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Methods in
Molecular Biology 1758
Kanika Chawla Editor
Biomaterials
for Tissue
Engineering
Methods and Protocols
Methods in Molecular Biology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes

http://www.springer.com/series/7651
Biomaterials for Tissue
Engineering
Methods and Protocols
Edited by
Kanika Chawla
Cellerant Therapeutics, San Carlos, CA, USA
Editor
Kanika Chawla
Cellerant Therapeutics
San Carlos, CA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7739-0 ISBN 978-1-4939-7741-3 (eBook)
https://doi.org/10.1007/978-1-4939-7741-3
Library of Congress Control Number: 2018935279
© Springer Science+Business Media, LLC, part of Springer Nature 2018

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Dedication
To my father—who instilled in me my love of science, taught me to never stop asking questions,

and inspires me still.
Preface
Biomaterials offer widespread opportunities for treating disease, and are being implemented in
new tissue engineering and regenerative medicine-based therapies. The field of biomaterials
had its inception in the late 1930s, with the development and application of poly(methyl meth-
acrylate) (PMMA) in dentistry1. This was followed by joint and cardiovascular implants in the
1970s and 1980s and, with the advent of tissue engineering in the late 1980s, implantable
organs by the early 2000s. The field itself is incredibly diverse with various materials (natural
and synthetic) and cell types utilized and subjected to a wide set of stimuli (biochemical, bio-
physical), in order to generate an appropriate treatment for repair/replacement of biological
tissues. Biomaterials are also emerging as extracellular-mimicking platforms designed to pro-
vide instructive cues to control cell behavior and, ultimately, be applied as solutions for biologi-
cal problems.
Given an increasing aging population worldwide, an increase in the incidence of disease
and procedures, as well as the development of new technologies, the global biomaterials
market is estimated to be nearly US $150 billion by 2021. With this growth and need for
applicable biomaterials for tissue engineering and regenerative medicine purposes, the
design of future functional biomaterials will require collaboration with clinicians and physi-
cians in order to determine the best and most relevant designs, blending both practical
engineering and biological principles. A remaining challenge will be translation of the bio-
material (acellular or cell-laden, depending on the application) from the in vitro to the
in vivo environment. Evaluation of the interaction of the biomaterial with the in vivo envi-
ronment will be critical to successful implantation.
This volume of Methods in Molecular Biology—Biomaterials for Tissue Engineering:
Methods and Protocols—contributes to that effort. In it, researchers provided step-by-step
protocols for generation of various biomaterials for tissue engineering and regenerative
medicine applications. The protocols described review a range of biomaterials including
hydrogels and other matrices (natural, synthetic, self-healing), biomaterials for drug and
gene delivery, surface modification and functionalization of biomaterials, and techniques
for controlling biomaterial geometry, such as three-dimensional printing and electrospin-
ning. The protocols also describe a variety of characterization techniques. Applications
utilizing multiple cell types are also described. The target audiences of this book are scien-
tists and engineers working in the areas of biomaterials, tissue engineering, and regenerative
medicine. Biomaterials for Tissue Engineering is part of a larger, critically acclaimed Methods
in Molecular Biology series. The entire series provides step-by-step protocols aimed at
assisting the biological scientist or engineer in performing relevant studies in a reproducible
manner.
1
Ratner B, Hoffman A, Schoen F, Lemons J (2012) Biomaterials science: an introduction to materials in
medicine, 3rd edn. Academic Press, 1573 pp.
vii
viii Preface
The book comprises 15 chapters covering the aforementioned topics. I am pleased to

provide a summary of author contributions below:
●● Masehi-Lano and Chung describe a method for bone regeneration using citric acid-
based scaffolds. The method is novel in that it utilizes low-pressure foaming to
synthesize a scaffold with tailorable mechanical properties, similar to an elastomeric
tissue.
●● Shi et al. explore a versatile molecular design via self-assembling peptide-based micelles
containing a cancer targeting sequence on their surface for targeted intracellular deliv-
ery, with potential application as a novel cancer therapeutic. Through rational design,
these peptide amphiphiles can self-assemble into a variety of carrier nanostructures
with controlled shape, size, and biological functionality to deliver therapeutic and
imaging molecules to target cells.
●● Chow describes an electrospinning methodology with functionalization strategy for
generating scaffolds incorporating multiple bioactive cues for tissue engineering appli-
cations. The chapter provides a useful method for modifying poly(ε-caprolactone)
(PCL) with peptides and electrospinning these peptide-PCL conjugates to functional-
ize a scaffold surface in a single step.
●● Papastavrou et al. provide a unique low-temperature deposition modeling technique
for preparing hierarchical ceramic scaffolds with tailorable porosity for applications in
bone tissue engineering. The authors have developed a material with viscoelastic prop-
erties which can be tuned as well as a modified 3D printer to extrude the scaffolds at
subzero temperatures.
●● Zhou and Shikanov and Mendez examine two methodologies for studies involving
ovarian follicles. The first focuses on a hydrogel-based method for studying the effects
of toxicants on ovarian follicles while the second utilizes a polyethylene glycol (PEG)
hydrogel-based system for engineering the ovarian follicle environment in order to
study folliculogenesis.
●● Chandrawati explores a versatile layer-by-layer assembly technique for generating poly-
mer capsules as carriers for therapeutic intracellular delivery. Various materials can be
incorporated in the multilayer design based on the properties and functionalities
desired.
●● Sproul et al. provide a methodology for modifying fibrin-based materials in order to
control and modulate polymerization dynamics and promote cell infiltration, an aspect
currently lacking in the application of fibrin tissue sealants. The chapter also describes
methods for characterizing fibrin network morphology and polymerization and degra-
dation dynamics.
●● Paez et al. contribute a protocol for preparing thin, mechanically defined
poly(acrylamide)-based hydrogels for use as model substrates in studying the cell-
matrix interface. The methodology described can be used to incorporate a variety of
instructive signals in order to more closely mimic in vivo scenarios.
●● Gu et al. describe a method for fabricating neural tissue by three-dimensional printing
neural stem cells with a bioink (comprised of alginate, chitosan, and agarose), and sub-
sequent gelation of the bioink for cell encapsulation and eventual differentiation into
functional neurons and neuroglia. The bioink strategy enables greater control over the
three-dimensional architecture of the construct and supports high cell viability with
efficient induction to neurons and other cells in the constructs.
Preface ix
●● Vorwald et al. provide a high-throughput methodology for encapsulating mesenchymal

stem cells (MSCs) within alginate spheroids in situ. The method results in improve-
ment of retention and survival of cells upon transplantation, localizes payloads at the
defect site, and provides cells with continued exposure to the properties of the
biomaterial.
●● Chao reports a robust method using electrospinning and post-processing to generate
parallel polymer fibers with crimp to simulate the structure-function relationship of col-
lagen fibers. The resulting platform can be used to study cell-material interactions in a
biomimetic physical microenvironment mimicking native structure-function
relationships.
●● Witherel et al. contribute a protocol for culturing macrophages (a major component of
the immune system and contributor in the in vivo response to implanted biomaterials)
on three-dimensional biomaterials in order to study cell-material relationships which
may influence the success of implantable biomaterials.
●● Paschuk provides a relevant methodology for generating self-assembled peptide-based
hydrogels by solid phase peptide synthesis, including suggestions on how to improve
synthetic yield and purity of self-assembling peptides.
●● Kaur et al. describe a peptide amphiphile hydrogel designed to release hydrogen sul-
fide, an endogenously produced cell signaling molecule that has a role in several physi-
ological processes including angiogenesis and inflammation, in a controlled manner.
I would like to acknowledge the authors for contributing their time and energy to this
edition. I wish to acknowledge John Walker for his guidance in editing the book chapters.
I am also very thankful for my husband Nimeesh’s encouragement; this book would not
have been possible without his support. Our sons, Deven and Arjun, represent significant
inspiration in working to develop novel biomaterials for tissue engineering and regenerative
medicine applications—in the hopes of benefiting the next generation. I would also like to
express my gratitude to my parents, brother, and family for their encouragement.
San Carlos, CA, USA Kanika Chawla

Contents
1 Engineering Citric Acid-Based Porous Scaffolds

for Bone Regeneration�� 1
Jacqueline J. Masehi-Lano and Eun Ji Chung
2 Multifunctional Self-Assembling Peptide-Based Nanostructures
for Targeted Intracellular Delivery: Design, Physicochemical
Characterization, and Biological Assessment�� 11
Yejiao Shi, Ran Lin, Honggang Cui, and Helena S. Azevedo
3 Electrospinning Functionalized Polymers for Use
as Tissue Engineering Scaffolds�� 27
Lesley W. Chow
4 Low-Temperature Deposition Modeling of β-TCP Scaffolds
with Controlled Bimodal Porosity�� 41
E. Papastavrou, P. Breedon, and D. Fairhurst
5 Three-Dimensional Hydrogel-Based Culture to Study the Effects
of Toxicants on Ovarian Follicles�� 55
Hong Zhou and Ariella Shikanov
6 Layer-by-Layer Engineered Polymer Capsules for Therapeutic Delivery�� 73
Rona Chandrawati
7 Controlling Fibrin Network Morphology, Polymerization,
and Degradation Dynamics in Fibrin Gels for Promoting Tissue Repair�� 85
Erin P. Sproul, Riley T. Hannan, and Ashley C. Brown
8 Biofunctionalization of Poly(acrylamide) Gels�� 101
Julieta I. Paez, Aleeza Farrukh, Oya Ustahüseyin,
and Aránzazu del Campo
9 Synthetic PEG Hydrogel for Engineering the Environment
of Ovarian Follicles�� 115
Uziel Mendez, Hong Zhou, and Ariella Shikanov
10 Engineering Human Neural Tissue by 3D Bioprinting�� 129
Qi Gu, Eva Tomaskovic-Crook, Gordon G. Wallace, and Jeremy M. Crook
11 High-Throughput Formation of Mesenchymal Stem
Cell Spheroids and Entrapment in Alginate Hydrogels�� 139
Charlotte E. Vorwald, Steve S. Ho, Jacklyn Whitehead, and J. Kent Leach
12 Crimped Electrospun Fibers for Tissue Engineering�� 151
Pen-hsiu Grace Chao
13 In Vitro Model of Macrophage-Biomaterial Interactions�� 161
Claire E. Witherel, Pamela L. Graney, and Kara L. Spiller
xi
xii Contents
14 Synthesis of Self-Assembling Peptide-Based Hydrogels for Regenerative

Medicine Using Solid-Phase Peptide Synthesis�� 177
E. Thomas Pashuck
15 H2S Delivery from Aromatic Peptide Amphiphile Hydrogels�� 193
Kuljeet Kaur, Yun Qian, and John B. Matson
Index�� 209
Contributors
Helena S. Azevedo · School of Engineering and Materials Science, Institute of

Bioengineering, Queen Mary University of London, London, UK
P. Breedon · School of Science and Technology, Nottingham Trent University, Nottingham,
England, UK
Ashley C. Brown · Joint Department of Biomedical Engineering, North Carolina State
University and The University of North Carolina at Chapel Hill, Raleigh, NC, USA
Aránzazu del Campo · INM—Leibniz Institute for New Materials, Saarbrücken,
Germany; Chemistry Department, Saarland University, Saarland, Germany
Rona Chandrawati · School of Chemical Engineering, The University of New South Wales,
Sydney, NSW, Australia
Pen-hsiu Grace Chao · Institute of Biomedical Engineering, National Taiwan
University, Taipei, Taiwan
Lesley W. Chow · Department of Materials Science and Engineering, Lehigh University,
Bethlehem, PA, USA; Department of Bioengineering, Lehigh University, Bethlehem, PA,
USA
Eun Ji Chung · Department of Biomedical Engineering, University of Southern
California, Los Angeles, CA, USA
Jeremy M. Crook · ARC Centre of Excellence for Electromaterials Science, Intelligent
Polymer Research Institute, AIIM Facility, University of Wollongong, Wollongong, NSW,
Australia; Illawarra Health and Medical Research Institute, University of Wollongong,
Wollongong, NSW, Australia; Department of Surgery, St Vincent’s Hospital, The
University of Melbourne, Fitzroy, VIC, Australia
Honggang Cui · Department of Chemical and Biomolecular Engineering, Institute for
NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA
D. Fairhurst · School of Science and Technology, Nottingham Trent University,
Nottingham, England, UK
Aleeza Farrukh · INM—Leibniz Institute for New Materials, Saarbrücken, Germany
Pamela L. Graney · Biomaterials & Regenerative Medicine Laboratory, School of
Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia,
PA, USA
Qi Gu · ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer
Research Institute, AIIM Facility, University of Wollongong, Wollongong, NSW,
Australia
Riley T. Hannan · Department of Pathology, University of Virginia, Charlottesville, VA,
USA
Steve S. Ho · Department of Biomedical Engineering, University of California—Davis,
Davis, CA, USA
Kuljeet Kaur · Department of Chemistry, Virginia Tech University, Blacksburg, VA, USA
xiii
xiv Contributors
J. Kent Leach · Department of Biomedical Engineering, University of California—Davis,

Davis, CA, USA; Department of Orthopaedic Surgery, School of Medicine, University of
California—Davis, Sacramento, CA, USA
Ran Lin · Department of Chemical and Biomolecular Engineering, Institute for
NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA
Jacqueline J. Masehi-Lano · Department of Biomedical Engineering, University of
Southern California, Los Angeles, CA, USA
John B. Matson · Department of Chemistry, Virginia Tech University, Blacksburg, VA,
USA
Uziel Mendez · Department of Biomedical Engineering, University of Michigan, Ann
Arbor, MI, USA
Julieta I. Paez · INM—Leibniz Institute for New Materials, Saarbrücken, Germany
E. Papastavrou · School of Science and Technology, Nottingham Trent University,
Nottingham, England, UK
Eva Tomaskovic-Crook · ARC Centre of Excellence for Electromaterials Science,
Intelligent Polymer Research Institute, AIIM Facility, University of Wollongong,
Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute,
University of Wollongong, Wollongong, NSW, Australia
E. Thomas Pashuck · Department of Materials Science and Engineering, Imperial
College London, London, UK
Yun Qian · Department of Chemistry, Virginia Tech University, Blacksburg, VA, USA
Yejiao Shi · School of Engineering and Materials Science, Institute of Bioengineering,
Queen Mary University of London, London, UK
Ariella Shikanov · Department of Biomedical Engineering, University of Michigan, Ann
Arbor, MI, USA; Department of Macromolecular Science & Engineering, University of
Michigan, Ann Arbor, MI, USA
Kara L. Spiller · Biomaterials & Regenerative Medicine Laboratory, School of Biomedical
Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA, USA
Erin P. Sproul · Joint Department of Biomedical Engineering, North Carolina State
University and The University of North Carolina at Chapel Hill, Raleigh, NC, USA
Eva Tomaskovic-Crook · ARC Centre of Excellence for Electromaterials Science,
Intelligent Polymer Research Institute, AIIM Facility, University of Wollongong,
Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute,
University of Wollongong, Wollongong, NSW, Australia
Oya Ustahüseyin · INM—Leibniz Institute for New Materials, Saarbrücken, Germany
Charlotte E. Vorwald · Department of Biomedical Engineering, University of
California—Davis, Davis, CA, USA
Gordon G. Wallace · ARC Centre of Excellence for Electromaterials Science, Intelligent
Polymer Research Institute, AIIM Facility, University of Wollongong, Wollongong, NSW,
Australia
Jacklyn Whitehead · Department of Biomedical Engineering, University of California—
Davis, Davis, CA, USA
Claire E. Witherel · Biomaterials & Regenerative Medicine Laboratory, School of
Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia,
PA, USA
Hong Zhou · Department of Biomedical Engineering, University of Michigan, Ann
Arbor, MI, USA
Chapter 1
Engineering Citric Acid-Based Porous Scaffolds

for Bone Regeneration
Jacqueline J. Masehi-Lano and Eun Ji Chung
Abstract
Tissue engineering aims to develop scaffolds that are biocompatible and mimic the mechanical and
biological properties of the target tissue as closely as possible. Here, we describe the fabrication and char-
acterization of a biodegradable, elastomeric porous scaffold: poly(1,8-octanediol-co-citric acid) (POC)
incorporated with nanoscale hydroxyapatite (HA). While this chapter focuses on the scaffold’s potential
for bone regeneration, POC can also be used in other tissue engineering applications requiring an elasto-
meric implant. Because of the relative ease with which POC can be synthesized, its mechanical properties
can be tailored to mimic the structure and function of the target elastomeric tissue for enhanced tissue
regeneration.
Key words Hydroxyapatite, Tissue engineering, Tissue regeneration, Osteogenicity, Composites,

Mechanical properties
1 Introduction
The objective of tissue engineering is to develop biological scaf-

folds that restore, maintain, or improve the function of damaged
tissue. Generally, damaged tissue is replaced by autografts (tissue
derived from the patient) or allografts (tissue taken from a donor
and implanted in a patient) [1, 2]. However, both techniques have
major drawbacks such as donor-site morbidity in the case of auto-
grafts, and the possibility of disease transmission in the case of
allografts. The advent of tissue engineering shifted the focus from
tissue replacement to tissue regeneration by developing biological
substitutes that act as templates to guide the formation of new tis-
sue produced by the own body. More specifically, tissue substitutes
are synthesized in vitro, implanted at the injured site, and gradually
replaced with new native tissue by the body’s own regenerative
capacity in vivo [1].
Despite the recognized abundance of tissues of the body that
have elastomeric properties such as bone, research into the
Kanika Chawla (ed.), Biomaterials for Tissue Engineering: Methods and Protocols, Methods in Molecular Biology, vol. 1758,
https://doi.org/10.1007/978-1-4939-7741-3_1, © Springer Science+Business Media, LLC, part of Springer Nature 2018
1
2 Jacqueline J. Masehi-Lano and Eun Ji Chung
evelopment of compliant biodegradable scaffolds has been highly

d
underserved and 90% of bone grafting procedures today still
require autografts or allografts [2]. This is because few biocompat-
ible synthetic materials meet the requirements of bone grafts and
the materials reported in literature are too costly for clinical and
commercial implementation [3]. For example, while existing bio-
degradable scaffolds such as poly(L-lactide) (PLL),
poly(caprolactone) (PCL), or combinations of these polymers
show favorable in vivo compatibility with progenitor cells and
osteoblasts, PCL and PLL show mechanical properties that are
either too weak for bone integration or have degradation rates that
hamper tissue integration. Here, we describe the synthesis of a
porous biodegradable elastomer, poly(1,8-octanediol-co-citric
acid) (POC) incorporated with nanoscale hydroxyapatite (HA).
POC offers many advantages compared to the other polymeric
biodegradable scaffolds, namely, the use of nontoxic monomers,
relatively easy synthesis that can be carried out under mild condi-
tions, controllable mechanical and degradation properties, cost
efficiency, and compatibility with several cell types [3].
Bone is a natural composite that is made up of HA crystals
embedded within an elastic collagen matrix. HA makes up 60–70%
of native bone by weight and incorporation of HA confers the scaf-
fold with osteogenicity and osteoconductivity [4–7]. POC is an
ideal polymer for the matrix phase, as it has previously been shown
to accommodate up to 60–70% of HA particles by weight [3, 8].
In contrast to other polymer-HA composites, the elastomeric and
binding properties of POC enable the HA component to make up
the majority of the composite [4]. This, in turn, maximizes the
osteoconductivity and more closely mimics the native components
of bone [3, 8].
Incorporating porosity to the POC-HA scaffold further mim-
ics the native structure of bone, which is 50–90% porous [4, 9–12].
Current methods to achieve porosity require the use of organic
solvents, supercritical carbon dioxide, or porogen leaching, which
are not applicable to many polymers. In contrast, interconnected
pores in the POC-HA scaffolds can be fabricated using low-
pressure foaming (LPF). In LPF, the air bubbles that arise during
the polymer mixing step act as pore nucleation sites [13]. Applying
vacuum expands the nucleation sites into interconnected pores
that are stabilized through cross-linking.
Citric acid-based nanocomposites in both short- (6 weeks) and
long-term (26 weeks) studies have previously shown favorable
bone-implant and bone-cartilage response in rabbits [14, 15].
Furthermore, the degradation rate of porous POC-HA enables
optimal tissue ingrowth and regeneration. In addition to the fabri-
cation and characterization of porous POC-HA scaffolds, this
chapter describes methods to observe human mesenchymal stem
cell (hMSC) attachment and alkaline phosphatase activity to con-
firm scaffold biocompatibility and osteogenicity.
Engineering Citric Acid-Based Porous Scaffolds for Bone Regeneration 3
2 Materials
2.1 POC-HA 1. Citric acid (Sigma-Aldrich, St. Louis, MO, USA).

2. 1,8-Octanediol (Sigma-Aldrich, St. Louis, MO, USA).
3. 250 mL Round-bottom flask (Synthware Glass, Pleasant
Prairie, WI, USA).
4. Magnetic stir bar (VWR, Radnor, PA, USA).
5. Hot plate with magnetic stirrer (VWR, Radnor, PA, USA).
6. Silicon oil bath (Sigma-Aldrich, St. Louis, MO, USA).
7. Paper towels.
8. Ethanol (200 Proof) (Sigma-Aldrich, St. Louis, MO, USA).
9. Parafilm M laboratory film (Bemis, Neenah, WI, USA).
10. Orbital shaker (VWR, Radnor, PA, USA).
11. Large beaker (VWR, Radnor, PA, USA).
12. Pasteur pipette (VWR, Radnor, PA, USA).
13. Rubber bulb for pipette (VWR, Radnor, PA, USA).
14. Lyophilizer (Labconco, Fort Scott, KS, USA).
15. Spatula (VWR, Radnor, PA, USA).
16. Amber glass bottle.
2.2 Viscosity 1. Ubbelohde viscometer (Cannon Instrument Co., State

College, PA, USA).
2.3 HA 1. Hydroxyapatite nanoparticles (Berkeley Advanced Biomaterials,

Inc., Berkeley, CA, USA).
2. Ethanol (200 Proof) (Sigma-Aldrich, St. Louis, MO, USA).
3. X-ray diffractometer.
4. Scanning electron microscope (SEM).
5. Teflon dishes (100 mL) (VWR, Radnor, PA, USA).
2.4 Compression 1. Sintech mechanical tester model 20/G (Research Triangle

Testing Park, NC, USA).
2. Milli-Q water.
2.5 Porosity 1. Water bath (Branson, North Olmsted, OH, USA).

Measurements 2. Milli-Q water.
Method 1
3. Density bottle.
4. Thin wire.
5. Weigh scale.
6. Ethanol.
7. Kimwipes (VWR, Radnor, PA, USA).

8. Vacuum hose.
2.6 Porosity 1. Autopore IV (Micromeritics, Norcross, GA, USA).

Measurements
Method 2
2.7 Preparation 1. hMSCs (Lonza, Walkersville, MD, USA).

of POC-Based 2. Expansion media: Prepare low-glucose DMEM (1 g/mL glu-
Scaffolds for Human cose) with 10% fetal bovine serum (FBS) and 1% penicillin/
Mesenchymal Stem streptomycin.
Cell (hMSC) Culture 3. 24-Well plate.
4. AN74j/Anprolene ethylene oxide sterilization system
(Anderson Sterilization, Inc., Haw River, NC, USA).
5. 2.5% Glutaraldehyde.
6. Ethanol.
7. Lyophilizer.
8. Sputter coater.
9. SEM.
10. Quant-iT Pico Green dsDNA Reagent (Invitrogen, Carlsbad,
CA, USA).
11. 0.1% Triton-X 100.
12. Sonicator.
13. 10 mM p-Nitrophenyl phosphate.
14. 50 mM Glycine buffer.
15. 0.5 mM MgCl2 (Sigma Aldrich, St. Louis, MO, USA).
16. Spectrophotometer.
3 Methods
3.1 Poly(1,8- 1. Add equimolar amounts of citric acid (0.5 mol, 96.06 g) and
octanediol-co-citric 1,8-octanediol (0.5 mol, 73.11 g) to a 250 mL round-bottom
acid) Synthesis flask. Put a magnetic stir bar inside the flask and place the flask
in a silicon oil bath on a magnetic stirrer with a hot plate. Stir
at 155 °C and 140 rpm for approximately 9.5 min to melt the
mixture. Lower the temperature to 140 °C and stir the mixture
at 140 rpm for 45 min to create the prepolymer solution.
Continue to polymerize until the magnetic stir bar can no lon-
ger spin even at low speeds (see Note 1). While the polymeriza-
tion process is taking place, wash a large beaker with ethanol
and place a magnetic stir bar inside it. Weigh the beaker and
magnetic stir bar and record the empty weight. This will be
used in the purification steps later.
2. Remove the flask from the oil bath. Wipe off excess oil from
the flask with paper towels. Immediately place the flask under
cold running water for 5–10 min until the flask cools to room
temperature. Next, add ethanol to the flask such that the
ratio of ethanol to POC is 3:1. Cover the flask with parafilm.
Place the flask on an orbital shaker to dissolve the POC
polymer (see Note 2).
3. Fill the previously weighed beaker containing the magnetic
stir bar with water. Place the beaker on a magnetic stirrer.
After the POC has dissolved, use a Pasteur pipette with a bulb
to add the POC dropwise to the large beaker of water under
magnetic stirring. The ratio of water to POC in ethanol should
be approximately 2:1 so that any unreacted monomers and
low-molecular-weight species can be dissolved in the water.
Once all the POC prepolymer has been added to the water,
stop mixing and allow the POC prepolymer to settle to the
bottom of the beaker. Discard the water without letting any of
the POC prepolymer pour out from the bottom of the beaker
(see Note 3). After discarding the water for the final time,
place the beaker with POC prepolymer in a −80 °C freezer.
After freezing, place the beaker in a lyophilizer for 2–4 days to
remove all the water (see Note 4).
4. Following the freeze-drying process, weigh the beaker con-
taining the magnetic stir bar and POC to obtain the final
weight. Subtract the empty weight from the final weight to the
get the mass of POC recovered. Determine the recovery using
the below equation:
Mass of POC recovered
Recovery = ×1
100
Mass of POC originally polymerized
5. Determine the mass of ethanol that must be added to the bea-

ker to make a 30% (w/w) solution of POC in ethanol using the
following equation:
Mass of POC recovered
0.30 =
Mass of POC recovered + Mass of ethanol
Convert the mass of ethanol calculated from the above

equation to a volume by using the density of ethanol (ρethanol
~0.789 g/cm3 at room temperature). Add the correct amount of
ethanol to the beaker. Cover the beaker with parafilm and place on
an orbital shaker to dissolve the POC (see Note 5). After dissolv-
ing, pour the contents of the beaker into an amber glass bottle and
store at room temperature until further use.
3.2 Fabrication 1. Dissolve 10 g of POC prepolymer in 18 g ethanol at 80 °C. Mix

of Porous POC-HA with the wt.% of HA powder. Warm Teflon dishes to 80 °C. Place
Nanocomposites the POC-HA mixture in the dishes. Stir the mixture manually
via Low-Pressure with a spatula for 30 s every 5 min for 2 h. Mix manually for
Foaming 30 s every hour for 24 h. During POC-HA mixing, the sample
can be left covered overnight at room temperature and finished
mixing the next day. This is only after the POC-HA mixture is
more solid vs. liquid mixture. After the POC-HA becomes a
solid that can be rolled into a ball, place the mass at 80 °C
under vacuum at 2 Pa for 3 days. Then place the mass at 120 °C
under vacuum at 2 Pa for 1 day (see Note 6). To obtain nonpo-
rous POC-HA composites, insert the POC-HA mass into
Teflon molds to obtain the desired shape and polymerize at
80 °C for 2 Pa for 3 days without vacuum (Fig. 1).
3.2.1 Tuning 1. To fabricate pre-POC with varying viscosities and degrees of

of Prepolymer Viscosity polymerization, synthesize the pre-POC under one of the fol-
on POC-HA Scaffolds lowing conditions: 9.5 min at 155 °C, 15 min at 155 °C, or
15 min at 155 °C followed by 35 min at 140 °C. Stir all three
types of pre-POC at 140 rpm during synthesis.
2. Measure the kinematic viscosities at 25 °C with 2% pre-POC
solution in ethanol (w/v) using an Ubbelohde viscometer, fol-
lowing the manufacturer’s instructions. Stir the POC mixture
manually as specified above and post-polymerize under the
same conditions.
3.3 Compression Prepare at least three samples for each POC-HA scaffold. To pre-
Testing pare samples under wet testing, soak some of the samples in
Milli-Q water for 24 h at room temperature before testing. Use a
mechanical tester (i.e., Sintech mechanical tester model 20/G) to
measure compression moduli of samples under dry and wet condi-
tions according to American Society for Testing and Materials
(ASTM) d695.
Fig. 1 Digital images of (a) solid POC, (b) porous POC, (c) solid 20% by weight HA nanocrystals in POC (POC-
20HA), (d) porous POC-20HA, (e) solid POC-40HA, and (f) porous POC-40HA. (g) The porous scaffolds contain
an outer nonporous layer (reproduced from [13] with permission from Mary Ann Liebert, Inc.)
3.3.1 Porosity 1. Fill the water bath with water. The level of water in the bath
Measurement of POC-HA should cover the neck of the density bottle but not high
Scaffolds Using enough that water will enter the bottle. Warm the bath to
Archimedes Principle 30 °C. Place the thin wire in the plug of the cap of the density
(Method 1) bottle to enable hanging of polymer samples from the end of
the wire.
2. Cut out polymer samples and weigh them on a scale. Record
the mass of each scaffold (Ws). Ensure that the polymer sample
is small enough to fit through the neck of the density bottle
but large enough to be easily placed on the hook of the density
bottle. Fill the density bottle with ethanol. Place it in the water
bath at 30 °C.
3. Equilibrate the density bottle containing ethanol in the water
bath. When the bottle is initially placed in the water bath, the
ethanol will expand as the temperature rises. This expansion
will push ethanol out the hole in the top of the cap and form a
droplet on the surface of the cap. When ethanol stops rising
out the top of the cap and the top becomes dry, the bottle has
reached equilibrium. After equilibrating to 30 °C, remove the
density bottle from the water bath. Dry the outside with a
Kimwipe. Place the density bottle on scale to obtain its mass
filled with ethanol (W1).
4. Place the scaffold on the hook of the density bottle such that it
is hanging above the ethanol in the bottle. Attach a vacuum
hose to the bottle cap and through repeated vacuum cycles,
ensure that ethanol is filling up all the pores. The pores are
filled when air bubbles no longer escape from the scaffold
under vacuum.
5. Refill the density bottle with ethanol until it is full. Place it
back in the water bath at 30 °C. When the bottle equilibrates
again, immediately remove it from the water bath. Dry the
bottle with a Kimwipe and place it on the scale to obtain the
mass of the density bottle with the scaffold (W2). Remove the
scaffold from the bottle and weigh the bottle one more time to
obtain the mass of the bottle after removing the scaffold (W3).
The porosity of the scaffold can be calculated using the following
equations (see Note 7):
(W2 − W3 − WS )
Vp =
ρe
(W1 − W2 + WS )
VS =
ρe
WS
ρS =
VS
VP
ε=
VP + VS
●● Vp = Volume of scaffold pores

●● Vs = Volume of scaffold skeleton
●● ρs = Scaffold density
●● ε = Porosity
●● ρe = Density of ethanol
3.3.2 Porosity 1. Use an Autopore IV to determine the incremental intrusion of

Measurement of POC-HA mercury with pressure for POC-HA samples. Calculate a vol-
Scaffolds Using Mercury ume percentage for a particular pore diameter using the fol-
Intrusion Porosimetry lowing equation:
and SEM (Method 2)
Incremental intrusion × Porosity
Volume percentage = ×100
Total intrusion
2. Sputter-coat the cross section and the surface of the scaffolds

and observe using SEM. Measure the average pore size and
pore wall thickness manually using the SEM images. Perform
at least four measurements for each parameter (Fig. 2).
3.4 hMSC Culture 1. Culture and expand hMSCs in expansion media at 37 °C in

on POC-HA humidified air containing 5% CO2 (see Note 8).
and Osteogenesis 2. Place each sample in a 24-well plate. Sterilize the samples using
an AN74j/Anprolene ethylene oxide sterilization system that
performs a 2-h degassing step under vacuum after 12 h of gas
exposure. Prepare the samples as 10 mm cubes. Condition the
samples in supplemented media at 37 °C for 24 h (see Note 9).
Next, suspend 40,000 cells in 15 μL of media before seeding.
2 h post-seeding, aliquot 2 mL of media into each well.
3.4.1 Morphology 1. After 24 h, fix the samples with 2.5% glutaraldehyde. Then
and Attachment of hMSCs dehydrate the samples in graded series of ethanol (i.e., 50, 60,
on POC-HA Scaffolds 70, 80, 90, 95% ethanol) and freeze-dry them. Sputter-coat
the samples and observe the morphology of the cells using
SEM.
2. For hMSC attachment, quantify total DNA using Quant-iT
Pico Green dsDNA Reagent. Lyse cells using 0.1% Triton-X
100. Sonicate for 20 min and use the lysate. Use at least three
samples for each material.
3.4.2 Intracellular Add one aliquot of the cell lysate to an equal amount of reaction
Alkaline Phosphatase buffer containing 10 mM p-nitrophenyl phosphate in 50 mM gly-
Activity of hMSCs cine buffer at pH 10.5, supplemented with 0.5 mM MgCl2. After
on POC-HA Scaffolds 30 min at 37 °C, add 0.05 M NaOH to stop the reaction. Measure
the reaction solution by spectrophotometry at 410 nm. Construct
a standard curve from different dilutions of p-nitrophenol stock
solution. Use at least three samples for each scaffold type for each
media condition and normalize results by total DNA.
Fig. 2 SEM cross-section images of POC-40HA prepolymer synthesized under (a) 9.5 min at 155 °C, (b)
15 min at 155 °C, and (c) 15 min at 155 °C followed by 35 min at 140 °C. The distance between pores
increased with increased polymerization time. Scale bar: 1 mm (reproduced from [13] with permission from
Mary Ann Liebert, Inc.)
4 Notes
1. Stop at 100 rpm, when the stir bar can no longer spin, but
moves very slowly with brief jolts. For 0.1 moles/monomer,
the polymerization time should be between 1 and 1.5 h.
2. If the POC does not dissolve after 2 days and instead swells,
the POC was over-cross-linked on the hot plate and the reac-
tion was not stopped in time. Reduce the polymerization time
on the hot plate next time and remove the hot plate before the
stir bar can no longer spin.
3. POC can be redissolved in ethanol depending on the desired
purity of the polymer.
4. The time in the lyophilizer will vary depending on the amount
of POC prepolymer synthesized.
5. Some of the POC may stick to the sides of the beaker above
the level of the ethanol. If the beaker is sealed tightly with
parafilm then the POC on the sides will eventually dissolve
from the ethanol vapor.
6. Perform SEM or transmission electron microscopy (TEM) to
confirm the size of the HA microcrystals.
7. This method can be highly variable. Slight variations in the
temperature, volume of ethanol in the density bottle when full,
or whether or not the pores are completely filled with ethanol
can cause large variations in the calculated porosity. This
method will need to be repeated many times so that the tech-
nique can be mastered and the variability reduced. It is advis-
able to have an experience member of the lab guide you
through this protocol the first time. In addition, if more accu-
rate data is needed, mercury intrusion porosimetry (Subheading
3.3.2) should be implemented for porosity measurements.
8. Cells at passage five were used in the studies and media was
changed every 3 or 4 days.
9. Make sure that media color does not change due to mono-
meric leaching.
Acknowledgments
This work was supported by NIH grant R00HL124279 granted to

EJC.
References
1. O’Brien FJ (2011) Biomaterials and scaffolds for for orthopedic implants. Biomaterials 27:
tissue engineering. Materials Today 14:88–95 5845–5854
2. Chung EJ, Sugimoto M, Ameer GA (2011) 9. Móczó J, Pukánszky B (2008) Polymer micro
The role of hydroxyapatite in citric acid-based and nanocomposites: structure, interactions,
nanocomposites: surface characteristics, degra- properties. J Indus Eng Chem 14:535–563
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Biomater 7:4057–4063 Biomaterials and bone mechanotransduction.
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ric acid-based biodegradable elastomers for tis- 11. Temenoff JS, Lu L, Mikos AG (1999) Bone
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apatite nanocomposites for bone and ligament (2001) Biomechanics of trabecular bone. Annu
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Development of nanocomposites for bone for the fabrication of porous scaffolds for tissue
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6. Ignatius AA, Betz O, Augat P, Claes LE (2001) 18:113–121
In vivo investigations on composites made of 14. Chung EJ, Kodali P, Laskin W, Koh JL, Ameer
resorbable ceramics and poly(lactide) used as GA (2011) Long-term in vivo response to citric
bone graft substitutes. J Biomed Mater Res acid-based nanocomposites for orthopaedic tis-
58:701–709 sue engineering. J Mater Sci Mater Med
7. Rizzi SC, Heath DJ, Coombes AG, Bock N, 22:2131–2138
Textor M, Downes S (2001) Biodegradable 15. Chung EJ, Qiu H, Kodali P, Yang S, Sprague
polymer/hydroxyapatite composites: surface SM, Hwong J, Koh J, Ameer GA (2011) Early
analysis and initial attachment of human osteo- tissue response to citric acid-based micro- and
blasts. J Biomed Mater Res 55(4):475–486 nanocomposites. J Biomed Mater Res Part A
8. Qiu H, Yang J, Kodali P, Ameer GA (2006) A 96:29–37
citric acid-based hydroxyapatite composite
Chapter 2
Multifunctional Self-Assembling Peptide-Based

Nanostructures for Targeted Intracellular Delivery: Design,
Physicochemical Characterization, and Biological
Assessment
Yejiao Shi, Ran Lin, Honggang Cui, and Helena S. Azevedo
Abstract
Peptide amphiphiles (PAs), consisting of a hydrophobic alkyl chain covalently bound to a hydrophilic
peptide sequence, possess a versatile molecular design due to their combined self-assembling features of
amphiphile surfactants and biological functionalities of peptides. Through rational design, PAs can self-
assemble into a variety of nanostructures with controlled shape, size, and biological functionality to deliver
therapeutic and imaging agents to target cells. Here, we describe principles to design multifunctional PAs
for self-assembly into micellar nanostructures and targeted intracellular delivery. The PA micelles are
designed to display a tumour targeting sequence on their surfaces and direct their interactions with specific
cells. This targeting sequence includes an enzymatic sensitive sequence that can be cleaved upon exposure
to matrix metalloproteinase 2 (MMP-2), an enzyme overexpressed in tumor environment, allowing the
presentation of a cell-penetrating domain. The presentation of this domain will then facilitate the delivery
of therapeutics for cancer treatment inside targeted cells. Methods to characterize the key physicochemical
properties of PAs and their assemblies, including secondary structure, critical micelle concentration, shape
and size, are described in detail. The enzyme responsiveness of PA assemblies is described with respect to
their degradation by MMP-2. Protocols to evaluate the cytotoxicity and cellular uptake of the micellar
carriers are also included.
Key words Self-assembly, Multifunctional micelle, Enzyme-responsive, Cell-penetrating peptide,

Targeted intracellular delivery
1 Introduction
Current research in the area of drug delivery is focused on the

development of stimuli-sensitive systems that can perform multiple
functions and overcome diverse physiological barriers to optimize
delivery to target sites (organs, tissues, cells) [1]. The ubiquitous
distribution and great diversity of enzymes present in the human
body have motivated the design of nanocarriers responsive to
enzyme activities and thus translate the enzymatic modification
Kanika Chawla (ed.), Biomaterials for Tissue Engineering: Methods and Protocols, Methods in Molecular Biology, vol. 1758,
https://doi.org/10.1007/978-1-4939-7741-3_2, © Springer Science+Business Media, LLC, part of Springer Nature 2018
11
12 Yejiao Shi et al.
into a suitable response, such as material degradation or disassembly,

and the simultaneous release of entrapped drug. Targeted delivery
strategies exploit the selective affinity of ligands for tissue compo-
nents or cell-surface receptors for retention in the targeted tissue
and/or enhanced uptake by specific cells. It can reduce toxicity
and adverse side effects by accumulating the delivered therapeutics
specifically at the site of interest.
Molecular self-assembly provides the possibility to construct
multifunctional nano-architectures with precise shape, size and
functional control. It is a bottom-up approach which uses ratio-
nally designed molecular units to construct various types of nano-
structures (micelles, vesicles, and fibers) by self-organization
processes driven by non-covalent forces (electrostatic interactions,
hydrogen bondings, aromatic interactions, van der Waals forces,
and hydrophobic effects) [2]. Due to the involvement of non-
covalent interactions, self-assembly is also a reversible process, and
the self-assembly/disassembly can be triggered by an external or
internal trigger (pH, temperature, concentration or enzyme activ-
ity). Thus, this methodology offers the possibility of incorporating
different therapeutic agents during the process of self-assembly
while also controlling their release through reversible structure
transitions.
Among all the building blocks available for self-assembly, pep-
tides have gained huge attention over the past decades due to their
many advantageous properties. These include simple structure,
facile synthesis, biodegradability, biocompatibility and customiz-
able bioactivity. Diverse peptide-based self-assembling systems
(peptide amphiphiles, cyclic peptides, surfactant-like peptides, aro-
matic dipeptides, dendritic peptides) [2] have been extensively
studied to construct nanocarriers for the delivery of different ther-
apeutics (oligonucleotides, hydrophobic drugs). The release of
entrapped molecules could be easily triggered by protease hydroly-
sis of peptide bonds or by cues from the environment.
Peptide amphiphile (PA) molecules are typically composed of
a hydrophobic alkyl tail and a hydrophilic peptide sequence [3].
The hydrophilic peptide sequence can be designed for specific
interaction with other molecules and cells. For intracellular deliv-
ery, cell-penetrating peptides (CPPs), a family of short peptides
that are able to translocate across cell membranes [4], have been
widely used to facilitate cellular uptake of various therapeutic and
imaging molecules, such as proteins and peptides, differentiating
and constrast agents, DNA, siRNA, chemotherapeutic drugs and
quantum dots [5]. However, the non-specificity of CPPs could
lead to non-selective cytotoxicity and off-target side effects, result-
ing in a major limitation in systematic delivery. To overcome this
problem, numerous approaches have been developed to selectively
present CPPs at target sites [6]. In our work, multifunctional
micelles are designed to display the cell-penetrating domain only
Self-Assembled Micelles for Intracellular Delivery 13
Fig. 1 (A) Rational design of multifunctional PAs for micelle self-assembly and targeted intracellular delivery;
I: hydrophobic segment (e.g., CnH2nO2, 14 < n < 20); II: CPP sequence for mediating lipid membrane transloca-
tion (e.g., TAT peptide); III: enzyme cleavable peptide sequence (e.g., MMP-2 substrate GPX1G|LX2G, where |
denotes the expected cleavage site by MMP-2); IV: cell or tissue targeting peptide sequence (e.g., RGDS).
(B) PA self-assembly into micellar structure with simultaneous drug (yellow star shape) encapsulation and
presentation of CPP on the micelle surface triggered by enzymatic degradation
in situ where the enzyme matrix metalloproteinase 2 (MMP-2) is

overexpressed. The multifunctional micelles are obtained upon
self-assembly of rationally designed PAs, which consist of a
16-carbon alkyl tail (region I), a CPP domain (region II), a MMP-2
cleavable site (region III) and a tumor-targeting peptide sequence
(region IV) (Fig. 1).
The methods to synthesize [7] and purify [8] PA molecules
have already been reported in detail and will not be described in
this chapter.
In this chapter, we describe the procedure to analyze the sec-
ondary structure of PAs using circular dichroism (CD), which
also provides information on the type and stability of the assem-
bled PA structures [9]. We also describe a method to determine
the critical micelle concentration (CMC) using a solvatochromic
fluorescence dye (Nile Red) [10], which provides information on
the concentration that can trigger the PA self-assembly. Detailed
procedures for using transmission electron microscopy (TEM)
together with the negative staining technique are described to
examine the shape and size of PA assemblies, which can strongly
affect their blood circulation lifetime, bioavailability and cellular
uptake [11].
The conditions for assessing the enzymatic degradation of PA
micelles by MMP-2 in vitro are described, as well as methods to
analyze the peptide fragments after degradation and calculate the
degradation efficiency.
To assess the potential of the PA micelles as carriers for

intracellular delivery, the protocol to conduct a sulforhodamine B
(SRB) cytotoxicity assay [12] is described, as well as a qualitative
evaluation of cell internalization of the PA micelles by confocal
laser scanning microscopy (CLSM).
2 Materials
2.1 Preparation 1. PA (previously synthesized, purified and lyophilized) powder.

of PA Solution 2. Milli-Q water.
3. 0.1 M Hydrochloric acid (HCl) solution.
4. 0.1 M Sodium hydroxide (NaOH) solution.
5. Milli-Q water purification system.
6. pH meter.
7. 0.22 μm Syringe filter.
2.2 Characterization 1. 1 mm Path length quartz cuvette.

of PA Secondary 2. Chirascan™ circular dichroism (CD) spectropolarimeter.
Structure
2.3 Determination 1. 50 μM Nile Red acetone solution: Weigh out 0.50 mg Nile
of PA Critical Micelle Red and place it in a 50 mL centrifuge tube. Add 31.41 mL
Concentration (CMC) acetone to the centrifuge tube and the final concentration of
Nile Red solution should be 50 μM (see Note 1).
2. 0.5 mL Eppendorf tube.
3. Ethanol.
4. Vacuwash cuvette washer.
5. 10 mm Quartz cuvette.
6. Fluorolog® spectrofluorometer.
2.4 Morphological 1. 2% (w/w) Uranyl acetate aqueous solution: Weight out 0.2 g
Characterization of uranyl acetate (see Note 2) and add carefully into a 25 mL
of Assembled PA beaker. Measure 10 mL Milli-Q water and pour slowly into the
Nanostructures beaker. Then stir the solution for at least 30 min. Filter the
solution with a 0.22 μm syringe filter and then aliquot into
2 mL screw-cap brown bottle and label properly. The prepared
2% uranyl acetate solution should be kept at 4 °C in the dark
place and filtered again before using.
2. Carbon film coated copper TEM grid (400 mesh).
3. PELCO easiGlow™ glow discharge unit.
4. Reverse (self-closing) TEM tweezer.
5. TEM grid storage box.
6. Filter paper.
7. Technai12 TWIN TEM with SIS Megaview III wide-angle
CCD camera.
2.5 Evaluation of PA 1. TCNB buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl,

Degradation and 0.05% Brij™ L23, pH 7.4): Dissolve 0.24 g Tris, 0.04 g
by MMP-2 CaCl2, 0.35 g NaCl and 0.02 g Brij™L23 in 35 mL Milli-Q
water. After all the reagents are completely dissolved, adjust
the pH to 7.4 using 0.1 M HCl solution. Add Milli-Q water to
a final volume of 40 mL.
2. MMP-2 (active, human, recombinant, CHO cells) (see Note 3).
3. 0.1 mL Eppendorf tube.
4. Water bath.
5. Liquid nitrogen.
6. Trifluoroacetic acid (TFA).
7. Acetonitrile (ACN).
8. Milli-Q water.
9. Waters® clear glass 12 × 32 mm screw neck Qsert vial, 300 μL
volume
10. Alliance® high-performance liquid chromatography (HPLC)
system with Waters® e2695 separations module, 2489 UV/Vis
detector, Xbridge™ reverse phase column (C18, 4.6 × 150 mm,
3.5 μm), and Empower® chromatography data software.
11. Electrospray Ionization Mass Spectrometry (ESI-MS).
2.6 Evaluation of PA 1. Human fibrosarcoma HT-1080 cell line.

Cytotoxicity 2. Human embryonic kidney 293T cell line.
3. 10% (w/v) Trichloroacetic acid (TCA) aqueous solution: Add
22 mL of Milli-Q water to a bottle containing 15 g of solid
TCA to dissolve the TCA first and then continuously add
Milli-Q water until the final volume is 150 mL. This will give
150 mL of a 10% (w/v) TCA solution.
4. Sulforhodamine B (SRB) solution (0.4% in 1% acetic acid) (see
Note 4).
5. 1% (v/v) Acetic acid aqueous solution: Dilute the 10% (v/v)
acetic acid solution tenfold with Milli-Q water.
6. 10 mM Tris base solution: Weight out 0.121 g Tris and dis-
solve in 90 mL Milli-Q water first and continuously add water
to set the final volume of 100 mL.
7. 96-Well culture plate.
8. Sterile gauze.
9. SPECTROstar Nano® microplate reader.
2.7 Evaluation of PA 1. Human fibrosarcoma HT-1080 cell line.

Micelle Cell Uptake 2. Human embryonic kidney 293T cell line.
3. Coumarin-6.
4. Hexafluoroisopropanol (HFIP).
5. Dulbecco’s phosphate-buffered saline (DPBS) solution.
6. Hanks’ balanced salt solution.
7. 20 μM Lysotracker® Red staining solution: Dilute the
Lysotracker® Red stock solution (1 mM) 50-fold with DPBS
(see Note 5).
8. 2 mg/mL Hoechst® 33342 staining solution: Dilute the
Hoechst® 33342 stock solution (10 mg/mL) fivefold with
DPBS.
9. Collagen I coated chambered glass coverslip with 8 wells.
10. Leica TCS SP2 confocal laser scanning microscope (CLSM).
3 Methods
3.1 Characterization 1. Dissolve the PAs in Milli-Q water at a specific concentration

of PA Secondary (0.01–0.1 mM) (see Note 6) and adjust the pH to 7.4 using
Structure 0.1 M HCl or NaOH solution. The prepared samples are fil-
tered through 0.22 μm syringe filters to remove any scattering
particles before characterization.
2. Wash the cuvette (1 mm path length) with detergent first and
then rinse with Milli-Q water. Use pure nitrogen to dry the
cuvette.
3. Pipette 0.6 mL of Milli-Q water or prepared PA solution to the
cleaned cuvette.
4. Record the spectrum on a CD spectropolarimeter (see Note 7)
from 180 to 320 nm at 25 °C with a 10 nm/min scan rate, 1 s
integration time, 1 nm bandwidth, and 3 spectral acquisitions.
5. Record first the baseline by measuring the cuvette containing
only Milli-Q water and subtract this spectrum from each spec-
trum obtained for PA solution.
6. Convert the CD signals to the concentration-independent
parameter of mean residue ellipticity [θ]λ using the following
equation:
[q ]obs
[q ] l = (1)
10 × c × l × n
where [θ]λ is the mean residue ellipticity at λ in deg·cm2·dmol−1,

[θ]obs is the observed value of ellipticity from the instrument at
λ in mdeg, c is the concentration of PAs in M, l is the light path
Fig. 2 Illustration of CD spectra showing three conformations typically observed

in PA molecules: α-helix, β-sheet and random coil
Table 1
Positive and negative maxima in the CD spectra for typical PA conformations: α-helix, β-sheet, and
random coil
Wavelength, λ (nm)
Conformation Positive maximum Negative maximum Zero

α-Helix 190–195 208 and 222 202 and 250
β-Sheet 195–197 217–218 207 and 250
Random coil 218 197 and 240 211, 234 and 250
length of the cuvette in cm, and n is the number of amino acid

residues in the PA molecule.
7. Plot the mean residue ellipticity [θ]λ as function of wavelength.
Qualitatively compare the plotted spectrum with the spectra of
the three typical peptide secondary structures, α-helix, β-sheet,
and random coil (Fig. 2, Table 1), and assign the secondary
structure of the PAs.
3.2 Determination 1. Add 10 μL of Nile Red solution to a set number of 0.5 mL

of PA Critical Micelle Eppendorf tubes. Leave all the Eppendorf tubes open in the
Concentration (CMC) dark fume hood and wait for about 5 h to allow the acetone to
evaporate completely (see Note 8).
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sleet. We had sumptuous meals of trout, and tenderloin and heart of a Rocky
Mountain ram, which the scout had killed near the summit. He passed the time
telling me about his home and Indian tribe. He told about their ancient customs
and strange religious beliefs, Indian legends and tribal tales about the very
region where we were camped.
Now an Indian is generally slow to speak his innermost thoughts and to talk
about his religious beliefs. But during that big storm, the scout was in the mood
to talk. He said:
“The Sun is the Great Power. He is in the birds and wild animals, lakes and
streams, prairies and mountains. He brings the leaves in the spring-time. He
makes the grass and berries grow; and upon them the birds and animals
depend for life.
“The Thunder is a great bird. It flies with the clouds, and brings the rain. From its
eyes the lightnings flash.
“The blizzard is a person, who runs before the storm and shoots his arrows.
“Long ago an Indian, who camped in this valley, saw the Wind Maker rise from
the waters of a lake. He was like a monster bull elk. When he flapped his ears,
the wind blew hard; and when he sank again beneath the water, the wind went
down.
“My people are afraid of spirits. We believe they are everywhere—underground,

in the air, in the forest, in rocks and streams. We are afraid of ghosts which take
the form of [17]owls and come in the dark to harm people; ghosts of disembodied
relatives and friends often come around. The Blackfoot are happy on the open
plains. In the mountains they are afraid; the forests are dark and gloomy and
they hear strange sounds.
“Last summer an Under-Water-Spirit took a child of Bear Paw. He is my friend

and lives near me on Cutbank River. One day Bear Paw went into the mountains
to cut lodge-poles. He camped at the edge of the forest, near a bend in the river,
where a big rock stood and the water was deep. His wife went there for water
and saw the rock move; and that night she had a strange dream. The Rock
stood over her and said: ‘Give me your child.’ The woman was so frightened she
went to the river and sacrificed some of her ornaments; she threw them into the
water close to the Rock. Soon after that one of her children died. Now they
believe it was taken from them by the Spirit of the Rock.”
The scout related a story which Heavy Breast, another friend who lived in his
valley on Cutbank River, told him.
Heavy Breast and the Grizzly Bear
“When one of my children died last autumn, I felt so badly I did not want to see any
one. So I went alone to the forest on the mountain. It was dark and gloomy and I felt
lonely. But the only animal to be feared was the grizzly bear and I knew he would do
me no harm, because I am the guardian of the Bear Medicine. Through its wonderful
power I have cured many people.
“One night I came to a cave near the forks of a stream. It was raining and I decided to
stay there, because in the cave I would have shelter from the storm. I built a warm fire
and lay down to sleep. When I awoke the sun had not risen, but, through the mouth of
the cave, I saw that day was beginning to dawn. I heard a noise outside, like some
[18]animal sniffing the air. I thought one of the dogs had followed from camp and was
nosing around, trying to get my scent. Then I heard heavy footsteps and knew it was a
large animal. So I was careful. I made no sound; I scarcely even breathed.
“My back was towards the mouth of the cave, so I turned my head very slowly, very
carefully, and saw close to the entrance a huge grizzly bear. Then I said to myself: ‘If
this bear is angry, he has me caught in a trap.’ I have often laughed at animals in traps,
but I did not feel like laughing this time. Again I said to myself: ‘This grizzly can do me
no harm; my Bear Medicine will protect me; it has often helped me to cure the sick;
besides I have always had a friendly feeling for bears, as if they were my relatives; I
must be bold and make a strong talk; I must make this bear understand that I am his
friend.’
“Then I thought: ‘Perhaps he intends to play with me before he kills me.’ And this made
me feel very queer.
“Now, all this time the bear did not move. He stood with his head down and gazed into
the mouth of the cave. Oh! How big he looked! He stood high in front and had a broad
head; and his great feet had long sharp claws. He did not make a sound, but I knew he
was angry; his hair stood straight up on his back.
“Then I remembered an old medicine man saying, that a bear never harms a person
who does not move and talks to him in a friendly voice. So I lay with arms stretched out
and head on my hands, like a bear does. Thus I lay and looked straight into his eyes.
And then I began talking in a friendly way, using the softest and kindest voice I knew. I
flattered him the best I could. I said:
“ ‘Brother Bear, you are very good-looking; you have nice eyes and white teeth; you are
big and strong. I have never killed bears; I do not care to hunt them. Yes! I have always
liked bears. I look upon them as my relatives.’ [19]
“While I talked, his hair began to flatten, so I talked again harder than ever. I kept on
flattering him; I told him some of the secrets of my Bear Medicine. I saw that he liked
my talk; he was in a good humor; and then I began to pray, saying:
“ ‘Brother Bear, pity me! I am poor and in trouble.

Brother Bear, I am the keeper of the Bear Medicine.
Brother Bear, it is I who guard the Bear Secrets.
Brother Bear, I ask you to go away and to leave me in peace.’
“Now, the bear was no longer angry. The hair on his back all went down smooth. Soon
he turned and walked slowly from the cave; and after that I saw him no more.”
Thus my guide, an Indian belonging to a tribe of the stone age of thought, told
me about their religious faith. They believed in the power of the Sun, and that
birds and wild animals were endowed with his wisdom and supernatural power.
They communed with the wild animals, looking upon them as brothers; they
believed they had tribes like men, with head-chiefs, councils and dances; that
they were friendly, and had power to help people in trouble. Nor did they exclude
the animals from the spirit world, the place where they expected to go after
death.
We were storm-bound in our summit camp for several days. But, on the morning
of the fourth day, we awoke to find the heavens a vast expanse of blue. A foot of
snow had fallen. The surrounding mountains were covered with a white blanket.
After the great storm, the air was strangely clear and sparkled with myriads of
shining particles. The clouds had rolled away towards the east, revealing the
entire chain of Rocky Mountain peaks, their white summits glowing under the
bright rays of the rising sun.
Then we made ready to break camp and leave the snow and ice of the high
altitude for the milder climate of the [20]valley; but the devil was in our horses that
day. It took many weary hours to catch the herd. We made a series of corrals
with lariats and pack ropes. By the time we had the horses packed and ready to
start, the sun had long passed the meridian. The scout led the way down the
mountain, while I followed on foot with camera and tripod, driving the horses and
leading my saddle horse Kutenai, loaded with baggage, because one of our
pack horses had escaped us down the mountain. Then the contrary bell-mare
ran into the underbrush and bucked her pack loose, and the horse of the scout
ran away and threw him off. I found him lying senseless on the ground, with
blood flowing from nose and mouth. When he came to himself, he made light of
his accident; he said that he had been weakened by his former life of exposure
in the Indian wars.
We camped that night on the floor of the valley, in a park surrounded by a dark
forest of lodge-pole pine and spruce; the air was mild; bunch-grass grew
luxuriantly and many varieties of wild flowers—blue camas, orchids with pale
green flowers, and yellow columbine with lovely pendant blossoms.
TRIBAL CAMP OF BLACKFOOT ON THE PRAIRIE
Rocky Mountains in the distance
Our last day in the mountains, we followed a trail down the eastern slope, a well-
known Indian route across the Rocky Mountains, famous in legend and war
story. We passed through the long forest-covered valley of Cutbank River,
between two massive snow-covered mountain ranges, and rode through the
foothills with their lovely lakes and meadows, groves of aspen and thickets of
willows, crossing high grass-covered ridges, closely following one another like
great waves of the ocean.
Finally, from the crest of a ridge about twenty miles from the foot of the
mountains, we looked down upon a scene I shall never forget. On a broad
stretch of prairie and on the shore of a lake lay the tribal camp of the Blackfoot;
many [21]hundreds of smoke-colored tepees, pitched in the form of a great circle
more than a mile in circumference. In an open space near the center of camp
was a throng of Indians, taking part in the ceremony of the Sun Dance. The
surrounding meadows were bright with blue lupines, shooting stars, camas, and
yellow sunflowers. Smoke from the evening fires rose from the tepees. Many
horses were feeding contentedly on the hills. As we stood looking down at the
great camp, a light breeze carried distinctly the shouts of men and women,
crying of children, barking of many dogs, neighing of horses, and the rhythmic
beating of Indian drums in dances and ceremonial gatherings.
On that first night, we slept on the open prairie with only the sky for a roof. Late
in the night, I was wakened by Indian horsemen riding through the camp, singing
strange melodies, giving at intervals shrill war whoops, jingling bells keeping
time with the slow and measured trot of their horses. Their songs had a lilt and
wildness, and were sung with a vigor and enthusiasm that made me long to
record them.
Excitement was in the air. Flaring inside fires lighted up the lodges, casting weird
shadows of the inmates on the outside coverings. I heard the booming of drums,
shrill cries and shouts of dancers, laughter and cheers of the crowds. From the
center of camp came a solemn chanting of many voices, accompanied by heavy
beating of rattles on the ground. At intervals the low monotone of men singing in
unison, united with the shrill voices of women. Then the mysterious chanting
died away and I fell asleep. [22]
[Contents]
CHAPTER IV
HOME OF THE SCOUT
Next day the scout took me to the lodge of the head-chief White Calf
and his wife Catches-Two-Horses. These were the givers of the Sun
Dance ceremony. We talked with the venerable chief Running Crane,
and saw his wife who was fasting, because of a vow to the Sun. We
went to the tepees of the war chiefs, Little Plume and Little Dog, and
smoked a friendly pipe, also with the judges, Shoots-in-the-Air, Curly
Bear and Wolf Plume, and the medicine men, White Grass and Bull
Child. In this way I met some of the head men of the tribe, and
among them chief Mad Wolf, an orator of renown and the owner of
the ancient Beaver Bundle, an important religious ceremony. This
was the beginning of a friendship, unusual between an Indian and a
white man. It lasted as long as Mad Wolf lived, and had a strange
influence upon my life in the years to come.
When the Sun Dance came to an end and the big camp broke up, I
went with the scout to his ranch on the prairie, in the valley of
Cutbank River, near the homes of the chiefs, White Calf and Mad
Wolf, and of the medicine men, White Grass and Ear-Rings.
The scout had a cabin built of pine logs from the mountains, with
sod-covered roof and clay-chinked walls, also corrals and low-lying
sheds, a garden, and herds of cattle and horses. His wife was an
Indian woman named White Antelope, and they had a family of four
children.
She was young and good looking, but had a high temper. She liked
to take things easy, to dress in Indian finery and go visiting, leaving
ranch and children to the care of the scout. [23]But he was patient
with her; he was kind-hearted and always tried to keep things
smooth. She cooked and waited on the table, when she felt in the
mood; she and the children ate after the men. If she was moody, the
scout prepared the meals.
Their family all slept in one room and I in another. My bunk of rough
boards was built against the walls. But, in good weather, I slept
outside the cabin and under the stars, on the grassy bank of the
river, with a shady grove of cottonwoods near by, and a lovely
landscape of meadows and distant snow-capped mountains.
Siksikaí-koan was a good friend, honest and trustworthy. He stood

high in the councils of his tribe and was liked by all the people. He
was always ready to help any who came to his ranch, to advise his
people in their struggle towards civilization. Through him, I met
Indians both old and young. I made friends with them, and tried to
understand them and to see things their way.
Every morning before sunrise, the scout wakened me to go into the

hayfields. He mowed while I drove the horserake; and then came
days of pitching and stacking. Then every part of me seemed sound
and sane; I was light-hearted and happy, untrammeled and free. On
those broad prairies were no worries nor pessimists, no laws nor
creeds, nothing but a wonderful peace and contentment; something I
had longed for all my life.
The west wind blew fresh from pine forests on the mountains, from
meadows with odors of wild flowers, sweet grass, and ripe
strawberries. Bees hummed in the air, western meadow larks sang
on the prairie, willow thrushes and white-crowned sparrows in the
river valley.
But the scout could not stand heavy work in the hayfields. He
suffered from the hardships of his former life on the plains, from an
injury by a wild horse, and wounds received in the Indian wars. [24]
Then Yellow Bird came to help in the haying. He was a relative of the
scout, a young half-breed of my own age, strong, full of life, and a
good worker when he felt like it. But he was wild and could not be
depended upon. Like young men of the Blackfoot, he wanted to be
gay and craved excitement. He liked to wander, to hunt, to rope
cattle and ride wild horses, to see friends and visit new places, to be
always on the move; he liked jolly companions and people who gave
him a good time; but he loved to go with girls best of all.
He took me to Indian camps to dance and see the girls. On our way
home at night, he liked to gallop past ranches where they kept packs
of ferocious dogs. They rushed after us and he had the fun of riding
at a mad gallop, yelling and shooting at them on the run. He was
thrilled at the idea of being chased by their angry owners, and of
hearing bullets whizzing harmlessly in the dark.
We attended a meeting held by a white missionary in our valley. I led

the singing and sat in the front row with Bear Chief and Eagle Child,
who were prominent Indians. They listened gravely and attentively
but understood not a single word. They were broad-minded chiefs
and came as an example to other Indians; to show they approved of
the missionary and of his religious ceremony.
Thus with Yellow Bird I entered into the life of the people. I wanted to
see them natural and without restraint. With them I talked not of my
life in civilization, but of things of their everyday life, of horses and
cattle, hunting and wild animals, dancing and ceremonies. In this
way I became one of them, and they saw I was not critical of them
nor of their ways.
After we stacked eighty tons of hay at the scout’s ranch, Yellow Bird
and I rode the range after stray horses and cattle. We skirted the
base of the mountains, along the [25]foothills and edge of the forest,
until we came to a well-worn trail, which led to an open park far back
in the mountains. Many cattle were there, seeking refuge from the
swarms of flies and mosquitoes on the grass-covered prairies. Then
we found a herd of mares and geldings. Their leader, a fiery young
stallion, tried to drive us off. But we rounded them up with the cattle
and drove them back to the ranch, feeding them salt, that they might
not again stray away.
When we wanted to break a new team for the wagon, we drove that
wild herd of range horses into the corral and lassoed a roan and a
three-year-old sorrel. We tied them with ropes while we put on the
harness, then hitched them to the wagon and took blankets and
provisions; we knew not how far they might run.
At the start the broncos bucked and plunged; then ran and tried to
tear themselves loose from the rattling wagon, bounding over rocks,
swinging as though it would turn over. After running many miles, our
broncos broke into a stampede so wild that Yellow Bird turned them
up a butte and put the brakes on hard. They galloped up one side of
that steep butte and down the other, our wagon plunging over ruts,
stones, and badger holes, and into a swamp in the valley of a
stream, where they sank deep into soft muck and the wagon went
down over the hubs. But they soon freed themselves, and, with their
sides covered with foamy sweat, they pulled us through to firm
ground. Then they ran again and did not stop, until we were far out
on the open plains. That night we tied up our wild team and slept
peacefully under the wagon, twenty-five miles from the ranch.
Sometimes in the evening, after our work at the ranch, we saddled

our horses and rode down the river to see two sisters who were
home from school, Katoyísa and Nínake. Their father, Lone Wolf,
was dead, and they lived alone with their mother, a quiet, pleasant-
faced woman. Their log cabin of [26]three rooms had low ceilings,
and walls of hewn logs chinked and plastered, all whitewashed and
clean. The floor was spotless and covered with skins of wolf, bear,
and mountain goat; in the windows were grasses and ferns and wild
flowers, and a dish of fragrant red apples on a table.
Nínake, the younger sister, was the favorite of Yellow Bird. She was
lively, a great talker, and gave him a good time. But I liked Katoyísa
better, a quiet bashful girl of nineteen, with shapely head and good
features. Her black hair hung in two heavy braids almost to her
knees. She wore homemade cotton gowns of thin material which
showed her slender graceful form. From the look in her eyes and
expression of her face, I knew she had courage and character. In her
was the stuff of our bravest pioneers.
After we had finished with the hay of the scout, Yellow Bird and I
went to their ranch to help with their crops. The girls cooked and
gave us good food, fresh vegetables from their garden, beef, bread
and butter and milk.
Many years have passed, but they have not dimmed the memory of
those happy days without a care in the world, the primitive simplicity
of that family, and the way they made me one of them. We both
enjoyed our work, we were near the girls from morning till night, and
that kept us in a good humor.
Then the scout wanted timber from the mountains, so we took two
teams and made ready the wagons. We threw off their beds and
placed the wheels far apart by means of a long reach, to hold the
heavy logs. Yellow Bird drove one wagon and I the other. For me it
was a new thing to drive a team of broncos. I sat on the reach, on a
gunnysack stuffed with hay. I had to wield a whip with a long lash,
and had a heavy chain for binding the logs together.
We left the ranch soon after sunrise and went to a burned stretch of
timber on a slope of the Rockies. We felled only [27]trees that were
sound and well-seasoned, cutting them into logs and snaking them
down the forest trails to be loaded on our wagons.
At first it was hard to chop hour after hour with an axe. I blistered my
hands and was drenched with sweat; my arms and back ached; I felt
weak in the knees and had a consuming thirst. Then I became
accustomed to the work and had a feeling of exhilaration. I liked the
fresh odor of the wood, the ring of my axe and the feeling of a good
stroke, to know my sharp blade was cutting deep.
There was always danger of being cut with an axe, from felling trees
that had lodged, and from Yellow Bird; sometimes his trees fell
perilously near. Once I was nearly struck by a pine that let go at the
roots; I heard a sharp crackling, saw it coming towards me and
jumped just in time.
But for me the hardest work was the loading of the wagons. The
heavy logs were twenty-five feet long and from one to two feet in
diameter. The roads were steep and rough and our brakes would not
hold. But we always joked about hard work and danger, and had to
look out for ourselves.
Soon Yellow Bird tired of ranch work and wanted a change. He

proposed that we ride across the Montana line into Canada, to visit
relatives in a camp of Blood Indians, a northern division of the tribe.
So we rounded up the wild herd of range horses and drove them into
the corral. We each chose a saddle horse, Yellow Bird a brown with
silvery mane and tail, I a powerful sorrel. My gentle horse, Kutenai, I
left to graze at the ranch.
In handling horses that ran wild on the range, we were always ready
for trouble. To control them was a question of mastery; they took kind
treatment as a sign of weakness. When I tried to saddle my sorrel,
he rose on his hind legs and [28]with forefeet high in the air tried to
bring them down on my head. In mounting, I held his bit in one hand,
the pommel with the other, and made a flying leap upon his back.
Before I was in the saddle, he sprang forward like a race horse at a
desperate gallop. He had an easy motion and I kept my seat; but to
stop him baffled all of my endeavors.
We went north across the open plains, without fences or roads to bar
the way. Our horses ran like the wind; we gave them free rein and
held on. I rode Indian fashion, letting myself go freely with the motion
of my horse and kept a firm grip with my knees.
The first night we stopped at the cabin of a squaw man, near a rocky
peak which rose abruptly out of the prairie, standing apart from the
main range of the Rocky Mountains. Our host was a white man with
an Indian wife and four half-breed children, the oldest a girl of
seventeen. She and Yellow Bird were sweethearts; and while they
made love by the river, I went into the meadow to help the old man
with his hay. He was one of those pioneer settlers of early days,
short and sinewy in stature, and with a heavy beard. His life had
been filled with hardships, toil, and little pleasures. He was
suspicious by nature, and liked to talk about free silver; but at heart
he was a good fellow, resolute, brave, a hard worker and hospitable.
His Indian wife was a laughing, broad-faced woman, good-natured
and lazy. Their cabin was dirty and swarmed with flies. The second
daughter was strangely pretty, with flashing black eyes, jet black hair,
and marvelously clear olive skin. She had a pet colt which followed
her like a dog. He came into the cabin for supper. When they put him
out, he ran to the open window and poked me in the back with his
nose; he whinnied and grunted and made such a fuss that his young
mistress went to the window and gave him sugar.
Soon after sunrise on the following morning Yellow Bird [29]and I

saddled our broncos and moved on. To the west rose the mighty
frontier range of the Rockies. The rugged valleys and peaks still had
a thin veil of morning mist. In the cool air our horses had wonderful
speed. They chafed at the bit and were tireless, as though their
sinews were of steel. But, after that first day, they were not so hard
to hold.
We crossed buttes on the run, up hill and down, it was all the same;
in steep places our horses put their feet together and slid. They
jumped streams, rocks, and badger holes; galloped over ledges and
sharp volcanic rocks, across hills and ravines; it was beautiful to see
them go; they never stumbled, but lifted their feet cleanly up and
over, and always planted them securely and firmly. We passed lakes
and marshy ponds, starting noisy flocks of ducks and other water
birds, crossed Boundary Creek, and were over the Montana line into
the Province of Alberta.
We came to the ranch of a Frenchman named Big Steve, far out on

the prairie. He and his wife were pitching hay in a meadow. She was
buxom and smiling, with rosy cheeks and did the work of a man.
Both were friendly and wanted to talk, but we could not tarry; our
horses were wild and hard to hold.
At midday we dashed into a Mormon settlement, and a number of

men came to meet us. They all looked alike, over six feet in height,
with smooth faces and prominent features. They were good-natured
and hospitable and gave us all the food we could eat. But it was a
dreary place on a barren plain, a group of board shanties, without
trees or vegetation. I thought to myself: “How dull an existence
compared to our life in the Indian country, with dances and games,
feasts and ceremonies!”
Near the border line we entered a region of bandits and law-

breakers. We saw a white man who tried to hold us up, but we
spurred our broncos and they ran so fast he gave up [30]the chase;
then a band of Blood Indians closely muffled in their blankets; they
were on their way south and kept their faces hidden. We passed
another rider, who was followed by the North-West Mounted Police;
they said he was leader of a gang of cattle thieves.
We came that afternoon to the end of our journey in a camp of the

Blood Indians. Yellow Bird took me to the home of his relative, an
elderly man named Strong. In his lodge we met some of the head
men of the tribe—Thunder Chief, Spotted Calf, Running Coyote, and
Grasshopper. They were all friendly and glad to see us. They
greeted us with “How!” shook us by the hand and welcomed us to
their feast.
For refreshments, they had a meat stew and hot tea. Their manner
of eating was different from that of people in civilization. They ate
with their fingers, gulped down the food, sucked their teeth, and
drank with a hissing intake of the breath. But with them these were
not breaches of good manners; they were not sensitive to any of
these things. While eating they did not talk; and after the manner of
Indians showed no enthusiasm. None of them said the food was
good, or that they liked anything; nor, on the other hand, did any one
grumble or say the food was bad.
After the feast they smoked a large pipe of polished redstone, which
was handed stem first to each person. Then they talked, speaking
rapidly, in guttural voices that were not harsh, and making graceful
gestures with their hands. The Indian named Grasshopper had a
reputation as a wit. He kept them laughing—all but Thunder Chief,
who was head man and had his dignity to maintain.
Grasshopper wore a coyote-skin cap with the tail hanging down

behind and an eagle feather on top; slung over his shoulder was a
polished buffalo horn. He had beaded moccasins and leggings, and
a blanket coat with bright stripes. After we had eaten, he turned to
me and said with a laugh: [31]
“You look like an eagle. You sit straight and with your head up. Now
is the time to shake your tail feathers, like an eagle after it eats.” This
was Indian humor and made the others laugh.
Grasshopper was the life of the party. He said his parents died when
he was small. He was raised by a chief named Red Crow, who had
started many boys in life; he had become a successful man,
because he followed the advice of his adopted father.
Grasshopper was pleased when he saw me recording his

conversation in my notebook. He said: “Now I am going to tell you
some stories.” I sat waiting, but he did not begin; so I said: “Go
ahead; I am ready.”
To make the others laugh, he held out his hand saying: “How much
do you pay?” I took his outstretched hand, shook it and said: “That is
what I pay.”
Then every one laughed, even the dignified head chief joined in.
They liked the repartee and wanted more.
Grasshopper said: “Well, instead of paying he only shakes.” He

turned to the head-chief and said: “This white man is a great traveler.
I like him and want him for my partner. We had better keep him here
with us.”
And then he said to me: “Why don’t you join our tribe and stay with
us? You could take an Indian wife; you could hunt and trap and
make a good living.”
That evening we sat outside the lodge and watched the sun go down
fiery red, with its glow reflected in a near-by stream. Then the moon,
nearly full, rose over the distant hills of the prairie, like a ghostly
phantom in the twilight.
Then by the lodge-fire the Indians told stories of their hunting trips
and war expeditions of former days. They talked far into the night,
and next morning we saddled our horses early and rode back to
Montana. [32]
[Contents]
CHAPTER V

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Biomaterials and nanotechnology for tissue engineering

Bioinspired Biomaterials: Advances in Tissue

Chitosan Based Biomaterials Volume 2. Tissue

Regenerative Medicine from Protocol to Patient 3 Tissue

Smart Materials for Tissue Engineering Fundamental

Tissue Engineering Strategies for Organ Regeneration

Surface Engineering of Polymeric Biomaterials 1st

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Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Library of Congress Control Number: 2018935279

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Printed on acid-free paper

To my father—who instilled in me my love of science, taught me to never stop asking questions,

The book comprises 15 chapters covering the aforementioned topics. I am pleased to

●● Vorwald et al. provide a high-throughput methodology for encapsulating mesenchymal

San Carlos, CA, USA Kanika Chawla

1 Engineering Citric Acid-Based Porous Scaffolds

14 Synthesis of Self-Assembling Peptide-Based Hydrogels for Regenerative

Helena S. Azevedo · School of Engineering and Materials Science, Institute of

J. Kent Leach · Department of Biomedical Engineering, University of California—Davis,

Engineering Citric Acid-Based Porous Scaffolds

Key words Hydroxyapatite, Tissue engineering, Tissue regeneration, Osteogenicity, Composites,

The objective of tissue engineering is to develop biological scaf-

­ evelopment of compliant biodegradable scaffolds has been highly

2.1 POC-HA 1. Citric acid (Sigma-Aldrich, St. Louis, MO, USA).

2.2 Viscosity 1. Ubbelohde viscometer (Cannon Instrument Co., State

2.3 HA 1. Hydroxyapatite nanoparticles (Berkeley Advanced Biomaterials,

2.4 Compression 1. Sintech mechanical tester model 20/G (Research Triangle

2.5 Porosity 1. Water bath (Branson, North Olmsted, OH, USA).

7. Kimwipes (VWR, Radnor, PA, USA).

2.6 Porosity 1. Autopore IV (Micromeritics, Norcross, GA, USA).

2.7 Preparation 1. hMSCs (Lonza, Walkersville, MD, USA).

5. Determine the mass of ethanol that must be added to the bea-

Convert the mass of ethanol calculated from the above

3.2 Fabrication 1. Dissolve 10 g of POC prepolymer in 18 g ethanol at 80 °C. Mix

3.2.1 Tuning 1. To fabricate pre-POC with varying viscosities and degrees of

●● Vp = Volume of scaffold pores

3.3.2 Porosity 1. Use an Autopore IV to determine the incremental intrusion of

2. Sputter-coat the cross section and the surface of the scaffolds

3.4 hMSC Culture 1. Culture and expand hMSCs in expansion media at 37 °C in

This work was supported by NIH grant R00HL124279 granted to

Multifunctional Self-Assembling Peptide-Based

Key words Self-assembly, Multifunctional micelle, Enzyme-responsive, Cell-penetrating peptide,

Current research in the area of drug delivery is focused on the

into a suitable response, such as material degradation or disassembly,

in situ where the enzyme matrix metalloproteinase 2 (MMP-2) is

To assess the potential of the PA micelles as carriers for

2.1 Preparation 1. PA (previously synthesized, purified and lyophilized) powder.

2.2 Characterization 1. 1 mm Path length quartz cuvette.

2.5 Evaluation of PA 1. TCNB buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl,

2.6 Evaluation of PA 1. Human fibrosarcoma HT-1080 cell line.

2.7 Evaluation of PA 1. Human fibrosarcoma HT-1080 cell line.

3.1 Characterization 1. Dissolve the PAs in Milli-Q water at a specific concentration

San Carlos, CA, USA Kanika Chawla

evelopment of compliant biodegradable scaffolds has been highly