Study protocols involving animals were approved by the ethics committee of the contract research organizations which carried out the studies. The biocompatibility study was approved by NAMSA Ethical Committee (NAMSA, France) and was carried out in accordance with OECD Good Laboratory Practice regulations, ENV/MC/CHEM (98)17, European Good Laboratory Practice regulations, 2004/10/EC Directive, and with United States Food and Drug Administration Good Laboratory Practice regulations (21 CFR 58). The time-course biointegration study was designed as a non-Good Laboratory Practice, protocol-controlled study, carried out at the Medanex Clinic (Diest, Belgium), and was approved by Ethical Committee Animal Studies of Medanex Clinic.
Device description
MINIject is a small oblong glaucoma drainage implant (length = 5.0 mm, width = 1.1 mm, thickness = 0.6 mm), made of a soft, flexible material (Fig. 1). Structurally, the implant is composed of STAR® material, a precision porous construct made of medical grade silicone, i.e. NuSil MED-6215 silicone (NuSil Technology, Carpinteria, CA) designed to maintain long-term stability. The material has a uniform internal pore size of 27 μm. During manufacturing of the material, the sizes of the connections between pores are controlled with high uniformity throughout the entire volume (Fig. 1), to ensure a predictable fluid flow.
The device is intended to be implanted in the suprachoroidal space of the human eye through an ab-interno MIGS procedure under direct gonioscopic visualization with the help of a delivery system. Briefly, the delivery system is introduced into the anterior chamber through a small peripheral corneal incision and the implant is placed in the suprachoroidal space by insertion through the iridocorneal angle. To accomplish this aim, MINIject is designed with a 0.4 mm wide color marking intended to act as a visual clue to control the implantation depth. The marking material is made of long-term medical grade silicone ink, i.e. NuSil MED-6613-6 (NuSil Technology, Carpinteria, CA), chemically crosslinked to the surface of the silicone STAR material.
Animals and treatments
For both studies, only healthy animals without signs of any significant ocular irritation were selected. Animals were acclimated for a minimum period of 6 days. They were maintained with a 12-h light-dark cycle with free access to food and water. Throughout the in vivo phase of the study, animals were observed daily for any abnormal clinical event. Body weight was recorded weekly.
Biocompatibility study
Fourteen (14) female New Zealand White rabbits, with ages of 30 to 33 weeks, were purchased from Charles River Laboratories (Arbresle, France). The animals underwent a MINIject implantation in one eye (Test group) while the contralateral eye either had no operation (Control group), or a sham operation (an identical operation without implant placement, Sham group). Slit-lamp biomicroscopy of the anterior segment was conducted preoperatively, at postoperative days (POD) 1 and 3, and postoperative weeks (POW) 1, 2, 4, 12, and 26. Indirect ophthalmoscopy of the posterior segment was performed preoperatively and on POW 1, 4, 12 and 26. Ocular abnormalities, if any, were recorded preoperatively. At each timepoint after the surgery, all ocular changes were scored following the McDonald-Shadduck scoring system. Intra-ocular pressure was measured diurnally under local anesthesia (1% tetracaine), through tonometry (TONO-PEN VET, Reichert, Depew, NY) preoperatively and weekly after MINIject implantation surgery. Animals were euthanized on POW 12 (n = 6) or 26 (n = 8), and eyes were enucleated for histopathological analysis.
Time-course biointegration study
Twelve (12) female Dutch Belted rabbits, aged 4 to 5 months, were purchased from Covance (Denver, CO). MINIject was implanted in the right eye (Test group) of each animal, while the left eye had no implant nor other operation (Control group). Slit-lamp biomicroscopy was conducted preoperatively, at POW 1, 2, 3 and 4, then at postoperative months 2, 3, 4, 5 and 6 to examine the anterior segment. Intra-ocular pressure was measured diurnally on non-anesthetized corneas through rebound tonometry (TonoVet, Icare Finland Oy, Vantaa, Finland), preoperatively and bi-weekly after surgery. Animals were euthanized on POW 4 (n = 3), 12 (n = 3) or 26 (n = 6), and eyes were enucleated for histopathological analysis.
Implantation procedure
Similar surgical procedures by qualified glaucoma surgeons were used in both the biocompatibility and biointegration studies using standard aseptic techniques. In the human eye, the MINIject implant is placed through the ab-interno pathway with the help of a specially designed delivery system. However, rabbit eyes have a shallow anterior chamber and narrow iridocorneal angle compared to human eyes, rendering incompatible the use of the delivery system designed for the human eye with safe and optimal implant positioning in the rabbit eye. Thus, substantial changes were made to the surgical approach. These changes involved implant placement through an ab externo surgical procedure as described below.
After induction of general anesthesia, the periocular zone was disinfected, and local anesthetic was applied to the cornea. Each animal was positioned under the operating microscope and an eyelid speculum was utilized. Intrastromal corneal fixation sutures were placed to allow appropriate positioning of the eye during surgery.
A fornix-based conjunctival flap was created in the upper quadrant, followed by the creation of a superficial scleral flap (approximately 50% depth; 3 mm width × 3 mm length). An incision that reached the anterior chamber was made by passage of a blade through the trabecular meshwork. Viscoelastic material (1.2% sodium hyaluronate, Beaver Visitec International, Bidford-on Avon, UK) was injected to fill and maintain a deep anterior chamber during the surgery. Two mm posterior to the incision into the anterior chamber, the remaining layer of deep sclera was incised down to the choroid, reaching the suprachoroidal space. A scleral bridge of 1–2 mm was thus left between the incision into the anterior chamber and the incision into the suprachoroidal space.
Upon careful separation of the sclera from the choroid using an atraumatic spatula, the non-colored posterior portion of the MINIject implant was introduced through the scleral incision and guided posteriorly into the suprachoroidal space. The anterior portion of the device was introduced into the anterior chamber through the trabecular incision so permitting the device to create a connection between the anterior chamber and suprachoroidal space.
Implant positioning was adjusted so that the green mark present on the anterior portion of the implant was half visible in the anterior chamber and the other half remained under the scleral flange. The superficial scleral flap as well as the conjunctival flap were then sutured in a water-tight manner to avoid bleb formation; non-absorbable (Ethilon 9–0, Ethicon, Johnson and Johnson, Diegem, Belgium) and absorbable (Safil 8–0, B. Braun, Melsungen, Germany) sutures were used in the respective tissues. Fixation sutures were removed. The viscoelastic material was removed from the anterior chamber by irrigation with a balanced salt solution (BSS Plus, Alcon, Fort Worth, TX) through a paracentesis incision.
Both test and sham eyes were operated using the same surgical procedure, with the exception that no implant was inserted into the sham eyes; the control eyes remained unoperated. Post-operative medications including a topical drop of anti-inflammatory and antibiotic ophthalmic solution (dexamethasone, neomycin and polymyxin B) 3 times a day, and daily subcutaneous injection of a non-steroidal anti-inflammatory drug (meloxicam) were administered for 7 days.
Termination and histological processes
After the last examinations were conducted, rabbits were euthanized. In the biocompatibility study, euthanasia of the animals was performed by intravenous injection of 1 mL/kg of pentobarbital (182.20 mg/mL, Dolethal, Vetoquinol, France). Prior to euthanasia, animals were sedated by an intramuscular injection of 10 mg/kg ketamin hydrochloride (Ketamine 1000, Virbac, France) and 2 mg/kg xylazine chlorhydrate (Rompun 2%, Bayer, Germany). In the biointegration study, euthanasia of the animals was performed by intravenous injection of 0.1 mL/kg of T61 (4.39 mg/mL tetracaine chlorhydrate, 26.92 mg/mL mebezonium diiodure, 200.00 mg/mL embutramide, MSD, France). Prior to euthanasia, animals were sedated by an intramuscular injection of 2 mg/kg of xylazine chlorhydrate (Xyl M, V.M.D., Belgium), followed 5 min later by an intramuscular injection of 25 mg/kg ketamin chlorhydrate (Nimatek, Dechra, Netherlands) and 3.5 mg/kg xylazine chlorhydrate (Xyl M, V.M.D., Belgium). Sedation prior to euthanasia was performed for ethical purposes. The eyes were then removed in toto and immersed in appropriate fixative solution for further histopathological processing.
Biocompatibility study
Each eye with its proximal optic nerve was macroscopically observed followed by immersion for 24 h in 4% glutaraldehyde mixed 1:1 with 10% neutral buffered formalin. Fixation was then completed in 10% neutral buffered formalin. Eyes of the Test and Sham groups were trimmed along a plane parallel to the surgical site, to provide sagittal sections of the MINIject implant or of the sham-surgical site, respectively. Three sections, separated by approximately 200-μm intervals, were obtained for each eye. For eyes of the Control group, the superior quadrant of the eye was trimmed along a similar plane. The optic nerve was also bisected to obtain longitudinal and transverse sections. Each part of the eye and the optic nerve were dehydrated in a graded series of alcohol, cleared in xylene, and embedded in paraffin. Histological sections were prepared using a microtome followed by staining with Safranin Hematoxylin Eosin. The slides were examined by light microscopy by a qualified pathologist and semi-quantitatively evaluated (based on a scoring system described in ISO 10993-6).
Biointegration study
Each eye was macroscopically observed and placed in 10% neutral buffered saline for 3 days. Eyes of the Test group were trimmed along a plane parallel to the implant, with the aim of providing sagittal sections of the device. For eyes of the Control group, the superior quadrant of the eye was trimmed along a similar plane. All samples were processed using a standard paraffin-embedding procedure. Five-μm thick sections were prepared using a microtome for histological and immunohistological investigations. The sections were stained with Weigert’s iron hematoxylin kit (1.15973.0002, Merck, Darmstadt, Germany) and Masson-Goldner Trichrome Staining Kit (1.00485.0001, Merck, Darmstadt, Germany) as per manufacturer recommended instructions.
Immunohistochemical detection of endothelial cells (CD34), lymphatic endothelial cells (LYVE-1), alpha-smooth muscle actin (alpha-SMA), and Type III collagen was achieved with antigen specific target antibodies. Epitope retrieval was performed by heating tissue sections in an epitope retrieval solution (Ultra Cell Conditioning 2, Ventana, Basel, Switzerland) for 20 min at 80 °C in a water bath. The solution was cooled down to room temperature over a period of 20 min to avoid a fast temperature drop and sections were then rinsed twice in distilled water. Endogenous peroxidases were blocked through 15 min incubation in 3% H2O2 followed by the blockage of nonspecific sites by a 20 min incubation in normal horse serum (ImmPRESS HRP Anti-Goat Ig Polymer Detection Kit, MP-7405, Vector Laboratories, Burlingame, CA), or for 10 min incubation in protein block serum free (X0909, Dako, Heverlee, Belgium), for goat or mouse primary antibodies, respectively.
Sections were further incubated for 1 h at room temperature in polyclonal goat anti-CD34 (1/100; sc-7045, Santa Cruz Biotechnology, Heidelberg, Germany), polyclonal goat anti-LYVE-1 (1/100; AF2089, R&D Systems, Abingdon, UK), monoclonal mouse anti-type III collagen (1/100; NBP2–33328, Novus Biologicals, Cambridge, UK), or monoclonal mouse anti-alpha-SMA (1/500; M0851, Dako, Heverlee, Belgium), followed by 3 washes in phosphate buffered saline (PBS). Sections were then incubated for 30 min at room temperature in ImmPRESS HRP Anti-Goat Ig Polymer Detection Kit (MP-7405, Vector Laboratories, Burlingame, CA) or in anti-mouse EnVision+ System/HRP (K4001, Dako, Heverlee, Belgium), for goat or mouse primary antibodies, respectively. The peroxidase activity was revealed using DAB+ substrate chromogen (Dako, Heverlee, Belgium). Tissues were finally counterstained with Carazzi hematoxylin 0.1%, then dehydrated and mounted with Eukitt medium (VWR, Leuven, Belgium).
Virtual images were acquired with a fully automated digital microscopy system Nanozoomer 2.0-HT (Hamamatsu Photonics, Shizuoka, Japan) using a 40x magnification objective (0.23 μm/pixel) and NDP.scan software (Hamamatsu Photonics, Shizuoka, Japan). Images were visualized using Cytomine software [30]. Histological sections were analyzed independently by four reviewers (IG, DM, MKR and AJM).
Statistical analysis
Data were expressed as the means ± standard deviations (SD). No statistical analysis of the data was conducted.