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BIOMATERIAL APPLICATIONS
Macro to Nanoscales
BIOMATERIAL APPLICATIONS
Macro to Nanoscales

Edited by
Sabu Thomas, PhD, Nandakumar Kalarikkal, PhD,
Weimin Yang, MD, and Snigdha S. Babu

Apple Academic Press

TORONTO NEW JERSEY
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ABOUT THE EDITORS

Sabu Thomas, PhD

Dr. Sabu Thomas is the Director International and Inter University Centre
for Nanoscience and Nanotechnology Mahatma Gandhi University, Kot-
tayam, India. He is also a full professor in the School of Chemical Sci-
ences at the same University. He is a fellow of many professional bodies.
Professor Thomas has authored or co-authored many papers in interna-
tional peer-reviewed journals in the area of polymer processing. He has
organized several international conferences and has more than 420 publi-
cations, 11 books and two patents to his credit. He has been involved in a
number of books both as author and editor. He is a reviewer to many in-
ternational journals and has received many awards for his excellent work
in polymer processing. His h Index is 67. Professor Thomas is listed as the
5th position in the list of Most Productive Researchers in India, in 2008.

Nandakumar Kalarikkal, PhD

Nandakumar Kalarikkal, PhD, is Associate Professor and Head of Ad-
vanced Materials Laboratory, School of Pure and Applied Physics, Mahat-
ma Gandhi University, Kottayam, India. He is also the Joint Director of the
International and Inter University Centre for Nanoscience and Nanotech-
nology at the same university. He is a fellow of many professional bod-
ies. He has authored many professional journal articles and has co-edited
several books. He is actively involved in research, and his group works on
the synthesis, characterization and applications of various nanomaterials,
ion irradiation effects on various novel materials, and phase transitions.

Weimin Yang, MD
Dr. Yang Weimin is the Taishan Scholar Professor of Qingdao University
of Science and Technology in China. He is a Professor of the College of
Mechanical and Electrical Engineering and Director of the Department of
International Exchanges and Cooperation, Beijing University of Chemical
vi About the Editors

Technology, Beijing , China. In addition, he is a fellow of many profes-

sional organizations. Professor Weimin has authored many papers in in-
ternational peer-reviewed journals in the area of polymer processing. He
has contributed to a number of books as author and editor and acts as a
reviewer to many international journals. In addition, he is a consultant to
many polymer equipment manufacturers. He has also received numerous
awards for his work in polymer processing. His interests include polymer
processing and CAD/CAE/CAM of polymer processing.

Snigdha S. Babu
Snigdha S. Babu is doing doctoral research in tissue engineering at the
International and Inter University Centre for Nanoscience and Nanotech-
nology at Mahatma Gandhi University, Kottayam, Kerala, India.
CONTENTS

List of Contributors..................................................................................... ix
List of Abbreviations................................................................................. xiii
Preface..................................................................................................... xvii
1. Green Organic-inorganic Hybrid Material from Plant
Oil Polyol...................................................................................................... 1
Eram Sharmin, Mudsser Azam, Fahmina Zafar, Deewan Akram,
Qazi Mohd. Rizwanul Haq, and Sharif Ahmad

2. Bio-Hybrid 3D Tubular Scaffolds for Vascular Tissue

Engineering—A Materials Perspective.................................................... 15
Harsh Patel, Roman Garcia, and Vinoy Thomas

3. Polymers for Use in the Monitoring and Treatment of Waterborne

Protozoa...................................................................................................... 49
Helen Bridle and Moushumi Ghosh

4. Synthesis of Polypyrrole/TiO2 Nanoparticles in Water by Chemical

Oxidative Polymerization.......................................................................... 73
Yang Tan, Michel B. Johnson, and Khashayar Ghandi

5. Poly (Lactic Acid) Based Hybrid Composite Films Containing

Ultrasound Treated Cellulose and Poly (Ethylene Glycol)
As Plasticizer and Reaction Media......................................................... 101
Katalin Halász, Mandar P. Badve, and Levente Csóka

6. An Experimental Observation of Disparity in Mechanical

Properties of Turmeric Fiber Reinforced Polyester Composites......... 123
Nadendla Srinivasababu, J. Suresh Kumar and K. Vijaya Kumar Reddy

7. Wavelength Dependence of SERS Spectra of Pyrene........................... 135

F. Hubenthal, D. Blázquez Sánchez, R. Ossig, H. Schmidt, and H.-D. Kronfeldt

8. Emerging Therapeutic Applications of Bacterial

Exopolysaccharides.................................................................................. 145
P. Priyanka, A. B. Arun, and P. D. Rekha
viii Contents

9. Preparation and Properties of Composite Films from Modified

Cellulose Fiber-Reinforced With Different Polymers........................... 169
Sandeep S. Laxmishwar and G. K. Nagaraja

10. Natural Bio Resources: The Unending Source of Nanofactory........... 195

Balaprasad Ankamwar

Index........................................................................................................... 203
LIST OF CONTRIBUTORS

Sharif Ahmad
Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi–110025,
India

Deewan Akram
Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi–110025,
India. Department of Chemistry, Faculty of Science, Jazan University, PO Box 2097, Jazan, Kingdom
of Saudi Arabia

Balaprasad Ankamwar
Bio-Inspired Materials Science Laboratory, Department of Chemistry, University of Pune, Ganeshkh-
ind, Pune–411007, India. E-mail:[email protected]. Phone: +91-20-25601397, Ext.533; Fax:
+91-20-25691728
A. B. Arun
Yenepoya Research Centre, Yenepoya University, Deralakatte, Mangalore, Karnataka State, India
Mudsser Azam
Microbiology Research Laboratory, Department of Biosciences, Jamia Millia Islamia, New Delhi–110025,
India

Mandar P. Badve
Department of Chemical Engineering, Institute of Chemical Technology, Mumbai, India

Dr. Helen Bridle

Royal Academy of Engineering/EPSRC Research Fellow, Institute of Biological Chemistry, Biophys-
ics and Bioengineering, Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS, Scotland

Levente Csóka
Department of Composite and Paper Technologies, Institute of Wood Based Products and Technolo-
gies, University of West Hungary, Sopron, Hungary, E-mail: [email protected]

Khashayar Ghandi
Department of Chemistry and Biochemistry, Mount Allison University, NB, Canada, E4L 1G8 E-mail:
[email protected], Tel: 506.961.080

Dr. Moushumi Ghosh

Thapar University, India

Katalin Halász
Department of Composite and Paper Technologies, Institute of Wood Based Products and Technolo-
gies, University of West Hungary, Sopron, Hungary, E-mail: [email protected]

Qazi Mohd. Rizwanul Haq

Microbiology Research Laboratory, Department of Biosciences, Jamia Millia Islamia, New Delhi–110025,
India
x List of Contributors

F. Hubenthal
Universität Kassel, Institut für Physik and Center for Interdisciplinary Nanostructure Science and
Technology-CINSaT, Heinrich-Plett-Str. 40, 34132 Kassel, Germany, E-mail: [email protected]
kassel.de

Michel B. Johnson
Institute for Research in Materials and Department of Chemistry, Dalhousie University, NS, Canada,
B3H 4R2

H. D. Kronfeldt
Technische Universität Berlin, Institut für Optik und Atomare Physik, Hardenbergstr. 36, 10623
Berlin, Germany, E-mail: [email protected]

J. Suresh Kumar
Professor, Department of Mechanical Engineering, JNTUH College of Engineering, Kukatpally,
Hyderabad-85, A.P., India

Sandeep S. Laxmishwar
Department of studies in Chemistry, Mangalore University, Mangalagangothri, Karnataka-574199,
India

G. K. Nagaraja
Department of studies in Chemistry, Mangalore University, Mangalagangothri, Karnataka–574199,
India
R. Ossig
Universität Kassel, Institut für Physik and Center for Interdisciplinary Nanostructure Science and
Technology-CINSaT, Heinrich-Plett-Str. 40, 34132 Kassel, Germany, E-mail: [email protected]
kassel.de

P. Priyanka
Yenepoya Research Centre, Yenepoya University, Deralakatte, Mangalore, Karnataka State, India

K. Vijaya Kumar Reddy

Professor, Department of Mechanical Engineering, JNTUH College of Engineering, Kukatpally, Hy-
derabad-85, A.P., India

Dr. P. D. Rekha
Deputy Director, Yenepoya Research Center, Yenepoya University, University Road, Deralakatte,
Mangalore–575018, Karnataka State, India. Tel: +91 824 2204668; Mobile: +91 9741501821; Fax:
+91 824 2204673. Email: [email protected], [email protected]

D. Blázquez Sánchez
Universität Kassel, Institut für Physik and Center for Interdisciplinary Nanostructure Science and
Technology-CINSaT, Heinrich-Plett-Str. 40, 34132 Kassel, Germany, E-mail: [email protected]
kassel.de

H. Schmidt
Universität Bayreuth, Forschungsstelle für Nahrungsmittel qualität E. C. Baumann-Str. 20, 95326
Kulmbach, Germany, E-mail: [email protected]

Eram Sharmin
Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi–110025,
India. Department of Pharmaceutical Chemistry, College of Pharmacy, Umm Al-Qura University,
Makkah Al-Mukarramah, Po Box 715, Postal Code: 21955, Kingdom of Saudi Arabia. E-mail: eram-
[email protected]
List of Contributors xi

Nadendla Srinivasababu
Professor, Department of Mechanical Engineering, Vignan’s Lara Institute of Technology and Sci-
ence, Vadlamudi-522 213, A. P., India. E-mail: [email protected]

Yang Tan
Department of Chemistry and Biochemistry, Mount Allison University, NB, Canada, E4L 1G8
Fahmina Zafar
Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi–110025,
India. Inorganic Materials Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New
Delhi–110025, India
LIST OF ABBREVIATIONS

AD Aldehyde
BC Bacterial Cellulose
BMSC Bone Marrow Stem Cell
BS Barbated Skullcup
BT Biotechnology
CABG Coronary Artery Bypass Graft
CB Conduction Band
CNF Cellulose Nanofibres
CNMB Center for Nanoscale Materials and Biointegration
CNW Cellulose Nanowhiskers
COWP1 Cryptosporidium (oo)cysts Wall Protein 1
CT Chemically Treated
CVD Cardiovascular Disease
DBSA Dodecylbenzenesulfonic Acid
DEA Diethanolamine
DEAA Diethylacrylamide
DLVO Derjaguin, Landau, Verwey and Overbeek
DMAA Dimethylacrylamide
DNA Desoxyribonucleic Acid
DSC Differential Scanning Calorimetry
DTG Derivative Thermogravimetric
dUTP Deoxynucleotidyl Transferase
EC Endothelial Cell
EPS Exopolysaccharides
e-PTFE Expanded Poly Tetra Flourethylene
FGF Fibrinogen Growth Factor
FTIR Fourier-Transform Infrared
Gf Grams-force
HAEC Human Aortic Endothelial Cell
HCAEC Human Coronary Artery Endothelial Cell
HUVSMCs Human Umbilical Vein Smooth Muscle Cells
ICAM Intercellular Adhesion Molecule
xiv List of Abbreviations

ICR Information Collection Rule

IMS Immunomagnetic Separation
IPN Interpenetrating Polymer Network
IT Information Technology
LAB Lactic Acid Bacteria
LRR Leucine Rich Repeats
LUMO Lowest Unoccupied Molecular Orbital
MA Methacrylate
MCC Microcrystalline Cellulose
MFC Microfibrillated Cellulose
NIR Near Infra Red
NPs Nanoparticles
NT Nanotechnology
OP Oxygen Permeability
OTR Oxygen Transmission Rate
PCL Polycaprolactone
PCR Polymerase Chain Reaction
PDO Polydioxanone
PEG Poly (Ethylene Glycol)
PGA Poly Glycolic Acid
PGC Polyglecaprone
PLA Poly Lactic Acid
PMBU Phosphorylcholine-co-Methacryloyloxyethyl Butyl-
urethane
PO Plant Oils
POH Prepared Hybrid Materials
PPy Polypyrrole
PU Polyurethane
QMRA Quantitative Microbial Risk Assessment
SEM Scanning Electron Microscopy
SERS Surface-Enhanced Raman Scattering
sHPMC Silylated Hydroxypropylmethycellulose
SIS Small intestinal sin mucosa
SMC Smooth Muscle Cell
SNR Signal-to-Noise Ratio
STM Scanning Tunneling Microscopy
TEM Transmission Electron Microscopy
TEOS Tetraethoxy Orthosilane
List of Abbreviations xv

TGA Thermogravimetric Analysis

TPF Turmeric Petiole Fiber
US Ultrasound
UTM Universal Testing Machine
VEGF Vascular Endothelial Growth Factor
WAXD Wide Angle X-Ray Diffraction
WVP Water Vapor Permeability
PREFACE

Natural polymers and biomaterials have always played a very important

role in our lives, and the research in this field has increased tremendously
over the last few decades and has led to many technological and commer-
cial developments. ICNP 2012 – Third International Conference on Natu-
ral Polymers, Bio-Polymers, Bio-Materials, Their Composites, Blends,
IPNs, Polyelectrolytes and Gels: Macro to Nano Scales took place at Ma-
hatma Gandhi University, Kottayam, Kerala, India, on October 26, 27,
and 28, 2012. It was jointly organized by Beijing University of Chemical
Technology, China. The conference aimed to emphasize interdisciplinary
research on processing, morphology, structure, and properties of natural
polymers, biomaterials, biopolymers, their blends, composites, IPNS, and
gels from macro to nano scales and their applications in medicine, auto-
motive, civil, chemical, and aerospace, computer, and marine engineer-
ing. During the three-day conference, distinguished scientists specializing
in various disciplines discussed recent advances, difficulties, and break-
throughs in the field of natural polymers and biomaterials. The conference
included keynote addresses, a number of plenary sessions, invited talks
and contributed lectures focusing on the diverse aspects of natural poly-
mers and biomaterials. It included discussions on recent advances, difficul-
ties, and breakthroughs in the field of natural polymers and biomaterials.
Bio-macromolecules, bio-polymers, bio-materials, natural polymers, bio-
composites, bio-nanocomposites, micro and nano blends based on natural
polymers, bio-degradable polymers, interphases in composites and blends,
IPNs, rheology of natural polymers, processing of natural polymers, deg-
radation and stabilization of natural polymer systems, biomass, recycling
of natural polymers were among the wide array of topics covered in the
conference. Additionally, there was a poster session with more than 50
posters to encourage budding scientists and researchers in this field. The
conference had over 200 delegates from all over the world.
This book, titled Biomaterial Applications: Macro to Nano Scales, is
a collection of chapters from the delegates who presented their papers
xviii Preface

during the conference. The book chapters include a wide variety of topics in
natural polymers, biomaterial, composites, and their applications.
We appreciate the efforts and enthusiasm of the contributing authors
for this book, and acknowledge those who were prepared to contribute
but were unable to do so at this time. We trust that this book will help in
stimulating new ideas, methods, and applications in the dynamic area of
research. The guest editors are Dr. Sabu Thomas, International and Inter
University Centre for Nanoscience and Nanotechnology, Mahatma Gan-
dhi University, Kottayam, India; Dr. Yang Weimin, Beijing University of
Chemical Technology, China; Dr. Nandakumar Kalarikkal, International
and Inter University Centre for Nanoscience and Nanotechnology,
Mahatma Gandhi University, Kottayam, India; and S. Snigdha, Interna-
tional and Inter University Centre for Nanoscience and Nanotechnology,
Mahatma Gandhi University, Kottayam, India.
We would like to thank all who kindly contributed chapters for this
book and the editors of Apple Academic Press, Inc., for their kind help and
cooperation. We are also indebted to the Apple Academic Press, Inc., edi-
torial office and the publishing and production teams for their assistance
in preparation and publication of this book.
—Dr. Sabu Thomas
CHAPTER 1

GREEN ORGANIC-INORGANIC
HYBRID MATERIAL FROM PLANT OIL
POLYOL
ERAM SHARMIN, MUDSSER AZAM, FAHMINA ZAFAR, DEEWAN
AKRAM, QAZI MOHD. RIZWANUL HAQ, and SHARIF AHMAD

CONTENTS

Abstract...................................................................................................... 2
1.1 Introduction....................................................................................... 2
1.2 Materials........................................................................................... 3
1.3 Synthesis of ALP and POH............................................................... 4
1.4 Instrumentation and Methods........................................................... 4
1.5 Results and Discussion..................................................................... 5
1.6 Conclusion...................................................................................... 12
Acknowledgments.................................................................................... 12
Keywords................................................................................................. 12
References................................................................................................ 13
2 Biomaterial Applications: Macro to Nanoscales

ABSTRACT

Plant oils (PO) are considered as environment friendly, domestically abun-

dant, cost effective renewable resources. They contain functional groups
(double bonds, active methylenes, hydroxyls, oxiranes), which are capable
of chemical transformations, yielding host of monomers or polymers. PO
polymers generally lack properties of rigidity, strength and thermal stabil-
ity. In this context, over the past few decades, several modifications of
PO polymers as organic-inorganic hybrid, composite and nanocomposite
materials have been accomplished, which have shown proven high per-
formance characteristics over their virgin and petro-based counterparts. In
this chapter, we have presented hybrid materials (POH) from PO derived
polyol and tetraethoxy orthosilane (TEOS) by microwave (MW) assisted
synthesis at lower preparation time relative to their counterparts synthe-
sized through conventional method. Some preliminary investigative stud-
ies have been carried out to assess the structure, morphology, water solu-
bility and antibacterial behavior of the said POH. These POH are foreseen
as prospective greener hybrid materials for coatings and paints.

1.1 INTRODUCTION

Recent advances in polymer science have been focused on the develop-

ment of cost effective, environment friendly and high performance poly-
mers from green resources en route Green Chemistry. The “green resourc-
es” include starch, cellulose, chitosan, plant oils [PO], and others. PO
are considered as the most environment friendly, domestically abundant,
cost effective bio-based precursors to polymers. They contain functional
groups (double bonds, active methylenes, hydroxyls, oxiranes), which are
capable of undergoing several derivatization reactions, yielding a host
of monomers/polymers, for example, diols, epoxies, polyols, polyesters,
polyester amides, polyurethanes. These polymers find versatile applica-
tions as adhesives, lubricants, antimicrobial agents, paints and coatings [1,
2]. PO polymers generally lack properties of rigidity, strength and thermal
stability. In the past few decades, several modifications of PO polymers
as organic-inorganic hybrid, composite and nanocomposite materials have
appeared. These have shown improved performance characteristics over
their virgin as well as petro-based counterparts [3, 4].
Green Organic-Inorganic Hybrid Material From Plant Oil Polyol 3

PO based organic-inorganic hybrids [POH] consist of nano sized in-

organic reinforcements in PO derived organic matrix (e.g., alkyds, epox-
ies, polyols, polyester amides and polyurethanes). POH may be prepared
by chemical reactions such as hydrolysis-condensation reaction between
organic matrix and metal alkoxides, for example, Zr(OR)4, Al (OR)3, Ti
(OR)4, Sn (OR)4, Si (OR)4), by mixing preformed metal nanoparticles, for
example, Ag, Cu, Au, by in situ generation of inorganic nano reinforce-
ments or by the inclusion of layered silicates (nanoclay) [5–12] in PO
derivatives. They exhibit excellent coating properties (mechanical proper-
ties, hardness, corrosion resistance, flexibility, and impact resistance) at-
tributed to the organic and inorganic components, respectively. Literature
scanning reveals that the preparation of POH generally occurs via cum-
bersome processes involving multistep syntheses and cure schedules at
elevated temperatures with prolonged synthesis and processing times and
consuming ample of solvents [6–16]. Thus, to overcome the aforemen-
tioned disadvantages, a greener, energy efficient, environment friendly ap-
proach is urgently needed, aimed at reducing multiple preparation steps
and time, minimizing or eliminating the use of hazardous solvents or per-
suading the use of greener solvents such as water [17–25], complying with
the principles of Green Chemistry.
In this chapter, we have prepared Linseed oil (LO) based POH through
an energy efficient process, that is, microwave (MW) assisted synthesis
technique, using polyol from LO [ALP] [26] as organic and tetraethoxyor-
thosilane (TEOS) as inorganic precursor via typical hydrolysis-condensa-
tion reaction. The approach presents a green protocol for the synthesis of
hybrid materials, that is, (i) the use of bio-resource PO as a raw material,
(ii) through MW (dielectric) heating which minimizes energy consump-
tion and reduces preparation time [27], and (iii) using an environmentally
benign solvent- water (in minimum quantity) [28], representing an excel-
lent example of “green,” “nano” hybrid material.

1.2 MATERIALS

LO (Pioneer in-organics, Delhi), hydrogen peroxide (30%), sulfuric acid,

glacial acetic acid, benzene (Merck, India), sodium metal, diethyl ether,
diethanolamine (DEA) (s.d. fine chemicals), (TEOS) (Merck, Germany)
were used as received.
4 Biomaterial Applications: Macro to Nanoscales

1.3 SYNTHESIS OF ALP AND POH

ALP was prepared by a two-step process consisting of hydroxylation of

LO, followed by amidation [26]. In this method, firstly, a typical hydrox-
ylation procedure was carried out with hydrogen peroxide (30%), sulfuric
acid, and glacial acetic acid that produced LO polyol from virgin LO [29].
LO polyol, so obtained, produced ALP, by simple amidation process in
presence of sodium methoxide and DEA [29, 30].
About 10 g of ALP, 5%, 10%, 15% of TEOS (percent by weight of
ALP) (prehydrolyzed at room temperature in presence of water and HCl
maintaining the pH of TEOS-water-acid mixture as 1.35) and 1 mL dis-
tilled water were taken in an Erlenmeyer flask and placed in the MW oven
for two minutes maintaining the temperature at 50 °C. The reaction mix-
ture was observed for clarity, perceptible changes such as abnormal rise in
viscosity or phase separation, if any, throughout the course of the reaction.
POH were obtained as clear, free flowing and yellowish brown colored
liquids. POH were named as 5-POH, 10-POH and 15-POH, where the
numbers indicate the percent loading of TEOS.

1.4 INSTRUMENTATION AND METHODS

The synthesis was carried out in LG microwave oven (model LG MS

1927C, MW frequency – 2500 MHz; power source –230 V ~50 Hz, energy
output – 800 W; input power – 1200 W).
FTIR spectra were taken on IR Affinity–1 CE (Shimadzu corporation
analytical and measuring instrument division, Kyoto, Japan) by placing
the samples in between two zinc selenide windows. Spectra were taken
from an average of 40 scans for each sample.
Morphological studies were carried out by transmission electron mi-
croscopy (TEM) (Morgagni 268-D TEM FEI instrument, Netherlands) on
a carbon coated copper grid. The test sample was diluted well in ethanol
and submerged in an ultrasonic bath for sonification for 30 min. Next, a
drop of this solution was placed on a Carbon Type-B (carbon film sup-
ported) 200 mesh copper grid by a micro pipette. The grid was allowed to
dry well before being used for TEM analysis.
Green Organic-Inorganic Hybrid Material From Plant Oil Polyol 5

Solubility in water was assessed by dissolving the samples in wa-

ter (percent by weight) in standard sample bottles at room temperature
(28 °C–30 °C) monitored through timely vigilant observation.
Transparency characteristic was visually observed by simply applying
these POH on glass slides.
Preliminary antibacterial studies of POH were performed by disk dif-
fusion assay against Staphylococcus aureus (MTCC 902) and Pseudomo-
nas aeruginosa (MTCC 2453).

1.5 RESULTS AND DISCUSSION

Hydroxylation of LO was carried out in situ that produced plain LO polyol

by the introduction of (Trans) hydroxyl groups on LO backbone. Plain LO
polyol further by base (sodium methoxide) catalyzed amidation reaction
with DEA, resulted in the formation of ALP and glycerol. The advantage
of this step was the introduction of amide groups along with additional
hydroxyl functionalities in the polyol backbone.
The synthesis of POH was accomplished in two steps in the presence
of water and glycerol (obtained during the preparation of ALP), as sol-
vents [26]. The first step involved hydrolysis of TEOS wherein silanols
were generated that condensed with hydroxyl groups of ALP and glycerol
in the second step, resulting in the anchorage of –O–Si–O– in LO ALP
forming POH (Fig. 1.1) [5, 31]. Here, 5-POH and 10-POH were obtained
as clear, glossy liquids while 15-POH was relatively foggy attributed to
higher content of TEOS. The said POH prepared through MW technique
were obtained in 2 min, while their conventionally prepared counterparts
were produced in 2 h at 50 °C. In MW assisted reactions (dielectric heat-
ing), due to rotation, friction and collision of (polar) molecules, under ap-
plied electromagnetic field, excessive heat is produced; the chemical reac-
tion occurs in much reduced time.
6 Biomaterial Applications: Macro to Nanoscales

FIGURE 1.1 Structure of POH.

1.5.1 FTIR

FTIR spectra of 5-POH, 10-POH and 15-POH (Fig. 1.2a–c) show absorp-
tion bands characteristic for –OH appearing at 3375–3360 cm–1 with slight
depression as the content of TEOS increased, correlated to the chemical
reaction (hydrolysis–condensation) between –OH of polyol and TEOS
[31, 32]. Relatively intense bands are observed at 862 cm–1, 1066 cm–1 and
1120 cm–1. These absorption bands gradually appear more intense with
increased TEOS content, supporting the formation of –Si–O–Si– network
in polyol [32], which are engulfed by the remnant polyol backbone. These
absorption bands support the incorporation of –O–Si–O– in ALP back-
bone forming POH [5, 26, 31–33].
Green Organic-Inorganic Hybrid Material From Plant Oil Polyol 7

FIGURE 1.2 FTIR spectra of (a) 5-POH, (b) 10-POH and 15-POH.
8 Biomaterial Applications: Macro to Nanoscales

1.5.2 TEM

TEM micrographs of POH are given in Fig. 1.3a–c. The visible gray re-
gion is the ALP matrix while the scattered dark points or globules observed
are the silica nanoparticles. The nanoparticles reveal size ranging from
4 nm–24 nm, with the highest distribution of particles of dimensions 14
nm–16 nm. These exist as spherical particles with distinct boundaries and
seem to be embedded within the matrices or engulfed by aliphatic fatty
chains of parent PO. These occur as unagglomerated particles since a por-
tion of polyol backbone is chemically bonded to nanosilica particles (also
supported by FTIR analysis) preventing their agglomeration [34–38]. The
study confirms the formation of organic-inorganic nanohybrids from LO.

FIGURE 1.3 TEM micrographs of (a) 5-POH and (b), (c), (d) 10-POH.

1.5.3 SOLUBILITY IN WATER

Freshly prepared 5-POH and 10-POH solutions (100%–20%) in water

(Fig. 1.4a) were obtained as clear solutions at room temperature (28 °C–
30 °C). These remained as clear homogenous solutions even after 12 h of
vigilant observations at the given temperature.
Green Organic-Inorganic Hybrid Material From Plant Oil Polyol 9

FIGURE 1.4 Solubility of 5-POH, 10-POH and 15-POH in water.

10 Biomaterial Applications: Macro to Nanoscales

1.5.4 TRANSPARENCY CHARACTERISTIC

POH were applied on glass slides and placed on a plain white sheet with
letters marked as “N,” “A” and “R.” These letters were clearly visible
through POH coated glass slides. Thus, POH produced fully transparent
films as evident in Fig. 1.5.

FIGURE 1.5 Transparency characteristic of 5-POH, 10-POH and 15-POH.

1.5.5 ANTIBACTERIAL BEHAVIOR

Determination of antibacterial sensitivity pattern of POH was performed

by Kirby Bauer method (i.e., disk diffusion assay) against Staphylococcus
aureus (MTCC 902) and Pseudomonas aeruginosa (MTCC 2453) [40,
41]. Mueller-Hinton agar plates were inoculated by the test organisms for
a uniform loan of culture. Discs containing 40 mg of the test sample were
placed at appropriate distance from the center. Para-film sealed plates
were incubated for 12–14 h at 37 °C. After incubation, zone of inhibi-
Green Organic-Inorganic Hybrid Material From Plant Oil Polyol 11

tion was measured and found to be 10 mm and 9 mm for Staphylococcus

aureus and Pseudomonas aeruginosa, respectively. Sensitivity pattern of
same isolates were tested for wide range of antibiotics having different
mode of action (Table 1.1).

TABLE 1.1 Antibiogram of Bacterial Isolates for Tested Antibiotics

Antibacterial agent MTCC 902 MTCC 2453

POH 10 9

Ampicillin/sulbactum 41 (S) 6 (R)

Levofloxacin 28 (S) 28 (S)

Rifampicin 26 (S) 6 (R)

Tetracycline 26 (S) 15 (S)

Polymixin B 8 (R) 15 (S)

Trimethoprim 20 (S) 6 (R)

Ofloxacin 21 (S) 21 (S)

Aztreonam 24 (S) 30 (S)

Tobramycin 23 (S) 35 (S)

Ciprofloxacin 25 (S) 36 (S)

Cefozolin 32 (S) 6 (R)

Ertapenem 29 (S) 14 (R)

Ceftazidime 16 (R) 20 (R)

Cefotaxime 33 (S) 24 (R)

Zone of Inhibition: diameter in mm S–Susceptible; R–Resistant.

These results showed that POH was efficient enough to inhibit the
growth of the given microbes, resistant to a number of antibiotics tested
for their sensitivity pattern (Table 1.1). Our preliminary investigations on
antibacterial behavior of POH by disc diffusion method revealed that this
bactericidal activity may be attributed to the structural build-up of POH.
Further studies on the mode of antibacterial action of POH are still needed
to be carried out.
12 Biomaterial Applications: Macro to Nanoscales

1.6 CONCLUSION

LO based nanostructured POH were prepared by MW assisted prepara-

tion method from amidated Linseed polyol and TEOS. The approach dealt
with the use of a bio-resource, simple, safe, less time consuming and en-
ergy efficient MW assisted preparation method, without the use of any
hazardous solvents but water, with no complex chemical transformations
or side reactions complying with the principles of Green Chemistry. Our
studies revealed that the said LO based POH could be used as water sol-
uble materials for time period not exceeding 24 h at ambient temperature
(28 °C–30 °C). Thus, what still remains on tenterhooks and unanswered
is the 100% solubility and 100% stability (as water soluble materials) of
POH for largely extended time periods at room temperature.

ACKNOWLEDGMENTS

Dr. Eram Sharmin (Pool Officer) and Dr. Fahmina Zafar (Pool Officer) ac-
knowledge the Council of Scientific and Industrial Research (New Delhi,
India) for Senior Research Associate ships against Grant Nos. 13(8464–
A)/2011 Pool and 13(8385-A)/Pool/2010, respectively. Dr. Fahmina Zafar
is also thankful to University Grant Commission, India for Dr. D. S. Ko-
thari Post Doctoral Fellowship, Ref. # F.4/2006(BSR)/13-986/2013(BSR).
Dr. Eram Sharmin and Dr. Fahmina Zafar are thankful to the Head, De-
partment of Chemistry (Jamia Millia Islamia, New Delhi) for providing
facilities to carry out the research work. Mudsser Azam is thankful to the
Council of Scientific and Industrial Research (New Delhi, India) for finan-
cial assistance against Grant No. 09/466(0136)/2011-EMR-I

KEYWORDS

•• Organic-Inorganic Hybrids
•• Polyol
•• Vegetable Oils
Green Organic-Inorganic Hybrid Material From Plant Oil Polyol 13

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CHAPTER 2

BIO-HYBRID 3D TUBULAR
SCAFFOLDS FOR VASCULAR TISSUE
ENGINEERING—A MATERIALS
PERSPECTIVE
HARSH PATEL, ROMAN GARCIA, and VINOY THOMAS

CONTENTS

2.1 Introduction..................................................................................... 16
2.2 Bio-Hybrid Vascular Graft Based on Collagen/Synthetic
Polymers......................................................................................... 19
2.3 Bio-Hybrid Vascular Grafts Based on Elastin................................ 23
2.4 Bio-Hybrid Vascular Graft Based on Collagen/Elastin/
Synthetic Polymers......................................................................... 25
2.5 Bio-Hybrid Vascular Grafts Based on Gelatin/Synthetic
Polymers......................................................................................... 26
2.6 Bio-Hybrid Vascular Grafts Based on Fibrin/Fibrinogen............... 30
2.7 Bio-Hybrid Vascular Grafts Based on Bacterial Cellulose............. 32
2.8 Bio-Hybrid Vascular Graft Based on Silk Fibroin/Synthetic
Polymers......................................................................................... 34
2.9 Multi-Layered Bio-Hybrid Vascular Grafts.................................... 37
2.10 Summary......................................................................................... 38
2.11 Acknowledgments.......................................................................... 40
Keywords................................................................................................. 41
References................................................................................................ 41
16 Biomaterial Applications: Macro to Nanoscales

2.1 INTRODUCTION

Cardiovascular disease (CVD) remains one of the leading causes of death

around the world [1]. This global health concern has prompted advances
in surgical and medical technologies. However, an increasing percentage
of the population continues to be at risk and eventually fall victim to this
debilitating disease. While the most common cause of blood vessel failure
in this case, is atherosclerosis, whereby plaque buildup within the lumen
results in hindered blood flow, injury, disease, or inflammation can also
result in a compromised vessel [2]. Routinely, a comparable native vessel
(ex. internal thoracic arteries, radial arteries, and saphenous veins) will
be excised and relocated to replace the occluded vessel in a procedure
known as a coronary artery bypass graft (CABG) surgery. These autolo-
gous grafts have limited availability and depend on donor site morbidity
[3]. For example, with close to a half a million coronary artery bypass
operations performed annually in the US alone, more than 20% of the
patients do not possess healthy graft veins for CABG [4]. Addressing this
concern, researchers have increasingly looked towards scaffold-based vas-
cular tissue engineering to produce readily available vascular grafts [5].
While synthetic polymers such as polyester (Dacron) or expanded poly
tetra flourethylene (e-PTFE) have been used successfully to fabricate large
diameter (>6 mm) blood vessels [6], these materials have resulted in loss
of vessel patency due to thrombosis in small diameter (<6 mm) applica-
tions [7]. Furthermore, as these synthetic vascular grafts will ultimately be
implanted into the patient, the biocompatibility of component materials
is an important factor for the future success of this technology. To this
end, vascular tissue engineers have shown a general movement toward
small diameter (<6 mm) scaffolding comprised of biopolymers, those that
naturally exist within vascular tissue matrix as well as naturally occurring
biomaterials.
The arterial wall structure of native vessels consists of three distinct
layers, intima, media, and adventitia, each having its own specific cell and
protein composition. The innermost layer (intima) surrounding the ves-
sel lumen contains a monolayer of endothelial cells (ECs), collagen type
IV, and elastin. The cellular physiology of the intima aids in the preven-
tion of thrombosis, infection, and inflammation of the blood vessel and
any underlying tissue [2]. The middle layer (media) is contains collagen
type I, III, elastin, proteoglycans, and smooth muscle cells (SMCs). This
Bio-Hybrid 3D Tubular Scaffolds for Vascular Tissue 17

layer provides the unique mechanical properties necessary for the vessel
to withstand in vivo pressures and forces. The outermost layer (adventitia)
is primarily comprised of collagen type I along with fibroblasts. As the
outermost layer, the adventitia’s primary function is to provide stability
to the vessel by anchoring it to nearby tissue as well as to connect with
body systems via vascular and nerve networks. Each distinct matrix/cell
layer works in tandem to withstand the physiologically rigorous pressure
conditions of the body and maintain homeostasis. Table 2.1 lists the me-
chanical properties for common native vessels used for synthetic vascular
graft comparison.

TABLE 2.1 Mechanical Properties for Some Native Vessels

Native vessel Tensile Strength Tensile Modulus Strain (%) Reference

(MPa) (MPa)

Femoral artery 1–2 9–12 63–76 [8]

Fresh carotid artery 1.76–2.64 - 110–200 [9]

Coronary artery 1.4–11.14 - 45–99 [10]

The three dimensional tubular structure of vascular scaffolds must pro-

vide a platform for cell migration, proliferation, and differentiation while
mimicking the structural and mechanical integrity of native vascular ECM.
The ideal scaffold should be biocompatible, biodegradable, bioresorbable
and a cellular-growth enhancing matrix. As new tissue formation starts,
the scaffold should gradually degrade leaving enough space for tissue
in-growth. Further, material degradation rates must be considered as any
premature hydrolysis of these polymers prior to complete cellular prolif-
eration will compromise the mechanical integrity of the scaffold. Vascular
tissue scaffolds can be divided into three types based on the nature and
origin: natural, synthetic, and bio-hybrid. Examples of natural scaffolds
would be those made of collagen, elastin, fibronectin etc., materials which
are structural and/or specialized proteins of native vascular ECM. Syn-
thetic scaffolds can be made of different biodegradable polyester polymers
such as polycaprolactone (PCL), poly lactic acid (PLA), poly glycolic acid
(PGA), copolymer form of a poly D, L lactic-coglycolic acid (PLGA) etc.
Bio-hybrid scaffolds use a combination of natural and synthetic polymers
to achieve adequate elastic mechanical properties attainable through syn-
18 Biomaterial Applications: Macro to Nanoscales

thetic polymers while increasing biocompatibility and cellular recognition

through the use of natural materials.
Various methods have been used to create tubular scaffolds for vascu-
lar tissue engineering. For example, researchers have engineered a com-
pletely biological vascular graft by incorporating collagen type I into a
gel scaffold through the use of molds prepared with the help of silicone
sleeves and hollow glass tubes [32]. Further, a procine derived small intes-
tinal sin mucosa (SIS) has been used to generate a biological vascular scaf-
fold or xenograft, since it is a decellularized tissue or ECM [33]. However,
concerns of disease transmission and negative immune response continue
to arise for vascular graft made from SIS. Another approach used to cre-
ate a completely biological tissue engineered blood vessel was developed
by the Augur group [34]. They made single layer cell sheets comprised
of human vascular SMCs wrapped around a tubular support. Afterward,
other cell sheets comprised of human fibroblasts were wrapped around of
the previous SMC sheet. After a few days of cell growth, the tubular sup-
port was removed leaving a tubular-shaped biological tissue engineered
vascular graft. In all these methods, the mechanical integrity of the graft
has not been adequate. Furthermore, these methods produce scaffolds with
low porosity limiting cell-growth and proliferation.
While many methods do exist to produce vascular scaffolds, electro
spinning, a nanotechnology enabling technique, has attracted much atten-
tion due to its simplicity to fabricate tubular 3D conduits and replicate na-
tive ECM-like formation of random oriented nanofibers with large surface
area to volume ratio for enhanced cell adhesion and proliferation [35].
Electro spinning is a facile technique for producing ultra-thin polymer mi-
cronano fibers using an electrostatic force [36]. Detailed reviews on the
electro spinning process and the applications of electro spun biomimetic
scaffolds can be seen elsewhere [37]. Electrospun scaffolds are highly po-
rous with interconnecting pore-networks (75–90%), providing a large sur-
face area with surface functional groups for protein and cell attachment.
Electrospinning of any polymer requires optimization of two main pa-
rameters: System parameters and process parameters. System parameters
consists of molecular weight, molecular weight distribution, polymer so-
lution viscosity, and conductivity, whereas process parameters consist of
electric potential, flow rate, distance between the capillary and collector,
temperature, and humidity [36]. These parameters can be varied to achieve
bead-free quality of fibrous mats for tissue engineering applications.
Bio-Hybrid 3D Tubular Scaffolds for Vascular Tissue 19

This process allows for the formation of nanofibers composed of polymer

blends, natural and synthetic, all done in a rather straightforward, cost
effective setup [17]. Therefore, an increased emphasis is given to review
electrospun bio-hybrid scaffolds, their mechanical integrity, biocompat-
ibility, and native cell adhesion and proliferation in this chapter.

2.2 BIO-HYBRID VASCULAR GRAFT BASED ON COLLAGEN/

SYNTHETIC POLYMERS

Since the introduction of biopolymers in vascular tissue engineering, col-

lagen has been a favored material due its prevalence in native vascular
make-up. It is made up of groups of fibrous proteins and naturally pro-
duced by mammalian cells. It is also one of the most plentiful proteins
found in human body. There are almost 28 different types of collagen in
the human body [38]. Collagen is a preferable biopolymer because it’s
low antigenicity, low inflammatory and cytotoxic response, and desirable
biological and hematological properties [39]. Collagen has been used in
various biomedical applications due to its biocompatibility and biodegrad-
ability [40]. As mentioned previously, it is a main component in the blood
vessel matrix-structure. Therefore, researchers have strived to compose
vascular grafts using collagen and gelatin (denatured collagen) with or
without other biodegradable polymers. The basic structure of collagen is a
three-parallel polypeptide chains formed by glycine, proline and hydroxyl
proline repeating units arranged in a triple helix structure [41]. The me-
chanical integrity of collagen type I can be different than collagen type III
or others and different tissue types have different types of collagen. In ad-
dition, collagen origin can also play a role in the final characteristics of the
collagenous tissue-scaffolds derived from animal or cadaver tissues [11].
Being a vascular matrix protein, researchers started to fabricate hy-
brid grafts made of collagens and biodegradable synthetic polymers to
impart bioactivity and to prevent scar-tissue fibrosis. Weinberg and Bell
have incorporated collagen with Dacron (nonbiodegradable) to create a
model for an artificial blood vessel in vitro [42]. Thereafter, the biode-
gradable grafts were introduced since they would degrade in vitro and
in vivo as the native tissue formation starts. Widely used aliphatic poly-
esters in tissue engineering applications take several months to a few
years to degrade. The biodegradation and cell-interaction characteristics
20 Biomaterial Applications: Macro to Nanoscales

of them can be tuned with the incorporation of collagen. For example,

when collagen fibers were incorporated onto electrospun PLA fibers
to mimic the properties of blood vessel [43], promising SMCs growth
and mechanical integrity improvements were observed. To mimic the
smooth muscle cells arrangement in the arterial wall, a preliminary graft
scaffold based on PLLA was created by electrospinning method on 4 mm
diameter mandrel with a single helical wind of collagen fibers embedded
in biodegradable matrix [43]. After 10 days culture with human aortic
SMCs, the vascular graft exhibited extensive SMCs proliferation and the
orientation of the SMCs.
Because of rapid degradation and low shear-stress resistance of pure
collagen scaffolds, there is a need for reliable and biocompatible syn-
thetic polymer for preventing premature degradation of scaffolds. The
longer degradation time is a useful characteristic when combined with
collagen in a vascular tissue graft, as cells need longer time to prolifer-
ate and to generate ECM to mimic native tissue structure. PCL is an
aliphatic polyester with a low glass transition temperature of 60 °C and
low melting point 60 °C [39] and slower in degradation (half-life is ~2
years). Atala’s group has combined PCL with collagen type I and seeded
with bovine ECs (bECs) and bovine SMCs (bSMCs) [12]. To fabricate
the scaffold, PCL and collagen type I (1:1 ratio) were dissolved in the
same solvent and then the solution was electrospun into tubular conduit.
They noticed formation of the confluent layer of bECs on the lumen
and bSMCs on the outer surface of the scaffold, which exactly mimic
the native artery structure. The mechanical properties were shown to
be comparable to native vessels (listed in Table 2.2). An important me-
chanical characteristic which is useful in understanding whether or not
the graft will survive in the arterial environment for vascular graft is its
burst pressure. The gold standard for CABG is a saphenous vein, which
has a burst pressure of 1680 ± 370 mmHg [44]. The burst pressure of
the composite PCL/collagen (1:1) scaffold was 4912 ± 155 mmHg with
an acceptable suture retention strength (above 2.0 N) [45]. Figure 2.1
details the vascular graft with uniform wall-thickness made of PCL/col-
lagen fibers with electrospinning technique [12]. It also illustrates the
surface morphology of the scaffold exhibiting randomly oriented fibers
and interconnected pores throughout the thickness.
Bio-Hybrid 3D Tubular Scaffolds for Vascular Tissue 21

TABLE 2.2 Mechanical Properties of Different Bio-Hybrid Compositions Used in

Vascular Grafts

Scaffold Diameter of Tensile Tensile Strain at Refer-

the fibers Strength Modulus break ence
Composition
(MPa) (MPa) (%)
Collagen type I 100–730 nm 1.5 ± 0.2 52.3 ± 5.2 – [11]
PCL/collagen (wet) 520 nm 4.0±0.4 2.7 ± 1.2 140 ± 13 [12]
PCL/collagen I/ – 18 7.79 – [13]
collagen III
P(LLA- CL)/col- 470 ± 130 nm 6.27 ± 1.38 43.99 ± 4.04 176 ± 49 [14]
lagen I
PDO/collagen I 4.6–6.7 7.6–18.0 56.5–186.4 [15]
Gelatin 460 ± 148–517 8–12 426 ± 39 80–100 [16]
± 241 nm
Gelatin/PCL – 1.29 30.8 1.38 [17]
PLCL/gelatin 200 nm – 1.3 5.1 ± 0.8 340 ±8.5 86 ± 19 [18]
(50:50) µm
PLGA/gelatin/ 317 ± 46 nm 130 ± 7 770 ± 131 – [19]
elastin
PGA/gelatin (30%) 863.96 ± 265.09 – 32 32 [20]
nm
Polyglyconate/ 200–400 nm 2.71 ± 0.2 20.4 ± 3 140 ± 3 [21]
gelatin/elastin
Polydioxanone/ 300–500 nm 1.77 ± 0.2 5.74 ± 3 75.08 ±10 [22]
gelatin/elastin
Fibrinogen 80–700 nm 2 80 – [23]
Fibrinogen/ PLLA- 305 ± 78 nm 10.1 ± 0.4 – 118.8 ± [24]
CL (20:80) 12.3
PLGA/fibrin 50 nm - 4 µm 0.651 ± 0.24 – 105 ± 10 [25]
Bacterial Cellulose 311 ± 29 – – [26]
B. mori Silk 9.3 ± 0.3 µm 740 10000 20 [27]
B. mori silk/ PEO – 2.42±0.48 2.45±0.47 – [28]
Silk fibroin/gelatin 143 ± 36 nm 1.12 ± 0.11 – 30.55 ± [29]
3.46
PLA/SF – gelatin – 1.28 ± 0.21 – 41.11 ± [29]
2.17
Collagen/chito- 409 ± 120 nm 16.9 ± 2.9 10.3 ± 1.1 112 ± 11 [30]
san/ PLLA-CL
(25:5:75)
PEUU/PMBU 525±162 nm 7±1 7±3 342 ± 43 [31]
(15%)
22 Biomaterial Applications: Macro to Nanoscales

FIGURE 2.1 (a) The gross appearance and SEM images of electrospun PCL/collagen
composite scaffolds: (b) entire (x18), (c) surface (x6.0K), and (d) cross-sectional (x4.0K)
morphologies. Reprinted from Biomaterials, Volume 29/Issue 19, Sang Jin Lee, Jie Liu,
Se Heang Oh, Shay Soker, Anthony Atala, James J. Yoo, Development of a composite
vascular scaffolding system that withstands physiological vascular conditions, 2891–2898,
2008, with permission from Elsevier.

In another study, collagen was combined with the poly (L-lactide) co-
poly (caprolactone) (PLLA-CL) and electrospun into nanofibers to create
a mesh like structure to mimic native ECM both morphologically and bio-
chemically [46]. In contrast with the previous method, the PLLA CL fibers
were electrospun first and then treated with air plasma treatment for 5 min.
Plasma treatment has been used to successfully increased hydrophilicity
of the material, which aids in cell attachment [47]. The scaffold was sub-
sequently coated with a collagen solution. The scaffold exhibited porosity
between 60–70%. Fiber diameter, tensile strength, and tensile modulus
(listed in Table 2.2) were promising. Human coronary artery endothelial
cells (HCAECs) were seeded on these nanofibers to promote endotheli-
alization, and it provided promising results for potential use for vascular
tissue engineering. Further regeneration studies using bioreactor (HAECs
seeded inside the lumen of the tube) and implantation in rabbits, the tubu-
lar scaffolds coated by the collagen type 1 [48] exhibited promising results
Bio-Hybrid 3D Tubular Scaffolds for Vascular Tissue 23

that scaffolds were maintained structure integrity up to 7 weeks in vivo.

Figure 2.2 illustrates the human aortic endothelial cells (HAECs) spread-
ing and attachment on the large surface area of the electrospun scaffold
made of PCL/collagen [48].

FIGURE 2.2 (A) FC1, 0.27 ± 0.09, (B) FC2, 1.00 ± 0.15, (C) FC3, 2.39 ± 0.69, and (D)
FC4, 4.45 ± 0.81 mm. Scale bar indicates 50 mm (magnification; x1K). EC morphologies
on the scaffolds with various fiber diameters were observed. Cell adhesion and spreading
can be guided by the direction of the smaller fibers. Reprinted from Biomaterials, Volume
31, Issue 15, Young Min Ju, Jin San Choi, Anthony Atala, James J. Yoo, Sang Jin Lee,
Bilayered scaffold for engineering cellularized blood vessels, 4313–4321,2010, with the
permission from Elsevier. SEM images of HAEC adhesion and spreading onto electrospun
PCL/collagen fibrous scaffolds with four different fiber diameters at 3 days after cell
seeding.

2.3 BIO-HYBRID VASCULAR GRAFTS BASED ON ELASTIN

Elastin is a native protein widely used in electrospun vascular tissue scaf-

folding as it is a critical component in the complex architecture of native
vascular extracellular matrix. The protein is found within the intima and
media layers of the blood vessel wall. Elastin fibers can maintain their
structure even after extension of approximately 140% [49]. It plays a criti-
cal role in preserving the shape and elasticity of tissues under stress, there-
fore integral in maintaining the biomechanical properties of native vascular
24 Biomaterial Applications: Macro to Nanoscales

tissue [50]. Scaffolds were made by using pure elastin [51]. While pure
elastin has been electrospun to achieve fiber diameters within range of na-
tive matrices [50], scaffolds comprised of pure elastin alone do not exhibit
adequate properties of a successful graft. Gelation and hydrolysis rapidly
degrade proteins such as elastin when placed in culture media [52]. There-
fore, elastin-based constructs alone do not withstand the mechanical stress
common to vascular tissue [52]. Alternatively, elastin is used as a com-
ponent in a blend of polymers, allowing elastin’s unique properties to be
expressed in relation to other materials. For example, an elastin-collagen
blend in varying concentrations was electrospun in order to fabricate a
tubular scaffold with layered structure [50]. A solution of 80:20 collagen
type I: elastin was electrospun around a 4 mm tubular mandrel, which
formed the outermost adventitia layer. At this ratio and at a concentration
of 0.083 g/ml an average fiber diameter of 0.49 ± 0.22 µm was observed.
A subsequent layer of 30:70 collagen type I: elastin was formed around a
2 mm tubular scaffold. The insertion of this smaller tube into the 4 mm
tubular scaffold formed the vascular graft [50].
Expanding on Boland’s preliminary study of electrospun elastin and
collagen, Li et al. compared solubilized alpha-elastin and recombinant hu-
man tropoelastin [16]. Increasing the concentration of elastin and tropo-
elastin from 10% to 20% in spinning solution has yielded continuous, uni-
form fibers free of beads. Both biopolymers produced fibers of a greater
size than gelatin and collagen. Tropoelastin fibers exhibited widths 2–3
times greater than those of elastin under similar conditions. Elastin and
tropoelastin fibers under SEM imaging appeared to be flat and ribbon-
shaped which was observed to be independent of variant electrospinning
parameters. The native wave-like pattern of elastin remained after electro-
spinning. Tensile testing showed a tensile strength of 1.6 MPa for elastin
and 13 MPa for tropoelastin. Li et al. concluded that tropoelastin would
be a more advantageous biopolymer than soluble alpha elastin when engi-
neering vascular grafts. Cell-scaffold interactions were favorable in elas-
tin and tropoelastin scaffolds. Another study of electrospinning of elastin,
NaCl, and PEO (poly ethylene oxide) blend comprised of elastin 5% (w/v)
with a 5:1 ratio of elastin to PEO at a voltage of 10 kV and infusion rate of
50 µL/min, a fibrous matt with an average fiber diameter was 500 nm was
obtained [53]. Substantial splaying of elastin/PEO fibers had contributed
to the small fiber diameters observed in this experiment. Increasing the
concentration of elastin has decreased the surface tension of the jet during
Bio-Hybrid 3D Tubular Scaffolds for Vascular Tissue 25

electrospinning, which leads to decreased bead formation. However, an

increase in fiber diameter was observed with increasing elastin concentra-
tion [53].

2.4 BIO-HYBRID VASCULAR GRAFT BASED ON COLLAGEN/

ELASTIN/SYNTHETIC POLYMERS

Collagen has been combined with the elastin to recapitulate the biome-
chanical and biochemical properties of native ECM. An artery’s mechani-
cal stability greatly depends on the amount of collagen present; however,
the elasticity and the ability to recover from the deformation are depen-
dent on the presence of elastin. Therefore, a hybrid type of scaffold was
prepared by electrospinning method but, incorporating polydioxanone
(PDO), elastin, and collagen [54] with different compositions 50:25:25
and 45:45:10 (PDO:elastin:collagen). Buttafoco et al. have spun collagen
type I (from calf skin) and elastin (from bovine neck ligament mixed)
together with the high molecular weight of poly (ethylene oxide) PEO to
electrospun nanofibers (ranging from 220 to 600 nm diameter) scaffolds
[53]. After electrospinning, the meshes were stabilized by crosslinking
using a mixture of N-(3-dimethylaminopropyl)-N”-EDC and N-hydroxy-
succinimide (N-NHS) [55]. Interestingly, another arterial vascular graft
composed by multi lamellar structure from synthetic collagen microfibers
and a recombinant elastin like protein [56] in which the synthetic collagen
fibers settled into parallel arrays embedded within the elastin, and then
the entire matrix is rolled into tubular form to create a vascular graft. The
mechanical characteristics such as burst pressure and suture retention were
measured to be 239–2760 mm Hg and 35–192 grams-force (gf), respec-
tively. In short, postfabrication methods such as cross-linking affect the
mechanical strength due to fiber architecture, fiber spacing and orienta-
tion.
Electrospun nanofibers scaffolds made with Poly (D, L-lactide-cogly-
colide, 50:50 ratio) PLGA and incorporated collagen type I from calf skin
and elastin were examined for vascular graft application [57]. Figure 2.3
shows the scaffold made of PLGA, collagen and elastin with different ra-
tios and the SEM image of their nanofibers [57]. Important to note, when
subjected to a burst pressure 12 times higher than normal systolic blood
pressure, the scaffold did not rupture [57].
26 Biomaterial Applications: Macro to Nanoscales

FIGURE 2.3 Characterization of the electrospun scaffold. (a) Electrospun nanofiber

scaffold before crosslinking: collagen, elastin, and PLGA composite. (b) SEM image
of electrospun nanofiber scaffold before crosslinking at 1800X magnification. (c)
Immunohistochemical analyzes using antibodies specific to collagen type I in the scaffold.
(d) The electrospun scaffold with 15% elastin demonstrated a uniform elastin matrix
throughout the vascular scaffold wall. Reprinted from Biomaterials, Volume 27, Issue 7,
Joel Stitzel, Jie Liu, Sang Jin Lee, Makoto Komura, Joel Berry, Shay Soker, Grace Lim,
Mark Van Dyke, Richard Czerw, Controlled fabrication of a biological vascular substitute,
1088–1094 2006, with permission from Elsevier.

2.5 BIO-HYBRID VASCULAR GRAFTS BASED ON GELATIN/

SYNTHETIC POLYMERS

Gelatin is the denatured form of collagen with many of the same attractive
qualities such as biological origin, biocompatibility, biodegradability and
commercially available at the low cost, all owing to its popularity as a bio-
polymer [17]. Since the very similar behavior as the collagen, gelatin has
been used as a biopolymer for the vascular tissue engineering application.
Electrospinning of gelatin into nano-micro scale fibers (50–500 nm) and
a comparison of its mechanical characteristics with collagen and elastin
were reported elsewhere [16]. Results have shown that the gelatin fibers
have higher tensile modulus than collagen fibers, but have similar tensile
strength and ultimate elongation [16]. This data ensures that gelatin can be
electrospun in nano-micro scale fibers when proper solvent and concen-
tration were used. As in the case of other biopolymers, gelatin fibers once
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