vision loss and complications of blindness.
  • Example 10 Applicability as a Mediator for Transplantation of Corneal Epithelial Stem Cells Located in the Corneal Lesion
  • the transparency of the cornea is the most basic and important factor in the proper functioning of the eye.
  • These epithelial cells of the cornea are constantly maintained healthy by stem cells located in the limbal area of the cornea, but are fatal and irreparable vision when damaged by diseases such as heat or chemical damage, Stevens Johnson syndrome, multiple surgeries, and infectious diseases.
  • it is necessary to treat ocular surface diseases because corneal limbal cell damage may occur even in normal patient groups such as long-term wear of contact lenses.
  • This example was to investigate the possibility of using the CD-ECM material of the present invention as a basic material for transplantation of corneal epithelial stem cells in ocular surface disease causing severe visual loss.
  • the limbal epithelial stem cells of the cornea were collected from about 2.5 kg of white rabbit, and the cells were grown on the CD-ECM material of the present invention and transplanted again into the corneal tissues from which the epithelium was removed. .
  • the corneal limbal tissue collected from the white rabbit was divided into 12 pieces, divided into tissue culture vessels, and treated with dispase buffer to separate epithelial cells and then passaged. Thereafter, in order to transplant the cultured epithelial cells onto a CD-ECM cut in a circular shape of 6 mm in diameter, the cut CD-ECM was laid on the bottom of the tissue culture vessel, and the cultured cells were dispensed and cultured to make a cell sheet.
  • FIG. 12 it was confirmed that epithelial cells previously labeled with PKH26 were well identified in red and formed in a dense state (see A of FIG. 12). It can be confirmed that is well engrafted in one or two layers (see FIG. 12B).
  • the white rabbit cornea was then cut out with a tubular saw with the same size of 6 mm to obtain corneal tissue. Then, all epithelial cells were removed by the method of 20% ethanol and abrasion.
  • the epithelial cell sheet prepared above was implanted into the epithelial region of the corneal tissue, and the dorsal subcutaneous tissue of the athymic Balb / C mouse After transplantation for 3 weeks, the engraftment of the transplanted tissue was observed.
  • the CD-ECM material of the present invention is used in the treatment of intractable ocular surface diseases causing corneal blindness, epithelial stem cells, oral mucosa cells of the limbal limbus of another or others. It can be said to be a useful material that can be used for the treatment of non-mucosal cells and cell sheet transplantation.
  • a novel composition for treating corneal neovascular disease such as pterygium by using an angiogenesis-inhibiting effect of chondrocyte-derived extracellular matrix membrane secreted from cartilage-derived chondrocytes of cultured animals and formed into a membrane-like support; Corneal or conjunctival implants may be provided.

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Abstract

La présente invention concerne une nouvelle composition pour traiter des maladies angiogéniques à l'aide d'une membrane de matrice extracellulaire d'une cellule dérivée de cartilage, sous forme de membrane ou de film, qui est sécrétée par des cellules de cartilage en culture qui ont été isolées du cartilage d'un animal et mises en culture monocouche, ainsi qu'un matériel pour greffe de cornée ou de conjonctive. La composition pour le traitement des maladies angiogéniques et le matériel pour greffe de cornée ou de conjonctive selon la présente invention empêchent la fixation ou la prolifération des cellules endothéliales et empêchent le mouvement des cellules endothéliales, inhibant ainsi la croissance de l'endothélium vasculaire et la pénétration vasculaire, et par conséquent l'angiogenèse.
PCT/KR2013/008018 2012-09-05 2013-09-05 Composition pour traitement des maladies angiogéniques à l'aide d'une membrane de matrice extracellulaire de cellules dérivées de cartilage, et matériel pour greffe de cornée ou de conjonctive Ceased WO2014038866A1 (fr)

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CN201380057870.6A CN104837494A (zh) 2012-09-05 2013-09-05 使用软骨来源细胞的胞外基质膜的治疗血管生成疾病的组合物、以及角膜或结膜的移植物

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KR1020130106092A KR101539700B1 (ko) 2012-09-05 2013-09-04 연골세포 유래 세포외 기질막을 이용한 신생혈관질환 치료용 조성물 및 각막 또는 결막 이식재
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US9217016B2 (en) 2011-05-24 2015-12-22 Symic Ip, Llc Hyaluronic acid-binding synthetic peptidoglycans, preparation, and methods of use
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US9512192B2 (en) 2008-03-27 2016-12-06 Purdue Research Foundation Collagen-binding synthetic peptidoglycans, preparation, and methods of use
US10689425B2 (en) 2008-03-27 2020-06-23 Purdue Research Foundation Collagen-binding synthetic peptidoglycans, preparation, and methods of use
US9217016B2 (en) 2011-05-24 2015-12-22 Symic Ip, Llc Hyaluronic acid-binding synthetic peptidoglycans, preparation, and methods of use
US9200039B2 (en) 2013-03-15 2015-12-01 Symic Ip, Llc Extracellular matrix-binding synthetic peptidoglycans
US9872887B2 (en) 2013-03-15 2018-01-23 Purdue Research Foundation Extracellular matrix-binding synthetic peptidoglycans
US10772931B2 (en) 2014-04-25 2020-09-15 Purdue Research Foundation Collagen binding synthetic peptidoglycans for treatment of endothelial dysfunction
CN105770994A (zh) * 2016-05-09 2016-07-20 拜欧迪赛尔（北京）生物科技有限公司 一种生物工程脱细胞眼结膜的制备及用途
US11529424B2 (en) 2017-07-07 2022-12-20 Symic Holdings, Inc. Synthetic bioconjugates
CN117065096A (zh) * 2023-10-17 2023-11-17 山东瑞安泰医疗技术有限公司 一种生物膜的快速制备方法及其应用
CN117065096B (zh) * 2023-10-17 2023-12-22 山东瑞安泰医疗技术有限公司 一种生物膜的快速制备方法及其应用

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