Chris Lynch (cslynch)

BME 203 Fall 2014

Collagen Hydrogels for Vocal Fold Restoration
Vocal folds, often referred to as vocal cords, are composed of twin infoldings of mucous
membrane stretched horizontally across the larynx [as shown in Figure 1 (7)]. They vibrate,
modulating the flow of air being expelled from the lungs during phonation (1). Phonation is the
modification of the vocal folds for speech. During speech, the degree of tension and space of
each vocal fold is modified to create a slit like hole that creates resistance to the air flow and sets
up a series of laryngeal sound waves with various pitch and intensity (2). Males tend to have a
larynx that is 40% larger than the female larynx and this difference in size is called the laryngeal
prominence (also known as the Adams apple) (3). Vocal fold resonating frequency varies
through each stage of life for both males and females. Baby cries have a fundamental frequency
of around 500 HzChild speech ranges from 250-400 Hzadult females tend to speak at
around 200 Hz on averageand adult males around 125 Hz (3). Repairing vocal folds proves to
be challenging because there are three main considerations specific to the therapeutic response
of vocal folds which must be addressed when designing and evaluating gel-based injectables:
inflammatory response, ECM remodeling response, and biomechanical response (45).

Figure 1. Vocal Fold Orientation

With the constant abuse vocal cords endure, injury is likely to occur. Vocal fold
scarring, a specific vocal fold injury is accompanied by a marked decrease in voice quality and
control secondary to pathophysiological [functional changes associated with or resulting from
disease or injury (5)] changes of the vocal fold lamina propria [allows for ease of vibration
throughout vocal fold (51)] extracellular matrix (ECM) (4). Living tissue is not a solid mass of
tightly packed cells. Much of a tissues volume is made up of extracellular spaceThis void is
filled with a complex meshwork called the extracellular matrix [ECM]the extracellular matrix
largely determines how a tissue looks and functions. The extracellular matrix is made up of
proteoglycans, water, minerals, and fibrous proteins. A proteoglycan is composed of a protein

core surrounded by long chains of starch-like molecules called glycosaminoglycans (6). There
were two main hydrogels tested to aid in restoration, hyaluronic acid (HA) based hydrogels and
polyethylene glycol (PEG)-diacrylate based hydrogels/semi-IPNs, which were found to
approximate the viscoelastic mechanical properties of the native human vocal mucosai (vibratory
component) and the vocal ligament (strain component), respectively (46). Many researches
have conducted several experiments to determine the most effective way of repairing vocal fold
scarring.
Vocal fold scarring [shown in Figure 2 (24)] is a term that refers to fibrous tissue
replacing normal tissue and causes multiple layers of the vocal folds to be fixed together (18).
When the vocal folds are fixed together, they lose the ability to be able to vibrate independently.
Vocal fold scarring usually results from blunt laryngeal trauma or, more commonly, as the
result of surgical injury after excision or removal of vocal fold lesions (20). Vocal fold scars
prevent the mucosa [mucous membrane (23)] from vibrating freely as a result of being fixed to
underlying tissue (22). A vocal fold scar needs to be evaluated by stroboscopy. A stroboscopy is
a laryngoscopy [examination of throat, voice box (larynx), and vocal cords (19)] with
synchronized strobe light that provides a slow motion-like view of vocal fold vibration (mucosal
wave) (16). Treatment of vocal fold scars is one of the most difficult voice problems to treat
and depends on the cause of the scar and how long the scar has been present (21). Surgery for
mature scars is a hassle because of the difficulty in restoring the layers to normal separation.
Normal separation is imperative for normal phonation (17).

Figure 2. Stroboscopy image of scarred vocal fold

Past research and experiments on vocal fold scarring have revealed changes in the
organization and distribution of extracellular matrix components (ECM), such as dense and/or
disorganized type 1 collagen deposition decreased elastin and decorin, increased fibronection,
and occasional decreases in hyaluronic acid (HA)Given that these histological changes stiffen
the properties of the vocal fold, phenotypic changes of vocal fold fibroblasts and a correction of
the distribution of ECM components is needed to restore the vocal fold after scarring (8).
Fibronectin functions, not only, as an adhesion molecule by anchoring cells to collagen substrate,
but also to organize cellular interactions with the extracellular matrix by binding to different

components of the extracellular matrix (9). Many hydrogels have been researched to test the
ability of restoration.
Hydrogels [used in drug release and tissue engineering] are cross-linked, insoluble threedimensional networks of polymer chains with the empty space being composed of water. Crosslinking ensures the hydrogel is insoluble in water and provides the required mechanical strength
and physical integrity (11). PEG diacrylate-based hydrogels exhibited linear stress-strain
response and elastic modulus consistent with the properties of the vocal ligament at low strains
(0-15%), but did not replicate the non-linear behavior observed in native tissue at higher strains.
Methacrylated hyaluronic acid hydrogels displayed dynamic viscosity consistent with native
vocal mucosa, while elastic shear moduli values were several-fold higher. Cell culture studies
indicated that both hydrogels supported spreading, proliferation and collagen/proteoglycan
matrix deposition by encapsulated fibroblasts throughout the 3D network (10).
Poly(ethylene) glycol (PEG) is a biomaterial that is inherently resistant to non-specific
cell adhesion and protein adsorption, thus providing a blank slate upon which ECM-derived
signals can be systematically introduced as well as spatially and temporally manipulated to
control cell behavior and tissue regeneration (12). In order to create PEG hydrogels, PEG chains
functionalized with multiple crosslinkable groups, are dissolved in aqueous solution, mixed with
a photoinitiator such as 2,2 dimethoxy-2-phenyl acetophenone, and exposed to UV or visible
light (13). A hydrogel system composed of collagen and multi-armed PEG (represented in Figure
2 (15)) has been developed to inhibit tissue regeneration. The collagen component enables
cellular adhesion and permits enzymatic degradation, while the multi-armed PEG component has
amine-reactive chemistry that also binds proteins/tissue and is hydrolytically degradable (14).
PEG hydrogels are highly elastic, which is important to the vocal folds which must sustain
prolonged high frequency stresses, and their mechanical properties are tunable, meaning that
their properties can be tailored to patient needs (43). The elasticity and ability to be tuned make
PEG hydrogels a viable material for vocal fold restoration.

Figure 2. Step-growth PEG hydrogel

The extracellular matrix [Figure 3 (36)] provides the cell with a microenvironment in
which the cell can exist. The ECM is responsible for cell anchorage, serves as a tissue scaffold,
guides cell migration during wound repair, has a key role in tissue morphogenesis [shaping of an
organism according to genetic blueprint (34)], and is responsible for transmitting environmental

signals to cell, which affects cell proliferation [rise in number of cells resulting from cell growth
and division (35)] (33). Communication of ECM occurs from molecules bound to the plasma
membrane or intracellular receptors. The receptors are deciphered by molecular pathways that
eventually converge on transcription factors inducing gene modification and in turn creating
chemical signals (37). Blood clotting is an example of ECM communication. When the cells
lining the blood vessel are damaged, they display a protein receptor called tissue factor. When a
tissue factor binds with another factor in the extracellular matrix, it causes platelets to adhere to
the wall of the damaged blood vessel and stimulates the adjacent smooth muscle cells in the
blood vessel to contract (thus constricting the blood vessels). Subsequently, a series of steps are
initiated which then prompt the platelets to produce clotting factors (36).

Figure 3. Extracellular Matrix

Injectable hydrogels have recently been a major research topic in tissue engineering for
the repair of vocal folds. Injectable hydrogels are showing promise as scaffolds in regenerative
medicine because they can be injected in liquid form and transform in situ [in the normal
location (48)] into the gel state (47). The hydrogels or compositions thereof are injected into
the superficial lamina propria or phonatory epithelium to restore the phonatory mucosa of the
vocal cords, thereby restoring a patient's voice (44). There are three reasons as to why injectable
hydrogels are strongly preferred. First, an injectable material could be formed into any desired
shape at the site of injury upon injection. Second, crosslinkable polymer mixtures would adhere
to the tissue during gel formation and the resulting mechanical interlocking would strengthen the
tissue-hydrogel interface. Third, introduction of a crosslinkable hydrogel could be accomplished
by injection, thereby minimizing the invasiveness and potential trauma of the procedure (25).
Hydrogels have been injected into scaffolds to reduce the healing time.
Scaffolding has been researched to aid in the reconstructive abilities of the vocal fold. In
order to aid in tissue regeneration, a scaffoldwill act as a temporary matrix for cell
proliferation and extracellular matrix deposition, with subsequent ingrowth until the tissues are
totally restored or regenerated (28). The scaffold needs to be designed to withstand and
effectively transfer local stresses evenly across the area of implantation during the degradation
period. Also, the degradation properties are catered to match the cell differentiation rates and
extracellular matrix deposition rates of the implant site in order to provide continuous support

throughout the repair process (49). Scaffolding is a key process in the ability to one day grow
new organs. Bioengineers use scaffolds to mimic the bodys extracellular matrix, which
supports the growth and maintenance of living cells. Synthetic scaffolds are used as frameworks
to form replacement tissues and, perhaps someday, regenerate entire organs from a patients own
cells. Once their work is done, the scaffolds are designed to degrade and leave only natural,
healthy tissue behind (50). Hydrogels are used as scaffolding materials for drug and growth
factor delivery, engineering tissue replacements, and a variety of other applications (27). Tissue
regeneration requires materials that not only serve as bulk mechanical and structural
requirements, but as well as enabling interactions with cells at the molecular level (29). Collagen
meets the requirements to aid in tissue regeneration.
Collagen hydrogels have shown promising results in ability to regenerate tissue,
specifically vocal fold tissue. Collagen is the most widely used tissue-derived natural polymer,
and it is a main component of extracellular matrices of mammalian tissues including skin, bone,
cartilage, tendon, and ligament (26). Collagen hydrogel fibers are formed in a hydrated
environment by self-aggregation [blood cell clumping process (30)] and crosslinking of the
collagen molecules [represented in Figure 4 (32)]. Collagen is formed in a triple helix structure.
A repeated sequence of three amino acids forms this sturdy structure. Every third amino acid is
glycine, a small amino acid that fits perfectly inside the helix. Many of the remaining positions in
the chain are filled by two unexpected amino acids: proline and a modified version of proline,
hydroxyproline (31). Typically, hydrogels based on polymers from natural origins such as
collagen are advantageous in tissue engineering applications due to their intrinsic characteristics
of biological recognition, including presentation of receptor-binding ligands and the
susceptibility to cell-triggered proteolytic remodeling and degradation (32). One example of a
collagen hydrogel is in the product Salvecoll. When the Salvecoll gel is injected into the skin,
fibroblastsbegin to migrate from the surrounding tissues and invade the collagen gel.
Additionally, the injection has the immediate effect of mechanically filling the defects, then a
transition matrix is formedwhich stimulates the immune system and activation of
granulocytes, macrophages and fibroblasts, accompanied by enhanced transport of growth
factors released from cells, which in turn leads to increased migration and proliferation of
fibroblasts and epithelial cells. The purpose of the collagen gel is to provide a matrix for the
cells, allowing them to form new tissue (40). Collagen is the protein that gives the skin its
tensile strength while also playing a large role in wound healing (41).

Figure 4. Basic Structure of Collagen Hydrogel Fibers

Collagen-alginate hydrogels have proven to be the most effective at restoring tissue

within the extracellular matrix to promote the restoration of the vocal folds. These particular
hydrogels were experimented because of their ability to support ECM synthesis with limited
hydrogel compaction and/or resorption. Collagen-alginate compositesresisted scaffold
compaction and mass losswhile allowing for ECM synthesis (38). On the contrary, the
matrix protein levels detected in collagen-HA gelswere comparable to those in porcine vocal
fold tissue (39). HA-based hydrogels have been studied more closely than collagen hydrogels
because the half-life of native HA is short, synthetic HAhas been developed to control HAs
turnover rate through crosslinking and chemical modification (42). Vocal fold restoration
[Figure 5 (27)(28)(40)(47)(50)] still proves to be a very challenging task due to the vocal folds
being delicate while being strong enough withstand much force.

Figure 5. Vocal Fold Restoration with Collagen Hydrogel

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