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Yang et al. Plast Aesthet Res 2020;7:8 I http://dx.doi.org/10.20517/2347-9264.2019.63 Page 11 of 13

assistance. The primary driver of fat particle size manipulation is to avoid “large” fat particles in subsequent
grafting procedures. These particles may clog injection cannula or have insufficient oxygen and nutrient
diffusion to the interior particle core, resulting in large necrosis and oil cyst formation. These oil cysts are
eventually resorbed through macrophage clearance resulting in graft tissue loss and inferior long-term fat
grafting outcomes.

Clinically, it has been difficult to parse out the impact of fat particle size on graft retention from other
confounding variables such as trauma associated with harvest or injection. Our study aimed to isolate the
variable of fat particle size to determine the impact of fat particle size on in vivo survival and viability of
fat grafts. To do this, an immunocompetent, inbred mouse model was selected to avoid donor variability
with human adipose tissue. Adipose particles for grafting were prepared from whole subcutaneous fat pads
by mincing tissue with surgical scissors. This allowed for consistent particle samples of known diameter
while eliminating possible trauma from harvest cannulas. Study outcomes included graft retention, tissue
histology, and measured genetic markers of inflammation, apoptosis, and tissue regeneration (qPCR).

Our data suggest that fat particles below 7 mm produce similar fat grafting outcomes despite increased
hypoxia in 5-7-mm particles compared to 2-4-mm diameter particles. The histological findings from our
study show that the small fat particles group led to milder necrosis and more neovascularization (especially
in the early stages) while larger particles experienced increased necrosis and areas with irregular adipocyte
morphology. Interestingly, while small fat particles showed superior architecture at the one-week study
timepoint, this was reversed at the 12-week timepoint, with large fat particle grafts containing larger
adipocytes and a better-organized histological structure. This may suggest that early adipocyte necrosis
leading to extracellular matrix deposition through fibrosis results in a scaffold for tissue regeneration
[29]
from circulating progenitor cells, as was also proposed by Del Vecchio et el. , who found that smaller
particles may lack connective tissue for structural support of adipocytes and proliferating stem cells. The
regenerative role of fascia, connective tissue, and extracellular matrix components of the fat graft is often
overlooked and yet to be fully understood. This study, along with our previous published work, aims to fill
this knowledge gap.

Cumulatively, our studies suggest that, independent of adipose tissue quality, regenerative adipogenesis
is highly dependent on the tissue scaffold, followed by angiogenesis, and lastly adipose induction or
adipogenesis with all components playing important sequential and dynamic roles. It is hypothesized that
finding the balance of these components will depend largely on the recipient site and the amount and
nature of the host tissue in that region. As previous studies have demonstrated the angiogenic nature of
proteins present in the fascia/connective tissue which angiogenesis occurs prior to adipogenesis, future
studies on particle size could also include immunohistochemistry for CD31 to see if the larger particle
group goes through this phase of angiogenesis at a different rate from small particles.

The results from this study have been corroborated elsewhere where particle size has shown no significant
[30]
impact on long-term outcomes when the particle diameter is less than or equal to 7 mm [11,12] . Fisher et al.
transplanted lipoaspirate filtered with 500 and 800 µm filters, whose average sizes were approximately 5.9
[31]
and 3.2 mm, and found there was no statistically significant difference between each other. Ozsoy et al.
compared lipoaspirate from human pannus harvested with 2-, 3-, and 4-mm diameter cannulas, and showed
[15]
that the 4-mm cannulas had the highest level of adipocyte viability. Erdim et al. evaluated viability of fat
grafts, which was harvested from the abdomen of 10 consecutive patients using 2-, 4-, and 6-mm cannulas,
by using colleganase digestion, supravital staining, and adipocyte counting using a haemocytometer.
The results demonstrate a higher number of viable adipocytes from the aspirate occurred with the 6-mm
cannula as opposed to the 2- and 4-mm cannulas. Therefore, the impact of harvesting cannula diameter
may not be a critical factor on ultimate graft retention when fat particles are ≤ 7 mm in diameter, which we
found to be the case in four commonly used lipoplasty harvesting cannula.