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6 Ecacy andSafety ofCell-Enriched Fat Grafting intheBreast
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Fig. 6.6 Clinical Case: fat grafting for correction of breast contour irregularity. One year post-op after two procedures
6.5.3 Complications
Some studies showed a higher complication incidence with
CAL compared with traditional lipotransfer (8.4% vs
1.5%), while others showed that this increased complication rate appeared to be signicant only for breast fat grafting [18, 37].
Currently, the majority of the studies comparing CAL
with traditional fat grafting did not show any signicant
difference in terms of classical complications like oil
cysts, infections, fat necrosis, calcications, nodules, and
brosis.
6.5.4 Oncological Safety
Today, it is generally accepted by the scientic community
that fat grafting is a safe technique when cautiously performed and, most importantly, when performed after complete cancer remission [39].
The subject of CAL is obviously still much debated due to
the enhancement of its regenerative effect and the greater
rate of ASCs.
Several invitro trials demonstrate that ASCs can promote
tumor growth and increase the risk of cancer recurrence
[40–42].
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V. Cervelli et al.
Fig. 6.7 Clinical Case: fat grafting for correction of congenital breast asymmetry. One year Post-op after three procedures
On the other hand, invivo trials so far have not shown that
CAL can increase the risk of breast cancer incidence or
recurrence. Only one trial demonstrated the incidence of a
single local malignancy, but further characterization showed
that to be a secondary localization of pelvic metastasis which
was independent from the treatment [43].
Since cancer occurrence is a long-term complication,
there is still a lack of evidence about the oncological potential of CAL as those studies are still ongoing.
6 Ecacy andSafety ofCell-Enriched Fat Grafting intheBreast
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6.6 Conclusion
The identication of ASCs and their clinical application are
quite recent concepts and it is very likely that in the coming
years there will be a signicant increase of clinical randomized trials on this subject that can hopefully bring a standardization of the technique. Currently, there is evidence that
CAL is a useful and promising technique that in the future
could lay the foundation for several clinical applications in
regenerative surgery and breast surgery.
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Waugh JM, Shenaq SM, Spira M.Increased free fat-graft survival
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T, Aiba-Kojima E, Iizuka F, Inoue K, Suga H, Yoshimura
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RS, Glovinski PV, etal. Enrichment of autologous fat grafts with exvivo expanded adipose tissue-derived stem cells for graft survival: a
randomised placebo-controlled trial. Lancet. 2013;382:1113.
20. Sterodimas A, de Faria J, Nicaretta B, Boriani F. Autologous fat
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22. Peltoniemi HH, Salmi A, Miettinen S, Mannerström B, Saariniemi
K, Mikkonen R, et al. Stem cell enrichment does not warrant a
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23. Lee SK, Kim D-W, Dhong E-S, Park S-H, Yoon E-S.Facial soft tissue augmentation using autologous fat mixed with stromal vascular
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24. Tiryaki T, Findikli N, Tiryaki D.Staged stem cell-enriched tissue
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25. Yoshimura K, Sato K, Aoi N, Kurita M, Inoue K, Suga H, etal.
Cell-assisted lipotransfer for facial lipoatrophy: efcacy of clinical
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26. Zuk P, etal. Multilineage cells from human adipose tissue: implications for cell-based therapies. J Tiss Eng. 2001;7:211.
27. Doi K, Tanaka S, Iida H, Eto H, Kato H, Aoi N, etal. Stromal
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29. Rohrich RJ, Sorokin ES, Brown SA. In search of improved fat
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31. Laloze J, Varin A, Bertheuil N, Grolleau JL, Vaysse C, Chaput
B. Cell-assisted lipotransfer: current concepts. Ann Chir Plast
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32. Van Dongen JA, Tuin AJ, Spiekman M, Jansma J, van der Lei B,
Harmsen MC.Comparison of intraoperative procedures for isolation of clinical grade stromal vascular fraction for regenerative purposes: a systematic review. J Tissue Eng Regen Med. 2018;12:e261.
33. Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T, Harii K.Cellassisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells. Aesthet Plast Surg.
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35. Tonnard P, Verpaele A, Peeters G, et al. Nanofat grafting:
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36. Koh KS, Oh TS, Kim H, Chung IW, Lee KW, Lee HB, et al.
Clinical application of human adipose tissue-derived mesenchymal stem cells in progressive hemifacial atrophy (parry-Romberg
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37. Laloze J, Varin A, Gilhodes J, Bertheuil N, Grolleau JL, Brie J,
Usseglio J, Sensebe L, Filleron T, Chaput B. Cell-assisted lipotransfer: friend or foe in fat grafting? Systematic review and metaanalysis. J Tissue Eng Regen Med. 2018;12:e1237–50.
38. Li M, Chen C.The efcacy of cell-assisted lipotransfer versus conventional lipotransfer in breast augmentation: a systematic review
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39. Raj S, Abu-Ghname A, Davis MJ, Izaddoost SA, Winocour
SJ. Safety and regulation of fat grafting. Semin Plast Surg.
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40. Chaput B, Foucras L, Le Guellec S, Grolleau JL, Garrido
I.Recurrence of an invasive ductal breast carcinoma 4 months after
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41. Chaput B, Grolleau JL, Bertheuil N, Eburdery H, Chavoin JP,
Garrido I. Another suspected case of breast cancer recurrence
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42. Alharbi M, Garrido I, Vaysse C, Chavoin JP, Grolleau JL, Chaput
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Enrichment oftheFat Graft
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withVascular Stem Cells
MaximGeeroms, MoustaphaHamdi, andRicaTanaka
Contents
7.1 Introduction 57
7.2 Evolution of Fat Grafting 57
7.3 The Vascular State of the Fat Graft 59
7.4 Endothelial Progenitor Cells (EPCs) 61
7.5 Fat Grafting Enriched with QQ-cultured MNCs 62
7.6 Reections on the Use of QQ-cultured MNCs in Clinical Fat Grafting 64
7.7 Comparison to Other Enrichment Techniques 66
7.8 Conclusion 67
References 67
7
7.1 Introduction
Despite its clinical application since 1893 [1], we still struggle with the fact that part of the fat graft perishes postoperatively. Ischemia is known to be the limiting factor in graft
survival. Several strategies [2] have been investigated to create an early vessel network in order to avoid this tissue loss
[3–5]. In this chapter, we analyze the vascular state of the fat
graft which has led to an experimental enrichment modality.
M. Geeroms (*)
Department of Plastic and Reconstructive Surgery, Vrije
Universiteit Brussel—Universitair Ziekenhuis Brussel,
Brussels, Belgium
Department of Plastic and Reconstructive Surgery, Juntendo
University School of Medicine, Tokyo, Japan
M. Hamdi
Department of Plastic and Reconstructive Surgery, Vrije
Universiteit Brussel—Universitair Ziekenhuis Brussel,
Brussels, Belgium
R. Tanaka
Department of Plastic and Reconstructive Surgery, Juntendo
University School of Medicine, Tokyo, Japan
Department of Regenerative Therapy, Juntendo University
Graduate School of Medicine, Tokyo, Japan
Specically, we describe the development of a highly vasculogenic cell population containing endothelial progenitor
cells (EPCs), which can be added to the fat grafts, followed
by its advantages, limitations, and future perspectives.
7.2 Evolution ofFat Grafting
7.2.1 From 1920 to2020
“Regardless of the technique used, it does not matter what ultimately happens to the transplanted fat as long as it fulls its
goal of permanently lling a space.” (Passot, 1920)
We are one century past this remarkable quote [6, 7], which
highlights two facts. On the one hand, a lot happened since the
early twentieth century. The main goal still is lling a space
with a stable product. But we have understood the composition of the adipose tissue that we harvest, we are aware of its
potential regenerative properties, we know the common principles to follow when we perform the injection, and we have
learned its limitations. This evolution is based on a better scientic understanding, and it has led to clinical progress.
Nowadays, fat grafting is part of our armamentarium and a
plastic surgery practice without fat grafting is outdated.
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
A. Di Giuseppe et al. (eds.), Fat Transfer in Plastic Surgery, https://doi.org/10.1007/978-3-031-10881-5_7
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Pubmed-listed research articles on "fat grafting"
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On the other hand, it is confronting how little we have
advanced. The fat remains in the rst place a ller. But a fat
graft which ultimately consists of a high percentage of calcications, thick brosis, or oil cysts is not a desirable outcome, even if it maintains its volume. Despite the rapidly
growing number of research experiments, inconsistencies in
clinical technique and outcome are the norm, and at conferences we often have to lter the truth out of unsubstantiated
theses and claims, which are not rarely company-inuenced.
Therefore, Aristotle’s popular quote “the more we know, the
more we realize we do not know” is applicable to fat grafting
research as well.
7.2.2 Historical Milestones
Neuber is a recurring name in manuscripts on fat grafting, as
he was the rst to transfer adipose tissue [1]. In 1893, he corrected an osteomyelitis-induced depressed scar in the face.
Czerny did the rst autologous breast reconstruction by
grafting a st-sized lipoma from the buttock en bloc to the
breast after a partial mastectomy [8]. In 1909, fat transfer by
injection was born thanks to Holländer, who injected fat to
contour deformities [9]. Halfway through the twentieth century, fat grafting became in disuse due to its tissue loss, transformation to oil cysts, and unpredictability [10]. It was
demonstrated by Peer that half of the transferred adipocytes
did not survive the process, and that a lack of vascularization
caused cyst formation [11].
Three key revolutions led to the revival of fat grafting.
First, Illouz developed the technique of liposuction in the
late seventies as a tool to reduce the subcutaneous tissue
layer, which caught the attention of American plastic surgeons, as well as the press [12, 13]. He injected this semiliquid aspirate of adipocytes again to ll dimples,
depressions, hollows, atrophied areas, the breast and wrinkles [13].
Second, in the late eighties, Chajchir pointed out the
importance of the delicate manipulation of the adipose tissue
prior to re-injection, in order to not destruct fat globules; the
relevance of the elimination of broken-down fat cells, oil and
blood, in order to obtain a pure fat graft; and the value of the
vascularity of the recipient site which correlates inversely
with the resorption rate of the grafted fat [14]. Coleman was
further responsible for making the procedure of harvesting
(gentle extraction by means of a blunt liposuction cannula),
processing (centrifugation, followed by removal of the aqueous and oily fraction), and injection (placement of the graft
in tiny aliquots) standardized for facial recontouring [15].
Nowadays, we still refer to the Coleman method, even
though numerous modications of his technique are being
applied. The combined work of Chajchir and Coleman lowered the complication rate and renewed interest in fat grafting, allowing the procedure to regain popularity.
Third, at the beginning of the twenty-rst century, and in
an effort to better understand the cellular basis of adipose
tissue, Zuk [16, 17] and Mizuno [18] described the abundant
presence of adult mesenchymal stem cells in the adipose tissue. These adipose-derived stem cells (ASCs) are an undifferentiated self-renewing cell population, capable of
multilineage differentiation. Since the discovery of ASCs,
the regenerative potential of adipose tissue became a hot
topic in our eld, opening doors for new research projects
(Fig.7.1).
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0
0
0
0
0
0
0
0
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Fig. 7.1 PubMed-listed research articles on “fat grafting” from 1928 to 2019
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To date, fat grafting has permeated many aspects of plastic and reconstructive surgery, as well as other medical specialties. Due to its rich composition, it has regenerative
potential beyond simple volume augmentation [19–28].
Benecial effects of fat grafting have been reported in wound
healing [29, 30], peripheral nerve regeneration [31], scarring
[32, 33], scleroderma [34, 35], radiotherapy damage [36],
ulcerations [37], rheumatoid arthritis [38], osteoarthritis
[39], dermal atrophy [40], and brosis [41].
7.2.3 The Limitations ofFat Grafting
Independent from the technique, and regardless of the indication, the major drawback of fat grafting remains its volume
loss due to fat necrosis [42]. Small areas of non-viable adipocytes are reabsorbed or can lead to calcication, and larger
zones of fat necrosis can turn into oil cysts or hard brosis,
which can cause confusion in breast imaging for inexperienced radiologists [43–47]. Patients are informed that fat
graft success is unpredictable and never equal to 100%, and
that subsequent procedures are often required, but these
operations lead to additional costs, donor site morbidity, hospital admissions, anesthesia, patient discomfort, and psychological burden.
One of the main indications for fat grafting is breast
reconstruction. Approximately 13% of women will be diagnosed with invasive breast cancer in their lifetime according
to the currently available data. The average 5-year and
10-year survival rates for female patients with invasive breast
cancer have increased to 90% and 83%, respectively [48].
Thus, patients live a longer time with the consequences after
the breast cancer treatment and, therefore, the quality of life
of these women should not be neglected. Whether the patient
requires a partial or complete reconstruction, with an implant
or autologous tissue, reconstructive surgeons can choose a
breast reconstruction modality which is tailored to each
patient, and fat grafting often plays a role, as it offers several
advantages to the outcome and patient satisfaction. By creating a stable fat graft, we can eventually nd a solution for its
shortcomings, i.e., the unpredictable graft resorption and
multiple procedures.
The engraftment of the fat graft depends on various
parameters which can all be studied separately and be clinically inuenced. Technical variables consist of the tumescent solution being inltrated prior to liposuction, the
harvesting method, the manipulation of the fat, and the
injection technique. Different approaches lead to mainly
non-signicant differences in outcomes, and there is no consensus for a universal clinical protocol [49–59]. No high
level of evidence exists that support a preferred technique of
inltration, harvesting, or processing [60]. Patient-related
variables that may inuence graft yield and viability are
recipient site properties [61], age [62, 63], smoking [64],
and comorbidities.
To overcome the tissue loss and volume reduction due to
fat graft resorption, overcorrection has been applied and
advocated by clinicians. Overcorrection means the injection
of a volume of fat that exceeds the requirement for correcting
a volume depletion or contour defect. However, based on scientic evidence [65] and clinical experience [66], we know
now that overgrafting does not lead to an improved survival
of the grafted tissue. On the contrary, because of increased
interstitial pressure, capillary perfusion can be restricted to
the adipocytes, and the individual cells will be more prone to
apoptosis. By consequence, overcorrecting causes complications such as necrosis and oil cysts. We cannot expect more
adipose tissue to survive than what the recipient site can
accommodate, and, therefore, grafting beyond the capacity
of the recipient site is avoided.
7.3 The Vascular State oftheFat Graft
7.3.1 Grafted Tissues Depend
onRevascularization
The most essential requirement of fat graft survival is blood
ow, because we need circulation for all tissues to live.
Adipose tissue has an inherent rich vascular network in
which adipocytes are connected to capillary vessels. By consequence, the tissue oxygen tension is very high in the subcutaneous adipose tissue [67]. When liposuction is
performed, the fragile parts of adipose tissue are aspirated,
but the brous honeycomb-like network remains intact in the
donor area. This network consists of connective tissue as
well as the relatively large vessels. Thus, individual adipocytes are disrupted from their original blood supply once
they enter the suction cannula. Upon injection, the adipose
tissue will lack a vascular network.
Harold Gillies already mentioned in 1957 “a (skin) graft is
a piece of detached skin which is dead when you put it on and
comes to life later” [68]. By “life” and “death,” Gillies meant
the presence or absence of a blood vessel network. Similarly,
because a fat graft lacks a blood supply at the moment of
injection, it goes through ischemia. The fat graft depends initially on plasmatic imbibition from the surrounding recipient
tissue to obtain its nutrients and oxygen, until adequate revascularization is established to supply metabolically active
cells. Adipocytes in the graft are prone to cell death within 24
hours in this hypoxic situation, and, inevitably, a percentage
of the adipocytes will die [65]. Other cells in the adipose tissue, such as bone marrow-derived mesenchymal stem cells,
are less susceptible to cell death under hypoxic circumstances,
which makes them a preferable cellular source for cell-based
therapy in ischemic tissues [69].
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7.3.2 Vascularization Determines Fat Graft
Survival
The adequacy of blood perfusion early after the grafting
appears to be key to the fate of the grafted fat. Yamaguchi
proved in a murine experiment that the administration of an
angiogenesis inhibitor led to less blood ow from the recipient tissue toward the graft, resulting in a lower graft weight
and impaired adipocyte function [4]. Lu demonstrated a link
between vessel density and graft retention in a murine experiment with VEGF-transduced ASCs [70, 71]. In rabbits,
Karacaoglu had the best outcome when the graft is placed in
the supramuscular plane, presuming this is a consequence of
being a more appropriate and highly vascular recipient site,
due to the musculocutaneous perforators emerging from the
outer muscular surface [3]. Also Lee has shown that by preconditioning and increasing the recipient site’s vascularity,
an increased graft weight can be obtained [5]. Taking into
account these ndings, the establishment of an early blood
supply is of great importance for the fat graft.
7.3.3 Vascularization Determines
Adipogenesis
The vascular supply of oxygen and nutrients is essential to
adipose function, including their contribution to metabolism,
and their secretion of soluble factors for adipocyte survival
and adipocyte regulation [72]. Not only the survival of the
grafted fat but also the de novo adipogenesis is regulated by
factors that drive angiogenesis. Evidence indicates that blood
vessel development inuences adipocytes and adipogenesis
[73]. During fetal development, arteriolar differentiation precedes adipocyte development, and blood vessel extracellular
matrix (ECM) differentiation precedes differentiation of adipocyte ECM [74, 75]. Administration of anti-angiogenic factors to mice decreased fat pad weights by 12–22% and
decreased body weights in a dose-dependent manner [76].
Kawaguchi injected bFGF and Matrigel subcutaneously in
mice for neovascularization which resulted in migration of
adipose precursor cells and their differentiation into adipocytes. Their results demonstrated that enhancement of de
novo adipogenesis was due to increased neovascularization
[77]. Rophael showed how combinations of angiogenic cytokines enhanced early angiogenesis and cell inltration which
led to synergistically increased adipose tissue growth [78].
Fukumura described a reciprocal regulation of adipogenesis
and angiogenesis in which inhibition of angiogenesis by
blocking VEGFR-2 reduced angiogenesis but also blocked
pre-adipocyte differentiation [79]. Based on these reports, an
adequate blood vessel network in the fat graft can drive adipogenesis after adipocyte apoptosis.
7.3.4 The Graft’s Three Layers
andtheFarmer’s Four “S”
The importance of the vascularity has been reected in publications by Yoshimura, who provided us with great insight
on how the fat graft can be subdivided in three areas from the
periphery toward the graft center: surviving zone, regenerative layer, necrotic core [80]. The fate of the individual adipocytes depends on their location in the graft:
1. Outer (surviving) layer: Cells survive the grafting process
because of the proximity of the surrounding ingrowing
capillaries.
2. Intermediate (regenerative) layer: Adipocytes suffer from
ischemia and will be phagocytized. ASCs repair the ischemic and injured tissue, together with stem and progenitor cells, that originate from the bone marrow and migrate
in response to ischemia-related paracrine signaling. ASCs
are activated by adjacent adipocytes undergoing apoptosis to differentiate to adipocytes, and contribute to the cell
replacement, if the microenvironment, including the capillary network and oxygen tension, is improved within 3
days after the grafting. If vessel ingrowth from surrounding host tissue does not reach this area within 3 days, the
ASCs will also degenerate, thereby aborting the regenerative process. In that case, this regenerative layer becomes
part of the necrotic area [80].
3. Innermost (necrotic) layer: The vasculogenic delay
impairs the survival of all cell types which undergo
destruction. No adipogenesis or regeneration occurs in
this necrotic core. The inner layer will undergo resorption, brogenesis, and cicatrization, or can develop into
sand-like calcications or oil cysts, which can lead to
lumps with never-ending inammation and tenderness
[66, 67, 81]. Thus, this model emphasizes the early and
abundant vascularization for successful retention of fat
grafts.
A demarcation in the fat graft, between the surviving
outer layer and the regenerating middle layer, becomes rapidly apparent. Based on immunohistochemistry experiments,
viable adipocytes can be distinguished from the intermediate
regenerative zone at 1 week [67, 80]. Two to four weeks after
the grafting process, the demarcation between the regenerative zone and the necrotic center becomes clear [67, 80]. At
2 weeks post-grafting, immature adipocytes appear between
moribund adipocytes [67, 80]. After the inammatory and
regenerative phase, a stable graft results, with capillaries
consisting of endothelial cells, which are derived equally
from the graft and the host bone marrow [82].
Based on experimental work, the regenerative zone has a
600- to 1 200-μm thickness, whereas the radius of the super-
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cial surviving zone measures maximum 300 μm, which
became approximate numbers to remember for clinicians
[67, 80]. This indicates that a close relationship should be
respected between adipocytes and surrounding host tissue in
order to avoid necrosis, and stresses the biological violation
of injecting fat particles larger than 2 mm. Khouri had
launched the analogy with how a farmer plants seeds in a
eld and has to respect the four “S”: soil, sowing technique,
seeds, and support [83, 84]. Surgical craftsmanship aims for
a close contact between fat particles and the recipient tissue.
Through the diffuse fan-like pattern injection of small noodles of adipose tissue without overgrafting, we optimize the
contact and potential vascular ingrowth in the graft. However,
even when the fat grafting is properly executed (sowing tech-
nique), when the recipient site is well-vascularized (soil:
absence of radiotherapy sequelae, peripheral vascular disease, diabetes mellitus-induced micro-angiopathy, tobacco
use, etc.), and when the postoperative care is optimal (sup-
port: avoidance of trauma, shear force, high pressure, or tight
compression), there remains room for technical improvement. Maximizing the potential of the fat graft can be done
through cell enrichment (seeds). On the other hand, overloading a fat graft with stem cells while neglecting the other
three “S” would add little benet to the outcome, similar to
adding horsepower to a car stuck in trafc.
7.4 Endothelial Progenitor Cells (EPCs)
and ow cytometric analyses, nearly half of vascular endothelial cells in the grafts originated from host bone marrow,
and a chimeric capillary network was seen constructed with
graft-derived and host-derived cells [82]. This has two interesting consequences:
• Graft-derived vascular structures are involved in revascu-
larization, but EPCs mobilized from bone marrow make
an equal contribution. As in situations of ischemia, EPCs
will be released from the bone marrow and migrate toward
ischemic sites where it can differentiate into endothelial
cells and incorporate in newly formed vessels [87].
• A rapidly established vascular network toward and in the
fat graft is necessary to deliver these EPCs as well as
ASCs.
The creation of a vascular network takes 3–7days [88–
92], and even longer to reach the innermost core of the graft,
where they might not even reach. The addition of EPCs to
the fat graft is a legitimate manipulation because we skip the
transportation step, and we deliver building blocks for vessels. This is especially useful since conventional fat grafts
have relatively low stem cell numbers compared to excised
fat [93, 94], because the viable endothelial cell component
in lipoaspirates decreases quickly as opposed to ASCs [65],
and because a positive signicant correlation exists between
the innate stem cell percentage in adipose tissue and graft
retention [95].
7.4.1 EPCs Target Ischemic Repair
Endothelial progenitor cells (EPCs) are precursors of endothelial cells, the squamous cells which line our circulatory
system, from heart and aorta to peripheral capillaries. EPCs
were rst discovered by Asahara [85]. After isolating CD34+
mononuclear cells (MNCs) from peripheral blood, he noticed
their fast proliferation, ability to form tubular structures and
CD31 expression. He demonstrated how these cells homed
to ischemic limbs after distant injection in the murine tail
vein, as they “sensed” the requirement of the ischemic tissues. The integration of the labeled CD34+ MNCs in vascular
structures was conrmed histologically. EPCs were shown to
contribute to postnatal physiological and pathological endothelial repair and neovascularization. These ndings opened
the door to the concept of therapeutic vasculogenesis.
7.4.2 Role ofEPCs intheFat Graft
In conventional fat grafting, EPCs can originate from the
graft itself, from the surrounding tissues, or from the bone
marrow [86]. Doi described the cellular origin of regenerated
adipose tissue and vessels in the graft. Based on histologic
7.4.3 Previous Experiments onEPC-Enriched
Fat Grafting
In 2006, Yi conducted an animal experiment, in which human
EPCs were mixed with murine adipose tissue and grafted in
mice. A dual pro-vasculogenic and anti-brotic effect was
attributed to the EPCs. The EPCs originated from umbilical
cord blood CD133+ cells from a different human donor than
the adipose tissue donor, and there was no proof of their integration into vascular structures. A clinical application based
on this study would be impractical because human umbilical
cord blood is not readily available, and their application
would not be autologous [89].
Hamed also added EPCs to fat grafts and showed how
EPCs are required for fat graft vascularization, graft retention, and reduced adipocyte apoptosis [86]. In another experiment, Hamed has demonstrated how the induction of EPCs
in the bone marrow and their migration toward the graft contribute to graft retention [96].
Xu indirectly used EPCs as an enrichment modality in fat
grafting by adding CXCR4-transfected ASCs to the fat graft.
CXCR4 is a membrane receptor involved in the recruitment
of stem cells such as EPCs, neovascularization, and cell pro-
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liferation. In his animal experiment, Xu demonstrated a
higher capillary density in nude mice that received the grafts
mixed with CXCR4-transfected ASCs [97].
More recently, Pallua has described lipoconcentrate,
which is a processed lipoaspirate and obtained through a
modication of Tonnard and Verpaele’s nanofat protocol
[98, 99]. By including an extra centrifugation step, the lipo-
concentrate contains a higher amount of EPCs and it is currently being used clinically [33].
7.5 Fat Grafting Enriched
withQQ-cultured MNCs
7.5.1 Development oftheQQ Culture
The limitation of using EPCs hinges on its sparse presence in
peripheral blood [100]. With 70–210 EPCs per mL, EPCs
account for only 0.002% of the peripheral blood mononuclear cells (PBMNC), or for 0.4% of the total peripheral
blood CD34+ cell population [101]. EPCs are characterized
by even lower numbers, and impaired proliferation, adhesion, and vascular incorporation, in elderly patients, patients
with diabetes, hyperlipidemia, peripheral vascular disease
and ischemia, cardiac and lung disease, endocrine, gastrointestinal, and hematologic disorders, musculoskeletal problems, alcohol or drug abuse, tobacco use, the use of
immunosuppressive drugs and chemotherapy [102–110].
This quantitative and qualitative impairment of EPCs results
in a lack of clinical value for autologous EPC therapy, and
indicates the need to pre-treat these cells.
The Quality and Quantity culture (QQ) overcomes this
problem, by increasing cell counts and ameliorating the
functional decits of EPCs to a highly vasculogenic and
regenerative level. The QQ culture is a 1-week culture which
is based on an optimal cytokine and growth factor mix consisting of stem cell factor (SCF), thrombopoietin, vascular
endothelial growth factor (VEGF), interleukin-6 (IL-6), and
Flt-3 ligand.
In in vitro experiments in 2012, Masuda discovered both
a quantitative increase as well as a qualitative enhancement
(differentiation of EPCs into endothelial cell lineage as
shown by an increase in denitive EPC colony-forming
units; endothelial cell surface marker expression of
VEGFR- 2, CD146, and vWF; and VEGF and HGF secretion) of CD133+ EPCs that underwent QQ culture. In vivo
was demonstrated how the intramyocardially injected
QQ-cultured cells were able to preserve more left ventricle
function and contractility in a myocardial infarction model,
and more endothelial cells and cardiomyocytes were discovered on immunohistochemistry [106, 111].
In 2014, Masuda obtained QQ-cultured mononuclear
cells (MNC-QQ) enriched in EPCs from unfractionated
mononuclear cells (MNC) [112]. The MNC-QQ formed
blood vessels in an ischemic hind limb mouse model. Antiinammatory cytokines (IL-10) and genes related to angiogenesis and tissue regeneration (VEGF, IGF-1, MMP-2,
MMP-9) were up-regulated in MNC-QQ.
To date, the QQ culture is the only ex vivo culture system
with the ability to expand and improve the vasculogenic
potential of EPCs for clinical usage [106]. The invitro tendency of MNC-QQ to develop into an EPC population with
pro-vasculogenic characteristics as cell adhesion, tube-like
structure formation, and endothelial lineage differentiation
was demonstrated through EPC colony-forming assay [113–
118]. In EPC culture assays, MNC-QQ were shown to be
adherent cells that fulll the three requirements of being
viable early EPCs in a more differentiated phase compared
to the colony-forming EPCs (DiI-acLDL uptake, UEA-1
binding, and DAPI staining) [116, 119, 120]. However,
describing the QQ-cultured MNCs as a pure EPC population
would be incomplete: the QQ culture also induces a phenotypical switch from classically activated pro-inammatory
M1 macrophages (CCR2+) to the alternatively activated antiinammatory M2 macrophages (CD206+), and activates
helper T-lymphocytes [107, 113, 120–122]. The higher
angiogenic potential of M2 macrophages can be explained
by their FGF and PlGF signaling [123], because of their
PDGF-BB and MMP9 signaling and their effect on anastomosing sprouting endothelial cells [124]. Phipps grafted adipose tissue with M2 macrophages and noticed an improved
graft retention which was attributable to vascular development [125]. Early depletion of macrophages resulted in
incompetent angiogenesis and eventually a poor retention
rate, whereas up-regulated macrophages allowed better
angiogenesis and survival [126].
Therapeutic vasculogenesis with QQ-cultured cells has
been shown more efcient than conventional modalities for
wound healing [107, 114], ischemic stroke [127], myocardial infarction [122], and acute kidney injury [121].
7.5.2 Current Scientic Findings onFat
Grafting Enriched
withQQ-culturedMNCs
In 2019, the addition of a homogenous QQ-cultured EPCpopulation to fat grafts was studied for the rst time in a
murine recipient model [128]. Our main nding was the signicantly increased vessel density in these fat grafts compared to conventional (non-enriched) fat grafts, fat grafts
enriched with non-cultured EPC, and ASC-enriched fat
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