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Fig. 24.7 Positioning and healing phases of split-thickness skin graft on dermal substitute
24 Skin Substitutes
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Fig. 24.8 Scar contracture after burn injuries
259
Fig. 24.10 Follow-up after skin grafting onto dermal
substitute
Fig. 24.9 Positioning of dermal substitute
medical devices while cell therapy has been classied as an advanced therapy medicinal product
(ATMP). In Europe, these therapies are controlled by the European Medicines Agency
(EMA), a monitoring institute of the European
Union, which is dedicated to the scientic evaluation and supervision of access to the medicines
market. The European regulation (EC) no.
1394/2007 provides the general framework on
the production and use of ATMP within the
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V. Cazzato et al.
European Union (EU). According to this regulation, an ATMP is a “medicine for human use
based on genes, cells or tissue engineering.” With
the aim of regenerating, repairing, or replacing
human tissue, some products may contain or consist of engineered cells or tissues and may also
contain viable or non-viable cells or tissues of
human or animal origin. This means that most
skin substitutes with living cells (both autologous
and allogeneic), adipose tissue and matrices containing human and/or xenogenic material, must
be considered according to these regulations
while. One of the main features of these regulations is that the production of ATMP for human
use must take place under conditions of good
manufacturing practice (GMP). Each GMP is
also linked to the single histological typology.
ATMPs are evaluated by at least three scientic committees of the EMA, the CAT (Committee
for Advanced Therapies), the CHMP (Committee
for Medicinal Products for Human Use), and the
PRAC (Pharmacovigilance Risk Assessment
Committee).
As required by the ATMP regulation (EC) n.
1394/2007, the scientic evaluation of MAA
applications for ATMP is mainly carried out by
the Committee for Advanced Therapies (CAT).
The CAT prepares a draft opinion on the quality,
safety, and efcacy of each ATMP subject to a
Marketing Authorization Application (MAA)
which is sent for nal approval to the Committee
for Medicinal Products for Human Use (CHMP).
The CHMP recommendation is then sent to the
European Commission, which adopts a binding
decision in all Member States.
These stringent regulatory and manufacturing
requirements imply intense collaboration
between centers to develop new ATMPs and promote their commercial exploitation (EMA
European Medicine Agency 2018) and determine
the great difference in terms of products and
materials between Europe and USA.
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Mesenchymal Cells fromAdipose
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Tissue
PaoloPersichetti, GiovanniFrancescoMarangi,
CarloMirra, MarcoGratteri, andLucreziaArcari
25
25.1 Introduction
Mesenchymal stem cells are tissue-specic stem
cells that meet the minimum criteria proposed by
the International Society for Cellular Therapy
(ISCT) in 2006:
1. plastic adhesion under standard culture
conditions;
2. expression of the surface markers CD73,
CD90, CD105, and the lack of expression of
the markers CD14, CD34, CD45, CD11b,
CD79a, CD19, and HLA-DR;
3. ability to differentiate into osteoblasts, chondrocytes, and adipocytes invitro [1].
Although these characteristics are common to
all mesenchymal stem cells, there are slight differences depending on the tissue of origin. In
fact, these cells, rst isolated in bone marrow,
have been found to reside in many other tissues
of the body, such as adipose tissue, synovial uid,
dental pulp, endometrium, peripheral blood, salivary glands, skin, placenta and fetal membrane,
P. Persichetti (*) · G. F. Marangi · C. Mirra
M. Gratteri · L. Arcari
Plastic Surgery Unit, Campus Bio Medico University,
Rome, Italy
e-mail: p.persichetti@unicampus.it;
g.marangi@policlinicocampus.it;
c.mirra@unicampus.it; m.gratteri@unicampus.it;
lucrezia.arcari@unicampus.it
Wharton’s jelly of the umbilical cord, and amniotic uid and membrane [2].
Although these cells can be obtained from
various sources, adipose tissue-derived stem cells
(ADSCs) are mainly used in regenerative medicine. These cells meet several requirements that
make them ideal for tissue engineering:
– they are present in abundant quantities (more
than 1 billion cells per individual);
– they can be harvested using minimally inva-
sive procedures;
– they are able to differentiate into multiple cell
lines in a controlled and reproducible manner;
and controlled and reproducible manner;
– can be transplanted autologously;
– can be expanded in accordance with CGMP
guidelines (current Good Manufacturing
Practice) guidelines [3].
Adipose-derived stem cells are autologous mesenchymal stem cells rst identied by Zuk etal. in
2001 from processed lipoaspirates [4]. These
authors identied broblast-like cells capable of
self-renewal and dened the multipotency of these
cells by their ability to differentiate in a chondrogenic, osteogenic, adipogenic, and myogenic direction. Recent studies have demonstrated the
pluripotency of these cells, as they can also differentiate into non-mesodermal cell lines [5]. The localization of ADSCs within the adipose tissue is not
yet clearly known. Traktuev etal. showed that these
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Maruccia et al. (eds.), Pearls and Pitfalls in Skin Ulcer Management,
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cells have a predominantly perivascular localization
(at the interface between the endothelium and adipocytes) and that they are rarely scattered between
adipocytes [6]. Maumus etal. identied their presence in the stroma of adipose tissue [7] while Lin
etal. identied ADSCs in the capillaries and adventitia tunica of the largest vessels [8].
25.2 Immunophenotype
The immunophenotype consists of the immunotyping of surface and intracellular molecules,
called differentiation clusters (CDs), to dene cell
populations at different stages of their development by means of ow cytometry and immunohistochemistry studies. ADSCs express typical
mesenchymal markers, i.e., CD13, CD29, CD44,
CD63, CD73, CD90, and CD105 whereas they
are negative for hematopoietic markers, such as
CD14, CD31, CD45, and CD144. As there are no
single markers to uniquely identify the cellular
subpopulations of SVF, the International Fat
Applied Technology Society (IFATS) in 2013
identied the minimum phenotypic criteria that
characterize uncultured stromal vascular fraction
and cultured ADSCs. In SVF, native ADSCs are
characterized by the lack of expression of CD45,
CD235a, CD31, while they are positive for CD34,
accounting for 20% of the entire SVF.Specically,
CD235a is used to assess contamination of erythroid lineage cells; CD45 is a marker conventionally used to identify cells of hematopoietic
origin (except red blood cells); and CD31 identies endothelial cells and their precursors.
ADSCs in culture are dened as positive for
CD73, CD90, CD105, CD44 and negative for
CD45 and CD31 [9].
More controversial is the expression of CD34;
its expression is positive in the rst passages of
culture and then becomes negative in subsequent
subcultures. The loss of CD34 expression according to Suga etal. would appear to be due to the
physiological process of commitment or differentiation of ADSCs. Indeed, these authors demonstrated that the CD34+ subpopulation at the
rst culture passages shows a higher proliferative
rate than the CD34- subpopulation, which is
characterized by for an increased differentiative
capacity in an adipogenic and osteogenic direction [10]. Other authors believe, however, that the
loss of CD34 expression is due to the lack of the
microenvironment present in culture invivo [11].
ADSCs thus represent a heterogeneous population of cells, which present a dynamic phenotype that changes during culture passages [12]. In
this regard, Li etal. conducted a study in 2011 to
dene the cell subpopulations in the stromal vascular fraction and assess their differentiation
potential in an adipogenic sense. Four subpopulations were identied:
1. CD146+/CD31-/CD34- pericytes
2. mature endothelial cells CD31+/CD34-
3. premature CD31+/CD34+ endothelial cells
4. CD31-/CD34+/CD146-/CD90+
preadipocytes
The subpopulation present in the greatest
quantity were the preadipocytes (67.6%), which
showed the greatest proliferative rate and differentiation in the adipogenic direction [13].
25.3 Secretion ofGrowth Factors
The ability to differentiate toward specic cell lines
represents one of the different mechanisms by
which ADSCs initiate tissue repair and regeneration. These cells have been found to possess paracrine activity, being able to secrete various growth
factors such as bFGF (basic broblast growth factor), VEGF (vascular endothelial growth factor),
IGF-1 (insulin-like growth factor 1), HGF (hepatocyte growth factor), and TGF-b1 (transforming
growth factor beta 1). This characteristic also
makes these cells particularly promising for the
treatment of ischemia. Indeed, hypoxia provides a
stimulus for the secretion of these growth factors,
which promote angiogenesis and tissue repair.
ADSCs are also responsive to the growth factors
bFGF and PDGF (platelet-derived growth factor),
released from damaged extracellular matrix and
activated platelets respectively, which stimulate
cell proliferation, leading to more efcient regeneration. ADSCs also express receptors for the
growth factors VEGF, HGF, and EGF (epidermal
growth factor). VEGF stimulates cell migration
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and promotes chondrogenic differentiation, HGF
promotes hepatogenic differentiation invitro, and
EGF inhibits adipogenic differentiation [14].
Also included in the secretome of ADSCs are
the cytokines IL-6, IL-7, IL-8, IL-11 TNF-a, the
factors G-CSF and M-CSF (granulocyte and
macrophage colony-stimulating factors), and adipokines, released in response to inammatory
stimuli [15].
25.4 Immunomodulatory
Properties
Similar to bone marrow-derived mesenchymal
stem cells, ADSCs have been shown to possess
immunomodulatory properties, including the
ability to restore immune tolerance. Indeed, these
cells have shown the following characteristics.
– the lack of expression of human leukocyte
antigens (HLA) class II;
– the ability to inhibit pro-inammatory
cytokines;
– the ability to stimulate the production of the
anti-inammatory cytokine IL-10;
– the induction of the antigen-specic response
of regulatory T lymphocytes.
These characteristics have enabled these cells
to be used in the treatment of immunological diseases and have made them safe for use in both
autologous and allogeneic transplantation [16,
17].
25.5 Dierentiation ofADSCs
ADSCs are capable of differentiating into multiple cell lines both invivo and invitro. The rst
studies conducted by Zuk et al. on ADSCs had
shown the ability of these cells to differentiate
into mesodermal cell lines; later studies, however, demonstrated the ability to differentiate into
cells of endodermal and ectodermal origin
through a process of cross-differentiation, which
is why they are now considered pluripotent cells
[18, 19] Fig.25.1.
Fig. 25.1 The differentiation capability of ADSCs
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25.6 Mesodermal Lines
25.6.1 Adipogenic Dierentiation
ADSCs show a greater capacity for adipogenic
differentiation than bone marrow-derived mesenchymal stem cells, which show a greater
capacity for osteogenic differentiation [20].
The culture medium used for adipogenic differentiation typically contains the following
induction factors: insulin, IBMX (3-isobutyl1-methylxanthine, a phosphodiesterase inhibitor that increases levels of the second messenger
cAMP), dexamethasone, rosiglitazone, and
indomethacin. At 7days after exposure to the
induction factors, the stem cell begins to acquire
the phenotype of a mature adipocyte through
the formation of multiple intracytoplasmic lipid
droplets, detectable by Oil Red O or Nile Red
staining. These fat accumulations gradually
increase in size to merge into a single lipid
droplet over 2–3 weeks, giving rise to the
mature unilocular adipocyte. During this
period, the extracellular matrix proteins bronectin, laminin, and various types of collagen
are expressed. The differentiation process
occurs through the expression of genes specic
to the mature adipocyte, such as PPAR-g2 (peroxisome proliferator-activated receptor), leptin,
aP2 (adipocyte protein 2), LPL (lipoproteinlipase), and glucose transporter type 4 [21]. In
vivo differentiation can be achieved through the
use of scaffolds containing specic biomolecules that stimulate the differentiation of
ADSCs in situ. The scaffolds used include type
I collagen, brin, alginate, hyaluronic acid, and
matrigel. ADSCs can also be associated with
matrices, making the differentiation process
even more efcient. From a clinical point of
view, ADSCs, combined with different biomaterials and implanted in the subcutis, can be
used to promote regeneration of the adipose tissue in the treatment of volume defects, breast
reconstruction, lipodystrophy, and congenital
malformations [22].
25.6.2 Chondrogenic Dierentiation
The chondrogenic differentiation capacity of
ADSCs has made it possible to use these cells as
a therapeutic alternative to autologous chondrocyte cultures in the repair of articular cartilage
damage. In vitro chondrogenic differentiation
can be achieved by supplementing the culture
medium with BMP-6, TGF- b, dexamethasone,
ascorbate-2-phosphate, and IGF-1. For this type
of differentiation, the cells require a threedimensional microenvironment, which is
achieved with a culture technique called “micromass pellet culture.” This technique mimics the
condensation of pre-cartilage that occurs during
embryonic development and promotes cell-cell
interaction and the production of cartilage-like
matrix. After 1–2weeks, differentiated chondrocytes express type II and type IV collagen, aggrecan, prolyl endopeptidase, and proteoglycan
sulfate, all components of the cartilage-like
extracellular matrix. These cells can be highlighted with Alcian Blue staining and staining for
type II collagen [23, 24]. The scaffolds used to
induce the differentiation of ADSCs into chondrocytes invivo are alginate, agarose, brin, gelatin, and chondroitin sulfate [25]. This
differentiation capacity makes ADSCs a promising cell therapy for reconstructive surgery of the
nose and ear and for regeneration of intervertebral disks, tendons, and articular cartilage damaged by acute trauma or chronic diseases such as
osteoarthritis [26, 27].
25.6.3 Osteogenic Dierentiation
Osteogenic differentiation of ADSCs in vitro is
achieved over 2–4 weeks using media enriched
with 1,25 dihydroxyvitamin D3, ascorbate- 2phosphate, BMP-2, and b-glycerophosphate.
Osteoblasts are characterized by the production
of calcium phosphate in the extracellular matrix,
which can be detected with Alizarin Red or von
Kossa staining. During osteogenesis, the follow-
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ing genes are expressed: alkaline phosphatase,
type I collagen, osteopontin, osteocalcin, bone
sialoprotein, Runx-1, BMP-2, BMP-4, the parathormone receptor, and BMP 1 and 2 receptors
[28].
In vivo differentiation into osteocytes is
achieved using bioceramics, such as hydroxyapatite and b-tricalcium phosphate, which mimic
bone architecture. Other biomaterials used are
collagen (being one of the fundamental components of bone tissue), or synthetic polymers, such
as PCL or PLA [29]. The osteogenic differentiation of ADSCs has given rise to a line of research
for their use in the regeneration of bone tissue
following trauma or congenital defects. The ability to repair defects of the cranial bones, mandible and maxilla, has been demonstrated [30, 31].
25.6.4 Myogenic Dierentiation
In vitro, ADSCs are able to differentiate into both
smooth and skeletal muscle cells. Differentiation
into skeletal muscle cells is achieved in medium
enriched with horse serum, hydrocortisone, and
dexamethasone through the expression of the
muscle-specic transcription factors MyoD and
myogenin. ADSCs fuse to form multinucleated
myotubes and express skeletal muscle lineage
surface proteins such as myosin heavy chain [32].
Differentiation into smooth muscle cells is documented by the expression of smooth muscle
a-actin, calponin, and SM22-a [33]. The capacity
for myogenic differentiation has also been demonstrated invivo. In mdx mouse models (which
have a mutation in the DMD gene coding for a
non-functioning dystrophin) treatment with
ADSCs showed the restoration of dystrophinexpressing muscle bers. These results support
the possible use of ADSCs as a cell therapy for
degenerative muscle diseases such as Duchenne
muscular dystrophy [34]. Differentiation into
smooth muscle cells invivo has also been demonstrated in mouse models by injecting ADSCs
into the urethra and bladder, and morphologically
and phenotypically documenting the incorporation and differentiation of these cells into smooth
muscle [35] In 2003, Rangappa etal. showed the
ability of ADSCs to differentiate into cardiomyocytes in culture medium containing 5- azacytidine.
After 3weeks of culture, the adipose stem cells
acquire a cardiomyocyte-like morphology and
spontaneous contractile activity. These cells are
immunostained positive for myosin heavy chain,
troponin I, and a-actinin [36]. ADSCs are also
capable of spontaneously differentiating into
myocardiocytes. After 11–14 days of culture,
some cells begin to show contractile activity reminiscent of cardiomyocytes and myotube-like
structures appear. At 20–30 days of culture,
branched cardiac muscle bers are observed.
During cardiomyogenesis, cardiomyocytespecic transcription factors GATA-4 and
Nkx2.5, ventricular and atrial myosin light chains
(MLC-2v and MLC2-a), and atrial natriuretic
peptide are expressed [37]. ADSCs may, therefore, promote myocardial regeneration, also considering the positive contribution of paracrine
activity, which stimulates neoangiogenesis and
immunomodulatory properties. In vivo, however,
only a small percentage of stem cells are able to
undergo cardiomyogenic differentiation. In this
regard, it has been shown that exposure to bronectin, which occurs 12 hours after myocardial
infarction, stimulates the engraftment and proliferation of ADSCs in the infarcted tissue [38].
25.7 Non-mesodermal Lines
25.7.1 Neuronal Dierentiation
Neurogenesis showed the ability of ADSCs to
give rise to cells derived from ectodermal tissue.
The culture medium used to achieve neuronal differentiation is enriched with butyl hydroxyanisole, valproic acid, insulin, EGF, and FGF.The
stem cells acquire neuronal morphology through
the expression of the following markers: neuronspecic enolase, nestin, b-tubulin III, the MAP2,
and NeuN intermediate laments. It has also
been shown that ADSCs can also differentiate
into glial lineage cells by expressing the corresponding markers, i.e., S100, p75 NGFR (nerve
growth factor receptor), NG2 (nerve glial antigen
2), and the glial brillary acidic protein. They are
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