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P. Persichetti et al.
therefore able to initiate the myelinization
process in peripheral neurons by differentiating
into Schwann cells [39, 40].
In 2010, Di summa et al. demonstrated how
ADSCs in combination with biosynthetic conduits could be a potential cell therapy for peripheral nerve regeneration, replacing autologous
nerve grafting [41]. Another exciting result was
obtained by transplanting ADSCs into canine
models with spinal cord injury and showing the
resulting functional recovery [42]. Again in animal models, ADSCs were also shown to be promising in the treatment of ischemic brain damage,
both for their ability to differentiate neurons and
for their stimulation of neoangiogenesis and
immunomodulating properties [43].
25.7.2 Hepatocyte Dierentiation
ADSCs can undergo endodermal transdifferentiation and give rise to hepatocyte cells.
In vitro, cells are grown in hepatogenic induction
medium containing the following substances:
HGF (hepatocyte growth factor), oncostatin M,
and dimethyl sulfoxide (DSMO). The cells
acquire a polygonal morphology, typical of the
mature hepatocyte, and are positive for albumin
and a-fetoprotein. Functionally, they also take on
the characteristics of the hepatocyte, becoming
capable of capturing LDL (low-density lipoprotein) and producing urea [44]. In vivo, preclinical
studies in mouse models have shown the therapeutic capacity of ADSCs in liver damage, nominating these cells as a future alternative therapy
to liver transplantation. ADSCs injected into the
portal vein resulted in a decrease in the enzymes
indicative of liver damage, ALT (alanine aminotransferase) and AST (aspartate aminotransferase), and an improvement in serum albumin
levels [45].
25.7.3 Pancreatic Dierentiation
In high glucose culture medium enriched with
the differentiation factors activin-A, exendin-4,
HGF, nicotinamide, and pentagastrin, ADSCs
showed the ability to differentiate into insulinsecreting cells. Stem cells in assuming this phenotype express pancreatic endocrine transcription
factors such as Isl-1 and the pancreatic developmental transcription factors Pax- 6, Ipf-1, and
Ngn-3. The differentiated cells are able to produce the endocrine pancreatic hormones insulin,
glucagon, and somatostatin [46].
In vivo transfer of the Pdx-1 (pancreatic duodenal homebox 1) gene, a key transcription factor
in the differentiation of pancreatic b-cells, into
ADSCs of mouse models with induced diabetes
promotes their differentiation into insulinsecreting cells, resulting in decreased glucose
levels and restoration of normal pancreatic function. This study lays the foundation for the development of a cell therapy for type 1 diabetes
mellitus [47].
25.7.4 Endothelial Dierentiation
Although endothelial cells are part of the heterogeneous SVF population derived from lipoaspirate samples, the endothelial differentiation
capacity of ADSCs has been demonstrated both
invitro and invivo. CD31-, CD34-, CD106-, and
k-1+ cells cultured with VEGF are able to differentiate into endothelial cells, through the
expression of the endothelial marker CD31 and
the von.
Willebrand factor. These cells form a branched
network, consistent with the formation of vascular structures [48].
In vitro data were supported by invivo data. In
mouse models with hind limb ischemia, transplantation of CD31- ADSCs enhances neoangiogenesis and the resulting blood supply.
Endothelial differentiation is one of several
mechanisms by which ADSCs stimulate neoangiogenesis. The secretion of VEGF and HGF
contributes to angiogenic properties and is
increased under hypoxic conditions [49]. This is
why cell therapy with ADSCs has been successfully used in the treatment of lower limb ischemia [50].
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25.7.5 Epithelial Dierentiation
After discovering the ability of ADSCs to give
rise to endothelial cells, the epithelial differentiation potential of these cells was studied. It was
seen that, in retinoic acid-enriched medium or
conditioned with renal tubular epithelial cells,
ADSCs are able to give rise to epithelial cells.
ADSCs thus treated reduce the expression of
CD90 and vimentin (a mesenchymal marker),
while they begin to express de novo cytokeratin
18, one of the rst epithelium-specic structural
proteins. Specically, ADSCs are able to give
rise to epidermal, renal, and retinal epithelial
cells [51].
In vivo differentiation of ADSCs toward the
epithelial lineage has been demonstrated in a
model of renal tubular damage and in wound
healing. In both cases, differentiated epithelial
cells are identied through the expression of pancytokeratin [52, 53].
25.7.6 Hematopoietic Dierentiation
Unlike bone marrow-derived mesenchymal stem
cells, ADSCs are not able to undergo a complete
program of hematopoietic differentiation; however, they can support this differentiation. In 2003,
Cousin etal. demonstrated the ability of ADSCs to
reconstitute major hematopoietic cell lines in
lethally irradiated mice [54]. Such cells, in fact,
are able to stimulate the differentiation of hematopoietic progenitors into myeloid and lymphoid
B-lineage cells, but are unable to maintain the survival and self-renewal of hematopoietic stem cells.
They are therefore a source of cells for reconstituting hematopoiesis in the short term [55].
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Peripheral Blood Mononuclear
https://t.me/medicina_free
Cells
SaraCarella andMariaGiuseppinaOnesti
26
26.1 Background
The prevalence of chronic wounds increases each
year and is associated with a variety of conditions, such as old age, obesity, vascular disease,
and diabetes [1] Chronic wounds are dened as
wounds stalled in a constant and excessive
inammatory state. Considerable evidence
revealed that chronic wounds are closely associated with impaired phenotype transition of proinammatory macrophages to anti-inammatory
phenotypes in wounds. Persistent hyperinammation is a distinguishing pathophysiological
characteristic of chronic wounds, and macrophage malfunction is considered as a major contributor thereof. Management of chronic wounds
remains a critical issue because continuous
inammation in these setting is very difcult to
control. One side bacterial biolms interact with
the host immune system by activating neutrophils
and pro-inammatory macrophages, resulting in
the accumulation of inammatory cytokines like
TNF-α, IL-6, and MMP [2]. On the other hand,
the unbalanced immune environment of chronic
wounds supports the proliferation of bacteria,
increasing macrophage activation in inammatory M1 phenotype which lead to the vicious
cycle of biolm growth and continuous inam-
S. Carella (*) · M. G. Onesti
Sapienza University of Rome, Rome, Italy
e-mail: mariagiuseppina.onesti@uniroma1.it
mation. Several strategies have been proposed to
improve the healing of chronic wounds such as
bioactive molecules that stimulate neovascularization and re-epithelization. Combining immunomodulatory biomaterials with active molecules
that promote the polarization of macrophages to
M2 phenotype may achieve a better pro-healing
function than using materials or cells or active
molecules alone [3]. Another therapeutic strategy
for wound healing is represented by cell-based
technologies The immune system is a key contributor to the resolution of acute wounds as well
as the persistence of chronic wounds, providing a
strong rationale for utilizing immunomodulation
to improve the tissue healing [4, 5].
26.2 Introduction
26.2.1 Autologous Peripheral Blood
Mononuclear Cell-Based
Therapy
Cell-based therapies are rapidly emerging in
regenerative medicine as dynamic treatments that
perform multiple therapeutic functions. The
treatment of non-healing wounds with bloodderived macrophages has been widely described
[6]. Danon etal. treated pressure ulcers in elderly
patients by injecting blood macrophages from
healthy donors into the wound periphery and
adding a part of the cell suspension on the wound
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Maruccia et al. (eds.), Pearls and Pitfalls in Skin Ulcer Management,
https://doi.org/10.1007/978-3-031-45453-0_26
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bed [7]. In the macrophage-treated group, 27%
patients healed, while only 6% healed in the control group (p<0.001). Moreover, the macrophagetreated group showed a faster healing (p<0.02),
and no side effects were observed [7]. A second
prospective controlled trial was designed to compare macrophage injections from healthy donors
(66 patients) to standard care treatments (38
patients) for stage III and IV pressure ulcers in
elderly patients [8]. This study observed a
signicant percentage of completely healed
wounds in the macrophages-treated group in
comparison with standard care [8] . Magenta
etal. recently published an extensive review on
autologous cell therapy from different tissue
sources (blood, bone marrow, and adipose tissue)
to treat critical limb ischemia in diabetic patients,
reporting data from basic science to clinical trials
[9]. These trials suggest that autologous PBMNCs
represent a promising therapy for non-healing
wounds.
PBMNCs consist of a heterogeneous population of lymphocytes and monocytes, CD34+
hematopoietic stem cells and circulating endothelial progenitor cells, EPCs. They represent a
promising autologous cell therapy [10].
Circulating CD14+ monocytes are originated in
the bone marrow and consist of 5–10% of circulating white blood cells in humans. They are resident in all tissues as macrophages under
homeostatic conditions(M0). These cells are
known for their immune functions, such as host
defense, the promotion and resolution of inammation, and the support of cell proliferation and
tissue restoration following injury. It has been
demonstrated that they are also progenitors at
high plasticity. PBMNCs, in fact, invitro differentiate into a multitude of mature functional cell
types in specic microenvironments, including
mesodermal and neuroectodermal lineages, as
mesenchymal stem cell [11]. The plasticity of
macrophages plays a decisive role in tissue repair
and regeneration.
Studies performed to characterize wound
macrophage phenotypes suggest that macrophage polarization state at the repair site is highly
dynamic and is dependent on the wound environment. In fact, dependent on the microenvironment stimuli, resident macrophages can
differentiate into specic subpopulations with
distinct phenotype: a pro-inammatory phenotype, antibacterial “M1,” involved in initiating
and sustaining inammatory processes, and an
anti-inammatory regenerative one “M2.”
Specically, the onset of wound healing, designated the inammatory phase, can be subdivided
into an early and late inammatory phase. After
injury, macrophage in the quiescent state (M0)
arrives at the site of injury from the systemic circulation. In the early phase which occurs at
1–4 days post-injury, the inammatory milieu
drives macrophage toward M1 polarization,
induced by some cytokines such as IFN-γ, tumor
necrosis factor -alfa (TNF-α), granulocyte/macrophage colony-stimulating factor (GM-CSF),
and bacterial lipopolysaccharide (LPS) [12]. In
the normal healing process, in the early phase,
the macrophage population switches from a proinammatory (M1) to an anti-inammatory phenotype (M2), promoting the migration and
proliferation of broblasts, keratinocytes, and
endothelial cells to repair the dermis, epidermis,
and vasculature [12]. In particular, M1 macrophages show an inammatory phenotype, known
as the classically activated M1 macrophage and
possess potent microbicidal properties and support IL-12-mediated type 1 helper T-cell
responses, which are essential in the early stages
of wound healing. They produce numerous proinammatory cytokines and chemokines, such as
the TNF-a, interleukin-1β (IL-1β), monocyte
chemoattractant protein-1 (MCP-1), and chemokine (C-C motif) ligand 2 (Ccl2), to attract
defense components and stimulate proliferation
of wound cells, such as broblasts and keratinocytes [13, 14]. M1 macrophages associated with
the early inammatory phase are highly phagocytic and produce pro-inammatory cytokines,
proteases, and reactive oxygen species (ROS)
with the goal of pathogen control and removal of
necrotic cell and tissue debris. In the late inammatory phase, which occurs at 5–7 days postinjury, the change in wound microenvironment
and the process of clearance of apoptotic cells
drive the M1 macrophages toward M2 polarization. M2 macrophages are polarized by IL-4,
IL-10, IL-13, TGF-alfa, and macrophage colonystimulating factor (M-CSF). As wounds heal, the
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local macrophage population transitions from
predominantly pro-inammatory M1 phenotype
to anti-inammatory M2 phenotypes [13]. The
M2 phenotype is known as the healing-associated
macrophage with downregulated inammatory
factors and upregulated anti-inammatory cytokines, including TGF-b, IL-10, and growth factors, such as platelet-derived growth factors
(PDGFs), resistin-like molecule-a (RelmA), epidermal growth factor (EGF), and vascular endothelial growth factor-a (VEGF-A) that drive
tissue repair. M2 macrophages have a potent
phagocytosis capacity, scavenge debris, and
apoptotic cells and promote tissue repair and
wound healing. During the granulation tissue formation and wound contraction phase of wound
healing at 7–10 days post-injury, M2 macrophages continue to release anti-inammatory
cytokines and pro-angiogenic factors, thus facilitating the inammation resolution. These cells
recruit broblasts into the wound site and promote myobroblast differentiation. Fibroblasts
are responsible of deposition of new ECM.The
activated myobroblast bridges the wound gap
and develops contractile forces to facilitate
wound contraction. M2 macrophages together
with myobroblasts induce keratinocytes to proliferate, thus facilitating re-epithelization. The
matrix deposition and tissue remodeling phase,
which occurs after 10days post-injury, is characterized by a decrease in macrophage numbers
populating the wound site.
By contrast, in chronic wounds, the inammatory phase did not resolve, leading to poor and
unsuccessful healing [15]. Persistent inammation in such wounds is characterized by excessive
quantities of pro-inammatory M1 macrophages,
whereas the amount of macrophages with antiinammatory M2 phenotypes is reduced [16, 17].
In addition, macrophages found in chronic
wounds showed a reduced capacity to eliminate
dead neutrophils. This process is responsible of a
highly inammatory environment with an excess
of inammatory molecules, such as TNF-α and
IL-1β [16, 17]. Chronic wound macrophages also
release high levels of matrix metalloproteinases
(MMP), such as MMP-2 and MMP-9, which
degrade the ECM and inhibit the proliferative
stage of healing. So, over-activated M1 macro-
phages contribute to the hyperinammation state
by secreting high levels of pro-inammatory factors, inducing a premature senescence of resident
broblasts, thus impairing restoration and the
switch M1-M2 is highly reduced and/or incomplete [18].
Perturbation of the M1 macrophage phenotype during the early inammatory phase, either
by conditional depletion or due to impaired
recruitment, results in delayed granulation tissue
formation and wound closure [19]. Depletion of
M2 macrophages during the late inammatory
phase results in prolonged inammation and
impaired wound repair [19, 20]. These M2 macrophage depleted cutaneous wounds resemble
chronic wounds typically associated with the
pathogenesis of chronic venous ulcers (CVU)
and diabetes. In fact, studies have shown that failure of cutaneous wound macrophages to undergo
the M1 to M2 phenotypic transition represents a
hallmark of these chronic inammatory diseases
[21]. These studies highlight the importance of
functional macrophage heterogeneity and the
extent to which immunomodulatory effects of
M2 macrophages are critical for efcient wound
healing and tissue remodeling.
It has been demonstrated that, by promoting
the M2 phenotype, either via specic recruitment
or local polarization, the inammatory response
may instantly be directed toward healing instead
of inammation. It has also been observed that
paracrine factors realized by M1 macrophages
induce a pro-inammatory broblast phenotype,
which is able to degrade the ECM while broblasts stimulated by paracrine factors released by
M2 macrophages show an increased proliferation
rate [22, 23] .
26.2.2 New Therapeutic Strategies
May Induce ImmuneModulation M1-M2
Several papers support this innovative regenerative strategy, which exploit immune cells like the
PBMNCs [24–27]. While it is well recognized
that some stem cell types have an immunomodulatory effect, the immune system also considerably modulates the regenerative activity of stem
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S. Carella and M. G. Onesti
cells. Therefore, components of the immune system could be exploited to improve regenerative
therapies. Importantly, signals derived from
immune cells have been shown to modulate tissue stem cells in a positive or negative manner,
leading to cell activation and dampening of quiescence or inhibition of regenerative activity [4,
5, 28] Therefore, one of the most effective ways
to promote tissue regeneration [4, 29] could be
represented by modulation of the immune system. The modulatory role of macrophages on
stem cell regenerative capacity is evident across
multiple species and plays a pivotal role in tissue
repair and regeneration [4]. Some studies have
shown that the control of inammation is crucial
to allow tissue regeneration by resident stem cells
[30]. Actually, the presence of inammation
inhibits the regenerative action of tissue-resident
mesenchymal stem cells [30]. Thus, nextgeneration regenerative therapies need an
immune-centric approach instead of the use of
stem cells [4, 24, 25, 31–33]. Innovative regenerative strategies could be developed to stimulate
macrophage polarization or to recruit subpopulations of pro-healing macrophages. Mordechai in
2013 [34], Pinto in 2014 [35], and Vagnozzi in
2020 [36] have shown that the regeneration of
myocardial tissue after ischemia was induced by
macrophages that regulate resident stem cells and
promote regeneration, suggesting that targeting
macrophages could be a new strategy to improve
infarct healing and repair. The regenerative and
stem-cell-controlling capacity of macrophages
has also recently been demonstrated in other tissue, such as bone [37, 38], tendons [39], and
muscle [40] . In addition to an indirect action
through the activation of resident stem cells,
monocytes and lymphocytes act directly on tissue
regeneration through angiogenesis and macrophage polarization.
It is well known that the innate immune system plays a key role in the tissue healing process,
while the adaptive immune system has only
recently emerged as a key player. Lymphocytes T
and in particular regulatory T cells (Treg) have
been shown to promote regeneration of various
tissues [41]. Treg can indirectly regulate regeneration by promoting neutrophil apoptosis, regu-
lating helper T cells, inducing macrophage
polarization, and also triggering resident stem
cells locally [42]. Treg can also facilitate cutaneous wound healing, mainly by the secretion of
anti-inammatory/immunosuppressive cytokines, including IL-10, IL-35, and TGF-β [42].
Tregs accumulate in skin early after wounding,
decreasing both IFN-γ production and proinammatory M1 macrophage accumulation
through the induced expression of the epidermal
growth factor receptor (EGFR) and in addition
also promote angiogenesis in ischemic tissue
though apelin-mediated sprouting in diabetic
patients [43, 44] . It has been observed that others
lymphocytes population such as NK lymphocytes and CD4-T-cells modulate arteriogenesis in
a murine ischemia model [45] . Moreover, CD8+
T-cell plasticity seems to regulate vascular regeneration [46]. Recent data have shown that naïve B
lymphocytes injected in acute or chronic diabetic
skin lesions can act as effective modulators of tissue regeneration, both in acute and in chronic
diabetic skin lesions, accelerating wound healing
associated with better-quality collagen deposition and reduced scar formation [47]. The
enhanced healing was reinforced by a higher proliferation of broblasts, together with a diminished level of apoptosis, a regenerative
modulation of cytokines, and matrix metalloproteinases [47]. Interestingly, the same process was
not observed by the injection of disrupted B cells
or hematopoietic stem cells [47].
All these relevant insights suggest a potential
development of innovative cell therapies based
on immune system cells such as monocyte/macrophages and lymphocytes to target non-healing
wounds.
26.2.3 Role ofthePBMNCs
inInducing New Vessel
Formation
In the last few years, a huge number of papers
studying the mechanism of action of PBMNCs
have been published, both on their characteristic
angiogenic potency and on their regenerative and
immunomodulatory capacity through the polar-
26 Peripheral Blood Mononuclear Cells
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ization of macrophages. Olingly et al. demonstrate that circulating monocytes are directly
recruited within wounds, where they home to a
perivascular niche and generate M2 wound healing macrophages, suggesting that blood-derived
monocytes represent the major contributors to
alternatively activated M2 macrophages and
highlighting them as key regulators of inammatory response and regenerative outcome [48]. The
M2 polarization event induces the migration and
proliferation of broblasts, keratinocytes, and
endothelial cells to repair the dermis, epidermis,
and vasculature, a cross-talk that is impaired in
diabetic wounds [12, 49]. Macrophages are
responsible for the vascularization process, rst
creating new vessels through sprouting mediated
by VEGF then forming anastomoses between
newly formed vessels [50, 51]. The macrophages’
ability to create a functional anastomosis has
been observed in invivo imaging, showing that a
macrophage arrives at the lesion, extends lopodia to physically adhere to vessels’ endothelial
ends, and through direct physical adhesion and
mechanical traction repairs brain vasculature
rupture [52]. This conclusion has been conrmed
by the observation that macrophages secrete high
concentrations of VEGF to stabilize tip cell
fusion and increase vascular complexity [50, 51].
Gurevich etal. showed, through invivo imaging,
that after tissue injury in both mice and zebrash,
macrophages could form angiogenic sprouts and
drive wound neo-angiogenesis and consequent
vessel remodeling [53].
26.2.4 PBMNC Wound Healing
inCritical Limb Ischemia
andDiabetic Foot
Diabetes has a detrimental effect on wound healing capacity, increases oxidative stress and
inammation, and causes proliferation decrease,
apoptosis, and increased senescence of endothelial cells, cutaneous keratinocytes, and broblasts. Non-healing wounds in diabetes have
been associated with an increased number of
wound monocytes/macrophages, as well as an
impaired transition from pro-inammatory into
pro-healing wound. Moreover, the migration of
cutaneous keratinocytes and broblasts is
decreased in type 2 diabetes mellitus. Similarly,
stem and progenitor cells are decreased in number due to an increase in apoptosis and senescence and exhibit reduced migration. This effect
is aggravated by growth factor demise and functional impairment, caused by growth factor
glycation.
The wound healing process is impaired in diabetic foot ulcers and plays a causative role in
limb amputations. One devastating complication
of diabetes is peripheral artery disease (PAD)
including critical limb ischemia (CLI) which
may result in limb loss.
CLI is the most severe form of PAD and is
often associated with diabetes, age, hypercholesterolemia, and smoking. CLI promotes the development of non-healing ulcers with consequent
tissue necrosis with gangrene and rest pain.
Patients with CLI are mainly treated with surgical bypass or endovascular procedures in order to
restore perfusion, thus preventing limb amputation. Recently, PBMNCs-based therapy has
become an attractive alternative for the treatment
of patients with no-option CLI (NO CLI), as well
as for patients with diabetic ulcers. The primary
goal of this therapy is to induce therapeutic
angiogenesis and neovascularization, promoting
collateral vessel formation, and tissue regeneration in non-healing wounds.
PBMNCs showed promising results to treat
NO CLI patients and diabetic foot patients [54–
56]. Huang etal. [57] showed at the angiography
of the lower limb a signicantly increased formation of new collateral vessels in diabetic patients
affected with CLI thanks to PBMNCs-based
therapy. De Angelis et al [55] documented by
angio-resonance magnetic imaging new vessel
formation and a recanalization of a double stenosis after PBMNC injection in the ischemic tissue
in NO CLI diabetic patients.
Rigato etal. on a recent meta-analysis on NO
CLI patients showed that PBMNCs were associated with a signicant decrease in amputation
and increase in amputation-free survival [58].
The same results were observed by Liew etal. in
a meta-analysis of 16 randomized trials where
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S. Carella and M. G. Onesti
PBMNCs lowered the risk of major amputation
and increased ulcer healing signicantly [59].
Others meta-analyses on autologous cellular
therapy including PBMNCs on diabetic foot
patients showed a benet of wound healing and
reduced amputation associated with transcutaneous oxygen pressure (TcPO2) increase and
reduced pain [60, 61]. Dubsky etal. compared 28
patients affected by diabetic foot disease (17
treated with bone marrow cells and 11 with
PBMNCs) with a control group treated with standard care. At 6months, authors reported a statistical increase in TcPO2 with no signicant
differences between bone marrow cells and
peripheral blood cell groups, while no change in
TcPO2in the control group was observed [62]. In
addition, at 6months, major amputation rate was
signicantly lower in the cell therapy group compared with that in the control group (11.1% vs.
50%), with no difference between bone marrow
cells and peripheral blood cells [63]. Interestingly,
the same group reported a comparable improvement of CLI major amputation with autologous
cell therapy in diabetic foot patients compared
with repeated endovascular revascularization and
a more effective healing of foot ulcers in the cell
therapy group [64]. A user-friendly point-of-care
device based on peripheral blood selective ltration to be used for intra-operatory use in human
cell therapy has been developed to produce fresh
autologous PBMNCs, with evidence in terms of
adequate potency in therapeutic angiogenesis
invitro and in vivo [62] . Recently, Scatena etal
[56] reported a positive clinical outcome at
2 years follow-up in patients with diabetic foot
and critical non-revascularizable ischemia.
PBMNC-based therapy signicantly reduced the
amputation rate and improved survival and
wound healing in 76 NO CLI patients (38 control
group, 38 treated with PBMNC). Only 4 out of
38 amputations (10.5%) were reported in the
PBMNCs group versus 15 out of 38 amputations
in the control group (39.5%) (p = 0.0037).
Furthermore, Kaplan-Meier curves and log-rank
test results showed a signicantly lower amputation rate in the PBMNCs group than in the control group (p=0.000). Furthermore, at 2 years’
follow-up, almost 80% of the PBMNCs group
were still alive compared to only 20% of the control group (p=0.000). In the PBMNCs group, 33
patients were healed (86.6%), whereas only one
patient was healed in the control group
(p=0.000). Persiani etal. have observed a 9.4%
decrease in major amputation in 18 no-option
patients with diabetes treated by PBMNC
together with an increase in TCPo2 and a pain
reduction at 2 years [33]. A similar result in terms
of major amputation has also been previously
reported on CLI non-option patients, including
diabetic and Burgers patients [65]. Interestingly,
it has been demonstrated by a histological examination of incisional biopsies of diabetic nonhealing ulcers that autologous PBMNC implants
produced by selective ltration point-of-care system and injected in the perilesional area of diabetic non-healing wounds polarize M1
macrophages in M2. Moreover, the implantation
of PBMNCs promotes relevant changes in the
overall molecular setting over time [66]. The
consequent cellular and biochemical adaptations
favor the establishment of conditions similar to
physiological ones that progressively support the
regeneration of damaged tissues and nally
wound healing measured as inhibition of HIF,
NF-KB, and TNF-alpha, progressive polarization
of M1 into M2, increase in VEGF, and newly
formed capillaries [66]. As the regenerative processes occur, an increase in the vascular network
formation is clearly seen [66]. These preliminary
data conrm in the ability of fresh, naïve, autologous PBMNCs to induce immunomodulation
through macrophage polarization and that this
results in complete wound healing in a diabetic
ulcer. On the contrary, the delivery of macrophages polarized invitro into M2a and M2c phenotypes and then injected into mouse wounds did
not accelerate healing in wild-type mice and
delayed healing in diabetic mice [67]. It seems
that to produce a positive clinical outcome in
terms of wound healing, polarization should
occur in the patients in the wounded tissue which
sends the right microenvironmental signals to
PBMNCs. A similar results were observed for the
rst time by De Angelis etal. in no-option CLI
patients, including a subset of diabetic patients,
after PBMNCs implant [55]. Histological data
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