Team 8

CHEN 5970, Spring 2016

Engineered Corneal Stroma for use in Corneal Transplants

Kelsey Henderson

Matthew Lopez

Quinn Otte

Executive Summary

The objective of the proposed project is to create a potential direction for a tissue engineered

corneal stroma for either direct transplantation or combination with other tissue engineered

corneal layers prior to transplantation. The need for corneal transplants is increasing, just as the

number of viable tissue donors is decreasing. The rise of LASIK is widely decreasing the number

of useable corneal tissues in the US, due to the LASIK process thinning the cornea and making it

unusable for transplants.1 For many pathologies, replacing only the stroma is an effective

treatment.2 For a total cornea transplant, the proposed product can be combined with engineered

versions of the remaining corneal layers for a final product. The final design uses a silk fibroin

scaffold seeded with keratocytes differentiated from dental pulp stem cells.

Background

The eye is an incredibly complex organ, acting as a biological transducer to turn light into

electrical signals to the brain via the optic nerve. The cornea is the most anterior part of the eye,

and is the first part of the eye that comes into contact with visible light. Part of its function is as

the first lens, directing light toward the interior lens that exists behind the iris. It is also

protective, selectively allowing small molecules like oxygen to diffuse through but keeping out

larger proteins and debris.3 These two main functions are critical to the overall function of the

eye. The loss of one or both of these functions of the cornea will lead to a damaged eye, which

nearly always leads to a decreased quality of life by inducing chronic pain, loss of eyesight,

and/or aesthetic degeneration.

The cornea is comprised of three main layers: the epithelium, stroma, and endothelium. The

epithelium is the outer layer; it is highly innervated and comprised mostly of tightly packed

stratified squamous cells. These cells are highly proliferative and are attached to the stroma by

the Bowman’s membrane: an acellular collagen membrane. The endothelium is a single layer of

flattened, highly specialized cells. These cells are held together by tight junctions and control

mass transport between the cornea and the interior of the eye. As such, it is responsible for

maintaining the slightly dehydrated state of the stroma necessary to maintain optical clarity. The

endothelium remains attached to the stroma via the Descemet membrane: also an acellular

collagen layer. These acellular layers permit the epithelium and endothelium to be surgically

removed from the stroma, allowing for the independent transplantation of engineered stromal

tissue.3

The stroma makes up 90% of the cornea’s thickness; it is about 465μm on average. As the

thickest part of the cornea, it is responsible for the optical properties of the tissue. It is comprised

mostly of ECM components, the vast majority being collagen. The organization of this collagen

is the key to maintaining optical clarity. Collagen is an individual fibril, which bundle together in

parallel “ropes” called lamellae. The fibrils must be parallel within the lamellae, which are in

turn arranged orthogonally to one another, with about 55µm spacing between fibrils.4 If this

organization breaks down (as it often does with some of the diseases listed), then optical clarity

is compromised and eyesight suffers. The collagen is produced by keratocytes, which only

occupy 5-10% of the stroma’s total volume. This is an advantageous property for tissue

engineers, as target cell density to maintain a functional tissue is relatively low compared to

other tissues. Keratocytes have a low rate of proliferation; they are highly specialized

descendants of mesenchymal stem cells of the neural crest.5

The cornea is vulnerable to several infirmities that can be remedied by a corneal transplant,

either total or exclusively stromal. Mechanical injury to the eye sometimes can be require a

transplant, as well as corneal ulcers which are usually a side effect of severe infections. Ulcers

from infections are highly likely to form if the infection is left untreated, due to the relative

absence of immune system cells in the cornea. However, this locally suppressed immune system

is helpful during a transplant, as an inflammatory response and foreign body rejection is easier to

avoid. Keratoconis is describes any pathology that causes a cornea to become misshapen over

time. Some conditions are genetic, such as Fuch’s dystrophy or macular corneal degradation.

Fuch’s dystrophy causes a failure of the endothelium, leading to over-hydration of the stroma

and a loss of optical clarity and shape. Macular corneal degeneration affects patients early in

life—usually before ten years of age. It is a painful malfunction of keratocytes, and is only

treated by a transplant.6 This project aims to create a viable replacement for donor tissue—the

only treatment for most of these ocular diseases.

Review of existing clinical therapies

Currently, there exists two main types of corneal transplants, known as keratoplasty, which differ

in the way and the amount of the cornea that is transplanted. The first type, penetrating

keratoplasty (PK), is the oldest surgical method used for corneal transplants. All three main

layers of the cornea (epithelium, stroma, and endothelium) are surgically removed and replaced

with a donor cornea. After the donor cornea is positioned correctly, it is then immobilized with

sutures so that healing can ensue. When this method is used, the average recovery time for

complete restoration of the patient’s vision (assuming no complications) is about 15 months.

The second surgical method, known as anterior lamellar keratoplasty (ALK), is the preferred

method for corneal transplants. The success of this method is due to limiting the layers of the

cornea that are surgically removed. This method involves dissecting the cornea into two layers

then removing and subsequently replacing the outer layer. A refinement of ALK, known as deep

anterior lamellar keratoplasty (DALK), removes the stroma, leaving behind both the endothelium

and epithelium. Both ALK and DALK reduce the necessary amount of healing time to mere

weeks rather than months.

As always when using donor tissue, there exists the possibility for disease transmission.

Although this has been reported as a rare event, the potential is nevertheless present.7

Complications associated with corneal transplants include cornea graft rejection and infection of

the eye. The more serious of these two complications is graft rejection, occurring in 5 to 30

percent of patients.8 However, the use of ALK and DALK avoid the possibility of this

complication due to the ability of the epithelium and stromal cells to regenerate. Another

problem is that donated corneas usually only last about 10 years.9 Perhaps the most severe

limitation to the use of corneal transplant methods is the availability of donated corneas. In future

years, the supply of donated corneas will decrease due to the increasing use of LASIK surgery.

LASIK surgery corrects a person’s vision but eliminates the cornea’s ability to be donated

posthumously.10 Therefore, any person who has undergone LASIK surgery is not considered a

possible donor.

Review of existing corneal tissue engineering strategies

Due to the decreasing supply of donated corneas for the use in corneal transplants, research and

development of engineered corneal tissue as a replacement to donor corneas has become an area

of high interest.

There are many different approaches being used to develop a viable scaffold for use as a corneal

allograft tissue. These approaches vary from the use of regenerative medicine techniques to the

development of synthetic polymers.

Natural polymer techniques provide vast potential for use as a scaffold material due to the

inherent biocompatibility, biofunctionality, and vast availability. Therefore, their use as a

scaffold material averts the need to focus attention on these considerations, allowing the

appropriation of this attention to other, more specific concerns. Synthetic polymer techniques, on

the other hand, offer benefits in other areas such as the potential for customized properties, the

lack of batch to batch variability, and the lack of potential for disease transmission. Many of the

current studies combine both natural and synthetic polymers. Uchino et al designed a scaffold

material composed of an amniotic membrane (AM) immobilized polyvinyl alcohol (PVA) in

order to combine the stability of synthetic polymers with the biocompatibility of natural

polymers.11 Control over the stability of the AM (natural component) proved to be the most

difficult obstacle to overcome. Another combination of natural and synthetic polymers, collagen-

immobilized polyvinyl alcohol (PVA-COL), was used as a scaffold material.12 Mayashita et al

determined that through the use of PVA-COL, that a stratified human epithelium was found in 6

of the 9 trials and that the stratified epithelium had the same histological and functional

characteristics of a healthy epithelium. However, the studies performed by Uchino et al

determined that the use of PVA-COL to support the growth of rabbit epithelium tissue resulted in

defects in approximately half of the trials.13 Collagen vitrigel (CV) was also tested for the

potential viability as a scaffold material.14 This study showed that keratocytes cultured onto CV

exhibited the correct morphology with both endothelial and epithelial cells exhibiting adhesive

structures as well. This technique shows that perhaps the most promise for a viable material for

use as a corneal scaffold is one derived from silkworm fibroin.

Using silk as a biomaterial has been extensively studied.15 Madden et al demonstrated that an

endothelial layer can be grown on silk fibroin membranes.16 Liu et al demonstrated that silk

fibroin supports corneal epithelial cells to proliferate, differentiate, and retain the normal

epithelium phenotype.17 It was also shown that silk fibroin exhibited transparency comparable to

the native corneal tissue, making it the most ideal candidate tested thus far for application in

keratoplasty. This material has been shown to exhibit biocompatibility, sufficient transport

properties (including the ability for oxygen to diffuse into the material), optical clarity, and the

ability to degrade without creating an immune response. Silk fibroin has also been shown to

support the growth of the various cell types that are found in the human cornea. Therefore, silk

fibroin is proposed as the scaffolding material that will be used in the development of the

proposed material.

Extensive study of different methods of culturing keratocytes through the use of various types of

stem cells has been done. It has also demonstrated how effectively human limbal epithelial

(HLE) cells can be cultivated through the use corneal stromal stem cells (CSSC).18 In this study,

it was shown that a mixed population of HLE cells and CSSCs were successfully cultivated

while maintaining stem cell phenotype for both types of cells. Other research has been successful

in differentiating neural crest-derived adult dental pulp cells into keratocytes in vitro and in vivo

as well as generating a tissue-engineered corneal stromal-like tissue.19 This provides a great

option for the type of cells as well as the source of the cells that could lead to a viable tissue-

engineered corneal allograft.

Engineering Considerations

As with designing any tissue engineered product, there are various aspects that must be

considered prior to making the choice for materials and cell sources. The most important

consideration is always biocompatibility. If the proposed product is not viable due to

biocompatibility reasons, it is not likely to succeed no matter the amount of adjustments that are

made. For the proposed product, degradation plays a key role. The engineered material will need

to degrade at the same rate that cells are proliferating. This process mocks how the cornea

naturally operates, as it is continually renewing itself. A major factor that plays a role in

determining if the engineered product will be commercially viable is the cell source. Since an

engineered tissue will be composed of billions of cells on average, if the cell source that is used

is not capable of supplying the necessary amount of cells, production simply cannot ever take

place. The process of deciding on a choice of material involves considerations that a more

specific to the tissue that is being proposed. Due to an absence of a vascular network, the oxygen

necessary for cellular function will need to diffuse into the material from the surrounding air.

Therefore, our material choice will need to be sufficiently permeable with respect to oxygen.

Since our proposed material will be used to treat problems associated with a person’s vision,

optical clarity is paramount when choosing a material. It was found that the optical clarity of the

material was mostly dependent on these properties: parallel fibrils within lamella, orthogonally

aligned lamellae, constant diameter of lamellae.

Our Proposed Design

A primary measure of success of any engineered tissue is the ability of the material to support

cell attachment and proliferation. When human corneal fibroblasts were cultured on multiple,

stacked silk sheets, random cell and ECM orientation was observed. Because of the necessary

level of organization required of the stroma, a new strategy must be used. Studies have shown

that coupling silk biomaterials with RGD enhances cell attachment, proliferation, and alignment

as well as ECM production. The complex structure of the stroma can be replicated by covalently

linking arginine-glycine- aspartic acid (RGD) in the correct lamellar structure, and the cells can

then be cultured onto the biomaterial. Studies have shown that this will cause the cells to

properly align on the surface of the silk. The RGD coupling also increases cell proliferation and

production of ECM components, specifically collagen I and II and proteoglycans. 15

Another important consideration is degradation properties of our design; the degradation rates

need to match the regeneration rate of the corneal stroma. Silk is made up of pseudo-crystalline

lattice structures called β-sheets, which determine its degradation rate. Increased β-sheet content

increases the density of the silk material and provides fewer spaces for protease XIV and α-

chymotrypsin to contact the silk and induce hydrolysis. β-sheet content also is responsible for the

mechanical stiffness of the biomaterial. Thus, an optimum β-sheet content is required: too much

will prevent the silk from degrading fast enough and too little will not provide enough support

for the engineered tissue. Multiple processes are used to generate the physical crosslinks

necessary to induce β-sheet production, but the most easily tunable is water vapor annealing and

slow drying.20 Annealing is the process by which a substance is heated past its recrystallization

temperature and then cooled, resulting in the removal of internal stresses.21 Shang et al have

developed a novel method of water vapor annealing followed by enzymatic pretreatment that

allows the tunability of the β-sheet content and therefore degradability. The β-sheet content was

optimized at 17-18% which can be achieved by water vapor annealing for 30-45 minutes. This β-

sheet content will support the structure of the engineered tissue while still allowing for

degradation at the desired rate. Additionally, this low β-sheet content increased the transmittance

of light in the visible range through the silk biomaterial.20

Most of the research previously done exploring silk fibroin has used human corneal stromal stem

cells or human corneal fibroblasts to examine the viability and proliferation within the scaffold.

However, because of the lack availability of healthy corneal cells and the desire for

personalization of this therapy, we decided to seek other stem cell options. Dental pulp is a

vascularized connective tissue at the center of the tooth and contains multipotent mesenchymal

stem cells. These stem cells are neural-crest derived and thus share developmental origins with

keratocytes. They can be harvested through minimally invasive endodontic procedures and then

differentiated into keratocytes. After differentiation, dental pulp stem cells yield the

characteristic gene and protein expression. Additionally, keratocan and keratin sulfate were

expressed which are responsible for the uniform spacing of collagen fibrils in the native

microenvironment. Ideally, this would allow the use of an autologous cell source; dental pulp

stem cells could be harvest from banked exfoliated deciduous teeth or routine extraction of third

molars. If neither of these options were available, cells could be harvested from the patient’s own

second molars or from allogenic dental sources. Typical protocol calls for the cells to be

differentiated in vitro, then seeded onto the scaffold of choice.22 Although dental pulp cell

derived keratocytes have not been combined with silk fibroin biomaterials, we feel that the

success of this material and these cells will be additive when combined.

Previously tissue engineered stroma from silk fibroin sheets have been synthesized in 2-4 μm

thicknesses. Sheets have been stacked by using applied pressure on the edges of the scaffold, but

200 of these sheets would be needed to generate a full thickness cornea; so far research has only

extended to layer a few sheets at a time.15 We would propose attempting to make several stacks

of silk sheets and then layer those stacks on one another or attempt to increase the thickness of

one sheet, but little research on these methods was found.

Design Tests

Many tests would have to be performed on our proposed strategy to determine its efficacy and

viability for treatment. Initially, we would want to perform as many acellular, in vitro tests as

possible. The silk sheets would be synthesized, including RGD coupling and the short time water

vapor annealing. Degradation tests would be performed: the mass of the material would be

determined over a period of time and the degradation products would be tested for toxicity.

Additionally, the mechanical stiffness would be quantified to ensure adequate support of the

tissue. Gas diffusion tests would also be necessary to determine if the required amount of oxygen

can diffuse through the material in the desired amount of time. Layering the stacks of silk sheets

could also be tested acellularly, at first. Eventually, we would probably want to culture cells on

each of the sheets before stacking them because the cells could not easily migrate the entire

400μm depth. Of course, all of these tests would need to be repeated once cells were introduced

into the scaffold to verify the results.

After harvesting the dental pulp stem cells and differentiating them, we would need to test gene

and protein expression to ensure successful differentiation. Our keratocytes would then be

seeded onto the silk scaffolds and live/dead assays would be performed to ensure the viability of

the cells since dental pulp-derived keratocytes have not been combined with silk fibroin before.

We would also want to ensure that key ECM components—collagen I, V, and VI, proteoglycans,

keratocans, lumican and keratan sulfate—were being produced in the biomaterial in the same

concentrations as in the native microenvironment. To ensure the clarity of the tissue, we would

perform image analysis to examine the size and spacing of the lamellae and the orientation of the

fibrils and lamellae. Furthermore, the amount of visible light wavelengths that pass through the

biomaterial would be quantified.

Initial biocompatibility tests could be performed in vitro but would ultimately need to be moved

to in vivo models. The activation of keratocytes to corneal fibroblasts and the deposition of scar

tissue are both indicative of rejection. The formation of vascular networks and a hazy appearance

also denote complications with biocompatibility as well as interfere with optical transparency15.

Eventually, tests would be performed in vivo, beginning with mouse models and hopefully

working up to clinical trials. The end goal would be a replacement for allogenic donor tissue that

would be at least as effective for the patient and easy for the surgeon to transplant by already-

familiar methods.

Conclusions

The need for a viable engineered corneal tissue is increasing due to the decreasing number of

usable donor corneas. A majority of the corneal transplants performed each year are performed

by replacing the epithelium and stroma layers of the cornea. A silk fibroin scaffold has been

shown to be biocompatible, have inherent transport properties that are necessary for corneal

function, and been shown to support the proliferation of endothelial cells.23,24 Furthermore, the

use of dental pulp stem cells as a cell source for keratocytes has been shown to be a viable

option.25 Therefore, a novel use of a silk fibroin scaffold seeded with keratocytes differentiated

from dental pulp stem cells has been proposed for the use in cornea transplants.

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