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14 Biomimetics: ANew Abstraction forBone Implant Design
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14.7 Conclusions
In order to medically complete bone regeneration, the current therapies must evolve in accordance with current research. Biomimetics drives
creativity and innovation to emulate the healing
mechanisms already in place. Due to the biological nature of much of the research mentioned in
this chapter, product development still faces considerable regulatory hurdles. However, the baseline knowledge derived from experiments and
research listed herein is a great platform upon
which therapies can be built. The biomimetic
boundaries to be pushed in bone regeneration are
collagen or collagen analogs, soluble minerals,
and active cell populations.
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The Role ofMicrobiota inSkin
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Regeneration
AngelaFaga, MarcoMarioTresoldi,
andGiovanniNicoletti
15
15.1 Introduction
Regeneration can be dened as the regrowth of a
damaged or missing organ part from the remaining tissue. It is a common feature in different animal species, as amphibians; on the contrary, in
humans the wound healing process does not lead
to regeneration but to a scar. Actually humans are
not lizards; therefore, they pay their position at
the endpoint of phylogeny with an almost total
loss of the regenerative attitude, except for liver
and, sometimes, for ngertips [1].
Everyday plastic surgeons face disguring or
aesthetically impairing scars, huge loss of sub-
A. Faga (*)
Advanced Technologies for Regenerative
Medicine and Inductive Surgery Research Center
(T.A.Me.Ri.C.I.), University of Pavia, Pavia, Italy
e-mail: angela.faga@unipv.it
M. M. Tresoldi
Plastic Surgery Unit, Department of ClinicalSurgical, Diagnostic and Pediatric Sciences,
University of Pavia, Pavia, Italy
I.C.S. Maugeri Research and Care Institute,
Pavia, Italy
e-mail: marcomario.tresoldi@unipv.it
G. Nicoletti
Advanced Technologies for Regenerative
Medicine and Inductive Surgery Research Center
(T.A.Me.Ri.C.I.), University of Pavia, Pavia, Italy
Plastic Surgery Unit, Department of ClinicalSurgical, Diagnostic and Pediatric Sciences,
University of Pavia, Pavia, Italy
e-mail: giovanni.nicoletti@unipv.it
stance or difcult to heal wounds. Despite the
current high sophisticated technical skills, the
problem of regeneration is impairing severely the
treatment of all of these clinical problems.
Therefore, current plastic surgery is increasingly
turning to regenerative medicine, an emerging
interdisciplinary eld of research and clinical
applications focused on the repair, replacement
or regeneration of cells, tissues or organs [2].
The wound healing process consists of highly
integrated and overlapping phases, whose steps
are clot formation, inammation, broblasts
recruitment, epithelization and scar remodelling.
Many types of immune and non-immune
cells, including macrophages, neutrophils, platelets, broblasts, vascular endothelial cells and
keratinocytes, contribute to wound healing. The
inammatory response begins immediately upon
injury and leads to the secretion of a variety of
growth factors and cytokines, which regulate the
cellular and tissue movements that are required
for repair [3, 4]. It is proven that the proliferative
phase of wound healing is inversely proportional
to the quantity of inammatory post-traumatic
reaction [5].
Multiple local and systemic factors can interfere with one or more of these phases, thus causing improper or impaired tissue repair; the main
local barriers are necrosis, bacteria and exudates.
Regarding the negative role of bacteria, surgeons have slowly learned from Semmelweis’
time [6] to fear and ght microbes by scrubbing
their hands before surgery, treating the operating
© Springer Nature Switzerland AG 2019
D. Duscher, M. A. Shiffman (eds.), Regenerative Medicine and Plastic Surgery,
https://doi.org/10.1007/978-3-030-19958-6_15
159

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A. Faga et al.
eld with antiseptics and administering
antibiotics to the patients. Actually, the infection
of a wound turns to dehisce and to a clinical
disaster.
In the past, the role of microorganisms was
investigated only during pathologic events; however, the availability of upgraded technologies is
currently allowing a more sophisticated and thorough analysis of their association with the host.
Microorganisms are present in every type of
environmental niche and interplay with all its
components.
The term microbiota is used currently to dene
the microbial community, composed of bacteria,
viruses, fungi and Archaea, hosted by the human
body [7]. Actually the microbiota might be considered as a ‘superorganism’ [8], ruled by specic inter-microbial communications and cell
signalling associations with the human organism.
Host-microbe interactions are essential for various aspects of normal mammalian physiology,
ranging from metabolic activity to immune
homeostasis [8–11].
Every area of the human body hosts a unique
microbial community.
Microbial cells living in the human body outnumber the totality of cells of our organism by a
factor of ten, and the genes of those microbial
cells outnumber the human genes by a factor of
hundred [12]; the term microbiome is currently
used to indicate the whole genome of the microbial community.
From 2008, starting with the U.S.A. Human
Microbiome Project (HMP), different studies
were performed to establish any possible associations between the human microbiota and health
and disease conditions [13, 14].
The difculty of recreating the same conditions invitro as invivo is a major humper for the
microbiota characterization [15]. Thanks to current metagenomics techniques, it is possible to
screen the whole genes of an environmental sample and to create a genetic library of the proteins
expressed by the community [16]. Moving from
conventional cultures to metagenomics, a perspective change is occurring, focusing on the
activity and products of a whole community,
rather than of a single community member [17].
15.2 Skin Microbiota
Skin is the rst defensive line of the human body
and houses different populations of microorganisms. Every squared centimetre of skin (including hair follicles and sebaceous glands) contains
~1 billion bacteria [18].
The acquisition of the microbiota begins at
birth [19], with the transfer of maternal microbiota, and differs according to vaginal or caesarean delivery. The skin microbiota evolves
over the years, becoming similar to that of
adults by the age of 12–18 months [20].
Basically, the microbiota composition is related
to the skin structure and physiology [21] and to
the adnexa-regulated microenvironment.
Furthermore, microbiota composition changes
according to ethnicity, geography and lifestyle
[12]. Individuals modify their own microbiota
through contact with other individuals, visiting
different places, and eating food. The microbiota composition is dependent on the anatomical
area too. Microbiota α-diversity expresses the
difference in community composition, comparing a specic area of the body to other areas of
the same individual. Microbiota β-diversity
expresses the difference in community composition comparing a specic area of the body
between different individuals. Notably, antecubital fossae have the highest β-diversity, but the
lowest α-diversity [22]. The differences among
different individual microbiotas are due to the
less represented bacteria.
Most skin microorganisms are commensal or
temporary passing members. The following four
bacterial phyla are present on human skin:
Actinobacteria, Proteobacteria, Firmicutes and
Bacteroidetes. Staphylococcus, Propionibacterium
and Corynebacterium are the dominant genera,
and their inter-individual distribution is constant.
Furthermore, on normal human skin viruses
(human papillomavirus, human polyomavirus,
circovirus and bacteriophages) and eukaryotes
microbes (fungi and protists) are present [23].
Considering the community composition in different individuals, it is possible to state that the
microbiota of an individual is as unique as a ngerprint [24, 25].

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Microorganisms do not inhabit just the epidermis. Metagenomic techniques demonstrate
bacterial DNA deep in the dermis too, although
such a technology may not assess the viability
of the associated microorganisms [12, 23].
Phagocytic cells might translocate the supercial microbiota to a sub-epidermal level and
epidermal physical barriers or antimicrobial
peptides (AMPs) would serve as key regulators
in the maintenance of dermal microbiome
homeostasis [26]. The action of microorganisms below the basement membrane might be
in correlation with the immunological properties of the skin. The skin is an active immune
organ where the keratinocytes can no longer be
considered as the sole barrier against the external environment, but as components of a complex immune-regulatory network [27]. The
resident skin bacteria provide the rst line of
defence against potentially dangerous pathogens, and produce small molecules that inuence the growth and behaviour of their microbial
neighbours. The major innate mechanism of the
antimicrobial defence of the skin consists of
AMPs, such as defensins, cathelicidin LL-37
and dermicidin [28]. These peptides are emerging as important tools in the control of skin
pathogenic bacteria. It is proven that skin commensal bacteria have a promoting effect on T
cell response, controlling nuclear factor-κB signalling and the production of cytokines, such as
tumour necrosis factor (TNF)-α and
interleukin-1β [29, 30] although they can act
both in synergy and in opposition to the immune
system [31].
Germ-free mice without commensal skin
microbes have been demonstrated to produce
abnormal cytokine and cutaneous T cell populations, as they were unable to mount an appropriate immune response against the intradermal
Leishmania major infection; immunity could be
rescued by allowing Staphylococcus epidermi-
dis colonization on the mouse skin [30].
As skin microbiota is important in the development of a well-functioning immune system
and in the modulation of the inammatory processes, it may be signicant in the wound healing
process.
15.3 The Role ofMicrobiota
inWound Healing
Actually, although many animal species regenerate spontaneously in the wild life, this process
does not take place in a germ-free environment.
While the role of microbiota in controlling the
health-disease balance is being widely investigated as a source of novel therapeutic options,
however the local application of bacterial products to enhance wound healing has been reported
rarely.
Topical bacterial lipopolysaccharide is demonstrated to affect the wound healing process
by accelerating the resolution of inammation,
increasing macrophage inltration, enhancing
collagen synthesis and altering the secretion of
numerous mediators involved in skin regeneration [32]; moreover, inoculation with
Pseudomonas aeruginosa is demonstrated to
accelerate re-epithelialization and neovascularization in wound tissues through the production
of TNF-α [33]. All of these effects might be
related, although not exclusively, to some bacterial both anti-inammatory and antibioticlike actions, due either to microorganisms
metabolites, or to a virtuous balance among
strains, or both.
It has long been known that the recovery of
skin lesions and scar maturation can be boosted
by the topical applications of spring waters where
a rich presence of non-pathogenic microora is
established [34–38].
In recent years, researchers have developed
programmes to investigate the molecular mechanism underlying benecial effects of spring
waters. Aquaphilus dolomiae is a non-spore-
forming bacterium belonging to the Neisseriaceae
family, which is isolated from Avène thermal
water (France), historically used in the management of chronic inammatory skin diseases. The
incubation of human keratinocytes with
I-modulia, a biological extract from cultures of
Aquaphilus dolomiae, showed an upregulation of
the innate immune response [39, 40]. Similarly,
the lysate of Vitreoscilla liformis has been
shown to enhance skin defence mechanisms and
to decrease UV-induced sunburn cells in human

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skin, possibly by the activation of cutaneous regulatory T cells. Vitreoscilla liformis is a lamen-
tous Gram-negative aerobic bacterium belonging
to the Neisseriaceae family found in LaRochePosay thermal water (France), historically
applied to manage chronic inammatory skin
diseases [41, 42].
Our research group is originally assessing the
peculiar regenerative properties of a thermal
spring water (Comano, TN, Italy). In an experimental animal (rabbits) wound model, the areas
treated with this water healed faster than the areas
treated with conventional medical dressings and
demonstrated a network of collagen and elastic
bres comparable with the normal skin [37]. In
an in vitro trial on human skin broblasts cultures, we observed that the vitality of cells maintained in DMEM (Dulbecco enriched
conventional medium) 20% replaced with the
Comano water, at 72hours was 31% higher than
the control cultures maintained in conventional
DMEM [43]. Moreover, in an ex vivo human skin
experimental wound model, used to simulate the
physiological conditions in vivo, the adjunct of
this spring water to the culture medium induced a
faster repair of the wound, a more ordered reorganization of the newly formed collagen bres
and a higher number of actively DNA synthesizing epithelial cells [44].
In this spring water with conventional culture
methods nine non-pathogenic different strains
were isolated, whose common features are a
very rare virulence, an antibiotic-like activity
and the ability to control environmental pollution [38].The whole bacterial ora genoma is
being investigated by the research group of
CIBIO laboratories (Trento University, Italy)
too, integrating culturomics and shotgun metagenomics. Preliminary results demonstrate that
while the isolates are phylogenetically distributed among traditional phyla (Proteobacteria,
Actinobacteria and Firmicutes), most of the
metagenome- assembled genomes belong to
phyla that are typically recalcitrant to cultivation, and many new species have been isolated
or detected [45, 46]. The researchers are also
testing specic strains to detect any immune
modulating, proteolytic and anti-bacterial activity, likely related to the proven role of this water
in the wound healing process.
Thus, the role of microbiota in the skin health
maintenance appears to be of paramount importance and, therefore, indicates towards a hypothesis of exerting a positive effect in the skin
healing processes.
15.4 Conclusion
The mechanisms on which the microbial community structure and the association between host
and symbiont are based must become incorporated into the current denition of human health.
Medical intervention must aim to minimize or
avoid damage to health-associated homeostasis
between humans and their microbiota; therefore,
the therapeutic strategies to maintain healthy skin
may require the inhibition of the growth of pathogenic bacteria, as well as the promotion of a balanced microbiota. Ignoring the association
between host and microbiota in therapeutic planning is a shortsighted conduct, as demonstrated
by the spread of antibiotic-resistant microorganisms [47]. Thus, the role of microbiota is resulting in novel and fascinating scenarios in
regenerative medicine and surgery, promoting
unexpected progress in the development of novel
clinical proposals, not only for tissue regeneration and wound healing but for anti-ageing purposes, too.
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https://doi.org/10.1186/

Erythropoietin: AnInnovative
https://t.me/medicina_free
Therapeutic Approach inThermal
Trauma
ChristinaIreneGünter
andHans-GüntherMachens
16
16.1 Introduction
Erythropoietin (EPO), whose existence had been
postulated already more than a century ago [1], is
an endogenous hormone that is produced by broblasts of the renal parenchyma depending on the
partial pressure of oxygen in the peripheral blood.
It controls the differentiation of erythrocytes in
the bone marrow. The rst isolation of the protein
succeeded half a century ago (1953) [2]. However,
it took more than a quarter of a century (1984) to
develop genetically modied hamster cells, which
produced recombinant human EPO.Only thereafter the pharmaceutical mass-production could
start. In the meantime, EPO has become indispensable in clinical routine: in anemia of endstage renal failure, in tumor-induced anemia, and
before donating autologous blood [3]. About
15years ago, rst papers were published on the
non-erythropoietic effects of EPO [4].
16.2 EPO Eects After Trauma
Since then, numerous publications have demonstrated that EPO plays a key role in the response
to both acute and chronic tissue damage. EPO
inhibits the initial inammatory response in a
C. I. Günter (*) · H.-G. Machens
Department for Plastic Surgery and Hand Surgery,
Klinikum rechts der Isar, Technical University of
Munich, Germany
variety of tissues, thereby facilitating healing [
Also in the regenerative phases of wound healing, various EPO effects have already been published. These include, for example, the inhibition
of apoptosis [6, 7], which has been described in
particular for endothelial cells of the capillaries
and other small blood vessels that run in the
immediate vicinity of the trauma zone. These are
often damaged by the reactions of the inammatory phase and because of apoptosis. This in turn
means that thromboses develop, and thus the
trauma zone is ultimately enlarged.
The stem cell recruitment [8] is described, for
example, for endothelial progenitor cells, which
are recruited locally and from the bone marrow.
Effects on mesenchymal stem cells have also
been described. In vitro experiments conrmed
the effect of EPO on human dermal mesenchymal stem cells cultured under hypoxia conditions. In addition, increased concentrations of
IL-6 were added to the culture medium, which
alone had a markedly antiproliferative effect. In
addition, EPO added to the culture medium
showed an increased proliferation rate [
The release of different growth factors [10] is
also inuenced by EPO. Angiogenesis is stimulated on the one hand by the release of relevant
growth factors and on the other hand by the
above-described effects of stem cell recruitment
and inhibition of apoptosis. The increase in reepithelialization is also caused by the secreted
growth factors [10]. Comprehensive reviews of
the acute and longer-term anti- inammatory
5].
9].
© Springer Nature Switzerland AG 2019
D. Duscher, M. A. Shiffman (eds.), Regenerative Medicine and Plastic Surgery,
https://doi.org/10.1007/978-3-030-19958-6_16
165
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