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conrmed dermal granulation tissue and an
increased number of monocytes (CD68+) and
newly formed microvessels (CD31+). After the
PBMNC treatment in the healed epidermis, the
presence of the new vessels was observed,
whereas dermal inammation and monocyte
inltration were reduced.
26.2.5 PBMNCs inAutoimmune
Disease
PBMNCs could have an indication of use in autoimmune- based diseases where there is a vascular
and/or microcirculation alteration not only for
their angiogenic capacity but also for their ability
to regenerate tissues and restore the correct M1/
M2 balance, always compromised in the nonhealing lesions of patients suffering from these
pathologies, as recently published [68–73].
In particular, PBMNC implants have been
successfully used to treat vasculopathy-related
manifestations of Scleroderma patients [69] The
authors also obtained successful results regarding
the healing of the digital ulcers in scleroderma
patients [69].
26.2.6 PBMNC-Based Therapy
andPain
Pain with non-healing wounds very often experience pain, which impact on their quality of life.
Recent increasing evidence underlines the role of
macrophage as a peripheral pain regulator. A
number of substances derived from the primary
afferent neurons and from macrophages have
been identied as mediators for the neuroimmune cross-talk, which is involved in somatic
and visceral pathological pain including inammatory and neuropathic components.
Immunity not only controls pain development
and maintenance but is also essential for pain
resolution. Macrophages and regulatory T cells, a
subpopulation of T lymphocytes with immune
regulatory function, were shown to be important
contributors to pain recovery [74].
Macrophage inltration into the nerve is an
essential step to allow nerve regeneration. In particular, anti-inammatory/reparative M2 macrophages have been indicated to play a role for
repair processes after nerve injury [75]. Further,
it has been demonstrated that tissue-resident
macrophages, as well as bone marrow-derived
inltrating macrophages, interact with the primary sensory neurons in the peripheral tissues
and regulate not only inammatory responses but
also pain signals. Thus, the PBMNC injection
represents a valid therapeutic solution to alleviate
pain in chronic ulcers.
26.2.7 Methods toObtain
Autologous PBMNCs
PBMNCs can be obtained by apheresis, Ficoll
centrifugation, and selective ltration.
Compared to other autologous cell therapies
that require bone marrow or adipose tissue harvesting, PBMNCs are particularly easy to obtain
through a simple peripheral blood collection. The
non-invasiveness of the procedure also allows for
repeated implantation instead of one shot. This is
a great advantage as it has been shown that the
frequency of implantation is more important than
the number of cells implanted.
Moreover, in the past, PBMNCs were primarily isolated and concentrated through therapeutic
apheresis [57, 65]. Today, it is possible to concentrate PBMNCs directly in the operating room
using dedicated point-of-care systems.
Autologous PBMNC concentrate can be produced with a blood ltration system. It is a pointof- care medical device for intra-operative use, for
the rapid preparation of total nucleated cells,
TNC/PBMNCs concentrate from 20 to 120mL
of anticoagulated blood for use in human cell
therapy applications [76]. Characteristic of the
ltration system is the separation technology
based on gravity ltration. This lter is able to
separate cell populations across the membrane
potential, useful for the concentration of autologous TNCs from low volumes of peripheral blood
(20–120 mL). The system is easy to use, not
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operator-dependent, single-use. It separates cells
by gravity and requires no dedicated instrumentation and nor centrifugation. PBMNCs are produced in approximately 10 min, in three steps:
load, lter, and recover. Filtration takes place in
8–12min, PBMNCs remain trapped in the lter,
and after a backwashing of the lter with 10mL
of sterile physiological saline which allows to
collect the cells in an empty syringe the cells are
ready to be implanted. Cells are not further
manipulated, stored, or frozen. Implantation
takes place immediately after ltration in a single
surgical procedure. The cell concentrate produced with this system has been extensively
characterized by Spaltro etal [76] In particular,
total nuclear cell TNCs are concentrated 2.9-fold
(16.24 +/−3.97 × 103/μL) with an average
implanted dose of 2.08 +/−0.53 × 10,8 while
MNCs (monocytes and lymphocytes) are concentrated 4.2-fold (8.3+/−2.31×103/μL) with an
average implanted dose of 1.06 +/−0.28× 108).
The cell concentrate contains neither plasma nor
serum and does not concentrate platelets (from
226,000 platelets/μL in peripheral blood to
292,000/μL in the cellular concentrate) and cannot be traced in any way to platelet-rich plasma
(PRP). Moreover, the system also concentrates
CD34+ stem cells, which are enriched by
5.6%±4.2% compared to peripheral blood with
an average implanted CD34+ cell count of
1.37×10 [6], which corresponds to 0.7%–1% of
total implanted cells. Moreover, the CD34+
hematopoietic stem cell enrichment efciency of
this selective ltration system is comparable with
the CD34+ concentration obtained from the use
of the point-of-care device for bone marrow cells
(BMAC 2) [77]. PBMNCs isolated by this ltration system have also been shown to secrete a
panel of angiogenic factors and are able to
migrate in response to a gradient of VEGF and
stromal-derived factor 1, SDF-1 [76].
Interestingly, ltration preserves and optimizes
the release of paracrine factors, which is signicantly reduced when the cell concentrate is produced by centrifugation [76]. In addition, after
PBMNC injection into a mouse model of hind
limb ischemia, the cell concentrate obtained by
this system from healthy donors induces neo-
vascularization by increasing the number of capillaries, arterioles, and regenerative bers [76]
suggesting that this ltration system represents a
new, effective, and reliable point-of-care device
to obtain an autologous cell product from peripheral blood with adequate potency for therapeutic
angiogenesis.
Key Points
PBMNC treatment protocol.
The procedure is performed in opera-
tions room as follows:
• In a sterile-operating environment,
PBMCs are obtained from a 100–
120 mL venous blood sample
(Fig.26.1).
• 120 mL of acid citrate dextrose, ACD
anticoagulated blood is transferred to
the upper bag of the system which is
hang up to let the blood ow by gravity
through the lter below (Fig.26.2).
• The selective membrane retains the total
nuclear and mononuclear cells and the
residual blood owed to the waste blood
bag under the lter.
• After ltration, which takes around
10–15min, a 10 mL of saline solution
backwash allows to harvest the
PBMNCs from the lter, resulting in
8–10mL of cell concentrate collected in
a cell recovery bag and ready to be
injected (Fig.26.3).
Sedation and/or loco-regional anesthesia are performed in order to facilitate inltration of the cell concentrate
After appropriate surgical cleansing of
the wound bed, the injection is performed perilesionally and intralesionally using a 21-G needle under the
lesion bed. A gauze is applied upon the
wound and a bandage is performed.
After the autologous PBMNC implant,
the wound may be covered with a with a
hyaluronic acid-based medication. A
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gauze is applied upon the wound and a
bandage is performed when indicated.
The session may be repeated for
three times each 3weeks, depending on
the clinical state of the ulcer.
Post-operatory: treatment with antibiotics and subcutaneous anticoagulants
are administrated when indicated.
281
Fig. 26.1 Peripheral blood collection
Fig. 26.2 Filtration system
Fig. 26.3 Cell concentrate collected in a cell recovery
bag and ready to be injected
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26.3 Tip andTricks
• Local anesthesia should be avoided in order to
not inuence cell vitality. A loco-regional
anesthesia must be preferred.
• In order to improve the cell vitality, a homogeneous distribution of the cell concentrate is
necessary. For this reason, a grid composed of
squares of 1cm for each late may be designed
around the lesion. Moreover, 0.25mL of the
cell concentrate may be injected at the center
of each square perilesionally and
intralesionally at intervals of 1 cm, using a
21-G needle. (Fig.26.4).
• Using a 21-G needle is mandatory. A diverse
caliber of the needle may inuence cell
vitality.
• Pressing the point from which the needle is
extracted may be necessary to avoid blood
leaking.
• Mononuclear cells reveal a half-life of
30days. The session should be performed at a
distance of no more than 30days from each
other session, in order to have the possibility
to exploit the effect of the therapy.
• After PBMNCs implant, the lesion should be
covered with a hyaluronic acid medication or
lipido-colloidal-based dressings. The use of
an interface dressing is essential in order to
avoid any traumatism which can occur with
the removing of the dressing at the following
medication.
• After the rst session, follow-up should be
performed at 3weeks, because the healing of
the wound can occur even before the second
session (Figs. 26.4, 26.5, 26.6, 26.7 and
26.8). Authors experience a complete healing
always after only one session of the PBMNC
injection in the treatment of non-healing
wounds.
Fig. 26.4 Cell concentrate injection
Fig. 26.5 PBMNCs injection for the treatment of a
37-year-old patient affected with pressure ulcer
Fig. 26.6 Complete Healing at 21days
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Fig. 26.7 A 75-year-old patient affected with a 5-month
non-healing diabetic ulcer
Fig. 26.8 Complete healing at 20days
26.4 Discussion
Although the various treatments for chronic
wound we have at our disposal, there is still a signicant number of patients suffering from lower
limb amputation due to the further deterioration
of the wounds. Even more unfortunately, the
occurrence of chronic wounds is rises at a higher
rate than the emergence of novel and effective
treatment strategies (Fig.26.8).
Several treatments are aimed to contrast the
major obstacles to restoration such as necrotic
tissue, biolm and infection, excess of metalloproteinases. Moreover, many strategies are available to improve and accelerate the healing
process. Acting at the basis of the problem, that
is pathophysiology cause of the non-healing
wound, it is crucial. Recently, several therapeutic approaches aimed at restoration of macro-
283
phage function have been developed. These
methods may restore the correct M1/M2 balance, always compromised in the non-healing
wounds. They include mesenchymal stem cells
(MSCs)-based therapy, autologous PBMNC
implants above described, exosomes, and
biomaterials.
26.4.1 MSCs
MSCs are multipotent cells benecial in regenerative medicine and tissue repair. Their antiinammatory and immunomodulatory effect has
been widely described. Evidence indicates that
MSCs exert powerful modulating effects on the
immune system, in particular with regard to the
immunoregulatory function on macrophages.
MSCs can be isolated from different tissues
besides bone marrow, such as adipose tissue,
umbilical cord blood, placenta, synovium, periosteum, and muscle.
Several papers showed that a main mechanism
of action of MSCs from adipose tissue, is to promote tissue regeneration through M2 polarization, but hypoxia reduces their capability to
polarize macrophages in the M2 phenotype while
for PBMNC hypoxia is a physiological trigger
for angiogenesis [78].
On the other hand, several papers highlight the
role of MSC-derived exosomes in the polarization of M2 macrophages invitro [79, 80].
26.4.2 Exosomes
A promising approach for therapeutic angiogenesis and wound healing resides in the study of
exosomes derived by MNCs. Healing capacity is
accelerated by paracrine activity of exosomes
secreted by all cellular types allowing intercellular communication. Exosomes are one group of
extracellular vesicles with 50–150nm in diameter. They consist of membrane-contained particles naturally released by cells, not containing a
nucleus. They contain peptides/proteins, microRNAs (miRNAs), and messenger RNAs (mRNAs)
that may have immunomodulatory and anti-
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inammatory effects and pro-survival effects.
They are formed after the fusion of endosomes
membrane with the plasma membrane. MSCderived extracellular vesicles (MSC-EVs) can
transfer functional proteins and nucleic acids,
including miRNA and mRNAs to other cells
without cell-to-cell contact. It was demonstrated
that exosomes played a pivotal role in enhancing
the proliferation and migration of broblasts of
both normal donors and patients with chronic
wound [81]. Moreover, these exosomes induce
angiogenesis invitro, since endothelial cells can
uptake them. Exosomes are fundamental for
angiogenic improvement and similar to miRNAs,
are useful in activating signaling pathways
involved in angiogenesis. The exosomes can also
contain active transcription factors, such as
STAT3, able to induce the transcriptional upregulation of different growth factors; i.e., SDF1,
IL-6, HGF, and nerve growth factor (NGF) that
are all compromised in chronic wounds, particularly in diabetic patients. Further, it was shown
that exosome-depleted conditioned medium had
impaired angiogenesis response [82]. MSC provokes M2 polarization and could accelerate
wound healing by releasing exosome-derived
microRNA. Li et al. [83] conrmed that
macrophage- derived exosomes exercised antiinammatory effects through the inhibition of the
secretion of inammatory enzymes and cytokines and provided the healing of diabetic wound
by signicantly quickening angiogenesis and
improving repair. A similar method can be used
with exosomes as a therapeutic approach in
chronic wounds, using autologous MNCs engineered to overexpress a specic miRNA or a
transcription factor useful for angiogenesis and
wound healing improvement. The latter could be
injected subcutaneously around wound sites to
ameliorate healing.
26.4.3 Biomaterials
Biomaterials can provide suitable environments
that enhance inherent biological activities and
functions in repairing cells through appropriate
biochemical cues (e.g., composition and surface
chemistry) and biophysical cues (e.g., stiffness
and surface topography). It is now well recognized that the ability of these materials to promote constructive remodeling is tied to their
ability to modulate the host macrophage response.
In particular, they can inuence the exible
nature of macrophages in wounds. The decellularized dermal scaffold (DDS) can regulate the
transition of macrophages from the M1 proinammatory phenotype to the M2 pro-repairing
phenotype, thus promoting macrophage
polarization.
Ideal scaffolds and tissue substitutes including
skin matrices should have a strong capacity to
promote M2 polarizations [84]. It is essential to
know the immunomodulatory effects of different
biomaterials, especially when implanted in
chronic wound. It has been observed that brous
collagen scaffolds with box-shaped pores and
precise inter-bers spacing from 100μm down to
only 40μm facilitate primary human macrophage
elongation accompanied by polarization into M2
phenotype [85]. Scaffold pore size can inuence
the macrophage: a relatively larger size (~360μm)
leads to enhanced blood vessel formation, with
higher levels of VEGF+ cells and a lower level of
M1 macrophages [85]. Also, collagenfunctionalizing additives could play a role on
macrophage activation. Chondroitin sulfate (CS)
at an increasing dose range (from 10 to 1000μg/
mL) was found to signicantly increase the
phagocytic activity and ROS production as well
as the secretion levels of nitric oxide (NO), TNFα, IL-6, and IL-10 in a monocyte/macrophage
lineage [86]. Recently, it has been demonstrated
that a dermal substitute consisting of a threedimensional porous matrix of type 1 bovineorigin collagen and a layer of reinforced silicone
may inuence the inammatory inltrate inducing M2 macrophages polarization in diabetic foot
ulcers [87]. At 6-month follow-up after the
implant, 6 patients (60%) of the dermal substitute
group completely healed, while only 1 patient
(20%) healed in the control group, suggesting
that this dermal substitute induce tissue reparative processes through macrophage activation
and M2 reparative polarization in diabetic lesions
[87]. The same positive clinical outcome of this
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dermal substitute was previously observed in 41
patients with chronic diabetic wound [88].
Take Home Message
• Resident and recruited macrophages are
key regulators to ensure proper healing.
• Macrophages play essential roles in the
persistence of the initial inammatory
process in non-healing wounds. Hence,
the treatment of chronic wounds lies in
immunomodulation.
• PBMNCs are able to stimulate the resident stem cell pools, induce angiogenesis, polarize inammatory M1 into
healing M2 macrophages.
• Autologous PBMNC injection represents a new safe and effective strategy to
transform a “non-healing wound” in
“healing wound.”
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Platelet-Rich Plasma (PRP)
https://t.me/medicina_free
ValerioCervelli andAndreaA.Pierro
27
27.1 Introduction
Chronic wounds are a common chronic medical
condition predominantly affecting the aging population, which causes a signicant reduction in
the quality of life, while creating a signicant
economic burden for healthcare systems.
The development of chronic lesions often
results from predisposing conditions, the most
common being arterial and venous diseases, diabetes, and pressure injuries. The standard of care
for chronic wounds involves correction and management of underlying predisposing conditions,
repeated debridement of the lesions, and the use
of appropriate dressings; however, there is ongoing research on alternative approaches aimed at
enhancing and accelerating the healing of such
complex lesions.
These treatments entail different innovative
strategies thought to promote the regeneration
and replacement of damaged cells and tissues,
which have already been applied to different
elds of medicine. The value of regenerative
medicine in the treatment of complex non- healing
ulcers has been recognized, and the application
of innovative approaches is under active investigation. Among these, platelet-rich preparations
have been outlined as valuable tools for the treatment of chronic non-healing wounds due to the
V. Cervelli (*) · A. A. Pierro
University of Rome “Tor Vergata”, Rome, Italy
richness of growth factors and bioactive molecules they contain. Platelet-released growth factors induce and accelerate the healing process by
promoting cell migration, proliferation, and
angiogenesis. In this chapter, the authors provide
an overview of platelet-rich plasma (PRP) biological activity and its use in chronic wound
management including the most up-to-date evidence on the use, safety, and efcacy of PRP in
the treatment of diabetic, venous, and pressure
ulcers.
27.1.1 Platelet-Rich Plasma
The platelet-rich plasma (PRP) is a preparation
of plasma obtained from the centrifugation of
peripheral venous blood and characterized by a
higher-than-baseline concentration of platelets.
Although variations in PRP preparation protocols
exist, the generic sequence of preparation
involves blood collection, centrifugation, and
separation of RBC, a second centrifugation to
obtain concentrated platelets, and the potential
addition of activating agents. There is no consensus on whether platelet activation yields better
results compared to non-activated PRP. Some
authors prefer utilizing PRP activated with
thrombin or calcium chloride, while others utilize
platelets without previous activation. By varying
the preparation technique, different platelet-rich
products may be obtained.
© 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_27
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