Bioengineered corneal tissue for minimally invasive vision restoration in advanced keratoconus in two clinical cohorts

Abstract

Visual impairment from corneal stromal disease affects millions worldwide. We describe a cell-free engineered corneal tissue, bioengineered porcine construct, double crosslinked (BPCDX) and a minimally invasive surgical method for its implantation. In a pilot feasibility study in India and Iran (clinicaltrials.gov no. NCT04653922), we implanted BPCDX in 20 advanced keratoconus subjects to reshape the native corneal stroma without removing existing tissue or using sutures. During 24 months of follow-up, no adverse event was observed. We document improvements in corneal thickness (mean increase of 209 ± 18 µm in India, 285 ± 99 µm in Iran), maximum keratometry (mean decrease of 13.9 ± 7.9 D in India and 11.2 ± 8.9 D in Iran) and visual acuity (to a mean contact-lens-corrected acuity of 20/26 in India and spectacle-corrected acuity of 20/58 in Iran). Fourteen of 14 initially blind subjects had a final mean best-corrected vision (spectacle or contact lens) of 20/36 and restored tolerance to contact lens wear. This work demonstrates restoration of vision using an approach that is potentially equally effective, safer, simpler and more broadly available than donor cornea transplantation.

Main

Loss of corneal transparency and poor refractive function are among the leading causes of blindness globally^1,2,3,4. Although corneal blindness can be treatable by transplantation, an estimated 12.7 million people await a donor cornea, with one cornea available for every 70 needed³. With an incidence of over 1 million new cases of corneal blindness annually⁵, the severe shortage of donor corneas presents an unequal burden of blindness heavily skewed towards low- and middle-income countries (LMICs) in Asia, Africa and the Middle East^2,3. Over half of the world’s population does not have access to corneal transplantation owing to a lack of infrastructure for tissue donation, harvesting, testing and eye banking in LMICs^1,3. The access problem is complex, involving economic, cultural, technological, political and ethical barriers^2,4. Additionally, infectious diseases and pandemics bring donor tissue procurement and use to a virtual standstill, necessitating further measures to ensure donor tissue safety^6,7.

For these reasons, intense research effort has focused on bioengineering tissue for corneal transplantation^8,9,10. To date, however, no biotechnological advance has been able to address the burden of corneal blindness or improve access to transplantable corneal tissue. In many parts of the world including Europe and Australia, keratoconus—a corneal disease characterized by stromal thinning, weakening and scarring¹¹—is the leading indication for corneal transplantation^2,12. Keratoconus affects both men and women and all ethnic groups, with highest prevalence reported in China (0.9%, or 12.5 million)¹³, India (2.3%, or 30 million)¹⁴ and Iran (4% of the rural population, or 3.4 million)¹⁵.

Keratoconus is progressive, but with a complex etiology that is not well understood. With proper screening and access to specialist care, keratoconus progression can be detected and halted in its early stages while vision is still good; however, if not addressed early and in LMICs where keratoconus is highly prevalent and access to healthcare is limited, the disease often progresses. In advanced stages, it requires transplantation to prevent blindness, using techniques such as penetrating keratoplasty (PK) or deep anterior lamellar keratoplasty (DALK)^16,17,18,19. These techniques, however, are subject to the limited supply of donor corneas, risk of graft rejection, post-operative complications associated with sutures and wound healing, risk of corneal neovascularization and/or infection, high astigmatism after suture removal, need for long-term immunosuppression and necessity for long-term patient follow-up²⁰. To partially address these issues, newer and less invasive techniques such as stromal lenticule addition keratoplasty²¹ and Bowman layer transplantation²² have been introduced. While promising and still developing, these techniques stabilize the condition but offer only marginal vision improvement^21,23, and rely on availability of donor corneas and tissue banking infrastructure and are thus inapplicable in many regions of the world.

To address these limitations, we bioengineered a cell-free implantable medical device as a substitute for human corneal stromal tissue. As a raw material we used natural type I collagen, the main protein in the human cornea²⁴. For an abundant yet sustainable and cost-effective supply of collagen, we used medical-grade collagen sourced from porcine skin, a purified byproduct from the food industry already used in FDA-approved medical devices for glaucoma surgery²⁵ and as a wound dressing²⁶. In a previous clinical study^27,28, we evaluated implants engineered from recombinant human collagen that had several limitations: the collagen could be produced only in small quantities, implants were mechanically weak and required invasive suturing, implants were not evaluated for long-term stability, and surgery was invasive and led to a strong wound-healing response and partial implant melting. Here we addressed these limitations by using type I medical-grade porcine dermal collagen, developing a new method of double crosslinking to improve implant strength and stability, and using a new minimally invasive surgical implantation technique to promote corneal thickening, reshaping and rapid wound healing.

Pure collagen is a soft material prone to degradation, so we applied dual chemical and photochemical crosslinking to form a transparent implantable hydrogel, termed the bioengineered porcine construct, double crosslinked (BPCDX). BPCDX is an improvement on our earlier porcine collagen-based materials^29,30,31 that has additionally been photochemically crosslinked with the UVA–riboflavin crosslinking procedure³². BPCDX, fabricated in a good manufacturing practices (GMP)-certified clean room according to stringent quality processes, was tested to evaluate optical and mechanical properties, enzymatic degradation and cell compatibility and underwent a panel of third-party certified medical device tests compliant with ISO standards to assess biocompatibility, toxicity, carcinogenicity, sensitization and irritation using in vitro and in vivo assays in mice, guinea pigs and rabbits.

A further challenge is to provide devices to different regions and potentially to rural areas without biobanking or storage and tissue preparation facilities. We addressed this by developing compatible packaging and sterilization processes and testing packaged devices in accelerated and real-time ISO shelf-life stability studies, to compare optical, mechanical, chemical and sterility properties of the fully packaged BPCDX as-made and after storage for up to two years.

Conventional transplantation techniques for advanced keratoconus remove and/or damage corneal epithelium, endothelium and nerves. On the basis of earlier studies in rabbits^29,31, we developed a minimally invasive surgery for advanced keratoconus, inserting a thick, large-diameter BPCDX into an intrastromal pocket within the recipient cornea to counteract pathologic stromal thinning and normalize refraction by reshaping the central and peripheral cornea, without removing recipient tissue. The intrastromal surgery is suture-free and leaves corneal nerves and cellular layers intact, promoting rapid wound healing²⁹. We specifically adapted previous intrastromal methods^21,33,34 to use a single corneal incision half the size of previous techniques^21,34 without disrupting the sclera or anterior chamber²², to significantly thicken and reshape the central cornea by inserting a 280–440-µm-thick BPCDX to achieve substantial flattening (>10 diopters (D)) of the steepest corneal curvature in keratoconus.

We first evaluated BPCDX implantation by this intrastromal method in a minipig model of advanced keratoconus, using surgical tools and protocols that different surgeons could replicate. To obtain human safety and feasibility data to justify a controlled clinical trial, we undertook a pilot feasibility study in India and Iran. Here we report safety and efficacy results in the first 20 advanced keratoconus subjects receiving the BPCDX. No intra- or post-operative complications or adverse events were noted in any subject during 24 months of clinical follow-up. Significant and stable corneal thickening and flattening of keratometry, maintenance of corneal transparency and improvement of best-corrected visual acuity (BCVA) by a mean of 7.6 logMAR lines to a mean of 20/58 in Iran and by a mean of 15.1 logMAR lines to a mean of 20/26 in India was achieved. These represent equivalent outcomes to standard corneal transplantation but with a simpler surgical technique and without the need for human donor tissue or tissue banking infrastructure.

Results

Manufacturing of collagen scaffolds

BPCDX is a corneal implant manufactured from purified medical-grade type I porcine collagen produced under GMP-compliant processes and conditions. No cells or viable biological material are present within BPCDX, and it is a Class III medical device designed to mimic properties of the natural cornea. The collagen in BPCDX is double crosslinked, both chemically and photochemically, imparting strength and resistance to degradation. The crosslinkers do not become integrated within the final device as they are water-soluble and rinsed out of the implant during manufacturing, resulting in an entirely natural, transparent hydrogel (Fig. 1a)…