Ön Çapraz Bağ Yırtıklarında Epidermal Büyüme Faktörünün (Egf) Bağın İyileşmesi Üzerine Etkilerinin Araştırılması

Abstract

Anterior cruciate ligament (ACL) tears are observed by approximately 40-50% in knee injuries. In addition to conventional treatment methods, effects of certain biological agents such as platelet-rich plasma, growth factors, hyaluronic acid and mesenchymal stem cell therapies have been investigated on ACL tear repair. Epidermal growth factor (EGF) increases cell proliferation, collagen and fibroblast production, has an influence on cell differentiation and stimulates angiogenesis. Recent studies suggest that EGF also promotes wound healing. We aimed to investigate the effects of EGF on ligament healing in anterior cruciate ligament tears. All procedures were performed after animal research ethical committee approval. A tear model was formed by a full-thickness incision in the mid-part ACL of 17 knees of 17 New Zealand rabbits. The distal cut-edge of ACL was sutured with nonabsorbable suture material for the intent of marking. Rabbits were randomised into two groups. In the study group consisting of 9 rabbits; a standardized, 3x3 mm porous policaprolactone foam scaffold carrying 15 μg of human recombinant EGF (Heberprot-P®) was administered in intercondylar notch of the joint space. No intervention or medical device were employed in the control group. The joint capsules and skins of all rabbits were closed. A total of 5 rabbits one of whom was in the control group and four of whom were in the study group deceased during the postoperative follow-up period. Animals were sacrificed at 8th week and tissue samples of ACL were examined histologically. Extracted tissue samples were fixated in buffered, 10% neutral formaldehyde solution. Tissues embedded in paraffin blocks were sampled continously in 5-micron thickness with a microtome and a number of 100 sections were granted. Having been deparaffinized, slides were stained with Masson’s Trichrome and Hematoxylin-Eosin dyes for examination via light microscope. Slides sampled in a 1 out of 10 fashion in compliance with the systematic random sampling method were examined by means of StereoInvestigator® (MicroBrightfield) software after utilizing “virtual slice” to obtain a 10 fold magnified image of the whole tissue. Volumes of the estimated vascular and ligamentous fractions of ACL tissue samples were assessed with Cavalieri volume computation method which is a stereological process. Since even tissue samples were not able to be excised among rabbits, vessel portion within the ligament was calculated through proportioning the vessel volume by the total of vessel and ligament volume. During dissection, cut edges of ACLs in both groups were observed to adhere to the surrounding tissue. An increase in volume of extracted ACL distal segments of the study group was observed in comparison to the control group. On microscopic examination; substantial neovascularization, namely angiogenesis, were noted in the ACL tissue samples of the study group. Moreover, active fibroblasts and profound stem cell increase adjacent to perivascular spaces were detected. Type 3 collagen synthesis were found in regions where active fibroblasts were observed. In addition, fat cells which are noticed in neo-tissue formation were noted. In the control group, scarce vascularization and stem cells due to incision were observed, however no active fibroblasts or fat cells were noted. Average vessel volume ratios was significantly higher in the study group (24.51%) than in the control group (1.36%) (p = 0.003). Our hypothesis that EGF application increases angiogenesis and promotes tissue repair in ACL tears was supported histologically and statistically. Further investigation will better enlighten the mode of EGF action and will settle its role as an alternative method in the ACL tear treatment. Biomechanical tools should be added to the next step.