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 Seunghee Cha Editor

Salivary Gland
Development and
Regeneration

Advances in Research and

Clinical Approaches to
Functional Restoration

123
Salivary Gland Development
and Regeneration
Seunghee Cha
Editor

Salivary Gland
Development and
Regeneration
Advances in Research and Clinical
Approaches to Functional
Restoration
Editor
Seunghee Cha
Oral and Maxillofacial Diagnostic Sciences
University of Florida
Gainesville, Florida
USA

ISBN 978-3-319-43511-4    ISBN 978-3-319-43513-8 (eBook)

DOI 10.1007/978-3-319-43513-8

Library of Congress Control Number: 2017933465

© Springer International Publishing Switzerland 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
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Preface

Rome was not built in a day, as the English playwright John Heywood
famously wrote. Innovation and advancement in the field of salivary gland
regeneration is one of the great examples that reflects this sentiment. The first
research article available on this topic through the US National Library of
Medicine dates back to 1950. The article, entitled “Regeneration in the
Submaxillary Gland of the Rat,” by B.B. Milstein in 1950, cites Van
Podwyssozki as the first to describe regeneration of organs of small animals
as long ago as 1886 and regeneration of the salivary glands (Die Regeneration
an den Speicheldrusen) in 1887.
Since the 1950s, an ever-expanding literature and diversified approaches
aimed at functional restorations have mirrored strong interest and attention to
this particular subject of research. Journal articles dealing with autoimmune
Sjögren’s syndrome, effects of radiation, and ductal ligation models in rats
and mice appeared in the early 1980s, followed by research on neural regula-
tion of secretion and effectiveness of epidermal growth factor in wound heal-
ing models and glandular regeneration in the late 1980s through the
mid-1990s.
In 1995, the late Dr. Michael Humphreys published his well-known review
article entitled “Saliva and growth factors: the fountain of youth resides in us
all,” which emphasized the vital importance of growth factors in oral/sys-
temic health and glandular repair/regeneration. Histological analyses of glan-
dular architecture and development were established by Dr. Robert Redman,
the author of Chap. 4 of this volume. With the turn of a new century, molecu-
lar and cellular mechanisms of branching morphogenesis and glandular
development were further investigated and pioneered by Drs. Kenneth
M. Yamada and Matthew P. Hoffman, whose work provided foundations for
the application of tissue engineering concepts and methodologies to salivary
regeneration. Outstanding contributions by Dr. Bruce J. Baum to the field of
tissue engineering and gene therapy have ultimately been solidified in appli-
cations such as clinical trials involving AAV2-mediated human aquaporin-1
delivery in recent years. Investigation of ductal ligation models, irradiation
models, and Sjögren’s syndrome NOD models dominated interest in the field
until around 2010, when stem cell research in vitro and in vivo reignited
research interest and passion in salivary gland regeneration.
In the current era, the authors and coauthors in this book, who are renowned
researchers, dentists, and surgeons in the field, have spearheaded efforts to
discover the underlying pathogenesis of xerostomia and innovative approaches

v
vi Preface

to restore secretory function. I am proud to present their collective efforts and

years of their research outcomes revealed in their book chapters, which will
establish another significant milestone in the history and tradition of studies
on glandular regeneration.
This book begins with the description of fundamental and molecular pro-
cesses occurring during salivary gland organogenesis/branching morphogen-
esis and molecular communications among epithelial, mesenchymal,
endothelial, and neuronal cells for cellular differentiation and organ develop-
ment (Chap. 1, Dr. Lombaert). The importance of understanding the commu-
nications and simulating optimal environments in glandular repair and
regeneration is further discussed under Future Prospects.
With rapidly advancing biotechnology, the application of systems biology
has become an indispensable tool in this field. Chapter 2 discusses the defini-
tion and applications of systems biology for glandular tissues and saliva sam-
ples (Chap. 2, Dr. Larson et al.). Systems biology approaches in conjunction
with traditional approaches unveil the complex molecular, cellular, and phys-
ical processes in development, disease processes, and regenerative medicine
involving the salivary glands.
One of the underappreciated subjects in the field is the important role of a
large family of mucins in oral health. In Chap. 3, the authors summarize the
main structural and functional characteristics of salivary mucins, their expres-
sion patterns during salivary gland development and regeneration, and quali-
tative and quantitative changes in pathological processes in the salivary
glands due to irradiation, autoreactive immune cells, neoplasm, or inflamma-
tion (Chap. 3, Dr. Castro et al.).
Changes due to radiation are not limited to mucin expression profiles but
are also manifested in the parenchymal and stromal structures in the salivary
glands. These changes are detailed in Chap. 4 with photomicrographs and
transmission electron micrographs of rat and human salivary glands (Chap. 4,
Dr. Redman). Understanding the damage occurring in the glands before and
after radiation therapy will expedite the development of intervention strate-
gies to protect the salivary glands from the harmful radiation.
In Chap. 5, Dr. Tran’s group reviews recent advances from the years 2010
to 2015 in the treatment of salivary gland hypofunction with a special empha-
sis on mesenchymal stem cells (Chap. 5, Dr. Tran et al.). This chapter covers
in detail adipose tissue-derived stromal cells, mesenchymal stromal cells
derived from various sources, and finally the authors’ experience with the
soluble contents/factors in bone marrow soup extracted from a whole bone
marrow cell lysate.
As differentiation-inducing factors are crucial for initiating stem cell dif-
ferentiation from the state of quiescence, these extrinsic and intrinsic factors
(transcription factors) involved in pancreas, liver, and salivary gland regen-
eration are further detailed in Chap. 6 with a focus on directed-cell differen-
tiation and transdifferentiation (Chap. 6, Drs. Park and Cha).
Current cell models for bioengineering of the salivary glands are presented
in Chap. 7, along with the pros and cons of utilizing various salivary cell
lines. Practical tips on cell isolation and culture techniques in conjunction
with the use of scaffolds complement the use of stem, progenitor, and acinar
Preface vii

cells for salivary gland regeneration. Current trends in salivary gland bioen-
gineering deliver great promise in functional restoration of the salivary glands
(Chap. 7, Dr. Baker).
To explore further the subject of bioengineering, factors and elements
needed for successful development of a functional salivary gland are dis-
cussed in detail in Chap. 8, emphasizing the dynamic nature of the basement
membrane and the significance of the extracellular matrix and cell polarity
in salivary gland development and reconstruction. In addition, studies utiliz-
ing the salivary-derived stem cells/gland progenitor and three-dimensional
(3D) ­biomimetic scaffolds encompassing decellularization methods, various
matrices, and polymers are summarized for 3D culture technique, which
underpins ­ current knowledge on bioengineering of the salivary glands
(Chap. 8, Martinez et al.).
3D printing technology creates life-size body parts and tissues using living
cells as the ink. This technology has revolutionized the field of regenerative
and reconstructive medicine, enabling customized and personalized thera-
peutic approaches. In Chap. 9, Dr. Choi et al. describe basic principles and
different types of 3D technologies, patient-specific modeling, bioprinting,
and salivary gland regeneration (Chap. 9, Dr. Choi et al.).
A novel bioengineering method involves epithelial and mesenchymal stem
cell manipulation to generate a bioengineered organ germ. In Chap. 10,
Dr. Ogawa explains that the bioengineered glandular germs demonstrated
reciprocal interactions between epithelial and mesenchymal cells in one day
and invagination of epithelial tissue in three days in vitro. Once the germ was
engrafted into the parotid gland duct of salivary gland-defective mice, the
connection between the germ and the duct was established in a month, and
the mice exhibited restored salivary secretion after transplantation. This inno-
vative approach emphasizes that current advancement in the field promises a
therapeutic intervention for patients suffering from xerostomia (Chap. 10,
Drs. Ogawa and Tsuji).
Currently, functional restoration of the salivary glands is still challenging
to accomplish even with successful reconstruction of salivary cellular compo-
nents. Therefore, understanding the mechanisms of saliva secretion becomes
critical for positive clinical outcomes that we desire. Chapter 11 covers con-
siderations for establishing functional secretion by providing information on
stimuli for secretion, neural connection along with neurotransmitters and
receptors, protein secretion, and studies of neural agonists and antagonists.
The chapter also clarifies myths surrounding this topic with recent research
data (Chap. 11, Drs. Carpenter and Carvalho).
Thought-provoking renderings of the past, current, and future of gene
therapy in salivary gland diseases are provided by Dr. Passineau in Chap. 12.
In this chapter, current challenges in the field of salivary gland gene therapy,
along with the author’s proposals to circumvent or overcome the hurdles, are
forthrightly discussed (Chap. 12, Dr. Passineau).
Last, but not least, the chapter on surgical management of salivary gland
disease reveals the critical considerations for glandular regeneration from the
perspectives of otolaryngologists and surgeons (Chap. 13, Drs. Varadarajan
and Dziegielewski). The extensive description in this chapter includes, but is
viii Preface

not limited to, glandular anatomy, pathology, surgical advances for neoplastic
and nonneoplastic diseases of salivary glands, and recent discoveries in the
field such as salivary gland transfer and salivary duct repositioning. The
importance of understanding the expected sequelae in human patients follow-
ing radiation or surgery cannot be overemphasized as none of the existing
laboratory approaches would come to fruition for patients without such
knowledge.
Based on the cutting-edge information offered in this book, it is undoubt-
able that many more innovative strategies for salivary gland regeneration will
emerge in upcoming years. Research that unlocks the complex processes of
organ development would be fundamental to develop such approaches. With
the current enthusiasm and growing interest in the field, it will just be a matter
of time before we build another Rome.

Gainesville Seunghee Cha, DDS, PhD

FL, USA
Contents

Part I Updates on Salivary Gland Development

1 Implications of Salivary Gland Developmental Mechanisms

for the Regeneration of Adult Damaged Tissues. . . . . . . . . . . . . . . 3
Isabelle M.A. Lombaert
2 Systems Biology: Salivary Gland Development, Disease,
and Regenerative Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Melinda Larsen, Petko Bogdanov, Ravi Sood,
Hae Ryong Kwon, Deirdre A. Nelson, Connor Duffy,
Sarah B. Peters, and Sridar V. Chittur
3 Mucins in Salivary Gland Development,
Regeneration, and Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Isabel Castro, María-José Barrera, Sergio González,
Sergio Aguilera, Ulises Urzúa, Juan Cortés
and María-Julieta González

Part II Glandular Damage and Cell Replacement Therapy

4 Histologic Changes in the Salivary Glands

Following Radiation Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Robert S. Redman
5 Adult Stem Cell Therapy for Salivary Glands,
with a Special Emphasis on Mesenchymal Stem Cells . . . . . . . . . 93
Simon D Tran, Yoshinori Sumita, Dongdong Fang,
and Shen Hu
6 Directed Cell Differentiation by Inductive Signals
in Salivary Gland Regeneration: Lessons Learned
from Pancreas and Liver Regeneration . . . . . . . . . . . . . . . . . . . . 103
Yun-Jong Park and Seunghee Cha

Part III Bioengineering of Salivary Glands

7 Current Cell Models for Bioengineering Salivary Glands . . . . . 133

Olga J. Baker

ix
x Contents

8 Matrix Biology of the Salivary Gland:

A Guide for Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Mariane Martinez, Danielle Wu, Mary C. Farach-­Carson,
and Daniel A. Harrington
9 3D Printing Technology in Craniofacial
Surgery and Salivary Gland Regeneration. . . . . . . . . . . . . . . . . . 173
Jong Woo Choi, Namkug Kim, and Chang Mo Hwang
10 Functional Salivary Gland Regeneration
by Organ Replacement Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . 193
Miho Ogawa and Takashi Tsuji

Part IV Therapeutic Considerations for Restoration of

Salivary Function

11 Regulation of Salivary Secretion. . . . . . . . . . . . . . . . . . . . . . . . . . 207

Guy Carpenter and Polliane Carvalho
12 Salivary Gland Gene Therapy in Experimental
and Clinical Trials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Michael Passineau
13 Surgical Management of Salivary Gland Disease . . . . . . . . . . . . 229
Varun V. Varadarajan and Peter T. Dziegielewski
Contributors

Sergio Aguilera, MD INDISA Clinic, Santiago, Chile

Olga J. Baker, DDS, PhD School of Dentistry, University of Utah, Salt
Lake City, UT, USA
María-­José Barrera, PhD Institute of Biomedical Sciences, Faculty of
Medicine, University of Chile, Santiago, Chile
Petko Bogdanov, PhD Department of Computer Science, University at
Albany, SUNY, Albany, NY, USA
Guy Carpenter, PhD Mucosal and Salivary Biology Division, King’s
College London Dental Institute, London, UK
Polliane Carvalho, MSc, PhD Mucosal and Salivary Biology Division,
King’s College London Dental Institute, London, UK
Isabel Castro, MSc, PhD Institute of Biomedical Sciences, Faculty of
Medicine, University of Chile, Santiago, Chile
Seunghee Cha, DDS, PhD Department of Oral and Maxillofacial
Diagnostic Sciences, University of Florida College of Dentistry, Gainesville,
USA
Sridar V. Chittur, PhD Center for Functional Genomics, University at
Albany, SUNY, Albany, NY, USA
Jong-Woo Choi, MD, PhD, MMM Department of Plastic and
Reconstructive Surgery, Ulsan University College of Medicine, Asan
Medical Center, Seoul, South Korea
Juan Cortés, PhD Institute of Biomedical Sciences, Faculty of Medicine,
University of Chile, Santiago, Chile
Connor Duffy Biological Sciences, University at Albany, SUNY, Albany,
NY, USA
Peter T. Dziegielewski, MD, FRCS(C) University of Florida Department
of Otolaryngology, Gainesville, FL, USA
Dongdong Fang, BDS, MSc McGill Craniofacial Tissue Engineering and
Stem Cells Laboratory, Department of Biomedical Sciences, Faculty of
Dentistry, McGill University, Montreal, Quebec, Canada

xi
xii Contributors

Mary C. Farach-Carson, PhD BioScience Research Collaborative,

Department of Biochemistry and Cell Biology, Houston, TX, USA
Departments of BioSciences and Bioengineering, Rice University, Houston,
TX, USA
María-­Julieta González, MSc Institute of Biomedical Sciences, Faculty of
Medicine, University of Chile, Santiago, Chile
Sergio González, MSc Mayor University, Santiago, Chile
Daniel A. Harrington, PhD Departments of BioSciences and
Bioengineering, Rice University, Houston, TX, USA
Shen Hu, PhD, MBA Division of Oral Biology and Medicine, School of
Dentistry, University of California, Los Angeles, CA, USA
Chang Mo Hwang, PhD Biomedical Engineering Research Center,
University of Ulsan College of Medicine, Asan Medical Center, Seoul,
South Korea
Namkug Kim, PhD Department of Convergence Medicine, Biomedical
Engineering Research Center, University of Ulsan College of Medicine,
Asan Medical Center, Seoul, South Korea
Hae Ryong Kwon, PhD Biological Sciences, University at Albany, SUNY,
Albany, NY, USA
Oklahoma Medical Research Foundation, Oklahoma City, OK, USA
Melinda Larsen, PhD Biological Sciences, University at Albany, SUNY,
Albany, NY, USA
Isabelle M.A. Lombaert, PhD University of Michigan, School of
Dentistry, Ann Arbor, MI, USA
Biointerfaces Institute, Ann Arbor, MI, USA
Mariane Martinez Department of BioSciences, Rice University, Houston,
TX, USA
Deirdre A. Nelson, PhD Biological Sciences, University at Albany, SUNY,
Albany, NY, USA
Miho Ogawa, PhD Organ Technologies Inc., Tokyo, Japan
Laboratory for Organ Regeneration, RIKEN Center for Developmental
Biology, Kobe, Hyogo, Japan
Yun-Jong Park Department of Pharmacology, The University of North
Carolina at Chapel Hill, Chapel Hill, NC, USA
Michael Passineau, PhD Gene Therapy Program, Allegheny Health
Network, Pittsburgh, PA, USA
Sarah B. Peters, PhD Biological Sciences, University at Albany, SUNY,
Albany, NY, USA
Department of Cell, Developmental, and Integrative Biology, University of
Alabama at Birmingham, Birmingham, AL, USA
Contributors xiii

Robert S. Redman, DDS Oral and Maxillofacial Diagnostic Science,

University of Florida, Gainesville, FL, USA
Washington DC VA Medical Center, Washington, DC, USA
Ravi Sood Department of Computer Science, University at Albany, SUNY,
Albany, NY, USA
Yoshinori Sumita, DDS, PhD Department of Regenerative Oral Surgery,
Nagasaki University, Nagasaki, Japan
Simon D. Tran, DMD, PhD McGill Craniofacial Tissue Engineering and
Stem Cells Laboratory, Department of Biomedical Sciences, Faculty of
Dentistry, McGill University, Montreal, Quebec, Canada
Takashi Tsuji, PhD Organ Technologies Inc., Tokyo, Japan
Laboratory for Organ Regeneration, RIKEN Center for Developmental
Biology, Kobe, Hyogo, Japan
Ulises Urzúa, PhD Institute of Biomedical Sciences, Faculty of Medicine,
University of Chile, Santiago, Chile
Varun V. Varadarajan, MD University of Florida Department of
Otolaryngology, Gainesville, FL, USA
Danielle Wu, PhD Department of BioSciences, Rice University, Houston,
TX, USA
 Part I
Updates on Salivary Gland Development
 Implications of Salivary Gland
Developmental Mechanisms 1
for the Regeneration of Adult
Damaged Tissues

Isabelle M.A. Lombaert

Abstract
The convergence of the fields of tissue engineering and regenerative
medicine provides a potential blueprint to repair damaged tissues.
Accordingly, a range of therapeutic applications have emerged that hold
great potential to regenerate branching organs, such as salivary glands.
This unique saliva-secreting organ is required for proper oral health,
lubrication, immunity, and food digestion but is susceptible to damage
either by co-­irradiation as a side effect of radiotherapy cancer treatment,
autoimmune-­related Sjögren syndrome, disease-related medications, or
surgical resection. This chapter focuses on fundamental cellular and
molecular processes occurring during organ ontogenesis and in develop-
ing branching glands. We cover the growth of the epithelial compart-
ment, which is the major functional component of the gland, but also
how surrounding niches such as mesenchymal, endothelial, and neuronal
cells communicate, intertwine, and influence the formation of glands
and other branching organs. Finally, we highlight how this key informa-
tion has created new regenerative-related approaches and how these
impact future clinical translation.

1.1 Introduction

Increasing our knowledge of how organs develop

has profound implications for the design of thera-
pies to regrow and/or repair injured tissues.
I.M.A. Lombaert, PhD
University of Michigan, School of Dentistry,
Understanding the mechanisms regulating cell
2800 Plymouth Road, Ann Arbor, MI 48109, USA survival, expansion, specification, movement,
Biointerfaces Institute, 2800 Plymouth Road,
communication with neighboring cells, as well as
Ann Arbor, MI 48109, USA how they respond to damage is critical to navigat-
e-mail: [email protected] ing the landscape of future therapy designs.

© Springer International Publishing Switzerland 2017 3

S. Cha (ed.), Salivary Gland Development and Regeneration, DOI 10.1007/978-3-319-43513-8_1
4 I.M.A. Lombaert

In order to appropriately translate informa- o­ utlined in depth in the following chapters. In

tion gathered from studies on organ develop- this review, we focus on different salivary gland
ment, we need to compare molecular and cellular cell types and their supportive environment that
processes during embryonic development with is needed to form the fully functional secretory
adult homeostasis and when repair initiates and/ branching organ. Subsequently, we outline how
or fails after each damaging event. Each of these this knowledge can render future therapeutic
stages correlates with specific cellular responses, implications and/or what potential complications
activation of specific signaling pathways, and might arise.
accumulation of environmental cues. Thus,
developmental-related information is instrumen-
tal to stimulate regrowth within an existing dam- 1.2  pithelial Growth Driven
E
aged in vivo organ or to initiate de novo growth. by Stem Cells
The majority of our current knowledge on
salivary gland organogenesis derives from exper- Branching organs such as salivary, lacrimal, and
imental animal models, primarily mice and rats. mammary glands are comprised of different cell
While rodent biology is not identical to that of types, including epithelial and the surrounding
humans, many processes and pathways are very mesenchymal, endothelial, and neuronal cells
similar. As such, developmental biologists have (Fig. 1.1). Intertwined within these tissues are
been and continue to be a valuable resource to circulating hematopoietic-related blood and
other disciplines such as engineering, oral sur- immune cells. The major component of develop-
gery, and oncology to translate conceptual ideas ing and adult salivary glands (SGs) is the epithe-
into therapeutic designs. lia, which is responsible for saliva secretion and
The advantages of specific biomaterials, gene transportation to the oral cavity. Here, we
therapy, and surgical in vivo approaches are describe how the epithelial compartment of three

a c

Fig. 1.1 Developing salivary glands in mice. (a) Bright (arrow). Confocal image of E-cadherin stained epithelia
field picture represents E13 submandibular (SMG) and and Tubbulin-3 stained PSG. (c) Different niches sur-
sublingual gland (SLG). The epithelial compartment is rounding the epithelium in adult mouse submandibular
comprised of a distal endbud and proximal duct area. (b) gland. Confocal 30 μm projected image of stained SMG
Epithelia (blue) innervated by the parasympathetic with epithelial marker E-cadherin (blue), neuronal
nerves (PSG, red) during embryonic SMG development. marker Tubbulin-3 (red), and endothelial protein CD31
The PSG releases neurotransmitters via varicosities (green)
1 Implications of Salivary Gland Developmental Mechanisms for the Regeneration of Adult Damaged Tissues 5

major glands, which provide 90 % of total saliva, they are not yet fully functional and must still
becomes established by tightly controlled mecha- undergo specific acinar-lineage maturation so that
nisms of cellular interactions. by birth, in both humans and mice, the organ is
comprised of functional secretory compartments.

1.2.1 Morphological Development

of Salivary Glands 1.2.2  ontribution of Stem Cells
C
and Their Differentiating
Salivary glands originate as an invagination of the Progeny
oral epithelium from a placode at embryonic day
(E) 11.5 in mice or Carnegie stage 18 (~44 days, The stem/progenitor cell theory asserts that all
weeks 6–7) in humans. This thickening epithe- salivary gland cells are initiated from and main-
lium arises on the side of the tongue outside of the tained by stem and progenitor cells. By defini-
lamina dentalis at the anlage of the dental arch. tion, these cells are characterized by their ability
Each major gland initiates at slightly different to expand themselves, i.e., self-renew, as well as
locations: the serous parotid gland (PAR) in the to propagate multiple more defined cell types,
labiogingival sulcus, the mucous sublingual such as acinar cells. Stem cells are further clas-
(SLG) in the paralingual sulcus, and seromucous sified as being more potent than progenitor cells
submandibular gland (SMG) in the linguogingival in their self-renewal and differentiation potential.
sulcus. Even though glands arise in the tongue When both these cellular processes are tightly
area, they grow out during development toward controlled, stem/progenitor cells not only give
the back of the mouth below the ears, floor of the rise to tissues but also maintain and repair organ
mouth near the mandibular bone, and the anterior structures during adulthood. Any deregulation in
floor of the mouth. While in mice, SMG, SLG, this regulatory network during development can
and PAR initiate around E11.5, E12, and E13, lead to malformation/absence of the organ and in
respectively, human SLGs initiate later than SMG the adult may cause cancer formation. Over the
and PAR at the ninth embryonic month. More past years, remarkable progress has been made
detailed descriptions on anatomic locations of wherein multiple stem/progenitors have been
human glands have been recently reviewed [41]. classified based on their ability to (1) form mul-
The developmental origin of each gland has not tiple cell types (mouse genetic lineage tracing,
been clear, with some classifying the PAR as ecto- ex vivo culturing), (2) alternate quiescence with
dermal derived and SMG and SLG as endoder- proliferation (BrdU incorporation or genetically
mal, while genetic experiments in mice suggest labeled DNA tracing), and (3) restore radiation-­
they are all ectodermal [87]. induced damaged SGs (in vivo transplantation
Once the epithelial thickening arises, a cell assay). One new consensus gathered from this
population, termed endbud or tip, forms dis- data is that different stem/progenitor cells con-
tally from an elongating stalk (Fig. 1.1a), which tribute to the growing SG and that these cells
developmentally progresses to form major ducts may originate at different time points during
termed Wharton’s (SMG), Bharton’s (SLG), and development. Importantly, this permits the organ
Stensen’s (PAR) ducts. The unique SG branch- to compensate for any losses in specific stem/pro-
ing pattern is created by repetitive clefting of the genitor cells and still allows proper development
initial and subsequently formed endbuds. Ductal [88]. Known stem/progenitors contributing to
structures gradually mature by elongation, lumen SG organogenesis include cells marked by their
formation, and expansion. The clefting endbuds expression of intracellular cytokeratin 5 (CK5,
mature by E16 in mice and 19–24 weeks (7th K5) and CK14 [50, 57], receptors KIT (c-Kit,
month) in humans to form polarized pro-acinar CD117) and FGFR2b [57], and transcription fac-
and pro-myoepithelial cells. While pro-acinar tors SOX2 [3] and ASCL3 [10]. Remarkably,
cells do express some secretory-related proteins, stem/progenitors contributing to development
6 I.M.A. Lombaert

Fig. 1.2 Signaling

pathways influencing
epithelial growth. Cartoon
represents known signaling
pathways that influence SG
epithelial cell survival,
proliferation, expansion,
and differentiation. Green:
expressed by epithelial
cells; orange: expressed by
mesenchyme; red:
expressed by neuronal
cells; black: expression by
multiple compartments

might not serve a similar role during adult to ­significantly increased saliva levels [58, 62, 72,
homeostasis. Recent studies observed active pro- 103]. This does not, however, exclude the poten-
liferation of cells within specific compartments, tial of other SG-specific epithelial cells, non-SG
such as acini and intercalated, striated, and excre- specific cells, and/or their bioactive cell lysate to
tory ducts, wherein these cells self-duplicate to contribute to the repair of damaged SGs. These
replenish their own entity, as reviewed in [4]. To options will be surveyed in following chapters,
what extent these adult compartmental “reser- and their impact on when to use them in different
voir” cells contribute to recovery after injury is damaging situations has been recently reviewed
a focus of ongoing research. At least after severe [61]. In this chapter, we will further outline our
radiation-induced damage, which leads to irre- current understanding of how SGs are structur-
versible hyposalivation, there is no active repair ally built by various cell types (Fig. 1.1b, c) and
initiated by remaining SG cells. This is often a how their continuous interactions are informing
combinatorial result of (a) drastic loss of acinar the design of current and future therapies.
and duct “reservoir” cells or stem/progenitor
cells, (b) decrease in signaling pathways required
to activate surviving “reservoir” or stem/pro- 1.2.3  essons from Developmental
L
genitor cells, and/or (c) severely damaged cells Regulatory Mechanisms
that can no longer contribute to self-duplication Guiding the Epithelium
or differentiation. In such cases, multiple strat-
egies ranging from constructing a new gland to Often disorders in humans and genetic rodent
gene therapy and stem/progenitor cell transplan- model systems can provide critical information
tations may aid in restoring the functional and on what signaling pathways are essential for epi-
morphological components of the gland. Thus thelial cell survival, proliferation, differentiation,
far, transplantations of cells selected for their and movement (Fig. 1.2). Major examples are
expression of receptor KIT, EPCAM, CD24, and/ Fgf10−/− and Fgfr2b−/− mice, which are related to
or CD29 (Integrin β1, ITGβ1) were shown to human loss-of-function mutations in FGF10 and
restore acinar and ductal compartments, leading FGFR2 that result in hereditary diseases ­including
1 Implications of Salivary Gland Developmental Mechanisms for the Regeneration of Adult Damaged Tissues 7

lacrimal and SG-related aplasia of lacrimal and branching via epithelial ITGβ1 [84]. MMPs also
salivary glands (ALSG), lacrimo-auriculo-­dento- cleave pro-HB-EGF into an N-terminal and
digital (LADD) syndrome, and lung-­ related C-terminal fragment at the membrane so that the
chronic obstructive pulmonary disease (COPD). latter fragment can move to the nucleus to acti-
In these conditions, SG development is stalled, as vate cell proliferation via cyclin A [95].
FGFR2b+ epithelium no longer receives survival Conversely, another member of the EGF family,
and proliferative cues from the surrounding neuregulin (NRG), binds ERBB3 on endbuds to
FGF10-producing mesenchyme [80]. Thus, when aid in their local expansion [70]. NRG1 is further
invading oral epithelial cells at gland ontogenesis essential for innervation as Nrg1−/− mice are
receive FGF10, they initiate an endbud and duct devoid of nerves and show aberrant duct forma-
formation. From then on, FGFR2b signaling tion and lumenization [73].
expands KIT+ progenitors in the continuously Similar to FGF10, Fgf8 hypomorphic and
clefting endbuds in combination with stem cell Fgfr2c heterozygous mice exhibit hypoplastic
factor (SCF)/KIT signaling [57]. As FGF10 has a glands due to reduced communication between
heparan-binding (HB) core, it evokes and expands FGF8-producing epithelia and FGFR2c-receiving
more rapid responses once it is bound to specific mesenchyme. In both FGF-deficient mice, initial
3-O-sulfated heparin sulfate (3-O-HS). This HS epithelial invagination occurs, but subsequent SG
belongs to a group of heparan sulfate proteogly- growth does not occur. To date, FGF8 has been
cans (HSPGs) located in the basement membrane described as a potential target of the EDA path-
or at cell surfaces. Interestingly, KIT+ endbud way. Human mutations in ectodysplasin-A
progenitors highly express HS3ST3, the 3-O-HS- (EDA) or its receptor EDAR result in hypohi-
specific modifying enzyme 3-O-sulfotransferase, drotic ectodermal dysplasia (HED). Defects in
to rapidly increase their expansion during devel- teeth, hair, sweat, and salivary glands are notice-
opment [80]. A similar function remains during able due to reduced cell proliferation and differ-
adult homeostasis. Regulating this FGFR2b sig- entiation [45, 74]. In SGs, EDA and downstream
naling pathway is of crucial importance so that target NF-kB aid in ductal lumenization and end-
every epithelial cell does not undergo extensive bud branching, presumably by inducing ductal
proliferation. Ductal cells therefore express FGF maturation within the center of endbuds. Early
antagonists, Sprouty 1 and 2, to lower FGFR2b on, mesenchyme-produced EDA is downstream
signaling and upregulate WNT [48]. Both canon- of mesenchymal WNT and upstream of epithelial
ical WNT/β-catenin and noncanonical WNT5b SHH (sonic hedgehog) signaling. As such, SHH
pathways drive ductal formation via upregulation treatment can rescue SGs deprived of EDA [100].
of Tfcp2l1 while inhibiting endbud development. After E13, EDA does not seem to correlate with
In turn, endbuds repress duct development by WNT anymore, based on their different expres-
FGF-mediated Wnt5b repression and secretion of sion pattern located in the epithelial or mesen-
WNT ­ligand-­sequestering protein SFRP1 [78]. chymal compartment [31, 78]. SHH’s important
Evidently, a tight FGF-WNT gradient allows for role in SG development has been confirmed, as
KIT+ progenitor expansion in endbuds, while SHH-deficient SGs are hypoplastic with unpolar-
ductal cells prepare for upcoming lumenization ized epithelial cells and underdeveloped lumen
and maturation. In this process, ERBB1 (EGFR)+ formation [36, 43]. SHH is also linked to FGF8
ductal K5+ progenitors proliferate in response to as both can upregulate each other [43]. Therefore,
HB-EGF to give rise to maturing K19+ cells [50]. FGF8 is able to rescue Hedgehog inhibition but
One mechanism of action is via induction of surprisingly not EDA deficiency [43]. As such,
membrane-type-2 matrix metalloproteinase EDA-FGF8’s precise signaling interaction still
(MT2-MMP) and FGFR expression in epithelial needs to be determined.
cells. MT2-MMP is crucial to release bioactive FGF signaling, in particular via FGFR1 (vari-
NC1 domains from extracellular matrix (ECM) ant b in the epithelium and c in the mesenchyme),
protein collagen IV, which in turn promotes can also upregulate bone morphogenetic protein
8 I.M.A. Lombaert

(BMP) ligands. BMPs are part of the TGFβ sig- spatial proliferation, differentiation, and clefting.
naling family and signal via BMP receptors. Initiation of some of those embryonic signaling
FGFR1 signaling regulates BMP7 directly and pathways has been observed in active repair situ-
BMP4 indirectly to regulate epithelial growth. ations, such as ductal ligation settings where aci-
BMP4, which is mesenchyme specific, inhibits nar atrophy and hyposalivation is temporarily
epithelial branching, while BMP7, released by induced by restricting salivary flow from the
both epithelium and mesenchyme, increases it major duct [17]. We can thereby try to manipu-
[90]. The role of another member of the TGF late the activation and/or repression of specific
family, TGFβ1, is still inconclusive. While developmental pathways to stimulate in vivo
TGFβ1-deficient mice have normal SGs, over- repair of damaged SGs, as is outlined further in
stimulation of TGFβ1 results in acinar loss, elon- this chapter.
gated ducts, and/or fibrosis [35, 42].
Additionally, ECM and epithelial integrin cell
interactions are just as essential for branching 1.3 Environmental Cues
morphogenesis. These ECM molecules line up Patterning Epithelial
the basement membrane (BM) separating the epi- Branching and Maturation
thelium from mesenchyme. Interestingly, iso-
lated epithelial cells can easily grow without the SGs are highly vascularized and innervated, all of
physical presence of mesenchymal cells but not which integrate within a condensed mesenchyme.
without ECM component(s), such as laminin, Developmentally, SG epithelia invade a con-
fibronectin, perlecan, collagen, or mouse densed mesenchymal placode already containing
sarcoma-­ derived reconstituted BM “Matrigel.” a complex endothelial network and parasympa-
Deposition of unique ECM components along thetic neuronal bodies awaiting cues for innerva-
the clefting endbuds and elongating ducts plays a tion [48]. Both signals for epithelial invasion into
role in correct branching. Impairing these con- the mesenchyme and subsequent branching are
nections will lead to reduced clefting, endbud transmitted via direct cell-cell contact and/or
number, cell movement, and/or growth. Detailed indirect paracrine signaling pathways, which are
descriptions of disruptive ECM cell outcomes discussed below.
were recently reviewed in [79].
Finally, an underexplored area contributing to
SG formation are microRNAs (miRNAs), which 1.3.1 Guiding Neurons
are small, noncoding RNAs that specifically tar-
get mRNAs to globally regulate gene expression. Different cranial nerves innervate the pre- and
Epithelial endbud progenitors highly express postnatal SG where they exert different func-
miR200c to reduce FGFR-dependent prolifera- tions. While the autonomic nervous system regu-
tion. miR200c downregulates the autocrine ree- lates the SG at an unconscious level and in stress
lin/very low-density lipoprotein receptor conditions, sensory neurons respond to mechani-
(VLDLR) pathway, which positively regulates cal, thermic, and light signals.
FGFR signaling [83]. Additionally, it was found For decades, both the parasympathetic and
that EGF can specifically induce mesenchymal sympathetic nervous system have been acknowl-
production of miR-21, which decreases multiple edged as the driving stimulant to release saliva
target mRNA candidates. One of these, RECK, from acinar cells into ducts. While parasympa-
inhibits MMPs, which subsequently influences thetic stimulation results in serous secretion and
ECM degradation to enhance SG branching [37]. ion release, sympathetic activation stimulates
In conclusion, various signaling pathways mucous or protein-containing saliva and can also
instruct different cell types within the epithelial play role in local inflammation and blood flow
compartment and not surprisingly interact with [23, 67]. Both parasympathetic and sympathetic
and regulate each other to safeguard temporal-­ nerves are part of the autonomic nervous system,
1 Implications of Salivary Gland Developmental Mechanisms for the Regeneration of Adult Damaged Tissues 9

Fig. 1.3 Signaling

pathways driving neuronal
survival and innervations.
Illustrated signaling
pathways for
parasympathetic nervous
system were demonstrated
in prenatal glands,
sympathetic nervous system
in postnatal SGs. Green:
expressed by epithelial
cells; red: expressed by
neuronal cells; blue:
expressed by endothelial
cells; black: undefined
which compartment takes
part in it

and their neuronal guidance is ensured by axons initiating SG epithelia to localize into ganglia
that sprout along unique paths within the tissue. around the primary duct and send out axons
While the former innervates along the epithelia, toward the endbuds [48]. Sympathetic nerves
the latter follows the vasculature. This directional innervate SGs along the blood vessels during
guidance is driven by neurotrophic factors, later stages of development when final epithelial
secreted by cells in the periphery, as well as the maturation is needed. As such, developmental
presence of specific receptors on the axons that experiments can clearly dissect the role of neu-
allow or block their adhesion to the adjacent rotransmitters and neurotrophic factors affecting
ECM. The most notable trophic molecules include the PSG. It is now well appreciated that the PSG
neurotrophins (e.g., NGF, BDNF, NT-3), netrins, establishes a communication loop with specific
semaphorins, ephrins, and myelin inhibitors. epithelial cells to allow outgrowth of both com-
Similarly, axons secrete neurotransmitters in their partments. When the PSG is absent, the pool of
proximity, exerting a variety of effects through K5-expressing epithelial progenitors is signifi-
specific receptors on their target cells (Fig. 1.3). cantly reduced [50], which influences down-
Parasympathetic nerves signal via the cholinergic stream K19+ ductal luminal differentiation and
acetylcholine (ACh) pathway, targeting musca- subsequent epithelial outgrowth. This is medi-
rinic receptors on neighboring cells, as well as ated via a loss in ACh-CHRM1 (muscarinic
water channels such as aquaporin 5 (AQP5). In receptor 1) signaling from the PSG to K5+ cells
contrast, sympathetic nerves release epinephrine and resulting in a subsequent reduction of
and norepinephrine (i.e., noradrenaline, NA) that HB-EGF/EGFR pathway signaling that initiates
bind to β-adrenergic receptors (adrenoceptors) on maintenance and differentiation of K5+ progeni-
acini. Other non-ACh, non-­NA neurotransmitters tors. Lumenization, which marks further ductal
can be produced by both parasympathetic and maturation, is also coordinated by the PSG but
sympathetic nerves and may include vasoactive not via the ACh pathway. The neurotransmitter
intestinal peptide (VIP), substance P (SP), neuro- VIP activates a cAMP/protein kinase A (PKA)
peptide Y (NPY), neurokinin A, pituitary adenyl- pathway to induce epithelial duct cell prolifera-
ate cyclase-activating peptide (PACAP), and tion and formation of a single lumen by the fusion
calcitonin gene-related peptide (CGRP). of multiple microlumens. After initial lumen for-
Developmentally, parasympathetic ganglia mation, VIP remains essential to expand the
(PSG) neuron cell bodies migrate along the lumen size via the cystic fibrosis transmembrane
branches of mandibular arteries [91] to cues from (CFTR) pathway [73].
10 I.M.A. Lombaert

Organ development also requires proper bidi- does lead to ­hypoplasia of the gland [82] and
rectional communication. Feedback signaling thus must involve a direct or indirect role for
from epithelial cells toward the PSG stimulates sympathetic nerves in either epithelial cell main-
cell survival, migration, and innervation. At SG tenance or maturation. In the adult gland, RET
ontogenesis, WNT-producing epithelia, particu- signaling is also known to be essential for sym-
larly K5+ progenitors, maintain PSG neuron sur- pathetic neuron survival, but likely via the ligand
vival and proliferation [48]. At later stages of artemin instead of NRTN. SGs also produce high
branching morphogenesis, the neurotrophic fac- amounts of NGF and genetic ablation of NGF or
tor neurturin (NRTN), which is mainly secreted its TrkA receptor leads to defective sympathetic
by endbud progenitors, not only promotes neuro- innervation, indicating its crucial role in sympa-
nal survival via GFRα2/RET but also maintains thetic neuron survival [22, 27]. Depletion of non-
axon outgrowth along ducts toward the endbuds canonical WNT5a in WNT1-derived neural crest
[49]. In the developing lung, there also appears to cells further leads to incomplete sympathetic
be a link between nerves and blood vessels. innervation and branching in prenatal SGs. While
Denervation, in this case via physical cell abla- the authors suggest this is due to an autocrine
tion, resulted in reduced endothelial prolifera- WNT5a/retinoid-related orphan receptor (ROR)
tion, leading to hypo-vascularized lungs [9]. It is pathway in sympathetic neurons, it doesn’t rule
unclear whether this is a direct or indirect out that epithelial WNT5a-producing cells might
neuronal-­endothelial effect and whether similari- be stimulating sympathetic neurons as well [89].
ties exist within the developing SG. Similarly, endothelial-released endothelin 3
Detailed anatomical descriptions of nerves in (EDN3) is also suggested to be a cue for a sub-
adult SGs are outlined in a recent review [40]. It set of EDN receptor A+ sympathetic neurons to
is assumed that similar communication between innervate the prenatal SG along the nascent exter-
nerves and epithelium persists into adulthood as nal carotid arteries [63]. The specific role of other
denervation of SGs, via ductal ligation or neurec- neurotransmitters from the GDNF and NPY fam-
tomy, reduces epithelial content that regenerates ily as well as other neurotrophic factors are still
after ligation removal if the nerve is intact or being explored. While semaphorins are involved
reconnected [46, 55, 65]. A morphological differ- in axon pruning and neuronal migration in the
ence of early development with later stages and central nervous system, they also appear to have a
adulthood is that smaller ganglia are found dis- role in developing SGs. Semaphorin (SEMA) 3A
persed within adult tissue [40], presumably to and 3C bind co-receptors neuropilin and plexin.
reach their target cells more easily as distances Neuropilin is expressed by epithelial endbuds
are much larger compared to embryonic and by activation with SEMA3A and 3C cleft
development. formation is induced without changing prolifera-
Even though tyrosine hydroxylase (TH)- tion and, most likely, by affecting cell movement
expressing sympathetic ganglia are presumed [15]. However, additional FGF7/10 growth-pro-
not to be present at SG ontogenesis, some moting signals from surrounding mesenchyme
mRNA expression levels of its unique recep- were required to mediate this cleft formation.
tor neuropeptide Y receptor 2 (NPY2R) were Whether additional participation of SEMAs on
detected early during development at low levels receptive nerves is required for cleft formation or
that increase before birth [23]. Since NPY2R is SG development still remains unclear.
also present on endothelial cells, some, if not At adulthood, it remains to be determined how
all, of the mRNA expression could be related to sensitive sympathetic nerves are to injuries such
blood vessel formation within the SG. However, as radiation. In rodents, sympathetic nerve func-
TH-expressing neuronal cells were detectable tion was retained after radiation [52], and
by E16.5, which might indicate there is a pre- increased levels of TH as well as NGF/NGFR and
natal presence of sympathetic ganglia [89]. adrenergic receptor 2 (ADRA2B) were detected
Nevertheless, postnatal sympathetic denervation in radiated human SMGs [49]. Whether a
1 Implications of Salivary Gland Developmental Mechanisms for the Regeneration of Adult Damaged Tissues 11

r­ eestablishment of the balance between parasym-

pathetic and sympathetic nervous system is nec-
essary for regeneration is not known.
Furthermore, it is assumed that sensory neuro-
nal cells are present along the sympathetic and
parasympathetic nerve tracks in adult SGs [51],
although they have not yet been studied in detail.
While sensory neurons can be defined into mul-
tiple subtypes based on different criteria such as
their origin and molecular expression patterns,
they often are loosely classified as unmyelinated
capsaicin-sensitive TRPV1+ receptor expressing
neurons or myelinated glutamate receptor-­
expressing neurons. Upon activation, sensory
nerves can secrete various neuropeptides, such as
Substance P and CGRP. At least in the lung and
pancreas, literature indicates that sensory neu-
rons release neurotransmitters in the periphery to
Fig. 1.4 Potential communications initiated by and to
serve as direct mediators for recruiting and acti- endothelial cells. Described pathways were majorly found
vating inflammatory cells [68, 86]. Whether a in other branching organs. Whether they exist in SGs
similar mechanism occurs in salivary glands is needs to be determined. Green: expressed by epithelial
not known. cells; orange: expressed by mesenchyme; blue: expressed
by endothelial cells
In conclusion, innervation plays an essential
role for organ development, homeostasis, and
repair after injury. Studies on SG biogenesis have lial secretory factors, termed angiocrines, which
been highly informative for defining the involve- impact organ development (Fig. 1.4). While
ment of the PSG in branching morphogenesis, research on blood vessels in SGs remains limited,
not only of SGs but also other organs such as much can be learned from other branching organs.
prostate and lungs. The existence and/or loss of Overall, complex cross-communication between
bidirectional communication with epithelial epithelial and endothelial cells appears to regu-
stem/progenitor cells have been reported to occur late both epithelial differentiation and angiogen-
in rodent and human SG homeostasis and postra- esis. The initial cues to form an endothelial cell
diation. The inhibition of parasympathetic neuro- plexus around a condensed mesenchyme do not
nal function influences adult epithelial K5+ require epithelia. This is observed in Fgf10−/−
progenitors [49] but it is not known yet whether mice that don’t form initial SMG epithelia but
postradiation regeneration due to epithelial stem/ where the placode of mesenchyme, blood ves-
progenitor transplantation repairs neuronal func- sels, and neuronal bodies are present [48]. After
tion, even though morphological repair has been SG ontogenesis, however, epithelial-­ derived
suggested [71]. angiogenic factors, such as VEGF-A, do play a
role as null mutations in Vegf-A or its endothelial-­
expressed receptor (Vegfr) show v­ ascular defects
1.3.2 The Role of Blood Vessels in tissues, reduced epithelial budding, and ulti-
mately embryonic lethality [13, 107]. Another
The vasculature in branching organs develops in epithelial-induced angiogenic mechanism may
close proximity to the epithelia, although its spa- include the vitamin D pathway. The enzymes
tial pattern differs from parasympathetic nerves. CYP27B1/24A1 that activate and catabolize
Not only are endothelia important for mediating vitamin D are highly upregulated just before
gas exchange but also as a source of endothe- birth and in postnatal lung. Exogenous vitamin
12 I.M.A. Lombaert

D positively influences lung growth by inducing ­laminins. These components in turn served as
maturation in vitamin D receptor-­expressing epi- scaffolds for increased axon outgrowth.
thelial cells (VDR) [64]. As VDR is also present In addition to blood vessels, we must not for-
on endothelial cells, this enhanced growth might get the circulating cells within them. White blood
be due to direct effects of vitamin D on endothe- cell monocyte-derived macrophages and den-
lial cells and/or indirect effects from epithelia to dritic cells arise from the bone marrow and colo-
endothelia. Nevertheless, it is clear that epithe- nize tissues via blood vessels to phagocytose
lial-endothelial communication requires a tight cellular debris and help in the innate non-specific
balance as any hyper-­vascularization inhibits epi- and specific adaptive immune defense. While
thelial growth [14]. macrophages normally develop in the bone mar-
Apart from endothelial-epithelial cross-­ row via granulocyte-macrophage colony-­
communication, there is also endothelial-­ stimulating factor (GM-CSF), mesenchymal
mesenchymal communication, as recently cells in tissues can also release GM-CSF to
reviewed [94]. The early endothelial cells pro- induce a similar differentiation effect on circulat-
mote survival of pancreatic mesenchymal cells, ing monocytes. While it is clear that both macro-
which in turn have a pivotal role in organ devel- phages and dendritic cells may be involved in
opment. A similar complex paracrine signaling organ morphogenesis, their exact functions are
network was also found in the lung. Retinoic not always fully understood. Also in adult tissues,
acid (RA), which is produced by endothelial for example, in the lung, there is conflicting data
cells, induces VEGF-A expression in lung epi- on their specific role: antigen-sensing dendritic
thelia. Evidently, endothelial cells are recruited cells might induce different immune responses
via VEGF-A, and thus angiogenesis is stimu- depending on their physical location in the tissue
lated via this endothelial-epithelial communica- while surrounding different epithelial cell types
tion loop. Furthermore, endothelial-released RA [54]. Similarly, various macrophages invade the
also stimulated mesenchymal cells to produce mesenchyme where they can interact with den-
more FGF18 and ECM component elastin, thus dritic cells, lymphocytes, and epithelia to regu-
increasing epithelial alveolar formation [108]. late immunity. Macrophages suppress immune
Other organ-specific angiocrine factors that may responses by inhibiting both dendritic-mediated
follow this paracrine loop include HGF, WNT, T-cell activation and inactive TGFβ production.
NOTCH, and BMP ligands. Mesenchymal cells Subsequent activation of this inactive TGFβ into
also signal back to endothelial cells to stimulate bioactive TGFβ by lung epithelia is essential in
survival, proliferation, migration, and autoph- order to prevent spontaneous inflammation after
agy via production of ECM components, such acute injury. Lung alveolar cells in turn secrete
as the perlecan/heparan sulfate proteoglycan various ligands to receptive macrophages to
(HSPG2) fragment endorepellin, decorin, and ensure this prevention of inflammatory responses.
endostatin [18, 75]. Whether a similar action or disruption in this
While blood vessels and nerves can indepen- communication is occurring in adult SGs after
dently respond to their own set of signaling fac- radiation remains to be determined.
tors, there also seems to be a paracrine connection Apart from immune regulators, macrophages
via epithelial-released VEGF. Even though further shape the branching patterning of organs
VEGFR is absent on nerves and not required for by remodeling the ECM around the ducts to
innervation, VEGF overexpression in pancreas allow outgrowth as well as survival of endbud
not only led to hyper-vascularization but also to stem/progenitor cells [11, 102]. They also regu-
hyper-innervation [85]. Interestingly, endothelial late angiogenesis by instructing endothelial cells
cells did not produce any known neurotrophic to undergo apoptosis via WNT signaling, coun-
factors, but the effect appeared to be related to terbalancing a pro-survival factor produced by
their upregulated expression of basement mem- pericytes, which wrap around endothelial cells to
brane components, such as collagens and influence functions such as blood flow [2].
1 Implications of Salivary Gland Developmental Mechanisms for the Regeneration of Adult Damaged Tissues 13

Fig. 1.5 Described

signaling interactions with
the SG mesenchyme. Most
known communications
are between the SG
mesenchyme and
epithelium. Green:
expressed by epithelial
cells; orange: expressed by
mesenchyme; blue:
expressed by endothelial
cells; black: undefined
which compartment takes
part in it

In sum, blood vessels play important roles 29, 99]. This indicated that SG mesenchyme has
during development in other branching tissues strong and unique multicomponent instructional
not only for oxygen supply but also to maintain properties. One of them is the high production of
essential communication signaling pathways FGF10, which is also essential for lung, lacrimal,
with epithelia and surrounding mesenchyme. The and mammary gland initiation. Notably, non-SG
bone marrow-derived cells circulating in the epithelia only adapted SG-like branching patterns
blood vessels also aid in tissue branching mor- when they were placed in close vicinity to high
phogenesis and evoke or suppress immune FGF10-expressing mesenchymal tissue, confirm-
responses after injury. Whether similar mecha- ing the importance of FGF10’s spatiotemporal
nisms exist in the developing and adult SGs dosage [99]. With this in mind, it is important to
remains to be determined. understand that the SG mesenchymal component
in these experimental conditions contains mesen-
chymal cells as well as ganglia and blood vessels
1.3.3 Supporting Mesenchymal albeit disconnected from the rest of the body. It
Cells can therefore not be excluded that SG-specific
neuronal cells and/or blood vessels may have addi-
Embryonic SG mesenchymal cells are WNT1+ tional contributions to this specific SG patterning.
neural crest-derived cells [44, 105] and provide Interestingly, early-stage E11.5–12.5 SG epithe-
supportive cues such as growth factors, proteases, lia, but not later stages, are able to instruct E10.5
and ECM proteins to guide and activate epithelial, mesenchyme from different sources to produce
neuronal, and endothelial cells (Fig. 1.5). In vitro FGF10. However, not every mesenchyme is as
recombination experiments show that the SG mes- competent to receive this signal as only SG and pha-
enchyme induces an SG-like branching pattern in ryngeal second arch mesenchyme responded and
various epithelia such as a pancreatic, mammary, limb mesenchyme, for example, did not. This indi-
and pituitary gland [96]. This property, however, is cates that this initial signal is exclusively located
not found in mesenchyme from non-SG tissues, as within specific regions of the embryo, likely to
E11.5–13 SG epithelium does not properly branch restrict specific organ outgrowth to the correct loca-
unless it is recombined with SG mesenchyme [28, tion in the body. What this initial epithelial signal is
14 I.M.A. Lombaert

remains a subject of debate. Whereas early limb and in the first step, is exclusively expressed in early
lung epithelia secrete FGF8 or FGF9 to initiate this SG mesenchyme, while RA activity is mainly
process, it is unlikely that FGF8 serves a similar observed in RA receptor+ epithelia. Disruption in
function in SGs [99]. Neither is the signal FGF4, Rdh10 results in early embryonic lethality, often
BMP2, SHH, TGFβ1, or WNT6 [47]. One unex- before SG epithelial invagination. However, when
plored candidate is platelet-derived growth factor E13 SGs were treated with an RAR inhibitor
(PDGF). During development, PDGF-A and reduced branching was observed [101]. In contrast,
PDGF-B ligands are mainly produced by epithelia mesenchymal cells can also secrete signaling
and mesenchyme, respectively [105], while inhibitors to slow down branching. DLK1, a non-
PDGFR-A and PDGFR-B receptors are expressed canonical NOTCH1 ligand produced by the mes-
in the mesenchyme. By adding exogenous PDGF, enchyme, inhibits branching and subsequent
epithelial proliferation can accelerate via upregula- innervation, presumably to modulate cleft forma-
tion of mesenchymal Fgf7, Fgf10, Fgf1, and Fgf3 tion [25]. Furthermore, DLK1 appears to regulate
and downregulation of growth inhibitory factor the epithelial balance of K14+ progenitors,
Fgf2. While this induction was observed during SG although the precise mechanism is unclear [26].
morphogenesis (E14), it has not been confirmed The role of TGFβ1 is also unclear; there is some
that epithelial PDGF-A is a potential FGF10 inducer evidence that epithelial-secreted TGFβ1 enhances
at SG ontogenesis. Nevertheless, once FGF10 collagen production from Coll1α1+ mesenchymal
expression is initiated, it persists and becomes inde- cells to inhibit SG acinar formation [42].
pendent from epithelial cues. It is also interesting to Other mesenchymal signaling pathways that
note that mesenchymal condensation at placode ini- may influence epithelial branching are hepato-
tiation is independent of this FGF10 activation. As cyte growth factor (HGF)/c-MET and SDF1/
such, the mesenchyme can condense around a net- CXCR4 signaling [38]. Additional cellular
work of blood vessels and resting PSG cells before instruction mechanisms also include microRNAs
SG initiation and in the absence of FGF10 as seen in (miRNAs). miRNAs are small noncoding RNA
Fgf10−/− mice. This mesenchyme presumably molecules that function to silence other mRNAs.
awaits signal(s) from the invading oral epithelia to While most research has focused on
initiate FGF10, which in turn promotes SG-specific mesenchymal-­ epithelial interactions, there may
epithelial growth. also be mesenchymal-endothelial interactions.
Even during branching morphogenesis, mesen- When the mesenchymal factor SDF-1 was specifi-
chymal cells continue to play a part in multiple cally inhibited from binding CXCR7+ endothelial
bidirectional signaling pathways. Early on, WNT/ cells, SG epithelial branching was decreased [38],
β-catenin signaling is exclusively induced in the thus suggesting a tri-directional loop between
mesenchyme before it is expressed in lumenizing mesenchymal, endothelial, and epithelial cells. It
ductal cells [78]. This mesenchymal WNT can is also possible that mesenchymal cells may play a
activate EDA and, at least in part, influence SG role in axonal guidance. As outlined earlier, mul-
morphogenesis via epithelial EDAR [31]. tiple studies have verified that mesenchymal cells
Branching epithelia also regulate local FGF10 aid in cellular migration, clefting, and differentia-
expression to reduce aberrant cell proliferation. tion via regulation of ECM production.
Lung epithelia release BMP ligands as well as
SHH to spatially downregulate FGF10 and specifi-
cally induce secondary bud formation [12]. A 1.4  ranslation into Future
T
recent study further points to an important role for Therapies to Repair
mesenchymal retinoic acid (RA) to enhance Damaged Salivary Glands
branching. RA is a small diffusible hormone-like
molecule generated by a two-step enzymatic oxi- There is a tremendous need for long-term thera-
dation of dietary vitamin A via RDH10 and pies to restore salivary flow. Clinical impacts of
ALDH1A. RDH10, which metabolizes vitamin A dry mouth, or xerostomia, not only include difficulty
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Using a Manikin for the Study of Anatomy The best way to

study anatomy is to set up a book of anatomical diagrams before
you, and set up an art-store manikin alongside it. Draw the action
from the manikin and the muscles from the book. You can also make
rough sketches of the manikin itself for general bulk and action only.
Just copying anatomical diagrams docs not seem very helpful to
most students. The muscles must somehow be built upon a frame or
figure in order to get their proportion and relationship to the figure
as a whole. The joints of the manikin are usually balls of some kind,
and of course such joints must eventually be covered up. For this
reason it is well to concentrate on the muscles of the shoulders, and
those of the thighs, especially at the hips. Then study the chest,
waist, and buttocks. Next get the back, then the arms and the whole
leg. To balance the manikin on its feet requires about the same
arrangement of limbs and torso that the human needs to hold its
balance. The manikin is intended for line only, not for the study of
the figure in light and shadow. The lighting on these simplified forms
is not enough like that on live models. We consider later the figure in
light and shadow. Work in the life class should be done with the
anatomy book open. It is difficult to start drawing the figure from life
without any previous preparation. Upon entering a life class the
student should have a fairly accurate idea of the proportions of the
figure in heads, and in sixths, as illustrated on page 107. I have tried
to cover most of the problems of figure drawing in a previous
volume. Figure Drawing for All It's Worth. Some instructors object to
the use of the wooden manikin, since the action is only an
approximation at best, and there is no actual play of muscle to go
by. This objection is sound, provided the person studying drawing
has life classes available, the time for them, and the funds to pay for
them. I gladly agree that any young person who intends to make a
living at art should by all moans attend life classes. However, I
believe that the manikin has an important use for the study of
action, since a live model cannot hold an action pose for any length
of time. Working from the manikin tends to loosen up the student's
figure drawing. When an artist gets out into the active practice of his
art, he can seldom draw a figure posed as it would be posed in a life
class for twenty to twenty-five minutes at a time. The static poses of
the art class should be much more for the study of light on form,
values, and color. To get figures in action the artist is almost forced
to use the camera, and many present-day artists have high-speed
cameras for this purpose. However, for an action picture, the artist
should have a wcll-dcvclopcd knowledge of the figure under the
drapery. It does no harm to make the figure do something besides
stand or sit, or perhaps hold a rod or pole. The pose or gesture of
the figure docs much to make it tell a story. If you intend to be an
illustrator, you must have action in your work, or it will not be very
successful. The manikin helps particularly in making preliminary
sketches or developing rough ideas which hardly warrant the
expense of a model. A model can be hired for the last stage of the
work or for the material from which the final work will be drawn. The
student should of course use his own judgment. If he finds that the
manikin helps, let him use it. 106
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DRAWING FROM THE MANIKIN Manikins are a great help in

developing action in figure drawing, in that they can be put into
"still" poses no live model could hold. They can be purchased at
most art dealers. Their approximate construction is shown by the
figure at the right. For comparison, the figure at the left shows the
ideal proportions of the male human figure. The line at the extreme
left shows divisions of the height of the figure of ideal proportions.
One side of the line is divided into sixths and the other side into
eighths. These two sets of divisions indicate the important points of
the figure. Memorize these scales. 107
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The Figure in Light For some reason many students seem

to have their greatest difficulty in what they call "shading." Tin's is
probably because there is no such thing as "shading," in the sense
they mean. The term "modeling" is more accurate. The student
wants to add tone to outline, so he is likely to put in a lot of
meaningless grays and darks between the outlines. What he must do
with tone is the same thing the sculptor docs. The shadow is a tone
that is governed in the first place by the value in the light. Things
have what wc call local values, which means the material or
substance is light, gray (or a color darker than white), or dark. Put
them in any light and (lie values keep their relationship to one
another. A dark suit, for example, would never be as bright in value
as flesh, if both were rendered truly. When working with pencil we
seldom attempt to get all the values in scale as we would with paint.
When there is bright light and strong shadow we take some leeway,
but we suggest some tone in the lighted areas of dark materials,
making the shadow quite dark. For flesh, which usually is fairly light
in value, we leave the lights as white paper, for the pencil does not
give 08 quite the range of tones from light to dark that paint does.
So in pencil drawing the best effects come from keeping the strongly
lighted areas very delicate in modeling. Getting effects in die lighted
areas too dark makes drawings appear muddy or heavy. When
working in pencil, I try to think of about four tones, starting just
beyond white, or as light as you can state a gray, then a gray, a dark
gray, and a black. Thus the whites are extreme lights, the delicate
grays give some form to the lighted areas, the grays become the
halftones, and the dark grays and blacks are reserved for the
shadows. There can be no formula, because every subject has its
own particular values, determined by the light, its direction, its
brilliance, and its particular effect upon the local values. But die
student can gain much understanding of light very quickly if he can
learn to distinguish the differences between areas of light, halftone,
and shadow, and set them down. Even if the values in a drawing are
not true, the correct separation will give solidity to the drawing.
Instead of trying to match all the grays of a photograph which you
are using as copy, just look for the shapes of light, halftone, and
shadow. Sometimes there are tones within a shadow where light is
reflected; you must draw these also, even though they are
submerged in a lower all-over tone. It is foolish to try to fake the
lighting on a seriously drawn figure. Lighting is much too
complicated and subtle to guess at. Either have a figure to draw
from or get some good photographic material. It may be helpful to
work from copy first, and from life later. The ideal thing is to enter a
class in life drawing. Most classes work in charcoal, which is even
more flexible than pencil as a material, for it can be easily erased. If
you are studying the figure at home, get some charcoal, charcoal
paper, plastic or kneaded rubber. You will also need a drawing board.
Hemembcr to keep darks and blacks out of the lighted areas, except
where you find accents of shadow within or alongside of these
areas. Keep a long point on your pencil or charcoal so that you can
use the tip for line and the sides for tones. Get some good books on
figure drawing, and some on anatomy. If you practice a good deal
on still-life drawing, too, you will draw the figure much better. Light
is light no matter what it falls upon, and it always follows the form
with light, halftone, and shadow. 112
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