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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 7 of 13

Figure 5. Measurement of VEGF concentration in ex vivo culture media with ELISA. Data are presented as mean (n = 2 independent
biologic replicates per group) with no statistical comparison performed due to low sample size. VEGF: vascular endothelial growth factor
ELISA: enzyme-linked immunosorbent assay

were determined using P-value less than 0.05. Statistical modeling of intra-animal reproducibility was
performed in consultation with the University of Pittsburgh Department of Statistics.

RESULTS
In vitro analysis of human adipose particle size and hypoxia
Liposuction cannulas and harvested fat particles analysis
Semi-automated measurement of particle size was verified using glass calibration beads. The results from glass
particles demonstrated that the method was consistent, with 8% and 6.5% standard deviation for 3.35- and
2-mm particles, respectively, and the method was able to separate particle diameter differences of 1.35-mm
diameter with statistical significance [Figure 4A].

The radius of adipose particles harvested with the Bucket, Khouri, Mercedes Tip, and Shippert cannulas
were 3.03 ± 0.78, 1.98 ± 0.92, 2.31 ± 0.85, and 1.9 ± 0.59 mm, respectively [Figure 4B]. The range of fat
particles diameters was approximately 2-7 mm [Figure 2]; therefore, the range was divided into two groups
for future studies, with small (2-4 mm) and large (5-7 mm) sized particles.

VEGF expression in ex vivo model
An ex vivo study was conducted with fat particles of variable diameter to evaluate the impact of fat particle
size on oxygen diffusion limitation and tissue hypoxia. Adipose tissue obtained during panniculectomy was
minced with surgical scissors into small and large particle sizes and cultured in equal total amounts in cell
culture media. VEGF is an important growth factor for particle revascularization and is expressed by adipose
due to hypoxia. Quantification of VEGF concentration in media with ELISA showed increased expression in
both particle groups every consecutive day for seven days of culture [Figure 5]. VEGF media concentration
was significantly higher in 5-7-mm group compared to the 2-4-mm group at Days 4 and 6 (5928.73 ±
2572.74 pg/mL vs. 1507.18 ± 313.16 pg/mL and 23,950.61 ± 2946.86 pg/mL vs. 15,126.37 ± 3846.77 pg/mL,
respectively, P < 0.01), suggesting that larger particles experience increased hypoxia compared to smaller
particles.

In vivo analysis of fat particle survival in mouse autograft model
Fat particles preparation and transplantation
All experimental animals receiving fat grafts survived well without complications and all surgical incision
wounds healed normally. At each timepoint, grafted particles were easily discernable in the subcutaneous