Fish derived collagen for corneal regeneration.

1
Fish derived collagen scaffolds in Corneal Regeneration
Group E
Banoth Sneha, Brijesh Chandrakar, Hemant Meharchandani, Pooja Chaukimath, Prajita Roy
BSBE IIT Kanpur
BSE 411A, Biomaterials
1st November, 2014

[Fish derived collagen scaffolds in corneal regeneration] 2

Abstract
Loss of vision due to corneal disease or trauma affects over 10 million people worldwide. For
many, corneal transplantation is the only treatment but the supply of good quality donor tissue is
much less than the demand. Corneal substitutes clinically tested in humans have been fully
synthetic prostheses (keratoprostheses), [2][3]. But there are various complications in
keratoprostheses including retroprosthetic membrane formation, calcification, infection, and
glaucoma, [1]. Alternatively, we can support the inherent regenerative capacity of human cornea
to promote the in-vivo reconstruction of healthy viable corneal tissue. To serve this purpose, we
need to design a scaffold to mimic corneal extra cellular matrix (ECM). Cornea has dual role –
transparency and protection of inner eye-tissues. Corneal ECM is especially rich in collagens, [4].
Among different sources of collagen, we have to carefully look for a source which doesn't elicit
immune response and is biocompatible. The material should also have clarity (refractive index),
dimensions and biomechanical properties similar to that of Corneal ECM. We recommend
scaffolds derived from marine sources with the special case of jelly fish. In the reference [5]; the
biocompatibility of collagen derived from jelly fish is compared with that from popular bovine
collagen and gelatin. The results showed that jellyfish collagen elicit least immune response
among these three.
In the reference [6], it has been shown that engineered collagen substitutes successfully
induced corneal regeneration (in 10 patients) without any signs of rejection. Substitutes were
stable and improved vision in six patients than the preoperative conditions after 24 months.
Reference [7] shows improvement in adhesion and proliferation of human corneal epithelial cells
by incorporating YIGSR and RGD into the scaffolds. We suggest optimizing the corneal
substitute used in [6] with fish derived, engineered (incorporating YIGSR and RGD) collagen
scaffolds which can possibly lead to better results.
Keywords: Corneal regeneration, tissue engineering, scaffold, collagen
Introduction
The cornea is the transparent front part of the eye that covers iris, pupil and anterior chamber.
Fig 1.
Cornea location on eye
[By Mikael Häggström
(Image :Schematic_diagram_of_the_human_eye_en.svg)
[CC0], via Wikimedia Commons]

The function of cornea depends on its optical clarity. The refractive index of the cornea is not
uniform. The calculated dioptric power of the corneal epithelium is approximately -1.40 diopters
(D), [8].
The number of cases of corneal blindness is second only to cataract. Out of total 45 million blind
people in the world, 20 million are cataract cases, followed by trachoma which blinds 4.9 million
individuals mainly as a result of corneal scarring and vascularization. Ocular trauma and corneal
ulceration are significant causes of corneal blindness, may be responsible for 1.5–2.0 million
new cases of monocular blindness every year, [9]. Approximately, corneal trauma affects 10
million individuals annually.
In India, it is estimated that there are approximately 6.8 million people who have vision less than
6/60 in at least one eye due to corneal diseases; of these, about a million have bilateral
involvement, [15][16]. It is expected that the number of individuals with unilateral corneal
blindness in India will increase to 10.6 million by 2020, [16]. According to the National
Programme for Control of Blindness (NPCB) estimates, there are currently 120,000 corneal blind
persons in the country. According to this estimate there is addition of 25,000-30,000 corneal
blindness cases every year in the country, [17]. The burden of corneal disease in our country is
reflected by the fact that 90% of the global cases of ocular trauma and corneal ulceration leading
to corneal blindness occur in developing countries.
Currently, the successful and accepted treatment of corneal blindness is full-thickness
replacement of the damaged tissue with a human donor cornea in a procedure known as
Fig 2. Formation of the cornea. The cornea begins to develop when the surface ectoderm
closes after the formation of the lens vesicle and its detachment from the surface ectoderm.
Mesenchymal cells (neural crest cells) invade the cornea and form the corneal stroma after
condensation.
From: Corneal endothelium: developmental strategies for regeneration
J Zavala, G R López Jaime, C A Rodríguez Barrientos and J Valdez-Garcia

Penetrating Keratoplasty. The process is successful, but the fundamental problem with corneal
replacement is a severe shortage of donor tissue, resulting in approximately 10 million untreated
patients worldwide, and 1.5 million new cases of blindness annually, [9]. The other recent
developments include synthetic prostheses (keratoprostheses), tissue-engineered cell based
constructs, and hydrogels that also permit the integration of the implant and regenraiton of host
tissue. Only keratoprostheses is approved for human uses which suffer from complications such
as retroprosthetic membrane formation, calcification, infection, and glaucoma, [1].
To overcome these complications, a good idea is to adopt a regenerative medicine approach and
induce regeneration of the damaged cornea. This idea was used in paper [6]. They engineered an
acellular matrix that facilitated regeneration by emulating the functions of the highly bioactive
natural extracellular matrix (ECM) scaffolding of the cornea. They used clinical-grade traditional
recombinant collagen for scaffolding.
Our hypothesis is based on the results of reference [5]. This paper shows the superior
biocompatibility of collagen derived from jelly fish compared to traditional bovine collagen and
gelatin. So, we hypothesized that collagen derived from jelly fish, if used as a starting material
for scaffold preparation, will result in less immune response and better and long lasting corneal
regeneration. Also, bio-functionalization of scaffolds by incorporating YIGSR and RGD will
help mimicking corneal extra cellular matrix better, [7].
Our project is specifically aimed to address the following points for a better viable regenerative
cornea:
 Extracting collagen from jellyfish.
 Fabricating scaffold suitable for corneal regeneration by using collagen extracted as the
starting material.
 Bio-functionalizing the scaffold with peptide sequences for laminin and the integrin-
binding sequence (found in fibronectin).
 Keratocyte culture on the scaffold.
 Pre-clinical trials and clinical trials.
Materials and Methods
The materials and methods described are standard protocols previously followed in literature.
We have used different protocol for different procedures, but the quantity of the final materials to
be used in actual experimentation and testing may vary depending on requirements.
Extraction of collagen from jellyfish, [5] [14]
Preparation of jellyfish mesoglea
The mesoglea, the major component of the jellyfish umbrella, is desalted by washing
with cold distilled water at 4 degree Celsius for 3 days with two changes per day and then
lyophilized.

Isolation of acid-soluble collagen from mesoglea
The lyophilized mesoglea is cut into small pieces and suspended in 0.5-M acetic acid.
Acid soluble proteins are extracted twice with 0.5-M acetic acid for 3 days; extracts collected by
filtering are extensively dialyzed against 0.02-M Na2HPO4. The resultant precipitates are
harvested by centrifugation at 15000g for 1 hr and dissolved in 0.5-M acetic acid. After
centrifugation, solid NaCl is added to the supernatant to a final concentration of 0.9M. The 0.9M
NaCl- precipitated fraction, hereafter termed acid-soluble collagen, is dissolved in 0.5-M acetic
acid, dialyzed against 0.1-M acetic acid, and lyophilized.
Preparation of atelo-collagen by pepsin digestion
Acid-soluble collagen is digested with 5 percent pepsin at 4 degree Celsius in 0.5-M
acetic acid. After 24 hr of cold incubation, mixtures are centrifuged at 15000g for 1 hr and the
supernatant dialyzed against 0.02-M Na2HPO4 in order to inactivate the pepsin. The precipitate
was salted out by addition of NaCl to a final concentration of 0.9-M acetic acid, dialyzed against
0.1-M acetic acid, and lyophilized.
Fabrication of scaffold by electrospinning, [12]
In electrospinning a high voltage is applied to a polymer solution, which overcomes surface
tension and creates a fine charged jet. The charged polymer is then ejected, dried, and solidified
onto an oppositely charged collector plate. The ejected polymer solutions repel each other during
travel to the collector, which forms thin fibres after solvent evaporation. An electrically
grounded rotating drum results in the formation of preferentially oriented fibres. A
programmable syringe pump was used to transfer the polymer solution to the needle at a flow
rate of 1.0 mL/h under the conditions of 25°C and 40% relative humidity. A high-speed rotated
drum collector was placed 15 cm below the needle to collect fibers. The polymer jet was
stretched by the force generated by the high voltage (12–14 kV) between the needle and the
collector and deposited on the surface of the drum.
Biofunctionalization of scaffold, [13]
YIGSR was added to aqueous solutions of Tris-succinimidyl amino triacetate containing 1-ethyl-
3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) and the mixture
reacted overnight at room temperature with stirring. The molar ratio of YIGSR to the cross linker
was 1:1. A ratio of 5:5:1 EDC: NHS: NH2 of YIGSR was used. The YIGSR modified cross
linker product was purified by dialysis using membranes against water for 2 days. The same
procedure was carried out to get RGD modified crosslinker product.The electrospun mats were
crosslinked by using solution containing EDC: NHS: NH2 of the YIGSR and RGD modified
crosslinkers in ethanol for 4hrs.
Keratocyte culture on scaffolds, [19] [20]

Keratocytes are isolated from human corneas removed in accordance with ethical regulations and
stored at 31 °C in organ culture. These corneas should be unusable for treatment (because their
endothelial density is too low). Isolation is performed with 3 mg/ml collagenase A for 3 h at 31
°C, with stirring at 200 rpm. The digest is purified through a 70 μm cell sieve and immediately
placed in monolayer culture. Keratocytes are seeded at a density of 10,000 cells/cm2 then
cultured in a specially designed medium providing optimal growth and preservation of the
phenotype as follows: DMEM/Ham-F12 1:1, 10% NCS, 5 ng/ml bFGF, antibiotics. The medium
is changed every 2 days until cell confluence is reached. At confluence, cells are resuspended
using trypsin–EDTA (0.5 g trypsin/l and 0.2 mg/ml EDTA) and amplified over 3 passages (from
P0 to P2) then seeded at P3 on the matrix.
Scanning electron microscopy, [21]
Scanning electron microscopy is used to examine the morphological characteristics of corneal
cells cultured onto the scaffold from the previous step. Dried samples are sputtered with gold
using a SEM coating system, and the probes are examined by scanning electron microscopy.
Confocal microscopy
Image quantification of scaffold area and cell area is accomplished by confocal microscopy.
Evaluation of mechanical properties
The tensile strength, elongation at break (elasticity), elastic modulus (stiffness), and energy at
break (toughness) of tissue-engineered scaffolds can be determined using Mechanical testing
system. To avoid breakage and slippage of the samples in the jaw, tabs on the end of each dumb-
bell sample can be adhesively coated and reinforced with tapes on both sides. Every reported
value is average of atleast 3 measurements. The same method can be applied for testing the
mechanical properties of cornea.
Evaluation of optical properties
Light transmission measurement is to be made at 21 degree Celsius, both for white light (quartz-
halogen lamp source) and for narrow spectral regions (centered at 450, 500, 550, 600 and
650nm) for corneal materials, human eye bank. Samples are hydrated in 0.5 percent PBS before
and during the measurement.
Clinical Trials and Implantation, [10] [11]
After preparing the scaffold, the remaining tasks are clinical trials, testing, implantation and
authorization from the appropriate authorities.
Animal implantation and clinical evaluation, [22]
Implantation
In accordance to The International Committee for Laboratory Animal Science (ICLAS),
the corneal scaffolds are to be implanted into the one cornea of each of the tested animal (pigs)

by utilizing deep lamellar keratoplasy (DLKP) with overlying sutures. Prior to surgery and
follow-up examinations, animals are to be anaesthesized. The un-operated, contralateral eye of
each animal will serve as a control.
Clinical evaluation
Daily basis examination for the first week after surgery and then weekly examinations are
to be made. Slit-lamp examinations are needed to ensure the optical clarity of cornea. In vivo
confocal microscopy (IVCM) examinations can be used to examine cell and nerve in-growth,
and determine total corneal thickness in live animals. IVCM images from central cornea are
acquired at different period to visualize and examine corneal thickness.
Implantation:
The size of cornea made after culturing in scaffold is 500um thick having a expected
transmittance of white light around 95% with a pre-operative refractive index of 1.36. They are
trephined to size and implanted by anterior lamellar keratoplasty (ALK).After implantation the
size of cornea is reduced to 400 um and refractive index expected around 1.3.
To implant cornea inside, ALK is performed on patients. The patients cornea is first cut around 6
mm in diameter and then a depth upto 370-400 um. Then the cultured cornea is deepened
manually in place of the original cornea.
No suture corneal grafting, [23]
In the previous trial [6], thinning of cornea was observed around sutures. Hence we are
suggesting the use of overlaying suture and fibrin glue described in [23].
Overlay sutures and fibrin glue are to be used to secure the implant and then a bandage contact
lens was inserted. No sutures are required to directly suture the implant to host tissue. All
securing sutures can be removed 4 weeks after the surgery.
Fig 3. Fabricated cornea and implantation method. (A) An example of optically clear,
biosynthetic corneal substitutes used in these studies. (B) These were trephined to prepare a
button for corneal implantation. Damaged host tissue was removed to a similar depth and
diameter, and replaced by this button. (C) After implantation, the button was held in place with
three overlying sutures over the bandage lens (not shown in the figure).
Post-operative clinical trials:
After pre clinical trials, some keratoconus or central scar patients (around 10-12) are being
operated and being analysed on weekly basis for a year. Along with this, a detailed follow up
must be done after 4 each 4 weeks. The assessment that has to be done are: Central corneal
thickness for refractive index and transparency ( using ultrasound pachymeter) ; intraocular
Image source: Reference [6]

pressure (Goldman applanation tonometer), studying the surface or topography (using OrbScan),
Focal lengths (IVCM).
Following table can be made and efficacy can be hence be calculated:
Patient
No.
Time for full
epithelization
Pre
operative
Refraction
Post
operative
refraction
Tear
(per
micron)
Nerve
Density
(per
square
micron)
Visual
acuity
These are some parameters that are needed to be noted.
 Approximate nerve density after an year of implantation must be around 22000-
25000 micron/mm^2.
 Expected visual acuity must be more than 24/40 after a year.
Fig 4. Sample slit lamp biomicroscope photographs of the eyes of different patients at 24 months
after implantation with biosynthetic corneal substitutes. Implants are well-integrated into
recipient corneas, with implant boundaries only barely visible.
Expected Outcome
The implantation carried out by researchers of reference [6] had the mean corneal thickness of
(403 ± 109m) after 24 months. The normal thickness of human cornea is around 534m, [24].
Since, the collagen derived from jelly fish showed better biocompatibility and lesser immune
response, [5] & biofunctionalization of scaffold with YIGSR and RGD is expected to promote the
adhesion and proliferation of human corneal epithelial cells as well as neurite extension from
dorsal root ganglia, [7]. These two modifications in previous methodology should certainly
improve corneal thickness over the range of 24 months during follow-up. The optically clarity of
the implant is expected to be normal (transmittance 95%) with the refractive index of 1.3 as in
the previous test. Following are the other expected results of various characterization tests:
Image source: Reference [6]

 The Intraocular pressure may not be affected by the corneal implantation. It is expected
to be in the normal range of 9 – 20 mm Hg.
 Decrease in dosage and time of immunosuppressant as no sutures are used and jelly fish
collagen had less immunogenic response, [5].
 The biosynthetic implant supports human regeneration nerves. These nerves are noted
initially at the basal epithelium and substantially increased, [5]. With this substantial
increase in the regeneration nerves, the sensitivity of corneal implant is gradually
improved which can be measured with the help of Cochet-Bonnet aesthesiometer.
 The seeding of keratocytes enhances the migration of stromal cells into the central
implant.
 The tear production and tear osmalarity are expected to be normal.
 Corneal transparency over time can be verified by fundus photography.
Fig 5. Results of reference [6]. Fundus images of the retina as photographed through the
implanted biosynthetic corneas of all 10 patients at 24 months after surgery. Proper morphology
of the retinal vessels was observed, demonstrating transparency of the implants even after
regeneration of corneal tissue and nerves.
Expected Timeline (Approx.)
-The desaltation of Mesolgea is done by washing with cold water for 3 days and lyophilized.
-The Isolation of acid-soluble collagen from mesoglea for 3 days.
-Preparation of atelo collagen, by cold incubation for 24 hrs followed by centrifugation for 1 hr.
-Electrospinning in order to obtain the fibres and the addition of peptides like YIGSR and RGD
is done in 2 to 3 days.
-Keratocyte Culturing: The medium was changed every 2–3 days. Constructs are harvested for
analysis after 2, 3 and 4 weeks in culture.
-SEM: corneal debris are plated on scaffold and cultured for 2, 3 and 7 days and the samples are
examined under the scanning electron microscope.
-The expected time for the preparation of required biosynthetic corneal implant is expected to be
completed in 7 to 8 weeks.
-Prior to the use of this corneal implant by general population, pre-clinical and clinical tests are
conducted for 52 to 54 weeks. Then, if the results are as expected, these implants are implanted
by suture free DLKP method.
-So, the entire process of scaffold fabrication and implantation would take about 59 to 61 weeks.
-Follow-up of atleast 24 months till when corneal thickness is stable.
Image source:
Reference[6]

Conclusion
Corneal blindness is the second major cause of blindness next only to cataract. There is a
successful cure in the form of implantation of healthy cornea from a donor. However, number of
patients far exceeds the availability of donor corneas. Current artificial corneas aren’t very
successful and requires immunosuppressant and are highly prone to infection. The work by
group of Linköping University, [6] and their co-researchers suggest that collagen based
engineered scaffold could be a biocompatible, viable medium for corneal regeneration. The
results of this group were encouraging and we continued on that line to improve their design
based on their results and results of few other literatures. While the results of [6] showed
biocompatibility and acceptance of regenerative implant, even without immunosuppressant (after
7 weeks), there was thinning in corneal layer. The results of [5] introduced us to an easily
accessible collagen source which has less immune response and has better biocompatibility. And
the results of [7] showed the importance of YIGSR and RGD peptides in cell adhesion in
collagen scaffolds. These two results can be used in advantage to design a better biocompatible,
regenerative corneal substitute. Some simple improvements in implantation techniques like
avoiding suturing directly by using a bandage lens can reduce the possibility of thinning of
cornea and infection.
We expect a better outcome on our proposed design compared to the outcomes of previous
design. These outcomes can be one of the many steps towards building a final successful
regenerative cornea which can be used in treatment of millions of individuals suffering from
corneal blindness worldwide.

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