Amniyon membran ekstraktının hazırlanması, içeriğinin biyokimyasal analizi ve hayvan deneyinde etkinliğinin gösterilmesi
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Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Tıp Fakültesi
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
AMAÇ: Liyofilizasyon ve gama sterilizasyon uygulanmış amniyon membran ekstraktı (AME) elde edilmesi, büyüme faktörleri açısından biyokimyasal analizinin gerçekleştirilmesi ve AME’nin etkinliğinin deneysel kornea epitel defekti hayvan modelinde gösterilmesidir. GEREÇ VE YÖNTEM: Bu çalışma Ege Üniversitesi Tıp Fakültesi Göz Hastalıkları Anabilim Dalı’nda gerçekleştirildi. Elektif sezaryen ile term doğum yapmış, serolojik tarama testleri negatif 5 sağlıklı gebeye ait 5 amniyon membrandan bir havuz oluşturuldu. Amniyon membranlar yıkama solüsyonu ile yıkanarak küçük parçalara ayrıldı. Homojenizasyon, santrifüj ve liyofilizasyon işlemlerine tabi tutulduktan sonra gama radyasyon ile sterilize edildi. PBS ile sulandırılarak AME damla elde edildi. AME’nin total protein miktarı ve büyüme faktörlerinin (EGF, FGF, HGF, KGF, NGF ve TGF-b) konsantrasyonu analiz edildi. Transplantasyon için ise kriyoprezerve amniyon membran kullanıldı. Çalışmanın hayvan deneyi aşamasına 15 adet Yeni Zelanda tavşanı dahil edildi. Tüm tavşanların bir gözünde merkezi kornea epiteli 6,5 mm çapında debride edildi. Tavşanlar, 4’er tavşandan oluşan 3 çalışma ve 3 tavşandan oluşan 1 kontrol grubu olacak şekilde gruplandırıldı. Tüm gruplara topikal antibiyotik verildi. Buna ek olarak Grup 1’deki tavşanlara AME damlası, Grup 2’dekilere amniyon membran transplantasyonu (AMT), Grup 3’tekilere prezervansız suni göz yaşı damlası uygulandı. Kontrol grubu olan Grup 4’teki denekler ise topikal antibiyotik dışında tedavisiz bırakıldı. Tüm damlalar günde 4 kez damlatıldı. Altı gün süresince her gün, tüm grupların ön segmentleri fotoğraflandı. Farklı gruplardaki tedavi etkinliği; korneal reepitelizasyonun süresi ve günlük re-epitelizasyon hızı ile değerlendirildi. Yedinci günde tavşanlar sakrifiye edildi ve ışık mikroskobisi altında yara iyileşmesi açısından histopatolojik olarak incelendi. BULGULAR: AME’nin total protein içeriği 0,149 mg/ml olarak bulundu. Büyüme faktörlerinin konsantrasyonu EGF, FGF, HGF, KGF, NGF ve TGF-b için sırasıyla 274,8; 287,6; 1358,9; 2188,8; 1387,4; 3382,5 pg büyüme faktörü/mg total protein idi. Epitel defekti modelinin oluşturulmasını takiben, kornea re-epitelizasyonu Grup 1’de ortalama 2,75±0,50 (2-3); Grup 3’te 3,5±1 (3-5) ve Grup 4’te 3,33±1,52 (2-5) günde tamamlandı. Grup 2’deki 3 tavşanın 7. günde amniyon membranların eksplante edilmesi sonrası epitel defektinin tamamen kapandığı görüldü. Grup 2’deki 1 tavşanda ise 3. günde amniyon membran
dislokasyonu izlendi, bu deneğin epitelinde tamamen iyileşme 6. günde tamamlandı. Histopatolojik incelemede en yüksek epitel tabakası sayısı (5 tabaka) Grup 1’de saptandı. Grup 1, diğer gruplara kıyasla daha organize kollajen ve daha düzenli epitel hücre yapısına sahipti, bu grupta inflamasyon belirteçlerine rastlanmadı. Grup 2, Grup 3 ve Grup 4’e göre daha düzenli epitel hücre yapısına sahip iken, kollajen organizasyonu ve epitel tabakası sayısı Grup 3 ve 4 ile neredeyse benzer düzeydi. Fakat yangısal hücre infiltrasyonu gibi inflamatuar belirteçlerin Grup 2’de diğer üç gruba göre daha belirgin olduğu izlendi. SONUÇ: Amniyon membranın oftalmoloji alanında en sık kullanım şekli olan AMT’nin cerrahi gerektirmesi ve yarı-opak yapısı nedeniyle postoperatif dönemde ön segmentin objektif klinik gözleminin yapılmasını engellemesi gibi dezavantajları bulunmaktadır. Liyofilize ve sterilize AME, yüksek protein ve büyüme faktörü içeriği ile AMT’nin kısıtlılıklarını ortadan kaldıran potansiyel bir tedavi seçeneğidir. Bu çalışmada AME’nin kornea epitel iyileşmesinde suni gözyaşından ve AMT’den daha etkili olduğu bulunmuştur. Bu öncü çalışma, AME’nin AMT’ye alternatif bir tedavi ürünü potansiyeli taşıdığını vurgulaması açısından önemlidir.
PURPOSE: To prepare lyophilized and gamma sterilized amniotic membrane extract (AME) and to analyze the concentration of growth factors and to investigate the efficacy of AME in an experimental corneal epithelial defect animal model. METHOD: This study was conducted at Ege University Department of Ophthalmology. Amniotic membranes were obtained from 5 healthy seronegative pregnant donors during elective cesarean deliveries and were collected in a pool together. After washing the amniotic membranes with solutions containing antibiotics, they were cut into small pieces. Homogenization, centrifugation and lyophilization processes were performed, followed by sterilization with gamma radiation. AME eye drops were obtained by diluting the lyophilized powder of AME with PBS. Total protein and growth factor (EGF, FGF, HGF, KGF, NGF and TGF-b) concentrations were analyzed. Cryopreserved amniotic membranes were used for transplantation. Fifteen New Zelland rabbits were randomly divided into 4 groups. The central 6.5 mm diameter corneal epithelium was debrided in the right eyes of all rabbits. All eyes received topical antibiotics 4 times a day. Group 1 (4 eyes) received AME drops 4 times a day, Group 2 (4 eyes) received AMT and Group 3 (4 eyes) received preservative free artificial eye drops 4 times a day. Group 4 (3 eyes) was assigned as the control group and did not receive any treatment except topical antibiotics. Anterior segments of all groups were examined and photographed daily for six days. Corneal re-epithelialization time and reepithelialization rate were evaluated to determine the efficacy of the treatment. On the 7th day, the rabbits were executed and wound healing was examined histopathologically under the light microscope. RESULTS: The total protein content of AME was found to be 0.149 mg/ml. Growth factor concentrations were expressed as pg of growth factor per mg of total protein. The concentration of each growth factor were as follows: EGF 274.8 pg/mg, FGF 287.6 pg/mg, HGF 1358.9 pg/mg, KGF 2188.8 pg/mg, NGF 1387.4 pg/mg, TGF- b 3382.5 pg/mg. Following epithelial defect modelling, the mean time to completion of re-epithelialization were 2.75±0.50 (2-3) days in Group 1, 3.5±1 (3-5) days in Group 3, and 3.33±1.52 (2-5) days in Group 4. The mean duration of epithelial healing was significantly decreased in Group 1 compared to the other 2 groups. The amniotic membranes of 3 rabbits in Group 2 were explanted on the 7th day, and the epithelial defects were completely healed. In 1 rabbit in Group 2, the amniotic membrane was displaced on day 3 and the model was completely healed on day 6. In the histopathological examination, corneal epithelium was thicker (5 layers) in Group 1 compared to the other groups. Group 1 had more organized collagen and epithelial cell polarity compared to the other 3 groups, and had no markers of inflammation. Group 2 had a more organized epithelial cell polarity than Group 3 and Group 4, while collagen organization and epithelial layer number were almost the same as in Group 3 and Group 4. Inflammatory markers such as inflammatory cell infiltration were observed to be more prominent in Group 2 than other groups. CONCLUSION: AMT is the most common technique for use of the amniotic membrane in ophthalmology. However, the AMT procedure has disadvantages such as requiring surgery and its semi-opaque structure that hides anterior segment details postoperatively. On the other hand, lyophilized and sterilized AME with its high total protein and growth factor concentration is a promising treatment option that can overcome all these limitations of the AMT procedure. In the present study, AME was found to be more effective than AMT and preservative free artificial tears in promoting corneal re-epithelialization. The findings of this pioneering study are important in emphasizing that AME may be an alternative treatment option to AMT in promoting epithelial healing.
PURPOSE: To prepare lyophilized and gamma sterilized amniotic membrane extract (AME) and to analyze the concentration of growth factors and to investigate the efficacy of AME in an experimental corneal epithelial defect animal model. METHOD: This study was conducted at Ege University Department of Ophthalmology. Amniotic membranes were obtained from 5 healthy seronegative pregnant donors during elective cesarean deliveries and were collected in a pool together. After washing the amniotic membranes with solutions containing antibiotics, they were cut into small pieces. Homogenization, centrifugation and lyophilization processes were performed, followed by sterilization with gamma radiation. AME eye drops were obtained by diluting the lyophilized powder of AME with PBS. Total protein and growth factor (EGF, FGF, HGF, KGF, NGF and TGF-b) concentrations were analyzed. Cryopreserved amniotic membranes were used for transplantation. Fifteen New Zelland rabbits were randomly divided into 4 groups. The central 6.5 mm diameter corneal epithelium was debrided in the right eyes of all rabbits. All eyes received topical antibiotics 4 times a day. Group 1 (4 eyes) received AME drops 4 times a day, Group 2 (4 eyes) received AMT and Group 3 (4 eyes) received preservative free artificial eye drops 4 times a day. Group 4 (3 eyes) was assigned as the control group and did not receive any treatment except topical antibiotics. Anterior segments of all groups were examined and photographed daily for six days. Corneal re-epithelialization time and reepithelialization rate were evaluated to determine the efficacy of the treatment. On the 7th day, the rabbits were executed and wound healing was examined histopathologically under the light microscope. RESULTS: The total protein content of AME was found to be 0.149 mg/ml. Growth factor concentrations were expressed as pg of growth factor per mg of total protein. The concentration of each growth factor were as follows: EGF 274.8 pg/mg, FGF 287.6 pg/mg, HGF 1358.9 pg/mg, KGF 2188.8 pg/mg, NGF 1387.4 pg/mg, TGF- b 3382.5 pg/mg. Following epithelial defect modelling, the mean time to completion of re-epithelialization were 2.75±0.50 (2-3) days in Group 1, 3.5±1 (3-5) days in Group 3, and 3.33±1.52 (2-5) days in Group 4. The mean duration of epithelial healing was significantly decreased in Group 1 compared to the other 2 groups. The amniotic membranes of 3 rabbits in Group 2 were explanted on the 7th day, and the epithelial defects were completely healed. In 1 rabbit in Group 2, the amniotic membrane was displaced on day 3 and the model was completely healed on day 6. In the histopathological examination, corneal epithelium was thicker (5 layers) in Group 1 compared to the other groups. Group 1 had more organized collagen and epithelial cell polarity compared to the other 3 groups, and had no markers of inflammation. Group 2 had a more organized epithelial cell polarity than Group 3 and Group 4, while collagen organization and epithelial layer number were almost the same as in Group 3 and Group 4. Inflammatory markers such as inflammatory cell infiltration were observed to be more prominent in Group 2 than other groups. CONCLUSION: AMT is the most common technique for use of the amniotic membrane in ophthalmology. However, the AMT procedure has disadvantages such as requiring surgery and its semi-opaque structure that hides anterior segment details postoperatively. On the other hand, lyophilized and sterilized AME with its high total protein and growth factor concentration is a promising treatment option that can overcome all these limitations of the AMT procedure. In the present study, AME was found to be more effective than AMT and preservative free artificial tears in promoting corneal re-epithelialization. The findings of this pioneering study are important in emphasizing that AME may be an alternative treatment option to AMT in promoting epithelial healing.
Açıklama
Anahtar Kelimeler
Amniyon Membran Ekstraktı, Amniyon Membran Transplantasyonu, Deneysel Kornea Epitel Defekti, Yara İyileşmesi, Amniotic Membrane Extract, Amniotic Membrane Transplant, Experimental Corneal Epithelial Defect Model, Wound Healing
