Bioengineered corneal tissue for minimally invasive vision ...

Manufacturing of collagen-based corneal stromal equivalent

We developed a collagen-based corneal stromal substitute, BPCDX. BPCDX was manufactured following principles of good manufacturing practices (GMP), in a Class 5 (according to ISO-14644-1) air quality laminar flow clean-room facility in Linköping, Sweden by LinkoCare Life Sciences AB. Although the BPCDX is subject to an end-stage sterilization process, substantial efforts are made throughout the manufacturing process to maintain raw materials, intermediate products and the final product as aseptic as possible.

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In brief, the fabrication procedure was as follows. Medical-grade purified freeze-dried type I porcine dermal atelocollagen was purchased from SE Eng Company (South Korea). The collagen was dissolved in PBS at room temperature to form a 5% collagen solution. The collagen solution was then exposed to a controlled vacuum evaporation29, and crosslinkers 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide methiodide (EDCM), N-hydroxysuccinimide (NHS) and riboflavin (vitamin B2) (Care Group, Vadodara) were added at 1% (w/v) ratios. The solution was mixed thoroughly and dispensed into custom curved contact lens molds with spacers of 280 µm and 440 µm used to delineate the final device thickness. Molds were clamped and samples were cured at room temperature. Removal from molds was achieved by immersion in PBS for 1 h at room temperature. Finally, BPCDX was rinsed with sterile PBS to extract any reaction residues. Following this chemical crosslinking, a second photochemical crosslinking of samples was performed by exposure to ultraviolet A (UVA) light, following the protocol of UVA–riboflavin corneal collagen crosslinking commonly used clinically32. We employed this second photochemical crosslinking step aiming to further improve the BPCDX mechanical strength, resistance to degradation and long-term stability. Notably, the BPCDX differs from single-crosslinked recombinant human-collagen-based implants evaluated previously in humans27,28,62 in several important ways. Firstly, we used medical-grade and FDA-approved porcine dermal collagen as a starting material, the ultra-purity of which results in a more mechanically robust hydrogel and reproducible batch-to-batch mechanical properties. The use of a novel vacuum evaporation technique enables a high collagen content (12–18%) to be controllably achieved29, relative to previous human-tested implants limited to 10% collagen by weight (the native human cornea is 13.6% collagen)27. We additionally increased the ratio of chemical crosslinkers to collagen from 0.4:1 to 1:1 to achieve a higher degree of crosslinking in comparison to earlier implants. Further, our use of the crosslinker EDCM29 (as opposed to EDC27) and subsequent UVA–riboflavin crosslinking with optimized dose and exposure parameters, differentiates the present BPCDX from previous collagen-based hydrogels, imparting additional strength and resistance to degradation (see Supplementary Table 1 for full comparison of manufacturing parameters and material properties).

Packaging of BPCDX

After manufacture, BPCDX is extracted from any reaction residues and rinsed thoroughly with sterile phosphate-buffered saline (PBS) in class 5 (Class 1000) laminar flow hoods and stored in sterile PBS in a sterilized, sealed blister-packed container as shown in Supplementary Fig. 4a. Primary blister packs are labeled according to ISO 15223-1:2012 and EN 1041:2008 as shown in Supplementary Fig. 4b. The entire blister cup package and instructions for use are inserted into a small outer packaging box for protection during transportation and storage (Supplementary Fig. 4c).

Sterilization of BPCDX

To provide an additional level of device safety beyond an aseptic manufacturing process, we implemented an additional end-stage sterilization procedure. Conventional sterilization techniques (for example, dry heat, steam, ethylene oxide, gamma irradiation and electron beam irradiation) widely used for rigid medical device sterilization have not been tested or validated for soft, tissue-engineered devices such as hydrogels, where they are likely to result in denaturation and loss of device integrity. For this reason, we developed a sterilization procedure using a pulsed UVC irradiation system (Xenon Z-1000 Flash UV Lamp System). Pulsed UV light is known for its ability to inactivate microbes, is widely used in the food industry and is gaining popularity as a sterilization method63,64.

Sterilization validation of BPCDX implants was performed according to ISO 14937:2016 and ISO 11137-1-3:2017 sterilization standards. Two sets of product samples were exposed to pulsed UVC light at two sterilization doses separately, and then tested for key properties and sterility to investigate if the UVC exposure at these dosages impacted BPCDX properties and/or packaging. Sterility tests were both performed internally using tryptic soy broth sterility test method and externally by an ISO-certified microbiology lab (MIKROLAB Stockholm AB) for independent sterility and bioburden tests.

Quality control of samples and their packaging was conducted by visual inspection, mechanical, optical, water content, analysis of physical dimensions and collagenase degradation tests on the UVC-exposed samples and non-sterilized samples as controls. Energy intensity of the pulsed UVC light at all positions of the sterilization tray was measured using a UVC Nova II Laser Power/Energy Meter (Ophir Spiricon Europe GmbH) and a LiteMark-XL light intensity monitor.

Optical transparency

Light transmission through BPCDX was measured across the UV and visible light spectrum (200–700 nm) at room temperature, using a High Performance USB4000 UV-Vis Spectrophotometer (Mettler Toledo). To enable direct comparison with light transmission through the native human cornea, BPCDX samples were 550 µm thick. Samples were immersed in PBS during measurement, and light transmission was compared with published data from healthy human corneal tissue35. Measurements were made for three independent samples per spectrum.

Mechanical properties of scaffolds

Tensile strength, elongation at break (elasticity), elastic modulus (stiffness) and energy at break (toughness) of BPCDX were measured with an Instron Automated Materials Testing System (Model 5943 Single Column Table Frame) equipped with BlueHill software (v.3), a load cell of 50 N capacity and pneumatic metal grips at a crosshead speed of 10 mm min−1. Test specimens were made by molding and curing the samples into dumbbell-shaped Teflon molds followed by equilibration in PBS. Specimens were attached to the grips and tensile force was applied until the sample break point. Data were automatically recorded by the software, and 22 dumbbell-shaped samples were used for each mechanical test.

Characterization by scanning electron microscopy

Morphology of BPCDX and the native porcine cornea was investigated using a Zeiss SEM (LEO 1550 Gemini). PBS-equilibrated samples were washed in water, frozen overnight at −80°C and lyophilized for 12 h. Samples were cut and attached onto metal holders using conductive double-sided tape, and sputter coated with a gold layer for 60 s at 0.1 bar vacuum pressure (Cressington Sputter Coater, 108) before SEM examination. SEM micrographs were taken at various magnifications at 25 kV and 5 kV for porcine cornea and BPCDX samples, respectively.

Enzymatic degradation test

Test samples of BPCDX, a version (BPC) that was singly crosslinked with EDC-NHS29 and donor human cornea, all with a thickness of 500 µm, were exposed to collagenase Type I (from Clostridium histolyticum) and their residual mass percentage as a function of time was measured. Trizma base (Tris base), 2-amino-2-(hydroxymethyl)-1,3-propanediol was used for preparing Tris-HCl buffer. Test samples were equilibrated in 5 ml 0.1 M Tris-HCl buffer (pH 7.4) containing 5 mM CaCl2 at 37 °C for 1 h. Subsequently, 1 mg ml−1 (288 U ml−1) collagenase solution was added to give a final collagenase concentration of 5 U ml−1 (17 µg ml−1). The solution was replaced every 8 h to retain sufficient collagenase activity. Samples were weighed at various time intervals after gently blotting away the surface water. Three replicates of each sample type were tested. The percent residual mass of the sample was calculated according to the ratio of initial sample weight to the weight at each time point.

In vitro cell biocompatibility

Culture of human corneal epithelial cells (HCE-2 50.B1 cell line, lot no. 70015331, ATCC) was established according to the manufacturer’s instructions. In brief, serum-free keratinocyte growth medium (1×, Gibco) was supplemented l-Glutamine, 5 ng ml−1 epidermal growth factor and 0.05 mg ml−1 bovine pituitary extract (BPE), 500 ng ml−1 hydrocortisone and 0.005 mg ml−1 insulin (Gibco). A T-75 cell culture flask was pre-coated with a mixture of 0.01 mg ml−1 fibronectin, 0.03 mg ml−1 bovine collagen type 1 and 0.01 mg ml−1 bovine serum albumin (BSA), and was incubated overnight at 37 °C. The next day, the excess coating was aspirated, and the flask was allowed to stand for 15 min before seeding the cells. The HCE-2 cell vial was thawed, and cells were seeded at a density of 104 cells per cm2 on the pre-coated flask. Cells were incubated at 37 °C at 5% CO2, and growth medium was changed every other day.

BPCDX (300 µm thick) pre-cut to 8 mm diameter were rinsed in PBS and equilibrated in the complete keratinocyte serum-free medium for 2 h in a humidified cell culture incubator. BPCDX samples were then laid down (concave side down) onto the bottom of a 48-well cell culture plate and allowed to adhere to the bottom of the plate for 2 h in an incubator at 37 °C. Three wells were used for BPCDX samples, and three were used as controls (that is, no biomaterial attached to the bottom of the well).

Upon confluence of the seeded HCE-2 cells in the culture flask, the cells were trypsinized with Trypsin-EDTA, then trypsinization was stopped with a complete growth medium and cells were harvested, counted and seeded into the six prepared wells. Cells were seeded at a density of 105 cells per well for the control and BPCDX-covered wells and incubated for 1 h before adding additional medium to reach a final volume of 200 µl growth medium per well. Growth medium was changed every other day, and cells were maintained in culture for 16 days. On day 16, the cells were washed and covered with fresh medium. Cells were stained with NucBlue Live cell stain (Hoechst 33342, Thermo Fisher Scientific) according to the manufacturer’s instructions. Images of stained cells were captured using a Leica DMi8 inverted live-cell microscope under ultraviolet light excitation (385 nm) to detect live cells with fluorescent blue nuclear stain. Brightfield images were additionally obtained to observe cell morphology.

Biological evaluation according to ISO 10993-1:2018

Biocompatibility testing of the BPCDX was performed in conformance to GLPs as per US FDA CFR Title 21 Part 58 and according to ISO 10993 and ISO 11979 standards by an external GLP-certified contract research organization (BioNeeds Private Limited, India). BPCDX underwent the following in vitro and in vivo biocompatibility studies:

1. 1.

   ISO 10993-3: genotoxicity, carcinogenicity and reproductive toxicity (bacterial reverse mutation tests (ref. report no. BIO-GT-348))

2. 2.

   ISO 10993-3: genotoxicity, carcinogenicity and reproductive toxicity (in vitro mammalian chromosome aberration test (ref. report no. BIO-GT-349))

3. 3.

   ISO 10993-3: genotoxicity Ames Test (ref. report no. BIO-GT-358)

4. 4.

   ISO 10993-3: genotoxicity, carcinogenicity and reproductive toxicity (mammalian erythrocyte micronucleus test in Swiss albino mice) (ref. report no. BIO-GT-350)

5. 5.

   ISO 10993-4: in vitro hemolysis test (ref. report no. BIO-TX-1575)

6. 6.

   ISO 10993-5: in vitro cytotoxicity:

   1. a.

      In vitro cytotoxicity (direct contact method) (ref. report no. BIO-GT-346)

   2. b.

      In vitro cytotoxicity (elution method) (ref. report no. BIO-GT-347)

7. 7.

   ISO 10993-10: skin sensitization in guinea pigs (ref. report no. BIO-TX-1574)

8. 8.

   ISO 10993-10: in vivo ocular irritation in rabbits (polar + non-polar) (ref. report no. BIO-TX 1584)

9. 9.

   ISO 10993-11: acute systemic toxicity in Swiss albino mice (ref. report no. BIO-TX-1576)

Bacterial endotoxin test according to ISO 11979-08

To ensure the sterile BPCDX would be safe for human use, endotoxin testing was performed internally and by an independent laboratory (S2 Medical AB) using an Endosafe-PTS endotoxin analyzer (Charles River) with a rapid, point-of-use spectrophotometer and USP/BET-compliant disposable cartridges for real-time endotoxin testing. The limulus amebocyte lysate cartridges used in the tests are FDA-licensed for in-process and final product release testing, ensuring regulatory compliance of the output results. In brief, the tests were performed and reported according to ISO 11979-8 (ophthalmic implants-intraocular lenses-part 8: fundamental requirements amendment 1) and ISO 15798. The acceptable endotoxin limit for ophthalmic devices as specified in ISO 15798 is 0.2 EU per device against which BPCDX test results were evaluated.

Shelf-life stability tests according to ISO 11607

Verification of stability is a time-consuming and resource-intensive task in the development of new medical devices and is often overlooked in research studies. For wide distribution of the BPCDX to regions with the greatest unmet need, shelf-life studies are critical to ensure the device produced in a normal manufacturing process functions as intended despite logistical, storage and other barriers that may occur in the distribution chain. Often devices are tested under accelerated conditions to increase the rate of chemical and/or physical degradation and therefore decrease the time required to obtain stability data. Long-term or real-time studies are still needed however, as the accelerated and long-term results may differ. For packaged and sterilized BPCDX, we therefore performed an accelerated shelf-life stability study by incubating devices at 28 °C for 6 months, and we performed a real-time shelf-life stability study by incubating devices at 7 °C for 2 years. Control BPCDX samples not subjected to aging (time-zero samples) were prepared and tested for visual appearance, mechanical properties, optical transmission, water content, size, collagenase degradation, in-house tryptic soy broth sterility test and for sterility tests conducted by an independent GMP-certified laboratory (MIKROLAB Stockholm AB). The accelerated and real-time aged samples were also tested for the above properties and compared with the control samples.

In vivo biocompatibility, subcutaneous implantation in rats

To test compatibility of BPCDX after surgical implantation in vivo, we used a model of subcutaneous implantation in rats as described previously43. Three male Wistar rats aged 8 weeks were given a pre-operative analgesic (0.01 mg buprenorphine) by intraperitoneal injection the day before surgery, day of surgery, and 1 and 2 days after surgery. Under general anesthesia (25 mg ml−1 ketamine and 0.5 mg ml−1 dexmedetomidine hydrochloride), a 2-cm long paravertebral cutaneous incision was made into the dorsal flank of the rat, after which a subcutaneous pocket was created by blunt dissection. A 1-cm square piece of the BPCDX was inserted into the pocket, and the pocket was sealed with three absorbable 9-0 Vicryl sutures. Eight weeks post-implantation, rats were euthanized, and the tissue region surrounding the implant zone was excised and prepared for immunohistochemical analysis. The procedures were performed after obtaining ethical approval from the Linköping Animal Ethical Review Board (permit ID 585), with procedures in compliance with EU Directive 2010/63/EU on the protection of animals used for scientific purposes.

Minimally invasive BPCDX implantation in minipigs

To evaluate implantation of BPCDX in vivo in the cornea, we designed a model of keratoconus in the Göttingen minipig to create an artificially thin native corneal stroma mimicking the pathologic thinning in advanced keratoconus. To achieve this, we used an ophthalmic femtosecond laser (iFS 150, Abbott Medical Optics) and modified the technique we previously reported in rabbit models, termed FLISK29,31. Animal experiments were performed after receiving approval from the Linköping Animal Ethical Review Board (permit ID numbers 153 and 37–16) and adhered to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and the Directive 2010/63/EU. All surgeries were performed in a licensed large-animal surgical suite with controlled temperature and humidity at the Linköping University Translational Medicine Center, under supervision of the university veterinarian and animal care team. Ten female Göttingen minipigs (Ellegaard Göttingen Minipigs A/S) aged 6 months and weighing 15 kg were pre-medicated with sedatives (3 mg kg−1 tiletamine HCl + 3 mg kg−1 zolazepam HCl, Zoletil and 0.06 mg kg−1 medetomidine HCl, Dexdomitor), administered intramuscularly. Anesthesia was initiated with 0.2 mg kg−1 Propofol, administered intravenously through a venous catheter placed in the ear vein. Thereafter, the animals were intubated with anesthesia maintained using 0.5–3.0% isoflurane gas. During anesthesia, hydration was maintained by intravenous administration of Ringer’s acetate solution at 10 ml kg−1 h−1. Immediately before surgery 0.02 mg kg−1 atropine was given by intramuscular injection. Topical anesthesia (0.5% tetracaine HCl eye drops) was given before laser surgery, and all surgeries were performed in a single eye per animal.

To achieve a thinner stroma mimicking advanced keratoconus, we used the femtosecond laser to cut a central mid-stromal button in the cornea 7 mm in diameter and 250 µm in thickness, with the anterior surface of the button located 200 µm below the corneal surface (Supplementary Fig. 2). To achieve this we pre-programmed the laser to perform cuts in the following order: a posterior circular lamellar planar cut of 7.1 mm diameter located 450 µm below the corneal surface, a 360° circular side cut of 7.0 mm diameter extending from the posterior lamellar plane 250 µm anteriorly, and an anterior circular lamellar plane of 7.1 mm diameter at a depth of 200 µm below the corneal surface, followed by a final 90° arc-shaped access cut, oriented at 45° to the lamellar planes and extending to the epithelial surface. Details of the femtosecond laser protocols are given elsewhere65. Following the laser procedure, we used a blunt hockey blade tool to separate the access cut and lamellar planes, and surgical forceps to extract the button of native stromal tissue through the access cut. This resulted in a cornea approximately two-thirds the thickness of the normal porcine cornea, mimicking the cornea in advanced keratoconus, albeit with a uniform reduction in thickness across the central 7 mm of cornea.

We thereafter conducted minimally invasive surgery to treat advanced keratoconus. A sterile, packaged BPCDX implant of 280 µm thickness and 10 mm diameter was opened in the operating room, and we cut the device to a 7 mm diameter using a Barron corneal punch trephine. Next, we used surgical forceps to grip the BPCDX, which was then inserted into the intrastromal pocket through the access cut65. Although the FLISK surgery was previously performed without the use of surgical sutures29, as a precautionary measure we decided to close the region of the access cut in the stroma anterior to the implant using two 10-0 nylon surgical sutures (Supplementary Fig. 2).

Immediately post-operatively, minipigs were placed on a ventilator and once spontaneous breathing resumed, 0.05 mg kg−1 intravenous buprenorphine (Temgesic) was given as an analgesic. Post-operatively, topical anesthetic eye drops were again instilled, followed by a combination topical corticosteroid-antibiotic (0.1% dexamethasone + 0.3% tobramycin, Tobrasone eye drops) given three times daily the first post-operative week and twice daily during the following three weeks. Post-operative analgesia consisted of intramuscular injection of 0.05 mg kg−1 buprenorphine every 12 h for the first five post-operative days.

Post-operative assessment and corneal imaging

Six months after surgeries, minipigs were placed under general anesthesia as described above, and we performed in vivo examinations and photo documentation in operated eyes. Examinations consisted of digital photography (Nikon D90 digital camera), anterior segment optical coherence tomography (iVue, Optovue) and in vivo confocal microscopy (Heidelberg Retinal Tomograph 3 with Rostock Corneal Module, Heidelberg Engineering) using a previously described microscopy protocol66. Following examinations and data collection, minipigs were euthanized while under general anesthesia and deep sedation, by intramuscular injection of 7 mg kg−1 Zoletil and intravenous injection of 100 mg ml−1 pentobarbital.

Histology and Immunohistochemistry

Following euthanasia, rat tissue and porcine corneas (to the limbal margin) were dissected under an operating microscope, embedded in optimal cutting temperature compound, and snap-frozen in liquid nitrogen. Corneas were stored at −80 °C until further use. For histology, rat and pig tissue were thawed, fixed in 4% paraformaldehyde solution, embedded in paraffin and sectioned to a thickness of 4 µm, followed by staining with hematoxylin and eosin (H&E). For immunohistochemistry, sections 10 µm thick were produced using Leica CM1510 cryostat (Leica AB). The resulting sections were mildly fixed with 2% paraformaldehyde (VWR Life Science) for 10 min, permeabilized by incubation in 0.05% Triton X-100 for 10 min and blocked with 5% BSA for 1 h at room temperature. The samples were then incubated overnight at 4 °C with primary antibodies β-III-tubulin (1:100, ab7751, clone TU-20, lot GR3238448-11, Abcam), α-SMA (1:25, ab7817, clone 1A4, lot GR119216-7, Abcam), type III collagen (1:100, Acris AF5810, clone III-53, lot A130097BH) and CD45 (1:100, ab23, clone UCH-L1, lot GR3189280-2, Abcam) in 2.5% BSA. Control sections were incubated with 2.5% BSA alone without the addition of the primary antibody. Sections were washed in PBS-T and visualized by goat anti-mouse IgG (H + L) Alexa Fluor 488 (1:1,000, A32723, polyclonal, RRID AB_2633275, Thermo Fisher Scientific) or DyLight 488 (1:1,000, 35503, polyclonal, RRID AB_844397, Thermo Fisher Scientific) and DyLight 550 (1:1,000, SA5-10173, polyclonal, RRID AB_2556753, Thermo Fisher Scientific) secondary antibodies. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI); (1:5,000; Sigma). The slides were mounted with a ProLong Diamond antifade reagent (Invitrogen, Thermo Fisher) and imaged with a laser fluorescence confocal microscope (LSM700, Zeiss).

Ethics statement

BPCDX was implanted intrastromally in human subjects in local investigator-driven pilot feasibility studies in Iran and India. BPCDX was implanted in cases of advanced keratoconus causing severe visual impairment or corneal blindness. Before subject recruitment, ethical approvals for the studies were obtained in Iran (Institutional Review Board, Farabi Hospital, Tehran University of Medical Sciences, Tehran, Ethics Approval Code IR.TUMS.FARABIH.REC.1395.442) and in India (Institute Ethics Committee, All India Institute of Medical Sciences, New Delhi, ref. no. IEC/NP-47/10.04.2015, RP-23/2015). Studies were conducted following the tenets of the Declaration of Helsinki, and signed informed consent was obtained from all participants before inclusion. LinkoCare Life Sciences AB sponsored the pilot studies, which were funded by LinkoCare Life Sciences AB in kind (both sites), Care Group India (Indian study) and the local investigators in India and Iran. No European Union funding was used for clinical activities outside of the EU. The study was an exploratory, non-randomized, non-blinded and non-controlled pilot case series whose primary goal was to test feasibility of a new surgical method and detect possible adverse reactions in the host or in the BPCDX device using different implanted device thicknesses, analogous to an initial dosing study to determine drug tolerance. The rationale for conducting the pilot study was to obtain first feasibility and safety/tolerability data to determine if it would be ethically acceptable to randomize visually impaired patients (a vulnerable group) to possibly receive the experimental treatment and not a standard donor cornea and surgery when available, as would be required for a future randomized controlled trial. Broad dissemination of pilot safety and feasibility data was not initially foreseen, and local investigators initiated surgeries immediately upon receiving local ethical approvals, without pre-registration of the pilot series in a public registry. Interim analysis of pilot data indicated safety and revealed an unexpected efficacy comparable to outcomes of standard transplantation surgery (DALK or PK) with possible additional clinical benefits from the milder surgery. On the basis of the interim data, approvals for randomized controlled studies were granted in the EU (see below). For these reasons, publication of initial pilot feasibility results including details of the proposed minimally invasive surgery would be in the interest of the medical and scientific community, before initiating a randomized controlled trial. To ensure the reporting of the pilot studies in Iran and India would be consistent with best practices for the conduct of investigational studies of medical devices, the ongoing pilot clinical study was registered in the ClinicalTrials.gov database in December 2020 (Clinicaltrials.gov: NCT04653922). The BPCDX manufacturing process and device test results presented here, along with preclinical data and pilot clinical results at 6–12 months of follow-up, were submitted to Regulatory Authorities in Sweden (Medical Products Agency) and the Czech Republic (State Institute for Drug Control), and approvals were granted for a randomized controlled study protocol according to EU Directive 93/42/EEC on Medical Devices (decision 5.1-2018-44565, Sweden; file no. sukls 21920/2020, Czech Republic) and for the BPCDX device manufacturing, sterilization and packaging methods (file no. sukls 21920/2020, Czech Republic). Additionally, an institutional ethical review committee in Sweden reviewed a randomized clinical trial protocol and approved it for use in Sweden (Linköping Regional Ethics Committee, decision 2017/34-31). Because BPCDX device development and preclinical studies were partially funded by the European Union (Project ARREST BLINDNESS, grant no. 667400), before any reporting of results the clinical studies conducted in Iran and India were subject to further review by an independent contracted biomedical ethics expert (H. Draper, University of Warwick, UK) and by an independent Ethics Review Panel at the European Commission, Division of the Director General—Research and Innovation. The outcome of these reviews was favorable, confirming that the conduct of the clinical studies was consistent with accepted ethical practices within the EU and that the studies were conducted without exploitation of the research subjects or local investigators.

Pilot feasibility study according to ISO 14155

To investigate the safety and feasibility of BPCDX use in LMICs, we conducted pilot studies in Iran (Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran) and India (All India Institute of Medical Sciences, Dr. R. P. Centre for Ophthalmic Sciences, New Delhi, India). The aim of these pilot studies was to set broad guidelines for the use of the BPCDX device (such as inclusion and exclusion criteria and parameters for intrastromal surgery) to determine the feasibility to implement the proposed treatment in local patient populations, while allowing surgeons the flexibility to adapt to local clinical procedures, protocols and surgeon experience and preferences. Several parameters therefore varied between sites such as the size of BPCDX and size of intrastromal pocket, choice of post-operative medications and timing and conduct of follow-up examinations using instruments and diagnostic methods available to investigators at the local clinics. The goal of these initial case series was to obtain initial safety, feasibility and efficacy data, which if successful, would support the design and implementation of future prospective, randomized, controlled clinical trials.

Ethical permission was granted for the pilot studies to treat up to 20 subjects with advanced keratoconus at each site (40 subjects total), based on the ability to detect adverse events in 10% of cases. We report results of 24-month follow-up of the first 12 patients treated in Iran and the first 8 patients treated in India (20 patients total). For the results reported here, clinical data collection occurred during February 2017–January 2020 in Iran and during November 2016–March 2020 in India. In addition to safety and feasibility of BPCDX implantation in advanced keratoconus using the minimally invasive FLISK procedure, the study sites collected data to enable assessment of possible efficacy in terms of rehabilitation of corneal curvature, corneal thickness and BCVA. A future definitive trial would be based upon occurrence of adverse events (for example, inflammation and rejection) leading to implant removal in a maximum of 10% of cases, along with evidence of efficacy in the form of at least 60% of operated eyes having sustained decrease of keratometry at 6 months, sustained increase in central corneal thickness at 6 months, and minimum visual acuity improvement of one Snellen line of vision at 6 months.

Subject recruitment and study endpoints

Potential study subjects at both sites were identified on the basis of clinical history, previous consultation visits and fulfillment of study inclusion/exclusion criteria. Potential candidates were contacted by telephone by the study nurses, then sent the study information and consent form by postal mail. If a subject decided to participate in the study, the consent form was signed by the subject and the local investigator-physician in charge of the study, and the subject was formally included. Study participants were not compensated for their participation in the study, monetarily or otherwise. The primary endpoint for this study was to determine the safety and stability of BPCDX implantation, defined as retention of the device without degradation, loss of transparency, inflammation or vascularization of the implant or host cornea during the first 6 post-operative months. Primary outcome measures included safety (determined by maintenance of corneal transparency and absence of rejection, scarring, inflammation or neovascularization detected during clinical examinations up to 6 months post-operatively) and efficacy measures to assess reduction in maximum keratometric power, increase in corneal thickness and improvement in uncorrected and BCVA at 6 months. The secondary endpoint was to determine safety during a 12-month post-operative period, while secondary outcomes were the same safety and efficacy measures as above, but at 12 months. Here we report the longer-term 24-month results for the primary study endpoint in the first 20 operated subjects.

Inclusion and exclusion criteria for subject selection

Subjects were eligible for study inclusion if the following criteria were all met in at least one eye:

1. 1.

   Grade 3 or higher advanced keratoconus (according to Amsler–Krumeich classification).

2. 2.

   No corneal scar.

3. 3.

   Male or female aged ≥18 years.

4. 4.

   Subjects indicated for a first corneal transplantation.

5. 5.

   Corneal thickness (including epithelium) at least 300 µm centrally, as measured by OCT.

6. 6.

   Signed and dated informed consent.

Patients fulfilling at the selection visit one or more of the following exclusion criteria were not included in the study:

1. 1.

   Ophthalmic exclusion criteria

   In the affected eye:

   • Prior corneal surgery (for example, refractive surgery, cataract, collagen crosslinking, endothelial keratoplasty, etc.).

     In either eye:

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   • Dry eye/tear film pathology.

   • Active ocular infection.

   • Glaucoma/ocular hypertension.

   • Active corneal ulceration.

   • Acute or chronic disease or illness that would increase the operation risk or confound the outcomes of the study (immune-compromised, connective tissue disease, clinically significant atopic disease etc.).

   • Any other medical condition that in the judgment of the local clinical investigator was not compatible with the study procedures.

2. 2.

   Systemic/non-ophthalmic exclusion criteria

   • General history judged by the investigator to be incompatible with the study (for example, life-threatening patient condition or other condition where post-operative follow-up may be difficult).

   • Known diabetes or other neuro-degenerative disorder (as corneal nerves can be affected leading to impaired wound healing).

3. 3.

   Exclusion criteria related to general conditions

   • Inability of patient to understand the study procedures and thus inability to give informed consent.

   • Participation in another clinical study within the last 3 months.

   • Already included once in this study (can only be included for one eye).

Surgical method for clinical case series

We used the minimally invasive FLISK method at both sites. All study subjects were candidates for penetrating or lamellar keratoplasty. The diagnosis of advanced keratoconus was made according to clinical signs (Munson’s sign, Rizutti’s sign) slit-lamp biomicroscopic examination (Vogt’s striae, Fleischer’s ring, apical thinning), corneal topography (skewed asymmetric bow-tie, severe central or inferotemporal steepening, high keratometric power), tomographic signs (abnormal elevation maps, abnormal pachymetry maps, keratoconus detection by Belin/Ambrosio Enhanced Ectasia Display) and refractive results (loss of BCVA). Subjects with refractive errors that could not be corrected with eyeglasses or routine contact lenses, and those who were scleral or mini-scleral contact lens intolerant were included. After the corneal center was marked on the basis of the pupil center, a mid-stromal pocket was created through the marked margins of the temporal cornea using a femtosecond laser. The laser surgical parameters are summarized in Supplementary Fig. 3. Following the laser procedure, the corneal stroma was dissected, taking care to avoid perforation of the Descemet membrane while dissecting the thinnest part of the cornea. Next, BPCDX was removed from the sterile packaging and trephined at 7.5–8.5 mm size and inserted into the intrastromal pocket using surgical forceps to grip the BPCDX across its entire diameter. In Iran, two different thicknesses of BPCDX were used, 280 µm and 440 µm, on the basis of the pre-operative corneal thickness at the thinnest point. Subjects with a thinnest value above 400 μm received 280-μm-thick BPCDX while those with thinnest value below 400 µm received 440-μm-thick BPCDX. In India, all subjects received 280-µm-thick BPCDX regardless of initial corneal thickness. No suturing was performed in any subject. Immediately post-operatively, a soft silicone hydrogel bandage contact lens (Comfilcon A, CooperVision) was placed on the operated cornea for 3 days in all treated subjects.

Post-operative medications and follow-up examinations

Post-operatively, medications were administered as deemed appropriate by the local surgeons. On the basis of the post-operative wound healing observed in the minipig model (where post-operative medications were administered for 4 weeks) and in a previous rabbit study where the intrastromal implantation model indicated complete wound healing within 8 weeks29, an 8-week regimen was instituted. In Iran, patients received artificial tears (carboxymethylcellulose 0.5%) as needed pre-operatively and three times daily post-operatively for 8 weeks, and betamethasone eye drops (0.1%) were given three times daily for 8 weeks post-operatively. Additionally, a bandage contact lens was placed over the operated cornea for 1 week. In India, patients received pre-operative moxifloxacin eye drops (0.5%) three times daily for 3 days prior to surgery. Post-operatively, patients received moxifloxacin eye drops (0.5%) three times daily, prednisolone eye drops (1%) four times daily and artificial tears (carboxymethylcellulose 0.5%) six times daily.

Surgeons assessed the eye immediately following surgery and at 1 day, 1 week and 1, 3, 6 and 12 months post-operatively. An additional 24-month visit was subsequently scheduled. Slit-lamp biomicroscopic evaluation was used to grade the corneal transparency according to a previously published scale67, uncorrected visual acuity and BCVA were determined by the Snellen eye chart and spectacles, and expressed in the logMAR scale, Scheimpflug-based anterior segment tomography was performed to assess corneal steepness (Pentacam HR, Oculus, Optikgerate GmbH), and refraction was assessed at various post-operative visits. Corneal thickness was assessed using anterior segment FD-OCT (Iran: Casia, Tomey; India: iVue, OptoVue). Pre-operative and post-operative corneal imaging examinations were performed by the same experienced optometrist at each site. Data was entered into a spreadsheet for later analysis (Microsoft Excel 365 for Windows, 32-bit).

Statistical analysis

Statistical analysis was performed using Statistical Package for Social Sciences software (v.22, SPSS). Normality in the distribution of the parameters was assessed using the Shapiro–Wilk test. For shelf-life studies, t-tests were performed comparing aged to non-aged samples. To examine differences in post-operative values for clinical parameters at 24 months relative to the pre-operative values, a two-tailed paired t-test was performed. Where results across two different groups were compared, an independent t-test was performed. A two-tailed critical value of α < 0.05 was considered statistically significant for all tests, unless otherwise stated. Visual acuity comparisons were made in logMAR units, with each line of improvement in acuity corresponding to a logMAR reduction of 0.1.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Despite LASIK’s accuracy, enhancements simply come with the territory. Every once in a while a target is missed or a patient from years ago will show up with visual complaints and refractive surgeons must consider time, technique and potential risks of retreatment. We asked some veteran refractive surgeons for their insight and pearls for these rare but unavoidable situations.

 

Reasons for Enhancements

Enhancements typically fall into two categories: early and late. Early enhancements take place within the first few months of the initial LASIK procedure, usually due to a refractive miss that was noticed quickly following surgery. Late enhancements happen years down the line as a result of changes in the patient’s natural lens.

Regarding early enhancements, “If the patient’s unhappy with their vision in the early postop period, and you missed the target by 0.75 to 1 D, our objective at that point is to get them through that period,” says Scott MacRae, MD, the director of refractive services in the department of ophthalmology at the University of Rochester. “Many times we’ll fit them with a soft contact lens so they can see better, particularly for patients who are overcorrected and hyperopic in the presbyopic age group. Those patients can really struggle, but fitting them with a hyperopic soft contact lens in that early postop period can help them get back to a much more functional state for going back to the office/daily activities. We try to nurse them through that two to three month period.”

Reliable measurements are critical in this early enhancement phase, continues Dr. MacRae. “If somebody’s really having a hard time, you could consider retreating at two months postop but you probably want two or three measurements before you actually go ahead and do your retreatment, particularly in patients who don’t have a real clear endpoint. Sometimes they’re still changing. That’s more true for dry eyes and hyperopic correction than with myopic correction. Usually myopic correction is relatively stable after the first week or two after the dryness issues clear up,” he says. “If the patients are still dry at two or three months postop and you’re not getting reliable refractions, make sure that you aggressively treat the dryness before you get your final numbers and proceed with retreatment.”

Figure 1. In his “Dry and Depress” technique for lifting a LASIK flap, Richard Mackool, MD, uses a dry sponge and gently presses on the central cornea until he spots the flap. 

Sometimes, the neuroadaptation of the patient needs to be considered and explained. “My tendency is that if the patient is 20/happy and I’m really close to the target, then I don’t encourage the patient to have retreatment unless they’re really insistent,” says Dr. MacRae. “If they’re insistent, then there are some tricks to that. Typically I’ll go ahead and refract them myself, or have my trusted staff refract the patient. Then we put that prescription into a spectacle trial frame and actually have the patient look at the difference compared to their uncorrected vision in each eye. If they notice a pretty significant visual improvement subjectively then I’ll go ahead and retreat, even if it’s relatively minor. 

“The optics are more complicated than just simple numbers—there’s image quality, which can be affected by higher order aberrations or other subtleties that the patient is perceiving,” he continues. “Certain patients are much more neuroadaptive to their vision than others, so some patients can tolerate a small amount of refractive error, while other patients are really insistent that their image quality isn’t good enough and they want an improvement.”

Setting them up in a spectacle trial can be helpful in comparing each uncorrected eye to the best corrected vision with the trial lens. “If they don’t notice a difference, then I say, ‘If we try to go back and retreat, I don’t think it’s going to make any improvement,’ ” says Dr. MacRae. “Sometimes that’s enough for the patient to say, ‘My vision is pretty good. I’m happy. The doctor tried to do everything they can to get me the best vision I can get and I can settle for this.’ Doing the trial lens is actually really helpful because it avoids that situation where you go ahead and retreat, but then the patient doesn’t notice any difference and you’ve gone through this whole process. It really saves everybody time.”

In cases of late enhancements, Louisville, Kentucky’s Asim Piracha, MD, says, “You have to consider why they need an enhancement two, three or five years later. It’s not that the LASIK ‘wore off’ or that the initial treatment didn’t work—it’s that something has changed to make them either myopic or hyperopic. For myopes, it’s even still more uncommon because their vision is pretty stable long term, and for hyperopes, they tend to get a bit more hyperopic over time. So, if you have a hyperope, usually they have a lot of accommodation and, as they get older, they lose accommodation which makes them more hyperopic. This may be the reason for the enhancement.”

Figure 2. Scott MacRae, MD, developed an instrument called the MacRae Flap Flipper that allows him to gently lift and separate the LASIK flap, which he says has made epithelial ingrowth a rare occurrence.

Topography will reveal a wealth of information. “If they’re beyond a couple years out and they have a myopic astigmatic change, my first thought is to see if there is any ectasia,” Dr. Piracha says. “Before any enhancement you must be really careful to make sure their topography is normal and there’s no sign of even subtle ectasia. We like to look at the preop topography and postop topography to see what’s changed. If there’s any progressive myopia, astigmatism or steepening of the inferior cornea, bullous keratopathy on the surface or posterior surface, then we’re concerned about ectasia. 

“For those patients you have to be cautious and have them come back in six months and make sure there are no progressive changes,” he continues. “If it’s completely stable and there’s any concern at all about ectasia, then I would only enhance with surface ablation, I wouldn’t lift the flap. If there are signs of ectasia progression I wouldn’t enhance them at all and I would do a cross-linking treatment to stabilize the cornea and then consider going back and doing a surface treatment to enhance them (ASA or PRK) once they’re stable, which I’m pretty cautious in waiting at least a year but ideally 18 months after cross-linking before doing any enhancement.”

Dr. Piracha takes a different approach if the patient is hyperopic. “Usually it’s not a sign of ectasia,” he says. “Typically it’s a loss of accommodation as they get older or their near vision is an issue. If they’re over 40 and hyperopic I’d possibly offer an enhancement, monovision or even a refractive lens exchange to enhance them vs. laser vision correction. In all patients after 40 and definitely after 45, I do wavefront measurements on the iTrace of their eyes and look for any dysfunctional lens syndrome—aberrations in the cornea vs. aberrations from their natural lens. That influences whether we pursue a lens exchange or not.”

Cataract development could also be contributing to their vision changes, notes Dr. MacRae. “If it’s an early cataract and we think that it’s progressing, we’ll recommend waiting for six to 12 months and seeing whether their refraction changes,” he says. “If the refraction is changing then it’s not a good time to go ahead and treat—you want to make sure that the refraction is stabilized. If they continue to progress and their refraction continues to change then we recommend cataract surgery or refractive lens exchange.”

 

Treatments and Techniques

Surgeons traditionally treat LASIK enhancements with PRK or by re-lifting the flap and ablating the bed. Whether the patient falls under the early or late enhancement category plays a major role in the decision.

“If they’re less than two years out, I typically lift the flap and enhance them,” says Dr. Piracha. “If the patient’s beyond two years there are definitely several studies and reports to show a higher rate for epithelial ingrowth.” 

Dr. MacRae concurs. “I’ll go back and retreat with a flap lift up to about two years postop but avoid it after that since epithelial ingrowth is more common with flap lifting after the first few years postop,” he says. “After two years post LASIK, typically I’ll go and do PRK on a retreat. Sometimes, we’ll see patients that are 10 or 15 years postop and they still have a little residual refractive error and they come back for just a small tune up, in which case I’ll do PRK on those eyes as well.”

 

PRK

For his PRK retreats, Dr. MacRae has a process, beginning with an 8.5-mm corneal trephine to demarcate the epithelium he wants to remove centrally vs. what he wants to preserve peripherally. “It very accurately demarcates the edge of the epithelium that you’re taking off so you don’t have scrolls and other irregularities,” he says. He uses an 8.5-mm well filled with 20% alcohol over the area and lets it sit on the cornea for 40 seconds. “Before removing the well, I use a Weck-Cel and allow it to swell. Then I gently twirl the Weck-Cel,” he continues. “The twirling motion causes those lateral adhesions of the epithelium to continue to adhere but the vertical epithelium anchor actually breaks or dehisces, in which case you’ll have a swirl of epithelium that comes off very easily. Then, I just take a Maloney spatula and gently remove the epithelium until I get to the edge of the area.”

One of the problems with treating or retreating with PRK is that patients can get recurrent erosions from loose epithelium, says Dr. MacRae. “PRK (or LASIK) can create mild erosion symptoms from the epithelium not vertically anchoring well. On all PRK and PTK retreats that I do, I use mitomycin-C prior to inserting the TSCL. Even if there’s a 1 percent haze rate we’d rather not have to have to deal with that at all.”

If you do notice scrolling of the epithelium, or it’s breaking down and not doing well, Dr. MacRae uses a Johnston applanator to smooth and stretch the flap. “If you notice the epithelium starting to loosen, use the Johnston applanator to applanate and stretch the flap, rather than trying to stretch the flap with Weck-Cels,” he says. “This instrument is a great option with friable epithelium and eyes with unrecognized anterior basement membrane disease to avoid damaging the epithelium further.

“Following treatment or retreatment with the excimer laser, I insert a bandage soft contact lens for seven to 10 days—sometimes, we might even leave it on longer,” he continues. “We used to worry about infection with a therapeutic soft contact lens but that’s an extremely rare event, as long as the patients are treated with antibiotics and they’re well instructed in terms of good hand-washing, rinsing the eye out in the morning with artificial tears and avoiding dust and dirt for that first week. After the bandage lens is removed, we put them on Muro 128 or a generic equivalent hypertonic ointment at bedtime for two months. This prevents recurrent erosion.”

 

Re-lifting the Flap

However, PRK doesn’t always make sense, even for late enhancements, notes Richard J. Mackool, MD, the founder and director of The Mackool Eye Institute and Laser Center in Queens, New York, as well as a professor at the New York Eye and Ear Infirmary and NYU Medical Center. “There are reasons the patient didn’t opt for PRK in the first place, including slow recovery, risk of haze and often discomfort,” he says. “The patient probably doesn’t want to go that route for retreatment.”

Another example of Richard Mackool, MD, finding the LASIK flap with the “Dry and Depress” technique. Dr. Mackool reports success using this technique on flaps created as far back as 29 years prior.

Dr. Mackool developed his own flap elevation technique that has since proven successful even for a LASIK flap created 29 years earlier. Dubbed the “Dry and Depress” technique, it requires nothing more than the standard tools a refractive surgeon would already have on hand, and consists of the following steps:

• With the surgeon seated superiorly at the excimer laser microscope, the patient looks upward, bringing the inferior cornea into view. At the 8 to 10 mm optical zone, the inferior cornea is gently dried with a sponge to improve the surgeon’s ability to detect areas of epithelial irregularity, depression or elevation.

• With a dry sponge, gently depress the adjacent central region of the cornea to flatten the mid-peripheral cornea and cause the corneal light reflex to shift peripherally. Dr. Mackool says this is the ‘a-ha’ moment when you then see the junction of the LASIK flap (Figure 1).

• A blunt spatula is run back and forth along the groove, penetrating the epithelium and Bowman’s membrane, and passed beneath the flap to continue the standard flap elevation. (A video of the procedure is available below.)

“We’ve used this technique in 50 of 51 eyes (all had their original LASIK flap created by a microkeratome) that were between six months and 29 years post-LASIK. In one eye we could not penetrate the incision, so we performed PRK,” Dr. Mackool says. “Most notably, none of these patients developed epithelial ingrowth. We believe it’s because of the minimal disruption of the epithelium, similar to primary LASIK.” 

Dr. Mackool advises surgeons to take their time looking for the flap. “If you don’t immediately find it down at 6 o’clock, look a little toward 7 or 8 o’clock, or 4 or 5 o’clock,” he says. “Just do a careful examination. We find it 100 percent of the time.”

Dr. Piracha’s technique for finding the flap varies. “You can mark the edge of the flap at the slit lamp—not the laser—with a 25- or 27-gauge needle,” he says. “That way, when you lift the flap, you’re not creating a corneal abrasion. Another way is with the current lasers with a slit lamp attached, and the third way is to actually press on the cornea with a Merocel sponge and the margin of the flap shows up. At that point, I take a very fine Sinskey hook and cleanly dissect it and initiate the flap. Then I’ll take a cannula or LASIK spatula and go underneath that space and open the flap. I’m very particular about initiating that because if you’re rough identifying the margin and entering the central space from the flap, you can get epithelial ingrowth and the ingrowth can be recurrent and persistent. If it keeps returning you may have to suture the flap, which I’ve done on patients with recurrent epithelial ingrowth.”

Dr. MacRae’s flap-lifting technique was adapted from Steve Wilson, MD, of the Cleveland Clinic. “He described this technique at one of the meetings, and showed a video where he took a platypus type forceps, made a small notch at the flap edge, and then just lifted the entire flap open,” he says. “I was somewhat shocked at how quickly he lifted it. It created a really clean separation of the epithelium at the edge of the flap with very little epithelial scrolls or epithelial defects around the edge of the flap. Prior to this, I used to just push the instrument horizontally to separate the flap edge, which caused more epithelial scrolls.”

It inspired Dr. MacRae to develop an instrument called the MacRae Flap Flipper (Storz). “I score the flap edge at the slit lamp with a Sinskey hook or the Flap Flipper,” says Dr. MacRae. “The leading edge of the Flap Flipper looks like a dandelion digger that allows me to gently lift and separate the flap while moving forward and lifting around the flap edge. Dr. Wilson was absolutely correct that lifting actually separates those horizontal or lateral adhesions of the epithelium, clearly avoiding epithelial tears.”

As all of these surgeons attest, nurturing the epithelium is critical. “Epithelium tends to adhere more laterally than it does vertically where it’s anchored, so the real challenge is to get those side adhesions separated,” says Dr. MacRae. “Dr. Wilson’s technique confirms that concept that the edges are cleaner if you tug vertically as you’re lifting rather than driving horizontally. That’s actually a key to performing retreats without having epithelial ingrowth. Sparing the epithelium and epithelial defects will dramatically reduce the rates of epithelial ingrowth in my experience. Using that technique, it’s extremely rare for me to have an epithelial ingrowth. If you do see a little tag of epithelium slip into the interface after you’ve lifted, then just gently tease it back away from the interface or from the edge of the flap and tease it peripherally away from the flap margin.”

Finally, Dr. Piracha advises the close consideration for lens exchange as an alternative to PRK and lifting the flap. “You have to look at the epithelial thickness and take an OCT and explain to the patient that if we do a surface treatment, it’s going to take a long time for vision to come back, and it might be worse for the first six months,” he says. “Then you have the argument to consider a lens replacement as opposed to surface treatment. That’s an option, especially with post-LASIK IOL calculations (Barrett True-K formula) and OCT imaging, and they have several options—monofocal lenses, multifocal lenses, EDOF lenses, the IC-8. It’s appealing because you don’t have the risk of an epithelial defect and it doesn’t take long to heal.” 

Dr. Mackool has no relevant disclosures. Dr. MacRae is the creator of the MacRae Flap Flipper. Dr. Piracha is a consultant to Johnson & Johnson Vision and Zeiss.

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