High-throughput Model for Adipocyte Preservation
Background: Soft tissue defects secondary to traumatic injury, oncologic resection, and repair of congenital malformations remain a major source of morbidity in modern surgical practice. Autologous adipose tissue transplantation has been used to reconstruct these defects; however, it remains unpredictable. In order to identify agents or techniques that will improve fat graft survival traditional animal models can take weeks to months for results. The purpose of this study was to develop a model for the rapid identification of agents using apoptosis specific fluorescence, which would correlate to results seen months later using traditional endpoints. By comparing various polymers and controls as cell resuscitation agents in our early apoptosis model we can demonstrate the ability to predict long term fat graft survival within a ten day period.
Methods: Human Fat was obtained from liposuction aspirates, washed with saline and centrifuged. The fat was then treated with one of five agents: P188, T1107 (surfactant polymers), polyethylene glycol (PEG 8000), dextran (molecular weight control), or saline. Fat lobules where explanted over a 10 day period every third day and then at six weeks. The explanted groups were incubated with fluorescent labels specific for apoptosis (SR-FLIVO or Mito PT). Samples were weighed and tested for G3PH activity, DNA content, cell counts, and examined using confocal microscopy.
Results: P188 treated fat displayed the lowest levels of apoptosis (see Chart 1). ATP levels and G3PH levels remained stable across all groups in the 10 day period, as did weights. At 6 weeks the saline and dextran controls exhibited up to 30% resorption; however, fat grafts treated with P188 demonstrated a 75% decrease in resorption (p<0.001). When P188 was compared to other polymers (T1107 and PEG 8000) it demonstrated a 6% re-absorption compared with 35% re-absorption in PEG 8000 and 20% re-absorption in T1107 and saline treated samples at 6 weeks (see Chart 2, p = 0.04). Additionally, at 6 weeks DNA and fluorescent count analysis demonstrated similar percent reductions in cell number that correlated to levels of apoptosis seen in the first ten days.
Conclusions: Treatment of cells with the membrane stabilizing agent P188 demonstrates a significant decrease in adipocyte apoptosis in the first 10 days post treatment, this correlates with our data at 6 weeks. This new model can be used as a high-throughput method for screening protection agents. Using this model we can predict long-term adipocyte survival and graft take during the first 10 days post-implantation.