In a recent study published in Nature Biotechnology, researchers bioengineered corneal tissue for minimally invasive vision restoration.
Poor refractive function and loss of corneal transparency are the leading causes of blindness worldwide. Although treatable by corneal transplantation, an estimated 12.7 million people await donors, with one cornea available for every 70 needed. Most people do not have access to corneal transplantation due to the lack of infrastructure. As such, research efforts have focused on bioengineering corneal tissue for transplantation.
Keratoconus, a disease characterized by stromal thinning, remains the leading indication for corneal transplantation in many regions, including Australia and Europe. Keratoconus is progressive, and its complex etiology is poorly understood. In advanced stages of keratoconus, transplantation is required to prevent blindness by performing deep anterior lamellar keratoplasty (DALK) or penetrating keratoplasty (PK).
However, these techniques are subject to graft rejection risk, limited donors, postoperative complications, risk of neovascularization and infection of the cornea, and the need for long-term immunosuppression and follow-up. Although several less-invasive techniques have been introduced to (partially) address these concerns, they are still under development and rely on the availability of donors and tissue banking infrastructure.
The study and findings
In the present study, researchers bioengineered a cell-free implant as a substitute for human corneal stroma using medical-grade collagen from porcine skin and described a minimally invasive surgery for its implantation. Because pure collagen is soft and prone to degradation, collagen was subject to chemical and photochemical crosslinking to generate a transparent hydrogel termed the bioengineered porcine construct, double-crosslinked (BPCDX).
BPCDX was manufactured from type I porcine collagen under good manufacturing practices (GMP)-compliant conditions and protocols. No viable biologic materials or cells were present in BPCDX. Cross-linkers were rinsed out during manufacturing to create a natural, transparent hydrogel. BPCDX had visible light transmission comparable to the human cornea, with enhanced mechanical properties to previously bioengineered constructs.
Degradation of BPCDX, human cornea, and single-crosslinked BPC was tested by collagenase. Fifty percent degradation required 18 hours for BPC, 24 hours for BPCDX, and 45 hours for human tissue. The biocompatibility of BPCDX was evaluated by seeding human corneal epithelial cells on the BPCDX surface. Sixteen days later, live adherent cells with normal morphology were detected on the BPCDX surface at a higher density than controls, indicative of biocompatibility.
An independent good-laboratory practice (GLP)-certified lab tested the biologic safety of BPCDX. BPCDX was non-irritant, non-toxic, non-cytotoxic, non-pyrogenic, non-genotoxic, non-sensitizing, and well-tolerated. Real-time shelf-life stability was tested by storing BPCDX for 24 months at 7 °C, and accelerated shelf-life stability was assessed by incubating BPCDX at 28 °C for six months.
Real-time stability examination revealed that, after 24 months, BPCDX maintained enzymatic resistance, transparency, water content, and mechanical properties comparable to non-aged controls, indicating a minimum of two years of shelf-life stability.
The authors did not observe any postoperative infection, wound abscess, or suture-related complications in Wistar rats after subcutaneous implantation of BPCDX under the dorsal flank. Next, 10 Gottingen minipigs underwent a femtosecond laser-enabled intrastromal keratoplasty (FLISK) to remove native stromal tissue (250 μm thick and 7 mm in diameter) in one eye, replicating a thin corneal stroma as in keratoconus.
Subsequently, the removed native tissue was replaced in five minipigs (auto-graft controls), and BPCDX (280 μm thick and 7 mm wide) was inserted in the remaining minipigs. Six months later, the central cornea in the eye was transparent in four autograft controls and all BPCDX recipients. Central corneal thickness was 657 μm preoperatively and 650 μm postoperatively with BPCDX.
Microscopy and optical coherence tomography indicated partial thinning and lower transparency in the access cut region with sutures in controls and BPCDX recipients. Because of partial thinning and haze due to suturing the access cut in minipigs, the team used a suture-free FLISK implementation with smaller access cuts in human subjects in a pilot study to minimize complications.
In humans with advanced keratoconus without scarring, the native corneal tissue was not removed. Only BPCDX was introduced, which simplified the surgery to a single lamellar cut and access cut. Ethical approvals were obtained in India and Iran to conduct the pilot study of BPCDX implantation. BPCDX was implanted into a laser-dissected intrastromal pocket in 20 subjects without removing native tissue.
Slit-lamp biomicroscopy, OCT pachymetry, and Fourier-domain OCT (FD-OCT) confirmed the placement of BPCDX. An eight-week medication was followed postoperatively. No intraoperative complications were noted. Dislocation/extrusion of BPCDX and thinning/scarring in the access cut region were not observed.
Two years postoperatively, corneal transparency was at the highest level (4+) in all subjects, without vascularization, inflammation, rejection, or other adverse events. In the Indian cohort, the team found a transient haze in five subjects during the first postoperative week, decreasing the transparency grade to 3+. Transparency increased to 4+ after the first postoperative week follow-up and was stable.
OCT imaging revealed similar light scattering in the native cornea and BPCDX. Intraocular pressure, measured in Indian subjects, increased slightly, not requiring any medication. The central corneal thickness increased by several hundred microns in all subjects and was sustained after two years. All subjects who were contact lens-intolerant preoperatively tolerated contact lenses for an extended period after 24 months.
Eleven subjects of the Iranian cohort and all Indian subjects had substantial gains in visual acuity. The final corrected acuity was 20/58 for subjects in the Iranian cohort and a remarkable 20/26 for those in the Indian cohort. Of the 14 subjects who were legally blind preoperatively, none were blind in the operated eye postoperatively.
In summary, researchers demonstrated that intrastromal implantation of cell-free BPCDX was safe and feasible to reverse the pathologic corneal thickening/deformation in advanced stages of keratoconus. The visual gains observed in the study were equivalent to historical results of standard penetrating corneal transplantation surgeries.
The results suggested that the final acuity after BPCDX implantation could exceed PK or DALK outcomes; however, more clinical studies are required to test this assertion. Overall, the safety and efficacy outcomes and the potential for benefits relative to the risk of adverse effects are promising and encourage the need for further randomized controlled studies.