The filtration membranes were made of terpolymer PTFE-PVDF-PP (Aldrich Chemical Co., Milwaukee, WI, USA). To prepare the porous membrane, the fluoro-based terpolymer, that is, polytetrafluoroethylene-co-polyvinylidene fluoride-co-polypropylene (PTFE-PVDF-PP) consisting of 56% PTFE, 27% PVDF, and 17% PP, was applied. A method for preparing an open porous polymeric membrane comprised preparation of a polymer solution with a solvent content (dimethylketone) of approximately 90 wt% and addition of 2 wt% amount of a water-soluble porogen (chopped sodium alginate microfibers), followed by removal of solvent from the polymer material. The polymer in the form of a thin foil was rinsed with water to remove the porogen. The details of the method and the membrane's characteristics have been published elsewhere.
29–32 Before animal research was conducted, cytotoxicity tests were performed. Embryonic 3T3/Balb fibroblast cell lines (BioConcept, Allschwil, Switzerland) and human epithelial cell line A549 (Lonza, Geneva, Switzerland) were used. All morphological characteristics of the cells (shape, size, and distribution) in the study and control frames were normal. The cells adhered to the entire surface of the studied material. All cell cultures exhibited cell division. No agglutination, vacuolization, separation from the test surface, or cell lysis was observed.
29,32 The viability of cells was assessed with human HS-5 fibroblast cell line (LGC Standards, Lomianki, Poland) and osteoblast hFOB 1.19 cell line (LGC Standards, Lomianki, Poland) using MTT Cell Viability Assay (Sigma-Aldrich, Poznan, Poland). Results on viability showed that the implant material could best support the osteoblasts for adhesion and proliferation. The fibroblasts in contact with the implant surface had the same viability as the control samples (tissue culture grade polystyrene [TCPS]). The implants cut from the thin porous membrane had the form of an equilateral triangle with 3.6-mm side length and thickness of 0.6 mm. In the middle of the implant, a circle-shaped opening with diameter of 1.0 mm was used to secure the implant in place; the opening also facilitated the flow of aqueous humor between the ciliary body and the superficial scleral flap. The shape of the implant and its microstructure are shown in
Figure 1.
All animal procedures were performed in accordance with European Standard EU ISO 10993-6 and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, which regulates both animal selection and the evaluation of early and late tissue reactions. Animal experiments were conducted in accordance with the ethical framework regarding animal research (Part 2: Animal welfare requirements, EN 30993). The study was approved by the Bioethics Committee in Cracow, Poland. In vivo tests were conducted in accordance with resolution No. 544/2008 of the Local Ethics Committee for Animal Experiments in Cracow. Nonpenetrating very deep sclerectomy was performed in 38 eyeballs of 19 New Zealand white rabbits. In one eye of each rabbit, an implant was placed during NPVDS (study group). In the other eye of each rabbit, NPVDS was performed but no implant was placed (control group). The age of the rabbits ranged from 9 to 12 months, and their weight was between 3 and 4 kg. Histopathological evaluation was carried out at 2, 4, 12, 24, and 52 weeks after the operation. We assessed inflammatory infiltrate, neovascularization, and stromal edema as well as a connective tissue attachment to the implant and adjacent tissues. The histopathological reaction was evaluated around the implant, in the area of the corneal limbus, in the ciliary body, within the suture area, and in the lacrimal gland as well as in the entire preparation. The reaction was graded for severity on a scale from 0 to 3, using a modified scoring system adopted according to Hoekzema et al.
34 and Erkiliç et al.
35 The absence of changes was marked as 0, mild changes as 1, moderate changes as 2, and severe changes as 3. Tests were conducted by one researcher but were checked by three independent external observers. Quantitative results were subjected to statistical analysis. From the obtained values evaluating the condition of each eyeball the median was determined. We assumed a value
P < 0.05 to be statistically significant. The statistical analysis was performed by using a nonparametric Mann-Whitney
U test.