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Fig. 10.1 Schematic representation of the three identiable zones of the fat graft. The outer zone
(surviving zone) is the zone adjacent to the vascularized recipient tissue. The intermediate zone is
the regenerating zone where cell remodeling is expected. The inner zone is the necrosis zone,
where all the grafted cells will die. The diameter of this zone is related to the amount of fat grafted.
(See text for details)
G. A. Mecott and S. G. Hernández-Soto
Depending on several variables, the size of each zone might vary, and nonregenerated cells will end as brosis or oil cyst formation. After the third month, no
cell regeneration is observed, but the remodeling could last several more months.
Oil drops larger than 8mm form oil cysts and become perpetual, whereas smaller
drops will be absorbed or replaced with brotic tissue [35]. This latter process might
be the responsible for the loss of volume seen during the rst year after grafting.
The adipose tissue is the organ with highest tissue partial oxygen tension among
all the organs [35], and perhaps, one of the main reasons for the poor resistance of
the adipocytes to ischemia. It seems that the threshold of partial pressure oxygen for
the fatty tissue would be between 30 and 35mmHg. However, other authors have
proposed that most adipocytes will die with a partial pressure oxygen of 15mmHg
[16]. In animal models of fat ischemia, it has been shown that apoptosis increases in
the rst day after the insult, whereas necrosis is seen predominantly on days 3–7. By
the day 28, little apoptosis or necrosis was observed [36]. Adipocytes are likely to
die within the rst 24h if the oxygen pressure is lower than the mentioned thresholds [16, 32].
Ischemia produces not only apoptosis and necrosis of the cells, but also angiogenesis, adipogenesis, and cell proliferation [36]. These processes appear to be
mediated, at least partially, by ASCs. These cells can remain alive for up to 3days,
even under severe ischemic conditions [16]. As we mentioned previously, if the
ASCs survive, they can proliferate and regenerate the lost cells.
In conclusion, based on these studies, we can assume that the process of necrosis
of the grafted cells occurs during the rst week, cell regeneration is completed after
3months, and the nal remodeling process might last for the rest of the rst year
after the graft. Also, we should agree that the fatty graft should be small enough in
order to avoid brosis and oil cysts. Specically, less than 3mm (1400–3000μm)
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of diameter, assuming that it is surrounded by viable and well vascularized tissue.
Grafts larger than 8mm will have a large necrosis zone and will have fat necrosis
that might cause oil cysts.
10.3 Donor Site
Patients usually seek liposuction from different areas of the body, and then, they are
amenable for being used as graft. However, at least theoretically, the fat from different areas of the body could have different characteristics that might result in different viability when used for grafting purposes.
In the late 1990s, it was accepted that the fat obtained from well-vascularized
areas, such as the periumbilical region, would be contaminated with blood, which
would lead to increased risk of infection. Thus, less vascularized donor sites, such
as the trochanteric area or the upper part of the inner thigh were preferred [12].
Rohrich et al. compared the fat removed from different body locations (i.e.,
thigh, knee, ank, and abdomen) and reported similar viability between the samples
[37]. In this article, the authors assessed the viability of the cells invitro, but emphasized that clinical studies were needed in order to determine the long-term viability.
These studies were performed later by other authors, either in animal models (i.e.,
human fat grafted into nude mice) [38, 39] or in clinical studies [40]. These studies
also concluded that the choice of the donor site is not relevant in terms of viability
of the adipocytes.
Padion etal. found that the fat collected from the medial thigh and lower abdomen had a higher concentration of stem cells compared with fat obtained from other
parts of the body [41]. However, no analysis was made about the inuence of the
stem cells in the viability of the adipocytes.
Age of the patient has been studied as another factor that might affect adipocyte’s
viability. Geissler etal. found that patients under 45years old had greater viability
in fat from the lower abdomen, whereas fat from the ank resulted with better viability in patients over 45years old [42]. Due to the scarcity of the available data
about this subject, it is unclear if the differences observed in age or concentration of
stem cells would have a relevant impact in clinical practice.
Then, as of today there is no denite evidence about whether the fat from one
donor site would be better, in terms of viability, than that obtained from other site.
Lidocaine is often used as part of the inltrating solution during the liposuction
and was often neglected as an important variable that could affect fat viability.
However, some concern arose after the report that lidocaine inhibited the growth of
adipocytes in culture [43]. However, this effect was transient and reversible once
lidocaine was removed from the culture media. More recently, Cucchiani and
Corrales found decreased viability in samples treated with lidocaine (1mL of lidocaine added to 9mL of fat) [44]. However, in both studies the addition of the lidocaine was different to its clinical use in liposuction. Thus, these ndings don’t
appear to have clinical relevance.
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G. A. Mecott and S. G. Hernández-Soto
10.4 Harvesting
As mentioned previously, the initial attempts of fat grafting consisted in removing
small fragments of fatty tissue and placing them in the desired recipient sites [17–
19]. It was only after 1980’s that liposuction became the preferred method of fat
harvesting [45].
Kononas etal. compared the changes in volume of fat, either obtained by liposuction or by surgical excision in an animal model (i.e., ear of rabbits). After
9 months, they measured size and weight on both groups and found that, even
though both showed signicant reduction in volume, surgically excised samples
retained more volume that the lipoaspirated ones [46]. No data about the intensity
of the negative pressure or the management of the fat was provided.
Smith etal. compared viability and histology of adipocytes obtained by syringe
or liposuction and didn’t nd any difference between both methods [47]. However,
they did not mention the intensity of the negative pressure in any of the studied
techniques. This is important because, as we will discuss later in the following section, the intensity of the suction could be the main variable and not the device used
for extracting the fat.
10.5 Negative Pressure
Nowadays, the fat is obtained through negative pressure, either with syringe or with
liposuction devices. However, that doesn’t mean that these are the only two variables involved. The negative pressure exerted with the syringe depends on the
amount of negative volume produced when pulling the plunge, and the negative
pressure in a liposuction device could be adjusted as well.
The negative pressure exerted by pulling a syringe plunger (at −1 cm increments) increases linearly. Furthermore, the intensity of the suction with a syringe
depends on the volume (in milliliters) that the plunger is pulled, independently of
the volume and diameter of the syringe [48]. In other words, the negative pressure
exerted by pulling the plunger up to the mark of 1cc, is exactly the same if we use
a 1cc syringe or a 3cc, 5cc, 10cc, or 60cc syringes. Smith etal. found that pulling
the plunger up to the maximum capacity of the syringe (−10cc), exerted the same
negative pressure than their liposuction device [47].
In 1988, Coleman described his technique of harvesting with a needle. He preferred to pull the plunger up to the mark of 1mL (1cc of negative pressure) on a
10mL syringe and to keep a low pressure all the time. He believed that high negative pressure would destroy the adipocytes [49]. That was demonstrated by
Tambasco, when he described that the number of disrupted adipocytes increased
linearly (from 0 to >75%) with increments of the negative pressure by pulling the
plunger at 5, 10, 15, and 30cc [50].
Several other authors have demonstrated that the intensity of the negative pressure has a deleterious effect on the viability and architecture of the adipocytes.
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Shiffman and Mirrafati found that harvesting with −700mmHg of negative pressure
damaged 10–15% of the cells, whereas lesser vacuum (from −250 to −500mmHg)
resulted in 98–100% intact cells [51]. Adanali et al. described that samples at
−700mmHg showed trauma and membrane rupture, whereas pressures of −250 to
500mmHg were not disrupted [52]. Nguyen etal. found 90% of adipocytes injured
at −760mmHg whereas only 5% of the adipocytes were injured after gentle syringe
aspiration [10] On the other hand, Lee etal. did not nd any signicant difference
in the architecture or the weight of fat the lobules obtained at −0.5atm. versus
−0.83atm. [53], although no actual viability methods where performed.
Based on the reviewed evidence, we can conclude that most of the literature
favors low negative pressure, below −500 mmHg, in order to better preserve the
adipocytes.
10.6 Processing
After harvesting, the fat is usually processed before it is used as a graft. The main
purpose of processing the fat after liposuction is to remove blood, oil, and inltrated
solution. Evidently, the lipoaspirate could be injected without processing, and this
approach would have the minimum aggression to the adipocytes [54]. However, the
actual volume of fat would be only a fraction of the volume that is injected, since all
the blood, oil, and inltrated solution would be injected as well.
Other reason for removing blood and the tumescent solution is that these portions are thought to be deleterious for the fat [13, 55]. However, well-designed
studies have proved that there is little benet in removing blood or oil in terms of
graft take [56].
The most commonly processing methods include decantation, concentration,
centrifugation, and washing with different solutions [57]. The wide variation in fat
grafting outcomes is accompanied by numerous descriptions of different processing
methods [58]. According to a national consensus survey of plastic surgeons who
perform fat grafting in the USA, 45% use gravity separation (decantation), 34% use
centrifugation and ltering, 11% use gauze rolling, and 3% do not process fat before
injection [59].
The literature contains contradictory results regarding the superiority of each of
these techniques. In the following paragraphs, we will discuss most of the available
literature about the most commonly methods of processing.
10.7 Decantation
Decantation is one of the simplest methods to prepare the fat for grafting, and one
of the most used in clinical settings. This is an ancient and widely known process,
based on gravity, aimed to separate the different components in mixtures or
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G. A. Mecott and S. G. Hernández-Soto
suspensions. However, it requires some time, depending on the nature of the elements of the suspension, to achieve a good separation of all the components. With
regard to fat grafting, it does not require sophisticated equipment and can be easily
replicated in most operating rooms with predictable results.
The main advantage about decantation is that the processing of the cells is minimal, and then, we could expect no physical damage to the cells. Conde-Green etal.
assessed the effect of decantation, centrifugation, and washing in the adipocytes.
They noted that the decanted samples had better preservation of their architecture
compared with the other two methods [31, 33]. Ferraro et al. demonstrated that
decanted cells were not damaged, as they observed an intact cell architecture in
decanted samples, compared to centrifuged ones [60]. On the other hand, once
removed from their vascular bed, adipocytes are deprived of blood and oxygen. As
we mentioned previously, adipocytes are very fragile cells with low resistance to
hypoxia. Thus, one of the main drawbacks with decantation is that adipocytes
decrease their viability over time while they are left in decantation.
We have demonstrated that decanted adipocytes decrease their viability and
increase their apoptosis rate during the rst 2h after harvesting. In this study, we
decanted adipocytes and measured viability at 0, 60, and 120min after recollection.
Viability decreased to 51% at 60min after liposuction and further decreased to 46%
at 120min after liposuction. Apoptosis increased up to 62% at 120min after decantation [57]. Based on those results, it was evident that the lipoaspirate shouldn’t be
left in decantation for long periods of time, and we recommended injecting the fat
within the rst hour after aspiration.
However, it became obvious that shorter decantation times improve the viability
of the adipocytes, but also might impair the separation of the elements of the
lipoaspirate. So, based on that notion, we performed a newer study to determine the
ideal time of decantation that provides the best separation of the components without compromising the viability of the adipocytes. We measured the volume of puried fat (i.e., volume of fat after removing oil and remnant solution) and its viability
at 0, 30, and 60min. It was observed that, indeed, the separation of the components
was better with longer decantation times, but viability decreased accordingly. We
found that 30min of decantation is the timepoint at which we found better relationship between viability and separation of the components [61].
In the same study, we observed that the actual volume of fat decreased over time
(6.6mL at baseline, 5.5 at 30 min, and 5.26 at 60min after decantation) from an
initial sample of 10mL, which seemed to make no sense at all. We then realized that
the reason for this was because we removed about 0.6mL of liquid and oil at baseline, 2.3mL at 30min and 3.1mL 60min after harvesting. In other words, decantation allowed to separate that amount of liquid. Hence, less “fat” was obtained over
time. Then, from the remaining 9.4mL of “fat” from the baseline fat, we further
removed 2.8mL of solution after centrifuging the sample that was not possible to
remove by decantation. That resulted in the 6.6mL of actual fat from the initial
10mL of aspirate. That accounted for 70.2% of actual fat and 29.8% of remnant
liquid. At 30min, we got 7.7mL of decanted fat, and after centrifuge, 5.5mL of
actual fat were obtained (71.5% of actual fat and 28.5% of remnant liquid). At
60min, we obtained 6.9mL of decanted fat, which turned into 5.2mL of actual fat
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after centrifugation (75.3% of fat and 27.7% of liquid). However, the viability of the
adipocytes was not signicantly different at baseline and 30min, but it was signicantly decreased at 60min, which allow us to recommend 30min as the best time
of decantation (Fig.10.2).
Based on these results, and making the pertinent post-hoc calculations, we can
calculate the amount of lipoaspirate needed to obtain 100mL of fat after decantation and the expected viability of it. That is, how many milliliters of viable fat we
are injecting for each 100mL of decanted fat (Tables 10.1 and 10.2). However, it is
also clear that when we inject decanted fat, we would be injecting a signicant
amount of liquid (about 30%) that might be absorbed and clinically accounted as
loss of the fat grafting.
Fig. 10.2 The upper row
represents the amount of
fat obtained after
decantation of 10mL of
lipoaspirate at 0, 30, and
60min. That fat was then
centrifuged and resulted in
pure fat (the amount
showed within the tubes)
for each 10mL of the
original lipoaspirate. In the
box is shown the viability
of that fat (see text for
details)
Table 10.1 Viable fat for each 100 of lipoaspirate in milliliters
Total aspirate: 100mL
Decantation time (min) Fat obtained (mL) Viability (%) Actual viable fat (mL)
0 94 73 68.62
30 77 72.5 55.825
60 69 59.3 40.917
Average values were set to 0 decimals. Based on the results of [61]
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Table 10.2 Viable fat for each 100 of decanted fat in milliliters
100mL of decanted fat
Decantation time (min) Required aspirate (mL) Viability (%) Actual viable fat (mL)
0 106 73 73
30 130 72.5 72.5
60 145 59.3 59.3
The second column is the theoretical amount of lipoaspirate that would be needed to obtain 100mL
of decanted fat at each timepoint. Average values were set to 0 decimals. Based on the results of [61]
G. A. Mecott and S. G. Hernández-Soto
In conclusion, if decantation is chosen as the preferred method of processing, our
recommendation is to decant for not more than 30min after harvesting, in order to
decrease mortality of the adipocytes. Further decantation would only increase apoptosis and mortality of the injected adipocytes.
10.8 Centrifugation
The main concern about centrifugation is that it is a mechanical aggression to the
cells that might affect their survival rates. After the widespread use of the Coleman’s
technique, it was of special interest to know if centrifugation could affect the viability of the grafted fat. Some authors have described that centrifugation leads to similar viability than decantation, washing, ltering or absorbing the liquid with sterile
cotton towels [37, 55, 62]. However, it is evident that many variables could inuence those results.
The main variables, related to centrifugation, which might affect cell viability,
are the centrifugal force and the time of centrifugation. The ideal way to report the
intensity of the centrifugation is gravity (g) and not revolutions per minute (rpm).
This is important since every centrifuge has different diameter of the rotors and then
the (g) variates for each device. Unfortunately, many studies describe only (rpm) in
their methods. In the present chapter, we will use (g) when available, otherwise we
will report the (rpm). There are many more variables involved in centrifugation that
makes difcult to compare results among different studies.
The detailed analyses and description of all the variables that could affect the
viability of the cells is beyond the scope of this chapter. In the following section, we
will revise the available information in regard to these variables and how they might
affect or not the viability of the adipocytes.
Coleman used centrifugation to remove undesirable elements from the lipoaspirate to improve the survival of grafted fat [63], in compliance with the cell survival
theory of Peer. He stated that after centrifugation, for each 10mL of harvested fat,
he obtained 4–6mL of “rened” fat, suitable for grafting [49].
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Fig. 10.3 After
centrifugation four
different layers are visible
(see text for details). The
puried fat is injected
while the rest of the layers
are disposed
After centrifugation, the lipoaspirate typically forms four identiable layers
(Fig.10.3):
1. Oil. Is the most supercial and is the result of disrupted adipocytes.
2. Puried fat. Consisting in mostly viable adipocytes.
3. Watery. Consists on saline solution, blood, and lidocaine/epinephrine.
4. Cell pellet. Cells and other tissue debris. It is found in the bottom of the tubes.
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The main purpose of centrifugation is the separation of the mentioned components. However, only few articles describe what parameters (intensity and time of
centrifugation) are most effective to achieve it. Salinas etal. clearly demonstrated
that the tumescence liquid is effectively removed at 1200g, whereas greater forces
(up to 23,000g), did not remove more uid. On the other hand, oil started to separate at 1200g and increased up to 23,000g [56].
Kurita etal. found that the adipocytes did not suffer disruption related to increased
centrifugal force even with 4200g, but the quantity of the remnant oil did increase
with increased centrifugation forces [64]. Thus, they suggested that the amount of
oil observed with increased centrifugation forces are the result of better separation
of the components of the lipoaspirate, and not a measure of cell damage, as other
authors have suggested [13, 65, 66]. Similarly, Pulsfort etal. studied the effect of
eight different centrifugal forces (0–20,627g) and found no difference in terms of
viability among all the samples. Also, they found that higher centrifugation forces
produced cleaner samples without impairing the viability of the cells [65].
Kurita etal. used four different forces (400, 700, 1200, 3000, and 4200g) to
assess their effect in separation of the components of the aspirate, morphologic
alterations to the adipocytes, graft take, and shift of red blood cells and ASCs
between the different portions of the lipoaspirate. They noted that after 700g, no
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G. A. Mecott and S. G. Hernández-Soto
difference was seen in terms of red blood cells or ASCs extraction from the fatty
portion of the aspirate, but ASCs were damaged after 3000g. Based on all these
measurements, the authors suggested that 1200g is the optimal centrifugal force for
the best results [64]. Shiffman described that centrifugation at 3000rpm for 10s or
1min resulted in compacting the cells, but no difference was seen in the architecture
of the cells [51].
Pu etal. studied the viability of adipocytes processed by the Coleman technique compared to the “conventional” technique. The conventional technique
consisted of using a liposuction machine and centrifuging the aspirate at 500rpm
for 10min. They found better viability of the cells processed with the Coleman
technique [67].
Conversely, other studies have reported that centrifugation, at least at high
speeds, adversely affects the viability of adipocytes [31, 53, 68]. Hoareau etal.
reported that centrifugation at higher forces produces more death of the adipocytes
[66]. However, they measure the amount of oil after the centrifugation as a measurement of damaged cells. As discussed previously, more oil after centrifugation
doesn’t necessarily mean damage to the cells due to centrifugation, but perhaps
better separation of the already present in the lipoaspirate. No actual viability measurements were made to support these ndings. Nevertheless, they found (in rodent
models) better take and less oil cysts in the samples centrifuged at 400g for 1min
compared to the Coleman’s technique and decantation. So, they recommended
those as the ideal settings for centrifugation.
Ferraro etal. found that centrifugation at 1300rpm (250g) for 5min had
similar results (72% viable cells) as decantation and centrifugation at 500rpm
(50g) for 10min. However, 250g for 5min had better concentration of progenitor cells, whereas centrifugation at 3000rpm (1500g) for 3min had more cell
damage and less graft take. Clinically, they found that processing the samples at
250g for 5min had better take than decanted fat and centrifugation at 1500g for
3min. So, they suggested the protocol of 1300rpm (250g) for 5min as the ideal
protocol for fat grafting [60] Boschert et al. reported that centrifugation can
stratify the percentage of adipocytes within the different layers of the lipoaspirate, the bottom layer having 250% more viability when compared with the
upper layer [13]. They also noted that centrifugation for more than 2min did not
change this number, so they did not recommend to centrifugate the samples for
more than 2min at 50g.
Kim and Cols studied the effect of centrifugal force and time on the viability of
the adipocytes. They found that centrifugation at more than 3000rpm (220g) was
detrimental regardless of the time of centrifugation. Conversely, more than 3min
was also detrimental regardless of the centrifugal force [69].
Most of the literature seems to agree that if centrifugation is chosen, lower speeds
and fewer times will give the best results in terms of cell viability [64, 69].
A representative scheme of the results of the articles is shown in Fig.10.4.
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Fig. 10.4 Representation
of the available data of the
effects of centrifugation in
the lipoaspirate. The left
numbers are the gravity
force (g) and the text the
observed effect. The scale
is representative, not a
perfect scale was intended
10.9 Washing
157
Washing the lipoaspirate with normal saline or lactated Ringer’s is based on the
belief that oil or blood is detrimental to adipocytes survival. Then, the efcacy of
washing should be evaluated as the efcacy to remove those components without
impairing the viability of the cells.
Conde-Green found that washed grafts (with normal saline) had similar volume
retention than centrifuged ones (98.8% vs. 99.3% respectively). However, the variability of the volume of the washed samples (standard deviation) was signicantly
greater (± 34.6%) than centrifuged samples (± 8.4%). Oil cyst with calcication was
present in half of the grafts [33].
Smith etal. found no difference between grafts washed with Ringer Lactate or
normal saline solutions in terms of viability, histologic characteristics, or weight of
the graft at 3months after grafting [47]. Hoareau etal. described a protocol consisting of washing the lipoaspirate (with Ringer Lactate) followed by “soft” centrifugation (400g for 1min) [66]. They found that the proposed protocol had better volume
retention and less oil cysts than decanted graft. These results were similar to the
ndings of Conde-Green [33].
As previously mentioned, the presence of oil and blood in the lipoaspirate doesn’t
seem to be deleterious for the graft. This premise is based on the results of Salinas
et al., who added oil and blood to fat samples, and they didn’t nd signicant
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