In a recent study published in Nature Biotechnology, researchers bioengineered corneal tissue for minimally invasive vision restoration.
Study: bioengineered corneal tissue for minimally invasive vision restoration in advanced keratoconus in two clinical cohorts. Image credit: Garna Zarina/Shutterstock
Poor refractive function and loss of corneal transparency are the leading causes of blindness worldwide. Although it can be treated by corneal transplantation, an estimated 12.7 million people are waiting for donors, with one cornea available for every 70 needed. Most people do not have access to corneal transplantation due to 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 necessary to prevent blindness by performing deep anterior lamellar keratoplasty (DALK) or penetrating keratoplasty (PK).
However, these techniques are subject to the risk of graft rejection, limited donors, postoperative complications, risk of corneal neovascularization and infection, and the need for immunosuppression and long-term follow-up. Although several less invasive techniques have been introduced to (partially) address these concerns, they are still in development and depend on the availability of donors and tissue banking infrastructure.
The study and conclusions
In the present study, the researchers designed 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, the collagen was subjected to chemical and photochemical cross-linking to generate a transparent hydrogel called bioengineered porcine construct, double cross-linked (BPCDX).
BPCDX was manufactured from porcine type I collagen under good manufacturing practice (GMP) conditions and protocols. No biological material or viable cells were present in BPCDX. The crosslinks were rinsed off during manufacture to create a natural, transparent hydrogel. BPCDX had visible light transmission comparable to the human cornea, with improved mechanical properties over previously bioengineered constructs.
Collagenase tested for degradation of BPCDX, human cornea, and single cross-linked BPC. 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 surface of BPCDX. Sixteen days later, live adherent cells with normal morphology were detected on the BPCDX surface at a higher density than controls, indicating biocompatibility.
An independent Good Laboratory Practice (GLP) certified laboratory tested the biological 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 enzyme resistance, transparency, water content and mechanical properties comparable to unaged controls, indicating a minimum of two years of useful life stability.
The authors observed no 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 femtosecond laser-enabled intrastromal keratoplasty (FLISK) to remove native stromal tissue (250 μm thick and 7 mm diameter) in one eye, replicating a thin corneal stroma as in the keratoconus
Subsequently, the removed native tissue was replaced in five minipigs (autograft controls) and BPCDX (280 μm thick and 7 mm wide) was inserted into the remaining minipigs. Six months later, the central cornea of the eye was clear in four autograft controls and in 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 reduced transparency in the sutured access cut region in controls and BPCDX recipients. Because of the partial thinning and fogging due to access cut suture in minipigs, the team used a sutureless FLISK implementation with smaller access cuts in human subjects in a pilot study to minimize complications
In humans with advanced keratoconus without scarring, 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 BPCDX implantation study. 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 BPCDX placement. A medication was followed for eight weeks after the operation. No intraoperative complications were observed. Dislocation/extrusion of BPCDX and thinning/scarring in the access cut region was not observed.
Two years postoperatively, corneal transparency was at the highest level (4+) in all subjects, with no vascularization, inflammation, rejection, or other adverse events. In the Indian cohort, the team found transient haze in five subjects during the first postoperative week, decreasing the degree of transparency to 3+. The transparency increased to 4+ after the first postoperative week follow-up and was stable.
OCT images revealed similar light scattering in native and BPCDX corneas. Intraocular pressure, measured in Indian subjects, increased slightly, without the need for any medication. Central corneal thickness increased by several hundred microns in all subjects and was maintained after two years. All subjects who were contact lens intolerant preoperatively tolerated contact lenses for an extended period after 24 months.
Eleven subjects in the Iranian cohort and all Indian subjects had substantial gains in visual acuity. 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 before the operation, none became blind in the operated eye after the operation.
Conclusions
In summary, the researchers demonstrated that intrastromal implantation of cell-free BPCDX was safe and feasible to reverse pathological corneal thickening/deformation in advanced stages of keratoconus. The visual gains seen in the study were equivalent to the historical results of standard penetrating corneal transplant surgeries.
The results suggested that the final acuity after BPCDX implantation could exceed the results of PK or DALK; however, further clinical studies are required to test this claim. Overall, the safety and efficacy results and the potential for benefit relative to the risk of adverse effects are promising and encourage the need for further randomized controlled studies.