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Biomaterials-enabled cornea regeneration in patients at high risk for rejection of donor tissue transplantation

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The severe worldwide shortage of donor organs, and severe pathologies placing patients at high risk for rejecting conventional cornea transplantation, have left many corneal blind patients untreated. Following successful pre-clinical evaluation in mini-pigs, we tested a biomaterials-enabled pro-regeneration strategy to restore corneal integrity in an open-label observational study of six patients. Cell-free corneal implants comprising recombinant human collagen and phosphorylcholine were grafted by anterior lamellar keratoplasty into corneas of unilaterally blind patients diagnosed at high-risk for rejecting donor allografts. They were followed-up for a mean of 24 months. Patients with acute disease (ulceration) were relieved of pain and discomfort within 1–2 weeks post-operation. Patients with scarred or ulcerated corneas from severe infection showed better vision improvement, followed by corneas with burns. Corneas with immune or degenerative conditions transplanted for symptom relief only showed no vision improvement overall. However, grafting promoted nerve regeneration as observed by improved touch sensitivity to near normal levels in all patients tested, even for those with little/no sensitivity before treatment. Overall, three out of six patients showed significant vision improvement. Others were sufficiently stabilized to allow follow-on surgery to restore vision. Grafting outcomes in mini-pig corneas were superior to those in human subjects, emphasizing that animal models are only predictive for patients with non-severely pathological corneas; however, for establishing parameters such as stable corneal tissue and nerve regeneration, our pig model is satisfactory. While further testing is merited, we have nevertheless shown that cell-free implants are potentially safe, efficacious options for treating high-risk patients.
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Biomaterials-enabled cornea regeneration in patients at high
risk for rejection of donor tissue transplantation
M. Mirazul Islam
1,2
, Oleksiy Buznyk
1,3
, Jagadesh C. Reddy
4
, Nataliya Pasyechnikova
3
, Emilio I. Alarcon
5
, Sally Hayes
6,7
,
Philip Lewis
6,7
, Per Fagerholm
1
, Chaoliang He
8
, Stanislav Iakymenko
3
, Wenguang Liu
9
, Keith M. Meek
6,7
, Virender S. Sangwan
4
and
May Grifth
1,4,10
The severe worldwide shortage of donor organs, and severe pathologies placing patients at high risk for rejecting conventional
cornea transplantation, have left many corneal blind patients untreated. Following successful pre-clinical evaluation in mini-pigs,
we tested a biomaterials-enabled pro-regeneration strategy to restore corneal integrity in an open-label observational study of six
patients. Cell-free corneal implants comprising recombinant human collagen and phosphorylcholine were grafted by anterior
lamellar keratoplasty into corneas of unilaterally blind patients diagnosed at high-risk for rejecting donor allografts. They were
followed-up for a mean of 24 months. Patients with acute disease (ulceration) were relieved of pain and discomfort within
12 weeks post-operation. Patients with scarred or ulcerated corneas from severe infection showed better vision improvement,
followed by corneas with burns. Corneas with immune or degenerative conditions transplanted for symptom relief only showed no
vision improvement overall. However, grafting promoted nerve regeneration as observed by improved touch sensitivity to near
normal levels in all patients tested, even for those with little/no sensitivity before treatment. Overall, three out of six patients
showed signicant vision improvement. Others were sufciently stabilized to allow follow-on surgery to restore vision. Grafting
outcomes in mini-pig corneas were superior to those in human subjects, emphasizing that animal models are only predictive for
patients with non-severely pathological corneas; however, for establishing parameters such as stable corneal tissue and nerve
regeneration, our pig model is satisfactory. While further testing is merited, we have nevertheless shown that cell-free implants are
potentially safe, efcacious options for treating high-risk patients.
npj Regenerative Medicine (2018) 3:2 ; doi:10.1038/s41536-017-0038-8
INTRODUCTION
In tissue engineering and regenerative medicine, exciting new
biomaterials and technologies such as 3D printing have produced
very promising results in animal models, showing regeneration in
a range of organs.
1
However, translation of these remarkable
accomplishments from animal models to patients in clinical
practice has been protracted. One problem is the failure to obtain
stable and functional integration of biomaterials into chronically
damaged, diseased or aged tissues, unlike the case with mostly
young, healthy animal models.
1
The limited predictive power of
pre-clinical animal studies, which typically involve the use of
rodents and rabbits, has indeed been identied as the primary
barrier to safe translation.
2
More recently, pigs have been
proposed as ideal pre-clinical models as the anatomy, physiology,
and biochemistry of many of their organs,
3,4
including their
corneas,
5
is similar to that of humans, allowing for greater
predictability of performance of new implants in human subjects.
The human cornea is a relatively simple tissue comprising three
main layers, an outer multilayered epithelium, a middle stroma
consisting of a largely collagenous extracellular matrix and cells
arranged in layers, and an inner single-layered endothelium. It is
highly innervated but avascular, and is optically transparent to
allow entry of light into the eye for vision. Irreversible loss of
transparency can result in corneal blindness. Corneal blindness is
estimated to affect 23 million individuals worldwide
6
and is
treated by corneal transplantation to restore clarity. However,
there is a severe worldwide shortage of donor tissues, as with
other transplantable organs. With only one donor cornea available
for every 70 needed,
7
it is evident that an alternative solution to
just increasing the donation rate is crucial. Furthermore, corneas
with inammation and severe pathologies have a high risk (up to
49%) for rejecting conventional donor allografts.
6
Over 90% of all
cornea blind individuals and in particular, high-risk individuals, live
in low to middle-income countries (LMICs).
6,8
Due to a lack of
resources in these countries, the treatment of these high-risk
patients with stem cell technology is not possible, and the limited
supply of donor tissues is prioritized for lower risk patients that
have a higher chance of successful recovery.
9,10
Articial corneas
Received: 17 July 2017 Revised: 6 December 2017 Accepted: 12 December 2017
1
Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden;
2
Schepens Eye Research Institute and Massachusetts Eye and Ear Inrmary, Harvard
Medical School, Boston, MA, USA;
3
Filatov Institute of Eye Diseases and Tissue Therapy of the NAMS of Ukraine, Odessa, Ukraine;
4
Tej Kohli Cornea Institute, LV Prasad Eye
Institute, Hyderabad, India;
5
Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, ON, Canada;
6
School of Optometry and Vision Sciences College of
Biomedical and Life Sciences, Cardiff University, Cardiff, UK;
7
Cardiff Institute for Tissue Engineering and Repair (CITER), Cardiff University, Cardiff UK;
8
Key Laboratory of Polymer
Eco-materials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China;
9
School of Materials Science and Engineering, Tianjin University,
Tianjin, China and
10
Department of Ophthalmology and Maisonneuve-Rosemont Hospital Research Centre, University of Montreal, Montreal, Canada
Correspondence: Keith M. Meek (MeekKM@cardiff.ac.uk) or Virender S. Sangwan (vsangwan@lvpei.org) or May Grifth (May.Grifth@umontreal.ca)
M. Mirazul Islam, Oleksiy Buznyk, Jagadesh C. Reddy, and Nataliya Pasyechnikova contributed equally to this work.
Keith M. Meek, Virender S. Sangwan, and May Grifth jointly supervised this work.
www.nature.com/npjregenmed
Published in partnership with the Australian Regenerative Medicine Institute
known as keratoprostheses have been developed but only two
have been successful in clinical use.
11
However, because of the risk
of severe side effects such as potentially blinding glaucoma, and
the need for immune suppression and lifelong antibiotics, these
are generally used only in end-stage diseased corneas.
12
Full-
thickness corneal grafting by penetrating keratoplasty (PK)
remains the mainstay of corneal transplantation globally, particu-
larly in LMICs
13
even though partial thickness grafts that address
only the affected layers are gaining in popularity. For damage
affecting the epithelium and stroma, partial-thickness anterior
lamellar keratoplasty (ALK) is performed. Given the magnitude of
the problem with an estimated 1.52 million new cases of corneal
blindness per year,
6
cost-effective, cell-free biomaterials implants
that promote endogenous regeneration while minimizing the
regulatory and scientic challenges of specialized cleanrooms
14
and immune rejection, are attractive clinical options.
Our team has previously shown that cell-free implants
comprising carbodiimide-crosslinked recombinant human col-
lagen type III (RHCIII) stimulated stable regeneration in conven-
tional cornea grafted patients.
15,16
However, for use in high-risk
patients, we incorporated a second network of 2-
methacryloyloxyethyl phosphorylcholine (MPC) as a structural
element within the implant. MPC is a synthetic lipid reported to
suppress inammatory responses,
17
and to minimize neovascular-
ization in rabbit models of corneal alkali burn.
18
In three pilot
patients, the use of small tectonic patches of RHCIII-MPC to
replace excised ulcerated tissue resulted in the successful
restoration of corneal integrity without any adverse effects.
19
Here, using the transplantation of RHCIII-MPC implants as a test
bed, we assessed the efcacy of the pig model at predicting
clinical outcomes. We examined the results of a pre-clinical mini-
pig study alongside those of a clinical study involving seven high-
risk patients with different pre-operative diagnoses. A pre-clinical
study of biosynthetic corneas comprising RHCIII-MPC was
performed in Göttingen mini-pigs to conrm previous safety
results
20
and to examine in detail the micro-architecture and
optical properties of regenerated neo-corneas. For our clinical
study, all recruited patients had been diagnosed with severe
corneal pathologies resulting from ocular trauma or infective
ocular ulceration and had consequently not been prioritized for
grafting due to their high risk of donor rejection.
21
The primary
endpoint of the clinical study was safety, i.e., no serious adverse
reactions such as excessive redness, pain or discomfort, swelling of
adjacent corneal tissues or clouding of anterior chamber uid. The
secondary endpoints were the restoration of corneal integrity and
regeneration of neo-corneas by mobilization of endogenous stem
cells. The potential benet to patients was the reduction of pain
and discomfort to those with active ulcers, and the possibility of
regaining eyesight in severely scarred eyes. The worst-case
scenario was no change in vision or the need for re-grafting with
a human donor cornea.
RESULTS
RHCIII-MPC implants
RHCIII-MPC implants comprising 8% RHCIII, 4% MPC and 1.3%
PEGDA were fabricated following Medical Devices Directive MDD
93/42/ECC and associated ISO standards in a certied and
monitored cleanroom at Vecura AB, Karolinska University Hospital,
Huddinge, Sweden. Only implants meeting quality control criteria
such as comparable optical quality to the human cornea were
used (Table 1A). Implants exhibited 92% light transmission,
22
which is above the minimum of 87% for healthy human corneas.
23
The refractive index of the implants was 1.334, similar to water
(1.333) and marginally lower than that of the human cornea at
1.373.
24,25
Denaturation temperature was 51 °C, lower than that of
the human cornea at 65 °C
26
but well above the highest body
temperature ever recorded at 45 °C.
27
Table 1. RHCIII-MPC implants and summary of pre-operative patient diagnoses: (A) Characteristics of corneal implants used in the study (n=3); (B)
Summary of pre-operative patient diagnoses
A
Properties Transmission in white light (%) Refractive index Denaturation temperature (°C) Water content (%)
Implants 92.4 ±0.1
22
1.334 ±0.0003
22
51.0 ±1.51 89.37 ±2.1
QC acceptance criteria 85 1.11.5 50 85
Human cornea 87.1 ±2.0
23
1.3731.380
24
65.1
26
78
25
B
Cause Age Gender Stage of
disease
Diagnosis LESCD Vascularised? Start of disease until
treatment (m)
Infection
Patient 1 64 F Acute Herpes simplex keratitis No No 5
Patient 2 36 F Scar Fungal keratitis No No 8
Patient 3 56 M Scar Herpes simplex keratitis No No 72
Burns
Patient 4 58 F Acute Alkali burn Yes Yes 480
Patient 5 73 M Acute Thermal burn (senile cataract in lens that
impeded vision)
Yes Yes 7
Other
Patient 6 76 M Acute Rejected graft (no light perception due to
glaucoma)
Yes Yes 3
Patient 7 47 M Acute Neurotrophic keratitis Yes No 13
Other underlying, pre-existing conditions are shown in brackets
Biomaterials-enabled cornea regeneration
MM Islam et al.
2
npj Regenerative Medicine (2018) 2 Published in partnership with the Australian Regenerative Medicine Institute
1234567890():,;
Implants in mini-pigs
RHCIII-MPC hydrogels (6.75 mm in diameter, 500 µm thick) were
implanted the corneas of Göttingen mini-pigs by ALK, replacing
the epithelium and anterior stroma but leaving the endothelium
and posterior stroma intact. Implanted corneas were optically
transparent like healthy, untreated control corneas at 12 months
post-operation (Fig. 1a). In vivo confocal microscopical examina-
tion of the neo-corneas showed regenerated epithelium, stroma,
and nerves (Supplementary Fig. 1), conrming the safety and
efcacy reported in previous animal studies.
18,20
Ultrastructural
imaging using serial block face scanning electron microscopy
(SBF-SEM) showed that both implanted corneas and unoperated
controls had very similar epithelia and stromas. The epithelia in
both had a well-dened layer of basal cells and layers of attened
interconnecting cells (keratocytes) were evident within the stroma
(Fig. 1a).
As assessment of visual acuity or best-corrected visual acuity
(BCVA) in mini-pigs was not possible, we examined the optical
similarity of regenerated neo-corneas to that of healthy controls.
Under white light illumination, the measured light transmission
and backscatter values of the regenerated neo-corneas at
12 months post-operation did not differ signicantly from that
of the healthy, unoperated corneas (Figs. 1b, c; P> 0.05).
Furthermore, when examined over a range of visible light
wavelengths (450650 nm), no signicant differences in light
transmission were detected between the operated and un-
operated corneas (P> 0.05). However, the gradual drop in light
transmission with decreasing wavelength, which was seen in both
the operated and un-operated corneas, appeared to be slightly
more pronounced in the regenerated neo-corneas (Fig. 1b). Values
for percentage backscatter of light in the operated and
unoperated corneas were almost identical over the entire visible
light spectrum (Fig. 1c).
Implants in patients
An open-label, rst-in-human observational study was conducted
following ISO 14971 - Medical devicesApplication of risk
management to medical devices, and ISO 14155:2011 - Clinical
investigation of medical devices for human subjectsGood
clinical practice. Clinical testing in Ukraine was performed in
following the Declaration of Helsinki and relevant laws of Ukraine,
following trial approval (ClinicalTrials.gov identier NCT02277054)
by the Bioethics Commission of the Filatov Institute of Eye
Diseases and Tissue Therapy of the National Academy of Medical
Sciences of Ukraine (FEI). In India, clinical testing was performed in
accordance with the Declaration of Helsinki, relevant laws of India
and after approval by the ethics committee (LEC 01-14-014) of the
LV Prasad Eye Institute (LVPEI) and trial registration at Clinical Trial
Registry-India (CTRI/2014/10/005114).
Seven unilaterally blind patients, aged 36 to 76 years old,
diagnosed with conditions putting them at high risk of rejecting
conventional corneal transplantation, and capable of providing
informed written consent were recruited. Supplementary Table 1
shows the inclusion and exclusion criteria. Patients were divided
into three groups based on the cause of corneal damage:
infection, burns (chemical or thermal) and other (immune/
degenerative disease) (Table 1B). The patients were also divided
into two groups based on their stage of diseaseacute phase
(with ulcers and erosions) and post-scarring (severe scarring in
Fig. 1 Regenerated corneas of Göttingen mini-pigs at 12-months
post-grafting with RHCIII-MPC compared to healthy, unoperated
control corneas. acontrol cornea versus RHCIII-MPC implanted
cornea both showing comparable optical clarity. Serial block face-
scanning electron microscopy (SBF-SEM) of single sections show
that the epithelium is multilayered with comparable morphology
including a layer of basal cells. Underlying the epithelium are
stromal keratocytes arranged in lamellae. Scale bars, 50 µm. 3D
reconstructions of the corneas show that both regenerated neo-
cornea and healthy control comprise stromas with keratocytes
arranged in highly ordered lamellae. bLight transmission prole of
regenerated neo-corneas compared to healthy contralateral cor-
neas. cBackscatter prole of regenerated neo-corneas compared to
healthy contralateral corneas
Biomaterials-enabled cornea regeneration
MM Islam et al.
3
Published in partnership with the Australian Regenerative Medicine Institute npj Regenerative Medicine (2018) 2
Table 2. Patient outcomes after RHCIII-MPC implantation: (A) Clinical outcomes after RHCIII-MPC implantation at last follow-up; (B) Symptoms before surgery and at last follow-up after RHCIII-MPC
implantation
A
Patient no. Graft
diam.
(mm)
Suture
removal
(weeks
after
surgery)
Full
epithelial
coverage
(weeks after
surgery)
BCVA
pre-op
LogMAR
BCVA at
last
follow-up,
LogMAR
IOP at last
follow-up in
the
operated
eye (mmHg)
IOP at last
follow-up
fellow eye
(mmHg)
Schirmer
test at last
follow-up
operated
(mm/5 min)
Schirmer
test at last
follow-up
fellow (mm/
5 min)
Corneal
pachymetry
Pre-op (µm)
Corneal
pachymetry at
last follow-up
(µm)
Neovascularization of
implant area at last
follow-up
Follow-up
(months)
1 6 8 4 LP 0·52 17 15 10 15+ 250 470 No 24
2 8 3 12 1.6 0.1 14 12 16 20 484 270 No 35
3* 8 3 1.6 1·0 NA NA NA NA 550 NA NA 1.5
4 7 12 50 LP 1.7 13 14 15+ 15+ 1200 260 Yes 24
5 5 6 7 LP 1·3 19 18 6 10 320 320 Yes 14
6 4 12 48 NLP NLP 25 15 14 14 410 560
**
Yes 2 4
7 4 n/r 4 LP 1.3 16 16 9 8 220 1400 Yes 24
B
No. Photophobia / pain Tearing Redness
Before After Before After Before After
1+ ++
2−−−−−−
3NA −−NA
4+ ++
5§ + ++
6+ ++
7+ ++
*Patient 3 dropped out of the trial due to an unrelated fungal infection that was treated by penetrating keratoplasty. His last follow-up examination was at 1.5 months post-operation
**18 months data
LPlight perception; NLP no light perception, n/r not removed
notes. +’—symptom is present, ’—symptom is not present, NA’—data not available
§Patient 5 did not come in for his 24 m follow-up but reported no symptoms when interviewed over the telephone by surgeon, OB at 24 m
Biomaterials-enabled cornea regeneration
MM Islam et al.
4
npj Regenerative Medicine (2018) 2 Published in partnership with the Australian Regenerative Medicine Institute
need of scar revision). Before implantation, all acute patients
suffered from chronic, recurrent episodes of pain accompanied by
redness, photophobia, and tearing related to corneal de-
epithelialization (Table 2B). Patients with severe scarring were
asymptomatic.
Apart from one patient who was excluded from the study early
on, all implants were well-tolerated over a follow-up period of 24
± 6.6 months (range of 1435 months) without immunosuppres-
sive steroids beyond four weeks of prophylaxis, compared to up to
12 months of steroids for conventional PK allografts and even
longer for high-risk grafts.
Clinical outcomes at the last follow-up varied depending on
initial diagnosis (Table 2A). Patients with ulcers or scarring due to
infection (Patients 13) showed the most improvement, followed
by those with burns (Patients 45). The two patients with immune
and degenerative disorders, i.e., previously rejected graft (Patient
6) and neurotrophic keratitis (Patient 7) performed most poorly.
Overall, epithelial cell migration over the implants took
450 weeks post-operation, being signicantly slower in patients
with diagnosed stem cell deciency, but the healed ocular surface
remained stable in all patients (Table 2A).
In Patients 13, who had an ulcer or severe scarring due to
infections, the implants were well-tolerated and stably incorpo-
rated. They remained relatively clear and edema-free (Fig. 2).
Patient 1s vision improved from near blindness (light perception)
to 1.3 LogMAR at 2 weeks post-operation, to 0.7 LogMAR at three
months and 0.52 LogMAR at eight months post-operation
(moderate vision loss). Her vision remained stable over the
24 months follow-up period (Table 2A). In vivo confocal
microscopical examination showed that she had fully regenerated
epithelium and stroma, and her endothelium remains healthy (Fig.
3). A few corneal nerves were visible (Fig. 3). At 1-week post-
operation, Patient 2s vision had improved from 1.6 to 1.1 LogMAR.
Complete epithelial coverage of the implant occurred over
12 weeks (Table 2A). The implant remained clear, free of edema
and neovascularization. BCVA improved to 0.1 LogMAR (normal
vision) by 6 months post-operation and remained stable over a
follow-up period of 35 months. Ultrasound biomicroscopy and
optical coherence tomography conrmed the preservation of the
cornea curvature in Patients 1 and 2 that was restored by
implantation (Supplementary Fig. 2). The vision of Patient 3
improved from 1.6 to 1.0 LogMAR at one-month post-operation
(Table 2A). Unfortunately, he developed fungal keratitis at six
weeks post-operation. Although this was deemed unrelated, as
pathology ndings showed the implant was untouched by fungus.
However, the patient required therapeutic penetrating kerato-
plasty and was excluded from the study. All three patients had
normal intraocular pressure (IOP).
In Patient 4, who had an alkali burn, healing was accompanied
by implant thinning due to delayed epithelialization. Vision
improved slightly from light perception to 2.0 LogMAR at 2 months
and 1.7 LogMAR at last follow-up at 24 months, but the patient
was still considered blind (Table 2A). Patient 5, who had a thermal
burn pre-operatively, showed improved vision from near blind-
ness (light perception) to 1.4 LogMAR at nine months post-
operation and slightly decreased to 1.52 LogMAR (severe vision
loss) at last follow-up at 14 months. It should be noted, however,
that this patient also had a senile cataract that had progressed
during the follow-up period, impeding vision despite an almost
clear cornea. IOP was normal in both patients 4 and 5. Supercial
vessels were seen in these corneas, which were previously
vascularized and with limbal epithelial stem cell deciencies (Fig.
2). These vessels were associated with invading adjacent
conjunctiva. However, ultrasound biomicroscopy conrmed the
preservation of the cornea curvature restored by implantation in
both patients (Supplementary Fig. 2).
Patient 6 was blind due to glaucoma and had no light
perception. He was grafted for symptom relief due to ulceration
of a rejected corneal graft. Although the implant remained
relatively clear, it was encircled by blood vessels, and these had
invaded the implant margin at the 67oclock position. In Patient
7, the implant site was thickened considerably by epithelial
hyperplasia causing the graft to become opaque, but the
regenerated corneal tissue remained stable. The vision of Patient
7 was 1.4 LogMAR at 1-month post-operation, and despite cornea
thickening remained stable throughout the follow-up and reached
1.3 LogMAR at last follow-up at 24 months post-operation. IOP
was within the normal range for Patient 7 but not Patient 6 who
had glaucoma before surgery (Table 2A).
Most notably, however, all ve patients with acute disease and
suffering from pain, irritation, and photophobia due to the
ulceration, became asymptomatic within 12 weeks post-
operation and remained as such at the last follow-up (Table 2B).
Before surgery, all acute phase patients had reduced touch
sensitivity from slight to the total absence of response (Fig. 3b).
After surgery, sensitivity in all patients was restored close to levels
observed in their healthy contralateral corneas, including the
individual with degenerative neurotrophic keratitis (Patient 5).
KruskalWallis test showed a signicant difference between
Fig. 2 Patient corneas before and after grafting with RHCIII-MPC implants at last follow-up. Patients are divided into three groups based on
their pre-operative diagnoses: infection (herpes simplex viral and fungal keratitis), burns (alkali and thermal) and other (failed graft and post-
stroke neurotrophic keratitis). Post-operation, regenerated neocorneas from Patients 1 and 2 are mostly clear. In Patients 3 and 4, where stem
cell deciency is present, some supercial vessels concurrent with conjunctival invasion are seen. Patient 5 has a mostly clear cornea encircled
by blood vessels but has invaded in one quadrant, while Patient 6s cornea remains hazy. Patient 2 has an unrelated nasal pterygium
Biomaterials-enabled cornea regeneration
MM Islam et al.
5
Published in partnership with the Australian Regenerative Medicine Institute npj Regenerative Medicine (2018) 2
average pre-operative sensitivity compared to contralateral eyes
(p< 0.05).
DISCUSSION
RHCIII-MPC implants have been evaluated in a range of animal
models including mice,
28
rabbits
18
and mini-pigs.
20
The mouse
implantation model is a rejection model and only provides
information on implant performance relative to allografting.
28
Rabbits have been used extensively in the pre-clinical testing of
new corneal implants. For example, in a well-established alkali
burn model that simulates severe pathology,
29
we were able to
show that the addition of the inammation suppressing MPC to
RHCIII biosynthetic corneas resulted in the implants remaining
stably incorporated and clear.
18
In contrast, burned corneas
grafted with RHCIII only, like allografts, were vascularized.
18
However, rabbit corneas differ from human corneas in that they
are thinner, have no Bowmans membrane and their endothelial
cells proliferate readily.
30,31
The pig cornea is structurally and
mechanically closer to the human cornea,
5,32
in that it possesses a
Bowmans membrane and an endothelium with minimal pro-
liferative capacity.
In the present study, ultrastructural examination of the
regenerated neo-cornea after RHCIII-MPC implantation in mini-
pigs showed a micro-architecture that resembled that of healthy,
unoperated corneas. The implants had stimulated the pigs
endogenous corneal progenitor cells to migrate into the implant,
proliferate and recreate a neo-cornea. Optically, the regenerated
neo-corneas also matched the healthy, unoperated corneas in
allowing light transmission through the tissue with minimal back
scatter.
As in the pre-clinical mini-pig study, the implants successfully
stimulated endogenous cells to affect corneal repair in all of the
six patients that completed the study. In most patients, the
restored corneal surface led to improvements in BCVA that were
maintained throughout the entire follow-up period of 14 to
35 months. The exception to this was Patient 6 who was blind due
to glaucoma and therefore not expected to regain vision. All of the
Fig. 3 Regenerated patient corneas. aIn vivo confocal images of the regenerated cornea from Patient 1 at 24 months post-operation,
showing the regenerated epithelium, regenerating nerve (arrowhead) and stroma. The unoperated endothelium remains intact. bChanges in
corneal touch sensitivity before and after RHCIII-MPC implantation as measured by Cochet-Bonnet aesthesiometry. The average pressure
required to elicit a blink response from corneas before surgery, after implantation, and in comparison to the normal, healthy corneas. Touch
sensitivity is inversely related to the pressure needed to elicit a blink response from the patients. Note: *p<0.05 compared to unoperated
contralateral eyes (KruskalWallis test with Dunns correction for multiple comparisons)
Biomaterials-enabled cornea regeneration
MM Islam et al.
6
npj Regenerative Medicine (2018) 2 Published in partnership with the Australian Regenerative Medicine Institute
other patients showed vision improvement, but only two showed
signicant improvement from legally blind to vision impaired, and
one gained normal vision. The vision of one of these patients was
hampered by an unrelated senile cataract in his lens. Stem cell
deciency and conjunctival invasion were the main barriers to
vision improvement. As the implants restored corneal stromal
integrity, shown by the relief from pain, discomfort and
photophobia, patients with stem cell deciency can potentially
undergo subsequent treatment to restore vision, e.g., if sufcient
funding can be raised. Nevertheless, the healing and in-growth of
cells to form neo-corneal tissue occurred and remained stable
over the entire follow-up period, as seen in pre-clinical mini-pig
model.
Epithelial coverage of RHCIII-MPC implants was signicantly
slower than reported for patients grafted with donor corneas by
ALK or human amniotic membranes (HAM) to treat corneal
thinning.
33
The delayed epithelial closure is most likely due to
retention techniques used
15
as overlying sutures or excess glue
retarded epithelial coverage creating an epithelial defect-like
situation that most likely initiated an early inammatory response.
This was likely followed by induction of metalloproteinases and
subsequent localized implant thinning. Corneal thickening due to
epithelial hyperplasia, such as seen in one patient has been
previously observed to a lesser extent in patients after refractive
surgery
34
and laboratory animals after corneal transplantation.
35
Nevertheless, we have shown that RHCIII-MPC implants can be
glued (albeit with caution with amounts used), opening up
possibilities for future use as patches that may circumvent the
need for transplantation.
A recent study comparing treatments for corneal ulceration
showed that 100% of patients grafted with donor corneas by ALK
became neovascularised.
33
HAM grafts suppressed neovascular-
ization but the membranes disintegrated within 6 months.
33
Here,
neovascularization was observed in the pre-operatively neovascu-
larised corneas with limbal epithelial stem cell deciencies.
Neovascularisation concurrent with conjunctival invasion is
common in corneas with limbal stem cell deciency.
36
These
patients had severely damaged or scarred corneas so it is not
surprising that their post-operative results and those of three
earlier pilot patients
19
were not as good as the outcomes seen
here and elsewhere in healthy mini-pig corneas
20
or even rabbit
corneas after alkali injury.
18
This discrepancy between animal data
and clinical outcomes is typical when translating promising animal
results into clinical application. Present results also show that
recovery from a severe pathology has a different course to that
seen in low risk patients.
11,15
In low-risk patients, vision restoration
was the indication for grafting but in high-risk patients, corneal
surface restoration and symptom relief were the main indications
for treatment of acute patients although vision improvement was
the goal for scarred patients.
Very few therapeutic interventions promoting nerve regrowth
into the cornea exist.
37
In donor corneas grafted by ALK or PK,
touch sensitivity remains low post-operatively.
37
In our pre-clinical
pig models, nerve regeneration was a main feature. We also noted
regeneration of the different corneal nerve sub-types in guinea
pigs grafted with collagen-MPC implants.
38
Aesthesiometry
performed on the acute phase patients showed that touch
sensitivity, which is correlated with nerve function,
39
was restored
to near-normal levels in all ve patients. Surprisingly, the
functional restoration was also observed in Patient 7 who had
no pre-operative touch sensitivity due to neurotrophic keratitis, a
degenerative condition. HAM treatment has been reported to
increase sensitivity in 9 out of 10 patients with similar proles to
our ve patients.
40
However, these patients had higher pre-
operative sensitivity than our patients and their nal sensitivity
was just below normal. HAM contains a high concentration of
growth factors and likely trophic factors that suppress inamma-
tion,
40
while the MPC used in our implants has reported anti-
inammation effects.
17
Taken together, both observations
strongly suggest that suppression of persistent inammation in
chronically ulcerated corneas facilitated nerve regeneration.
Following corneal wounding, elaboration of disorganized,
unaligned mainly type III collagen occurs to form a scar.
41
Here,
bridging the wound gape with an organized matrix comprising
aligned type III collagen,
22
however, appears to provide a template
for controlled in-growth of stromal cells that in turn allows for
regeneration of an optically clear cornea. Furthermore, complica-
tions such as graft rejection (45% in high-risk patients) are likely
elicited by the vascularized or inamed host cornea reacting
against the presence of allogeneic cells,
42
were circumvented by
use of cell-free implants. The inammation inhibiting MPC
networked within the implants most likely contributed to the
capacity of RHCIII-MPC implants to remain quiescent in the
immunogenic corneas, allowing stable restoration of the ocular
surface.
It would also be pertinent to mention that even though stem
cell replacement is an option in more afuent settings, there is still
an issue with allogeneic transplantation that has not been solved.
Systemic immune suppression is required for allografted corneal
limbal epithelial cells, with reported severe side-effects that
include anemia, hyperglycemia, elevated creatinine, and elevated
levels of liver function markers.
43
Furthermore, if the damage
extends into the stroma, as seen in our patients, a second surgery
requiring a human donor cornea is still needed.
43
Here, the cell-
free implants stimulated endogenous stem cells to affect the
repair in both stroma and epithelium, together with nerve
regeneration without immune suppression beyond prophylaxis.
With clinical application as the goal, synthetically-produced
recombinant human collagen was used to circumvent immuno-
genic reactions that can occur with animal-derived collagen in
susceptible patients due to their non-human protein composi-
tion,
44
and pathogen transmission risks. Furthermore, our
collagen-based biomaterials made for the cornea have been
modied for use in other systems,
4547
as similar conditions such
as skin ulcers in legs of diabetics, are enormous problems in
LMICs.
48
While conrmation in a larger number of patients is needed, we
nevertheless demonstrate that implantation with cell-free RHCIII-
MPC implants is a safe, reliable option for treating patients at high
risk of donor allograft rejection; providing pain relief, and
regenerating tissue and nerves. The clinical outcomes in humans
although not as ideal as those in pre-clinical studies, nevertheless
were predictable by use of wild-type mini-pigs as a model.
METHODS
RHCIII-MPC corneal implants
European Medical Devices Directive MDD 93/42/ECC and its associated ISO
standards were followed. For clinical evaluation, implants were made
within an EU Class A laminar ow hood located in a Class B certied and
monitored cleanroom at Vecura AB, Karolinska University Hospital,
Huddinge, Sweden. Aseptic working conditions and sterile chemicals and
reagents were used in the cleanrooms for implant production. Water for
injection (WFI, HyClone, Utah, USA) was used to make up all solutions. For
pre-clinical animal testing, implants were made under aseptic conditions in
certied tissue culture hoods approximating Class A conditions.
Very briey, implants were fabricated by mixing an 18% (wt/wt) aqueous
solution RHCIII (FibroGen Inc., San Francisco, CA) with 2-
methacryloyloxyethyl phosphorylcholine (MPC, Paramount Fine Chemicals
Co. Ltd., Dalian, China) and poly(ethylene glycol) diacrylate (PEGDA, Mn
575, Sigma-Aldrich) in a morpholinoethane sulfonic acid monohydrate
(MES, Sigma-Aldrich, MO) buffer. The ratio of RHCIII:MPC was 2:1 (wt/wt)
and PEGDA:MPC was 1:3 (wt/wt). Polymerization initiators ammonium
persulphate (APS; Sigma-Aldrich) and N,N,N,N-tetramethylethylenediamine
(TEMED, Sigma-Aldrich) at ratios of APS:MPC = 0.03:1 (wt/wt), APS:TEMED
(wt/wt) = 1:0.77, crosslinker, N-(3-dimethylaminopropyl)-N-ethylcarbodii-
mide (EDC; Sigma-Aldrich) and its co-reactant, N-hydroxysuccinimide
Biomaterials-enabled cornea regeneration
MM Islam et al.
7
Published in partnership with the Australian Regenerative Medicine Institute npj Regenerative Medicine (2018) 2
(NHS; Sigma-Aldrich) was then mixed in. The resulting solution was
dispensed into cornea-shaped moulds and cured. After demoulding, they
were washed thoroughly with phosphate buffered saline (PBS) and placed
into vials of aseptic PBS containing 1% chloroform, which were sealed to
maintain sterility.
During quality control, each implant was visually inspected for
manufacturing aws, discolouration or unwanted particulates under a
dissection microscope. Those with imperfections were rejected. Batch
controls were also performed on randomly selected implants, one from
each batch (1 out of every 4 samples). Implants tested were found sterile,
with endotoxin levels below the requirement of <0.5 EU/ml cut-off
requirement for implantable medical devices
49,50
by a Swedish Medical
Products Agency approved laboratory (Apotek Produktion & Laboratorier
AB, Stockholm, Sweden). Implants were tested to ensure their exibility.
Refractive index measurements were made using an Abbe 60 series
Refractometer (Bellingham & Stanley Ltd., Kent, UK) calibrated against a
silica test plate of known refractive index at room temperature. Light
transmission through implants was measured by a UV-VIS spectro-
photometer (U-2800 UV-VIS, Hitachi, Tokyo, Japan). Implant materials
(5 × 15 mm) were placed within a quartz cuvette and positioned within the
spectrophotometer in such a way that the beam was perpendicular to the
hydrogel. Light absorption by the hydrogel was measured in the visual
spectrum (400 to 700 nm). The equilibrium water content of hydrogels was
determined to ensure uniformity. Hydrated hydrogels were removed from
solution; the surface gently blotted dry and then immediately weighed on
a microbalance to record the wet weight (W
0
) of the sample. The hydration
of the hydrogels shown in Table S1 was calculated using a dry weight
obtained by drying the samples at 60 degrees until constant mass was
achieved (W). The equilibrated water content of the hydrogels (W
t
) was
calculated according to the following equation: W
t
=(W
0
W)/W
0
× 100%
The thermal stability of the implants was examined by measuring the
denaturation temperature using a differential scanning calorimeter (DSC,
Q20, TA Instruments, New Castle, UK). Heating scans were recorded in the
range of 10 to 80 °C at a scan rate of 1 °C min
1
. The samples ranging in
mass from 3 to 5 mg were surface dried and hermetically sealed in pans.
The denaturation temperature at the maximum of the endothermic peak
was measured. Implants needed to pass both the visual inspection and
batch sampling to be acceptable for clinical evaluation.
Pre-clinical evaluation in mini-pigs
The study was carried out by Adlego Biomedical AB (Solna, Sweden), an
approved GLP certied pre-clinical testing CRO. The methods performed
were approved by the regional ethics committee (Stockholm norra
djurförsöksetiska nämnd) and in compliance with the Swedish Animal
Welfare Ordinance and the Animal Welfare Act and OECD Principle of
Good Laboratory Practice, ENV/MC/CHEM (98) 17, 1997. Corneal implanta-
tion was performed in four female Göttingen SPF mini-pigs (Ellegaard,
Denmark), 56 months old. Two weeks before surgery the animals were
given a thorough clinical examination to establish a baseline for corneal
health. During the surgery, the right cornea of each pig was trephined with
a 6.5 mm diameter Barron Hessberg trephine to a depth of 500 μm. The
corneal button was dissected lamellarly with a diamond knife and
removed. A RHCIII-MPC implant, trephined to 6.75 mm in diameter was
put into the wound bed. Human amniotic membrane (HAM; from the
Cornea Bank, St. Eriks Eye Hospital, Stockholm, Sweden) was placed over
the implant to suppress undesired inammation and the implants were
held in place with overlying sutures. An antibacterial and anti-
inammatory ophthalmic solution (Tobrasone®, suspension with 3 mg/mL
dexamethasone and 1 mg/mL tobramycine, Alcon, Sweden) was adminis-
tered post-operatively. Each pig also received buprenorphine i.v. (0.05 mg/
kg Vetergesic®, Orion Pharma, Finland). Subsequently, the operated eyes
were treated 3 times daily with 1 drop Tobrasone®. Unoperated
contralateral corneas served as controls.
Another four mini-pig implantations in Canada were approved by the
University of Ottawa animal ethics committee (Protocol E-19) in
compliance with the animal care guidelines of the Association for Research
in Vision and Ophthalmology, and performed using similar methods. These
corneas were used for measurement of optical properties.
At 12 months post-operation, after clinical data acquisition, the animals
were euthanized. Both implanted and control corneas were harvested.
Histopathological evaluation of GLP animals were performed by a Swedish
MPA approved veterinary pathologist, BioVet AB (Sollentuna, Sweden).
Optical properties such as % light transmission through the regenerated
neo-corneas and the control unoperated eyes, and amount of back
scattered light (%) were determined by measuring freshly excised corneas
on a custom-built instrument equipped with a quartz halogen lamp for
white light measurements as we previously reported.
51
The mini pig corneal control and RHCIII-MPC implanted samples were
xed using 2.5% glutaraldehyde/2% paraformaldehyde in 100 mM
cacodylate buffer pH 7.2 at room temperature for 12 h. The samples were
then processed for the generation of high backscatter electron contrast for
SBF-SEM as previously described.
52
The samples were then transferred to a
Zeiss Sigma VP FEG SEM equipped with a Gatan 3View2XP system, where
data sets of 1000 images were acquired of the block surface every 100 nm
through automated sectioning. Each image on the SBF-SEM was acquired
at 4 K × 4 K pixels, at a pixel resolution 32 nm and a pixel dwell time of 8 µs,
using an accelerating voltage of 3.4 keV in low vacuum variable pressure
mode (28 Pa). Imaging data was acquired from a 134.93 µm × 134.94 µm
region of interest. Selected serial image sequences were extracted from
the image data and 3D reconstructions were generated using Amira
6.1 software (FEI Merignac, France).
Patient surgeries and follow-up
At FEI, after providing written informed consent, patients were each
grafted with a 350 µm thick RHCIII-MPC implant by ALK after manual
excision of 300 μm of pathologic epithelium and stroma, except for Patient
2 who had a swollen, calcied cornea, and required excision of 900 µm of
pathologic tissue. The excised pathological tissues, where available, were
processed for histopathological examination. In two patients, only detritus
was present so histopathology was not possible. The implants were
retained by overlying sutures placed peripherally.
15
After surgery, grafted
patients received antibiotic eye drops (ooxacin ophthalmic solution, 0.3%,
Bausch & Lomb GmbH, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH,
Berlin, Germany) 4 times daily, short-term mydriatic (cyclopentolate 1%,
Sentiss Pharma Pvt. Ltd., Gurgaon, India) and non-steroidal anti-
inammatory drug (Indometacin 0.1%, Bausch & Lomb GmbH) for 2 weeks,
followed by topical antiseptic (chlorhexidine bigluconate 0.02%, Farmacia,
Lugansk, Ukraine) and steroid (dexamethasone 0.1%, s.a. Alcon-Couvreur n.
v., Puurs, Belgium) 3 times per day for the rst week and tapered over
3 weeks to reduce post-operative inammation. Patients wore bandage
contact lenses until epithelial regeneration was complete. Sutures were
removed between 3 and 12 weeks post-operatively in all patients except
Patient 5, where the epithelium had grown over the sutures.
After providing written informed consent, LVPEI patients were each
grafted with an 8 mm diameter, 350 µm thick implant by ALK after
femtosecond laser (VisuMax, Carl Zeiss Meditec, Jena, Germany) assisted
excision of 350 μm of pathologic epithelium and stroma. The implant was
prepared using femtosecond laser and was retained using brin glue aided
by overlying sutures placed peripherally.
15
After surgery, patients were
given moxioxacin HCl ophthalmic solution 0.5% (Alcon, Fort Worth, USA)
4 times per day until re-epithelialization and topical 1 % Prednisolone
acetate ophthalmic solution, (Allergan, Irvine, USA) 4 times per day for the
rst week and tapered over 3 weeks to reduce postoperative inammation.
Patients wore bandage contact lenses until re-epithelialization. Sutures
were removed at 3 weeks post-operation.
All patients were assessed weekly until 1 month and then at 3 and
6 months, and then at 34 monthly intervals thereafter. Assessments of
BCVA, IOP, tear production (Schirmer test), were made and slit-lamp
microscopy was performed with and without uorescein to conrm
epithelial integrity. Patients also underwent ultrasound biomicroscopy
(Aviso, Quantel Medical, Cournon-dAuvergne, France) and in vivo confocal
microscopy (ConfoScan4, Nidek, Japan) at FEI. At LVPEI, patients were
examined by anterior segment optical coherence tomography (RTVue,
Optovue Inc, Fremont, USA) to assess the cornea and anterior chamber.
Nerve regeneration as evaluated by regaining of corneal touch sensitivity,
was assessed using a Cochet-Bonet aesthesiometer (Luneau Oftalmologie,
France). Very briey, the aesthesiometer uses a 0.12 mm diameter nylon
lament to obtain a blink response.
39
Statistical analyses
Quality control data in Table 1are expressed as means ± SD. For optical
properties measured for pig corneas, pairwise t-tests for white light and
each wavelength was performed, with a Bonnferroni correction. Measure-
ments of corneal nerve sensitivity were statistically evaluated using a
KruskalWallis test with Dunns correction for multiple comparisons. P
values of <0.05 were considered signicant.
Biomaterials-enabled cornea regeneration
MM Islam et al.
8
npj Regenerative Medicine (2018) 2 Published in partnership with the Australian Regenerative Medicine Institute
Data availability statement
Data analysed during the current study are included in this article and its
supplementary information les. Clinical trial protocols are available
through Clinicaltrials.gov (ID: NCT02277054) and the WHO International
Clinical Trials Registry Platform (http://apps.who.int/trialsearch/; ID: CTRI/
2014/10/005114). The individual patient data that support the ndings are
not publicly available due to patient condentiality. Datasets generated are
available from the corresponding authors (MG, KMM, NP, VSS) on
reasonable request.
ACKNOWLEDGEMENTS
We thank the following individuals from FEI: Borys Kogan, Olena Ivanovska, Galyna
Drozhzhyna for help with patient surgery and follow-ups, Oleksandr Artyomov and
Valeriy Vit for histopathology analyses, and Oleksandr Kovalchuk for US biomicro-
scopy analysis. We thank the following individuals from LVPEI: Savitri Maddileti and
Indumathi Mariappan. This work was supported by a Dept. of Biotechnology-Vinnova
Indo-Sweden collaborative health research project grant (BT/IN/Sweden/37/VS/2013
and dnr 2013-04645); Integrative Regenerative Medicine Centre (Linköping University
and Region Östergötland); Filatov Institute of Eye Diseases and Tissue Therapy; LV
Prasad Eye Institute; MRC program grant MR/K000837/1.
AUTHOR CONTRIBUTIONS
M.M.I., O.B., N.P., K.M.M., V.S.S., and M.G. planned the study. O.B., S.I., J.R., and V.S.S.
obtained the ethical/regulatory permission, signed informed consent for clinical
studies, performed the surgeries, follow-ups and analyzed clinical results. P.F., O.B., P.
L., and C.L.H. performed the pig studies and analyses. M.M.I., E.I.A., S.H., W.L., K.M.M.,
and M.G. were responsible for development, production following GMP and quality
control of implants used in the clinical study. M.M.I., O.B., J.R., and M.G. wrote the
manuscript, all authors participated in revisions and nal approval.
ADDITIONAL INFORMATION
Supplementary information accompanies the paper on the npj Regenerative
Medicine website (https://doi.org/10.1038/s41536-017-0038-8).
Competing interests: The Ottawa Hospital Research Inst., Canada has led a
biomaterials patent for collagen-MPC implants licensed to Eyegenix, USA and
LinkoCare, Sweden.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional afliations.
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Biomaterials-enabled cornea regeneration
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npj Regenerative Medicine (2018) 2 Published in partnership with the Australian Regenerative Medicine Institute
... A summary of recent clinical and preclinical studies of corneal bioengineering technologies is given in Supplementary Table 4. Most clinical studies, while having achieved significant advances in biomaterial properties, have addressed relatively rare causes of corneal blindness such as chemical burns 54,55 , infections [55][56][57][58] , ulcers 59 or high-risk cases 54,55 with the goal to stabilize the condition and avoid blindness, but not to optimize vision. Only two clinical studies to date have addressed keratoconus, a condition that impairs millions globally, aiming to provide vision gains comparable to standard PK or DALK. ...
... A summary of recent clinical and preclinical studies of corneal bioengineering technologies is given in Supplementary Table 4. Most clinical studies, while having achieved significant advances in biomaterial properties, have addressed relatively rare causes of corneal blindness such as chemical burns 54,55 , infections [55][56][57][58] , ulcers 59 or high-risk cases 54,55 with the goal to stabilize the condition and avoid blindness, but not to optimize vision. Only two clinical studies to date have addressed keratoconus, a condition that impairs millions globally, aiming to provide vision gains comparable to standard PK or DALK. ...
... A summary of recent clinical and preclinical studies of corneal bioengineering technologies is given in Supplementary Table 4. Most clinical studies, while having achieved significant advances in biomaterial properties, have addressed relatively rare causes of corneal blindness such as chemical burns 54,55 , infections [55][56][57][58] , ulcers 59 or high-risk cases 54,55 with the goal to stabilize the condition and avoid blindness, but not to optimize vision. Only two clinical studies to date have addressed keratoconus, a condition that impairs millions globally, aiming to provide vision gains comparable to standard PK or DALK. ...
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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.
... At 4 years follow-up, the corneas were well integrated into the host tissue. Interpenetrating polymer-based CACs have also been evaluated in a human pilot study 14 , and in a clinical trial 15 and showed the capacity to stabilize corneas and improve vision in aggressive disease condition. Although CACs are highly biocompatible, their mechanical properties do not permit suturing of the implant and require overlying sutures for transplantation 13 . ...
... The relative fold change of HAdV-D37 DNA is depicted in Fig. 7a. The analysis through immunofluorescence assay also revealed a reduction in HAdV-D37 in the Coll 15 -PyKC 1 group compared to the control Coll 15 . Notably, fluorescence was less in the construct Coll 15 -PyKC 1 , followed by Coll 15 -PyKC 2 at 48 hpi (Fig. 7b). ...
... To fill the gap between supply and demand, much research has focused on developing an artificial cornea. Toward this end, CACs have been transplanted into humans 13,15 , in most cases they consisted of collagen crosslinked with EDC and NHS. Carbodiimide-based chemical crosslinkers, such as EDC-based methods are considered a standard strategy with low cytotoxicity, but the mechanical strength of crosslinked collagen is less than satisfactory. ...
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Development of an artificial cornea can potentially fulfil the demand of donor corneas for transplantation as the number of donors is far less than needed to treat corneal blindness. Collagen-based artificial corneas stand out as a regenerative option, having promising clinical outcomes. Collagen crosslinked with chemical crosslinkers which modify the parent functional groups of collagen. However, crosslinkers are usually cytotoxic, so crosslinkers need to be removed from implants completely before application in humans. In addition, crosslinked products are mechanically weak and susceptible to enzymatic degradation. We developed a crosslinker free supramolecular gelation strategy using pyrene conjugated dipeptide amphiphile (PyKC) consisting of lysine and cysteine; in which collagen molecules are intertwined inside the PyKC network without any functional group modification of the collagen. The newly developed collagen implants (Coll-PyKC) are optically transparent and can effectively block UV light, are mechanically and enzymatically stable, and can be sutured. The Coll-PyKC implants support the growth and function of all corneal cells, trigger anti-inflammatory differentiation while suppressing the pro-inflammatory differentiation of human monocytes. Coll-PyKC implants can restrict human adenovirus propagation. Therefore, this crosslinker-free strategy can be used for the repair, healing, and regeneration of the cornea, and potentially other damaged organs of the body.
... For use in patients with severe pathologies that put them at high risk of rejecting conventional donor transplantation, RHCIII implants incorporating a synthetic lipid polymer, 2methacryloyloxyethyl phosphorylcholine (MPC) that suppresses inflammation, were successfully tested in high-risk patients with ulcerated and badly scarred corneas (Hackett et al., 2011;Islam et al., 2013Islam et al., , 2015Kakinoki et al., 2014). In these firstin-human clinical studies, the implants were manufactured aseptically under Class 100 or ISO 5 conditions and stored in phosphate-buffered saline (0.1 M) containing 1% chloroform (C-PBS) to maintain sterility (Fagerholm et al., 2010(Fagerholm et al., , 2014Islam et al., 2018). This storage solution required an extensive washing procedure to remove the chloroform before surgery, after which they were further washed in antibiotics before use to ensure their sterility. ...
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Sterilization of biodegradable, collagen-based implants is challenging as irradiation sterilization methods can alter their mechanical properties. Electron beam (EB) irradiation is a terminal sterilization method that has been used for biologically-derived implants. Here, recombinant human collagen type III-phosphorylcholine (RHCIII-MPC) hydrogels were irradiated with EB doses of 17, 19, or 21 kGy and their subsequent biocompatibility and ability to promote regeneration in rabbit corneas was evaluated. Unirradiated hydrogels stored in 1% chloroform in phosphate-buffered saline (C-PBS) were the controls. There were no significant differences between irradiated and non-irradiated samples in optical or physical properties (tensile strength, modulus, elasticity), or the ability to support cell growth. However, irradiated implants were more sensitive to high levels of collagenase than unirradiated controls and the C-PBS implants had increased cell growth compared to EB and controls at 72 h. Corneal implants e-beamed at 17 kGy or e-beamed and subsequently frozen (EB-F) to increase shelf-life showed no adverse biological effects of the irradiation. EB, EB-F, and C-PBS implanted corneas all rapidly re-epithelialized but showed mild neovascularization that resolved over 6 months. The regenerated neo-corneas were transparent at 6 months post-operation. In vivo confocal microscopy confirmed normal morphology for the epithelium, stroma, sub-basal nerves and unoperated endothelium. Histology showed that all the regenerated corneas were morphologically similar to the normal. Immunohistochemistry indicated the presence of a differentiated corneal epithelium and functional tear film. In conclusion, the e-beamed corneal implants performed as well as non-irradiated control implants, resulting in fully regenerated neo-corneas with new nerves and without blood vessels or inflammation that may impede vision or corneal function. Therefore, a complete validation study to establish EB irradiation as an effective means for corneal implant sterilization prior to clinical application is necessary as a next step.
... Similarly, the patients who had medium and long duration of diagnosis were reducing the high risk of blindness of glaucoma patients. is finding was consistent with another study (French and Margo, 2010) [36][37][38]. e result shown that, medium and long duration of diagnosis were reducing the hazard of death or blindness. Glaucoma pain should be reported to an eye specialist on a regular basis so that the condition can be detected and treated before a longterm visual loss occurs. ...
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Background: Glaucoma is one of the most frequent vision-threatening eye diseases. It is frequently associated with excessive intraocular pressure (IOP), which can cause vision loss and damaged optic nerves. The main objective of this study was to model time to blindness of glaucoma patients by using appropriate statistical models. Study Design. A Retrospective Community-Based Longitudinal Study design was applied. Materials and Procedures. The data were obtained from Ophthalmology Department of JUSH from the period of January 2016 to August 2020. The glaucoma patient's information was extracted from the patient card and 321 samples were included in the study. To discover the factors that affect time to blindness of glaucoma patients', researchers used the Accelerated Failure Time (AFT) model. Results: 81.3 percent of the 321 glaucoma patients were blind. Unilaterally and bilaterally blinded female and male glaucoma patients were 24.92 and 56.38%, respectively. After glaucoma disease was confirmed, the median time to the blindness of both eyes and one eye was 12 months. The multivariable log-logistic accelerated failure-time model fits the glaucoma patient's time to blind dataset well. The result showed that the chance of blindness of glaucoma patients who have absolute stage of glaucoma, medium duration of diagnosis, long duration of diagnosis, and IOP greater than 21 mmHg were high with parameters (ϕ = 2.425, p value = 0.049, 95% CI [2.249, 2.601]), (ϕ = 1.505, p value = 0.001, 95% CI [0.228, 0.589]), (ϕ = 3.037, p value = 0.001, 95% C.I [2.850, 3.22]) and (ϕ 0.851, p value = 0.034, 95% C.I [0.702, 0.999]), respectively. Conclusion: The multivariable log-logistic accelerated failure time model evaluates the prognostic factors of time to blindness of glaucoma patients. Under this finding, duration of diagnosis, IOP, and stage of glaucoma were a key determinant factors of time to blindness of glaucoma patients'. Finally, the log-logistic accelerated failure-time model was the best-fitted parametric model based on AIC and BIC values.
... This may indicate that our protocol to decellularize GI tissues and prepare ECM hydrogels can exclude contamination of SEM and IEM hydrogels from pathogens in the GI tissues. Given that the endotoxin level acceptable for implantable medical devices is 0.5 EU/ml in the guideline of the Food and Drug Administration (FDA) 23 , our decellularized GI tissue-derived ECM hydrogels would be able to serve biomaterials with clinical feasibility. Next, we checked the immunogenicity of decellularized GI tissue-derived ECM hydrogels. ...
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Matrigel, a mouse tumor extracellular matrix protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to-batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal tissue-derived extracellular matrix hydrogels are suitable substitutes for Matrigel in gastrointestinal organoid culture. We found that the development and function of gastric or intestinal organoids grown in tissue extracellular matrix hydrogels are comparable or often superior to those in Matrigel. In addition, gastrointestinal extracellular matrix hydrogels enabled long-term subculture and transplantation of organoids by providing gastrointestinal tissue-mimetic microenvironments. Tissue-specific and age-related extracellular matrix profiles that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that extracellular matrix hydrogels derived from decellularized gastrointestinal tissues are effective alternatives to the current gold standard, Matrigel, and produce organoids suitable for gastrointestinal disease modeling, drug development, and tissue regeneration. The culture of gastrointestinal organoids relies on Matrigel that has several drawbacks for clinical application. Here, the authors report the feasibility of gastrointestinal tissue-mimetic matrices as effective alternatives to Matrigel for organoid culture and transplantation.
Chapter
The human cornea acts as a protective covering for the eye and plays an important role in light transmission into the eye for vision. Corneal defects due to trauma, infection, or disease can have detrimental effects on the vision, and severe cases lead to vision loss. Twenty-three million people are estimated to be affected by corneal blindness worldwide. Treatment involves corneal transplantation surgery, but there is a severe shortage of donor corneas worldwide. Furthermore, patients with severe pathologies risk rejecting conventional corneal transplantation, thus leaving them untreated. Therefore, there is an urgent need to develop new therapies to replace traditional corneal transplant surgery. This review focuses on recent potential biomaterials development for corneal regeneration and repair. It includes cell-based therapies, cell-free regeneration-inducing biomaterials, and injectable or in-situ gelation-based biomaterials for patients with a high risk of graft failure. It also consists of the emerging role of exosomes and extracellular vesicles in corneal infections and regeneration.
Article
Tissue engineering cornea has shown great clinical potential for cornea reconstruction, but efficient recovery of natural structure and physiological function remains great challenges. In this study, the acellular porcine corneal stroma (APCS) was prepared by a phospholipase A2 decellularization method and further crosslinked with aspartic acid (Asp). The modified APCS-Asp scaffold showed significant increase of hydration degree, ultrastructure regularity, corneal viscoelasticity and anti-degradation ability compared to APCS. Autologous rabbit limbal tissue was pre-treated by tumor necrosis factor-alpha (TNF-α) and collagenase IV, and the pretreated primary limbal stem cells (LSCs) were cultured with embryonic stem cells conditioned medium (ESCM), and LSCs showed 3D cell sphere structure and improved stem cell properties compared to the control group. The auto-tissue engineering lamellar cornea (ATELC) was quickly reconstructed by using peptide hydrogel with a dynamic culture system. With intact and functional epithelial cell layer, the reconstructed ATELC quickly recovered natural optical characteristics 1 week post transplantation in the rabbit lamellar keratoplasty model and satisfying neural regrowth as well as favorable stromal repopulation were observed in the transplanted eyes in the 6 months following up post-surgery. In summary, this study provides a comprehensive optimized reconstruction strategy for ATELC, which maybe similar medical application to natural cornea.
Article
Natural biomaterials are crucial in ocular tissue engineering because they allow cells to proliferate, differentiate, and stratify while maintaining the typical epithelial phenotype. In this study, membranes as dressings were formed from silk fibroin and collagen (Co) extracted from fish skin and then modified with carbodiimide chemical cross linker in different concentrations. The samples were evaluated by different analyses such as structural, physical (optical, swelling, denaturation temperature, degradation), mechanical, and biological (viability, cell adhesion, immunocytochemistry) assays. The results showed that all membranes have excellent transparency, especially with higher silk fibroin content. Increasing the cross linker concentration and the ratio of silk fibroin to Co increased the denaturation temperature and mechanical strength and, conversely, reduced the degradation rate and cell adhesion. The samples did not show a significant difference in toxicity with increasing cross linker and silk fibroin ratio. In general, samples with a low silk fibroin ratio combined with cross linker can provide desirable properties as a membrane for corneal wound healing.
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Corneal diseases are the third most prevalent cause of blindness after cataract and glaucoma. It is estimated that about 5 million people in the world are affected by bilateral corneal blindness with an additional 23 million with unilateral blindness. Cornea transplantation is the standard practice for the management of various cornea related pathologies like fibrosis, ulcers, keratitis, etc. The high transplant cost, increased risk of graft failure/rejection, and long waiting list due to limited availability of good quality donor cornea imposes a huge clinical burden. Recently, biofabrication technologies are gaining a lot of attention because of their potential to direct hierarchical assembly of three-dimensional (3D) biological structures for tissue construction for various biomedical and clinical applications. In this regard, 3D bioprinting, which involves layer-by-layer deposition of acellular or cell-laden bioink in a specific pattern corresponding to the organotypic morphology of tissues/organs, has been extensively investigated for the fabrication of corneal substitutes. In addition to this methodology, novel biofabrication techniques have been explored for the fabrication of corneal tissues using bioinks with optical and mechanical performances comparable to native cornea tissue. In this review, we highlight the recent advances and offer future perspectives in the fabrication of corneal tissue equivalents that can be potentially employed for effective clinical repair, reconstruction, and regeneration of the cornea.
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Cornea as the outermost layer of the eye is at risk of various genetic and environmental diseases that can damage the cornea and impair vision. Corneal trans-plantation is among the most applicable surgical procedures for repairing the defected tissue. However, the scarcity of healthy tissue donations as well as transplantation failure has remained as the biggest challenges in confront of corneal grafting. Therefore, alternative approaches based on stem-cell transplantation and classic regenerative medicine have been developed for corneal regeneration. In this review, the application and limitation of the recently-used advanced approaches for regeneration of cornea are discussed. Additionally, other emerging powerful techniques such as 5D printing as a new branch of scaffold-based technologies for construction of tissues other than the cornea are highlighted and suggested as alternatives for corneal reconstruction. The introduced novel techniques may have great potential for clinical applications in corneal repair including disease modeling, 3D pattern scheming, and personalized medicine. K E Y W O R D S
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Regeneration of the corneal surface after an epithelial insult involves division, migration, and maturation of a specialized group of stem cells located in the limbus. Several insults, both intrinsic and extrinsic, can precipitate destruction of the delicate microenvironment of these cells, resulting in limbal stem cell deficiency (LSCD). In such cases, reepithelialization fails and conjunctival epithelium extends across the limbus, leading to vascularization, persistent epithelial defects, and chronic inflammation. In partial LSCD, conjunctival epitheliectomy, coupled with amniotic membrane transplantation, could be sufficient to restore a healthy surface. In more severe cases and in total LSCD, stem cell transplantation is currently the best curative option. Before any attempts are considered to perform a limbal stem cell transplantation procedure, the ocular surface must be optimized by controlling causative factors and comorbid conditions. These factors include adequate eyelid function or exposure, control of the ocular surface inflammatory status, and a well-lubricated ocular surface. In cases of unilateral LSCD, stem cells can be obtained from the contralateral eye. Newer techniques aim at expanding cells in vitro or in vivo in order to decrease the need for large limbal resection that may jeopardize the "healthy" eye. Patients with bilateral disease can be treated using allogeneic tissue in combination with systemic immunosuppressive therapy. Another emerging option for this subset of patients is the use of noncorneal cells such as mucosal grafts. Finally, the use of keratoprosthesis is reserved for patients who are not candidates for any of the aforementioned options, wherein the choice of the type of keratoprosthesis depends on the severity of the disease. In summary, limbal stem cell transplantation improves both vision and quality-of-life in patients with ocular surface disorders associated with LSCD, and overall, the use of autologous tissue offers the best results. Future studies aim at improving cellular expansion and finding different sources of stem cells.
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Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privilege of the eye. However, mechanisms originally thought to promote immune privilege, such as the lack of antigen presenting cells and vessels in the cornea, are challenged by recent studies. Nevertheless, the immunological and physiological features of the cornea promoting a relatively weak alloimmune response is likely responsible for the high survival rate in "low-risk" settings. Furthermore, although corneal graft survival in "low-risk" recipients is favourable, the prognosis in "high-risk" recipients for corneal graft is poor. In "high-risk" grafts, the process of indirect allorecognition is accelerated by the enhanced innate and adaptive immune responses due to pre-existing inflammation and neovascularization of the host bed. This leads to the irreversible rejection of the allograft and ultimately graft failure. Many therapeutic measures are being tested in pre-clinical and clinical studies to counter the immunological challenge of "high-risk" recipients. Despite the prevailing dogma, recent data suggest that tissue matching together with use of systemic immunosuppression may increase the likelihood of graft acceptance in "high-risk" recipients. However, immunosuppressive drugs are accompanied with intolerance/side effects and toxicity, and therefore, novel cell-based therapies are in development which target host immune cells and restore immune homeostasis without significant side effect of treatment. In addition, developments in regenerative medicine may be able to solve both important short comings of allotransplantation: (1) graft rejection and ultimate graft failure; and (2) the lack of suitable donor corneas. The advances in technology and research indicate that wider therapeutic choices for patients may be available to address the worldwide problem of corneal blindness in both "low-risk" and "high-risk" hosts.
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Purpose To investigate whether mesenchymal–epithelial cell interactions, similar to those described in the limbal stem cell niche in transplant-expired human eye bank corneas, exist in freshly enucleated rabbit eyes and to identify matrix molecules in the anterior limbal stroma that might have the potential to help maintain the stem cell niche. Methods Fresh limbal corneal tissue from adult Japanese white rabbits was obtained and examined in semithin resin sections with light microscopy, in ultrathin sections with transmission electron microscopy, and in three-dimensional (3D) reconstructions from data sets of up to 1,000 serial images from serial block face scanning electron microscopy. Immunofluorescence microscopy with five monoclonal antibodies was used to detect specific sulfation motifs on chondroitin sulfate glycosaminoglycans, previously identified in association with progenitor cells and their matrix in cartilage tissue. Results In the rabbit limbal cornea, while no palisades of Vogt were present, the basal epithelial cells stained differentially with Toluidine blue, and extended lobed protrusions proximally into the stoma, which were associated with interruptions of the basal lamina. Elongate processes of the mesenchymal cells in the superficial vascularized stroma formed direct contact with the basal lamina and basal epithelial cells. From a panel of antibodies that recognize native, sulfated chondroitin sulfate structures, one (6-C-3) gave a positive signal restricted to the region of the mesenchymal–epithelial cell associations. Conclusions This study showed interactions between basal epithelial cells and subjacent mesenchymal cells in the rabbit corneal limbus, similar to those that have been observed in the human stem cell niche. A native sulfation epitope in chondroitin sulfate glycosaminoglycans exhibits a distribution specific to the connective tissue matrix of this putative stem/progenitor cell niche.
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Importance Corneal transplantation restores visual function when visual impairment caused by a corneal disease becomes too severe. It is considered the world’s most frequent type of transplantation, but, to our knowledge, there are no exhaustive data allowing measurement of supply and demand, although such data are essential in defining local, national, and global strategies to fight corneal blindness.Objective To describe the worldwide situation of corneal transplantation supply and demand.Design, Setting, and Participants Data were collected between August 2012 and August 2013 from a systematic review of published literature in parallel with national and international reports on corneal transplantation and eye banking. In a second step, eye bank staff and/or corneal surgeons were interviewed on their local activities. Interviews were performed during international ophthalmology or eye-banking congresses or by telephone or email. Countries’ national supply/demand status was classified using a 7-grade system. Data were collected from 148 countries.Main Outcomes and Measures Corneal transplantation and corneal procurements per capita in each country.Results In 2012, we identified 184 576 corneal transplants performed in 116 countries. These were procured from 283 530 corneas and stored in 742 eye banks. The top indications were Fuchs dystrophy (39% of all corneal transplants performed), a primary corneal edema mostly affecting elderly individuals; keratoconus (27%), a corneal disease that slowly deforms the cornea in young people; and sequellae of infectious keratitis (20%). The United States, with 199.10−6 corneal transplants per capita, had the highest transplantation rate, followed by Lebanon (122.10−6) and Canada (117.10−6), while the median of the 116 transplanting countries was 19.10−6. Corneas were procured in only 82 countries. Only the United States and Sri Lanka exported large numbers of donor corneas. About 53% of the world’s population had no access to corneal transplantation.Conclusions and Relevance Our survey globally quantified the considerable shortage of corneal graft tissue, with only 1 cornea available for 70 needed. Efforts to encourage cornea donation must continue in all countries, but it is also essential to develop alternative and/or complementary solutions, such as corneal bioengineering.
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The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering. The resulting hybrid materials at [AgNPs] < 0.4 μM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 μM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant.
Article
Purpose: There are few studies comparing different surgical procedures for the treatment of corneal thinning. Lamellar corneal transplantation (LCT) has been reported to be efficient, but its results can be jeopardized by allograft rejection, opacification, or high astigmatism. Amniotic membrane transplantation (AMT) has been considered a good alternative, but it is not as resistant as LCT and the tissue can be reabsorbed after surgery. Methods: A prospective, randomized, interventional, and comparative study of consecutive patients with corneal thinning over 6 months was performed. Ophthalmological examination was performed before transplant surgery and then repeated 1, 7, 15, 30, 90, and 180 days after surgery and ultrasound biomicroscopy was performed before and then 30, 90, and 180 days after surgery to assess corneal thinning. Results: Herpes simplex infection was the main cause of corneal thinning (9 eyes), followed by surgery (cataract, glaucoma, 5 cases), rheumatoid arthritis (1), chemical burn (1), perforating trauma (1), previous band keratopathy treatment (1), and Stevens-Johnson syndrome (1). Although all patients showed significant increase in final thickness in the area of thinning, it was higher in those submitted to LCT at 180 days postoperatively. Regardless of the surgical technique, all patients showed epithelialization. Patients undergoing AMT showed an 89% decrease in neovascularization. Final corrected distance visual acuity was better in patients submitted to AMT. Conclusions: LCT proved to be the best option for treating corneal thinning. AMT represents an alternative that allows good visual recovery but does not restore corneal thickness as efficiently as LCT.
Purpose: To evaluate outcomes of anterior lamellar keratoplasty (ALK) and endothelial keratoplasty (EK) within the Singapore Corneal Transplant Study (SCTS), with the hypothesis that both ALK and EK are able to provide equivalent or improved clinical outcomes, compared to penetrating keratoplasty (PK), and to determine changing trends globally with other international databases. Methods: Clinical data on all transplants performed was derived from our SCTS database, a prospective national keratoplasty registry, and clinical outcomes (graft survival, endothelial cell loss, complications, visual acuity) were compared between PK, ALK, and EK. Global trends on indications and forms of keratoplasty performed in 2011/2012 were obtained from national keratoplasty or eye banking registries, corneal/ophthalmological societies, national eye banks, and national ophthalmic institutions. Results: Global rates of EK surgery vary widely, from 52% (Sweden) to 0% (South Africa), with higher adoption by industrialized countries. ALK adoption rates similarly vary from 28.7% (China) to 1.0% (Philippines). SCTS data show high adoption rates in Singapore: EK 44% and ALK 28%. Our surgical modifications to big-bubble deep anterior lamellar keratoplasty (DALK) surgery resulted in visual outcomes matching PK, and a low conversion to PK of 2.1%, whereas our evolving approaches to donor insertion in Descemet's stripping automated endothelial keratoplasty (DSAEK) show significant reduction in 1-year postoperative endothelial cell loss rates from 60% (folding), to 22% to 30% (Sheets Glide), to 15% (EndoGlide inserter). Conclusion: Improvements in various forms of ALK and EK surgery can lead to better visual outcomes, longer graft survival, and reduced complications, as compared to PK. Global trends suggest adoption of these procedures at different rates.
Article
The cornea and sclera together form the tough outer tunic of the eye, which withstands the intra-ocular pressure from within and protects the contents from mechanical injury from without. The cornea is the major component in the optical system of the eye; of the total dioptric power of the human eyeball, nearly three-quarters is contributed by the interface between the cornea and the air. To this end, it is curved and transparent, and its surfaces, particularly the external one, are smooth to good optical standards. Generally, the globe of the mammalian eye approximates to a sphere, sometimes rather flattened in the antero-posterior direction, so that the curvature of cornea and sclera is similar. Its radius ranges from 1.75 mm in the mouse to 25 mm in the horse, among the more common animals. In man, and to a lesser extent in many other species, the cornea is more curved than the eyeball as a whole.
Article
Organ and tissue loss through disease and injury motivate the development of therapies that can regenerate tissues and decrease reliance on transplantations. Regenerative medicine, an interdisciplinary field that applies engineering and life science principles to promote regeneration, can potentially restore diseased and injured tissues and whole organs. Since the inception of the field several decades ago, a number of regenerative medicine therapies, including those designed for wound healing and orthopedics applications, have received Food and Drug Administration (FDA) approval and are now commercially available. These therapies and other regenerative medicine approaches currently being studied in preclinical and clinical settings will be covered in this review. Specifically, developments in fabricating sophisticated grafts and tissue mimics and technologies for integrating grafts with host vasculature will be discussed. Enhancing the intrinsic regenerative capacity of the host by altering its environment, whether with cell injections or immune modulation, will be addressed, as well as methods for exploiting recently developed cell sources. Finally, we propose directions for current and future regenerative medicine therapies.
Article
Purpose: To evaluate the potential utility of collagen-based corneal implants with anti–Herpes Simplex Virus (HSV)-1 activity achieved through sustained release of LL-37, from incorporated nanoparticles, as compared with cell-based delivery from model human corneal epithelial cells (HCECs) transfected to produce endogenous LL-37. Methods: We tested the ability of collagen-phosphorylcholine implants to tolerate the adverse microenvironment of herpetic murine corneas. Then, we investigated the efficacy of LL-37 peptides delivered through nanoparticles incorporated within the corneal implants to block HSV-1 viral activity. In addition, LL-37 complementary DNA (cDNA) was transferred into HCECs to confer viral resistance, and their response to HSV-1 infection was examined. Results: Our implants remained in herpetic murine corneas 7 days longer than allografts. LL-37 released from the implants blocked HSV-1 infection of HCECs by interfering with viral binding. However, in pre-infected HCECs, LL-37 delayed but could not prevent viral spreading nor clear viruses from the infected cells. HCECs transfected with the LL-37 expressed and secreted the peptide. Secreted LL-37 inhibited viral binding in vitro but was insufficient to protect cells completely from HSV-1 infection. Nevertheless, secreted LL-37 reduced both the incidence of plaque formation and plaque size. Conclusion: LL-37 released from composite nanoparticle-hydrogel corneal implants and HCEC-produced peptide, both showed anti–HSV-1 activity by blocking binding. However, while both slowed down virus spread, neither was able on its own to completely inhibit the viruses. Translational Relevance: LL-37 releasing hydrogels may have potential utility as corneal substitutes for grafting in HSV-1 infected corneas, possibly in combination with LL-37 producing therapeutic cells.