Tissue Engineering Applications And Advancements Raj K Keservani

Tissue Engineering Applications And Advancements Raj K Keservani
Tissue Engineering Applications And Advancements Raj K Keservani
Tissue Engineering Applications And Advancements Raj K Keservani

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6. TISSUE ENGINEERING
Applications and Advancements

8. TISSUE ENGINEERING
Applications and Advancements
Edited by
Rajesh K. Kesharwani, PhD, MTech
Raj K. Keservani, MPharm
Anil K. Sharma, PhD, MPharm

9. First edition published 2022
Apple Academic Press Inc. CRC Press
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Library and Archives Canada Cataloguing in Publication
Title: Tissue engineering : applications and advancements / edited by Rajesh K. Kesharwani, PhD, MTech, Raj K. Keservani,
MPharm, Anil K. Sharma, PhD, MPharm.
Names: Kesharwani, Rajesh Kumar, 1978- editor. | Keservani, Raj K., 1981- editor. | Sharma, Anil K., 1980- editor.
Description: First edition. | Includes bibliographical references and index.
Identi�ers: Canadiana (print) 2021034444X | Canadiana (ebook) 20210344482 | ISBN 9781774630204 (hardcover) | ISBN 9781774638774
(softcover) | ISBN 9781003180531 (ebook)
Subjects: LCSH: Tissue engineering.
Classi�cation: LCC R857.T55 T57 2022 | DDC 610.28—dc23
Library of Congress Cataloging‑in‑Publication Data
Names: Kesharwani, Rajesh Kumar., 1978- editor. | Keservani, Raj K., 1981- editor. | Sharma, Anil K., 1980- editor.
Title: Tissue engineering : applications and advancements / edited by Rajesh K. Kesharwani, Raj K. Keservani, Anil K. Sharma.
Other titles: Tissue engineering (Kesharwani)
Description: First edition. | Palm Bay, FL : Apple Academic Press, 2022. | Includes bibliographical references and index. | Summary:
“This new volume on applications and advances in tissue engineering presents signi�cant, state-of-the-art developments in this
exciting area of research. It highlights some of the most important applied research on the applications of tissue engineering along
with its different components, speci�cally different types of biomaterials. It looks at the various issues involved in tissue engineering,
including smart polymeric biomaterials, gene therapy, tissue engineering in reconstruction and regeneration of visceral organs, skin
tissue engineering, bone and muscle regeneration, and applications in tropical medicines. Covering a wide range of issues in tissue
engineering, the volume Provides an overview of the ef�cacy of the different biomaterials employed in tissue engineering (such as
skin regeneration, nerve regeneration, arti�cial blood vessels, bone regeneration). Looks at smart polymeric biomaterials in tissue
engineering Discusses the hybrid approach of tissue engineering in conjunction with gene therapy Explores using tissue engineering
in the management of tropical diseases Considers various skin tissue engineering applications, including wound healing methods,
skin substitutes and other materials Reports on the use of various biomaterials in bone and muscle regeneration Describes the use
of tissue engineering in reconstruction and regeneration of visceral organs Covers polysaccharides and proteins-based hydrogels for
tissue engineering applications Providing an abundance of advanced research and information, Tissue Engineering: Applications
and Advancements will be a valuable resource for medical researchers, pharmaceutical manufacturers, healthcare personnel, and
academicians”-- Provided by publisher.
Identi�ers: LCCN 2021049654 (print) | LCCN 2021049655 (ebook) | ISBN 9781774630204 (hardback) | ISBN 9781774638774
(paperback) | ISBN 9781003180531 (ebook)
Subjects: MESH: Tissue Engineering--methods | Biocompatible Materials--therapeutic use
Classi�cation: LCC R857.T55 (print) | LCC R857.T55 (ebook) | NLM QS 525 | DDC 612/.028--dc23/eng/20211101
LC record available at https://lccn.loc.gov/2021049654
LC ebook record available at https://lccn.loc.gov/2021049655
ISBN: 978-1-77463-020-4 (hbk)
ISBN: 978-1-77463-877-4 (pbk)
ISBN: 978-1-00318-053-1 (ebk)

10. About the Editors
Rajesh K. Kesharwani, PhD, MTech
Nehru Gram Bharati (Deemed to be University),
Prayagraj, Uttar Pradesh, India
Rajesh K. Kesharwani, PhD, has more than 10 years of research and eight years
of teaching experience at various institutes of India, imparting bioinformatics
and biotechnology education. He has received several awards, including the
NASI-Swarna Jayanti Puruskar from The National Academy of Sciences
of India. He has supervised one PhD and more than 20 undergraduate and
postgraduate students for their research work, and has authored over 40 peer-
reviewed articles, 20 book chapters, and 11 edited books with international
publishers. He has been a member of many scientific communities as well
as a reviewer for many international journals. He has presented many papers
at various national and international conferences. Dr. Kesharwani received
his PhD from the Indian Institute of Information Technology, Allahabad, and
worked at NIT Warangal for two semesters. He has been a recipient of a
Ministry of Human Resource Development (India) Fellowship and a Senior
Research Fellowship from the Indian Council of Medical Research, India.
His research fields of interest are medical informatics, protein structure and
function prediction, computer-aided drug design, structural biology, drug
delivery, cancer biology, nanobiotechnology, and biomedical sciences.
Raj K. Keservani, MPharm
Faculty of B. Pharmacy, CSM Group of Institutions, Allahabad, India
Raj K. Keservani, MPharm, is a member of the Faculty of B. Pharmacy,
CSM Group of Institutions, Allahabad, India. He has more than 10 years
of academic experience from various institutes of India in pharmaceutical
education. He has published 30 peer-reviewed papers in the field of phar­
maceutical sciences in national and international journals, 30 book chapters,
two co-authored books, and 15 edited books. He is also active as a reviewer
for several international scientific journals. Mr. Keservani graduated with

11. vi About the Editors
a pharmacy degree from the Department of Pharmacy, Kumaun Univer­
sity, Nainital (Uttarakhand), India. He received his Master of Pharmacy
(MPharm) (specialization in pharmaceutics) from the School of Pharmaceu­
tical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, India.
His research interests include nutraceutical and functional foods, novel drug
delivery systems (NDDS), transdermal drug delivery/ drug delivery, health
science, cancer biology, and neurobiology.
Anil K. Sharma, PhD, MPharm
School of Medical and Allied Sciences, G. D. Goenka University, Sohna,
Haryana, India
Anil Kumar Sharma, PhD, MPharm, is an expert in the area of pharmaceu­
tics with a background in drug delivery. He has taught these subjects for
nearly 10 years at universities such as the Delhi Institute of Pharmaceutical
Sciences and Research, University of Delhi, and School of Medical and
Allied Sciences, G D Goenka University, India. Prior to taking up his current
role in 2018, Dr. Sharma served in academic positions such as Lecturer
(Pharmaceutics) at Delhi Institute of Pharmaceutical Sciences and Research,
University of Delhi. Dr. Sharma holds a PhD (Pharmaceutical Sciences)
from the University of Delhi; MPharm (Pharmaceutics) from the Rajiv
Gandhi Proudyogiki Vishwavidyalaya; and BPharm from the University of
Rajasthan, India.

12. Contributors.............................................................................................................ix
Abbreviations ...........................................................................................................xi
Foreword................................................................................................................. xv
Preface ..................................................................................................................xvii
1. Biomaterials in Tissue Engineering...............................................................1
Blessing Atim Aderibigbe and Shesan John Owonubi
2. Smart Polymeric Biomaterials in Tissue Engineering...............................59
Akhilesh Kumar Maurya and Nidhi Mishra
3. Gene Therapy in Tissue Engineering: Prospects and Challenges ............89
Soma Mondal Ghorai and Hardeep Kaur
4. Tissue Engineering and Application in Tropical Medicine .....................137
Sora Yasri and Viroj Wiwanitkit
5. Skin Tissue Engineering: Past, Present, and Perspectives ......................159
Sinem Selvin Selvi, Merve Erginer Hasköylü, and Ebru Toksoy Öner
6. Biomaterials in Bone and Muscle Regeneration ......................................203
Shesan John Owonubi, Eric G Ayom, Blessing A. Aderibigbe, and
Neerish Revaprasadu
7. Tissue Engineering in Reconstruction and Regeneration of
Visceral Organs...........................................................................................233
Soma Mondal Ghorai
8. Polysaccharides and Proteins‑based Hydrogels for Tissue
Engineering Applications ...........................................................................275
Roberta Cassano, Federica Curcio, Maria Luisa Di Gioia, Debora Procopio,
and Sonia Trombino
Index.....................................................................................................................309
Contents

14. Contributors
Blessing Atim Aderibigbe
Department of Chemistry, University of Fort Hare, Alice Campus, Eastern Cape 5700, South Africa
Roberta Cassano
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende,
Cosenza, Italy
Federica Curcio
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende,
Cosenza, Italy
Eric G Ayom
Department of Chemistry, University of Zululand, KwaDlangezwa, KwaZulu-Natal, South Africa, 3886
Soma Mondal Ghorai
Department of Zoology, Hindu College, University of Delhi, Delhi-110007, India
Maria Luisa Di Gioia
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende,
Cosenza, Italy
Merve Erginer Hasköylü
IBSB, Department of Bioengineering, Marmara University, 34722, İstanbul, Turkey
Hardeep Kaur
Department of Zoology, Ramjas College, University of Delhi, Delhi 110007, India
Akhilesh Kumar Maurya
Chemistry Laboratory Department of Applied Sciences, Indian Institute of Information Technology,
Allahabad, India
Nidhi Mishra
Chemistry Laboratory Department of Applied Sciences, Indian Institute of Information Technology,
Allahabad, India
Sudhanshu Mishra
Department of Advanced Science & Technology, Nims University Rajasthan, Jaipur-303121, India
Ebru Toksoy Öner
IBSB, Department of Bioengineering, Marmara University, 34722, İstanbul, Turkey
Shesan John Owonubi
Department of Chemistry, University of Zululand, KwaDlangezwa, KwaZulu-Natal 3886, South Africa
Debora Procopio
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende,
Cosenza, Italy
Neerish Revaprasadu
Department of Chemistry, University of Zululand, KwaDlangezwa, KwaZulu-Natal, South Africa, 3886

15. x Contributors
Sinem Selvin Selvi
IBSB, Department of Bioengineering, Marmara University, 34722, İstanbul, Turkey
Sonia Trombino
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende,
Cosenza, Italy
Viroj Wiwanitkit
Department of Community Medicine, Dr DY Patil University, Pune, India
Sora Yasri
Private Academic Practice, Bangkok, Thailand

16. Abbreviations
AAV adeno-associated virus
ADA adenosine deaminase ALL acute lymphoblastic leukemia ASC adipose-derived stem cells ASCs adipose stem cells BC bacterial cellulose BCP biphasic calcium phosphate CA cellulose acetate CAP cellulose acetate phthalate CEAs cultured epithelial autografts CMC carboxymethyl cellulose CMCS carboxymethyl chitosan CNC cellulose nanocrystals CNTs carbon nanotubes CPBs calcium phosphate biomaterials CPCs calcium phosphate cements CPTi commercially pure titanium CS chondroitin sulfate CSA chondroitin sulfate A CSGT cancer suicide gene therapy CT computed tomography DBCO dibenzyl cyclooctyne DEAE diethylaminoethyl dECM decellularized ECM DMAc dimethylacetamide DSM decellularized skeletal muscle EBs embryoid bodies EC ethyl cellulose ECM extracellular matrix ECs endothelial cells EGF epidermal growth factor ELRs elastin-like recombinamers EPCs endothelial progenitor cells ESC embryonic stem cells

17. xii Abbreviations
EXOs exosomes
FGF fibroblast growth factor
FNS fibronectins
GAGs glycosaminoglycans
GAM gene-activated matrix
GGT germline gene therapy
GI gastrointestinal
HA hydroxyapatite
HAC human artificial chromosomes
HCF heparin-conjugated fibrin
hKCs human keratinocytes
HPMC hydroxypropyl methylcellulose
HSV herpes simplex virus
IDD insulin-dependent diabetes
IEPs isoelectric points
IHC immunohistochemistry
IM intermediate mesoderm
INH isoniazid
IPNs interpenetrating networks
LCA Leber congenital amaurosis
LIF leukemia inhibitory factor
MC methyl cellulose
MeHA methacrylated HA
MM metanephric mesenchyme
MPS microphysiological system
MRI magnetic resonance imaging
MSCs mesenchymal stem cells
NaCMC sodium carboxymethyl cellulose
NAG N-acetyl-D-glucosamine
NGF nerve growth factor
NK natural killer
NMMO N-methylmorpholine-N-oxide
pDNA plasmid DNA
PEDOT poly(3,4-ethylendioxythiophene)
PEEk polyetheretherketone
PEG polyethylene glycol
PEO polyethylene oxide
PGA polyglycolic acid
PHAs polyhydroxyalkanoates

18. Abbreviations xiii
PLA polylactic acid
PLGA poly(lactide-co-glycolide)
PLL poly-L-lysine
PMMA polymethyl methacrylate
PIGS polyimmunoglobulin G scaffold
PRP platelet-rich plasma
PTMC poly(trimethylene carbonate)
PUs polyurethanes
PVA polyvinyl alcohol
PVP polyvinylpyrrolidone
PVPI povidone-iodine
RGD arginine-glycine-asparagine
RWM restrata wound matrix
SA sodium alginate
SCGT somatic cell gene therapy
SCID severe combined immunodeficiency
SDS sodium dodecyl sulfate
SF silk fibroin
SIS small intestinal submucosa
STZ streptozotocin
SVF stromal vascular fraction
TA tibialis anterior
TE tissue engineering
TEPS tissue-engineered pancreatic substitute
TK thymidine kinase
TNF tumor necrosis factor
TTCP tetracalcium phosphate
UCST upper critical solution temperature
VEGF vascular endothelial growth factor

20. Foreword
It is a matter of great pleasure for me to write foreword for this book, Tissue
Engineering: Applications and Advancements. Tissue engineering is a
specialized branch under biomedical engineering (bioengineering). Owing to
its outstanding advantages, tissue engineering is considered as an ultimately
ideal medical treatment to regenerate a patient’s own tissues and organs that
are entirely free of low bio-functionality and severe immune rejection.
Tissue engineering evolved from the �eld of biomaterials development
and refers to the practice of combining scaffolds, cells, and biologically
active molecules into functional tissues. It involves medical devices, tissue
replacement, repair, and regeneration. This book covers different applied
aspects in tissue engineering, such as for example, smart polymeric biomate­
rials, gene therapy, tissue engineering application in tropical medicines, skin
tissue engineering, bone and muscle regeneration, and tissue engineering in
reconstruction and regeneration of visceral organs.
The volume also provides an overview of the ef�cacy of different bioma­
terials employed in tissue engineering (such as skin regeneration, nerve
regeneration, arti�cial blood vessels, and bone regeneration) and varied
aspects of tissue engineering and gene therapy, their advantages and future
prospects as well as the challenges pertaining to the combination of the two.
This book also describe the bioengineering of visceral organs and the various
challenges that are faced in the �eld of tissue engineering and reconstructive
surgery.
All the contributing authors have provided advanced and rich informa­
tion that provide valuable insight for scientists, medical researchers, faculty,
and students working in the �eld of material and biomedical Sciences.
I congratulate the editors for bringing together experts of biomaterials
and tissue engineering and the authors for their excellent contributions.
—Prof. Krishna Misra
Honorary Professor, Indian Institute of Information Technology Allahabad,
Devghat, Jhalwa, Prayagraj 211015, Uttar Pradesh, India

22. Preface
This new book, Tissue Engineering: Applications and Advancements focuses
on the ever-growing and very important topics that give a state-of-art picture
of biomaterial and its application with the emphasis on its present, past, and
future. There have been significant developments in the field of biomaterial and
its advancement used in tissue engineering, and this volume highlights some
of the most important developments in the field. The book covers different
applied research involved in tissue engineering, biomaterial, smart polymeric
biomaterials, gene therapy, tissue engineering in reconstruction and regenera­
tion of visceral organs, skin tissue engineering, bone and muscle regeneration,
and application in tropical medicines.
With high demand for organs and tissues all over world, the research on
tissue engineering is of more concern, and this volume will be a valuable
resource for medical researchers, pharmaceutical manufacturers, healthcare
personnel, and academicians.
The present book includes eight chapters containing information about
the advanced research on biomaterial, and tissue engineering with different
applications for human welfare.
Chapter 1, entitled “Biomaterials in Tissue Engineering” authored by B.
A. Aderibigbe and S. J. Owonubi, focuses on biomaterials and their use to
replace the natural function of a defective organ/tissue, which can be clas­
si�ed as either synthetic (such as metals, polymers, ceramics) or biological
material (such as natural polymers, carbon-based nanomaterials). This
chapter gives an overview of the ef�cacy of different biomaterials employed
in tissue engineering (such as skin regeneration, nerve regeneration, arti�cial
blood vessels, and bone regeneration).
The role of smart polymeric biomaterials in tissue engineering is well
demonstrated by Nidhi Mishra and her coauthor in Chapter 2. Polymeric
biomaterials are one of the basics of tissue engineering. A wide range of
materials has been used in the medical �eld. The advancement of synthetic
and natural biomaterials has led to the development of stimuli responsive
biomaterials, known as “smart biomaterials.” These polymers have been
used in the development of various medical devices, arti�cial organs and
organ parts, targeted and controlled drug delivery with effect of speci�city
of biomaterials. More advanced and smart biomaterials will be developed

23. xviii Preface
in the future. This chapter is focused on development of smart biomaterials.
It discuses natural and synthetic polymers used in their preparation and the
most recent applications as biomaterials.
Chapter 3, “Gene Therapy in Tissue Engineering: Prospects and
Challenges,” written by Soma Mondal Ghorai and Hardeep Kaur, emphasizes
gene therapy holds great prospects for future clinical therapeutics as
it can be effortlessly united with tissue engineering and can repair the
damaged tissue at the cellular and molecular levels. This hybrid approach
of conjunction between tissue engineering and gene therapy provides the
optimum environment for therapeutic protein expression for regeneration of
cells and tissues; it thereby holds immense potential in areas of skin, bone,
and cartilage repair. The aim of this chapter is to provide readers with the
varied aspects of tissue engineering and gene therapy, their advantages, and
future prospects as well as the challenges pertaining to the combination of
the two.
Chapter 4, “Tissue Engineering and Application in Tropical Medicine,” is
authored by Sora Yasri and Viroj Wiwanitkit, with reference to application in
tropical medicine.The modeling of the natural tissue growth and development
is the basic concept and process of the novel tissue engineering. Introducing
the new technology such as tissue engineering for the management of tropical
diseases becomes a new challenging issue in tropical medicine.
Since skin is the outermost layer of human body and protects it from
external hazards, any damage of this tissue, like wounds and burns may
result in serious health problems. Chapter 5, “Skin Tissue Engineering:
Past, Present, Future Perspectives” is written by Sinem Selvin Selvi and his
colleague. Skin tissue engineering applications are found to be one of the
�rst ones in history because of their importance and urgency. Here in this
chapter, the authors discuss the history of tissue engineering, wound healing
methods, skin substitutes, materials used with the current and promising
applications.
The details about regeneration or repair of skeletal muscle tissue or bone
are well described in Chapter 6, “Biomaterial in Bone and Muscle Regen­
eration,” by Shesan John Owonubi and his associates. Biomaterials have
been employed over the years in the regeneration of bones and muscles.
The regeneration or repair of skeletal muscle tissue or muscles is necessary
when innate muscle or bones cannot on initiate their own repair. This chapter
introduces biomaterials and the advancement of biomaterials, is it indicates
exactly what both bone and muscle regeneration involves, and with numerous
research �ndings reported, it dwells on biomaterials in bone regeneration.

24. Preface xix
Chapter 7, “Tissue Engineering in Reconstruction and Regeneration of
Visceral Organs,” by Soma Mondal Ghorai and Sudhanshu Mishra, well
describes reconstruction and regeneration of visceral organs and limitations
due to imperding shortage of donor tissues/organs and low rates of successful
surgeries, mainly in pediatric patients. Tissue engineering is an innovative
�eld that combines engineering with biology and is the answer to the above
mentioned limitation. This strategy helps to reconstruct and regenerate
damaged and diseased tissues/organs. This chapter discusses bioengineering
of visceral organs and the various challenges that are faced in the �eld of
tissue engineering and reconstructive surgery.
Chapter 8, “Polysaccharides and Proteins Based Hydrogels for Tissue
Engineering Applications,” is written by Sonia Trombino and her colleagues,
in very precise manner with scienti�c fact. The authors describe speci�cally
the natural biomaterials based on polysaccharides and proteins used in tissue
engineering and their most important applications.
The present volume, Tissue Engineering: Applications and Advance ­
ments, provides detailed information on application of tissue engineering
and its different components especially different types of biomaterials, and
their applications in various types of tissue/organ regeneration. This book
provides advanced research and a wealth of information that will be valuable
to medical scientists, researchers, academicians, and students.

26. CHAPTER 1
Biomaterials in Tissue Engineering
BLESSING ATIM ADERIBIGBE
1*
and SHESAN JOHN OWONUBI
2
1
Department of Chemistry, University of Fort Hare, Alice Campus,
Eastern Cape 5700, South Africa
2
Department of Chemistry, University of Zululand, KwaDlangezwa,
KwaZulu-Natal 3886, South Africa
*
Corresponding author. E-mail: [email protected]
ABSTRACT
Biomaterials used to replace the natural function of a defective organ/tissue
can be classified as either synthetic (such as metals, polymers, and ceramics)
or biological materials (such as natural polymers and carbon-based nano­
materials). These materials interact with the biological system. They can be
modified to enhance their biostability, biocompatibility, physicochemical
properties, mechanical properties, and their interaction with the biological
system. Biomaterials have been employed in tissue engineering as regen­
erative scaffolds, for bone regeneration, as implants, etc. In the selection of
biomaterials for tissue engineering, several factors are taken into consider­
ation such as the nature and properties of the biomaterial, etc. This chapter
gives an overview and efficacy of the different biomaterials employed in
tissue engineering (such as skin regeneration, nerve regeneration, artificial
blood vessels, and bone regeneration).
1.1 INTRODUCTION
Tissue engineering is an interdisciplinary field, which is focused on the
regeneration of functional human tissues/organs (Keane and Badylak, 2014;
Tur, 2009; O’brien, 2011). These materials are developed for the regeneration

27. 2 Tissue Engineering
of tissues/organs and are in continuous contact with the body tissue cells
(Keane and Badylak, 2014). These biomaterials are combined with cells and
bioactive molecules (Keane and Badylak, 2014; Tur, 2009). Biomaterials
are classified as biological and synthetic depending on their sources. A
biomaterial scaffold provides shape, mechanical and structural support with
a tailored surface suitable for cell attachment, cell–cell interaction, and cell
proliferation and differentiation (Khan and Tanaka, 2017). Biomaterials are
placed within the body and as such, they must be biocompatible, nontoxic,
and have adequate physicochemical and mechanical properties (Tur, 2009).
They have been used in various biomedical applications such as skin regen­
eration, bone regeneration, tissue/organ regeneration, dental applications,
etc. The success of a biomaterial developed for implantation is determined
by the host response to the scaffold material after years of implantation.
There are advantages and disadvantages for each biomaterial used in tissue
engineering. This chapter will give an overview of biomaterials used in
tissue/organ regeneration.
1.2 CLASSES OF BIOMATERIALS
Biomaterials used in tissue engineering are classified as synthetic bioma­
terials and biological/natural biomaterials (Figure 1.1) (He and Lu, 2016).
Synthetic biomaterials are further classified as polymers, composites, metals,
ceramics, and glasses (He and Lu, 2016; Nigam and Mahanta, 2014). The
biological/natural biomaterials are further classified as carbon-based mate­
rials, cell, tissue, protein, peptides, etc. (Bhat and Kumar, 2012; Ha et al.,
2013). Some biomedical-based scaffolds are prepared from the combination
of synthetic and natural biomaterials resulting in scaffolds with excellent
physicochemical properties (Mengyan et al., 2005; Galler et al., 2018). The
biomedical applications of biomaterials include dental, orthopedic, skin
regeneration, etc. (Galler et al., 2018).
1.2.1౰ SYNTHETIC-BASED BIOMATERIALS USED IN TISSUE
ENGINEERING
1.2.1.1 METALS
Metals are used in the design of scaffolds for implantation due to their unique
feature such as excellent strength. However, they are prone to corrosion

28. 3 Biomaterials in Tissue Engineering
resulting in the release of metal ions that cause toxic reactions and their poor
biocompatibility limits their use as implants (Saini et al., 2015). Metal-based
biomaterials are used for the development of biomedical devices for ortho­
pedic, artificial organs, dental, bone applications, etc. (Bose et al., 2012;
Xiao et al., 2012). Some of the metals used are Ti- and Co-based alloys,
stainless steel, magnesium, and iron and the metal-based nanoparticles used
are Ag, Cu, Au, or Co nanoparticles (Prasad et al., 2017; Yoo et al., 2008).
FIGURE 1.1 Classification of synthetic biomaterials and biological/natural biomaterials.
w­ä x­ä w­ä w­ä w�p� ���‡�–�ƒ�Ž­æ�„�ƒ�•�‡�†� ���‹�‘�•�ƒ�–�‡�”�‹�ƒ�Ž�•� �ˆ�‘�”� ���‘�•�‡� �ƒ�•�†� ���‘�‹�•�–�
Replacement
Selected metals such as surgical cobalt–chromium (Co–Cr) alloys, stain less steel (316L), and titanium (Ti) alloys are commonly used metals for
fracture fixation and bone remodeling (Prasad et al. , 2017). They exhibit
long-term stability resulting from their low corrosion, excellent mechanical
properties, and friction. Surgical stainless-steel alloys (316L) made with
varying amounts of nickel, iron, and chromium have been used in the
­

29. 4 Tissue Engineering
manufacture of prostheses (Yoo et al. , 2008). The low carbon content in
surgical stainless steel reduces corrosion and metal allergic reactions (Yoo
et al., 2008). However, it is prone to stress corrosion and cracking. Its use
is often limited in biomedical application where strength is not required
for a prolonged period (Pruitt and Chakravartula, 2012). Stainless steels
are less expensive when compared to other alloys. However, some metals
lack a biologically active surface that can induce osteointegration or prevent
infections resulting in more research which involves the coating of implants.
In the selection of coatings for metal implants used for bone replacement,
factors such as biocompatibility, the capability to induce osteoblasts, good
mechanical stability, and antimicrobial activity must be considered (Godbole
et al., 2016; Kiel et al., 2008). Metal implants are used for artificial hip
joints, bone plates, spinal fixation devices, and artificial dental roots. The
mechanical biocompatibility of metals used for implants is measured by
Young’s modulus, which describes the response of a material to stress and
strain (Niinomi and Nakai, 2014). Metals with Young’s modulus, equal to
that of the bone, are ideal for metallic implants, thereby reducing the stress
shielding effect (Niinomi and Nakai, 2014).
Titanium alloys have been developed for bone replacement by several
researchers. They are employed in the development of orthopedic devices
such as knee and hip implants because of their excellent mechanical proper­
ties and resistance to infections. However, there have been concerns about
their long-term effect on human health. Powderized titanium–tantalum alloy,
which is highly biocompatible for bone replacements, was prepared using
selective laser melting by blending jagged tantalum powder with titanium
microspheres powder. The stress shielding effect, which is common in
metal-based implants, when they are too elastic, thereby transferring insuf­
�cient loads to the neighboring bones, was not observed in the powderized
titanium-tantalum alloy indicating their potential to improve patient care
(Nikels, 2016). Sumitomo et al. investigated the biocompatibility of low
rigidity titanium alloy (Ti-29Nb-13Ta-4.6Zr) in bone plate �xation in vivo
using rabbit (Sumitomo et al., 2008). Experimental fractures were made
in rabbit tibiae and �xed using the titanium alloy. It was compared to the
conventional bone plates of SUS316L and Ti-6Al-4V. The diameter of the
tibia bone was increased in the titanium alloy indicating bone remodeling
when compared to SUS316L and Ti-6Al-4V. The elastic modulus of the bone
plate in�uenced bone tissue reaction to the bone plate �xation. The elastic
modulus of the titanium alloy was 58 GPa when compared to SUS316L
and Ti-6Al-4V which was 161 GPa and 108 GPa, respectively. The low

30. 5 Biomaterials in Tissue Engineering
rigidity of the titanium alloy delayed the bone atrophy and enhanced bone
reorganization due to the mechanical stress, which is clinically useful for
long-term implantation in aged patients and high-risk patients with severe
complications (Sumitomo et al., 2008). Nakai et al. (2011) also investigated
titanium alloys by self-adjustment of Young’s modulus.
Young’s modulus of titanium alloys via deformation-induced phase
transformation was investigated. β-type titanium alloys exhibiting defor­
mation-induced ω-phase transformation in which only Young’s modulus
of the deformed part was increased while the nondeformed part remains
low was developed making them useful for spinal �xation devices (Nakai
et al., 2011). In the development of spinal �xation implants, implants with
high rigidity result in the stress shielding effect. Materials with low Young’s
modulus are often preferred for healthy spine formation (Shi et al., 2012;
Wang et al., 2013; Nakai et al., 2015). Implants are usually subjected to
bending during surgery in order to obtain the curvature of the spine indi­
cating that the implant must have a high Young’s modulus (Nakai et al.,
2011, 2015; Noshchenko et al., 2011). The degree of spring back of the
implant is in�uenced by the strength and the Young’s modulus of the spinal
�xation implant. Higher Young’s modulus will exhibit a smaller spring back
(Niinomi and Nakai, 2011). The aforementioned factor has resulted in the
development of deformation-induced ω-phase transformation in β-type Ti
alloys in which the deformed material exhibits high Young’s modulus, while
the nondeformed part exhibits low Young’s modulus thereby satisfying the
con�icting requirement of Young’s modulus (Nakai et al., 2015; Zhao et al.,
2011a, 2011b, 2012a, 2012b; Liu et al ., 2014).
In magnetic resonance imaging used for the diagnosis of various diseases,
metallic orthopedic devices implanted in the human body cause distortions
of the images of the organs and tissues around the implant thereby hindering
correct diagnosis. The distortion of the images formed is due to the differ­
ence in magnetic susceptibilities between living tissues and metallic mate­
rials (Nakai et al., 2015). This magnetic susceptibility of Ti is lower when
compared to ferromagnetic iron and Co. However, it is higher than water.
Zirconium, Zr, exhibits a smaller magnetic susceptibility of 1.3 × 10
−6
cm
3
g
−1
and some of its alloys have been employed for implants (Nakai et al.,
2015; Kondo et al ., 2011; Suyalatu et al., 2010, 2011).
Cobalt-chromium-based alloys have been used for prosthetic arthroplasty.
Their unique mechanical properties make them suitable for total hip and knee
joint arthroplasty (Bal et al., 2007). Cobalt is not a biocompatible biomaterial.
However, the addition of 15–30% chromium creates a passivating oxide �lm

31. 6 Tissue Engineering
which is stable in the body (Haynes et al., 2000; Rabiei, 2010). CoCr alloys
exhibit good corrosion resistance. However, they are dif�cult to fabricate due
to their brittle nature (Haynes et al., 2000; Rabiei, 2010). Ogawa et al. seeded
CoCr-based alloy implant surface with rabbit marrow mesenchymal cells
and left the other side unseeded (Ogawa et al., 2012). The CoCr implants
were then implanted in rabbit bone defects. After 3 weeks, the implantation
revealed new bone formation. However, no bone formation was detected
on the unseeded side indicating that early �xation of CoCr-based alloy can
result in cementless �xation in various joint arthroplasties (Ogawa et al.,
2012). The wear resistance of Co alloys is higher when compared to Ti alloys
and stainless steel alloys (Niinomi, 2002). The head of the joint in arti�cial
hip joints is subjected to wear. To increase the resistance of Co alloy to wear,
it has been overcome by dispersing carbide in Co alloys and by transforming
the metastable γ phase to the ε martensitic phase (Niinomi, 2008). Grand�eld
et al. studied in vivo response to the free-form fabricated CoCr implants
with and without hydroxyapatite (HA) plasma-sprayed coatings in the tibial
metaphysis of New Zealand white rabbits. After 6 weeks, the plasma-sprayed
coating of HA to implants enhanced in vivo interaction suggesting resorption
and increased interface strength (Grand�eld et al., 2011). The coating of
the implants also has the potential to prevent the release of metallic ions at
the implant surface, which can induce periprosthetic bone loss resulting in
implant failure (Haynes et al., 2000). The addition of a biocompatible layer,
such as HA, may act as a buffer to prevent the release of biologically harmful
metallic ions. There have been reports of high concentration of chromium
and cobalt in blood after implants resulting in neurological problems (Clark
et al., 2014; Bradberry et al., 2014; Campbell and Estey, 2013). Cobalt from
implants is found in two forms inside the body. The divalent (Co
II
) state is
more toxic when compared to the metallic (Co0) state. Chromium has three
states which are metallic (Cr
0
), trivalent (Cr
III
), and hexavalent (Cr
VI
). Triva­
lent chromium is mildly toxic, while the hexavalent chromium compounds
are strongly carcinogenic and can cause kidney damage (Swiatkowska et al.,
2018).
The most commonly used steel alloys are 316 and 316L grades. 316L
steel alloy is preferred for implant fabrications because of its reduced carbon
content which decreases the possibility of chromium carbide formation
which can result in intergranular corrosion, thereby making it resistant to
corrosion by physiological saline. However, it is prone to corrosion by chlo­
ride ions and reduced sulfur compounds (Manivasagam et al., 2010; Xu et
al., 2006). Microorganism on the metal surface can affect the concentration

32. 7 Biomaterials in Tissue Engineering
of the electrolytic constituents, pH, and oxygen levels negatively (Maniva­
sagam et al., 2010; Xu et al., 2006). Furthermore, studies have revealed that
over 90% of the failure of 316L stainless steel implants result from pitting
and crevice corrosion attack (Sivakumar et al., 1995). Ti alloy implants have
been reported to suppress the development of in�ammation when compared
to stainless-steel alloy implants (Akyol et al., 2017).
1.2.1.1.2�p���‡�–�ƒ�Ž­æ�„�ƒ�•�‡�†� ���‹�‘�•�ƒ�–�‡�”�‹�ƒ�Ž�•� �ˆ�‘�”� ���‡�•�–�ƒ�Ž� ���’�’�Ž�‹�…�ƒ�–�‹�‘�•
Metal-based biomaterials have been used as dental implants such as artificial
tooth root which is inserted in the jaw for tooth replacement (Duraccio et
al., 2015). The implant is surgically inserted and the shape of the implant
varies. The formation of a strong bond between the implant and jawbone is
known as osseointegration, which anchors the implant by the development
of bone tissue around the implant. However, it is important to mention that
an absolute bone-to-implant contact does not occur (Duraccio et al., 2015).
There are several factors that influence osseointegration such as the medical
state of the patient, habits, for example, smoking, the properties and the
design of the implant, quality of the bone, radiation therapy, and bacterial
contamination, etc. (Duraccio et al., 2015; Goutam et al., 2013; Park et al.,
2006). The common metals, which are used for dental implants, are pure
titanium and the alloy of titanium and zirconium dioxide. The unique proper­
ties of titanium, which include its nontoxicity, biocompatibility, resistance to
corrosion, good fatigue strength, controlled degradability, and modulus of
elasticity, are useful for dental applications (Duraccio et al., 2015). Titanium
implant surfaces are usually modified by roughening and coating in order to
enhance the rate of osseointegration (Duraccio et al., 2015).
Methods used for creating a rough surface of titanium implant include
titanium plasma spraying, anodization, blasting with ceramic particles, and
acid etching (Bauer et al., 2013). In titanium plasma-spraying method, tita­
nium powder is injected onto the implant resulting in the formation of a �lm
with enhanced surface area and tensile strength (Jemat et al., 2015). Knabe et
al. investigated a titanium surface having a porous titanium plasma-sprayed
coating on rat bone marrow cells. The titanium surface enhanced facilitated
rat bone marrow cells growth with high cell density which indicates that it
is suitable for dental implants (Knabe et al., 2002). Hung et al. developed
dental implants by plasma-sprayed HA coating on titanium (Ti-6Al-4V ELI)
surfaces with a coating thickness of approximately 120 µm. The implants

33. 8 Tissue Engineering
were uniformly covered by the HA coating and exhibited high biocompat­
ibility (Hung et al., 2013). Eom et al. also showed that the HA coating of
implants shortens the time of bone healing at poor bone quality sites and
they are useful for early loading after the placement of the implant (Eom et
al., 2012). Huang et al. reported the antibacterial effect of surface treatment
of titanium. ZrO
2
–silver (Ag) and ZrO
2
–copper (Cu) coatings were sprayed
onto titanium surface resulting in an improved antibacterial performance of
the coated titanium implant when compared to the pure titanium implants
(Huang et al., 2013). Titanium surface is also roughened by blasting with
hard ceramic particles. However, particles must be stable, biocompatible,
and should not interfere or hinder the osseointegration of the implants (Dura­
ccio et al., 2015). Alumina has been used for blasting of titanium surfaces
due to its insolubility in acid.
Gehrke et al. evaluated the effects of aluminum and titanium dioxide
(TiO
2
) microparticles for blasting during the sandblasting acid surface
treatment in titanium dental implants using a rabbit tibia model (Gehrke et
al., 2016). The residual blasting titanium particles on the surface of dental
implants did not affect the osseointegration of the titanium dental implants
but resulted in high osteoconductivity and good bone formation (Gehrke et
al., 2016). Diniz et al. characterized titanium surfaces blasted with aluminum
oxide particles followed by the treatment with a hydro�uoric acid-based solu­
tion. The treatment resulted in depressions up to 10 µm on the homogeneous
surfaces (Diniz et al., 2005). After the chemical treatment, the surfaces were
smooth, homogeneous with a regular microtopography when compared to
surfaces without chemical treatment which exhibited irregular morphology
and depressions (Diniz et al., 2005). In another report by Gehrke et al.,
in vivo host response of a surface sandblasted with particles of titanium
oxide followed by acid etching was studied. The implants were placed in
the tibia of six rabbits. The surface of the implants was homogeneous with
uniform irregularities and small residues of the metal oxides. Reduced risk
of contamination by the residual metal oxide from the blasting procedure
was observed. The titanium oxide blasting produced an enhanced effect on
the osseointegration and on the biomechanical features of the implants. The
residual blasting titanium particles on the surface of dental implants did not
affect the osseointegration of titanium dental implants (Gehrke et al., 2015).
Etching with strong acids is another approach for the roughening of tita­
nium dental implants, which creates micropits on the surface of the implant
(Duraccio et al., 2015). It also enhances osseointegration (Herrero-Climent
et al., 2013; Okazaki et al., 2017). The use of dual acid etching produces a

34. 9 Biomaterials in Tissue Engineering
microrough surface and enhances the osteoconductive process resulting in
bone formation directly on the surface of the implant (Al-Radha and Sahib,
2016; Giner et al., 2017). The titanium anodized surface results in micro- and
nanosurfaces, which increases blood clot retention and favors osseointegra­
tion (Traini et al., 2018). The anodization process is complex and is in�u­
enced by factors such as the concentration of acid used, the density of the
current, the composition, and the temperature of the electrolyte (Traini et al.,
2018; Shayganpour et al., 2015).
In titanium surface coating, different methods have been developed such
as sol–gel coating, plasma spraying, sputter deposition, etc. Hyun-Pil et al.
evaluated coated TiO
2
nanotube with HA by the sol–gel process on osteo­
blast-like cell and bone formation in rat tibia. Cell viability was studied after
1, 3, and 7 days of incubation in vitro. Implants were inserted into the tibia of
rats. After 7 days of implantation, there was high cell viability on the coated
TiO
2
nanotube with enhanced hydrophilicity and improved osseointegration.
The �ndings suggest that HA-coated TiO
2
nanotube can be used as dental
implants (Lim et al., 2018). Catauro et al. coated commercially pure titanium
(CPTi) grade 4 substrates by a sol–gel process with poly(ε-caprolactone)
(PCL) and silica.
Signi�cant HA nucleation was observed on the surface of the coated
substrates after soaking in a �uid simulating human blood plasma. The coating
improved the bioactivity and biocompatibility of the implants (Catauro et al.,
2018). The coating with PCL produced a crack-free surface (Catauro et al.,
2018; Teng et al., 2014). Harle et al. developed sol–gel coatings on titanium
substrates with various compositions under controlled processing condi­
tions and evaluated the biological ef�cacy of the coatings. In vitro studies
on primary human osteoblast cells revealed signi�cant proliferation and
attachment on the sol–gel-coated surfaces when compared to the uncoated
titanium (Harle et al., 2006).
Zirconia has also been used to fabricate dental implants because of its
properties such as tooth-like color, excellent mechanical properties, and
biocompatibility. The use of zirconia prevents complication such as gingival
recession (Depprich et al., 2008; Piconi and Maccauro, 1999). Despite its
excellent mechanical properties, its long-term durability is uncertain. There
have been reports of its failure in vivo caused by low-temperature degradation
or aging (Lughi and Sergo, 2010). In the presence of water, there is a slow
transformation from the tetragonal into the monoclinic phase resulting in its
progressive deterioration. The results of the aging process are surface and
strength degradation with grain pullout and microcracking (Lughi and Sergo,

35. 10 Tissue Engineering
2010). Some of its physical and mechanical properties include low thermal
conductivity, favorable fracture resistance, wear and corrosion resistance,
and high �exural strength (900–1200 MPa) (Cionca et al., 2016). Thoma et
al. placed zirconia dental implants (BPI, VC, ZD) in six dogs. The implant
systems demonstrated signi�cant loss of marginal bone between the baseline
and the crown insertion ranging between 0. 29 and 0.80 mm. The zirconia
implants were prone to fracture prior to and after loading with one-piece
zirconia implants when compared to the two-piece zirconia implants and
titanium implants (Thoma et al., 2015). The fracture was in�uenced by the
implant design. Gahlert et al. reported that zirconia implants with a diameter
of 3. 25 and 4 mm are prone to fracture. The fractured implants were located
at the anterior side of the maxilla and the mandibular. The fracture was caused
by strong bruxism. Mechanical overloading also caused the fracture of the
implants. The proper handling of the material, the surface modi�cation, and
the design had an impact on the strength of zirconia (Gahlert et al., 2011).
Bormann et al. compared zirconia implants with sandblasted and acid-
etched titanium implants. All implants had a diameter of 4.1 mm and a length
of 10 mm. The zirconia implants properties were similar to the roughened
titanium surface. There were no statistically signi�cant differences between
the two materials after a healing period of 4 and 12 weeks (Bormann et
al., 2011). Hempel et al. evaluated Saos-2 cells on either a sandblasted
or a sandblasted/etched zirconia and compared with a sandblasted/etched
titanium. A high adherent of cells after 24-h incubation on zirconia when
compared with titanium was observed. The rate of cell proliferation after
48 h was high with zirconia when compared with titanium. The result indi­
cated that zirconia has a signi�cant effect on cell adhesion, proliferation,
and differentiation when compared with titanium (Hempel et al., 2010).
Chung et al. evaluated and compared the osseointegration in rabbit tibiae
of smooth and roughened powder injection-molded zirconia implants with
or without (Ti, Zr)O
2
surface coatings. The coatings changed the surface
topography and chemical composition of the zirconia implants. The smooth
and roughly coated zirconia implants revealed enhanced bone to implant
contact when compared to the uncoated implants. The roughly coated and
uncoated implants exhibited high mechanical anchorage (Chung et al.,
2013). Montero et al. evaluated titanium and zirconia implants placed into
fresh extraction sockets in beagles without oral hygiene attention or a soft
diet during postoperative healing. The implant failure rate for the zirconia
implants was 3.5 times higher when compared to the titanium implants.
The zirconia implants were characterized by a less favorable topography, a

36. 11 Biomaterials in Tissue Engineering
signi�cantly lower roughness (Ra = 0.85 ± 0.04 pm), negative skewness of
the surface pro�le (–1.56 ± 0. 27), and higher kurtosis (7.88 ± 1.99) (Montero
et al., 2015). Schliephake et al. reported that after 4 weeks, the mean bone
to implant contact of sandblasted and sandblasted/etched zirconia surfaces
was comparable to sandblasted/etched titanium surfaces in vivo. After 13
weeks, there was a continuous increase in the mean bone to implant contact
in the titanium group. However, the mechanical anchorage was signi�cantly
lower in the zirconia implant when compared to the titanium implants after
4 weeks (Schliephake et al., 2010). However, it is important to mention that
most of the clinical studies on zirconia implants are short term indicting that
there is a need for substantial results from long-term clinical trials. More
research to prevent the aging, improve the structure, surface properties, and
osseointegration of zirconia implant is needed.
1.2­ä w­ä w­ä y�p� ���‡�–�ƒ�Ž­æ�„�ƒ�•�‡�†� ���‹�‘�•�ƒ�–�‡�”�‹�ƒ�Ž�•� �ˆ�‘�”� ���ƒ�…�‹�ƒ�Ž� ���‡�…�‘�•�•�–�”�—�…�–�‹�‘�•
The application of metal-based biomaterials for the restoration of the shape
and function of the maxillofacial region has been reported. Metals used for
maxillofacial surgery require specific mechanical properties such as good
tensile strength, shear stress, elasticity, and yield strength (Pacifici, 2016).
The commonly used metals for maxillofacial surgery are titanium, gold, stain
less steel, and vitallium. 3D-printed titanium TRUMATCH
®
maxillofacial
implants have been designed for jaw and facial reconstruction (Goble, 2017).
­
Clinical studies reported that the reconstruction of maxillectomy defects and
through cheek defects using a titanium mesh in combination with folded-free
anterolateral thigh flap are a feasible approach with high success rate and a
low incidence of complications (Wu et al., 2016). Titanium is chemically inert
with little hypersensitive reaction, it is easily trimmed, it resists mechanical
strains, and it maintains a proper shape (Wu et al., 2016). However, there is a
risk of infection after reconstruction. The use of titanium mesh implantation
for skeletal reconstruction after maxillectomy is beneficial for fragile and
aged patients (Wu et al., 2016). Costan et al. studied the efficacy of titanium
mesh panels for reconstructing the craniofacial skeleton between January
2015 and December 2017. The study on 26 patients in 20 trauma-related
cases and six cases of postablative defects when titanium mesh was covered
using locoregional flaps revealed restoration of facial dimensions (Costan et
al., 2018). The use of titanium mesh resulted in a proper reconstruction of the
orbital floor, without complaints of postoperative diplopia. The mesh was

37. 12 Tissue Engineering
not exposed postoperatively (Costan et al., 2018). There are other reports on
the application of titanium implant for facial reconstruction (Crainiceanu et
al., 2016; Yi et al., 2012; Gear et al., 2002; Schön et al., 2006). The titanium
mesh is a malleable system that provides good support where there is a lack
of bone support (Sixto et al., 2016). Nasal reconstruction can be performed
using titanium mesh and external skin reconstruction without the repair of
the intranasal lining, thereby decreasing operation time. However, titanium
mesh is not suitable for patients who had prior postoperative radiotherapy
(Kao et al., 2017).
Vitallium mesh has also been used for facial reconstruction as a replace
ment for the bony skeleton without complications or morbidity (Sengezer
and Sadove, 1992). However, its use is dependent on its rigid �xation
(Sengezer and Sadove, 1992). Study on animal models showed that it is
less biocompatible when compared to titanium which is characterized by an
increase in the accumulation of metal ions in the implant surrounding area
(Paci�ci, 2016; Jakobsen et al., 2010).
1.2.1.2 POLYMERS
Polymers used in regenerative medicine can be classified as natural or
synthetic polymers. They represent the most significant biomaterials
resulting from their inert nature. Their physicochemical properties are easily
tailored based on their applications. Synthetic polymers are characterized by
good mechanical properties. However, their biocompatibility is poor when
compared to natural polymers. Polymers have been used for the regeneration
of bone, cartilage, skin, etc.
1.2.1.2.1 Polymers Application for Skin Regeneration
Polymer-based materials such as hydrogels, films, composites, fibers, etc.,
have been developed for skin regeneration. Severe skin damage resulting
from injury can be life-threatening. Skin regeneration involves a combina
tion of complex biological processes such as inflammation, proliferation,
and remodeling. Despite the availability of a wide range of wound dress
ings, wound healing still remains a clinical challenge globally among the
aged people, people with chronic conditions, burn patients, etc. (Han and
Ceilley, 2017).
­
­
­

38. 13 Biomaterials in Tissue Engineering
Polymers have been designed for skin regeneration in chronic and acute
wounds. Synthetic polymers such as polyethylene glycol (PEG), poloxamer,
pluronic, polylactic acid (PLA), polyacrylic acid, PCL, polyvinylpyrrolidone,
and polyvinyl alcohol (PVA) have been used as biomaterials to develop
hydrogels for skin regeneration (Mogoşanu and Grumezescu, 2014; Sell et
al., 2010). Synthetic polymers have good mechanical properties but some
of them are not biocompatible. PEG-based materials have been designed
for skin regeneration and they are characterized by rapid wound closure and
bleeding control, tissue adhesion, controlled degradability, and elastomeric
mechanical properties suitable for skin regeneration (Chen et al ., 2018b).
They are also characterized by good biocompatibility, which provide
adequate moisture to the wound bed with gradual resorption and replacement
by structured skin tissue suitable for the management of complicated wounds
(Venzin and Jacot, 2016). Incorporating chemokines onto PEG-based wound
dressing materials is useful for skin regeneration in chronic wounds resulting
in the reduction of in�ammation, increased granulation tissue formation,
vascularization, and wound closure (Lohmann et al., 2017). PEG-based
wound dressings have been designed as injectable gels with excellent pH
response, rapid gelation time, excellent deformability, and good mechanical
strength with the capability to stop bleeding in severe traumatic injuries
(Zhang et al., 2018). PEG-based scaffold has also been designed as bilayered
microenvironment, which supports �broblasts on the top and keratinocytes
at the bottom (Tsao et al ., 2014).
The combination of polycaprolactone with poloxamer to develop nano­
�bers resulted in materials with excellent mechanical strength and good
biocompatibility. The nano�ber acted as a protective barrier and integrated
with the host skin tissue. The tensile strength and the hydrophilic property
of the nano�bers provided good resilience and compliance to movement as
a skin graft (Pan et al., 2014). Polycaprolactone scaffolds incorporated with
ZnO nanoparticles can act as skin substitute materials, thereby promoting
wound healing. It enhances cell adhesion and migration without the forma­
tion of a scar (Augustine et al., 2014). The combination of PEG, poly­
caprolactone, and natural polymers to produce scaffolds further enhanced
epithelial differentiation. In vivo ef�cacy studies on a second-degree burn
wound model in Wistar rats revealed an increased rate of wound contraction
(Bhowmick et al., 2018).
Polycaprolactone-based materials used to develop engineered skin
substitute exhibited good cell adhesion and inhibited early shrinkage of the
material after seeding with �broblasts (Bhowmick et al., 2018). Nano�bers

39. 14 Tissue Engineering
prepared from a combination of chitosan and PCL were evaluated in vivo on a
mouse cutaneous excisional skin defect model. An increased wound healing rate
and complete wound closure were observed when compared to the commercially
available occlusive dressing (Levengood et al. , 2017).
Other synthetic polymers have also been used to develop materials for
skin regeneration. However, due to the poor biocompatibility of synthetic
polymers, they are combined with natural polymers for enhanced biocom
patibility. Polyvinylpyrrolidone has been combined with gelatin for skin
grafts applications.
The skin grafts were biocompatible and biodegradable resulting from
gelatin, while polyvinylpyrrolidone is an inert component with good water
af�nity (Kenawy et al., 2014). PVA is one of the oldest synthetic polymers
used to develop materials for wound dressing due to its good biocompat
ibility (Kenawy et al., 2014). However, materials developed from PVA do
not have suf�cient elasticity and exhibit poor hydrophilicity properties when
used as a wound dressing polymeric material (Yang et al., 2010). PLA-based
nano�bers have been found to be effective in wound healing by protecting
the wound, preventing the loss of moisture, enhancing the proliferation of
cells, and the closure of large wounds (Sundaramurthi et al., 2014).
w­ä x­ä w­ä x­ä x�p� ���‘�Ž�›�•�‡�”�•� ���’�’�Ž�‹�…�ƒ�–�‹�‘�•� �ˆ�‘�”� ���‘�•�‡� �ƒ�•�†� ���ƒ�”�–�‹�Ž�ƒ�‰�‡�
2‡ß‡‡”ƒ–‹‘
­
­
The design of polymer-based scaffolds for bone and cartilage regeneration
is a potential approach, which can eliminate the problems of supply scarcity,
potential pathogen transfer, and immunogenicity (Pina et al., 2015). In the
design of scaffolds for bone and cartilage regeneration, the physicochemical,
structural, and biological properties of the scaffolds should mimic the natural
extracellular matrix (ECM) thereby providing desired environment for cell
proliferation, differentiation, and bone or cartilage regeneration (Chan and
Leong, 2008). The porosity of the scaffolds also plays a major role in main­
taining the diffusion of nutrients and metabolites (Loh and Choong, 2013).
Among the materials used for the preparation of scaffolds for bone and carti­
lage regeneration, polymers and their composites are the most promising
candidates due to their good biocompatibility and biodegradability (Puppi
et al., 2010).
Synthetic polymers such as PLA, polyglycolic acid, and polycaprolactone
have been used for the development of scaffolds for bone tissue regeneration.

40. 15 Biomaterials in Tissue Engineering
However, synthetic polymers are combined with inorganic materials and
bioactive molecules for optimized osteogenic outcomes (Puppi et al., 2010).
The synthesis of synthetic polymers is via controlled conditions resulting
in good mechanical properties, controlled rate of degradation, and micro­
structure, which are reproducible by the incorporation of selected functional
groups (Ma et al., 2016). Aliphatic polyesters such as PLA, polyglycolic
acid, etc., degrade in vivo by hydrolysis, which are nonenzymatic resulting
in products which are not toxic (Shi et al., 2016). PLA scaffolds prepared
by the interface of the Ti3C2Tz nanosheets with the hydrophobic matrix of
PLA exhibited a tensile strength of 72 MPa which is over 33% higher than
the pure PLA membrane.
The scaffolds exhibited excellent biocompatibility, increased cell adhe­
sion, proliferation, and osteogenic differentiation indicating their potential
application for bone regeneration (Chen et al., 2018a). PLA-based scaf­
fold, prepared from the combination of high concentration solvent casting,
particulate leaching, and room temperature compression molding, exhibited
good pore size and mechanical properties. The concentration of HA in the
scaffold in�uenced its mechanical properties and porous structure. The
scaffold with 20% HA exhibited porosity, contact angle, compressive yield
strength, and weight loss of 84. 28 ± 7.04%, 45.13 ± 2.40, 1.57 ± 0.09 MPa,
and 4.77 ± 0.32%, respectively, after 56 days (Mao et al., 2018). The afore­
mentioned properties revealed the effect of combining synthetic polymers
with ceramics. Poly(lactic-co-glycolic acid)-based scaffolds exhibited an
interconnected porous architecture with pore sizes of 200–400 µm. SBA-15
modi�cation provided the desired environment for the cell attachment,
spreading, and proliferation on the scaffolds. The scaffolds exhibited excel­
lent biocompatibility and biodegradability with effective osteogenesis in
vivo (Wu et al ., 2017).
Polycaprolactone-based scaffolds have also been used in bone regenera­
tion. The design of a 3D polycaprolactone scaffold with properties such as
porosity and biocompatibility with mechanical strength suitable for the
proliferation and regeneration of tissues has been reported. The pore size
was in a range of 200–400 µm and Young’s modulus (E) was in the range of
0.121–0.171 GPa, which are compatible with the modulus of natural bone.
The scaffolds cell proliferation capability revealed their ability to enhance
osseous tissue formation, which is suitable for bone tissue regeneration
(Gómez-Lizárraga et al., 2017). Polycaprolactone scaffolds seeded with
bone morphogenetic protein-7-transduced human gingival �broblasts and
implanted subcutaneously in immunocompromised mice for a period of 4 and

41. 16 Tissue Engineering
8 weeks indicated good bone penetration into high permeability scaffolds,
with visible blood vessel in�ltration at 4 weeks. The results indicate that
polycaprolactone scaffold with regular architecture and good permeability is
suitable in vivo for bone regeneration (Mitsak et al., 2011). Polycaprolactone
scaffolds have been utilized for the delivery of mesenchymal stem cells. The
scaffolds were not enriched with biologically active agents. The scaffold
provided a suitable environment for osteogenic differentiation of the mesen­
chymal stem cells illustrating the potential capability of polycaprolactone­
based scaffolds as bone grafts (Rumiński et al., 2017).
Other synthetic polymers such as PVA, polyphosphoester (PPE),
poly(trimethylene carbonate) (PTMC), polyurethanes (PUs), and poly-γ-
glutamic acid (γ-PGA) have also been used for scaffolds for bone regen­
eration. PVA-based scaffolds exhibit good water uptake indicating a good
hydrophilic property. They also possess uniform pore size with diameters
ranged between 142 and 519 µm and are nontoxic in nature which is suitable
for bone tissue regeneration (Pineda-Castillo et al., 2018). Scaffolds prepared
from a combination of modi�ed cellulose and PVA exhibit an average diam­
eter in the range of 117–500 nm; decreased degree of crystallinity and the
presence of glycosidic linkage further enhanced the biocompatibility of the
scaffolds (Chahal et al ., 2013).
Combining biphasic calcium phosphate (BCP) and PVA resulted in porous
scaffold with interconnected pore structure, good mechanical strength, good
biodegradation rate, and cytocompatibility essential for bone tissue regenera­
tion (Nie et al. , 2012). Coating scaffolds prepared from the combination of
PVA and hydroxyethyl cellulose with nano-HA enhanced the tensile strength
and decreased the elongation at breakage of the scaffolds. The coating in�u­
enced cell proliferation and differentiation (Chahal et al. , 2016). The release
mechanism of recombinant human bone morphogenetic protein-2 (rhBMP-2)
from scaffolds of PPE loaded with rhBMP-2 was a combination of burst
and sustained release. The cytotoxic effect of the scaffolds was reduced and
the scaffolds were biocompatible. The bioactivity of rhBMP-2 was retained
after the polymerization process of the scaffolds (Tian et al. , 2012). PPE is
a biodegradable polymer with excellent biocompatibility (Qiu et al. , 2006).
Polyurethane-based scaffolds exhibit homogeneous morphology and a
regular pore size of 407 µm. Biomineralization of the scaffolds enhanced
the mechanical properties of scaffolds and made the surface of the scaffolds
suitable for cell attachment and proliferation suggesting that it promotes
osteoconductivity and bone bonding (Meskinfam et al., 2018). PU scaffolds
with dense shell and a porous core have been reported for the treatment of

42. 17 Biomaterials in Tissue Engineering
oromaxillary bone defects. The scaffolds were biocompatible and suitable
for guided bone regeneration applications (Giannitelli et al., 2015).
Polyurethane scaffolds loaded with lovastatin exhibited porous structure
with interconnected pores suitable for cell attachment and proliferation. In
vivo studies revealed their good osteoconductive potential and biocompat­
ibility (Yoshii et al., 2010). γ-PGA-based scaffolds prepared from covalent
cross-linking using an organosilane promoted cell attachment and were not
cytotoxic (Poologasundarampillai et al. , 2010). γ-PGA is biodegradable with
carboxylic acid functional groups useful for modi�cation. It degrades enzymati­
cally from the surface and the degradation rate is in�uenced by its environment,
the concentration of enzymes present, and the surface area exposed to enzyme
activity (Poologasundarampillai et al. , 2010). γ-PGA-silica hybrid scaffolds
exhibit tailorable physiochemical properties useful for bone tissue regenera­
tion (Gao et al ., 2016).
Synthetic polymers, used for the design of scaffolds for cartilage
regeneration, are easily molded with microstructure, mechanical properties,
and degradation suitable for bone regeneration. PEG-based scaffolds have
been demonstrated to be a potential platform for cartilage regeneration.
PEG-based hydrogels prepared by Michael addition reaction were reported
to be biocompatible. Collagen type II and chondroitin sulfate accumulated
over time in the gels (Jin et al., 2010). PEG-based cross-linked hydrogels
prepared by Diels–Alder click reaction from hyaluronic acid and PEG exhib­
ited good mechanical properties and short gelation time suitable for cartilage
tissue engineering (Yu et al., 2014). Encapsulation of rabbit chondrocytes
into the injectable hydrogels prepared from a combination of γ-PGA and
alginate revealed the viability of the entrapped cells. The gel exhibited good
mechanical stability, cell ingrowth, and ectopic cartilage formation (Yan et
al., 2014). In another report in which hydrogels were fabricated for cartilage
regeneration by click reaction, the hydrogels exhibited fast gelation with
antifatigue properties, a storage modulus of 27 kPa, breakage strength of
109.4 kPa, and compressive strain of 81.9% with high metabolic cell viability
and proliferation in vitro suitable for cartilage tissue engineering (Yu et al. ,
2018). An electrospun scaffold of gelatin-incorporated poly(L-lactide- co-ɛ-
caprolactone) exhibited mechanical properties similar to natural cartilage.
In vivo studies on the scaffolds indicated good cartilage formation in
partial-thickness defects of rabbit knees (Kim et al., 2012b). PU-based
scaffold prepared for cartilage regeneration exhibited excellent water
uptake similar to natural cartilage with compressive modulus in the range of
1.9–14.4 MPa similar to the articular cartilage. The cell viability using human

43. 18 Tissue Engineering
chondrocytes showed 14% and 33% increase in cell viability (Bonakdar et
al., 2010). Poly(L-glutamic acid)-based scaffolds encapsulation with rabbit
chondrocytes revealed the viability of the entrapped cells with a rapid in vivo
gel formation and signi�cant mechanical stability and ectopic cartilage forma­
tion (Yan et al. , 2016). Swine auricular chondrocytes were photoencapsulated
into PEG dimethacrylate copolymer scaffolds with either a degradable or
nondegradable macromers. The photoencapsulated chondrocytes survived
with signi�cant cell proliferation. Scaffold with a 100% nondegradable
PEG dimethacrylate inhibited cell–cell interactions of the chondrocytes and
the formation of contiguous cartilage. Scaffolds with ratios of degradable
and nondegradable macromers of 60:40 and 70:30 were suitable for engi
neering auricular cartilage. The scaffolds supported in vivo chondrogenesis
by photoencapsulating auricular chondrocytes (Papadopoulos et al., 2011).
Degradable polymers are preferred for articular cartilage repair because they
form a temporary scaffold for mechanical support and when new cartilage
starts forming within the defect site, these polymer materials degrade leaving
behind the regenerated tissue (Seal, 2001).
w­ä x­ä w­ä x­ä y�p���‘�Ž�›�•�‡�”�•� ���’�’�Ž�‹�…�ƒ�–�‹�‘�•� �ˆ�‘�”� ���‹�•�•�—�‡����”�‰�ƒ�•� ���‡�‰�‡�•�‡�”�ƒ�–�‹�‘�•
Synthetic polymers have also been employed for the design of scaffolds for
organ regeneration such as nerve, liver, and cardiovascular system (heart,
arteries, lungs, veins, and capillaries). The human nervous system lacks the
capability to regenerate its components when damaged (Maiti and Díaz Díaz,
­
2018). Nerve injuries can cause partial or total loss of autonomic, sensory,
and motor functions (Navarro et al. , 2007). Nerve injuries are treated by
autograft method, which involves transplanting tissue from one part of the
body to another.
However, autograft method suffers from several limitations such as it
is expensive, time-consuming, there is a need for a secondary surgical
site, immunogenic rejection, the risk of transference of disease, a diameter
mismatch between defected nerves, and newly grafted nerves (Maiti and
Díaz Díaz, 2018; Pi et al., 2015). Polymers, which are biodegradable, have
been used to construct nerve guide channels, which avoid the need of a
second surgery to remove the nerve guide channels from the body, thereby
preventing chronic tissue responses or nerve compression (Maiti and Díaz
Díaz, 2018).
Polyphosphoester has been used for nerve regeneration. Polymer-based
microspheres loaded with nerve growth factors prepared from biodegradable

44. 19 Biomaterials in Tissue Engineering
PPE and loaded into PPE conduits provided prolonged and site-speci�c
delivery of nerve growth factors (NGFs). In vivo studies in rat sciatic
nerve model, 3 months after implantation, revealed higher values of �ber
diameter, population, and density when compared to the control conduits
loaded con�rming a long-term promoting effect of exogenous NGF on
the regeneration of peripheral nerves. This �nding con�rmed the potential
of polymers in enhancing the functional recovery in patients with injured
nerves (Xu et al., 2003). Poly(L-lactide- co-glycolide) has also been used for
nerve regeneration. Poly(L-lactide- co-glycolide)-based �bers were used for
the fabrication of the conduits. The degradation of the conduit was via �ber
breakage revealing the bulk hydrolysis of the polymer. In vivo studies in rat
sciatic nerve model with a 12-mm gap revealed the absence of in�ammatory
response after implantation, good biological response of the conduit which
was characterized by the formation of a �brin matrix cable.
After 4 weeks of implantation, nerve regeneration was observed in the
rats. The implants were �exible and permeable revealing their ef�cacy in
nerve regeneration and repair (Bini et al., 2003). Poly(lactic- co-glycolic
acid) (PLGA) conduits have been reported to have good plasticity, intensity,
and elasticity indicating that they are useful for the repair of sciatic nerve
injury (Yu et al., 2013). Poly(L-lactide- co-glycolide) biodegradable polymer
nano�bers fabricated to conduits functioned as nerve guidance channels. In
vivo studies on rat sciatic nerve model with a 10-mm gap length revealed
successful nerve regeneration and the absence of tube breakage and in�am­
matory response. The nano�ber nerve guidance conduits were also �exible
and permeable with no sign of swelling (Bini et al., 2004). Stem cells from
human exfoliated deciduous teeth seeded in poly(L-lactide-co-glycolide)
scaffolds and implanted in sciatic nerve transection rat model with a 7-mm
nerve gap bridge revealed an improved function in sciatic functional index,
signi�cant functional recovery, and nerve regeneration.
Scaffolds with stem cells accelerated nerve recovery (dos Santos et al.,
2019). The scaffold enhanced cells attachment, migration, and growth as
conditioning for axonal outgrowths, which is essential for nerve regeneration
(dos Santos et al., 2019). NGF-loaded poly(L-lactide- co-glycolide) micro-
spheres have been reported to be useful for nerve regeneration in peripheral
nerve injury with a signi�cant effect on functional recovery in the long term.
The release of NGF-loaded microspheres in small gap tubulization facili­
tated nerve regeneration (Kaka et al., 2017; Wang et al., 2014b; de Boer et
al., 2012). Biodegradable synthetic polymers such as PLA and PCL have
also been used as scaffolds for nerve regeneration.

45. 20 Tissue Engineering
Poly(ε-caprolactone) scaffold loaded with NGF in a nerve conduit used
in repairing the transected rat sciatic nerve by implantation into the 10-mm
right sciatic nerve defects revealed motor and sensory functional recovery
and signi�cant motor distal latency The scaffold mechanical properties and
biocompatibility improved sciatic nerve regeneration in rats (Mohamadi et
al., 2018). Nanocomposites prepared from a mixture of PCL and carbon-
based nanoparticles exhibited improved cell adhesion, facilitated a twofold
increased in the number of myelinated axons in the repaired nerves suggesting
their potential in nerve tubulization repair (Assaf et al., 2017). PCL �lms with
modi�ed surfaces with selected functional groups in�uenced their cellular
response, hydrophilicity, hydrophobicity, and mechanical properties. Hydro­
lyzed �lms exhibited signi�cant hydrophilicity and the aminolysis of the
�lms decreased their mechanical properties. Improved cell attachment was
signi�cant on the modi�ed �lms with amino groups on the material surface
(de Luca et al., 2012). PCL scaffold prepared possessed good hydrophilicity
and �exibility. In vitro study revealed that the scaffold promoted Schwann
cell adhesion, elongation, and proliferation. In vivo test revealed that the
porous nerve conduits supported nerve regeneration through an 8-mm sciatic
nerve gap in adult rats successfully.
The implanted polymer scaffold nerve conduits facilitated signi�cant
axons regeneration via the conduit lumen and gradually degraded which is
essential for longer nerve defect (Yu et al., 2011). PLA, an aliphatic polyester,
has low allergenic potential, low toxicity, and is biocompatible with predict­
able kinetics of degradation. PLA microporous hollow �ber application as a
conduit to bridge a nerve gap in a mouse sciatic nerve injury model revealed
a signi�cant increase in the number of myelinated �bers and blood vessels
in animals, good overall tissue organization, and a signi�cant improvement
in functional recovery indicating that PLA conduits are potential alterna­
tive to autograft (Goulart et al., 2016). PLA is used in nerve conduit due to
its biocompatibility, easily shaped properties, and degradation to low toxic
lactic acid. However, its use is restricted by its hydrophobicity and lack of
binding sites for cellular activities. Incorporation of graphene oxide into PLA
nano�bers enhanced its mechanical properties and hydrophilicity, thereby
making it suitable for peripheral nerve regeneration application (Öztatlı and
Ege, 2016).
Polymers have also been used for liver tissue regeneration. A nano�brous
scaffold was fabricated from polycaprolactone. The nano�bers were charac­
terized by few diameters, better pore, size, and good orientation (Semnani
et al., 2016). Polymers have also been employed for the regeneration of

46. 21 Biomaterials in Tissue Engineering
cardiovascular system. Their biocompatibility makes them to be used for
cardiovascular application such as prosthetic heart valves, vascular grafts to
stents, catheters, hemodialyzer, heart assist devices, etc. (Jaganathan et al.,
2014). Some of the polymers used are polytetra�uorethylene, polyethylene
terephthalate, PU, PVA, and polyamide (Helmus and Snyder, 2009). The
cardiovascular system is composed of the heart and the blood vessels. Poly­
mers used for the regeneration of cardiovascular system may have contact
with blood and a number of other cells. They interact with the heart, blood,
and blood vessels. However, they are susceptible to failure resulting from
thrombosis resulting in heart attacks, strokes, damage of blood cells, reduce
oxygen carrying capability of the blood, paralysis, and organ failure (Helmus
and Snyder, 2009).
Polyamides are used for the development of catheters and hemodialysis
membranes. Polytetra�uorethylene is a mono�lament plastic polymer, which
is durable with good �exibility, biostability, and breaking strength with nega­
tive charge-like native endothelium (El Khoury and Vohra, 2012). It exhibits
good hemodynamic properties but its use in the development of prosthetic
heart valves and vascular grafts is limited by calci�cation, thrombosis, and
stiffening of lea�ets (Hayabuchi et al., 2007; Saha et al., 2011; Mehta et
al., 2011). Polyethylene terephthalate has been used for the sewing cuffs of
prosthetic heart valves in vitro. The pretreatment of prosthetic heart valves
with antibiotics reduced the risk of bacterial adhesion and infections. The
soaking of prosthetic heart valves in antibiotic solutions before implanta­
tion and combination with �brin glue can prevent the development of early
prosthetic valve endocarditis (Mashaqi et al., 2011).
A �brous composite prepared from polyethylene terephthalate polyeth­
ylene exhibited physical and mechanical properties revealing its potential
application for the valve lea�et function (Amri et al., 2018). Woven poly­
ethylene terephthalate polyethylene has been reported to exhibit small pores
with the capability to reduce leakage of blood and good ef�ciency as vascular
grafts when compared to knit one (Jaganathan et al., 2014). Polyethylene tere ­
phthalate polyethylene grafts are also coated with protein such as collagen or
albumin resulting in reduced loss of blood and good biocompatibility (Kudo
et al., 2002). Textile polyester provides unique folding and strength proper­
ties with good biocompatibility. Its mechanical and hemodynamic proper­
ties were compared with biological tissue. The textile valves elasticity and
mechanical strength were superior when compared to the biological tissue.
The �exibility of polyester textile valve lea�ets was similar to the biolog­
ical lea�ets. The regurgitation and turbulent patterns of the polyester textile

47. 22 Tissue Engineering
valves were signi�cant resulting from its increased porosity and rapid tissue
ingrowth postimplantation when compared to the biological tissue revealing
the ef�cacy of polyester for the development of heart valve lea�et mate­
rial (Youse� et al., 2016). PCL has long degradation time, good �exibility,
and biocompatibility and its small-diameter grafts are promising alterna­
tives to polyethylene terephthalate polyethylene grafts resulting from its
healing characteristics. Tubular PCL reinforced with different polyethylene
terephthalate-knitted fabric structures to form multilayer composites was
reported to mimic human blood vessels with good mechanical properties,
suture retention, water permeability, and elastic recovery. The loop density
in�uenced the compressive strength, suture retention strength, and elastic
recovery. The mechanical properties were better than the commercially
expanded polytetra�uoroethylene (Wang et al., 2014a). After an average
implantation of 6 years, the expanded polytetra�uoroethylene/Gore exhibits
stronger resistance against dilatation when compared to polyester/Braun and
polyester/Vascutek tube grafts. No graft failure or rupture occurred with a
graft patency of 100% (Stollwerck et al., 2011).
Some of the commercially available vascular grafts are Vascutek
®
­
knitted polyester grafts, GORE-TEX
®
vascular grafts, CoreoGraft
™
, etc.
PU-based grafts have also been reported to signi�cantly increase endothelial
cell proliferation in vitro with 100% patency after implant with no signs
of aneurysmal dilatation. The thin-walled grafts were found to be desirable
form of a biodegradable vascular implant with the potential of long-term
performance characteristics (Bergmeister et al., 2015). PU-based grafts have
been designed with good cytocompatibility, porous architecture, average
�ber range of 1.5–2.0 µm, good tensile strength, and signi�cant biostability
suitable for vascular grafts (Arjun and Ramesh, 2012; Styan et al., 2012).
PVA-based cryogels coated with lyophilized decellularized vascular matrix
resulted in enhanced adhesion of human umbilical vein endothelial cells.
However, the implantation of the coated scaffold into the abdominal aorta
of Sprague Dawley rats resulted in the mortality of all the animals’ 3–4 days
after surgery indicating that the coating produced a thrombogenic surface.
The rat implanted with PVA without coating survived 12 months after
implantation suggesting the potential of PVA for the fabrication of arti�cial
vessels (Conconi et al., 2014). PVA hydrogel has excellent potential as a
vascular graft because of its unique properties such as mechanical properties
that are tunable, bioinert, high water content, and low thrombogenicity. PVA
hydrogel grafts, with submillimeter diameter and the mechanical proper­
ties, are similar to the rabbit femoral artery that was reported. The graft

48. 23 Biomaterials in Tissue Engineering
exhibited low thrombogenicity, improved perfusion of the distal limb, and
was patent over 2 weeks after implant without the use of anticoagulant or
antithrombotics.
On the luminal surface of the grafts, endothelial cells were visible indi­
cating their potential application as a vascular replacement in microvascular
surgery (Cutiongco et al., 2016). Moreover, vascular grafts composed of
weft knits have high porous connectivity and compliance similar to native
vessels (Lin et al., 2017). PVA tubes have also been reported to exhibit thick­
ness of 344 ± 13 µm almost similar to a human artery, which is in a range of
350–710 µm. Its suture retention was 140 ± 11 g, which was also close to that
of human vessels and the burst pressure was 507 ± 25 mm Hg which is more
than three times higher than the human healthy systolic arterial pressure. No
leakage was observed at arterial pressure, which was contrary to vascular
expanded polytetra�uoroethylene prostheses. Implantation in rats did not
result in thrombotic complications. These �ndings indicate that cross-linking
method of PVA results in mechanical properties, which are compliance and
compatible with the circulatory blood �ow with no thrombotic complica
tions (Chaouat et al., 2008).
w­ä x­ä w­ä x­ä z�p���‘�Ž�›�•�‡�”�•� ���’�’�Ž�‹�…�ƒ�–�‹�‘�•� �ˆ�‘�”� ���‘�•�•�‡�–�‹�…� ���—�”�‰�‡�”�›�
Polymers have been designed for cosmetic surgery, which includes breast
implant and facial reconstruction. Hydrogels prepared from 2.5% polyacryl
­
­
amide have been reported to be useful for the reconstruction of facial lipoat­
rophy resulting from combination of antiretroviral therapy. Clinical studies
in 110 patients, who received two to six injections every 4 weeks, resulted in
average cheek skin thickness of 9.7mm at baseline increased by an average
of 4.4mm at month 12 and a further increased by an average of 0.87mm at
month 24. No severe adverse effects related to the polyacrylamide hydrogel
were observed (Mole et al., 2012). Polyacrylamide hydrogel injections
significantly improved the quality of life of HIV-infected patients with facial
lipoatrophy. However, polyacrylamide hydrogel injections suffer from limi­
tations such as infections (Kalantarhormozi et al., 2008). Porous polymeth ­
ylmethacrylate implants filled with a gel that releases antibiotics to protect
the tissue from infection have been designed. The thermogel is composed
of block copolymer for the controlled release of colistin, an antibiotics and
turns into a gel at body temperature followed by a slow degradation over
the period of implantation up to 28 days (Mountziaris et al., 2016). Cranial

49. 24 Tissue Engineering
implants have been developed from a biocompatible polymer, ultrahigh
molecular weight polyethylene, by computerized tomographies of the
patient, converting them into a 3D model using the software InVesalius.
The use of polymer based for cranial implants has advantages
such as lightweight, low heat conductivity with mechanical properties
similar to bone, and low cost (Bagudanch et al. , 2018). Resorbable
polymer implants prepared from polylactide and polyglycolide polymers
are potential devices for the treatment of acquired and congenital cranio­
facial deformities (Cohen et al. , 2004). A porous shape memory-polymer
foam prepared from PCL had interconnected pores which promoted bone
cells to migrate in and grow. It is potentially useful for craniomaxillofacial
bone grafting. The foam coated with polydopamine locks the polymer into
place by inducing the formation of a mineral found in bone. It induces
osteoblasts to adhere and spread throughout the polymer. The biodegrad­
able nature of the foam results in its biodegradation leaving behind only
the new bone tissue (Mann, 2014).
Computed tomographic biomodeled, heat cured, and prefabricated
polymethyl methacrylate implants are well tolerated in patient in long
term. Their advantages are long-term biocompatibility, customized design,
and distinct esthetic results (Groth et al. , 2006). Polymethyl methacrylate
produces minimal thermal, electrical, and magnetic conductivity making
it useful for orbital implantation (Chiarini et al. , 2004). High-density
polyethylene has been used for dorsum nasi augmentation due to its long-
term structural stability and lack of resorption (Dresner and Hilger, 2008).
It is also used for chin and malar augmentation. The histopathological
results indicated soft-tissue ingrowth and collagen deposition with signi�­
cant vascularization. Complications were few and 91% of the implants
remained unchanged (Niechajev, 2012). Polyethylene terephthalate is used
for correction in nasal reconstruction (Rai et al. , 2013). However, it cannot
be used for structural support. It is a good alloplastic implant for facial
skeleton augmentation, if autogenous grafts cannot be harvested. However,
it can be rigid and infection is a complication which has been reported
(Ionita et al. , 2015).
Polymers have also been designed for breast implants. Polymeric �llers
synthesized from acrylamide by redox polymerization are biocompatible and
are permanent injectable �ller material for breast reconstruction (Kim et al.,
2018). Polymers are also alternative to the use of acellular dermal matrices
and are used as temporary reinforcement in patients undergoing breast
reconstruction (Becker and Lind, 2013). An arborescent block copolymer

50. 25 Biomaterials in Tissue Engineering
prepared and investigated as breast implant did not exhibit leakage that can
provoke in�ammatory responses in patients (Becker and Lind, 2013). It is
biocompatible when compared to silicone in the long term (Becker and Lind,
2013). Silicone gel-�lled breast implants with a silicone outer shell are �lled
with silicone gel. They are approved by FDA for breast augmentation and
reconstruction in women age 22 years or older. However, complications
associated with silicone breast implants are capsular contracture, implant
rupture, wrinkling, asymmetry, scarring, pain, infection, and anaplastic large
cell lymphoma (FDA, 2017). A branched arborescent polyisobutylene core
was reported to be an alternative biomaterial to silicone rubber. Implantation
in a rabbit model revealed that the material exhibited excellent tissue–mate­
rial interactions resulting from the lower surface energy of polyisobutylene
that forms a thin layer on the surface of the material. No acute in�ammation
was observed and the material was highly biostable (Teck Lim et al., 2013).
1.2.1.3 CERAMICS
Ceramics have been used widely in bone regeneration. They are designed
for a strong bonding to bone and are an alternative to metallic implants.
They are characterized by excellent corrosion resistance, biocompatibility,
hard brittle surface, and osteoconductivity. However, they are limited by
poor brittleness, fracture toughness, elasticity, and high stiffness (Salinas and
Vallet-Regí, 2013). Some examples of ceramics used in tissue regeneration
are calcium phosphate (CaP), HA, CaCO
3
(argonite), CaSO
4
∙2H
2
O (plaster
of Paris), Ca
3
(PO
4
)
2
[tricalcium phosphate (TCP)], etc.
A 3D-fabricated ceramic scaffold prepared from HA/TCP exhibited pore
sizes smaller than scaffolds prepared from HA. The release of calcium ions
from the scaffolds was higher in TCP scaffold. The HA/TCP scaffolds had a
greater capacity to enhance bone regeneration when compared to HA scaf­
folds. The calcium ion releasing effect of the scaffolds and the rough surface
morphology induced bone regeneration indicating their potential in complex
bone defects (Seol et al., 2014). (BCP ceramics are biocompatible, osteo­
conductive, bioactive, and can induce differentiation of stem cells. Ceramics
are easily tailored into matrix for bone regeneration (Lobo and Livingston
Arinzeh, 2010). Scaffolds composed of different concentrations of HA and
β-tricalcium phosphate (β-TCP) exhibit controlled bioactivity with a balance
between resorption/solubilization, which in�uences their stability and also
promotes bone growth (Daculsi et al., 1990).

51. 26 Tissue Engineering
The high release of calcium from ceramic scaffolds to the microenviron­
ment induces an in�ammatory response, resulting in a change of the pH and
it promotes �brous tissue formation (Hing et al., 2007). High calcium ion
levels in�uence osteoclastic activity and the controlled levels of calcium
ions promote the formation of an apatite layer useful for HA/β-TCP ceramics
bioactivity (Daculsi and LeGeros, 2008). Ceramic scaffolds can cause the
transformation of macrophages into an M2c phenotype, genes associated
with remodeling. These �ndings indicate that macrophages must be in direct
contact with the scaffold for tissue regeneration (Graney et al., 2016). The use
of dielectric barrier discharge plasma with oxygen can enhance the hydro­
philicity of nonporous HA surfaces. In vivo implantation of plasma-treated
interconnected porous calcium HA exhibited signi�cant bone ingrowth.
Plasma-treated interconnected porous calcium HA promoted osteogenic
differentiation of seeded marrow mesenchymal stem cells indicating the
osteoconductive potential of interconnected porous calcium HA when used
as bone substitutes (Moriguchi et al., 2018). Some physical properties of
ceramics when compared with the human natural bone include mechanical
properties. CaP ceramics promote bone repair, but have limited lifetime and
cannot adapt to skeletal changes (Oryan and Alidadi, 2017). Synthetic HA
exhibits a strong af�nity to the host bone tissue resulting from its chemical
similarity with mineralized human bone tissue (Oryan and Alidadi, 2017).
It resorbs slowly and undergoes little transforming into a bone-like material
after implantation when compared to β-TCP. β-TCP scaffolds often possess
lower strength when compared to the HA scaffolds (Oryan and Alidadi,
2017).
Several researchers have investigated the potential of ceramic-based
scaffolds in bone regeneration. HA scaffolds seeded with rabbit mesen­
chymal stem cells (rMSCs) with 54%–81% porosity exhibited signi�cant
bone regeneration in rabbit mandible defects (Guo et al., 2009). HA scaffolds
induced bone formation after implantation into immunode�cient male mice
(Teixeira et al., 2009). β-TCP scaffolds loaded with BMP-2 placed in rabbit
femoral defects induced bone formation signi�cantly (Sohier et al., 2009).
β-TCP scaffolds cocultured with rMSCs and rMSC-derived endothelial cells
followed by implantation into rabbit large segmental defects resulted in bone
regeneration at week 16 revealing enhanced osteogenesis and vasculariza­
tion (Zhou et al., 2010). Scaffolds composed of 60:4 HA:β-TCP ratio and
interconnected pores of 300–800 µm with 75%–85% porosity were cultured
in mouse mesenchymal stem cells. The scaffolds differentiate into osteoblast­
like cells and implantation in rat lower jaw bones and tibias resulted in bone
formation (Kim et al ., 2012a).

52. 27 Biomaterials in Tissue Engineering
1.3 BIOLOGICAL-BASED BIOMATERIAL IN TISSUE ENGINEERING
Biological-based biomaterials are used in tissue engineering due to their
unique properties such as excellent biocompatibility, nontoxic nature, biode­
gradability, and they are readily available. Some examples of biological-based
biomaterials are natural polymers, peptides, carbon-based materials, etc. They
have been used in the design of scaffolds for bone and cartilage regeneration,
skin regeneration, cosmetic surgery, soft-tissue regeneration, etc.
1.3.5౰NATURAL POLYMERS
Natural polymers such as silk, keratin, chitosan, alginate, fibronectin,
collagen, fibrin, hyaluronic acid, and amylose are used in tissue engineering.
They are biodegradable and can be replaced upon the generation of new
tissues. However, they are limited by their immunological integrity and poor
mechanical properties (Sproul et al., 2018). They can be further classified
based on their sources such as animal-based and plant-based polymers. They
are used for bone and cartilage regeneration, skin regeneration, cosmetic
surgery, and soft-tissue regeneration.
1.3.2 FIBRIN
Fibrin plays an important role in blood clotting. It is the primary component
in clots and is useful in hemostasis and tissue repair (Sproul et al., 2018).
It is used as a tissue sealant, biodegradable, and biocompatible with a high
cell binding and good signaling capacity (Sproul et al., 2018). The precur
sors of fibrin, fibrinogen and thrombin, can be isolated from patients’ blood
­
which limits immunogenicity (Janmey et al., 2008; Huang and Fu, 2010). Its
network exhibits properties useful for the development of skin substitutes
and the rate of polymerization, architecture, the pore size of the scaffolds,
and the thickness can be easily tailored (Huang and Fu, 2010; Bencherif et
al., 2017). Its cost of isolation is affordable when compared to other natural
polymers (Bencherif et al., 2017; Ahmed et al., 2008). Fibrin-based scaffolds
provide adequate time suitable for neomatrix formation with slow resorption
influenced by the action of proteases (Bencherif et al., 2017).
The aforementioned factors make it useful in wound healing and skin
regeneration. Heparin-conjugated �brin (HCF) scaffold was loaded with
�broblast growth factor 2 (FGF2). It was used as a vehicle for long-term

53. 28 Tissue Engineering
delivery of FGF2. It was implanted into full-thickness skin defects of mice
and the formation of neoepidermis was signi�cantly thick indicating the ef�­
cacy of �brin in skin regeneration mediated by FGF2 (Bhang et al., 2010).
Fibrin-based interpenetrating polymer networks prepared by photochemistry
provided good mechanical support for cellular growth. It was cultured in
human dermal equivalent cells and the surface of the network supported
cell growth revealing excellent biocompatibility. In vivo studies after
subcutaneous implantation in an animal model did not induce in�ammation
indicating that it is a potential scaffold for human skin substitute (Gsib et al.,
2018).
The application of engineered 3D PEGylated �brin scaffold loaded with
adipose-derived stem cells (ADSCs) in burn injury model showed the pres­
ence of stem cells at the wound site 2 weeks after application. Enhanced
vascularization and granulation tissue formation were signi�cant (Chung et
al., 2016). A PVA hydrogel loaded with granule-lyophilized platelet-rich
�brin exhibited good mechanical strength and degradation rate, which was
in�uenced by the concentration of the granule-lyophilized platelet-rich
�brin. However, the excellent elastic properties and biocompatibility of the
hydrogel were not in�uenced by the concentration of granule-lyophilized
platelet-rich �brin. Application of the scaffold to an acute full-thickness
dorsal skin wounds enhanced wound closure at days 7 and 9. The scaffold
enhanced the formation of granulation tissue, collagen deposition, and new
vessel (Xu et al. , 2018). Scaffold prepared from a combination of synthetic
polymers, �brin and loaded with platelet lysate exhibited sustained release
of the platelet lysate in vitro. The application of the scaffold on genetically
diabetic mouse full-thickness skin wound enhanced wound closure at day
15. Re-epithelialization, granulation tissue formation, and collagen deposi­
tion were increased indicating their potential use for the treatment of diabetic
foot ulcers (Losi et al., 2015). Fibrin scaffold has been developed into a
three-layered skin substitute by embedding ADSCs and mature adipocytes
in the hydrogel with �broblasts for the construction of the dermal layer. On
top of the two layers was loaded keratinocytes to mimic the epidermal layer.
After 3 weeks, the loaded stem cells and the �broblasts were viable and
proliferated and differentiated into matured adipocytes with morphology
similar to native adipose tissue. The keratinocytes formed an epithelial-like
layer (Kober et al., 2015). Fibrin scaffolds cultured in human �broblasts
and seeded with human keratinocytes on the top of the scaffold healing
potential was assessed in deep partial and full-thickness burns in vivo. Good
cell attachment and colony spreading of keratinocytes and �broblasts on the

54. 29 Biomaterials in Tissue Engineering
scaffold were visible. The appearance of the skin did not differ from the areas
of transplanted skin indicating good healing (Kljenak, 2016). Fibrin glue has
been applied for the closure of a meshed skin graft with reduced scarring and
decreased infection. Addition of angiogenic growth factor further enhanced
the growth of blood vessels from the wound bed into the graft (Feldman and
Osborne, 2018).
Fibrin has also been used in cosmetic surgery for facelift surgery. Aero­
solized �brin glue used in facelifts surgery resulted in less bruising, reduced
swelling, and a rapid healing response. The risk of hematoma was also
reduced with shortened operating times by 13.3 minutes when compared
to facelift surgery with the use of glue (Fezza et al. , 2002). Autologous
platelet-rich �brin matrix enhances patient’s natural wound healing ability.
It induces the formation of viable blood vessels, fat cells, and collagen
deposits. It is used with minimal invasive and for open surgical procedures
(Sclafani and Saman, 2012). In facial implant and uplift, it promotes rapid
integration of the implant into the surrounding tissues. Fibrin scaffolds have
also been loaded with �broblasts for regeneration of new facial nerve in
pigs indicating the potential application of �brin for facial nerve regenera­
tion (Lasso et al. , 2015).
Fibrin-based scaffolds have also been used in bone regeneration by
several researchers. Platelet-rich �brin enhanced osteoblast proliferation and
differentiation indicating their effect on bone regeneration with low risk of
complications (Kim et al., 2017). A comparison study on the effects of �brin
and collagen on proliferation and differentiation of osteoblasts and protein
adsorption revealed that �brin adsorbed approximately 6.7 times more when
compared to serum �bronectin. Fibrin stimulated the proliferation of larger
MC3T3-E1 preosteoblasts at a low cell density and promoted enhanced
osteoblast differentiation when compared to collagen revealing the superi­
ority of �brin to collagen in bone regeneration (Oh et al., 2014). Leukocyte-
platelet-rich �brin has been used in the reconstruction of surgical bone defect.
The closure of the bony defect by neo-osteogenesis was observed by CT
scan. However, the density of the new bone was less when compared to the
surrounding normal bone showing its capability to induce bone healing and
regeneration at the surgical site defect (Fredes et al. , 2017). Platelet-rich �brin
stimulated bone regeneration in diabetic rabbits with a mean percent of 16.87
and 29.59 at weeks 4 and 8, respectively (Durmuşlar et al. , 2016). Platelet-
rich �brin application in two bicortical skull defects in 20 New Zealand white
rabbits showed that it induced bone formation when used alone or when
combined with autogenous bone (Pripatnanont et al. , 2013).

55. Another Random Document on
Scribd Without Any Related Topics

56. and of violence, by which this divine sweetness is tainted, and this
peace broken by suspicion, by hatred, and heat of blood."
"The book says somewhere," said the Duke, turning over the
leaves, "that, as the penitent thief rose from the cross to Paradise,
so we, if we long after Christ with all the powers of our souls, shall,
at the hour of death, rapidly soar aloft from our mortal remains, and
then all fear of returning to earth and earthly desires will be at an
end."
"It must surely," said Inglesant after a pause, speaking more to
himself than to the Duke, "be among the things most surprising to
an angelic nature that observes mankind, that, shadows ourselves,
standing upon the confines even of this shadowy land, and not
knowing what, if aught, awaits us elsewhere, hatred or revenge or
unkindness should be among the last passions that are overcome.
When the veil is lifted, and we see things as they really are, nothing
will so much amaze us as the blindness and perversity that marked
our life among our fellow-men. Surely the lofty life is hard, as it
seems hard to your Grace; but the very effort itself is gain."
Inglesant left the presence of the Duke after his first interview
impressed and softened, but troubled in his mind more than ever at
the nature of the mission on which he was sent. Now that he had
seen the Duke, and had been touched by his eager questions, and
by the earnest searching look in the worn face, his conscience smote
him at the thought of abusing his confidence, and of persuading him
to adopt a course which Inglesant's own heart warned him might not
in the end be conducive either to his own peace or to the welfare of
his people, whose happiness he sincerely sought. He found that, in
the antechambers and reception rooms of the palace, and even at

57. the Duke's own table, the principal subject of conversation was the
expected cession of the dukedom to the Papal See; and that
emissaries from Rome had preceded him, and had evidently received
instructions announcing his arrival, and were prepared to welcome
him as an important ally. On the other hand, there were not wanting
those who openly or covertly opposed the cession, some of whom
were said to be agents of the Grand Duke of Florence, who was heir
to the Duchy of Umbria through his wife. These latter, whose
opposition was more secret than open, sought every opportunity of
winning Inglesant to their party, employing the usual arguments with
which, since his coming into Italy, he had been so familiar. Many
days passed in this manner, and Inglesant had repeated conferences
with the Duke, during which he made great progress in his favour,
and was himself won by his lofty, kindly, and trustful character.
He had resided at Umbria a little less than a month, when he
received instructions by a courier from Rome, by which he was
informed that at the approaching festival of the Ascension a
determined effort was to be made by the agents and friends of the
Pope to bring the business to a conclusion. The Duke had promised
to keep this festival, which is celebrated at Venice and in other parts
of Italy with great solemnity, with unusual magnificence; and it was
hoped that while his feelings were influenced and his religious
instincts excited by the solemn and tender thoughts and
imaginations which gather round the figure of the ascending Son of
man, he might be induced to sign the deed of cession. Hitherto the
Duke had not mentioned the subject to Inglesant, having found his
conversation upon questions of the spiritual life and practice
sufficient to occupy the time; but it was not probable that this

58. silence would continue much longer, and on the first day in
Ascension week Inglesant was attending Vespers at one of the
Churches in the town in considerable anxiety and trouble of mind.
The sun had hardly set, and the fête in the garden was not yet
begun, when, Vespers being over, he came out upon the river-side
lined with stately houses which fronted the palace gardens towering
in terraced walks and trellises of green hedges on the opposite bank.
The sun, setting behind the wooded slopes, flooded this green hill-
side with soft and dream-like light, and bathed the carved marble
façade of the palace, rising above it with a rosy glimmer, in which
the statues on its roof, and the fretted work of its balustrades, rested
against the darkening blue of the evening sky. A reflex light, ethereal
and wonderful, coming from the sky behind him, and the marble
buildings and towers on which the sun's rays rested more fully than
they did upon the palace, brooded over the river and the bridge with
its rows of angelic forms, and, climbing the leafy slopes, as if to
contrast its softer splendour with the light above, transfigured with
colour the wreaths of vapour which rose from the river and hung
about its wharves.
The people were already crowding out of the city, and forcing
their way across the bridge towards the palace, where the
illuminations and the curious waterworks, upon which the young
Duke had, during his short reign, expended much money, were to be
exhibited as soon as the evening was sufficiently dark. The people
were noisy and jostling, but as usual good-tempered and easily
pleased. Few masques or masquerade dresses had appeared as yet,
but almost every one was armed with a small trumpet, a drum, or a
Samarcand cane, from which to shoot peas or comfits. At the corner

59. of the main street that opened on to the quay, however, some
disturbing cause was evidently at work. The crowd was perplexed by
two contending currents, the one consisting of those who were
attempting to turn into the street from the wharf, in order to learn
the cause of the confusion, the other, of those who were apparently
being driven forcibly out of the street, towards the wharves and the
bridge, by pressure from behind. Discordant cries and exclamations
of anger and contempt rose above the struggling mass. Taking
advantage of the current that swept him onward, Inglesant reached
the steps of the Church of St. Felix, which stood at the corner of the
two streets, immediately opposite the bridge and the ducal lions
which flanked the approach. On reaching this commanding situation
the cause of the tumult presented itself in the form of a small group
of men, who were apparently dragging a prisoner with them, and
had at this moment reached the corner of the wharf, not far from
the steps of the Church, surrounded and urged on by a leaping,
shouting, and excited crowd. Seen from the top of the broad marble
bases that flanked the steps, the whole of the wide space, formed
by the confluence of the streets, and over which the shadows were
rapidly darkening, presented nothing but a sea of agitated and
tossing heads, while, from the windows, the bridge, and even the
distant marble terraced steps that led up to the palace, the crowd
appeared curious, and conscious that something unusual was in
progress.
From the cries and aspect of the crowd, and of the men who
dragged their prisoner along, it was evident that it was the intention
of the people to throw the wretched man over the parapets of the
bridge into the river below, and that to frustrate this intention not a

60. moment was to be lost. The pressure of the crowd, greater from the
opposite direction than from the one in which Inglesant had come,
fortunately swept the group almost to the foot of the steps. Near to
Inglesant, and clinging to the carved bases of the half-columns that
supported the façade of the Church, were two or three priests who
had come out of the interior, attracted by the tumult. Availing
himself of their support, Inglesant shouted to the captors of the
unhappy man, in the name of the Church and of the Duke, to bring
their prisoner up the steps. They probably would not have obeyed
him, though they hesitated for a moment; but the surrounding
crowd, attracted towards the Church by Inglesant's gestures, began
to press upon it from all sides, as he had indeed foreseen would be
the case, and finally, by their unconscious and involuntary motion,
swept the prisoner and his captors up the steps to the side of the
priests and of Inglesant. It was a singular scene. The rapidly
advancing night had changed the golden haze of sunset to a sombre
gloom, but lights began to appear in the houses all around, and
paper lanterns showed themselves among the crowd.
The cause of all this confusion was dragged by his persecutors
up the steps, and placed upon the last of the flight, confronting the
priests. His hair was disordered, his clothes nearly torn from his
limbs, and his face and dress streaked with blood. Past the curtain
across the entrance of the Church, which was partly drawn back by
those inside, a flash of light shot across the marble platform, and
shone upon the faces of the foremost of the crowd. This light shone
full upon Inglesant, who stood, in striking contrast to the dishevelled
figure that confronted him, dressed in a suit of black satin and silver,
with a deep collar of Point-de-Venice lace. The priests stood a little

61. behind, apparently desirous to learn the nature of the prisoner's
offence before they interfered; and the accusers therefore addressed
themselves to Inglesant, who, indeed, was recognized by many as a
friend of the Duke, and whom the priests especially had received
instructions from Rome to support. The confusion in the crowd
meanwhile increased rather than diminished; there seemed to be
causes at work other than the slight one of the seizure by the mob
of an unpopular man. The town was very full of strangers, and it
struck Inglesant that the arrest of the man before him was merely
an excuse, and was being used by some who had an object to gain
by stirring up the people. He saw, at any rate, however this might
be, a means of engaging the priests to assist him, should their aid
be necessary in saving the man's life.
That there was a passionate attachment among the people to a
separate and independent government of their city and state, an
affection towards the family of their hereditary dukes, and a dread
and jealous dislike of the Pope's government and of the priests, he
had reason to believe. It seemed to him that the people were about
to break forth into some demonstration of this antipathy, which, if
allowed to take place, and if taken advantage of, as it would be, by
the neighbouring princes, would be most displeasing to the policy of
Rome, if not entirely subversive of it. With these thoughts in his
mind, as he stood for a moment silent on the marble platform, and
saw before him, what is perhaps the most impressive of all sights, a
vast assemblage of people in a state of violent and excited
opposition, and reflected on the causes which he imagined agitated
them,—causes which in his heart he, though enlisted on the
opposite side, had difficulty in persuading himself were not

62. justifiable,—it came into his mind more powerfully than ever, that
the moment foretold to him by Serenus de Cressy was at last indeed
come. Surely it behoved him to look well to his steps, lest he should
be found at last absolutely and unequivocally fighting against his
conscience and his God; if, indeed, this looking well to their steps on
such occasions, and not boldly choosing their side, had not been for
many years the prevailing vice of his family, and to some extent the
cause of his own spiritual failure.
The two men who held the apparent cause of all this uproar
were two mechanics of jovial aspect, who appeared to look upon the
affair more in the light of a brutal practical joke (no worse in their
eyes for its brutality), than as a very serious matter. To Inglesant's
question what the man had done they answered that he had refused
to kneel to the Blessed Sacrament, as it was being carried through
the streets to some poor, dying soul, and upon being remonstrated
with, had reviled not only the Sacrament itself, but the Virgin, the
Holy Father, and the Italians generally, as Papistical asses, with no
more sense than the Pantaleoni of their own comedies. The men
gave this evidence in an insolent half-jesting manner, as though not
sorry to utter such words safely in the presence of the priests.
Inglesant, who kept his eyes fixed upon the prisoner, and
noticed that he was rapidly recovering from the breathless and
exhausted condition the ill-treatment he had met with had reduced
him to, and was assuming a determined and somewhat noble
aspect, abstained from questioning him, lest he should make his
own case only the more desperate; but, turning to the priests, he
rapidly explained his fears to them, and urged that the man should
be immediately secured from the people, that he might be examined

63. by the Duke, and the result forwarded to Rome. The priests
hesitated. Apart from the difficulty, they said, of taking the man out
of the hands of his captors, such a course would be sure to
exasperate the people still further, and bring on the very evil that he
was desirous of averting. It would be better to let the mob work
their will upon the man; it would at least occupy some time, and
every moment was precious. In less than an hour the fireworks at
the palace would begin, might indeed be hastened by a special
messenger; and the fête once begun, they hoped all danger would
be over. To this Inglesant answered that the man's arrest was
evidently only an excuse for riot, and had probably already answered
its purpose; that to confine the people's attention to it would be
unfavourable to the intentions of those who were promoting a
political tumult; and that the avowed cause of the man's seizure,
and of the excitement of the mob, being disrespectful language
towards the Holy Father, the tumult, if properly managed, might be
made of service to the cause of Rome rather than the reverse.
Without waiting for the effect of this somewhat obscure
argument on the priests, Inglesant directed the men who held their
prisoner to bring him into the Church. They were unwilling to do so,
but the crowd below was so confused and tumultuous, one shouting
one thing and one another, that it seemed impossible that, if they
descended into it again, they would be allowed to retain their prey,
and would not rather be overwhelmed in a common destruction with
him. On the other hand, by obeying Inglesant, they at least kept
possession of their prisoner, and could therefore scarcely fail of
receiving some reward from the authorities. They therefore
consented, and by a sudden movement they entered the Church, the

64. doors of which were immediately closed, after some few of the
populace had managed to squeeze themselves in. A messenger was
at once despatched to the palace to hasten the fireworks, and to
request that a detachment of the Duke's guard should be sent into
the Church by a back way.
The darkness had by this time so much increased that few of
the people were aware of what had taken place, and the ignorance
of the crowd as to the cause of the tumult was so general that little
disturbance took place among those who were shut out of the
Church. They remained howling and hooting, it is true, for some
time, and some went so far as to beat against the closed doors; but
a rumour being spread among the crowd that the fireworks were
immediately to begin, they grew tired of this unproductive
occupation, and flocked almost to a man out of the square and
wharves, and crowded across the bridge into the gardens.
When the guard arrived, Inglesant claimed the man as the
Duke's prisoner, to be examined before him in the morning. The
curiosity of the Duke in all religious matters being well known, this
seemed very reasonable to the officer of the guard, and the priests
did not like to dispute it after the instructions they had received with
regard to Inglesant's mission. The two artisans were propitiated by a
considerable reward, and the prisoner was then transported by
unfrequented ways to the palace, and shut up in a solitary
apartment, whilst the rest of the world delighted itself at the palace
fêtes.
The garden festivities passed away amid general rejoicing and
applause. The finest effect was produced at the conclusion, when
the whole mass of water at the command of the engines, being

65. thrown into the air in thin fan-like jets, was illuminated by various
coloured lights, producing the appearance of innumerable rainbows,
through which the palace itself, the orangeries, the gardens, and
terraces, and the crowds of delighted people, were seen illuminated
and refracted in varied and ever-changing tints. Amid these sparkling
colours strange birds passed to and fro, and angelic forms
descended by unseen machinery and walked on the higher terraces,
and as it were upon the flashing rainbows themselves. Delicious
music from unseen instruments ravished the sense, and when the
scene appeared complete and nothing further was expected, an
orange grove in the centre of the whole apparently burst open, and
displayed the stage of a theatre, upon which antic characters
performed a pantomime, and one of the finest voices in Italy sang
an ode in honour of the day, of the Duke, and of the Pope.
CHAPTER VII.
The Duke had engaged the next morning to be present at a
theatrical representation of a religious character, somewhat of the
nature of a miracle play, to be given in the courtyard of the "Hospital
of Death," which adjoined to the Campo Santo of the city.
Before accompanying his Highness, Inglesant had given orders
to have the man, who had been the cause of so much excitement
the evening before, brought into his apartment, that he might see
whether or no his eccentricity made him sufficiently interesting to be
presented to the Duke.

66. When the stranger was brought to the palace early in the
morning, and having been found to be quite harmless, was
entrusted by the guard to two servants to be brought into
Inglesant's presence, he thought himself in a new world. Hitherto his
acquaintance with Italian life had been that of a stranger and from
the outside; he was now to see somewhat of the interior life of a
people among whom the glories of the Renaissance still lingered,
and to see it in one of the most wonderful of the Renaissance works,
the ducal palace of Umbria. Born in the dull twilight of the north,
and having spent most of his mature years amongst the green
mezzotints of Germany, he was now transplanted into a land of light
and colour, dazzling to a stranger so brought up. Reared in the
sternest discipline, he found himself among a people to whom life
was a fine art, and the cultivation of the present and its enjoyments
the end of existence. From room to room, as he followed his guide,
who pointed out from time to time such of the beauties of the place
as he considered most worthy of notice, the stranger saw around
what certainly might have intoxicated a less composed and
determined brain.
The highest efforts of the genius of the Renaissance had been
expended upon this magnificent house. The birth of a new instinct,
differing in some respects from any instincts of art which had
preceded it, produced in this and other similar efforts original and
wonderful results. The old Greek art entered with unsurpassable
intensity into sympathy with human life; but it was of necessity
original and creative, looking always forward and not back, and
lacked the pathos and depth of feeling that accompanied that new
birth of art which sought much of its inspiration among the tombs

67. and ruined grottoes, and most of its sympathetic power among the
old well-springs of human feeling, read in the torn and faded
memorials of past suffering and destruction. This new instinct of art
abandoned itself without reserve to the pursuit of everything which
mankind had ever beheld of the beautiful, or had felt of the pathetic
or the sad, or had dreamed of the noble or the ideal. The genius of
the Renaissance set itself to reproduce this enchanted world of form
and colour, traversed by thoughts and spiritual existences mysterious
and beautiful, and the home of beings who had found this form and
colour and these mysterious thoughts blend into a human life
delicious in its very sorrows, grotesque and incongruous in its
beauty, alluring and attractive amid all its griefs and hardships; so
much so indeed that, in the language of the old fables, the Gods
themselves could not be restrained from throwing off their divine
garments, and wandering up and down among the paths and the
adventures of men. By grotesque and humorous delineation, by
fanciful representation of human passion under strange and
unexpected form, by the dumb ass speaking and grasshoppers
playing upon flutes, was this world of intelligent life reproduced in
the rooms and on the walls of the house through which the stranger
walked for the first time.
He probably thought that he saw little of it, yet the bizarre
effect was burning itself into his brain. From the overhanging
chimney-pieces antique masques and figures such as he had never
seen, even in dreams, leered out upon him from arabesque carvings
of foliage, or skulked behind trophies of war, of music, or of the arts
of peace. The door and window frames seemed bowers of fruit and
flowers, and forests of carved leaves wreathed the pilasters and

68. walls. But this was not all; with a perfection of design and an
extraordinary power of fancy, this world of sylvan imagery was
peopled by figures and stories of exquisite grace and sweetness,
representing the most touching incidents of human life and history.
Men and women; lovers and warriors in conflicts and dances and
festivals, in sacrifices and games; children sporting among flowers;
bereavement and death, husbandry and handicraft, hunters and
beasts of chase. Again, among briony and jasmin and roses, or
perched upon ears of corn and sheaves of maize, birds of every
plumage confronted—so the grotesque genius willed—fish and sea
monsters and shells and marine wonders of every kind.
Upon the walls, relieved by panelling of wood, were paintings of
landscapes and the ruined buildings of antiquity overgrown with
moss, or of modern active life in markets and theatres, of churches
and cities in the course of erection with the architects and scaffold
poles, of the processions and marriages of princes, of the ruin of
emperors and of kings. Below and beside these were credenzas and
cabinets upon which luxury and art had lavished every costly device
and material which the world conceived or yielded. Inlaid with
precious woods, and glittering with costly jewels and marbles, they
reproduced in these differing materials all those infinite designs
which the carved walls had already wearied themselves to express.
Plaques and vases from Castel Durante or Faience,—some of a
strange pale colour, others brilliant with a grotesque combination of
blue and yellow,—crowded the shelves.
Passing through this long succession of rooms, the stranger
reached at last a library, a noble apartment of great size, furnished
with books in brilliant antique binding of gold and white vellum, and

69. otherwise ornamented with as much richness as the rest of the
palace. Upon reading desks were open manuscripts and printed
books richly illuminated. Connected with this apartment by open
arches, was an anteroom or corridor, which again opened on a
loggia, beyond the shady arches of which lay the palace gardens,
long vistas of green walks, and reaches of blue sky, flecked and
crossed by the spray of fountains. The decorations of the anteroom
and loggia were more profuse and extravagant than any that the
stranger had yet seen. There was a tradition that this portion of the
palace had been finished last, and that when the workmen arrived at
it the time for the completion of the whole was very nearly run out.
The attention of all the great artists, hitherto engaged upon different
parts of the entire palace, was concentrated upon this unfinished
portion, and all their workmen and assistants were called to labour
upon it alone. The work went on by night and day, not ceasing even
to allow of sleep. Unlimited supplies of Greek wine were furnished to
the workmen; and stimulated by excitement and the love of art,
emulating each other, and half-intoxicated by the delicious wine, the
work exceeded all previous productions. For wild boldness and
luxuriance of fancy these rooms were probably unequalled in the
world.
In the anteroom facing the loggia the stranger found Inglesant
conversing with an Italian who held rather a singular post in the
ducal Court. He was standing before a cabinet of black oak, inlaid
with representations of lutes and fifes, over which were strewn roses
confined by coloured ribbons, and supporting vases of blue and
yellow majolica, thrown into strong relief by the black wood. Above
this cabinet was a painting representing some battle in which a

70. former Duke had won great honour; while on a grassy knoll in the
foreground the huntsmen of Ganymede were standing with their
eyes turned upward towards the bird of Zeus, who is carrying the
youth away to the skies, emblematical of the alleged apotheosis of
the ducal hero. Richly dressed in a fantastic suit of striped silk, and
leaning against the cabinet in an attitude of listless repose, Inglesant
was contemplating an object which he held in his hand, and which
both he and his companion appeared to regard with intense interest.
This was an antique statuette of a faun, holding its tail in its left
hand, and turning its head and body to look at it,—an occupation of
which, if we may trust the monuments of antiquity, this singular
creature appears to have been fond. The Italian was of a striking
figure, and was dressed somewhat more gaily than was customary
with his countrymen; and the whole group was fully in unison with
the spirit of the place and with the wealth of beauty and luxury of
human life that pervaded the whole.
The man who was standing by Inglesant's side, and who had
the air of a connoisseur or virtuoso, was an Italian of some fifty
years of age. His appearance, as has been said, was striking at first
sight, but on longer acquaintance became very much more so. He
was tall and had been dark, but his hair and beard were plentifully
streaked with gray. His features were large and aquiline, and his face
deeply furrowed and lined. His appearance would have been
painfully worn, almost to ghastliness, but for a mocking and
humorous expression which laughed from his eyes, his mouth, his
nostrils, and every line and feature of his face. Whenever this
expression subsided, and his countenance sank into repose, a look
of wan sadness and even terror took its place, and the large black

71. eyes became fixed and intense in their gaze, as though some
appalling object attracted their regard.
This man had been born of a good but poor family, and had
been educated by his relations with the expectation of his becoming
an ecclesiastic, and he had even passed some time as a novice of
some religious order. The tendency of his mind not leading him to
the further pursuit of a religious life, he left his monastery, and
addressed himself to live by his wits, among the families and
households of princes. He had made himself very useful in arranging
comedies and pageantries, and he had at one time belonged to one
of those dramatic companies called "Zanni," who went about the
country reciting and acting comedies. Combined with this talent he
discovered great aptitude in the management of serious affairs, and
was more than once, while apparently engaged entirely on theatrical
performances, employed in secret State negotiations which could not
so well be entrusted to an acknowledged and conspicuous agent. In
this manner of life he might have continued; but having become
involved in one of the contests which disturbed Italy, he received a
dangerous wound in the head, and on rising from his sick bed in the
Albergo in which he had been nursed, he was merely removed to
another as a singular if not dangerous lunatic. The symptoms of his
disease first manifested themselves in a very unpleasant familiarity
with the secrets of those around him, and it was probably this
feature of his complaint which led to his detention. As he improved
in health, however, he ceased to indulge in any conversation which
might give offence, but, assuming a sedate and agreeable manner,
he conversed with all who came to him, calling them, although
strangers and such as he had never before seen, by their proper

72. names, and talking to them pleasantly concerning their parents,
relations, the coats-of-arms of their families, and such other
harmless and agreeable matters.
What brought him prominently into notice was the strangely
prophetic spirit he manifested before, or at the moment of the
occurrence of, more than one public event. He was taken from the
hospital and examined by the Pope, and afterwards at several of the
sovereign Courts of Italy. Thus, not long before the time when
Inglesant met him in the ducal palace at Umbria, he was at
Chambery assisting at the preparation of some festivals which the
young Duke of Savoy was engaged in celebrating. One day, as he
was seated at dinner with several of the Duke's servants, he
suddenly started up from his seat, exclaiming that he saw the Duke
de Nemours fall dead from his horse, killed by a pistol shot. The
Duke, who was uncle to the young monarch of Savoy, was then in
France, where he was one of the leaders of the party of the Fronde.
Before many days were passed, however, the news reached
Chambery of the fatal duel between this nobleman and the Duke of
Beaufort, which occurred at the moment the Italian had thus
announced it.
These and other similar circumstances caused the man to be
much talked of and sought after among the courts of Italy, where a
belief in manifestations of the supernatural was scarcely less
universal than in the previous age, when, according to an eye-
witness, "the Pope would decide no question, would take no journey,
hold no sitting of the Consistory, without first consulting the stars;
nay, very few cardinals would transact an affair of any kind, were it
but to buy a load of wood, except after consultation duly held with

73. some astrologer or wizard." The credit which the man gained, and
the benefits he derived from this reputation, raised him many
enemies, who did not scruple to assert that he was simply a clever
knave, who was not even his own dupe. Setting on one side,
however, the revelations of the distant and the unknown made by
him, which seemed inexplicable except by supposing him possessed
of some unusual spiritual faculty, there was in the man an amount of
knowledge of the world and of men of all classes and ranks,
combined with much learning and a humorous wit, which made his
company well worth having for his conversation alone. It was not
then surprising that he should be found at this juncture at the court
of Umbria, where the peculiar idiosyncrasies of the aged Duke, and
the interest attached to the intrigue for the session of the dukedom,
had assembled a strange and heterogeneous company, and towards
which at the moment all men's eyes in Italy were turned.
"Yes, doubtless, it is an antique," the Italian was saying,
"though in the last age many artists produced masques and figures
so admirable as to be mistaken for antiques; witness that masque
which Messire Georgio Vassari says he put in a chimney-piece of his
house at Arezzo, which every one took to be an antique. I have seen
such myself. This little fellow, however, I saw found in a vineyard
near the Miserecordia—a place which I take to have been at some
time or other the scene of some terrible event, such as a conflict or
struggle or massacre; for though now it is quiet and serene enough,
with the sunlight and the rustling leaves, and the splash of a
fountain about which there is some good carving, I think of Fra
Giovanni Agnolo,—for all this, I never walk there but I feel the

74. presence of fatal events, and a sense of dim figures engaged in
conflict, and of faint and distant cries and groans."
As he spoke these last words his eye rested upon the strange
figure of the man so hardly rescued from death the night before,
and he stopped. His manner changed, and his eyes assumed that
expression of intense expectation of which we have spoken before.
The appearance of the stranger, and the contrast it presented to the
objects around, was indeed such as to make him almost seem an
inhabitant of another world, and one of those phantasms of past
conflict of which the Italian had just spoken. His clothes, which had
originally been of the plainest texture, and most uncourtly make,
were worn and ragged, and stained with damp and dirt. His form
and features were gaunt and uncouth, and his gesture stiff and
awkward; but, with all this, there was a certain steadiness and
dignity about his manner, which threw an appearance of nobility over
this rugged and unpleasing form. Contrasted with the dress and
manner of the other men, he looked like some enthusiastic prophet,
standing in the house of mirth and luxury, and predicting ruin and
woe.
At this moment a servant entered the room, bringing a
sottocoppa of silver, upon which were two or three stiff necked
glasses, called caraffas, containing different sorts of wine, and also
water, and one or two more empty drinking-glasses, so that the
visitor could please himself as to the strength and nature of his
beverage. Inglesant offered this refreshment to the Italian, who
filled himself a glass and drank, pledging Inglesant as he did so. The
latter did not drink, but offered wine and cakes to the stranger, who
refused or rather took no heed of these offers of politeness; he

75. remained silent, keeping his eyes fixed upon the face of the man
who, but a few hours before, had saved him from a violent death.
"I have had some feelings of this kind myself, in certain places,"
said Inglesant, in answer to the Italian's speech, "and very
frequently in all places the sense of something vanishing, which in
another moment I should have seen; it has seemed to me that,
could I once see this thing, matters would be very different with me.
Whether I ever shall or not I do not know."
"Who can say?" replied the other. "We live and move amid a
crowd of flitting objects unknown or dimly seen. The beings and
powers of the unseen world throng around us. We call ourselves
lords of our own actions and fate, but we are in reality the slaves of
every atom of matter of which the world is made and we ourselves
created. Among this phantasm of struggling forms and influences
(like a man forcing his way through a crowd of masques who mock
at him and retard his steps) we fight our way towards the light.
Many of us are born with the seeds within us of that which makes
such a fight hopeless from the first—the seeds of disease, of
ignorance, of adverse circumstance, of stupidity; for even a dullard
has had once or twice in his life glimpses of the light. So we go on. I
was at Chambery once when a man came before the Duke in the
palace garden to ask an alms. He was a worker in gold, a good
artist, not unworthy of Cellini himself. His sight had failed him, and
he could no longer work for bread to give to his children. He stood
before the Prince and those who stood with him, among whom were
a Cardinal and two or three nobles, with their pages and grooms,
trying with his dim eyes to make out one from the other, which was
noble and which was groom, and to see whether his suit was

76. rejected or allowed. Behind him, beyond the garden shade, the
dazzling glitter stretched up to the white Alps. We are all the
creatures of a day, and the puny afflictions of any man's life are not
worth a serious thought; yet this man seemed to me so true an
image of his kind, helpless and half-blind, yet struggling to work out
some good for himself, that I felt a strange emotion of pity. They
gave him alms—some more, some less. I was a fool, yet even now I
think the man was no bad emblem of the life of each of us. We do
not understand this enough. Will the time ever come when these
things will be better known?"
As the Italian spoke the stranger took his eyes off Inglesant and
fixed them on the speaker with a startled expression, as though the
tone of his discourse was unexpected to him. He scarcely waited for
the other to finish before he broke in upon the conversation,
speaking slowly and with intense earnestness, as though above all
things desirous of being understood. He spoke a strange and
uncouth Italian, full of rough northern idioms, yet the earnestness
and dignity of his manner ensured him an audience, especially with
two such men as those who stood before him.
"Standing in a new world," he said, "and speaking as I speak, to
men of another language, and of thoughts and habits distinct from
mine, I see beneath the tinsel of earthly rank and splendour, and a
luxury of life and of beauty, the very meaning of which is unknown
to me, something of a common feeling, which assures me that the
voice I utter will not be entirely strange, coming as it does from the
common Father. I see around me a land given over to idolatry and
sensual crime, as if the old Pagans were returned again to earth;
and here around me I see the symbols of the Pagan worship and of

77. the Pagan sin, and I hear no other talk than that which would have
befitted the Pagan revels and the Pagan darkness which overhung
the world to come. Standing on the brink of a violent death, and
able to utter few words that can be understood, I call, in these short
moments which are given me, and in these few words which I have
at command—I call upon all who will listen to me, that they leave
those things which are behind, with all the filthy recollections of
ages steeped in sin, and that they press forward towards the light,—
the light of God in Jesus Christ."
He stopped, probably for want of words to clothe his thoughts,
and Inglesant replied,—
"You may be assured from the events of last night, signore, that
you are in no danger of violent death in this house, and that every
means will be taken to protect you, until you have been found guilty
of some crime. You must, however, know that no country can allow
its customs and its religion to be outraged by strangers and aliens,
and you cannot be surprised if such conduct is resented both by the
governors of the country and by the ignorant populace, though
these act from different motives. As to what you have said
respecting the ornaments and symbols of this house, and of the
converse in which you have found us engaged, it would seem that to
a wise man these things might serve as an allegory, or at least as an
image and representation of human life, and be, therefore, not
without their uses."
"I desire no representation nor image of a past world of
iniquity," said the stranger, "I would I could say of a dead life, but
the whole world lieth in wickedness until this day. This is why I travel
through all lands, crying to all men that they repent and escape the

78. most righteous judgment of God, if haply there be yet time. These
are those latter days in which our Saviour and Redeemer Jesus
Christ, the Son of God, predicted that iniquity 'should be increased;'
wherein, instead of serving God, all serve their own humours and
affections, being rocked to sleep with the false and deceitful lullaby
of effeminate pleasures and delights of the flesh, and know not that
an horrible mischief and overthrow is awaiting them, that the pit of
Hell yawns beneath them, and that for them is reserved the
inevitable rigour of the eternal fire. Is it a time for chambering and
wantonness, for soft raiment and dainty living, for reading of old
play-books such as the one I see on the table, for building houses of
cedar, painted with vermilion, and decked with all the loose and
fantastic devices which a disordered and debauched intellect could
itself conceive, or could borrow from Pagan tombs and haunts of
devils, full of uncleanness and dead sins?"
"You speak too harshly of these things," said Inglesant. "I see
nothing in them but the instinct of humanity, differing in its outward
aspect in different ages, but alike in its meaning and audible voice.
This house is in itself a representation of the world of fancy and
reality combined, of the material life of the animal mingled with
those half-seen and fitful glimpses of the unknown life upon the
verge of which we stand. This little fellow which I hold in my hand,
speaks to me, in an indistinct and yet forcible voice, of that common
sympathy—magical and hidden though it may be—by which the
whole creation is linked together, and in which, as is taught in many
an allegory and quaint device upon these walls, the Creator of us all
has a kindly feeling for the basest and most inanimate. My
imagination follows humanity through all the paths by which it has

79. reached the present moment, and the more memorials I can gather
of its devious footsteps the more enlarged my view becomes of what
its trials, its struggles, and its virtues were. All things that ever
delighted it were in themselves the good blessings of God—the
painter's and the player's art—action, apparel, agility, music. Without
these life would be a desert; and as it seems to me, these things
softened manners so as to allow Religion to be heard, who otherwise
would not have been listened to in a savage world, and among a
brutal people destitute of civility. As I trace these things backward
for centuries, I live far beyond my natural term, and my mind is
delighted with the pleasures of nations who were dust ages before I
was born."
"I am not concerned to dispute the vain pleasures of the
children of this world," exclaimed the stranger with more warmth
than he had hitherto shown. "Do you suppose that I myself am
without the lusts and desires of life? Have I no eyes like other men,
that I cannot take a carnal pleasure in that which is cunningly
formed by the enemy to please the eye? Am not I warmed like other
men? And is not soft clothing and dainty fare pleasing to me as to
them? But I call on all men to rise above these things, which are
transitory and visionary as a dream, and which you yourself have
spoken of as magical and hidden, of which only fitful glimpses are
obtained. You are pleasing yourself with fond and idle imaginations,
the product of delicate living and unrestrained fancies; but in this the
net of the devil is about your feet, and before you are aware you will
find yourself ensnared for ever. These things are slowly but surely
poisoning your spiritual life. I call upon you to leave these delusions,
and come out into the clear atmosphere of God's truth; to tread the

80. life of painful self-denial, leaving that of the powerful and great of
this world, and following a despised Saviour, who knew none of
these things, and spent His time not in kings' houses gorgeously
tricked out, but knew not where to lay His head. You speak to me of
pleasures of the mind, of music, of the painter's art; do you think
that last night, when beaten, crushed, and almost breathless, in the
midst of a blood-thirsty and howling crowd, I was dimly conscious of
help, and looking up I saw you in the glare of the lanterns, in your
courtier's dress of lace and silver, calm, beneficent, powerful for
good, you did not seem to my weak human nature, and my low
needs and instincts, beautiful as an angel of light? Truly you did; yet
I tell you, speaking by a nature and in a voice that is more unerring
than mine, that, to the divine vision, of us two at that moment you
were the one to be pitied,—you were the outcast, the tortured of
demons, the bound hand and foot, whose portion is in this life, who,
if this fleeting hour is left unheeded, will be tormented in the life to
come."
The Italian turned away his head to conceal a smile, and even
to Inglesant, who was much better able to understand the man's
meaning, this result of his interference to save his life appeared
somewhat ludicrous. The Italian, however, probably thinking that
Inglesant would be glad to be relieved from his strange visitor,
seemed desirous of terminating the interview.
"His Grace expects me," he said to Inglesant, "at the Casa di
Morte this morning, and it is near the time for him to be there. I will
therefore take my leave."
"Ah! the Casa di Morte; yes, he will expect me there also," said
Inglesant, with some slight appearance of reluctance. "I will follow

81. you anon."
He moved from the indolent attitude he had kept till this
moment before the sideboard, and exchanged with the Italian those
formal gestures of leave-taking and politeness in which his nation
were precise. When the Italian was gone Inglesant summoned a
servant, and directed him to provide the stranger with an apartment,
and to see that he wanted for nothing. He then turned to the
fanatic, and requested him as a favour not to attempt to leave the
palace until he had returned from the Duke. The stranger hesitated,
but finally consented.
"I owe you my life," he said,—"a life I value not at a straw's
weight, but for which my Master may perchance have some use
even yet. I am therefore in your debt, and I will give my word to
remain quiet until you return; but this promise only extends to
nightfall; should you be prevented by any chance from returning this
day, I am free from my parole."
Inglesant bowed.
"I would," continued the man, looking upon his companion with
a softened and even compassionate regard, "I would I could say
more. I hear a secret voice, which tells me that you are even now
walking in slippery places, and that your heart is not at ease."
He stopped, and seemed to seek earnestly for some phrases or
arguments which he might suppose likely to influence a courtier
placed as he imagined Inglesant to be; but before he resumed, the
latter excused himself on the ground of his attendance on the Duke,
and, promising to see him again on his return, left the room.
Inglesant found a carriage waiting to convey him to the
"Hospital of Death," as the monastic house adjoining the public

82. Campo Santo was called. The religious performance had already
begun. Passing through several sombre corridors and across a
courtyard, he was ushered into the Duke's presence, who sat,
surrounded by his Court and by the principal ecclesiastics of the city,
in an open balcony or loggia. As Inglesant entered by a small door in
the back of the gallery a most extraordinary sight met his eyes.
Beyond the loggia was a small yard or burial-ground, and beyond
this the Campo Santo stretching out into the far country. The whole
of the yard immediately before the spectators was thronged by a
multitude of persons, of all ages and ranks, apparently just risen
from the tomb. Many were utterly without clothing, others were
attired as kings, bishops, and even popes. Their attitudes and
conduct corresponded with the characters in which they appeared,
the ecclesiastics collecting in calm and sedate attitudes, while many
of the rest, among whom kings and great men were not wanting,
appeared in an extremity of anguish and fear. Beyond the sheltering
walls which enclosed the court the dazzling heat brooded over the
Campo Santo to the distant hills, and the funereal trees stood, black
and sombre, against the glare of the yellow sky. At the moment of
Inglesant's entrance it appeared that something had taken place of
the nature of an excommunication, and the ecclesiastics in the
gallery were, according to custom, casting candles and flaming
torches, which the crowd of nude figures below were struggling and
fighting to obtain. A wild yet solemn strain of music, that came
apparently from the open graves, ascended through the fitful and
half-stifled cries.
The first sight that struck upon Inglesant's sense, as he entered
the gallery from the dark corridors, was the lurid yellow light

83. beyond. The second was the wild confused crowd of leaping and
struggling figures, in a strange and ghastly disarray, naked or decked
as in mockery with the torn and disordered symbols of rank and
wealth, rising as from the tomb, distracted and terror-stricken as at
the last great assize. The third was the figure of the Duke turning to
him, and the eyes of the priests and clergy fixed upon his face. The
words that the fanatic had uttered had fallen upon a mind prepared
to receive them, and upon a conscience already awakened to
acknowledge their truth. A mysterious conviction laid hold upon his
imagination that the moment had arrived in which he was bound to
declare himself, and by every tie which the past had knotted round
him to influence the Duke to pursue a line of conduct from which his
conscience and his better judgment revolted. On the one hand, a
half-aroused and uncertain conscience, on the other, circumstance,
habit, interest, inclination, perplexed his thoughts. The conflict was
uneven, the result hardly doubtful. The eyes of friends and enemies,
of agents of the Holy See, of courtiers and priests, were upon him;
the inquiring glance of the aged Duke seemed to penetrate into his
soul. He advanced to the ducal chair, the solemn music that
streamed up as from the grave, wavered and faltered as if
consciousness and idea were nearly lost. Something of the old
confusion overpowered his senses, the figures that surrounded him
became shadowy and unreal, and the power of decision seemed no
longer his own.
Out of the haze of confused imagery and distracting thought
which surrounded him, he heard with unspeakable amazement the
Duke's words,—

84. "I have waited your coming, Mr. Inglesant, impatiently, for I
have a commission to entrust you with, or rather my daughter, the
Grand Duchess, has written urgently to me from Florence to request
me to send you to her without a moment's delay. Family matters
relating to some in whom she takes the greatest interest, and who
are well known, she says, to yourself, are the causes which lead to
this request."
Inglesant was too bewildered to speak. He had believed himself
quite unknown to the Grand Duchess, whom he had never seen, but
as he had passed before her in the ducal receptions at Florence.
Who could these be in whom she took so great an interest, and who
were known to him?
But the Duke went on, speaking with a certain melancholy in his
tone.
"I have wished, Mr. Inglesant," he said, "to mark in some way
the regard I have conceived for you, and the obligation under which
I conceive myself to remain. It may be that, in the course that
events are taking, it will no longer in a few weeks be in my power to
bestow favours upon any man. I desire, therefore, to do what I have
purposed before you leave the presence. I have caused the
necessary deeds to be prepared which bestow upon you a small fief
in the Apennines, consisting of some farms and of the Villa-Castle of
San Georgio, where I myself in former days have passed many
happy hours." He stopped, and in a moment or two resumed
abruptly, without finishing the sentence.
"The revenue of the fief is not large, but its possession gives the
title of Cavaliere to its owner, and its situation and the character of
its neighbourhood make it a desirable and delightful abode. The

85. letters of naturalization which are necessary to enable you to hold
this property have been made out, and nothing is wanting but your
acceptance of the gift. I offer it you with no conditions and no
request save that, as far as in you lies, you will be a faithful servant
to the Grand Duchess when I am gone."
The Duke paused for a moment, and then, turning slightly to his
chaplain, he said, "The reverend fathers will tell you that this affair
has not been decided upon without their knowledge, and that it has
their full approval."
These last words convinced Inglesant of the fact that had
occurred. Although the Duke had said nothing on the subject, he felt
certain that the deed of cession had been signed, and that for some
reason or other he himself was considered by the clerical party to
have been instrumental in obtaining this result, and to be deserving
of reward accordingly. He had never, as we have seen, spoken to the
Duke concerning the succession, and his position at the moment was
certainly a peculiar one. Nothing was expected of him but that he
should express his grateful thanks for the Duke's favour, and leave
the presence. Surely, at that moment, no law of heaven or earth
could require him to break through the observances of civility and
usage, to enter upon a subject upon which he was not addressed,
and to refuse acts of favour offered to him with every grace and
delicacy of manner. Whatever might be the case with other men, he
certainly was not one to whom such a course was possible. He
expressed his gratitude with all the grace of manner of which he was
capable, he assured the Duke of his readiness to start immediately
for Florence, and he left the ducal presence before many minutes
had passed away.

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