Sklera Hasarlarının Rekonstruksiyonunda Otojenik, Allojenik Ve Zenojenik Greftlerin Karşılaştırılması: Yeni Bir Otolog Yaklaşım “Trombositten Zengin Fibrin Membran”

Abstract

The purpose of the presented thesis was to compare the effects of xenogenic, allogenic and autogenic graft materials on scleral healing in the repair of scleral tissue damage. For this purpose, scleral tissue damage model with 1/2 depth and 8x5 mm in diameter was formed in rabbit sclera and 5 different study groups were determined. Group A (n: 9): follow-up without defect model (negative control), group B (n: 9): follow-up without graft after formation of defect pattern (positive control), group C (n: 9): grafting with human amniotic membrane (HAM), Group D (n: 9): grafting with rabbit scleral tissue, group E (n: 9): grafting with platelet-rich fibrin (PRF) material. Platelet-rich fibrin was obtained by centrifugation of 5 mL blood samples from rabbits for 12 min at 2,700 rpm. Rabbits were followed for biomicroscopic examinations for 2 months after grafting. In addition, at the end of week 1, month 1, and month 2, an equal number of rabbit euthanasia was applied from each group, and the damaged scleral tissue was excised. Sclera samples were examined by immunohistochemical staining, Small and Wide-Angle X-ray Scattering (SWAXS) analysis and hydroxyproline assay.

In biomicroscopic examinations, edema and secretion in eyelids, conjunctival and scleral vascularization and scleral damage model were investigated. As a result of the examinations, in the group E with autogenous grafting, findings similar to those of healthy scleral tissue in group A were determined. Additionally, it was observed that the degree of inflammation of the ocular surface was at the highest level in group B, which was left to the primary healing, whereas the increase of inflammation and vascularization in the groups C and D was found near the group B. Autografts prepared in group E allow to be considered as safe biomaterials, because they do not damage any parts of rabbits’ body during the 2-month follow-up period. With these results, it was thought that the clinical recovery model of groups B, C and D was backwarded compared to groups A and E.

The status of collagen and elastic fibrils in the repaired sclera, cellular infiltration and formation of new vessels were examined by Hematoxylin & Eosin, Mallory Tricrom, and Verhoeff Acid Fuksin stains. In group A, findings similar to that of healthy sclera were detected, while in groups B and C, moderate to severe inflammation and large-calibrated and increased number of vessel formation were observed. Group D showed increased vascularity with mild to moderate cellular infiltration. In Group E, mild-to-moderate inflammation was noted in the first week, but later on the inflammation table became calm and a morphological appearance similar to that of healthy sclera was obtained.

Immunohistochemical staining for TGFR1, BMP2, FGF, MMP2, Collagen1, and Aggrecan showed the lowest expression levels for all markers except MMP2 in group B when compared to healthy group and other study groups. MMP2 was exposed in group B at the highest level. For all ratings, group E showed the closest values to group A.

In the SAXS analyzes, it was found that the scattering electron densities of the samples of the negative control group were the highest, and that the closest scatter values were obtained in the samples subjected to autografting. When the distance distributions of ideal nano-formations corresponding to autograft samples were examined, homogeneous nano-formation distributions were obtained for all samples. Furthermore, the most probable structure models obtained when examining the morphology of nano-scale formations showed that more compact and smooth morphologies were obtained in the negative control and autograft groups.

In the hydroxyproline assay, no significant difference was found between the groups at week 1. However, the amount of hydroxyproline increased significantly in groups C and E during the first and second months.

Although autogenous grafts are currently used as the gold standard in the repair of sclera, their use is restricted due to the limited autogenous sources and damage to individual's own tissues and organs. Whereas, the PRF biomaterial is a graft material that can be prepared using a simple protocol with a small sample of blood, which will not harm the body. In the presented thesis study, it has been shown that PRF, used as autograft, provides favorable support to wound healing steps in sclera and causes regeneration similar to morphologically and physiologically healthy sclera tissue at the end of follow-up period. These results suggest that autogenous PRF material is superior to allogenic sclera and HAM in scleral repair.