Effect of Human Amniotic Fluid and Membrane on Fracture Healing on Rat Fracture Model

ÖZGÜN ARAŞTIRMA ORIGINAL RESEARCH Med J SDU / SDÜ Tıp Fak Derg u 2020:27(1):105-112 doi: 10.17343/sdutfd.525766 THE EFFECTS OF HUMAN AMNİOTİC FLUİD AND MEMBRANE ON FRACTURE HEALİNG ON RAT FRACTURE MODEL İNSAN AMNİOTİK SIVISI VE MEMBRANININ SIÇAN KIRIK MODELİNDE KIRIK İYİLEŞMESİNE ETKİLERİ Alper GÜLTEKİN1, A. Meriç ÜNAL2, Mehtat ÜNLÜ3, Safa SATOĞLU4, 1 Derince Research Hospital, Orthopaedics and Traumatology Department, Kocaeli, Turkey 2 Suleyman Demirel University, Faculty of Medicine, Sports Medicine Department, Isparta, Turkey 3 Dokuz Eylul University, Faculty of Medicine, Pathology Department, Izmir, Turkey 4 Dokuz Eylul University, Faculty of Medicine, Orthopaedics and Traumatology Department, Izmir, Turkey Cite this article as: Gültekin A, Unal AM, Unlu M, Satoglu S. The Effects of Human Amniotic Fluid and Membrane on Fracture Healing on Rat Fracture Model. Med J SDU 2020; 27(1): 105-112. Öz Amaç İnsan amniotik sıvısı çeşitli büyüme faktörleri ve hyaluronik asit içermektedir. Tendon, sinir ve kemik yaralanmalarının iyileşmesi üzerine pozitif etkiye sahiptir. İnsan amniotik membranı da tendon ve sinir iyileşmesi üzerine etkili olmakla birlikte yanık ve kompleks yaralarda deri iyileşmesi üzerine de pozitif etkiye sahiptir. Çalışmanın hipotezi insan amniotik sıvısı ve insan amniotik membranının kırık iyileşmesini pozitif yönde uyarmasıdır. Çalışmanın amacı, rat tibia kırık modelinde insan amniotik sıvı ve membranının kırık iyileşmesine etkisinin araştırılmasıdır. Gereç ve Yöntem 36 erkek Wistar ratın her iki tibiaları kullanılarak 72 açık kırık modeli oluşturuldu ve üç gruba ayrıldı. Grup1 kontrol grubu olarak alındı. Grup-2’de, seronegatif gebelerin ikinci trimesterde yapılan amniosentezlerinden alınan insan amniotik sıvısı kırık bölgesine enjekte edildi. Grup-3’te ise seronegatif term gebelerin sezaryanları sonrası alınan insan amniotik membranı kırık bölgesine sarıldı ve sonrasında insan amniotik sıvı enjeksiyonu yapıldı. Uygulamalar sonrası üçüncü ve altıncı haftalarda radyolojik kırık kaynama paternleri ve Modifiye Lane-Sandhu sınıflaması kullanılarak yapılan histopatolojik ölçümler değerlendirildi. Bulgular Üçüncü hafta sonunda, Grup-2 ve Grup-3’te Grup1’e gore daha iyi histopatolojik sonuçlar elde edildi. Altıncı haftada ise Grup-2’nin sonuçları Grup-1’den daha iyi bulundu. Grup-3’ün de sonuçları Grup-1’den iyi olmakla birlikte istatistiksel olarak anlamlı fark saptanmadı. Üçüncü ve altıncı haftalarda Grup-2’nin histopatolojik skoları Grup-3’ten anlamlı derecede daha iyi bulundu(p=0.021, p=0.019). Sonuç Mevcut sonuçlara gore insan amniotik sıvısının kırık iyileşmesi üzerine pozitif etkisi olmakla birlikte insan amniotik membranının pozitif etkisi saptanamamıştır. Düşük immünitesi ve temin kolaylığı nedeni ile insan amniotik membranı da insan amniotik sıvısı ile kombine bir şekilde kırık iyileşmesinde farklı uygulama teknikleri ile kullanılabilir. Anahtar Kelimeler : Tibia kırık modeli; insan amniotik sıvısı; insan amniotik membranı; hayvan çalışması; kırık iyileşmesi Abstract Objective Human amniotic fluid contains several growth factors and hyaluronic acid. It has positive effect in healing İletişim kurulacak yazar/Corresponding author: Müracaat tarihi/Application Date: 11.02.2019 • Kabul tarihi/Accepted Date: 15.04.2019 Available online at http://dergipark.gov.tr/sdutfd Makaleye http://dergipark.gov.tr/sdutfd web sayfasından ulaşılabilir. t 105 Süleyman Demirel Üniversitesi Tıp Fakültesi Dergisi the tendon, nerve and bone injuries. Human amniotic membrane has positive effects on tendon, nerve healing and is also used for skin healing in burns and complex wounds. We hypothesized that human amniotic fluid and human amniotic membrane have positive stimulating effects on fracture healing. The aim of this study is to investigate the effects of human amniotic fluid and human amniotic membrane on bone healing in rat tibial bone fracture model. Materials and Methods A 72 open fracture model was created for both tibial bones of 36 male Wistar rats. They were divided into three groups. Group-1 was the control group. In Group-2, human amniotic fluid which was taken from amniocentesis of seronegative pregnants in their second trimester was injected to the fracture site. In group-3, human amniotic membrane which was taken from normal seronegative term pregnants after their caesarean sections was wrapped around fracture site and then human amniotic fluid was injected to the fracture site. Radiological fracture union patterns and histopathological evaluations with Modified Lane-Sandhu classification were performed with Modi- Introduction Bone fractures are common conditions that can affect the whole population. Besides bony involvement, surrounded soft tissues are also affected from fracture. Fracture healing is a specialized type of wound healing and results in a complex condition with involvement of cellular and biochemical processes(1). These processes may be affected positively or negatively from many factors. Fracture healing occurs close to original anatomical and functional properties of bone without scar tissue with conservative or surgical treatment modalities. Besides these treatment methods, in up to 10% of all fractures, union problems may be seen(2). Factors that enhance bone healing and accelerate fracture union are still investigated. It is known that human amniotic fluid (HAF) which is taken at second trimester has high levels of growth factors including fibroblast growth factor(FGF), insulin like growth factor I and II(IGF-I and IGF-II) and epidermal growth factor(EGF) and molecules like chondroitin 4- and 6-sulfate, dermatan sulfate and heparan sulfate(3). Besides these, it also contains high levels of hyaluronic acid(HA) and hyaluronic acid stimulating activators(HASA)(4,5,6). HASA stimulates HA release and provides bone healing with remodelation(7). HA, alone or with growth factors, was studied to enhance t 106 Human Amniotic Fluid and Membrane on Fracture Healing fied Lane-Sandhu classification at the end of third and sixth weeks. Results At third week, Group-2 and Group-3 had better histopathological results than Group-1. At sixth week, Group-2 had significantly better results than Group-1. Group-3 had also better results than Group-1 although there was no statistical significance. At third and sixth weeks, histopathological scores of Group-2 were significantly better than Group-3(p=0.021, p=0.019). Conclusion Our data suggested that human amniotic fluid had positive effect itself but with human amniotic membrane, it had no positive effect on bone healing. With its low immunity and easy delivery, human amniotic membrane can also be used to stimulate bone healing in combination with human amniotic fluid with different application techniques. Keywords: Tibia fracture model, human amniotic fluid, human amniotic membrane, animal study, fracture healing bone healing on animal fracture models(8). It was demonstrated that human amniotic fl (...truncated)