Patent Application: US-55401906-A

Abstract:
a method for making , storing and using a surgical graft from equine amniotic membrane in veterinary ophthalmology . the amniotic membrane is obtained from equine placenta , from which the chorion has been separated . sheets of the amniotic membrane are cut to size and mounted on filter paper . the cells of the amniotic membrane are killed , preferably while being frozen and thawed in the storage solution . the equine amniotic membrane can be used in a variety of ocular surgeries in horses but also other species such as food animals , dogs and cats . it represents a strong biomaterial that will give a good physical support to the ocular tissues while inducing a minimal amount of scarring which is primordial in ocular surgeries in order to obtain the best visual outcome .

Description:
the harvest and the use of the human amniotic membrane has been previously reported . however , nobody prior to my application has reported the harvest , making , storage and use of the equine amniotic membrane for ophthalmic surgeries in horses as well as dogs , cats and food animals . the equine placenta is harvested during an elective cesarean section for term pregnancy on a mare with no infectious disease . the allantoammnion is separated from the allantochorion and the amnion ( epithelium , stroma and basement membrane ) is then separated by blunt dissection from the allantois . the am was then placed on a 0 . 45 μm pores nitrocellulose paper ( with the allantoic ( stroma ) side against the paper ) and stored frozen at − 80 ° c . for years in dulbecco &# 39 ; s modified eagle medium and glycerol at ratio of 1 : 1 ( volume to volume ), also containing antibiotics and antifungal ( penicillin 50 μg / ml , streptomycin 50 μg / ml , neomycin 100 μg / ml , and amphotericin b 2 . 5 μg / ml ). after the storage in the freezer , the am epithelium is almost inexistent therefore the am graft material consists of a stroma and a basement membrane . it is very difficult to separate the amnion from the other placental envelops in dogs and cats and the canine or feline amniotic membrane is not as strong as the equine amniotic membrane and therefore does not represent a good biomaterial to use in ophthalmic surgeries . because the equine amniotic membrane stored frozen does not contains a lot of cells , if any , it is a biomaterial that can be used not only in horses but in other species such as dogs , cats and food animal as it will not induce any severe foreign body type or immune reaction . prior to ophthalmic surgery , the am is naturally thawed , and then rinsed with sterile saline during 30 minutes to wash off the glycerol . during the wash , the am is kept on the nitrocellulose paper with the allantoic ( stroma ) side against the paper . the amniotic membrane is then prepared and cut according to the size of the defect to cover . amniotic membrane grafts should simply cover the defect with little tension present before the suture placement to reduce premature graft retraction . like for any grafts , it is important that the graft site is properly prepared . the recipient bed for the amniotic membrane graft is prepared by removing loose epithelium and necrotic corneal tissues with a beaver no . 64 microsurgical blade . the equine amniotic membrane can be in many ophthalmic surgeries in pets , food animals and horses where a defect needs to be covered : corneal ulcers , corneal perforations , defects left after removal of scleral / conjunctival / corneal necrotic or neoplastic lesions . it is a strong material , easily available , in large size , that contains anti - angiogenic , anti - inflammatory , anti - fibrotic and growth factors . for these reasons the equine amniotic membrane represents an ideal biomaterial to use in ophthalmic surgeries in dogs , cats , food animals and horses . the purpose and the evolution of the am graft depends on how the am is placed on the defect site : if the allantoic ( stromal ) side of the am faces the defect , the am graft will adhere to the defect and the am is expected to be incorporated to the tissue ; on the other hand , if the basement membrane of the am faces the defect , the tissue epithelial cells will migrate along the membrane and therefore the am is used as a bandage . it is expected to slough off in 7 - 10 days . the amniotic membrane is then sutured to the graft site with simple interrupted or continuous sutures of 7 - 0 to 8 - 0 absorbable suture material . several layers of amniotic membrane can be used and piled up to fill the defect if necessary . the am may be placed limbus to limbus to cover the complete cornea as a bandage . a temporary tarsorrhaphy can be then placed to minimize blinking movement , prevent excessive lid trauma to the graft and its sutures , and to allow quick graft adherence to the defect site . 1 . brooks d e : equine ophthalmology , in gelatt k n ( ed ): veterinary ophthalmology ( ed 3 ). philadelphia pa ., lippincott williams & amp ; wilkins , 1999 , pp 1053 - 1116 2 . brooks d e : corneal ulceration , in brooks d e ( ed ): equine ophthalmology for equine practitioner ( ed 1 ). jackson w g , teton newmedia , 2004 , pp 58 - 87 3 . severin g a ( ed ): veterinary ophthalmology notes , ( ed 2 ). fort collins , colo ., colorado state university , 1976 4 . gilger b c , whitley r d : surgery of the cornea and sclera , in gelatt k n ( ed ): veterinary ophthalmology ( ed 3 ). philadelphia pa ., lippincott williams & amp ; 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