Abstract
Purpose:
The purpose of the study was to assess the biocompatibility of porous terpolymer (polytetrafluoroethylene-co-polyvinylidene fluoride-co-polypropylene, PTFE-PVDF-PP) membranes as an implant material to be placed during nonpenetrating very deep sclerectomy (NPVDS). Another study objective was to determine whether the polymer membrane under investigation could be used to manufacture a new-generation implant, which would actively delay the process of fistula closure and facilitate aqueous humor drainage.
Methods:
Histological response and tissue tolerance of the implant material were assessed. The study was performed on 38 eyeballs of 19 New Zealand white rabbits (19 implanted, 19 control). Histological assessment was carried out between 2 and 52 weeks after surgery. We routinely assessed inflammatory infiltrate, neovascularization, hemorrhage, and stromal edema as well as connective tissue attachment to the implant and adjacent tissues.
Results:
At 52 weeks of observation, a statistically significant difference was revealed between the study and control groups in terms of resorptive granulation, tissue, and the inflammatory infiltrate. No features of acute inflammatory response to the implant were observed, and there was an absence of histological features of acute inflammatory infiltrates and subsidence of chronic inflammatory infiltrates and resorptive granulation over time.
Conclusions:
Slight fibrotic response and insignificant changes in neighboring eye tissues all indicate good tolerance to bioimplant materials. This allows for some optimism regarding the use of hydrophobic terpolymer in the construction of new intrascleral implants. However, the ultimate decision regarding its usefulness and safety in the treatment of glaucoma requires further investigation.
Glaucoma is the second leading cause of blindness in the World after cataracts.
1,2 It is estimated that the number of people suffering from binocular blindness caused by glaucoma will rise from 8.4 million in 2010 to 11.2 million in 2020. Within this group, 5.86 million patients will suffer from primary open-angle glaucoma (POAG) and 5.25 million from closed-angle glaucoma (PCAG).
3 Primary open-angle glaucoma is a complex disease characterized by chronic progressive neuropathy of the optic nerve with characteristic damage to the nerve, changes in the visual field, and retinal nerve fibre layer thinning. Most frequently, the changes are chronic and progressive.
4–6 Patients often do not develop warning symptoms and are therefore unaware of the disease and the threat of resultant visual field loss, which over time can progress to blindness. An increase in intraocular pressure is a risk factor for chronic neuropathy of the optic nerve, which can be managed surgically.
6,7
Krasnov is believed to have been the forerunner of nonpenetrating surgery; he published his pioneering work on Schlemm's canal externalization, which he referred to as sinusotomy, in 1968.
8 This treatment has undergone and is still undergoing a number of modifications, leading to the introduction of other treatment alternatives. However, it is only in the last decade that nonpenetrating surgeries have been gaining acclaim and more popularity among ophthalmologists and patients. The basic advantage is an increase in the safety of glaucoma surgery because of fewer complications and their sequelae as compared to fistula-creating surgeries. However, leaving the trabeculo-Descemet's membrane intact can reduce the effectiveness and compromise the outcome of these procedures. A significant improvement in the effectiveness of nonpenetrating deep sclerectomy was achieved following the introduction of intrascleral implants, which helped maintain the intrascleral space and facilitated adequate filtration of the aqueous humor, thus allowing significant reduction of intraocular pressure and antiglaucoma drugs.
9–11
Viscocanalostomy is the first modern antiglaucoma procedure that aims to restore natural aqueous outflow.
12,13 Canaloplasty is one of several new surgical alternatives to lower pressure for patients with glaucoma; the procedure consists of Schlemm's canal catheterization to distend the trabecular meshwork with the aim of widening the canal. Using a special ultrasound imaging system, the microcatheter's pathway is monitored during and after surgery.
14,15 Nevertheless, deep sclerectomy and its modifications still remain the most established surgical procedures for open-angle glaucoma.
16–21 Further increase in the effectiveness of the nonpenetrating sclerectomy has been attempted by placement of different absorptive or biostable intrascleral implants.
22–27 Ates et al.
28 have demonstrated that T-flux implants made of hydrophilic acryl material do not significantly increase the effectiveness of nonpenetrating deep sclerectomy. In our study, we used a biostable implant made of terpolymer (PTFE-PVD-PP) with highly hydrophobic properties. Our previous studies as well as the studies of other authors have shown that the material's highly hydrophobic surface effectively delays inflammatory infiltration and adhesion of plasma components to the implant surface.
29–33
The aim of the investigations was to assess the biocompatibility of the new polymer material (PTFE-PVD-PP) used for the manufacture of intrascleral implants placed in rabbit eyes during nonpenetrating very deep sclerectomy (NPVDS). We aimed to determine whether this highly hydrophobic material might be used to manufacture intrascleral implants that would actively delay the process of fistula closure, enhance aqueous humor outflow, and decrease the pressure gradient. Histological response and tissue tolerance of the implant material were assessed. An analysis of histology-related challenge to implant function was also performed.
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.
Before general anesthesia, all animals were medicated intramuscularly with 0.06 mg/kg atropine, 10 mg/kg xylazine, and 3 mg/kg azaperone. After premedication, the animals were administered thiopental sodium (Biochemia Pharma GMBH, Carl-Zeiss, Germany) at a dose of 5 to 15 mg/kg into the marginal ear vein through an intravenous cannula. Subsequent doses of thiopental sodium were injected during surgery, depending on the drug effectiveness (the surgery lasted approximately 35 minutes). Before surgery, the operative field was rinsed with 5% povidone-iodine solution (Betadine). The conjunctiva was incised at the limbus from 10 to 2 o'clock. Afterward, a 5- × 5-mm superficial scleral flap of approximately half the scleral thickness was dissected 0.5 to 1.0 mm into clear cornea. During the excision of the deep scleral flap, the ciliary body was partially exposed, leaving a band of sclera 0.5 mm in width (modification of traditional nonpenetrating deep sclerectomy [NPDS] surgery). Then, a 0.5 to 1.00 mm window was excised in the transparent cornea (
Fig. 2). Peeling of the adjacent trabecular layers was then performed. In one eye of each rabbit, a terpolymer implant was placed and secured with a 9-0 polypropylene suture. The superficial scleral flap was then secured with two single 9-0 polypropylene sutures. Two single 9-0 polypropylene sutures were also put on the conjunctiva at 10 and 2 o'clock. In the other eye of each rabbit, NPVDS surgery was performed with the same method but no implant was placed. Analgesics and Maxitrol (Dexamethasonum, Neomycini sulfas, Polymyxini B sulfas; Alcon Laboratories (UK) Ltd., Firmley, UK) eye drops were administered until the wound healed.
Euthanasia was conducted by intravenous administration of sodium pentobarbital (Morbital) in a dose of 0.6 mL/kg, after previous premedication. Following enucleation, the eyeballs were placed in 10% formalin solution for no longer than 24 to 48 hours. Hematoxylin and eosin (H-E) stain was used for basic tissue preparation.
During the surgery, ciliary body hemorrhage occurred in one eye in the implant group and in two eyes of the control group, most often during the deep scleral flap dissection. No bleeding into the anterior chamber was observed.
Typical values of the test comparing the histopathological image of the eyes with and without the implant are presented in the
Table (at 52 weeks of implantation) and in
Figures 3 through 12. A similar analysis was performed at 2, 4, 12, and 24 weeks after surgery. Statistical differences between the eyes with and without implant with regard to histopathological changes within the corneal limbus, the incision line, the ciliary body, subconjunctival area of the entire eyeball, and the lacrimal gland were analyzed.
At 2 weeks after surgery (
Figs. 3,
4), a mild-to-moderate lymphocyte infiltration was observed around the implant. Control eyes showed a similar inflammatory response in the incision area. The study and control eyeballs exhibited granulation responses in the subconjunctival space with slight fibrosis and hyalinization.
At 4 weeks of observation (
Figs. 5,
6), resorptive granulation and dense lymphocytic infiltrations were found around the implant in the study eyes. One eyeball showed granulation tissue with a lymphocytic infiltration within the corneal limbus and the ciliary body. In another eye, the implant became dislocated behind the ciliary body. Incision areas in two of the four control eyes exhibited granulation and lymphocytic infiltration. In one of these eyes, the inflammatory infiltrate also involved the ciliary body and corneal limbus.
At 12 weeks after surgery (
Figs. 7,
8), resorptive granulation and mild- to moderate-density lymphocytic infiltrations were still seen in the peri-implant area of the study eyes. One of the experimental eyeballs exhibited a mild lymphocytic infiltration in the subconjunctival space. Except in one eyeball, neither granulation tissue nor lymphocytic infiltrations were seen within the incision line in the control eyeballs. Mild-density lymphocytic infiltrations and slight neovascularization were observed in the ciliary body adjacent to the operative field.
At 24 weeks after surgery (
Figs. 9,
10), implant eyes showed insignificant resorptive granulation and mild-density lymphocytic infiltrations. The subconjunctival space and ciliary body of two of the four implant eyes exhibited mild lymphocytic infiltrations in the area of the operative field. The incision line on the control eyeballs did not show any inflammatory lesions. Mild lymphocytic infiltrations with fibrosis and neovascularization were seen in the subconjunctival space and ciliary body in the area of the operative field.
At 52 weeks after surgery (
Figs. 11,
12), the area around the implant showed slight fibrosis with focal hyalinization, slight vascular proliferation reminiscent of granulation tissue, and isolated lymphocytic infiltrates. One experimental eyeball exhibited mild lymphocytic infiltrations and stromal edema within the ciliary body. The incision area on the control eyeballs did not show any inflammatory lesions (
P = 0.049). Mild lymphocytic infiltrations were observed in the ciliary body in the area of the operative field. Statistical evaluation of histopathological signs at 52 weeks is presented in the
Table.
The evaluation scale for histopathological signs ranged from 0 to 3 points.
Thin porous membrane implants have been used for glaucoma treatment. Such an implant has a number of advantages over existing implant solutions. An appropriate microstructure of polymer material was developed with a highly hydrophobic inner surface that allowed the construction of an implant capable of continuous drainage of the aqueous humor within the conventional and unconventional outflow route. The size of the canaliculi in the corneoscleral layer of the trabeculum suggests that an intrascleral implant should contain channels of 20 to 50 μm in diameter and 250 μm in length (
Fig. 1). An analysis of the trabecular meshwork indicates that such filtration membrane is more likely to show functional properties, allowing its use during NPVDS surgery or as a Schlemm's canal prosthesis or filtering Seton implant. Investigations of the physical properties of the material have revealed that even in the case of its considerable deformation, the porosity is maintained and even improved. Nonpenetrating very deep sclerectomy surgery performed on rabbits' eyes posed some difficulties and, consequently, caused intraoperative complications, mainly resulting from anatomical differences between rabbit and human eyes. The procedure of implant placement and fixation was uneventful. No implant eye developed acute inflammation, which confirms adequate implant sterilization and maintenance of an aseptic environment during surgery. Each and every implant placed in a body causes tissue reaction. The acute reaction observed after surgery consists of granulation, granulocyte infiltrates, and giant foreign body cell formation. With time, the surrounding tissues adapt to the implant. The granulation tissue regresses and the acute phase response with granulocyte infiltrates becomes chronic, with lymphocytic infiltrates and fibrosis around the implant. Focal vascular proliferation is left after the maturation of the granulation tissue. In our study, implant placement did not lead to fistula closure or marked deterioration of aqueous humor outflow. The statistical analysis did not reveal considerable differences between the implant and the nonimplant group.
In the implant group, at 52 weeks after the operation, the space between the superficial scleral flap and the ciliary body was maintained and contained the implant. The shape of the implant material did not show any alterations; also, no changes were observed on its surface. In the control group, the chronic inflammatory process and granulation were almost absent, which indicates a different wound healing mechanism in these eyes.
Implant placement helped maintain the uveoscleral space but also induced a mild chronic inflammatory response and slight connective tissue attachment to the implant. The control group did not show any intrascleral space. The sclera flap firmly adhered to the ciliary body surface; the healing process was faster and did not induce connective tissue attachment. In the implant group the healing process resembled healing by granulation, while in the control group it resembled healing by primary intention. On examining rabbit eye tissues after deep sclerectomy without implant placement, Erkiliç et al.
35 did not find any chronic inflammatory response, presence of granulation, or fibrous connective tissue.
An analysis of fibrous connective tissue attachment to the implant at 52 weeks after surgery showed that its layer was not thick and did not differ significantly from the control (
P = 0.116). These results confirm those of in vitro studies, which have shown that the examined polymer material decreased the activity of fibroblasts. This could positively influence the condition of the fistula and aqueous humor outflow.
29 Other investigations of materials used during NPDS indicate that the amount and type of connective tissue around the implant are of primary importance in evaluating and accepting or rejecting a given substance as an implant material.
26,35,36 The examined implant did evoke a slight foreign body reaction, which might hinder its selection as a material for construction of a new intrascleral implant. The study demonstrated that the space between the sclera and the ciliary body was filled by the granulation tissue and blood vessels. This observation suggests the need to change the shape of the implant in order for it to adhere more tightly to the trabeculo-Descemet's membrane. Rabbit research carried out by Jacob et al.
37 indicate that implants made of expanded polytetrafluoroethylene (e-PTFE) could be well tolerated by conjunctiva and the sclera of the rabbits' eyes. Although early edematous reaction and inflammatory cell infiltration was observed following membrane placement, the reaction was comparable to that in the control group (
P > 0.05).
In conclusion, the absence of histological features of acute inflammatory infiltrates, subsidence of chronic inflammatory infiltrates and resorptive granulation over time, slight fibrotic response, and nonsignificant changes in neighboring eye tissues all indicate good tolerance to bioimplant materials. This allows some optimism regarding the use of hydrophobic terpolymer in the construction of the third-generation intrascleral implants. However, the ultimate decision regarding its usefulness and safety in the treatment of glaucoma requires further investigation.
Supported by the Polish Ministry of Science and Higher Education, Poland, Grant N518 028 32/1769; and The Foundation for Polish Science in Domestic Grants for Young Scientists Program (ES-Z).
Disclosure: R. Leszczynski, None; T. Gumula, None; E. Stodolak-Zych, None; K. Pawlicki, None; J. Wieczorek, None; M. Kajor, None; S. Blazewicz, None