Tissue engineering hydrogels with tunable degradability Tissue Engineering Min Lee, Soyon Kim, University of California, Los Angeles; Zhong-Kai Cui, University of California, Los Angeles; Min Lee, University of California, Los Angeles Corresponding Author: Min Lee, University of California, Los Angeles, [email protected]Abstract Photopolymerizable hydrogels derived from naturally occurring polymers have a unique opportunity to regenerate damaged tissues due to their excellent biocompatibility, hydrophilic nature favorable for cell ingrowth, and ability to be cured in situ through a minimally invasive procedure. We have previously developed injectable hydrogels using photopolymerizable chitosan (MeGC) and riboflavin (RF), an aqueous initiator from natural vitamin, that allow gelation in situ upon exposure to visible blue light (VBL). Tissue engineering scaffolds must be designed to degrade to allow subsequent tissue regeneration or the release of encapsulated bioactive molecules. Although the MeGC hydrogel supported proliferation and deposition of extracellular matrix (ECM) by encapsulated mesenchymal stem cells, it showed relatively slow degradation that may hinder cell recruitment and delay tissue regeneration. In this work, we have developed MeGC hydrogels with tunable degradation kinetics by introduction of hydrogel-specific enzymes. Lysozyme is existed ubiquitously in human body and could degrade chitosan by cleaving its polysaccharide backbone. Thus, we investigated whether the degradation rate of MeGC hydrogels can be modulated by introducing lysozyme and varying the enzyme concentration. Briefly, glycidyl methacrylate was reacted with lysozyme solution to synthesize photo-reactive lysozyme. Methacrylated lysozyme was mixed with MeGC solutions and hydrogels were formed by VBL- crosslinking in the presence of RF. The incorporation of lysozyme to MeGC hydrogels significantly increased the degradation rate of the hydrogels in a dose dependent manner. The lysozyme-functionalized MeGC hydrogels were not cytotoxic and significantly enhanced proliferation of encapsulated bone marrow stromal cells (BMSCs) as confirmed by the alamar blue assay. The encapsulation of BMSCs improved hydrogel modulus over time in culture compared with cell-free hydrogels, indicating deposition of ECM on hydrogels as the hydrogel degrades. In addition, the lysozyme- functionalized hydrogels significantly upregulated alkaline phosphatase (ALP) expression and osteogenic gene markers in encapsulated BMSCs and increased mineral deposition. Hydrogel degradation and osteogenic activity was further confirmed in a mouse calvarial defect model. Histological examination of the in vivo samples demonstrated remarkable hydrogel degradation with high cell infiltration at day 7. The MeGC hydrogels functionalized with lysozyme significantly
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Tissue engineering hydrogels with tunable degradability
Tissue Engineering
Min Lee, Soyon Kim, University of California, Los Angeles; Zhong-Kai Cui, University of California, Los Angeles;
Min Lee, University of California, Los Angeles
Corresponding Author: Min Lee, University of California, Los Angeles, [email protected]
Abstract
Photopolymerizable hydrogels derived from naturally occurring polymers have a unique opportunity to regenerate
damaged tissues due to their excellent biocompatibility, hydrophilic nature favorable for cell ingrowth, and ability to be
cured in situ through a minimally invasive procedure. We have previously developed injectable hydrogels using
photopolymerizable chitosan (MeGC) and riboflavin (RF), an aqueous initiator from natural vitamin, that allow gelation
in situ upon exposure to visible blue light (VBL). Tissue engineering scaffolds must be designed to degrade to allow
subsequent tissue regeneration or the release of encapsulated bioactive molecules. Although the MeGC hydrogel
supported proliferation and deposition of extracellular matrix (ECM) by encapsulated mesenchymal stem cells, it
showed relatively slow degradation that may hinder cell recruitment and delay tissue regeneration. In this work, we
have developed MeGC hydrogels with tunable degradation kinetics by introduction of hydrogel-specific enzymes.
Lysozyme is existed ubiquitously in human body and could degrade chitosan by cleaving its polysaccharide backbone.
Thus, we investigated whether the degradation rate of MeGC hydrogels can be modulated by introducing lysozyme and
varying the enzyme concentration. Briefly, glycidyl methacrylate was reacted with lysozyme solution to synthesize
photo-reactive lysozyme. Methacrylated lysozyme was mixed with MeGC solutions and hydrogels were formed by VBL-
crosslinking in the presence of RF. The incorporation of lysozyme to MeGC hydrogels significantly increased the
degradation rate of the hydrogels in a dose dependent manner. The lysozyme-functionalized MeGC hydrogels were not
cytotoxic and significantly enhanced proliferation of encapsulated bone marrow stromal cells (BMSCs) as confirmed by
the alamar blue assay. The encapsulation of BMSCs improved hydrogel modulus over time in culture compared with
cell-free hydrogels, indicating deposition of ECM on hydrogels as the hydrogel degrades. In addition, the lysozyme-
functionalized hydrogels significantly upregulated alkaline phosphatase (ALP) expression and osteogenic gene markers in
encapsulated BMSCs and increased mineral deposition. Hydrogel degradation and osteogenic activity was further
confirmed in a mouse calvarial defect model. Histological examination of the in vivo samples demonstrated remarkable
hydrogel degradation with high cell infiltration at day 7. The MeGC hydrogels functionalized with lysozyme significantly
promoted calvarial repair at week 6 as demonstrated by micro-computerized tomography and histology, compared with
unmodified MeGC. These findings suggest a promising approach to modulate the degradation behavior of hydrogels via
enzyme functionalization for improved tissue remodeling.
D
Effects of Wettability and Surface Roughness of Hollow Fiber Membranes on Bacterial Adhesion
Biomaterials
Kaitlyn Anderson, Utah State University; David Britt, Utah State University
Corresponding Author: David Britt, Utah State University, Biological Engineering Department, [email protected]
Abstract
Hollow fiber membranes (HFMs) have a wide variety of applications, such as for hemodialysis filtration membranes and
as bioreactors, and, depending on the use, cellular adhesion to the HFMs may be discourage, the former, or encouraged,
the latter. Fortunately, HFMs can be created from many different spinmass mixtures of polymers and additives with
each mixture yielding a particular set of HFM characterizations. This research will be focusing on the properties of
wettability and surface roughness of a variety of commercially available HFMs and how they impact the adhesion of two
various polymer-modified carbon dots for inactivation of bacterial pathogens, bacterial spores, and viruses, inhibition of
biofilms, as well as for capturing and concentrating bacterial pathogens. The results indicated that SWNTs, their
composites or combination with other chemical and physical methods were very effectively inactivated various bacterial
pathogens including common Salmonella, E. coli cells, Bacillus anthracis cells in suspensions. Whereas MWNTs were not
effective in inactivating bacterial cells in suspension, but MWNTs modified surfaces, including MWNT forest on silicon
wafer and MWNT sheet on poly(methyl methacrylate) (PMMA) film, significantly increased surface hydrophobicity and
enhanced the attachment of spores on their surfaces compared to the uncoated substrates, respectively, showing their
potential as adsorbents for removal of pathogens from fluids.
Another class of notable antimicrobial carbon-based nanomaterials is carbon dots (CDots). CDots defined as small
carbon nanoparticles with various surface passivation schemes, with their optical properties and photocatalytic
functions. They have been evaluated for their photoinduced bactericidal functions, with the results suggesting that the
dots were highly effective in bacteria-killing with visible light illumination. Mechanistic implications of the results will be
discussed. Challenges and opportunities in further development of CDots into a new class of effective visible/natural
light-responsible bactericidal agents for bacteria control and other potential antimicrobial applications will be discussed.
Acknowledgement: This research was supported partially by the USDA grant 2011-68003-30395 and the NIH grant
R15GM114752.
D
The approaching renaissance for calcium phosphates in biomedicine
Biomaterials
Vuk Uskokovic, Chapman University, University of Illinois at Chicago
Corresponding Author: Vuk Uskokovic, Department of Bioengineering, University of Illinois, Department of Biomedical and Pharmaceutical Sciences, Chapman University, [email protected]
Abstract
Calcium phosphate was selected by Nature to be the firm foundations of our bodies. This has served as an invaluable
inspiration for materials scientists who have attempted to discover in this material more potential than meets the eye.
Efforts are currently being made to expand the application repertoire of calcium phosphates beyond their use as
traditional bone fillers or tissue engineering construct components that impart osteoconductivity and high compressive
strength. The application of calcium phosphates for sustained drug delivery, gene and anticancer therapies, antibiofilm
coatings and hard tissue regeneration has been intensely explored recently. All this plethora of applications for calcium
phosphates that are now in the R&D stage are the consequence of the immense structural complexity of this material,
which is being directly reflected in its ability to display an array of exciting properties under precise synthesis regimens.
Like water, the princess of peculiarities in the realm of liquids, calcium phosphate deserves the epithet of the prince of
peculiarities in the realm of solids. Its protean nature and the applicative potentials arising from this peculiar nature will
be described in this lecture. Foreseen on the horizon will be a new generation of materials for therapeutic and
regenerative applications, containing only precisely designed calcium phosphates and substituting for the role of
expensive bone growth factors, antibiotics, viral vectors and polymers.
D
Vitamin E-coated hernia repair meshes for the mitigation of the oxidative stress
Biomaterials
Dmitry Gil, Vladimir Reukov, Clemson University; William Cobb, Greenville Health System; Alexey Vertegel, Clemson University
Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regeneration capacity
of adult human hearts, human induced pluripotent stem cell (hiPSC) has emerged as a powerful cell source for cardiac
repair. However, the current hiPSC-derived cardiomyocytes (hiPSC-CMs) poses arrhythmogenic risk after transplantation
due to their unspecific and immature phenotype. In addition, the hiPSC-CMs are quickly redistributed to other organs
after transplantation due to the shear stress in the beating hearts. To address this, my lab recently pioneered the use of
the electrical conductive silicone nanowire to facilitate the self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac
spheroids. The addition of e-SiNWs into human cardiac spheroids creates an electrically conductive microenvironment
and improves the functions of hiPSC-CMs. In addition, 3D configuration of the nanowired spheroids can improve their
engraftment after transplantation. Further, our recent research showed electrical stimulation can synergize with silicon
nanowire to facilitate the ventricular lineage specification and cellular maturation of hiPSC-CMs. Here, I will discuss the
recent development of my lab in the use of the electrical stimulation to derive hiPSC-CMs with enhanced ventricular
maturity for transplantation applications.
D
Sulfonated chitosan hydrogel to stabilize bone morphogenetic protein activity for enhanced osteogenesis
Tissue Engineering
Min Lee, Soyon Kim, University of California, Los Angeles; Zhong-Kai Cui, University of California, Los Angeles;
Min Lee, University of California, Los Angeles
Corresponding Author: Min Lee, University of California, Los Angeles, [email protected]
Abstract
Although bone morphogenetic protein-2 (BMP-2) has demonstrated extraordinary potential in bone formation, its
clinical applications require supraphysiological milligram-level doses. The premature release of such high dose BMP-2
from conventional collagen carriers may lead to ectopic bone formation and numerous side effects such as well-
documented life-threatening cervical swelling and osteoclastic bone resorption. Heparin is widely used in controlled
release systems for many heparin binding growth factors including BMP-2 due to its strong binding property and
protective effect for these proteins. However, heparin suffers from natural variability in structure, difficult modification,
and many non-targeting bioactivities. Similar biological activities to heparin have been observed in small compounds
that contain groups similar to those in the heparin, in particular, sulfates or sulfonates. In this work, we developed a
hydrogel surface that can mimic heparin to stabilize BMP-2 and enhance osteogenesis by incorporating two heparin-
mimicking polysulfonates, Poly-vinylsulfonic acid (PVSA) and Poly-4-styrenesulfonic acid (PSS) into photo-crosslinkable
hydrogel already developed in our previous study using methacrylated glycol chitosan (MeGC) and riboflavin initiators
Incorporation of heparin-mimicking molecules was performed by dissolving PVSA or PSS in MeGC solution and
subsequently crosslinking MeGC to form a gel. The homogeneous distribution and high entrapment efficiency (>95%) of
PVSA or PSS was verified by toluidine blue staining. The protective effect of PVSA or PSS on BMP-2 stability was
confirmed in various therapeutically relevant environments presenting thermal, acidic and enzymatic stressors in
comparison to natural heparin by assessing the ability of BMP-2 to increase alkaline phosphatase (ALP) expression in
bone marrow stromal cells (BMSCs). Ability of the developed hydrogels to enhance BMP-2 signaling and osteogenic
activity was determined with or without the addition of exogenous BMP-2. The sulfonated MeGC hydrogels significantly
increased osteogenic differentiation of encapsulated BMSCs without BMP-2 supplementation as observed by increased
mineralization as well as upregulated osteogenic gene markers compared to unmodified hydrogel. This is mostly due to
the sulfonated hydrogel surface that sequestered and augmented endogenous BMP-2 activity as validated by
immunostaining for BMP-2. Moreover, the sulfonated hydrogels supported sustained release of loaded BMP-2 with
reduced initial burst and significantly increased osteogenesis of encapsulated BMSCs compared to unmodified MeGC.
These findings suggest a novel hydrogel platform that could stabilize and enhance BMP-2 in bone tissue engineering.
D
Understanding the Role of Chain Flexibility in Amyloid Protein Aggregation through Rationally Designed Protein Sequences
Biomaterials
S. Zeb Vance, University of South Carolina; Xavier Redmon, University of Arkansas; Rachel Hall, University of South Carolina, Jamie Crawford, University of South Carolina, Christa Hestekin, University of Arkansas; Melissa Moss, University
of South Carolina
Corresponding Author: Melissa Moss, University of South Carolina, [email protected]
Abstract
The aggregation of amyloid proteins is associated with a myriad of medical conditions including Alzheimer’s disease
(AD), diabetes, and Parkinson’s disease. While attributable to different amyloid proteins, these proteins all share a
common feature: a periodic glycine motif (GxxxG). This glycine motif, associated with increased backbone flexibility, is
extended in a number of familial mutations that significantly increase severity of AD. A better understanding of the role
that chain flexibility plays in protein aggregation will allow for new and innovative protein engineering platforms for
future nanotech development as well as give insight into therapeutic strategies.
In this study, the glycine motif is targeted via either extension, by introduction of additional periodic glycine, or
contraction, by replacement of glycine with a bulky or constrained amino acid. Modifications that extend periodic
glycine include those that align with familial AD mutations. To examine how these alterations to the glycine repeat motif
impact aggregation kinetics, aggregation was monitored via thioflavin-T fluorescence and fit using a novel kinetic
equation that accounts for unique features observed at late stages of aggregation. Aggregation products were visualized
using transmission electron microscopy to examine morphological features. In addition, aggregates were fractionated by
size exclusion chromatography for size analysis via light scattering and morphology analysis via surface hydrophobicity.
Results indicate that increased chain flexibility correlates with faster nucleation as well as a larger quantity of small,
intermediate aggregates that exhibit reduced fibril morphology with unchanged surface hydrophobicity. Taken together
with observations that smaller aggregates are more physiologically active, these results support the hypothesis that
increases in protein chain flexibility, including that associated with familial mutations, may contribute to disease
progression.
Future work will extend studies to other amyloid proteins, such as amylin and chaplin H.