Emergent Materials

https://doi.org/10.1007/s42247-020-00085-2

REVIEW

3D bioprinting for the endocrine glands

Mervesu Gokyurek 1 & Kerim Bora Yilmaz 2,3 & Pinar Yilgor Huri 1,4

Received: 18 December 2019 / Accepted: 3 March 2020

# Qatar University and Springer Nature Switzerland AG 2020

Abstract
Endocrine glands regulate homeostasis of the body with the hormones they secrete. Regeneration and/or replacement of
malfunctioning endocrine glands is a global need since the diseases associated with them affect more people year by year.
Tissue engineering is an emerging promising field for the patients who suffer organ failure with recent pioneering clinical
applications. Success in tissue engineering strategy depends on the use of proper cells, bioactive factors that stimulate the
activities of these cells, and scaffolds that are produced to recapitulate the structure and support the function of engineered
tissues. 3D bioprinting is a developing strategy for the production of living tissues by using bioinks to produce the cell-laden
scaffolds in a layer-by-layer procedure. In this review, major endocrine glands, their functionalities, and tissue engineering
strategies to produce them are covered with special emphasis to their biofabrication through 3D bioprinting.

Keywords Endocrine glands . Tissue engineering . 3D printing . Bioprinting . Parathyroid . Thymus

1 Introduction
Endocrine glands are responsible for hormone production.
These hormones target cells that affect their morphology and
functions resulting in the regulation of activities within the
body including reproduction, development, growth, balance,
and metabolic activities [1]. According to the report of
Endocrinology Society [2], endocrine diseases increase annu-
ally worldwide. More than 5% of the US citizens are affected
by these diseases every year [2, 3]. Routine clinical treatment
for the endocrine diseases related with hormone deficiency
includes hormone replacements and organ transplantation,
where necessary. However, there is an increasing gap between
the number of donors and patients who are waiting for organ
transplantations. Therefore, transplantation is becoming more
* Pinar Yilgor Huri
phuri@ankara.edu.tr
1
Department of Biomedical Engineering, Ankara University,
Ankara, Turkey
2
Gulhane Research And Training Hospital Department of General
Surgery, University of Health Sciences, Ankara, Turkey
3
Department of Medical and Surgical Research, Institute of Health
Sciences, Hacettepe University, Ankara, Turkey
4
MEDITAM, Ankara University Medical Design Research and
Application Center, Ankara, Turkey
and more insufficient to meet the demand for functional or-
gans [4]. Moreover, hormonal levels and healthy physiologi-
cal states do not always match with hormone replacement
therapies. For these reasons, the therapy methods that are cur-
rently in use in the clinics have inadequate, and sometimes
negative, effects on the quality of life of the patients [3].
Tissue engineering is an emerging interdisciplinary field of
research for the production of functional organs in the labora-
tory using biomaterials and autologous cells. Specifically, the
use of stem cells with varying potency to give rise to various
cell types is an indispensable tool in tissue engineering re-
search. Recent developments in the production of organoids
as building blocks bring the potential for the development of
functional tissues and organs with this technique. By this way,
tissue engineering carries the promise to overcome the re-
quirement for the need of donor organs.
3D printing, and more specifically 3D bioprinting, is be-
coming an increasingly accessed biofabrication method for
the production of complex functional tissues and organs. In
a more broader sense, 3D printing is a computer-aided design
(CAD)/computer-aided manufacturing (CAM) fabrication
method to produce any complex shape based on the slice
information of the object and its production in a layer-by-
layer manner. Although the technique has been in use for
almost half a century mainly for the production of engine
pieces for industrial use, its implementation in the medical
field is relatively new. The progress of 3D printing technology

in service for medical applications has become possible with
the advent in materials science, i.e., the possibility of use of
3D printable biocompatible biomaterials. More recently, the
development of 3D bioprinters and techniques that enable the
use of living cells as the bioinks led to the breakthrough ad-
vances in the production of functional tissues and organs
ready for transplantation, already with some pioneering clini-
cal applications.
In this review, the principles of tissue engineering and 3D
bioprinting, as well as the functions of the major endocrine
glands, are covered by special emphasis on how the 3D
bioprinting approaches have been used for the production of
these tissues.
2 Major endocrine glands and their functions
All cells take signals from their environments and create sig-
nals as a response. This is called as the cell-to-cell communi-
cation. During this process, information is transferred to the
cells with the help of various molecules. Some signals are
carried to far distances, and some of them are carried locally
to the neighboring cells [5].
Nervous system and endocrine system work together to
arrange and control physiological activities. Nervous system
gives quick, short-term responses. On the other hand, endo-
crine system regulates long-term metabolic activities like
growth, development, and reproduction [1, 6]. Chemical mes-
sengers (hormones) are used to transfer information between
cells in the endocrine system [7]. Endocrine glands are duct-
less glands, and endocrine cells release hormones into the
bloodstream to regulate metabolic activities. Hormones are
divided into three groups, namely, peptide hormones (or pro-
teins), steroid hormones, and amine hormones. This classifi-
cation is based on their biosynthetic pathways [6].
Hypothalamus, pituitary gland, pineal gland, thyroid gland,
parathyroid gland, thymus, suprarenal glands, and gonads
are the major members of the endocrine system (Fig. 1).
Their functions are broadly covered in Table 1 to enlighten
the upcoming discussions in this review.
2.1 Hypothalamus and pituitary gland
The hypothalamus is located above the pituitary gland in the
brain, and it has crucial roles to regulate homeostasis in the
body. It controls nearly all hormonal secretions of the pitui-
tary. Function of the posterior pituitary is controlled with ner-
vous signals. However, the function of the anterior pituitary is
controlled with the blood vessels [8].
Hypothalamus releases and controls various kinds of hor-
mones to maintain homeostasis. Thyrotropin-releasing hor-
mone (TRH), corticotropin-releasing hormone (CRH), growth
hormone-releasing hormone (GHRH), growth hormone-
emergent mater.
inhibitory hormone (GHIH), gonadotropin-releasing hormone
(GnRH), and prolactin inhibitory hormone (PIH) are the major
hormones which are secreted from the hypothalamus. These
hormones are also known as regulatory hormones and control
hormones which are secreted from anterior pituitary region
[8].
The pituitary gland is located in sella turcica in the base of
the brain. This pea-sized gland is protected by sella turcica,
and it controls the functions of most of the other endocrine
glands [9]. The gland has two parts which are the front
(anterior) lobe and the back (posterior) lobe. Both lobes are
connected to the hypothalamus by a stalk and controlled by
the hypothalamus. The posterior lobe has a direct connection
with the brain, and it is controlled with nervous impulses.
However, the anterior lobe and the hypothalamus have no
direct neural bonding, and it is controlled via the blood vessels
[1].
Six main hormones are produced and secreted in the ante-
rior lobe. These are growth hormone (GH), thyroid-
stimulating hormone (TSH), adrenocorticotropic hormone
(ACTH), prolactin (PRL), follicle-stimulating hormone
(FSH), and luteinizing hormone (LH) [1]. Body growth and
physical development is regulated by GH. It is also important
for metabolic actions. TSH stimulates the thyroid to secrete
thyroid hormones, and it is also responsible for the growth of
the thyroid gland [10]. Adrenal glands are stimulated by
ACTH to produce hormones. PRL targets mammary glands
to produce milk. FSH and LH are both known as gonadotro-
pins. They control the functions of the gonads (testes and
ovaries). While LH initiates gonadal hormone production,
FSH promotes gametes (sperm and egg) production [1].
Antidiuretic hormone (ADH) and oxytocin, which are pro-
duced in the hypothalamus, are stored and released in the
posterior lobe. Oxytocin secretion is higher in nursing women,
and it triggers the milk ejection. ADH, or vasopressin, regu-
lates the reabsorption of water in the body [1].
2.2 Pineal gland
Brain has two hemispheres. Pineal gland, a tiny pinecone-
shaped gland, is found in between the two hemispheres, in
the center of the brain [10]. The main cell types of the glands
are called pinealocytes, and they produce the melatonin hor-
mone [1]. Melatonin regulates the internal clock of the body
and the circadian rhythm associated with daylight [10].
2.3 Thyroid gland
Thyroid gland is found in the neck and has a butterfly shape. It
produces and secretes thyroid hormone (TH) and calcitonin.
Both thyroxine (T4) and triiodothyronine (T3), two iodine-
containing amine hormones, are components of the thyroid
hormone. T4 has four bound of iodine atoms, and T3 has three
emergent mater.
Fig. 1 Schematic representation Hypothalamus
of the endocrine glands (created
with BioRender)
Pituitary Gland
Thyroid Gland
Suprarenal
Glands
Gonads (Ovary
for females)
of them. Under normal circumstances, about 90% of the thy-
roid output is in the form of T4 and about 10% is T3 [11].
After secretion into the bloodstream, T4 which is prohormone
transforms into biological active form T3 within the cells. TH
increases basal metabolic rate, regulates tissue growth and
development, and maintains blood pressure [1].
Another cell population in thyroid glands, parafollicular
cells, or C cells secrete calcitonin. Calcitonin plays an impor-
tant role for calcium metabolism. When there is a rise in the
Ca2+ level in the blood, calcitonin is released from the thyroid
gland to reduce its concentration [1].
2.4 Parathyroid glands
Parathyroid glands are localized at the back of the thyroid
gland in the cervical region. The main function of these
glands is to balance the Ca2+ level in the blood. There are
Pineal Gland
Parathyroid
Glands
Posterior Surface of
Thyroid Gland
Thymus
Pancreatic
Islets
Gonads
(Testes for
males)
usually 4 parathyroid glands, each of which is 3 or 4 mm
in size. They have a spherical shape and a weight of ca.
40 mg (without fat) [12, 13]. Since their appearance re-
semble that of the thyroid gland and it is hard to distin-
guish them with the naked eye during surgery, it is a
common complication to remove them as well during op-
erations to the thyroid.
The specialized cells of the parathyroid gland are chief
(principal) and oxyphil cells [12]. These cells have calcium-
sensing receptors, and they can detect the changes in the Ca2+
level. They secrete parathyroid hormone (PTH) which con-
trols the Ca2+ level in the blood [14, 15]. A healthy parathy-
roid gland secretes PTH to control the decline in the Ca2+
level, while the gland shuts down the secretion of PTH for
the rise in the blood Ca2+ level. The skeleton and the kidneys
are the two major target organs that get stimulated by the PTH
to control the Ca+2 level [1, 16, 17].
Table 1 Major endocrine glands and the chemical classification and functions of the hormones secreted from them

Endocrine gland Secreted hormones Hormone chemical Hormone major functions Ref
classification

Hypothalamus Regulatory hormones (TRH, CRH, GHRH, GHIH, GnRH, Amine Controls secretion of anterior pituitary hormones 8, 10
PIH) PIH
Peptide (others)
Production of ADH Peptide Regulates reabsorption of the water
Production of oxytocin Peptide Triggers the milk ejection
Pituitary gland In anterior lobe GH Peptide Overall growth and physical development 1, 6, 9, 10
TSH Peptide Stimulates thyroid to secrete thyroid hormones
ACTH Peptide Stimulates adrenal glands for hormone secretion
PRL Peptide Stimulates secretion of milk in female breast
FSH Peptide Promotes gametes production
LH Peptide Initiates gonadal hormone production
Stored in posterior ADH Peptide Regulates reabsorption of the water
lobe Oxytocin Peptide Triggers the milk ejection
Pineal gland Melatonin Regulates body’s internal clock and circadian 1, 10
rhythms
Thyroid gland TH Amine Increases basal metabolic rate, regulates tissue 1, 6, 10
growth
and development, and maintain blood pressure
Calcitonin Peptide Reduce Ca2+ concentration in bloodstream
Parathyroid glands PTH Peptide Controls blood calcium level 1, 6,
10–17
Thymus Thymosin Peptide Stimulates to generate T cells 1, 18
Suprarenal In adrenal cortex Aldosterone Steroid Regulates mineral balance 6, 10
glands Cortisol Steroid Regulates glucose, protein and lipid metabolism
In adrenal medulla Epinephrine, norepinephrine Amine Regulate stress adaptation and blood pressure
Pancreas Insulin and glucagon Peptide Regulate blood glucose level 1, 6, 9, 10
Gonads (testes for males) Testosterone Steroid Maturation of the male reproductive organs and 1, 6, 8
male secondary sex characteristics
Gonads (ovaries for females) Estrogen Steroid Maturation of female secondary sex characteristics
and maturation of the reproductive organs
Progesterone Steroid Maturation during menstrual cycle and pregnancy
emergent mater.
emergent mater.
2.5 Thymus
Thymus gland is located behind the sternum and between the
lungs. It grows during childhood and reaches its highest weight
during this period. Following that, it shrinks throughout adoles-
cence [18]. It secretes thymosin hormone which stimulates the
generation of the T cells, either by stimulating lymphopoiesis or
by the induction of immune cell competence [1, 18, 19].
2.6 Suprarenal glands
Suprarenal or adrenal glands are located on top of the kidneys
and have triangular shapes. These glands consist of the adre-
nal cortex and the adrenal medulla parts. In the outer part,
there is adrenal cortex, and in the inner part, adrenal medulla
can be found. Each part produces different hormones [1].
Adrenal cortex produces several steroid hormones which are
mineralocorticoids and glucocorticoids. Mineralocorticoids are
mainly aldosterone, and they affect the mineral balance and reg-
ulate the circulating fluid volume. Glucocorticoids generate a
catabolic state that characterizes the body’s response to stress
and mostly cortisol which regulate the glucose, protein, and lipid
metabolism [10]. On the other hand, adrenal medulla secretes
epinephrine (adrenaline) and norepinephrine (noradrenaline).
These hormones are responsible for fight or flight response [1].
2.7 Pancreas
Endocrine pancreas has pancreatic islets, also known as
islands of Langerhans. There are two major types of
hormone-producing cells in the islets, namely, alpha and beta
cells. Alpha cells secrete glucagon, and beta cells produce
insulin hormone. These two hormones are antagonistic, but
they both regulate blood glucose levels [1, 9]. Somatostatin,
vasoactive intestinal peptide, substance P, gastrin, pancreatic
polypeptide, and ghrelin are the hormones that are secreted by
the other endocrine cells of the pancreas. They have major
roles in the gastrointestinal system.
2.8 Gonads (testes and ovaries)
Gonads are reproductive organs for males and females. They
produce gametes and sex hormones. There are different go-
nads, testes for males and ovaries for females. Hormones se-
creted from the ovaries are mainly estrogen and progesterone.
Estrogen is significant for the appearance of female secondary
sex characteristics and maturation of the reproductive organs
[1]. Progesterone is an important hormone for maturation dur-
ing menstrual cycle and pregnancy [7]. Hormones which are
secreted from testes are primarily testosterone. Maturation of
the male reproductive organs and male secondary sex charac-
teristics is initiated by testosterone [1].
3 Tissue engineering and 3D bioprinting
3.1 Tissue engineering
Diseased and malfunctioning tissues and organs are routinely
replaced with the healthy counterparts in the clinical applica-
tions. Several different types of replacement materials can be
used for these procedures including autografts, allografts, and
xenografts [20, 21]. However, none of these methods are with-
out complications. For example, although autografts, grafts
obtained from the patient, are the golden standard, their avail-
ability is limited, and their use creates additional problems like
donor site morbidity and increased operation durations.
Allografts, or organ donors in the case of solid organ trans-
plantations, are limited in availability as well. In fact, a great
number of lives are being lost because of donor shortage while
on waiting list [4, 21, 22]. Also, these procedures are limited
by the presence of immune compatibility. Xenografts, mostly
grafts of animal origin, are being used with the potential risk
of disease transmission between different species. Therefore,
development of an alternative strategy to generate tissues and
organs in the laboratory by using autologous cells and bio-
compatible materials is a major clinical need worldwide.
Tissue engineering holds this promise [23, 24]. Tissue engi-
neering has emerged as an interdisciplinary research field with
the hope to create a viable solution to the tissue/organ waiting
lists without the risk of immune rejection [25].
In 1993, Langer and Vacanti made the definition of tissue
engineering. Tissue engineering is an interdisciplinary field
and uses the principles of engineering and life sciences to
regenerate a functional tissue [19, 23, 26]. With this technique,
custom-made tissues and organs can be regenerated in labora-
tory conditions and can be used for the replacement and repair
of tissues in the clinic [27, 28]. The three main components of
the strategy are cells, scaffolds, and bioactive factors (Fig. 2)
[21, 29–32]. Ideally, cells should be isolated from the healthy
portion of the tissue to be generated. However, this approach
is often not practical or not feasible to apply. For example, the
cell population could be scarce or keeping them in culture in a
proliferative state could be hard. In such situations, stem cells
are used as an alternative cell source [33, 34]. Stem cells are
usually preferred in tissue engineering approach as they have
the inherent ability of self-renewal and differentiation into
various cell lineages based on their potency. As native actors
in tissue repair and regeneration, they have been used as an
indispensable tool in the regenerative therapies toward skin,
nervous system, and musculoskeletal system injuries [35].
Generally, there are 4 main stem cell sources for tissue engi-
neering applications. These are embryonic tissues, fetal tissues
(like fetus and placenta, amniotic fluid, and umbilical cord),
adult tissues (fat, bone marrow, skeletal muscle, blood, etc.),
and differentiated somatic cells or induced pluripotent stem
cells (iPSCs) [36, 37]. Each source has different potency

Fig. 2 Essential factors for tissue engineering strategy including cells,
scaffolds, and bioactive molecules to regulate the behavior of the cells
(created with BioRender)
levels and advantages/disadvantages for use. Among them,
adult stem cells including the cells isolated from fat tissue hold
great promise for translational therapies since these cells are
readily available and abundant and can be isolated with high
yield [36]. Scaffolds are another major component of the strat-
egy. The important factor in scaffold design and production is
that the scaffold should match the structural and mechanical
properties of the native tissue. Chemical composition of the
Fig. 3 Schematic representation
of the main workflow of tissue
engineering strategy
emergent mater.
scaffold is also a significant part; scaffold and natural extra-
cellular matrix should be similar [38]. Moreover, the scaffold
should be biocompatible and biodegradable, while biodegra-
dation characteristics should match the regeneration rate of the
target tissue. Bioactive molecules constitute the third compo-
nent of the tissue engineering cascade. They are administered
to the culture environment either in free or in the encapsulated
form to control the proliferation and differentiation character-
istics of the grafted and host cells.
The workflow for engineering tissues using tissue engi-
neering technique is as follows (Fig. 3): Cells are isolated from
the target tissue, or stem cells are isolated and cultured toward
the required cell type. Scaffolds are designed and produced to
match the tissue characteristics. At this stage, the important
factor is to mimic the composition, structure, and 3D architec-
ture of the extracellular matrix (ECM) of the native tissue as
much as possible. ECM is a natural physicochemical support
material for the cells [28], and it is responsible for cell fate
(like proliferation or differentiation). ECM is composed of
polysaccharides (glycosaminoglycans like hyaluronan) and
large macromolecules (collagens, fibronectin, laminin) [39].
For instance, in human fetal adrenal gland’s ECM, collagen
IV, fibronectin, and laminin are expressed [40]. Moreover, the
basement membrane of the human mammary gland ECM is
composed of laminin and collagens (except for type 1) [39].
By mimicking this 3D natural environment, cells are allowed
to attach, proliferate, and function as if they are in their natural
niche. Along with the structural and mechanical similarity to
the natural microenvironment, it is important to culture the
cell-laden scaffolds within bioreactor systems that can provide
the biomechanical cues to the growing tissue [41]. These cues
include tensile, compressive, and/or torsion forces to the cells.
Including these mechanotransduction cues to the culture envi-
ronment is especially important in the engineering of dynamic
emergent mater.
tissues, tissues that are under constant dynamic loading in
their natural microenvironment, such as bone, cartilage, and
skeletal muscle.
Scaffolds are generally produced from synthetic or natural
polymers. Synthetic polymers, including polyethylene glycol
(PEG), polylactic acid (PLA), polyglycolic acid (PGA), or
their copolymers (PLGA) and polycaprolactone (PCL), can
be processed easily, and their mechanical properties can also
be adjusted relatively easily compared to the natural polymers
[42]. By using these polymers, desired geometrical shapes can
be processed, and required surface modifications are done
afterward. Surface modification is generally needed for the
synthetic polymers since they lack motifs for cell adhesion.
Another possible drawback is that the biodegradation rate may
not be regulated well enough to match the natural tissue char-
acteristics. On the other hand, natural polymers including col-
lagen, fibrin, hyaluronic acid, alginate, gelatin, and agarose
are highly used. They have higher biocompatibility and bio-
degradability compared to synthetic polymers. Their major
disadvantage is that they possess weaker mechanical proper-
ties, and it is not easy to generate weight loading and/or stable
scaffold structures from them [4].
3.2 3D bioprinting
With the combination of the abovementioned biomaterials and
biofabrication techniques, the natural microenvironment of
the cells can be mimicked both structurally and functionally
[43]. This biomimetic design should provide proper physio-
logical conditions for the cells and enhance the biological
functions such as cell-to-cell and cell-to-matrix interactions
and should support ECM production [44]. There are several
biofabrication techniques to manufacture 3D scaffolds with
desired geometries and topologies [4]. Traditional methods,
including solvent casting, freeze drying, gas foaming,
electrospinning, etc., are generally insufficient to form
predetermined shapes with uncontrollable pore shape, size,
and distribution [45]. On the other hand, 3D printing technol-
ogy allows to fabricate personalized scaffold designs with
control over the pore structure that would in turn determine
the cellular behavior within the scaffolds [46]. 3D bioprinting
involves fabrication of 3D scaffolds with cell-laden biomate-
rials, also termed as “bioinks.” Also, bioink term is different
from biomaterial. To classify a biomaterial as a bioink, it
should contain cells in both formulation and process phases
[47]. At this point, we need to separate 3D printing and 3D
bioprinting methods, because not every 3D printing tech-
niques are suitable for bioprinting. When two approaches are
compared with each other, bioprinting process should be cell
friendly [45].
3D bioprinting is an additive biofabrication method which
uses biomaterials and cells to produce the scaffold with con-
trolled features like pore geometry, external shape, etc. [44,
45]. The promise of this technology is that it allows to produce
personalized designs to replace or repair damaged organs or
tissues. Also, complex shapes and microstructures can be fab-
ricated rapidly with high accuracy [24, 48]. In this rapid
prototyping technique, a material is built layer by layer to form
a 3D structure, with or without a support material depending
on the design. Printability of the cell-laden biomaterial is re-
lated to the viscosity and the surface tension of the solution
[45, 49].
3D bioprinting has two major steps: 3D modeling and
layer-by-layer production. In the modeling step, medical im-
aging technologies are used to obtain slice images of the tis-
sues, such as CT and MRI. By the use of various software
platforms, the slice images are stored as digital models, and
2D cross-sectional data is converted into a virtual 3D model
by reconstruction of these slices. In the second step, 3D
bioprinting is performed to fabricate the actual 3D model layer
by layer [44, 50, 51].
3D bioprinting strategies can be divided into three major
groups according to how they deposit the material. These are
inkjet printing, extrusion-based printing, and laser-based
printing (Fig. 4) [52–55].
Inkjet or drop-based 3D bioprinting uses micro-droplets to
create each layer to constitute the desired pattern [44]. This
technique is one of the earliest methods that uses a cartridge,
which is similar to traditional 2D inkjet printing. Complex
structures can be 3D bioprinted with high resolution by this
method. However, the technique requires fast-gelling mate-
rials which is a limitation for the biomaterial selection. Also,
fabricating large organs can be challenging considering the
small volume of the droplets [42, 55].
Extrusion-based bioprinting is the most common tech-
nique which is composed of a fluid dispensing system to
control the extrusion and a robotic system to control the 3D
organization for manufacturing [44, 56]. In this method, the
cell-laden or cell-free biomaterial is extruded from a syringe
tip with pneumatic pressure or mechanical pistons controlled
by a computer. The viscosity of the biomaterial should be
fine-tuned carefully, since it generally is the limiting step to
control the printability and also the resolution of the product.
The material should stay stable after extrusion from the noz-
zle tip, and it should be mechanically stable to support the 3D
shape after setting (cooling and/or cross-linking) [57].
Compared to the drop-based systems, extrusion-based pro-
duction allows for continuous filament formation. This tech-
nique is commonly preferred for tissue engineering applica-
tions in the production of 3D scaffolds. Biomaterials with
high viscosity and high cell densities can be printed by using
this method. Moreover, there is a relatively higher range of
materials that could be used as a bioink, although there is
great room for further development of proper bioink mate-
rials [55, 58]. A drawback is the relatively low resolution of
fabrication [42, 44].

Fig. 4 Major 3D bioprinting technologies. a Inkjet-based system, b extrusion-based system, and c laser-induced forward transfer (LIFT)-based system
Although laser-based 3D printing or selective laser
sintering is generally used for the production of metallic
custom-made implants and prosthesis, it was reported recently
that the technique can be also used for bioprinting. This is
made possible by the use of laser-induced forward transfer
(LIFT) method [31, 42]. The device used for LIFT is com-
posed of a laser beam in which pulses are controlled in deter-
mined periods to 3D bioprint the cells and a ribbon which is a
printable material (donor material). The laser energy is
absorbed with a transport layer, which is coated with a laser
absorbing layer such as polyimide membrane, titanium, or
gold and transferred to the material. LIFT can 3D bioprint
bioinks with high cell densities and high viscosities. This
technique does not use a nozzle or a cartridge. Thus, clogging
or raising of the temperature of ejectors is not among the
problems encountered with other bioprinting techniques.
However, the dimension of the printable material is small
compared to the other techniques due to the restricted width
of the laser beam. Moreover, the equipment cost is high com-
pared to the other systems [42, 57, 59].
emergent mater.
4 3D printing strategies for the production
of the endocrine glands
Hormone replacement therapy and organ transplantation are
major treatment options for the endocrine gland dysfunction.
Unfortunately, each of these methods has their own draw-
backs. In hormone replacement therapy, the treatment must
be continued for lifelong which decreases the quality of life
of the patients significantly. Moreover, it is not straightforward
to match the healthy physiological states [3]. Organ transplan-
tation, on the other hand, has some obvious drawbacks includ-
ing the scarcity of donor organs and possible problems of
mismatch.
Tissue engineering could be an ultimate solution to these
problems. Although successful clinical applications of the tis-
sue engineered endocrine glands are yet to come to reality,
recent progress in the field shows promise for the future.
More specifically, the advancements in the 3D bioprinting
techniques carry the potential to produce functional complex
organs including the endocrine glands.
emergent mater.

Table 2 Summary of the studies in the literature on the 3D bioprinting of the endocrine glands

Endocrine Cell type Biomaterial used Biofabrication method Application Ref

gland

Thyroid gland Embryonic tissue spheroids Collagen hydrogel Hanging drop/3D printing In vivo 60
Parathyroid Human tonsil-derived Matrigel Embedding in Matrigel In vitro and 63
gland mesenchymal stem cells In vivo
(TMSC)
Adrenal gland Primary human fetal ECM from decellularization of Seeded with ECM In vitro 65
adrenocortical (HFA) cells porcine adrenal glands
Adrenal gland Bovine adrenocortical cells Alginate hydrogel Encapsulation of bovine In vivo 66
adrenocortical cells
in alginate
Pancreas Islet cells Pancreatic tissue-derived dECM Extrusion based 3D printing In vitro 67
bioink
Gonad Leydig cells Neovascularized implantable cell Extrusion based 3D printing In vitro and 3
homing and encapsulation (NICHE) in vivo
device
Gonad Theca and granulosa cells Micro-molded Seeded into In vitro 68
agarose gels micro-molded gels
Gonad Immature follicles An alginate hydrogel matrix Encapsulated in alginate hydrogel In vitro 69
Gonad Ovarian follicles Microporous hydrogel scaffolds 3D printed microporous hydrogel In vivo 70
scaffolds

Here, we covered the studies in the literature that uses tis-
sue engineering technique and biomaterials to produce endo-
crine glands with special emphasis on the application of 3D
bioprinting strategies (Table 2).
4.1 Thyroid gland
Pathologies like cancer and Hashimoto thyroiditis (autoim-
mune disease) or surgical removal of the thyroid gland can
result in hypothyroidism. The loss of function of the thyroid is
generally treated with the synthetic hormone replacement
therapy that should continue throughout the lifetime of the
patient [60].
In a study, Bulanova et al. aimed to bioprint a functional
and vascularized mouse thyroid gland by using extrusion
bioprinting by using collagen as the carrier biomaterial [60].
For this, thyroid explants were obtained from the mouse em-
bryos, and two different types of tissue spheroids, namely,
thyroid spheroids and allantoic spheroids, were prepared.
Allantoic spheroids were used for the vascularization of the
thyroid spheroids through the capillary network generated.
These spheroids were then used for the bioprinting of the
mouse thyroid gland by 3D printing on a transparent PTFE
surface. Printed thyroid constructs were cultured for 4 days on
PTFE membranes and then were transplanted to the hypothy-
roid mice under the renal capsule where they were kept for
5 weeks. This pioneering study showed promise for the pro-
duction of vascularized and functional endocrine glands by
3D bioprinting method that can also be applied to clinical
trials in the future.
4.2 Parathyroid glands
After thyroidectomy, parathyroid glands and their vascular
components can be damaged because of the cauterization pro-
cedure or as an iatrogenic outcome [61]. As a consequence,
parathyroid glands lose function, and PTH levels may chance
within the body [62]. This condition is known as hypopara-
thyroidism, and it leads to several pathologies including bone
diseases [63].
Park and his colleagues conducted some studies by using
stem cells to overcome this struggle [63, 64]. In their first
study, they demonstrated that tonsil-derived mesenchymal
stem cells (TMSC) have a potential to differentiate into cells
of tissues which are derived from the endoderm including the
parathyroid. They isolated TMSC from the tonsillar tissues
obtained after tonsillectomy and differentiated those cells into
parathyroid-like cells. Cells were cultured in 3D Matrigel, and
the PTH expressions were assessed. Results showed that dif-
ferentiated TMSCs secreted PTH hormone regulated by dif-
ferent extracellular Ca2+ dosages. Therefore, their results in-
dicated that TMSCs could be used as a cell source to produce
functional parathyroid cells and tissues, which shows promise
as a possible treatment alternative for osteoporosis in the fu-
ture [63].
4.3 Suprarenal glands
Robert Allen and his colleagues try to focus on an alternative
option for adrenal insufficiency [65]. They mentioned that
patients who suffer from this disease generally need lifelong
hormone therapies. With this study, they aimed to reduce

mortality of this condition and increase the life quality of
patients. In their research, they used decellularized adrenal
extracellular matrix (ECM), a biologic scaffold, for 3D envi-
ronment and tried to produce a functional tissue engineered
adrenal gland. Primary human fetal adrenocortical (HFA) cells
were used, and they were seeded with decellularized porcine
adrenal ECM. Adrenocortical functionalization was assessed
by secretion of cortisol associated with adrenocorticotropic
hormone stimulation. Also, cell proliferation was obtained
by adenosine triphosphate assay. Moreover, they try to find
out laminin effect on HFA-ECM constructs. Thus, they coated
scaffolds with laminin, but not an important change could be
seen on HFA morphology, proliferation, or function. At the
end of the research, they obtained HFA cell attachment and
proliferation on adrenal ECM scaffolds and endocrine func-
tion in vitro.
To overcome limits of possible treatment applications for
adrenal insufficiency, Mariya and her colleagues generated a
bioartificial adrenal [66]. Bovine adrenocortical cells were
used with alginate. To increase the life span of cells, they
encapsulate them in alginate. Rats were used for in vivo tests,
and encapsulated cells were transplanted. Results showed that
encapsulated cells could be used for adrenocortical
insufficiency.
4.4 Pancreas
A 2019 study mentions on bioprinting of pancreatic cells by
using pancreatic tissue-derived extracellular matrix (dECM or
pdECM) as a bioink [67]. In this study, they suggested that
pdECM can act as a 3D native microenvironment for pancre-
atic tissue. Functionality of the islets was measured with
glucose-induced insulin secretion test. Implantable 3D con-
structs were bioprinted by using islet cells and pdECM with
extrusion-based bioprinting.
4.5 Gonads
In 2018, Farina and colleagues conducted a study on a 3D
printed cell encapsulation device which was named as the
neovascularized implantable cell homing and encapsulation
(NICHE) device for transplantation of endocrine cells. They
used a biocompatible polymer, namely, polylactic acid (PLA),
to create the NICHE device. After in vitro characterization,
in vivo evaluation of the NICHE device was made by subcu-
taneously implantation together with Leydig cells in castrated
mice to assess vascularization and functionality of testoster-
one secretion. Also, it contained a growth factor enriched ma-
trix, polyvinylpyrrolidone (PVP)-based platelet rich plasma,
to increase the vascularization. After vascularization, the cells
were injected into the device transdermally by using the load-
ing port. Immunohistochemical analysis showed that suffi-
cient Leydig cell colonization and physiological testosterone
emergent mater.
levels were obtained in the castrated mice. With further exper-
iments, they showed that NICHE device ensured a
vascularized 3D environment for long-term cell proliferation
and function. Their innovative approach might be applied for
other endocrine diseases or pathologies in the future [3].
Another study was made to overcome ovarian failure using
theca and granulosa cells with seeded into micro-molded aga-
rose gels [68]. Researchers aimed to establish a recreated 3D
artificial human ovary with this study. After cells were isolated
from reproductive-aged women, they seeded into gels, and 3D
microtissues were self-assembled. Live dead staining and im-
munohistochemistry tests were made to assess cell viability
and identity. In the paper, they mentioned that an artificial
ovary can be fabricated using a self-assembled microtissue
in vitro.
To maintain the oocytes, researchers designed a 3D envi-
ronment using alginate hydrogel matrix as a scaffold [69].
Immature mouse follicles are encapsulated in hydrogel, and
alginate matrix gives them the provided support. To see func-
tionality, several hormones were measured by immunoassay
kits. This approach showed that alginate bead encapsulated
follicle can be cultured in vitro; in another word, follicles
can grow and develop normally in alginate hydrogel-based
3D culture. Researchers believe that these studies can be used
as a potential treatment of infertility.
In another paper, researchers investigated the effects of
scaffold’s pore geometry on cell behavior and function, and
they developed a functional bioprosthetic ovary [70]. They
used ovarian murine follicles with a 3D printed hydrogel scaf-
fold in the study. They printed various scaffolds with different
pore designs to make the analysis between cell behavior
(survival) and scaffold interaction. For in vivo tests, follicles
seeded into the microporous scaffolds, and they implanted to
mice. Findings of the article showed that an in vivo functional
ovarian implant designed and the pore architecture for scaf-
folds are crucial in cell survival.
5 Conclusion and future prospects
Demand on transplantation and the gap between the number
of patients and donors are increasing day by day. Tissue engi-
neering is a regenerative approach that can offer a solution to
this problem. The key factors for tissue engineering are proper
cells, bioactive molecules, and the scaffolds. Scaffolds can be
obtained by various rather than conventional methods includ-
ing solvent casting and electrospinning; however, recapitula-
tion of the complex structure of the endocrine glands is chal-
lenged by these techniques. 3D bioprinting is used as a rapid
biofabrication technology which allows for precise control
over scaffold design, pore size, shape, and distribution of cells
within bioinks with high accuracy. Also, personalized
(custom-made) scaffolds can be produced with this method.
emergent mater.
Hormones regulate homeostasis in the body, and they are
responsible for the organ development. They also have sec-
ondary functions and implications on other tissues. For exam-
ple, there are studies which showed that PTH is beneficial for
fracture treatment and that it can be used for fracture healing in
the clinic for the fixation of orthopedic implants [16, 71].
For gland studies in literature, it is noticed that hydrogels
are the most preferred biomaterials. As glands are classified as
soft tissues, researchers generally selected agarose gels, algi-
nate, or collagenase instead of using synthetic polymers [72].
Currently, 3D bioprinting of functional endocrine organs is
still in its infancy. Although 3D bioprinting constitutes an
important advancement toward the production of viable tis-
sues and organs using cell-laden bioinks, the problems asso-
ciated with getting the bioprinted tissue to be functional still
remains. Similarly to the development in the tissue engineer-
ing field in general, engineering and/or bioprinting of tissues
and organs which are less vascularized, less innervated, and
less functional such as cartilage were the ones to be studied
first. Organs with more complex cellular organization and
functionality require specific design considerations such as
organomorphic scaffold and/or bioreactor systems. Even
though complex cellular organization can be closely mim-
icked with the state-of-the-art bioprinter systems, the prob-
lems associated with orchestrating different cell types toward
a specific function need better mimicking of embryonic de-
velopment and tissue repair mechanisms. Therefore, engineer-
ing of endocrine glands which are organs with complex cel-
lular organization and function through 3D bioprinting is a
field with great room for research and development [72].
Moreover, there are some difficulties in keeping the cells of
endocrine glands in culture which further complicates the pro-
cesses. For example, various reports emphasize the difficulty
of long-term culturing of parathyroid cells probably due to
their high differentiation level [12, 73]. For such reasons, 3D
bioprinting of endocrine glands is a field under constant de-
velopment. Nevertheless, there are pioneering studies in the
field that show promise for the future. Future studies may also
combine their use in the co-culture systems to study the inter-
connected relations between organ systems.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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