1. Andrea Marks, The Design and Development of an Enzymatically Degradable Hydrogel for Tissue Engineering
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The Design and Development of an Enzymatically Degradable
Hydrogel for Tissue Engineering
Andrea Marks, UROP Proposal, 2015
Abstract
Biodegradable scaffolds have shown to improve formation of tissue and allow for the elaboration of
extracellular matrix that is imposed by nondegradable scaffolds. One challenge with these biodegradable
scaffolds is designing them so that the scaffolds degradation is proportional to the matrix deposition. The
objective of this research is to design and develop an enzymatically degradable hydrogel for cartilage
tissue engineering such that the peptide linker can be degraded at a rate that enhances chondrogenesis of
hMSCs. Specifically, we will be focusing on using an MMP-7 degradable peptide linker which is
upregulated during chondrogenic differentiation, to allow for degradation proportional to matrix
deposition.
Introduction
Osteoarthritis is the most common joint disease with 48 million Americans currently diagnosed
and 67 million Americans projected to have it by 2030 (Hootman & Helmick, 2006). One of the leading
causes of osteoarthritis is cartilage defects either due to disease or injury that have been left untreated.
Due to the lack of regenerative properties of cartilage tissue (low cell density and avascularity), treatment
of these defects is necessary to prevent osteoarthritis, and cartilage tissue engineering is a promising form
of treatment (Elders, 2000). However, engineering natural, native cartilage tissue from one’s own cell
source, without any synthetic scaffold materials, to allow for full integration of the neotissue into the
defect has been found to be challenging (Nguyen, Kudva, Guckert, Linse, & Roy, 2011).
Hydrogels, specifically poly(ethylene glycol) hydrogels, are a promising scaffold material for
cartilage tissue engineering due to their high water content, tissue-like properties, biocompatibility, and
talorability (Slaughter, Khurshid, Fisher, Khademjosseini, & Peppas, 2009) (Kloxin, Kloxin, Bowman, &
Anseth, 2010). Biochemical moieties and peptides can be incorporated within the hydrogels, allowing for
the ability to engineer an environment that closely mimics that of the native cell environment. Human
mesenchymal stem cells (hMSCs) are an attractive cell source for cartilage tissue engineering because
they have the ability to differentiate into cartilage tissue, as well as other cell lineages, and can be isolated
in a less invasive procedure than a cartilage biopsy (Noth, Steinert, & Tuan, 2008) (Caplan, 1991)

2. Andrea Marks, The Design and Development of an Enzymatically Degradable Hydrogel for Tissue Engineering
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(Pttenger, 1999). The interaction of hMSCs with their environment plays an important role in their
differentiation, thus the incorporation of the biochemical cues and degradable peptides into hydrogel
scaffolds can be used to locally control the differentiation of hMSCs.
Current research within the Bryant lab has looked at the incorporation of different biochemical
cues and mechanical stimulation in hydrogels to enhance the chondrogenesis of hMSCs. Specifically,
results have shown that the incorporation of the negatively charged glycosaminoglycan, chondroitin
sulfate (ChS), which is found in native cartilage tissue, as well as the adhesion peptide, RGD, enhances
chondrogenesis (Villanueva, Gladem, Kessler, & Bryant, 2010) (Nuttelman, Tripodi, & Ansesth, 2005).
Further enhancement was found when the hydrogels were subjected to unconfined dynamic compression.
However, during hMSC chondrogenesis in the nondegradable hydrogels, the cells are limited in the
amount of tissue they can produce because they are surrounded by a synthetic polymer matrix. We
believe that by creating a hydrogel that can degrade at a rate that allows it to maintain its structural
integrity while still degrading enough to allow ample room to foster cartilage development; we can
enhance the chondrogenesis of hMSCs.
The objective of this research is to design and develop an enzymatically degradable hydrogel for
cartilage tissue engineering, such that the peptide linker can be degraded at a rate proportional to
chondrogenesis and extracellular matrix production of hMSCs. Specifically, we will be focusing on using
an MMP-7 degradable peptide linker. MMP-7 is upregulated during chondrogenic differentiation, making
its degradable peptide sequence a desirable linker, to allow for degradation proportional to matrix
deposition (Tibbit & Anseth, 2009). It is important that as the extracellular matrix has room to develop
and create an integrated tissue. That is why our hydrogel must be degradable at a rate proportional to
matrix production (Anderson, Lin, Kuntizer, & Anseth, 2011).
This research will be conducted in the Jenny Smoli Caruthers Biotechnology Building on the
University of Colorado east campus. I will be working under the immediate supervision of graduate
student Elizabeth Aisenbrey, who works in the Bryant Lab.
My degree is in Biological and Chemical Engineering. This type of research directly correlates
with the field I hope to go in to in the future. I hope to work in tissue engineering and stem call research.
This research emphasizes the biological engineering part of my degree. All but one of my classes so far
has strongly emphasized the chemical engineering side of my degree, such as learning about chemical
separations, processes and kinetics. Yet my true interest lies in the bio side. The one class that supported
the biological side of my degree was biomaterials. Here we learned about different types of drug delivery,
as well as the three types of materials used to create medical implants. Thus working in a lab that is more
relevant to what I want to do really augments my knowledge of the biological side of my degree, and
helps open up opportunities for me to continue this type of work in the future.

3. Andrea Marks, The Design and Development of an Enzymatically Degradable Hydrogel for Tissue Engineering
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Background
Since 1960, hydrogels have been used and tested as a means for controlled drug delivery.
Recently they have been used as a favorable environment to promote chondrogenic differentiation.
Hydrogel scaffolds are used to deliver cells to the defect area and provide them with the support and
biochemical cues necessary to become the desired tissue (in this case, cartilage) and they facilitate matrix
development (Kloxin, Kloxin, Bowman, & Anseth, 2010) (Nuttelman, Tripodi, & Ansesth, 2005).
Current research has looked at using hMSCs in hydrogel scaffolds as a treatment for cartilage defects.
Human mesenchymal stem cells are a promising cell source for cartilage tissue engineering because they
can be differentiated into multiple cell types, including chondrocytes (Caplan, 1991). The differentiation
can be controlled by incorporating different biomolecules within the hydrogel and applying mechanical
stimulation. As differentiation occurs, the cells begin to create their own extracellular matrix. In previous
research, biodegradable scaffolds have shown to improve formation of tissue because they remove the
interference of synthetic polymers and allows for the elaboration of extracellular matrix that is imposed
by nondegradable scaffolds (Tibbit & Anseth, 2009). With enzymatically degradable materials, the
scaffold is capable of providing initial support to the encapsulated cells, but as the cells begin to produce
their own matrix (a natural scaffold) they also release enzymes that are capable of breaking down the
synthetic scaffold which provides the room necessary for the elaboration of the cells own matrix. Yet, one
challenge with these biodegradable scaffolds is designing them in such a way that the scaffolds
degradation is proportional to the matrix deposition. Thus, this situation prompted the need for research
into a biodegradable scaffold that degrades at a rate proportional to cartilage matrix development.
I have worked in many labs previous to this one. Two of them, in Cleveland, have focused on
more clinically relevant research. Thus I am proficient in the later side of research, conducting animal
experimentation as well as basic implantation in the animal subjects. Therefore I am well knowledged in
the practice of sterile procedure and know how to conduct medical trials. Yet, what I have not yet had
experience in is being at the head of actually developing engineering materials that could be used in
medical treatments. In the future I hope to be active in the development stages of medical biomaterials,
rather than solely working on the latter side. Yet, my experience with animal studies has well provided
me with the skills to do animal studies of my own biomaterials once they are developed.
Over the past academic year I have been working with Elizabeth to learn how to conduct
a cell study as well as make hydrogels. Over the past semester and a half I have learned how to passage
and feed stem cells, make hydrogels with and without cells, conduct mechanical testing, stain cells for
protein cultures, clean all necessary laboratory equipment and how to synthesize peptides by hand and

4. Andrea Marks, The Design and Development of an Enzymatically Degradable Hydrogel for Tissue Engineering
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with the peptide synthesizer. The work I have done over the past months I feel have fully prepared me to
efficiently and effectively conduct the proposed study.
Methods
There will be three main goals of the research: determine and develop a degradable peptide linker,
incorporate the degradable peptide into a hydrogel network and determines the degradation kinetics, and
finally perform cell studies that investigate the affect the degradable linker has on the chondrogenesis of
encapsulated hMSCs. The
first aim of the study, again, is to create a peptide that degrades at a rate proportional to matrix
development to allow ample cell growth. This will begin by conducting a literature review to determine if
there has been previous research on degradable peptides, like the one we are looking for, and to gain
information of what amino acids to use in the peptide. By doing so, we hope to investigate the
biochemistry of peptide to be created. This can also be done using websites that calculate isoelectric
points as well as hydrophobicity of the peptides to aid in peptide optimization. Once we have modeled an
acceptable peptide we will synthesize it by hand, using pre written protocols that have been used
previously in the lab. Purity and yield of the peptide will be conducted via mass spectroscopy after each
amino acid addition, and for the final product.
The second aim will be to study the peptide in the hydrogel as well as determine its degradation
kinetics. Before determining the degradation kinetics it is necessary to determine if the peptide can be
properly incorporated into the hydrogel network. Proper incorporation will be tested by submerging the
hydrogels into a high concentration of MMP-7 enzyme. If the peptide is properly incorporated into the
hydrogel and is the only crosslinker, the hydrogel will degrade. This will be investigated by measuring
the elastic modulus of the hydrogel over time as it is submerged in the enzyme bath, as well as measuring
the wet weight of the hydrogel over time. If the peptide is the only crosslinker in the hydrogel, eventually
the entire hydrogel will degrade.
Once that aspect is investigated, degradation studies will be conducted. We will
investigate the degradation of the hydrogel with differing weight percent of peptide linker in different
enzyme concentration without cells. In this manner, we can determines appropriate proportions of peptide
to optimize the hydrogel degradation. Afterwards, we will investigate the degradation of the hydrogel in
cell cultured media. We will take the media from chondrogenically differentiated hMSCs which will be
secreting MMP7, and culture our acellular degradable hydrogels in this media. This will give us an idea
of the amount of MMP7 that is released from the cells during differentiation, and the rate at which our
hydrogels will degrade when cells are encapsulated. Degradation will be measured by comparing the wet
weight, dry weight, swelling ratio and compressive modulus of hydrogels congaing the peptide to those

5. Andrea Marks, The Design and Development of an Enzymatically Degradable Hydrogel for Tissue Engineering
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without. Degradation kinetics results will be gathered and analyzed to determine a hydrogel formulation
that will have the degradation proportional to the extracellular matrix production of the encapsulated
cells. This formulation will be used for further cell-based studies.
The third aim of the study, again, is to investigate the affect the degradable linker, peptide, has on
the chondrogenesis of encapsulated hMSCs. Investigation of this will begin by preforming a one week
viability study to ensure that the peptide and the degradation products are not cytotoxic. Once it is
confirmed that the peptide is not harmful to the cells, a 28 day differential study will be conducted as
follows;
o Cells will be encapsulated into the hydrogel and allowed to free swell for the first 7 days of the study
o Half of the hydrogels will then undergo unconfined dynamic compression for the following 21 days
o Differentiation of the hMSCs will be monitored by taking samples at day 7,14,21,28 and will be
measured by: gene expression of chondrogenesis markers collagen I, II, X, aggrecan, RunX2, and
Sox9 using quantitative RT-PCR, protein production using immunohistochemistry staining of
collagen I, II, X, aggrecan, RunX2, GAG production using a fluorometric assay, ELISAs for
quantitative protein production levels.
o Samples will also be taken at day 7, 14, 21, and 28 to measure the rate of degradation by measuring
the compressive modulus, wet weight, and swelling ratio
After data collection, data will be analyzed to determine the effect of the degradable peptide on
chondrogenesis with and without mechanical loading. Based on these results, further experiments will be
designed to further study the effects of the degradable linker.

6. 6
May June July August
Aim 1 Literature review of enzymatically degradable
peptides in hydrogels
X
Develop and build peptide linker X
Determine purity and yield of peptide using mass
spectrometry
X
Aim 2 Ensure the incorporation of the degradable peptide
linker in the hydrogel
X
Determine the degradation kinetics of the acellular
hydrogel with varying weight percents of the
degradable linker and enzyme concentration
X X
Determine the degradation rate of acellular
hydrogels using chondrogenically differentiated
hMSC cultured media
X
Aim 3 Conduct a viability study of hMSCs encapsulated in
the degradable hydrogel
X
Culture cells for degradation study X X
Conduct a 28 day differentiation study of
encapsulated hMSCs in the degradable hydrogel
X X
Analyze the differentiation of hMSCs using RT-
qPCR, immunohistochemistry, and ELISAs
X X
Write report and finalize project X

7. 7
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