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HEALTH AND MEDICINE
LiQD Cornea: Pro-regeneration collagen mimetics
as patches and alternatives to corneal transplantation
Christopher D. McTiernan1,2,3*, Fiona C. Simpson1,2*, Michel Haagdorens4,
Chameen Samarawickrama5,6, Damien Hunter5, Oleksiy Buznyk6, Per Fagerholm6,
Monika K. Ljunggren6, Philip Lewis7, Isabel Pintelon8, David Olsen9, Elle Edin1,2, Marc Groleau1,
Bruce D. Allan10†, May Griffith1,2†
Transplantation with donor corneas is the mainstay for treating corneal blindness, but a severe worldwide shortage
necessitates the development of other treatment options. Corneal perforation from infection or inflammation is
sealed with cyanoacrylate glue. However, the resulting cytotoxicity requires transplantation. LiQD Cornea is an
alternative to conventional corneal transplantation and sealants. It is a cell-free, liquid hydrogel matrix for corneal
regeneration, comprising short collagen-like peptides conjugated with polyethylene glycol and mixed with fibrinogen
to promote adhesion within tissue defects. Gelation occurs spontaneously at body temperature within 5 min. Light
exposure is not required—particularly advantageous because patients with corneal inflammation are typically
photophobic. The self-assembling, fully defined, synthetic collagen analog is much less costly than human recom-
binant collagen and reduces the risk of immune rejection associated with xenogeneic materials. In situ gelation
potentially allows for clinical application in outpatient clinics instead of operating theaters, maximizing practicality,
and minimizing health care costs.
INTRODUCTION
The cornea is the transparent front surface of the eye that provides
about two-thirds of the focusing power of the eye. Any permanent
transparency loss from injury or disease can result in blindness.
Currently, 23 million people globally have unilateral corneal blind-
ness, while 4.9 million are bilaterally blind (1). Transplantation with
human donor corneas has been the mainstay for treating corneal
blindness for a century. However, a global donor cornea shortage
leaves 12.7 million on waiting lists, with only 1in 70 patients treated (2).
Conditions requiring corneal transplantation include persistent
ulceration leading to scarring or perforation after corneal infection,
burns, autoimmune diseases, and physical trauma. Corneal perfo-
rations are an emergency, and in many centers, the cornea is tem-
porarily sealed using cyanoacrylate glue to maintain integrity and
avoid losing the eye (3). However, cyanoacrylate glue is toxic and can
cause local irritation and inflammation. Its incomplete polymeriza-
tion leaves behind toxic cyanoacrylate monomers, while its hydro-
lysis releases potentially toxic compounds like formaldehyde and alkyl
cyanoacrylate (4). These induce corneal scarring and vascularization.
Patients generally require follow-up corneal transplantation. Despite
these clear limitations, the use of cyanoacrylate glue to seal corneal
perforations has remained the established emergency treatment for
more than 50 years (5). Other interventions include corneal suturing
(6), tectonic corneal grafts (7), conjunctival flaps (8), multilayered
amniotic membrane transplantation (9), soft “bandage” contact lenses
(10), and tissue sealants.
Sealants examined include a variety of natural adhesives like fibrin,
gelatin, chitosan, and alginate (11), as well as a number of synthetic
polyethylene glycol (PEG) derivatives (12). Most of these interventions,
however, work only in a limited range of cases or require invasive
surgery with possible limitations for future visual rehabilitation (13).
PEG-based sealants have shown promise in sealing perforating
microincisions, but to the best of our knowledge, there is no study
which has looked at their efficacy in sealing macroperforations.
Furthermore, PEG-based sealants typically require multicomponent
mixing and suffer from short application windows. For example,
ReSure (Ocular Therapeutix Inc.), which requires two-component
mixing of PEG and a trilysine acetate solution, allows only a 20-s
window for application upon initiation of polymerization (14).
A new bioadhesive, GelCORE, was recently reported as an alterna-
tive to cyanoacrylate glue for corneal tissue repair in partial-thickness
corneal defects and corneal perforations. The authors used white
light, with Eosin Y, triethanolamine (TEA), and N-vinylcaprolactam
(VC) as initiators to gel a mixture of methacryloyl functionalized
gelatin in situ (11). The GelCORE report included a 14-day rabbit
study in which a 50% thickness wound was repaired. However, be-
cause of the short duration of the study, long-term effects could not
be evaluated. The use of animal-derived gelatin has an associated
risk of zoonotic disease transfer, and severe allergic reactions to both
bovine and porcine gelatin in vaccines have been reported (15).
Photocrosslinking may also be problematic in the clinical setting.
Patients with corneal inflammation are photophobic (light-sensitive)
and may not be able to tolerate intense visible light application more
than 4min without retrobulbar or general anesthesia. In a mecha-
nism analogous to corneal cross-linking for keratoconus, the creation
of free radicals in photocrosslinking may also be toxic to the corneal
endothelium in thinned or perforated corneas (16). Hyaluronic
1Centre de Recherche Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada.
2Department of Ophthalmology and Institute of Biomedical Engineering, Université
de Montréal, Montréal, QC, Canada. 3Division of Cardiac Surgery, University of
Ottawa Heart Institute, Ottawa, ON, Canada. 4Department of Ophthalmology, Visual
Optics and Visual Rehabilitation, University of Antwerp, Antwerp, Belgium. 5Centre
for Vision Research, The Westmead Institute for Medical Research, and Faculty of
Medicine and Health, University of Sydney, Sydney, Australia. 6Institute for Clinical
and Experimental Medicine, Linköping University, Linköping, Sweden. 7School of
Optometry and Vision Sciences, Cardiff University, Cardiff, UK. 8Laboratory of Cell
Biology and Histology, University of Antwerp, Antwerp, Belgium. 9FibroGen Inc.,
San Francisco, CA, USA. 10NIHR Biomedical Research Centre at Moorfields Eye Hospital
NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK.
*These authors contributed equally to this work.
†Corresponding author. Email: may.griffith@umontreal.ca (M. Griffith); bruce.allan@ucl.
ac.uk (B.D.A.)
Copyright © 2020
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rights reserved;
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American Association
for the Advancement
of Science. No claim to
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Works. Distributed
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NonCommercial
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acid–based materials have also been tested as alternative bioadhe-
sives in an invitro organ setting using excised porcine eyes (17). This
solution relied on hydrazone cross-linking of dopamine-modified
hyaluronic acid (HA-DOPA), where dopamine supplied the tissue
adhesive properties. While successful invitro, this material has not
been evaluated in animal models. Neither GelCORE nor HA-DOPA
was tested for repair of full-thickness corneal perforations, nor
have they been examined as alternatives to donor corneal tissue
for transplantation.
Over 10 years ago, our team members conducted a first-in-human
clinical trial on cell-free, biosynthetic hydrogels made from recom-
binant human collagen type III (RHCIII). These hydrogels promoted
stable corneal tissue and nerve regeneration, showing that they were
immune-compatible alternatives to donor cornea transplantation in
anterior lamellar keratoplasty (ALK) (18,19). Recently, we demon-
strated that hydrogel implants derived from a short collagen-like
peptide (CLP) conjugated to an inert, but mechanically robust, multi-
functional PEG are functionally equivalent to the RHCIII-based im-
plants when tested under preclinical conditions in mini-pigs (20).
The use of fully defined short synthetic peptides provides homoge-
neous materials that are easily modified and scaled up in comparison
to their full-length analogs. In addition to being fully synthetic, the
use of CLP-PEG collagen analogs circumvents the batch-to-batch
heterogeneity seen with extracted proteins, as well as potential allergic
reactions to xenogeneic proteins (21) and possible zoonotic disease
transmission (22). Despite being able to promote regeneration, these
solid implants require an operating theater for implantation, involving
costs for a full surgical team. Realistically, to reach the enormous
numbers of patients awaiting transplantation, most of them living in
low to middle income countries, we need a drastic paradigm change.
To date, vaccines have been vastly successful both in cost and
delivery, with every person receiving a vaccine delivered in a syringe.
By analogy, in dentistry, when someone has a cavity in a tooth, the
pathologic tissue is removed, and the tooth is filled. A similar para-
digm is likely needed to tackle this important global issue, where the
pathologic tissue is replaced by a regeneration-stimulating liquid
corneal replacement, LiQD Cornea, in a syringe that gels in situ.
Previously, we reported that CLP-PEG polymerizes in situ and can
form a seal in experimental invitro models of corneal perforation
when supported by an ab interno patch (23). In this study, we introduce
the LiQD Cornea, a new injectable hydrogel matrix with adhesive
properties. We examined the potential efficacy of our LiQD Cornea
comprising CLP-PEG-fibrinogen as a sealant/filler of full-thickness
corneal perforations and an alternative to lamellar corneal trans-
plantation that potentially allow treatments to be carried out in an
ophthalmologist’s office.
RESULTS
Physical and mechanical characterization
The CLP-PEG-fibrinogen LiQD Cornea formed a porous hydrogel
upon gelation in the presence of thrombin and a nontoxic cross-
linker, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMTMM) (fig. S1). LiQD Cornea hydrogel samples
showed a refractive index of 1.354±0.037, consistent with human
corneas and physical and chemical properties consistent with previous
generations of RHCIII and CLP-PEG hydrogels (Table1) (18,20).
In the visible spectrum (400 to 800 nm), LiQD Cornea samples
transmitted between 93 and 99% of incident light. The transmission
of light in the ultraviolet (UV) region decreased to a low value of
19% in the UV-C spectral region. Bursting pressure testing using
exvivo porcine corneas showed that the LiQD Cornea formulation,
although less robust than cyanoacrylate or fibrin sealant, nevertheless
withstood 170 mmHg of pressure. This was a 7.7-fold increase over
the average 11 to 21 mmHg of intraocular pressure within the
human eyeball (Table1).
In vitro characterization
Human corneal epithelial cells (HCECs) from an immortalized line
(24) adhered to and spread readily on invitro gelled matrices, indi-
cating that the LiQD Cornea supports epithelial growth (Fig.1A).
The materials were also found to be immune compatible. Precursors
of murine bone marrow–derived macrophage (BMDM) cells seeded
on LiQD Cornea hydrogels in the presence of macrophage differen-
tiation media showed higher levels of expression of CD206 (anti-
inflammatory M2 marker) in comparison to CD86 (pro-inflammatory
M1 marker) at the time points examined (Fig.1B). This showed a
polarization of the mononuclear macrophage precursors into anti-
inflammatory or tolerizing phenotypes. Exposure of bone marrow–
derived dendritic cells (BMDCs) to the LiQD Cornea hydrogel and
its components resulted in low expression of CD40, CD80, and
CD86, which are markers of activated, antigen-presenting dendritic
cells. This showed that overall, the LiQD Cornea formulation did
not activate dendritic cells, which are the main cells associated with
triggering graft rejection (25). By comparison, dendritic cells showed
significant activation marker expression when exposed to the positive
lipopolysaccharide (LPS) controls (Fig.1C).
In vivo rabbit perforation study
In accordance with the Association for Research in Vision and
Ophthalmology (ARVO) Statement for the Use of Animals in
Ophthalmic and Visual Research and with ethical permission from
the Western Sydney Local Health District Animal Ethics Committee
(Australia), conical perforations were made in one cornea each in
three New Zealand white rabbits. The perforations measured 3mm
in diameter on the external epithelial surface tapering to 1mm on
the internal endothelial surface. To seal the wound gape, we first
applied thrombin solution to the wound margins. Then, a mixture
of CLP-PEG-fibrinogen and DMTMM cross-linker was applied. As
the gel sets, the thrombin that was applied to the wound surface con-
verted the fibrinogen at the interface of the gel to fibrin, adhering
the gel firmly to the wound. DMTMM then cross-links the entire
mixture. The surgically created perforations were completely sealed
with the LiQD Cornea hydrogel, as indicated by the retention of an
air bubble placed within the anterior chamber (Fig.1E). Two rabbits
had a complete seal with the first application, while the third cornea
required additional material for an air-tight seal. All animals received
antibiotic (chloramphenicol) and anti-inflammatory (dexamethasone)
eye drops three times daily for 3 days. There was no incidence of
leakage or infection of the perforation sites in any animal, and by
7 days after surgery, all normal peri-surgical inflammation had
subsided. The hydrogels initially showed haze that began to recede
1 day after surgery. At 28 days follow-up, two of three rabbits had
transparent corneas (Fig.1E) and normal slit lamp exams. In the
third rabbit, the gel remained visible as a slight haze in the cornea.
Histopathology of the cornea showed epithelial hyperplasia and
reduced corneal stroma in the perforation site, indicating that the
perforation site of each cornea had undergone reepithelialization.
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There was also keratocyte infiltration, indicating the onset of corneal
stromal regeneration, and partial regeneration of Descemet’s mem-
brane. Furthermore, when LiQD Cornea was applied to the perfo-
rated corneas of the rabbits, the gel remained cohesive with itself
and generally did not leak into the anterior chamber. While there
was small degree of postoperative anterior chamber inflammation
on day 1, this subsided by day 3 and remained that way for the
duration of the study.
In vivo study in Göttingen mini-pigs
Genetically uniform Göttingen mini-pigs were used (26) in compli-
ance with the Swedish Animal Welfare Ordinance and the Animal
Welfare Act and with ethical permission from the local ethical
committee (Linköpings Djurförsöksetiska Nämnd). Anterior lamellar
keratoplasty wound beds, 6.5mm in diameter, 500 m deep (i.e.,
over 70% depth), were made in one cornea each of four mini-pigs
by trephination, followed by dissection with a blade. LiQD Cornea
was applied as for the rabbits. Figure S2A shows the progress of repair
and regeneration of all pigs receiving the LiQD Cornea compared to
syngeneic grafts and healthy unoperated controls. At 12 months, the
application of LiQD Cornea was successful in all pigs (Fig.1F),
although in all cases, the surgeon applied the LiQD Cornea at least
twice, removing the first material before reapplication to achieve the
desired curvature. One pig, which received four attempts at LiQD
Cornea application, underwent full corneal perforation and was given
a suture to bridge the unintended gape. Postsurgical optical coherence
tomography (OCT) of the LiQD Cornea application (fig. S2B) showed
that although the initial LiQD Cornea fills were imperfect, the anterior
corneal surfaces of all four pigs were smooth and followed the con-
tours of the host tissue by 3 months after operation. These results
also show that an easy-to-use point-of-care (POC) delivery device
(fig. S3) is merited for future clinical application.
Clinical follow-up showed that at 1-month follow-up, all pigs had
successfully reepithelialized. At 3 months after surgery, pachymetric
analyses showed that the standard corneal thickness was restored in
LiQD Cornea animals (Fig.3A and fig. S2B). Intraocular pressure
was normal at all postsurgical exams, indicating that the LiQD Cornea
successfully sealed the surgical site (Fig.2B). The LiQD Cornea pigs
showed more significant haze and neovascularization than syngeneic
grafts at all postsurgical time points, but haze was reduced in three
of four animals at 12 months after operation (Fig.2,CandD). The
fourth pig had poor surgical results with iritis and formation of
peripheral anterior synechiae (attachment of the iris to the cornea)
and infiltration of a large blood vessel into the surgical site, resulting
in a hazy cornea at 12 months after operation. This pig had inadver-
tently received a full-thickness corneal perforation that was re-
attached with a suture that trekked in a large blood vessel. This pig,
nevertheless, showed full regeneration of corneal tissue and nerves.
Esthesiometry performed to determine touch sensitivity showed the
restoration of the corneal blink response in all operated corneas,
indicating the presence of regenerated nerves within the graft site
(Fig.2E). Analysis of the density of corneal nerves over time showed
that by 12 months after operation, there were no statistically signif-
icant differences in the nerve density (Fig.2F) between the LiQD
Table 1. Optical, physical, and mechanical properties of LiQD Cornea hydrogels.
Tensile strength
(MPa)
Modulus (MPa) Viscosity (Pa.s) Transmission
(%)
Refractive index Water content
(%)
Collagenase
(mg/min)
Td (°C)
0.02 0.16 31.7 ± 27.6 19–93% (UV) 1.354 ± 0.037 91.2 ± 2.3 7.3 × 10−7 ± 6.1× 10−7 64 ± 8.5
93–99% (Visible)
Material Average bursting pressure (mmHg)* Representative image of sealed ex vivo perforation
model†
Cyanoacrylate glue >300
Fibrin sealant 259 ± 14.5
LiQD Cornea 170 ± 16.9
*Maximum pressure measured by the pressure transducer is 300 mmHg. †Photographs of ex vivo porcine corneas, which were mounted in an artificial
anterior chamber and perforated according to the described model and sealed/filled with the corresponding material. Red arrows highlight the interface of the
applied material and the perforated cornea.
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Cornea (3012.8±1613.7 m/mm2), syngeneic (2205.3±1162.4 m/mm2),
and unoperated control (4800.4±1964.9 m/mm2). However, it is
clear that the unoperated controls had a higher nerve density. There
were no marked differences in tear production in the three treatment
groups at any time point, as indicated by Schirmer’s test (Fig.2G).
Collagen content analysis of the central cornea demonstrated sig-
nificantly lower levels of high–molecular weight, -, -, 1(V)-, and
1(I)-type collagen in the LiQD Cornea pigs, as compared to the
syngeneic grafts and unoperated eyes (Fig.2H and table S5).
Hematoxylin and eosin sections of mini-pig corneas at 12 months
after surgery showed that LiQD Cornea–treated corneas had
regenerated their epithelia and stroma (Fig.3A) and resembled
Fig. 1. Biological evaluation of LiQD Cornea. (A) Immortalized HCECs cultured on
LiQD Cornea hydrogels and control tissue culture plastic, showing that the hydrogels
support epithelial growth. (B) Expression of T cell costimulatory molecules in BMDCs.
Expression of CD40, CD80, and CD86 was measured by flow cytometry, and data
are presented as a ratio of mean fluorescence intensity of the experimental samples
to untreated BMDCs. LPS acted as a positive control for BMDC activation; *P ≤ 0.05 by
Student’s t test. (C) Expression of pro-inflammatory M1 (CD86) and anti-inflammatory
M2 (CD206) phenotypic markers at 4 and 7 days after exposure of naïve BMDM
precursors to LiQD Cornea hydrogels. (D) Example of a human corneal perforation.
(E) Postsurgical photos of rabbits immediately after injecting LiQD Cornea into a
perforated cornea. The two-stepped surgically induced perforation can be seen. At
day 2 after surgery, the air bubble placed under the cornea during surgery is prom-
inent, indicating that the perforation was completely sealed. The perforated cornea
was completed healed by 28 days after operation. Photo credit: Damien Hunter,
University of Sydney. (F) Mini-pig corneas where the LiQD Cornea was tested as an
alternative to a donor allograft, showing the gross appearance of the LiQD Cornea,
syngeneic graft, and an unoperated eye at 12 months after surgery. Photo credit:
Monika K. Ljunggren, Linköping University.
Fig. 2. Clinical exam progression of LiQD Cornea in Göttingen mini-pigs.
(A) Pachymetry showing corneal thickness measured by OCT, showing no significant
differences in thickness compared to controls. There was a normal increase in corneal
thickness in unoperated controls as the pigs matured. (B) Intraocular pressures
were similar in all three groups, showing a slight overall increase over the normal
aging process of the pigs. (C) Central corneal haze measured using a modified
McDonald-Shadduck scoring system on a scale from 0 to 4. An increase of haze
corresponds to the period of in-growth of stromal cells into the cell-free implants.
By 12 months after operation, the cells appeared to have attained quiescence.
(D) Corneal neovascularization was seen in the LiQD Cornea, mainly from the animal
that sustained an unintended perforation. (E) Corneal blink response measured by
Cochet-Bonnet esthesiometry showed no significant differences among the three
groups. (F) Corneal nerve density in the LiQD Cornea group was significantly lower
than the unoperated corneas during months 3 to 9 after operation when the severed
nerves were regenerating. (G) Schirmer’s tear test showed similar responses in all
three groups tested. (H) Expression of high–molecular weight collagens (HMW, ,
and ), type V collagen, and type I collagen (1 and 2) in the central portion of the
cornea. Figures (A), (B), and (E) to (H) were assessed using a mixed-effects model with
a Tukey post hoc test for multiple comparisons. Figures (C) to (D) were analyzed us-
ing a Mann-Whitney U test for ordinal data. *P ≤ 0.05 for LiQD Cornea to unoperated,
†P ≤ 0.05 for LiQD Cornea to syngeneic graft, and ‡P ≤ 0.05 syngeneic graft to un-
operated. All data are plotted as mean ± SEM or mean with individual values.
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corneas in the syngeneic graft (Fig.3B) and untreated control groups
(Fig.3C). The unoperated endothelia remained healthy. Transmission
electron microscopy (TEM) confirmed the presence of healthy
electron-lucent epithelial cells in all three samples (Fig.3,DtoF).
There were no cells with condensed cytoplasm or pyknotic, shrunken
nuclei that are characteristic of apoptotic cells. TEM also revealed
that the basal epithelial cells in all samples had desmosomes between
them (Fig.3, Gto I), showing that regenerated cells in the LiQD
Cornea had tight junctions and were functional as a barrier. Immuno-
histochemical analysis showed the presence of mucin, indicating a
functional tear film in all samples (Fig.3,JtoL), and epithelial
cytokeratin 12, indicating terminal differentiation of regenerated
epithelium (Fig.3,MtoO). All samples contained very few CD163+
cells from the monocyte/macrophage lineage (Fig.3,PtoR).
Immunohistochemical staining for –smooth muscle actin (-SMA)
(fig. S4, A to C) and the lymph vessel marker, lymphatic vessel
endothelial hyaluronan receptor 1 (LYVE1) (fig. S4, D to F), respec-
tively, showed no increase in staining for myofibroblasts or lymphatics
in the LiQD Cornea, as compared to allografts.
TEM images of the epithelial-stromal junction show the presence
of small vesicles in the epithelial cells in all three samples (Fig.4,
AtoC). Immunohistochemical staining showed the presence of large
numbers of Tsg101+ vesicles in the epithelium and stroma of LiQD
Cornea–treated samples (Fig.4D). Tsg101 is an established marker
for extracellular vesicles (EVs), forming part of the endosomal sorting
complex required for transport-I (ESCRT-I), which is necessary for
exosome-dependent intercellular signaling and vesicular trafficking
(27). The staining is more diffuse in the syngeneic grafts (Fig.4E)
and minimal in the untreated controls (Fig.4F). The samples were
also stained for the tetraspanin, CD9, another established EV marker
that more specifically marks exosomes (28). Colocalization of Tsg101
with CD9 showed that exosomes were present in the basal epithelium
and upper stroma in the LiQD Cornea pigs (Fig.4G), to a lesser
extent in syngeneic grafts (Fig.4H) and only minimally in the un-
treated corneas (Fig.4I).
In vivo confocal microscopy showed that the epithelium in the
LiQD Cornea was fully regenerated at the 3-month examination
time point and remained stable at the 12-month end point (Fig.5A),
resembling that of the syngeneic (Fig.5B) and untreated corneas
(Fig.5C). Regenerated nerves were found within the sub-basal epi-
thelium of the LiQD Cornea starting at 3 to 6 months after surgery.
At 12 months after operation, the nerves present were in distinct
parallel bundles (Fig.5D), characteristic of the subepithelial nerve
plexus, similar to those found in the healthy unoperated control
corneas (Fig.5F). Nerves in the syngeneic grafts were not as well
defined in their configuration (Fig.5E). From 3 to 9 months, reflec-
tive keratocytes indicative of in-growing cells were seen within the
matrix. The presence of reflective cells corresponded with the in-
creased haze seen by slit lamp biomicroscopy (Fig.2C). At 12 months,
keratocytes grew into the cell-free matrix to reconstitute the stroma
(Fig.5G). Most of these keratocytes were not reflective and resemble
keratocytes in the syngeneic grafts (Fig.5H) and untreated controls
(Fig.5I). The decrease in reflectivity likely corresponds to the de-
crease in haze in Fig.2C.
DISCUSSION
To address the severe shortfall of donor tissue in the treatment of
corneal blindness, it is imperative that novel alternatives to corneal
transplantation and perforation repair are developed. While a number
of techniques and materials are currently available to treat corneal
defects and perforations, many of them involve complex procedures
and use materials with poor biocompatibility, mechanical mismatch,
Fig. 3. Histopathology, TEM, and immunohistochemistry of the LiQD Cornea
at 12 months. (A to C) Paraffin-embedded sections of porcine cornea stained with
hematoxylin and eosin (H&E) show multilayered, nonkeratinizing epithelia in all
three samples. (D to F) TEM images of corneal epithelium in all three samples. (G to
I) Epithelial cells showed abundance of desmosomes between cells (arrowheads).
(J to L) Fully regenerated corneal tear film mucin stained with fluorescein isothiocyanate–
conjugated lectin (green) from Ulex europaeus is seen in the LiQD Cornea. This is
similar to the tear film in the controls. (M to O) Cytokeratin 12 (red), a marker for
fully differentiated corneal epithelial cells, is present in the regenerated LiQD Cornea
as in controls. (P to R) CD163 staining (red) shows that a few mononuclear cells are
present in stroma of all three samples. Cell nuclei were stained blue with DAPI.
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and an inability to support regeneration. Ideally, any newly devel-
oped method should be easy to apply in a clinical setting, readily fill
corneal defects, and seal perforations. At the same time, it should
support tissue regeneration, limiting the need for further surgical
intervention and follow-up corneal transplantation.
Our results showed that LiQD Cornea behaved as an injectable
liquid at temperatures above 37°C, gelling as it cools down. In situ
gelation of the LiQD Cornea in animal corneas took 5min at body
temperature, after initiation with DMTMM, a nontoxic cross-linker
(23). Most patients with corneal perforations have inflamed eyes and
are photophobic. Unlike light-activated systems, LiQD Cornea did
not require a dedicated light source for curing. Without the require-
ment for light activation, no anesthetic will be needed in future clin-
ical application to render the exposure to an intense light source for
cross-linking tolerable. In addition, photoinitiated cross-linking has
been reported to have possible phototoxic effects on the corneal
endothelial cells (16). Considering that the initial perforation in
pathologic corneas would also affect the health of the endothelium,
it would be prudent not to further deplete the local population of
endothelial cells.
The incorporation of an approved surgical fibrin sealant permitted
adhesion of the LiQD Cornea during in situ gelation. Corneal per-
forations in exvivo corneas were completely sealed in situ with a
bursting pressure of 170 mmHg, which is several-fold higher than
normal intraocular pressures of 11 to 21mmHg (29). HCECs grew
readily on the LiQD Cornea hydrogels. The BMDC study indicated
that the LiQD Cornea did not activate dendritic cells unlike the pos-
itive control, LPS, which is a well-established activator of dendritic
cells. As the LiQD Cornea formulation does not activate dendritic
cells, the risk of graft rejection due to activation of CD4+ and CD8+
T cells is reduced (30). The BMDM assay indicated that naïve BMDMs
cultured in the presence of LiQD Cornea hydrogels primarily matured
into an M2 phenotype that is associated with tolerogenic activity (31).
These results taken together demonstrated that the LiQD Cornea
formed a seal that will withstand the pressures encountered within
the eye and will be fully biocompatible and immune compatible.
Injection of the LiQD Cornea into full-thickness corneal perfo-
rations in rabbits confirmed the ability to seal the wound gape. The
completeness of the seal was validated by the addition of a postsur-
gical air bubble. The bubble was present up to 2 days after surgery,
indicating that the material had created a complete seal that did not
allow the leakage of air. The rabbit histology showed that the patch
was completely reepithelialized. However, the 28-day duration of
the study did not provide time for full stromal, endothelium, and
nerve regeneration.
Fig. 5. In vivo confocal microscopy images of the LiQD Cornea compared to a
healthy unoperated cornea and a syngeneic graft at 12 months after surgery.
Regenerated corneal epithelial cells cover the surface of the LiQD Cornea (A) as with
the syngeneic graft (B) and untreated cornea (C). Regenerated nerves (arrowheads)
were found at the sub-basal epithelium within the LiQD Cornea (D), ran parallel to
one another, and were morphologically similar to those found in the unoperated
cornea (F). Nerves in the syngeneic graft were less distinct (E). Keratocytes were
present in all corneas (G to I). The unoperated endothelium remained intact and
healthy in all corneas (J to L). Scale bars: 100 m.
Fig. 4. EV and exosome secretion of the regenerated LiQD Cornea compared
to a healthy unoperated cornea and a syngeneic graft. (A) Transmission electron
micrograph of a LiQD Cornea sample showing the presence of basal epithelial cells
invaginations into the stroma. A basement membrane was present. Vesicles can be
seen inside the epithelial cell (an example is indicated with a red arrow). EVs are seen
(white arrows) in the underlying stromal compartment. (B and C) TEM of syngeneic
graft and untreated cornea, respectively. (D to F) Surface reconstructions of corneal
sections stained with the cytosolic, EV marker Tsg101 (red) and DAPI (blue). (G to
I) Surface reconstruction of colocalized CD9 and Tsg101 staining indicating the
presence of exosomes in the basal epithelium and upper stroma of the LiQD Cornea
sample. There was less staining in the syngeneic graft and minimal in the untreated
control. Scale bars: 500 nm (red) and 20 m (white).
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The Göttingen mini-pigs used were genetically coherent or
homogenous. Hence, grafts from one animal to another were con-
sidered syngeneic, i.e., they were sufficiently identical and immuno-
logically compatible to allow for transplantation. The 12-month
invivo pig study confirmed that LiQD Cornea allowed regeneration
of the corneal epithelium, stroma, and nerves. Even if the material
does not achieve the desired perfectly smooth surface directly after
application, OCT results showed that the corneal thickness and
curvature were restored to those matching the syngeneic grafts and
unoperated controls (fig. S2B). The primary difference in the clinical
performance of the LiQD Cornea and the syngeneic grafts was in-
creased haze in the surgical site between 3 to 6 months after opera-
tion, during the period of rapid keratocyte in-growth into the cell-free
matrix. Syngeneic grafts were already populated with donor cells, so
no rapid in-growth of host cells was expected. However, at 1 year
after surgery, the haze was reduced to a low grade in three of four
LiQD Cornea recipients, while the syngeneic graft outline was still
visible in the corneas. Neovascularization had accompanied the haze,
as we had previously reported for solid CLP-PEG implants during
the rapid cell population of cell-free grafts (20). However, as observed
in previous solid implant studies (32,33), the vessels receded over
the 12-month observation period as haze cleared in three of four
animals. While this small amount of vascularization and haze is not
ideal, it is unlikely to lead to immune rejection. LiQD Cornea does
not activate dendritic cells invitro and is acellular and repopulated
by the host cells, unlike traditional corneal transplants that bring
with them allogeneic cells (25) whose surface proteins can trigger
immune reactions. Vascularization could increase the risk of rejec-
tion of subsequent allografts (34), but LiQD Cornea is designed to
regenerate the eye wall without the need for subsequent transplanta-
tion, avoiding problems with induced irregular astigmatism, rejec-
tion, and lack of access to transplant donor material. Where corneal
perforations involve the central visual axis, at minimum, LiQD Cornea
aims to restore eye wall integrity as a viable pathway for future reha-
bilitation. In our pig model, steroid medication was only administered
for 5 days. In a clinical setting, it may be possible to modulate
neovascularization during healing through application of topical
steroids for a longer period.
We also found that LiQD corneas had lower expression of mature
type I collagen in the cornea. This is in keeping with the fact that the
LiQD Cornea matrix had no collagen, and hence, all collagen found
at 12 months after surgery was due to active remodeling of the gel,
in comparison to syngeneic grafts, which had a complete extracellular
matrix at the time of grafting. When considering the relative perform-
ance of the LiQD Cornea and the syngeneic grafts, it is important
to note that the syngeneic grafts are likely less inflammatory than a
standard clinical allograft, because of the genetic homogeneity of
Göttingen mini-pigs. As previously reported for CLP-PEG (20), the
LiQD Cornea also induced the production of copious amounts of
EVs that included exosomes, in comparison to the syngeneic grafts,
and the lack of EVs in the untreated, healthy controls. We currently
hypothesize that the presence of the EVs is linked to the production
of new extracellular matrix in the surgical site, as the reduced collagen
content in the LiQD Cornea suggests that the new tissue is still
undergoing extracellular matrix protein secretion to restore the matrix
at 12 months.
Overall, LiQD Cornea performed equivalently to syngeneic grafts,
indicating a possible role as an alternative to conventional donor
corneal transplantation for conditions treatable by lamellar trans-
plantation. However, as noted, it took the surgeon an average of two
attempts to achieve the desired curvature, indicating that an appro-
priate point-of-care delivery device (fig. S3) is needed for clinical
application. The self-assembling, fully defined, synthetic collagen-like
LiQD Cornea is considerably less costly than human recombinant
collagen and reduces any risk of allergy or immune rejection associated
with xenogeneic materials. In situ gelation potentially allows for clini-
cal application in an outpatient clinic instead of an operating theater,
thereby maximizing practicality while minimizing health care costs.
MATERIALS AND METHODS
Synthesis of eight-arm CLP-PEG
The synthesis and characterization of CLP-PEG through conjugation
of eight-arm PEG-maleimide to the 38 amino acid CLP via the for-
mation of a thioether linkage has been previously described (23,35).
Successful conjugation of the CLP to the PEG-maleimide was con-
firmed through the disappearance of the vinylic proton peak at 7
parts per million by 1H nuclear magnetic resonance spectroscopy
and the appearance of characteristic vibrations in the Fourier trans-
form infrared (FTIR) spectrum.
Here, CLP [CG(PKG)4(POG)4(DOG)4] (AmbioPharm, SC, USA)
was conjugated to a 40-K eight-arm PEG-maleimide with hexaglycerol
core (Sinopeg Biotech Co Ltd., Beijing, China) to give rise to CLP-PEG
(35). Briefly, 20ml of water was degassed by sparging with N2 for
20min. The flask was charged with 770mg of eight-arm PEG-
maleimide, and the solution was stirred until complete dissolution
was achieved. CLP (625 mg) was added to the stirring solution (molar
ratio of eight-arm PEG-maleimide:CLP is 1:8). The solution was
allowed to stir for an additional 20min (at this point, all materials
should be dissolved). The pH of the solution was adjusted to 4.5
through dropwise addition of 2M NaOH. As the pH of the solution
is adjusted, the reaction mixture becomes too viscous to be appro-
priately stirred. At this point, another 30ml of N2-purged water was
added. The reaction flask was covered in aluminum foil and allowed
to stir for 5 days. The pH of the reaction mixture was monitored
periodically during this time and adjusted accordingly. At the end
of the 5 days, an additional 50ml of water was added to the reaction
mixture, and again, the pH was adjusted to 4.5. The solution was
filtered through a 0.45-m syringe filter. The filtered solution was
transferred to dialysis tubing (molecular weight cutoff: 14,000). The
tubes were dialyzed against water (pH 4.5) for 7 days while ex-
changing the water every 12 hours. The contents of the dialysis bags
were transferred to 50-ml Falcon tubes as 25-ml aliquots. The solu-
tions were frozen overnight at −80°C and freeze-dried, resulting in
a cotton-like solid CLP-PEG conjugate.
Reconstitution of CLP-PEG and fibrinogen at 10 and
1% (w/w), respectively
The plunger of a 10-ml sterile syringe with luer lock was removed,
and the end was fitted with a syringe cap. CLP-PEG and fibrinogen
(clottable protein, Tisseel, Baxter International, Deerfield, IL, USA)
were added to the barrel of the syringe. HyPure molecular biology–
grade water (GE Life Sciences, Logan, UT) was added to give a final
dilution of 10 and 1% (w/w) for the CLP-PEG and fibrinogen, re-
spectively. The syringe was then sealed with parafilm, and the CLP-PEG
and fibrinogen was allowed to reconstitute at room temperature (RT)
for 2 to 3 weeks. To facilitate the reconstitution process, the mixture
was stirred periodically with a spatula and warmed up to 37°C in an
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incubator. Once completely resuspended, the solution was heated
above its melting temperature (>37°C) and centrifuged at 3000rpm
for 10min. This process was repeated until all bubbles had been
removed from the syringe.
Reconstitution of thrombin
Thrombin was reconstituted at 250 U/ml by the addition of 4ml of
10 mM phosphate-buffered saline (PBS) to the vial of thrombin
contained within the Tisseel kit. The solution was mixed at RT for
20min before use. The solution was either immediately used or ali-
quoted into several Eppendorf tubes and frozen at −20°C for future
use. Frozen samples were thawed to RT before use.
Mixing and application of LiQD Cornea
The solution within the syringe containing 10% (w/w) CLP-PEG and
1% (w/w) fibrinogen behaves as a liquid (injectable) at temperatures
above 37°C but sets as a gel when cooled to 25°C due to the templated
assembly of the CLPs. However, this sol-gel transition is reversible.
To make this sol-gel transition irreversible to obtain a hydrogel, a
solution of the cross-linker DMTMM in 10 mM PBS was added to
the mixture to obtain a final 2% (w/w) concentration of DMTMM
while cooling down the solution of CLP-PEG and fibrinogen from
50° to 25°C.
For application into the cornea, the stock solution of CLP-PEG/
fibrinogen was heated to 50°C and transferred to a 2-ml glass syringe
and assembled within a T-piece mixing system that had been primed
with 10 mM PBS. The T-piece system was heated to 50°C and mixed
until homogeneous. A 10% (w/w) solution of DMTMM in 10 mM
PBS was introduced through the injection port of the mixing system
to give a final concentration of 2% (w/w) DMTMM. The solution
was mixed within the T-piece system until homogeneous and then
dispensed into the wound bed to which the solution of thrombin
(250 U/ml) had been applied.
Physical and mechanical characterization
(i) Collagenase degradation assay
Collagenase from Clostridium histolyticum (Sigma-Aldrich) at
5 U/ml in 0.1M tris-HCl buffer containing 5 mM CaCl2 was used to
evaluate the stability of the hydrogels as previously described. Briefly,
samples were weighed after blotting off surface water at different
time points to determine the rate of loss of mass. The percentage of
residual weight was calculated using the following equation: Residual
mass %=Wt/W0 %, where Wt is the weight of hydrogel at a certain
time point and W0 is the initial weight of the hydrogel.
(ii) FTIR spectroscopy
Hydrogels were dried under vacuum for 3 days and measured
using a Nicolet iS5 FTIR spectrometer equipped with an iD7 atten-
uated total reflectance sampling accessory with 4cm−1 resolution; a
total of 300 individual spectra were collected for each sample.
(iii) Differential scanning calorimetry
Denaturation temperature of the hydrogels was measured using a
Q2000 differential scanning calorimetry (TA Instruments, New Castle,
DE, USA). Heating scans were recorded in the range of 8° to 210°C
at a scan rate of 10°C/min. Glass transition temperature (Tg) was
measured as the onset of the endothermic peak.
(iv) Refractive index
The refractive index of the hydrogels was measured at RT on an
Abbemat 300 (Anton Paar) refractometer.
(v) Young’s modulus and tensile strength
The Young’s modulus and tensile strength of a 500 M sheet of
cross-linked material was evaluated in an Instron electromechanical
universal tester (model 3342, Instron, Norwood, MA) equipped with
Series IX/S software using a crosshead speed of 10mm/min. The
hydrogel was equilibrated in 1× PBS for 1hour before being cut into
a 10 mm–by–5mm rectangular piece. To remove surface water, the
hydrogel was gently blotted with paper immediately before Instron
measurement.
(vi) Water content of hydrogels
The water content of hydrogels was evaluated by weighing the
“wet weight” (W0) of the samples and then comparing this to the
weight of the material after being dried at RT until a constant weight
was achieved (W). The total water content of the hydrogels (Wt) was
calculated according to the equation: Wt %=(W − W0)/W %.
(vii) Pore size of hydrogels
Pore size measurements were made from scanning electron
microscopy images obtained using a low-temperature scanning
electron microscope in a Tescan (model: Vega II-XMU) with cold-
stage sample holder at −50°C.
(viii) Viscosity of hydrogels
The viscosity of 500-m hydrogels was measured on a Brookfield
RS-CPS+ Rheometer (Brookfield Engineering Laboratories Inc.,
Middleboro, MA). The measurements were carried out at 37°C under
parallel-plate geometry.
(ix) Light transmission of hydrogels
The light transmission of hydrogels between 250 and 800nm was
evaluated by placing a 5 mm–by–10mm strip of hydrogel on the
inside wall of a quartz cuvette filled with PBS and reading its ab-
sorption in a SpectraMax M2e series plate/cuvette spectrophotometer
(Molecular Devices, San Jose, CA, USA). A cuvette filled with only
PBS was used as the baseline reference. Measured absorbances were
then converted to corresponding % transmission values.
Ex vivo model of corneal perforation and
sealing evaluation
Corneoscleral buttons were excised from porcine eyes obtained from
a local abattoir (Tom Henderson’s Meats and Abattoir Inc.,
Chesterville, ON, Canada). The corneoscleral buttons were mounted
on an artificial anterior chamber (Barron Artificial Anterior Chamber,
Katena, NJ), and standardized corneal defects were made. Briefly, a
4-mm punch was used to partially trephine test corneas centrally to
a depth of approximately 200 m. Lamellar dissection of the cap was
performed with a pediatric crescent blade, leaving a residual stromal
depth that was then trephined with a 3-mm punch to a depth of
200 m. A subsequent central full-thickness defect was created in the
central stromal bed with a 1-mm skin biopsy punch to mimic
a full-thickness corneal perforation commonly encountered in clin-
ical practice.
(i) Sealing methods
Once the standardized corneal perforations had been made, one
of three different materials was used to seal the defect. Each condi-
tion was repeated four times. Cyanoacrylate glue was injected to
completely fill the defect. After allowing the glue to dry, the infusion
was increased, and the bursting pressures were measured. For fibrin
glue evaluation, fibrinogen (2%, w/w) and thrombin (250 U/ml)
solutions were mixed (4:1) and transferred to completely fill the de-
fect. After allowing the glue to dry, the infusion was increased, and
the bursting pressures were measured. For the LiQD Cornea, the
defect was coated with thrombin (250 U/ml). The LiQD Cornea was
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injected to completely fill the defect. After allowing the glue to dry,
the infusion was increased, and the bursting pressures were measured.
(ii) Bursting pressure evaluation
Artificial anterior chambers were connected via an intra-arterial
blood pressure monitor (TruWave, Edwards Lifesciences) to a saline
infusion bag using a pressure cuff to regulate infusion pressure. After
application of test material, the infusion pressure was increased until
the seal gave way, resulting in fluid egress. Bursting pressure (mmHg)
was recorded as the peak in a continuous trace of infusion pressure
versus time.
In vitro evaluation of the LiQD Cornea
The invitro compatibility of the LiQD Cornea was tested using green
fluorescence protein (GFP)–transfected immortalized HCECs (24).
Briefly, three wells of a 24-well plate were coated with the LiQD
Cornea. The glue was allowed to set for 1 hour before it was washed
three times with 1ml of 1× PBS. GFP-HCECs were seeded into the
control wells and onto the materials at a density of 5000 cells per
well. GFP-HCECs were supplemented with keratinocyte serum-free
medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) con-
taining bovine pituitary extract (0.05 mg/ml), epidermal growth
factor (5 ng/ml), and penicillin/streptomycin (1 mg/ml), and their
growth was monitored for 7 days in a humidified incubator at 37°C
and 5% CO2.
BMDC culture and flow cytometric analysis
With ethical permission from the Animal Care and Use Committee
of Maisonneuve-Rosemont Hospital, bone marrow was isolated from
the tibia and femur of male, C57BL/6J mice (6 to 12 weeks old)
(36,37). Cells were seeded on suspension culture plates with 1 ×
106 million cells per well in RPMI 1640 containing 10% (v/v) fetal
bovine serum (FBS) (Wisent), penicillin-streptomycin-glutamine
(0.5 mg/ml), 10 mM Hepes, 1 mM sodium pyruvate, 55 m of
-mercaptoethanol, and granulocyte-macrophage colony-stimulating
factor (25 ng/l; GM-CSF) (all from Gibco, Thermo Fisher Scientific,
Waltham, MA, USA). BMDCs were cultured for 6 days. RPMI-C
containing GM-CSF (50 ng/ml) was exchanged for half of the media
on days 2 and 3 of culture. On day 6, the cells were collected, and
enlarged cells were selected using a Histodenz density gradient
(Sigma-Aldrich, St. Louis, MO). The selected cells were seeded at a
density of 1 × 106 cells per well on a 24-well plate for materials testing.
For materials testing, BMDCs were incubated for 24 hours with
a 6-mm, 500-m-thick hydrogel disk. Individual hydrogel compo-
nents CLP, PEG, CLP-PEG, DMTMM, fibrinogen, and thrombin
were applied to the cells at a concentration equivalent to the hydrogel
volume (table S1). LPS was used as a positive control for BMDC
activation. BMDCs were labeled with direct-conjugate antibodies for
CD11c, CD40, CD80, and CD86 (table S2) and Zombie Aqua Fixable
Viability Kit (BioLegend, San Diego, CA). All samples were acquired
using a BD LSR II and analyzed using FlowJo software (Becton,
Dickinson and Company, Franklin Lakes, NJ, USA). BMDCs were
selected using Zombie Aqua and CD11c as markers of a live,
dendritic cell phenotype. Mean fluorescence for CD40, CD80, and
CD86 was measured for the selected BMDCs and transformed into
a ratio over the untreated BMDC control for analysis.
Macrophage polarization assay
Macrophages were isolated as previously described (38) with ethical
permission from the Animal Care and Use Committee of the Ottawa
Heart Research Institute. Briefly, BMDMs were generated from the
tibial bones of C57BL/6 female mice (8 to 10 weeks old). BMDMs
were maintained for 1 week in DMEM with 10% FBS, 15% L929
media containing macrophage colony-stimulating factor and
penicillin-streptomycin.
For the assay, BMDM precursors from female C57BL/6 mice (8 to
10 weeks old) were used. The wells of a 24-well culture plate were
fitted with 18-mm circular glass coverslips. A portion of the coverslips
was then coated with the LiQD Cornea. The hydrogel was allowed
to set for 1 hour before it was washed three times with 1ml of 1×
PBS, followed by two additional 1-ml rinses with media before the
seeding of cells. The BMDMs were seeded into the control wells and
onto the material at a density of 200,000 cells per well. The plate was
then placed in a humidified incubator at 37°C and 5% CO2 with the
media in each well being exchanged every 48 hours up to 7 days. On
days 4 and 7, a subset of the wells was processed for immuno-
fluorescence analysis to determine their polarization toward either an
M1 or M2 phenotype. Briefly, media were removed, wells were
washed two times with Hanks’ buffer, and then, cells were fixed with
a solution of 4% paraformaldehyde in 1× PBS at 4°C in the dark.
Fixative was removed, and wells were washed two times with NH4Cl
in PBS, waiting 7min between washes. The samples were then washed
three times with 1× PBS. On the final wash, 0.2% NaN3 was added
from a 2% NaN3 stock (10l/1ml). When ready for staining, samples
were washed with PBS and then blocked and permeabilized using a
2% bovine serum albumin in PBS solution containing 0.5% Triton
X-100 for 1.5 hours at RT. Primary antibodies for CD206 and CD86
(table S1) were then diluted appropriately and added to the well
plate to incubate overnight covered in foil at 4°C. The next day,
wells were washed with 1× PBS. Secondary antibodies (table S1)
were diluted and added to the plate and incubated at RT covered in
foil for 1 hour. After 1 hour of incubation with the secondary anti-
bodies, the wells were washed three times with 1× PBS. The coverslips
were removed from the wells and mounted onto a glass slide using
Prolong Gold antifade reagent with 4′,6-diamidino-2-phenylindole
(DAPI; Invitrogen, P36931). Cells were imaged with a Zeiss Axiovert
200M Fluorescence microscope equipped with an AxioCam MR
camera using 63× oil immersion objective. The filters used were
DAPI blue filter (excitation: 352 to 402/emission: 417 to 477), GFP
green filter (excitation: 457 to 487/emission: 502 to 538), and Texas
Red red filter (excitation: 542 to 582/emission: 604 to 644).
Rabbit perforation study
All experiments had ethical approval from the Western Sydney
Local Health District Animal Ethics Committee. Three New Zealand
white rabbits underwent controlled surgical perforation of the right
eye under general anesthesia [sedation: medetomidine (25 mg/kg);
analgesia: buprenorphine (0.5 mg/kg); anesthesia: ketamine (50 mg/kg),
and 2% inhaled isofluorane]. A 3-mm surgical trephine was used
to make a partial-thickness incision before full perforation using a
15° stab knife. The full incision was then enlarged to create a 1-mm
full-thickness perforation. The perforation was filled with LiQD
Cornea hydrogel and allowed to cross-link in situ. Air was injected
into the anterior chamber to ensure that the perforation was com-
pletely sealed. For the 3 days after surgery, rabbits received 0.1%
dexamethasone (Maxidex, Alcon Laboratories Pty Ltd, Australia)
and 0.5% chloramphenicol eye drops (Chlorsig, Aspen Pharmacare,
Australia) three times a day. Animals were monitored daily for
signs of discomfort or glue leakage for the first week after surgery
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and then twice weekly for subsequent weeks. Rabbits underwent
follow-up clinical evaluation and slit lamp exams on days 1, 2, 3, 7, 14,
21, and 28 after surgery. At day 28 after surgery, rabbits were
euthanized, and corneas from all operated and unoperated eyes
were excised, fixed in 10% buffered formalin, and processed for
paraffin embedding for histopathological examination (33).
In vivo study in Göttingen mini-pigs
In compliance with the Swedish Animal Welfare Ordinance and
the Animal Welfare Act, and with ethical permission from the local
ethical committee (Linköpings Djurförsöksetiska Nämnd), Göttingen
mini-pigs underwent an anterior lamellar keratoplasty of the left eye.
The left corneas were cut with a 6.5-mm surgical trephine to a depth
of 500 m, followed by blunt dissection of corneal stroma with a
blade to create a wound bed. Four pigs received the LiQD Cornea
formulation, which was cross-linked in situ and subsequently covered
with human amniotic membrane that was secured with overlying
sutures. Four pigs received syngeneic grafts, i.e., they were grafted
with the tissue removed from another, albeit genetically coherent,
pig in the group. Syngeneic grafts were secured with conventional
interrupted sutures. The right contralateral corneas served as un-
operated controls. After operation, the operated eyes received
dexamethasone/tobramycin eye drops (Tobrasone, Alcon Labora-
tories, Sweden). Upon surgical completion, the pigs received a
maintenance dose of one drop, three times per day for 5 days after
surgery. Pigs were monitored daily for ocular health. Clinical exams
were conducted under anesthesia before surgery and at 6 weeks,
3, 6, and 12 months after surgery. Clinical exams included slit
lamp examination using a Kowa SL-15L portable slit lamp (Kowa
Company Ltd., Aichi, Japan), anterior segment OCT (AS-OCT)
to conduct corneal pachymetry (Optovue, Fremont, CA, USA),
Schirmer’s tear test (tear strips from TearFlo, Hub Pharmaceuticals,
Rancho Cucamonga, CA, USA), esthesiometry to determine corneal
sensitivity as a measure of nerve function (using a Cochet-Bonnet
esthesiometer; Handaya Co., Tokyo, Japan), measurement of intra-
ocular pressure (using a TonoVet tonometer, Icare Finland Oy,
Vantaa, Finland), and invivo confocal microscopy (Heidelberg
HRT3 with a Rostock Cornea Module, Heidelberg Engineering
GmbH, Dossenheim, Germany).
Central collagen content analysis
Central corneal biopsies (3 mm) were taken from each cornea and
snap-frozen. For analyses, the samples were thawed and resuspended
in 10 mM HCl at a ratio of 1:35 (w/v). Samples were digested using
pepsin (1 mg/ml; Roche, Basel, Switzerland) at 2° to 8°C for 96 hours.
The soluble fraction was recovered by centrifugation at 16,000g for
30min at 2° to 4°C. An aliquot of the pepsin-soluble fraction was
mixed with NuPAGE 4× LDS sample buffer (Life Technologies,
Thermo Fisher Scientific, Waltham, MA, USA) denatured at 75°C
for 8min and analyzed on 3 to 8% tris-acetate gels under nonreduc-
ing conditions. Proteins were visualized by staining with Gelcode
Blue (Pierce, Thermo Fisher Scientific, Waltham, MA, USA).
Prestained broad-range marker (New England Biolabs, P7712) and
porcine skin type I collagen (Koken Co. Ltd., Tokyo, Japan) were
used as molecular weight standards. To quantitate the amounts
of type I and type V collagens in control and operated corneas,
densitometric scans of the stained gels were made to obtain relative
numerical units using GE Healthcare ImageQuant 350 (GE Healthcare,
Chicago, IL, USA).
Histopathology and immunohistochemistry
After removal of a central biopsy, a quarter of each operated and
unoperated cornea was processed, paraffin-embedded, and stained
with hematoxylin and eosin as described previously (32). Another
quarter of each operated and unoperated cornea was treated with a
sucrose gradient and fixed in 4% paraformaldehyde. The samples
were frozen in optimal cutting temperature medium and sectioned
at 8 or 10 m before mounting on glass slides. Slides were washed
in PBS before permeabilization in PBS with 0.3% Triton X-100 for
15min. Slides were then washed in PBS. Sections stained using
Alexa Fluor 488 or 647 secondary antibodies were incubated for
30min in tris-buffered saline (TBS) containing 50 mM ammonium
chloride to reduce background fluorescence. All sections were
blocked for 1 hour at RT in PBS containing 5% normal goat serum
or FBS with saponin (0.1 g/ml). Sections were stained with primary
antibodies for cytokeratin 12, CD163, –smooth muscle actin, and
LYVE1 (table S3) overnight at 4°C in blocking solution. Slides were
washed in PBS or TBS buffer containing 5% FBS and incubated with
secondary antibodies (table S3) conjugated to Alexa Fluor 488 or
594 diluted at 1:1000in blocking solution for 1 hour at RT. Sections
stained using Alexa Fluor 488 or 647 secondary antibodies were
quenched for autofluorescence using the Vector TrueVIEW Auto-
fluorescence Quenching Kit (Vector Laboratories, Burlingame, CA,
USA). Slides were stained with DAPI (5 g/ml) for 10min before
mounting in Vectashield Antifade Mounting Medium or Vectashield
Vibrance Mounting Medium (Vector Laboratories, Burlingame, CA,
USA). Slides stained using lectin were washed in PBS, stained with
lectin overnight at 4°C, washed, and counterstained with DAPI
before mounting in Vectashield Antifade Mounting Medium. All
slides were imaged using a Zeiss LSM 880 confocal microscope
(Zeiss, Oberkochen, Germany). Two-dimensional images were pro-
cessed using FIJI (39). EV and exosome staining using CD9 and
Tsg101 was reconstructed as surfaces in Imaris v9.2.1 (Bitplane Inc.,
Concord, MA, USA) with an intensity threshold of 1.5 and 2 for
CD9 and Tsg101, respectively, with a minimum voxel threshold
of 10. A colocalization channel was built using the same intensity
threshold as the surface reconstructions and converted into surfaces
using a fluorescence intensity threshold of 0.5 and a minimum voxel
threshold of 2.
Transmission electron microscopy
For TEM, a quarter of each cornea was fixed in 2.5% glutaraldehyde
solution in 0.1M sodium cacodylate buffer (pH 7.4). Samples were
then cut in 1-mm-wide strips and postfixed in 1% OsO4 solution for
2 hours. After dehydration in an ethanol gradient (50-70-90-95-100%
ethanol), whole mounts were embedded in EMbed 812 (Electron
Microscopy Sciences, Hatfield, Pennsylvania). Ultrathin sections
were stained with lead citrate and examined using Tecnai G2
Spirit Bio Twin Microscope (FEI, Eindhoven, The Netherlands)
at 120 kV.
Central cornea nerve analysis
In vivo confocal microscopy examinations were performed at various
time points (before surgery and 3, 6, 9, and 12 months after surgery)
throughout the 12-month mini-pig study. For each examination, all
images with nerves were identified. For identification purposes,
nerves were defined as bright, slender, straight, or branched structures;
as substantially uniform in intensity along their length and width;
and as having a marked contrast difference from the background
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SCIENCE ADVANCES | RESEARCH ARTICLE
11 of 12
intensity level. Nerve tracing and analysis software NeuronJ was
used in combination with FIJI software to manually measure the
total length of nerves present in each image identified as containing
nerves (39). The central cornea nerve densities are reported from
the average of the single-frame image displaying the highest nerve
density for each treatment group and time point.
Evaluation of preliminary POC delivery system
Testing of the preliminary POC delivery system (fig. S3) was per-
formed using the previously described exvivo corneal perforation
model. Briefly, a stock solution consisting of 10% CLP-PEG and 1%
fibrinogen was heated to 50°C and transferred to a 1-ml disposable
BD syringe. An equal volume of 10% (w/w) DMTMM in 10 mM
PBS was added to a second 1-ml disposable BD syringe. The syringes
were then attached to a dual-syringe adapter (Medmix Systems AG,
Switzerland), which was fitted with a 1:1 static mixer (Medmix
Systems AG, Switzerland) and a 19 gauge–by–25mm flattened tip
cannula. The mixing system was heated to 50°C, and the material
was dispensed through the mixing system into the wound bed to
which a solution of thrombin (250 U/ml) had been applied.
Statistical analyses
The invitro statistical analysis for BMDCs was performed using an
unpaired, two-way t test with a confidence interval of 95% for each
marker (GraphPad Prism 8.3.0, GraphPad Software LLC., San Diego,
CA, USA). The unit of analysis was the mouse (n=6 per group).
The unit of analysis for the clinical statistics was the eye. The clinical
statistics were conducted on uneven population sizes (LiQD Cornea:
n=4; syngeneic graft: n=4; unoperated: n=8). For variables with
repeated measures over time, a mixed-effects analysis with Geisser-
Greenhouse’s correction was performed (= 0.05) with Tukey’s
multiple comparisons test for treatment effects by time point
(GraphPad Prism 8.3.0). Postmortem collagen content analysis was
performed using a one-way analysis of variance (ANOVA) for each
collagen type with a Tukey post hoc test (=0.05) (IBM SPSS Sta-
tistics version 25, IBM Corp., Armonk, NY, USA). All graphs were
prepared using GraphPad Prism, and data are displayed as mean
with individual data points or mean±SEM.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/6/25/eaba2187/DC1
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Acknowledgments: We thank S. Lesage and C. Audiger for help with the dendritic cell assay
design and J. Laganiere and B. Hosseinpour for assistance with histopathological processing.
Funding: We acknowledge research funding from the Euronanomed II–Swedish Research
Council (Dnr IKE-2014-00597), the Euronanomed III–FRQS (file no. 278653), and the Quebec
Vision Health Research Network–FRQS (file no. 2017-3). C.D.M. was supported by a Caroline
Foundation Research Chair for Cellular Therapy in the Eye to M. Griffith. B.D.A. receives partial
salary support for research from the NIHR Biomedical Research Centre (BRC-1215-20002) at the
Moorfields Eye Hospital NHS Foundation Trust and the UCL Institute of Ophthalmology. C.S.
acknowledges support from a Westmead Charitable Trust Early Career Research Fellowship
(CC368629-8245), and F.C.S. acknowledges support for her doctoral studies from the Faculté
des études supérieures et postdoctorales de l’Université de Montréal, Fonds de recherche en
ophtalmologie de l’Université de Montréal (FROUM), Fonds de recherche du Québec-Nature et
technologies (FRQNT), and the Natural Sciences and Engineering Research Council of Canada
(NSERC). Author contributions: B.D.A. and M. Griffith developed the LiQD Cornea concept.
C.D.M. and M. Griffith designed the material. C.D.M. performed all optical, physical,
mechanical, and chemical characterization and BMDM assays and assisted with animal
surgeries and analyses of the results. F.C.S. performed the dendritic assays,
immunohistochemistry, and analyses of the clinical results from the pig study. B.D.A. and C.S.
designed the LiQD Corneal animal studies for feasibility evaluation. C.S. and D.H. performed
the rabbit perforation study. O.B. and P.F. performed the pig surgeries and follow-ups. M.H.
and M.K.L. contributed to the pig study design and assisted with pig clinical exams. D.O.
conducted the collagen content analysis. M.H., I.P., and P.L. contributed to the transmission
electron microscopy and interpretation of the results. M. Groleau contributed to the
immunohistochemistry experiments. E.E. contributed in IHC protocol development and
statistical analysis. M. Griffith developed the overall study plan and supervised all research and
analyses. All authors contributed to the writing, revisions, and final approval of the
manuscript. Competing interests: M. Griffith is a named inventor on PCT PCT/IB2017/056342
Collagen and CLP-based hydrogels, corneal implants, filler glue, and uses thereof, which was
assigned to the Hyderabad Eye Research Foundation, and then subsequently assigned to
North Grove Investments Inc. wherein PCT national phase applications have been filed in the
United States, Europe, India, China, and Canada. C.D.M. and M. Griffith are named inventors on
a U.S. provisional patent application no. 62916765, subsequent to a disclosure to Univalor,
technology transfer agent to Maisonneuve-Rosemont Hospital and Université de Montréal.
The authors declare that they have no other competing interests. Data and materials
availability: All data needed to evaluate the conclusions in the paper are present in the paper
and/or the Supplementary Materials. Additional data related to this paper may be requested
from the authors.
Submitted 14 November 2019
Accepted 8 May 2020
Published 17 June 2020
10.1126/sciadv.aba2187
Citation: C. D. McTiernan, F. C. Simpson, M. Haagdorens, C. Samarawickrama, D. Hunter, O. Buznyk,
P. Fagerholm, M. K. Ljunggren, P. Lewis, I. Pintelon, D. Olsen, E. Edin, M. Groleau, B. D. Allan,
M. Griffith, LiQD Cornea: Pro-regeneration collagen mimetics as patches and alternatives to
corneal transplantation. Sci. Adv. 6, eaba2187 (2020).
on June 17, 2020http://advances.sciencemag.org/Downloaded from
transplantation
LiQD Cornea: Pro-regeneration collagen mimetics as patches and alternatives to corneal
Allan and May Griffith
Buznyk, Per Fagerholm, Monika K. Ljunggren, Philip Lewis, Isabel Pintelon, David Olsen, Elle Edin, Marc Groleau, Bruce D.
Christopher D. McTiernan, Fiona C. Simpson, Michel Haagdorens, Chameen Samarawickrama, Damien Hunter, Oleksiy
DOI: 10.1126/sciadv.aba2187
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