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Lo SCALPELLO (2021) 35:117-128
doi number: 10.36149/0390-5276-208
Arthroscopic surgery, sports medicine and biotechnology
117
Received: March 23, 2021
Accepted: July 5, 2021
Correspondence
Francesco Caravaggio
Center for Foot and Ankle Surgery, “Casa di Cura
Città di Parma”, Piazzale Athos Maestri 5, 43123
Parma, Italy. E-mail: dott.caravaggio@tin.it
How to cite this article: Caravaggio F, Antonel-
li M, Depalmi F. Regenerative medicine: poten-
tial applications for foot and ankle disorders. Lo
Scalpello Journal 2021;35:117-128. https://doi.
org/10.36149/0390-5276-208
© Ortopedici Traumatologi Ospedalieri d’Italia
(O.T.O.D.I.) 2021
OPEN ACCESS
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Regenerative medicine:
potential applications for foot
and ankle disorders
Francesco Caravaggio1,2, Michele Antonelli3, Fabio Depalmi1,2
1Center for Foot and Ankle Surgery, “Casa di Cura Città di Parma”, Parma, Italy; 2“G.
Pisani Ex-Alumni” Association, Verduno, Italy; 3Public Health Service, AUSL-IRCCS
Reggio Emilia, Italy
Summary
Introduction. Regenerative therapies have recently gained popularity in orthopedics and
we overview the available scientific evidence on the topic.
Methods. A narrative literature review with three relevant case reports.
Results. Scientific evidence on regenerative medicine is growing, but some established
findings can de underscored. First, a persistent inflammatory response plays a key role in
tissue reparation because it inhibits the activity of stem cells: therefore, regenerative therapy
is eective if it can reduce local inflammation, thus allowing stem cells to regenerate the
damage. Secondly, the regenerative potential of stem cells is regulated by local immunity
and, in particular, by macrophages, which are involved in damage response and tissue
regeneration. Among others, the concentrate of peripheral blood mononuclear cells (PB-
MNCs), rich in monocytes, lymphocytes, and CD34+ hematopoietic stem cells, appears an
interesting cell-based therapeutic strategy to promote tissue regeneration in several ortho-
pedic disorders.
Discussion. Regenerative medicine can oer new valuable therapeutic strategies. In par-
ticular, potential applications of PB-MNCs in foot and ankle disorders are discussed with
some explanatory cases from clinical practice.
Key words: regenerative medicine, mononuclear cells, limb ischemia, tendinopathy,
muscle lesion
Introduction
Recently, regenerative therapies have gained popularity as cutting-edge therapeutic
strategies in both orthopedics and other medical specialties. Some of these ther-
apies imply the use of cells derived from specic tissues (e.g. cartilage), rst ex-
panded in vitro and then implanted in specic body areas involved by the disease;
regenerative tissue scaffolds frequently associated with platelet gel; bone marrow
or fat tissue (nano fat grafting or stromal vascular fraction). Since these treatments
are usually expensive, the risk of an inappropriate extension of their clinical indica-
tions under the pressure of economic interests cannot be fully excluded. Therefore,
we overviewed the available evidence on the topic.
In the scientic literature, there is more than one denition of regenerative medi-
cine:
• regenerative medicine can encompass all interdisciplinary activities, both clin-
ical and research-related, aimed to repair and regenerate damaged cells and
tissues;
• regenerative medicine is a branch of medicine whose ultimate purpose is to
F. Caravaggio et al.
118
repair damaged cells, tissues and organs by regenerating
(rather than substituting) them.
Regenerative medicine is based on innovative therapeutic
techniques, including cellular therapy, stem cell implantation,
cellular reprogramming and tissue engineering. In particu-
lar, tissue engineering was rst invented by Eugene Bell at
the Massachusetts Institute of Technology and is dened as a
branch of regenerative medicine which studies how to regen-
erate, preserve, improve or heal a damaged tissue by properly
assembling some particular functional constructs.
Materials and methods
Since relevant scientic evidence is rapidly evolving and re-
generative medicine is not yet a well-structured discipline, we
performed a narrative literature review to provide the reader
with a streamlined, cutting-edge and practical overview, com-
bining current evidence with the authors’ experience and con-
siderations. The search was performed by two reviewers (F.C.
and F.D.) in PubMed/Medline and Google Scholar using the
following keywords: “Regenerative Medicine”, “stem cell*”,
“Peripheral Blood Mononuclear Cell*”, “macrophage polari-
zation”, “bone*”, “tendon*”, “muscle*”, “cartilage*”, “critical
limb ischemia”, and “diabetic foot”. Then, retrieved evidence
was synthetized and critically discussed. In order to offer some
useful practical examples, three relevant case reports were
described along with the qualitative synthesis of existing ev-
idence. Full informed consent was obtained from each patient
on a voluntary basis. Clinical data and images were completely
anonymized, privacy protection was guaranteed in accordance
with EU and Italian regulations, and strict condentiality was
ensured, thus avoiding any stigma or harm to involved subjects.
Tissue regeneration and its phases
Tissue regeneration
All living beings have the potential to regenerate parts of their
bodies and this ability varies from species to species, being
inversely correlated with bio-physiological complexity of each
organism: for example, mammals progressively experience a
reduction in their regenerative potential after the rst years of
life. Equally, the regenerative capacity of diverse organs and
tissues is different even within the same species. The corner-
stone of any tissue regenerative capacity is the stem cell, which
can generate other stem cells or cells with different character-
istics. After an asymmetric cell division, any stem cell can pro-
duce another stem cell and a progenitor, from which a mature
differentiated cell of a specic tissue will originate. Among
stem cells, it is possible to identify the following cell types:
• Embryonic Stem Cells (ESCs): are pluripotent and derive
from the embryo before germ layers develop;
• Tissue or Adult Stem Cells (ASCs): can be found in an
adult or fetal tissue and have the potential to only differenti-
ate into cells of that specic tissue (e.g. hematopoietic stem
cells or HSCs, which can give origin to all blood cells);
• Induced Pluripotent Stem Cells (IPSCs): originate from
ASCs and regain their pluripotency through genetic manip-
ulation.
Current scientic knowledge underscores the fundamental role
of the immune system in regulating tissue regeneration: in par-
ticular, macrophages and lymphocytes can promote healing
processes in a damaged tissue thanks to their capacity to inu-
ence the inammatory micro-environment in such a way as to
allow regeneration1,2. Monocytes/macrophages and regulatory
T (Treg) lymphocytes can modulate the activity of local stem
cells in both physiological and in pathological conditions3.
Moreover, monocytes and macrophages, found in all body tis-
sues, form a cell population with different functions and phe-
notypes, and can therefore regulate local homeostasis in vari-
ous ways. With regards to phenotypic modications associated
with specic functions, mononuclear phagocytes can be cate-
gorized into two groups4:
• M1, which are involved into initial stages of the response to
tissue damages, with a pro-inammatory and degenerative
role;
• M2 (further divided into two subgroups), which have an-
ti-inammatory properties and can regulate the repara-
tive-regenerative stage4.
Phases of tissue regeneration
If we consider wound healing, two fundamental phases can be
identied:
1. the inammatory phase, which is necessary to circumscribe
the lesion and remove damaged tissues: lesion-induced hy-
poxia can stimulate endothelial, stromal and white cells
to release granulocyte-macrophage colony-stimulating
factor (GM-CSF), with consequent attraction of circulat-
ing monocytes and activation of local macrophages. These
leucocytes can produce cytokines, chemokines, growth
factors, inammatory mediators, and can also phagocytize
damaged tissues, “non-self” substances, and microorgan-
isms5. Fundamental mediators released by these white cells
are interleukin-1 (IL-1) and tumor necrosis factor (TNF-α).
The inammatory phase tends to markedly decline 3 to 4
days after the injury.
2. the proliferative phase, which follows the inammatory
phase. M1-like macrophages are converted into M2-like
polarized macrophages (repair function), which become
prevalent at 7 to 8 days after the injury6.
M2-like macrophages can:
• release growth factors2,5;
• promote angiogenesis7.
• activate local stem cells through exosomes and nano-ves-
icles8,9.
Evidence -based regenerative medicine in orthopedics: a literature review with three case reports
119
Regardless of the specic cause, whenever inammation per-
sists in injured tissues, regenerative processes are hindered or
even inhibited.
Regenerative therapies
First of all, regenerative therapies should switch off inam-
mation to indirectly favor regenerative processes promoted by
local stem cells: in fact, it has been demonstrated that chron-
ic inammation can reduce the regenerative potential of these
cells10. Some new therapeutic strategies involve the modula-
tion of the immune system function, thus improving the local
recruitment of macrophages and their polarization from M1 to
M2, with the ultimate aim to increase the activation of local
stem cells11. Recent autologous therapeutic strategies can be
divided into two categories: non-cellular therapies (growth fac-
tors) and cell therapies.
Non-cellular therapies
Among non-cellular therapies, platelet-rich plasma (PRP), rst
proposed in the 1980’s, is probably the most popular. PRP is
obtained from venous blood, after centrifugation, and includes
platelets (namely cytoplasmic fragments of megakaryocytes)
capable of releasing growth factors (PDGF, VEGF, TGF-β,
EGF), or serum and plasma in which platelets have already
released these substances. When evaluating the therapeutic ac-
tivity of PRP, it appears that anti-inammatory effects prevail
over regenerative ones. In a recent meta-analysis by Franchini
et al., the efcacy of PRP was rated as “marginal”, and it was
concluded that, to date, PRP is not supported by strong evi-
dence in orthopedics, thus recommending further randomized
controlled trials to thoroughly assess its potential indications,
long-term benecial effects, and cost-benet ratio12.
Cell therapies
Cell therapies are various, and some are difcult to source and
apply because they require cellular expansion in authorized
cell factories. Among cell therapies, it is worthwhile to men-
tion the following:
• Bone marrow-derived mesenchymal stem cells (BM-
MSCs). In bone marrow, stem cells are mostly hematopoi-
etic (HCS), and, although only to a small extent, even mes-
enchymal (MSC). Regenerative techniques involving these
cells imply that the sample is sent to dedicated laboratories
for in vitro cellular expansion: critical issues are the ne-
cessity to maintain the cell sample sterility throughout the
entire process and to purify the nal product from all cy-
tokines used to promote in vitro cell replication. Extensive
and complicated cell manipulations in specialized labora-
tories, as well as the fact that this is a two-step procedure
(bone marrow sampling followed by implantation of stem
cells) explain its high costs.
• Adipose tissue-derived mesenchymal stem cells (AT-
MSCs). After liposuction, cells are enzymatically extracted
from the sample and then expanded in vitro13. It is inter-
esting to notice that the number of mesenchymal cells ob-
tained from a concentrate of adipose tissue is 500 times
higher than the quantity of mesenchymal cells derived
from the same amount of bone marrow14. However, in vit-
ro experiments showed that AT-MSCs may have a lower
chondro-genetic potential compared with bone marrow-de-
rived stem cells15 and the techniques involving the use of
AT-MSCs still require two steps. Other concentrates can be
directly obtained in operating/surgical theaters with point-
of-care systems.
• Bone marrow aspirate concentrate (BMAC). It is ob-
tained from the iliac crest, sampled in an operating theater
and then centrifuged: the concentrate is rich in bone mar-
row-mononuclear cells (BM-MNCs), but also includes
other cell types, such as granulocytes, lymphocytes, eryth-
rocytes, platelets, CD34+ (hematopoietic) stem cells (1.5-
1.9%), and MSCs (0.01-0.03%) with their specic con-
centration varying on the basis of the device used16. Since
the procedure required to prepare the BMAC is invasive,
its repeatability over time in the same patient is not easy
to achieve, and, moreover, recent studies have concluded
that, in order to obtain better clinical outcomes in subjects
with critical lower limb ischemia, the frequency/number of
implants is more important than the quantity of cells trans-
planted in every single procedure17.
• Adipose-derived stromal vascular fraction (AD-SVF).
All marketed systems for a direct intra-operatory use in-
volve nano-grafts of AD-SVF extracted through centrif-
ugation or ltration of a lipo-aspirate. The AD-SVF is
composed of a heterogeneous population of cell types, in-
cluding pericytes, endothelial cells, smooth muscle cells,
broblasts, macrophages, and MSCs, which markedly vary
on the basis of adipose tissue sampling site19,20.
• Peripheral blood mononuclear cells (PB-MNCs). These
are obtained by ltration of an anticoagulated sample of pe-
ripheral blood (100-120mL). This system shares a high se-
lectivity for monocytes, lymphocytes, and CD34+ cells21.
At the present state of knowledge, this autologous regener-
ative technique appears quite promising, especially if we
consider that monocytes/macrophages and Treg lympho-
cytes play a central role in tissue regeneration and in the ac-
tivation of MSCs. To date, the clinical efcacy of PB-MNC
concentrates has been studied for the treatment of critical
limb ischemia in no-option patients, for whom it has been
demonstrated to be the only autologous cell therapy capa-
ble of reducing major amputations22-24. This technique has
also been used for patients with diabetic foot25-28 or, in gen-
eral, with chronic lesions29. Considering the mechanism of
action of this therapeutic technique, its use has therefore
been extended to selected orthopedic illnesses.
F. Caravaggio et al.
120
Clinical applications
Bone regeneration
Monocytes/macrophages substantially and lengthily contribute
to healing processes of bone fractures, thus regulating the ho-
meostasis of bone tissues by releasing interleukins (IL-1 and
IL-6), oncostatin M, bone morphogenetic protein (BMP), and
angiogenetic factors like vascular endothelial growth factor
(VEGF) and nitric oxide (NO) 30-35. These osteotrophic sub-
stances can activate local MSCs, thus favoring osteogenesis
and stimulate the secretion of bone mineralization-promoting
mediators such as alkaline phosphatase and type 1 collagen.
Monocytes/macrophages, through several molecular pathways
and elaborated intercellular cross-talk, can also stimulate other
cells to produce cytokines capable of promoting bone regen-
eration 10,36,37. This complex network of cellular cross-talk is
currently studied by a specialized discipline called osteo-im-
munology38.
To further underscore the key role of the immune system in bone
regeneration, Henrich et al. have demonstrated that a bone le-
sion cannot be regenerated when injected with a monocyte-free
bone marrow concentrate, whereas regenerative processes can
be observed if stem cells are eliminated from the concentrate,
thus highlighting that monocytes (but not stem cells) have an
osteoinductive capacity39. Additionally, macrophages can reg-
ulate the recruitment and differentiation of MSCs10,43-46, and
recent evidence suggests that cell polarization from M1-like
to M2-like macrophages is essential for bone regeneration47.
Finally, it has been observed that even T and B lymphocytes
contribute to bone regeneration because they can induce re-
lease of BMP-2, modulate cell differentiation, promote bone
mineralization, and restore local homeostasis after inltrating
the callus40-42.
Tendon regeneration
Tendon diseases can be caused by acute trauma or, more often,
by overuse or chronic stress which usually result in character-
istic degenerative alterations of tendon tissues. The majority
of tendons are surrounded by a layer of epithelial cells which
can turn into a source of broblasts in case of damage. In fact,
under certain conditions, epithelial cells can trans-differentiate
into broblasts and regenerate the damaged extracellular ma-
trix (ECM). This process usually starts with the activation of
a signaling pathway called “Epithelial-to-Mesenchymal Tran-
sition” (EMT): changes in the macrophage phenotype along
with downstream pathways triggered by the EMT can, in the
rst place, contribute to the degradation of damaged tissues
and, after that, to subsequent repairing processes of a tendon48.
In particular, it has been observed that an acute tendon injury
can potentially evolve into a chronic disease and, even if to
date there is no clear explanation, it is plausible to hypothesize
a causal association with locally persistent chronic inamma-
tion49,50.
Inammation in damaged tendons is characterized by the in-
ltration of immune cells like neutrophils and macrophages.
The balance between cells with pro- and anti-inammatory
properties (M1/M2) and release of soluble factors capable of
inuencing wound healing can have a marked impact on the
outcome of the inammatory process: deregulation or poor-
ly effective modulation of such processes can lead to chronic
inammation and brosis. The polarization of M1/M2 mac-
rophages can play a role in it, since the long-lasting prevalence
of M1-like macrophages with pro-inammatory properties can
increase the chance of acute damage to evolve into a chronic
tendinopathy. Therefore, the injection of circulating autologous
monocytes, which favor M1/M2 macrophage polarization may
be a valuable cell therapy for healing promotion of damaged or
degenerated tendons51.
Case report 1: regenerative therapies for a tendon injury
This case report describes the use of autologous PB-MNCs in
the treatment of a tendon lesion (Fig.1).
Patient’s general characteristics: male, 62 years old, active life-
style (amateur sportsman – triathlon).
History: the patient, with no relevant comorbidities, com-
plained of a progressively worsening pain in his right ankle.
The pain was severe enough to force him to interrupt any phys-
ical activity. The subject did note report taking any medicinal
drug and did not recall any major trauma in his recent history.
Diagnosis, treatment and outcome: after clinical assessment
and NMR examination, the patient was diagnosed with a
traumatic-like lesion (probably due to long-lasting functional
overload) extending to up to the 50% of the right Achilles
tendon accompanied by an asymptomatic bilateral tendino-
sis of both Achilles tendons (Fig.1A). Therefore, with the
patient’s informed consent, it was decided to opt for injec-
tion of autologous PB-MNCs and to fully interrupt any sports
activity for 2weeks. Then, from the third week on, it was
recommended to do eccentric physical exercises twice a day,
and, among sports activities, only swimming and cycling. Lo-
cal pain disappeared at one month after intervention and, at
the 2-month follow-up visit, NMR showed full recovery of
the tendon lesion, even if with a persistent background ten-
dinosis (Fig.1B). The subject started again with his amateur
sports activity without any other right Achilles tendon symp-
tomatology.
Muscle regeneration
The potential regenerative role of monocytes, macrophages
and lymphocytes has been extensively described in the scien-
tic literature 52-58. Juban et al. have demonstrated that, after
muscle injury, circulating monocytes can reach the lesion and
turn into M1-like macrophages, which stimulate myogenic cell
Evidence -based regenerative medicine in orthopedics: a literature review with three case reports
121
proliferation and activate the apoptosis of bro/adipogenic
progenitors (FAPs, namely stromal cells capable of becoming
broblasts or adipocytes), thus promoting regenerative wound
healing and inhibiting brotic processes 59. Monocytes can
therefore reduce the number of local FAPs and can also inhibit
their differentiation into adipocytes, which usually occurs in
chronic muscle disorders60.
In the following stage of muscle wound healing, M1-like mac-
rophages become M2-like macrophages, the latter being able
to stimulate the development of myobrils from myogenic
cells61-64 and to stimulate the production of extracellular ma-
trix59,65,66. Macrophage polarization does not passively accom-
pany the temporal sequence of myogenic regeneration, but it
can markedly inuence and “drive” each step of the healing
process57,61,67,68.
Case report 2: regenerative therapies for a muscle injury
This case report describes the use of autologous PB-MNCs in
the treatment of a muscle lesion (Fig.2).
Patient’s general characteristics: male, 22 years old, active life-
style (competitive athlete – triple jump).
History: the patient reported the occurrence of pain in his left
femoral biceps during a training session. The symptom did not
resolve in the following days and the subject decided to seek
medical advice. The patient had no relevant comorbidities and
did not report taking any medicinal drug.
Diagnosis, treatment and outcome: after clinical assessment
and NMR examination, the patient was diagnosed with a trau-
matic muscle lesion of his left femoral biceps (Fig. 2A-C).
Therefore, after obtaining the patient’s informed consent, it
was decided to accelerate the recovery with a local injection
of autologous PB-MNCs, followed by a 2-week interruption of
sports training sessions. After 3weeks, the subject was allowed
to practice cycling and swimming, and reported a progressive
resolution of symptoms. Two months after treatment, before
starting again with triple-jump training sessions, a follow-up
NMR examination (Fig.2D-F) was performed, which showed
full healing of the traumatic lesion with no macroscopically
detectable brosis in muscle, which is very important for com-
petitive/professional athletes.
Cartilage tissue repairing
Synovial-released inammatory substances can inhibit the
chondro-genetic differentiation of MSCs in cartilages, and
this is probably related to an excessive prevalence of M1-like
macrophages in the lesion69-74. The modulation of macrophage
polarization towards an anti-inammatory M2 phenotype is of
vital importance in the development of regenerative cell thera-
pies for cartilages74-77. Recent studies have demonstrated that
PB-MNCs can promote the migration of chondrocytes into
damaged cartilage, thus potentially promoting its healing78,79.
Furthermore, both direct contact and indirect paracrine signal-
ing between isolated chondrocytes and PB-MNCs can increase
the number and speed of chondrocyte migration into the carti-
lage lesion with no modication of their chondro-genic pheno-
type78,79. In particular, it seems that indirect paracrine signaling
may be more important than cell-to-cell direct interaction in
inuencing chondrocyte migration. The stimulation promoted
by PB-MNCs can also up-regulate some chondro-genic genes
like COL2A1 and SOX977, and this is more pronounced in
cases of local hypoxia. In laboratory animal models, when con-
sidering the extension of healed areas after wounds, it has been
observed that PB-MNCs can better promote cartilage regener-
ation than MSCs80, and PB-MNCs can promote the migration
and chondro-genetic differentiation of MSCs from the adipose
tissue81 . In vitro experiments have conrmed these ndings
and have demonstrated that adipose-derived MSCs can only
marginally increase the speed of chondrocyte migration, while
the addition of PB-MNCs can increase the chondrocyte migra-
tion speed of up to 9-fold after 3 hours and improve the total
number of MSCs up to 25 times after 24 hours81.
Figure 1. A) partial lesion of the right Achilles tendon
(NMR image); B) right Achilles tendon at 2 months after
the injection of a PB-MNC concentrate (NMR image).
F. Caravaggio et al.
122
Critical limb ischemia and diabetic foot
In patients with arterial diseases, two types of compensatory
mechanisms usually occur as a physiological response to ob-
structive alterations of main arterial vessels:
• angiogenesis: the development of new capillary networks
due to the sprouting of blood vessels from pre-existing cap-
illaries in response to local hypoxia;
• arteriogenesis: when pre-existing arterioles evolve into
functional collateral arteries capable of compensating the
ow reduction thanks to an increase of their diameter (up
to 20 times).
Monocytes and macrophages can play an important role in ei-
ther the former and the latter above-mentioned processes, be-
cause they release cytokines (VEGF, bFGF)7,82,83 with pro-an-
giogenetic properties, thus favoring blood vessel sprouting and
interconnection84. Furthermore, monocytes and macrophages
can interact with endothelial cells in such a way as to promote
angiogenetic processes during wound healing, as demonstrated
in several animal models85-88. Th1 and Treg lymphocytes, along
with natural killer cells, can synergistically interact with mono-
cytes and increase their pro-angiogenetic activity89-90.
Lower extremity arterial disease can sometimes evolve into crit-
ical ischemia with foot ulcerations and necrosis. Several studies
have indicated that pro-inammatory M1 macrophages can be
found in tissues around foot ulcerative lesions of diabetic pa-
tients: the failure of macrophages to switch from the M1-like
Figure 2. A-C) traumatic muscle lesion of the left femoral biceps (NMR image); D-F) two months after local injection
of autologous PB-MNCs (NMR image).
Evidence -based regenerative medicine in orthopedics: a literature review with three case reports
123
to the M2-like phenotype, along with diabetic micro-angiopa-
thy, can be responsible for chronic inammation and, therefore,
for difculties in wound healing91,92. Additionally, it has been
shown that, in smokers with diabetes and dyslipidemia at high-
risk critical ischemia, the number of endothelial progenitor cells
(EPC), namely a sub-population of monocytes capable of releas-
ing pro-angiogenetic factors, can be signicantly impaired93-95.
Recent meta-analyses of the scientic literature suggest that,
in patients with critical ischemia who have (or even have not)
undergone surgical revascularization, regenerative cell thera-
pies based on the vascular or local injection of PB-MNCs are
associated with a reduction in the number of major amputations,
quicker healing of ulcers, and signicant improvement of trophic
lesion-related pain96-100. Usually, for safe administration of PB-
MNCs-based therapies, patients with critical ischemia are hos-
pitalized and the entire procedure is performed along with the
ulcer surgical debridement in an equipped operatory theater with
epidural or with “Bi/Tri-Block” (namely, the block of peripheral
nerves) anesthesia. PB-MNCs are injected both around the ulcer
and along the leg vascular axis, with each injection amounting
to 0.25ml of concentrate. The entire therapy usually includes 3
sessions and each session takes place every 30-40 days.
Case report 3: regenerative therapies for diabetic foot
This case report describes the use of autologous PB-MNCs in
the treatment of diabetic foot complications (Fig.3).
Patient’s general characteristics: male, 84 years old, sedentary
lifestyle.
History: the patient reported to suffer from diabetes, hyper-
tension, and dyslipidemia, and to regularly take insulin, oral
hypoglycemic agents, antihypertensives, antiplatelet and sta-
tin drugs. The subject had a history of surgically-treated lower
limb arteriopathy and developed an osteomyelitis in his fourth
and fth rays of the left foot, along with a calcaneal ulcer.
Therefore, the patient underwent a trans-metatarsal amputa-
tion followed by reconstructive surgery (Fig.3). After weeks
of antibiotic therapy and advanced wound medications, the
calcaneal ulcerative lesion persisted, leaving the bone exposed.
Despite resorting to vacuum-assisted closure (VAC) therapy,
no improvement was noticed.
Diagnosis, treatment and outcome: the patient was diagnosed
with long-term and advanced complications of diabetic foot
and, after obtaining informed consent, it was decided to opt for
a conservative approach with a regenerative cell therapy. He
was administered 4 injections of autologous PB-MNCs (once
every 40 days) coupled with regular wound cleansing and sur-
gical debridement (twice a week for 5 months) with good clin-
ical outcome.
Conclusions
Cutting-edge research in the eld of regenerative medicine
is constantly advancing, but a gap between actual potentials
of existing therapies and clamed benets of some marketed
products exists. In this regard, Professor Giulio Cossu, in his
popular book entitled The fabric of life (La trama della vita,
Marsilio Editore, 2018), stated that the present turning point
and rapid evolution of regenerative medicine is unfortunately
“tainted” by some biomedical corporations that have aban-
doned evidence-based medicine and have reverted to an old
ex-adjuvantibus approach for prot maximization. Therefore,
the risk of inappropriate overuse of available regenerative
therapies is considerable, with high costs for both patients
and healthcare systems, as well as with negligible-to-poor
clinical outcomes if evidence-based indications are not fol-
lowed. It is important to carry out further studies in order to
better investigate the actual benets and limitations of regen-
erative therapies, and to formulate a list of evidence-based
indications and contraindications to optimize their use in
clinical practice.
In the light of available scientic evidence regarding the pro-
cess of tissue regeneration, some key points should be high-
lighted:
• inammation control is fundamental in determining the ef-
cacy of regenerative therapies, because some inammato-
ry processes can inhibit local MSCs;
• although injecting stem cells into damaged tissues appears
to be a promising therapeutic approach, the regenerative
potential of stem cells is inuenced by the local immune
response and, in particular, by macrophages;
• monocytes and macrophages are “key players” of the re-
generative response, since they have not only a scavenger
function in damaged or necrotic tissues, but hey also coor-
dinate tissue repair, promote angiogenesis, stimulate local
stem cells, and produce growth factors.
• In consideration of available evidence, the use of PB-MNCs
can be promising in some ankle and foot diseases as an
adjuvant/integrative strategy to improve surgical outcomes
and to avoid surgery in selected patients, as demonstrated
in the three case reports described herein.
Ethical consideration
This study was conducted in accordance with the Declaration
of Helsinki. Full informed consent was obtained from all in-
volved patients on a voluntary basis. Data were completely an-
onymized to prevent any harm or stygma. As a short collection
of three case reports, this research was considered exempt from
Ethics Committee approval. The literature review did not re-
quire any Ethics clearance too.
Acknowledgement
We would like to acknowledge Dr. Laura Rehak for her kind
help with drafting the manuscript.
F. Caravaggio et al.
124
Figure 3. A) trans-metatarsal amputation of the fourth and fifth rays of the left foot with large loss of substance;
B) 1 month after the first injection of PB-MNCs; C) 4 months after amputation (at the time of the third PB-MNC in-
jection): note the well-represented granulation tissue; D) at the time of the fourth PB-MNC injection (40 days after
Picture 3C): progressive regeneration of the cutaneous epithelium initiated; E) 2 weeks after the fourth injection;
F) 4 weeks after the fourth injection.
Evidence -based regenerative medicine in orthopedics: a literature review with three case reports
125
Funding
This study was not funded.
Conflict of interest
No conict of interest was disclosed by the authors.
Author contributions
Conceptualization, F.C., M.A. and F.D.; Methodology, F.C.,
M.A. and F.D.; Validation, F.C., M.A. and F.D.; Investigation,
F.C., M.A. and F.D.; Resources, F.C., M.A. and F.D.; Data Cu-
ration, F.C., M.A. and F.D.; Writing – Original Draft Prepara-
tion, F.C., M.A. and F.D.; Writing – Review and Editing, F.C.,
M.A. and F.D.; Visualization, F.C., M.A. and F.D.; Supervi-
sion, F.C., M.A. and F.D.; Project Administration, M.A.
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