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 lack 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 each 70 needed. Most individuals do not need access to corneal transplantation because of the shortage of infrastructure. As such, research efforts have focused on bioengineering corneal tissue for transplantation.

Keratoconus, a disease characterised by stromal thinning, stays the leading indication for corneal transplantation in lots of regions, including Australia and Europe. Keratoconus is progressive, and its complex etiology is poorly understood. In advanced stages of keratoconus, transplantation is required to forestall blindness by performing deep anterior lamellar keratoplasty (DALK) or penetrating keratoplasty (PK).

Nonetheless, these techniques are subject to graft rejection risk, limited donors, postoperative complications, risk of neovascularization and infection of the cornea, and the necessity for long-term immunosuppression and follow-up. Although several less-invasive techniques have been introduced to (partially) address these concerns, they’re still under development and depend on the provision of donors and tissue banking infrastructure.

The study and findings

In the current study, researchers bioengineered a cell-free implant as an alternative choice to 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 liable 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 the next 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 twenty-four 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 didn’t 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 seven mm in diameter) in a single eye, replicating a skinny corneal stroma as in keratoconus.

Subsequently, the removed native tissue was replaced in five minipigs (auto-graft controls), and BPCDX (280 μm thick and seven mm wide) was inserted within the remaining minipigs. Six months later, the central cornea in the attention was transparent in 4 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 within the access cut region with sutures in controls and BPCDX recipients. Due to partial thinning and haze because of 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 attenuate 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 right 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 position of BPCDX. An eight-week medication was followed postoperatively. No intraoperative complications were noted. Dislocation/extrusion of BPCDX and thinning/scarring within the access cut region weren’t observed.

Two years postoperatively, corneal transparency was at the best level (4+) in all subjects, without vascularization, inflammation, rejection, or other adversarial events. Within the Indian cohort, the team found a transient haze in five subjects in the course of the first postoperative week, decreasing the transparency grade to three+. Transparency increased to 4+ after the primary postoperative week follow-up and was stable.

OCT imaging revealed similar light scattering within the native cornea and BPCDX. Intraocular pressure, measured in Indian subjects, increased barely, 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 prolonged period after 24 months.

Eleven subjects of the Iranian cohort and all Indian subjects had substantial gains in visual acuity. The ultimate corrected acuity was 20/58 for subjects within the Iranian cohort and a remarkable 20/26 for those within the Indian cohort. Of the 14 subjects who were legally blind preoperatively, none were blind within the operated eye postoperatively.

Conclusions

In summary, researchers demonstrated that intrastromal implantation of cell-free BPCDX was secure and feasible to reverse the pathologic corneal thickening/deformation in advanced stages of keratoconus. The visual gains observed within the study were akin to historical results of ordinary penetrating corneal transplantation surgeries.

The outcomes suggested that the ultimate acuity after BPCDX implantation could exceed PK or DALK outcomes; nevertheless, more clinical studies are required to check this assertion. Overall, the protection and efficacy outcomes and the potential for advantages relative to the chance of adversarial effects are promising and encourage the necessity for further randomized controlled studies.