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.

Conflict of interest statement

M.R. holds stock and relevant patents in LinkoCare Life Sciences AB, a spin-off company from Linköping University developing BPCDX and related products. He also serves on the board of directors of the company. The terms of his arrangements have been approved by Linköping University in accordance with its policy on objectivity in research. S.T. is business development manager of LinkoCare and serves on the board of directors of LinkoCare Life Sciences AB, as an unpaid board member. N.L., M.J. and N.S. serve on the scientific advisory board of LinkoCare Life Sciences AB, as unpaid advisory board members. Other co-authors have no competing interests.

© 2022. The Author(s).

Figures

Fig. 1. Biomaterial properties of BPCDX.

a, Appearance of BPCDX, indicating transparency and refractive nature of the curved device. b, Light transmission through 550-µm-thick samples of BPCDX, single-crosslinked BPC and the human cornea. The human cornea contains a layer of epithelial cells which absorb UV light, whereas the bioengineered materials are cell-free. Data shown represent mean and standard deviation of measurements from three independent samples. c, Mechanical properties of BPCDX relative to single-crosslinked BPC and previously published data of bioengineered constructs made from porcine collagen,, with human cornea reference values included for comparison. Data values for BPCDX represent mean and standard deviation of measurements from 22 independent samples per test (taken across different production batches, 550-µm-thick ‘dog-bone’ specimens). d, Scanning electron microscope images of the surface and bulk (cross-section) structure of BPCDX and a porcine cornea, indicating tightly packed collagen fibrils in BPCDX with diameter slightly thicker than the native porcine cornea (representative images from three samples per cornea type with similar results). e, Degradation of BPCDX, single-crosslinked BPC and a human donor cornea in 1 mg ml−1 collagenase (data represent mean and standard deviation of measurements from three independent samples for bioengineered materials (550 µm thick, 12 mm diameter) and two independent samples of human donor cornea). f, HCE-2 human corneal epithelial cell attachment and growth on BPCDX relative to the control culture plate surface after 16 days of culture. Cells adhered to BPCDX, with NucBlue staining indicating nuclei and morphology of live, viable cells in brightfield mode. BPCDX had greater cell density than cell culture plasticware (three control samples, six BPCDX samples; error bars represent mean and standard deviation, P = 0.003, two-sided independent t-test). Scale bars, 100 µm. Source Data

Fig. 2. Results 6 months after intrastromal BPCDX implantation in minipigs.

a, OCT images of the central 6 mm of cornea and corresponding photographs of operated eyes indicated localized thinning and loss of transparency in the access cut region in both groups (arrows in OCT scans and photographs). In the autograft group, one cornea (central image) exhibited loss of transparency, while another had partial loss of transparency (bottom image). In both cases, the implantation zone was skewed towards the limbus. The remaining three eyes were transparent with good thickness and only minor thinning at the access cut. In the BPCDX group, two eyes (second image from top, and bottom image) had a partially reduced transparency. In both cases, the implanted zone was skewed towards the limbus. In all eyes, transparency outside the access cut region was maintained. b, Pachymetry maps indicating corneal thickness 6 months post-operatively with color coding of thickness indicated by the grading scale, and mean thickness in µm indicated in each sector. BPCDX corneas exhibited similar thickness as the native porcine cornea. The native porcine cornea is shown for comparison purposes at the bottom of a,b. c, Table indicating pre-operative and post-operative mean corneal thickness and difference in corneal thickness in the central 2 mm zone as determined by OCT. Absence of positive fluorescein staining indicates complete epithelial wound healing. d, In vivo confocal microscopy images of porcine corneas at 6 months. In both groups, the epithelial cell mosaic appeared intact. Basal epithelium and sub-basal nerves (white arrows) were also observed, indicating preservation of the nerve plexus owing to minimal trauma during surgery. Anterior stromal nerves and keratocytes had normal morphology in both groups. The mid-stromal region appeared normal in autografts but BPCDX was devoid of keratocytes, except for individual cellular features (black arrows). Posterior stromal keratocytes and endothelial cells appeared normal and intact in both groups. All images in d are 400 × 400 µm2. Representative images are from five corneas per group with similar results obtained for each group. Source Data

Fig. 3. Postmortem histologic analysis of corneas in the minipig model.

a, Hematoxylin and eosin (H&E) staining revealed autografts with thickened epithelium and increased presence of anterior stromal cells relative to the native porcine cornea. Epithelial and stromal layers in BPCDX corneas were uniform, with maintenance of overall corneal structure and anatomy. Representative images shown from three corneas per group. b, Three different BPCDX-implanted corneas where host cells (arrows, left and center images) migrated into the BPCDX. The edge of the BPCDX had multiple tissue attachments (arrows, right image) with cells appearing to migrate towards the BPCDX. c, Immunohistochemical analysis indicated sub-basal nerves by the β-III-tubulin marker (immediately below the epithelium, arrows), while a preserved stromal nerve in a BPCDX cornea was apparent. Leukocyte marker CD45 indicated weak staining of stromal cells located at the BPCDX periphery (arrows), suggesting leukocyte-mediated remodeling that differed in extent in different corneas (sections from two different BPCDX corneas shown). Leukocytes were absent in native and allograft corneas. All markers are indicated by green fluorescence, while a blue DAPI counterstain indicates the presence of cell nuclei. Non-specific diffuse signal from the green channel indicated the implanted BPCDX (asterisk in all panels). All images are representative images chosen from three corneas per group. Scale bars, 100 µm (a,c) and 50 µm (b). Source Data

Fig. 4. Clinical data from subjects in Iran receiving BPCDX.

a, Keratometric and corneal thickness maps from the same subject indicate the thin and steep pre-operative cornea that was substantially thickened and flattened after intrastromal implantation of a 440-µm-thick BPCDX. The corresponding OCT cross-section scans indicate corneal thickness and shape before and after BPCDX implantation, with anterior and posterior borders of the BPCDX indicated by white arrows. The subject had an initial BSCVA of 20/200 that improved to 20/50 at 24 months post-operatively. b,c, Photographs of eyes from two subjects with the BPCDX four months post-operatively, indicating maintenance of corneal transparency. d,e, In vivo confocal microscopy images obtained from a single subject confirming the presence of sub-basal nerves (d) and endothelial cell mosaic (c) 6 months post-operatively. Endothelial cell density was 2,222 ± 62 cells per mm2 in the eye. As only this single subject was imaged by in vivo confocal microscopy, it is unknown if these images are representative. Images in d,e are 400 × 400 µm2.

Fig. 5. Clinical data from a subject in India receiving BPCDX.

a, Slit-lamp photographs pre-operatively (left) and one day post-operative (right) with arrows indicating immediate change in thickness and curvature in the central cornea. b, OCT scans indicating sustained thickening and regularization of corneal curvature following implantation of 280-µm-thick BPCDX (anterior and posterior surfaces of BPCDX indicated by white arrows). c, Topographic maps (left, values given are keratometric power in diopters), anterior surface elevation maps (center, values are in µm displacement from a best-fit sphere) and OCT pachymetric maps (right, thickness in µm) from the same subject indicated substantial flattening of the steepest pre-operative central region (black arrow), and substantial increase in corneal thickness post-operatively. The subject initially had a best contact lens-corrected visual acuity (BCLVA) of 20/600. At 24 months BCLVA improved to 20/30.

Extended Data Fig. 1. Real-time shelf-life stability test data for BPCDX after 24 months of storage at 7°C, relative to time zero samples.

(a) Light transmission was maintained throughout the visible range of wavelengths (10 samples per group, BPCDX thickness 300 µm, diameter 9 mm, two-sided independent t-test). (b) Enzymatic degradation was unchanged, indicating implant integrity after long-term storage (9 samples per time point from 3 separate batches, BPCDX thickness 300 µm, diameter 9 mm, two-sided independent t-test). (c) Table indicating preservation of hydration and mechanical properties after long-term storage, with no significant differences relative to non-aged samples (550µm-thick ‘dog-bone’ specimens, two-sided independent t-test for each parameter). Data values in the graphs and table represent mean and standard deviation of measurements (error bars) from the indicated number of independent samples (at time 0 and 24 months, respectively). Source data