Reply (2014)

Sir: Thank you for allowing us to respond to the letter by Cheng et al. The authors of this letter make certain claims regarding our original article published in the Journal in May of 2013.1 First, they discuss how histology using standard hematoxylin and eosin staining is not an adequate endpoint for use with fat grafting research. On the contrary, histology has been used in many peer-reviewed publications and is a widely accepted endpoint for adipocyte analysis. If done properly, it can be reliably used to distinguish the cell architecture of healthy adipocytes, vacuoles, and fibrosis/infiltrate. Healthy adipocytes are typically between 80 and 150 μm in size and are surrounded by other healthy adipocytes, whereas vacuoles are much larger and are usually surrounded by fibrosis/infiltrate. Cheng et al. used a glucose transport test and the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide metabolic assay as a surrogate for adipocyte viability. This makes the assumption that all cells have the same metabolic activity, regardless of the changes in experimental variables, and, thus, correlate with viability. While this can sometimes be assumed with fresh lipoaspirate, it should not be used with long-term in vivo experiments. In small animal models, injured fat grafts are infiltrated with neutrophils and fibroblasts. As a result, these grafts demonstrate very high metabolic activity but only reflect that of nonadipocytes, such as neutrophils and fibroblasts. Differences between healthy and injured fat grafts, however, can clearly be distinguished using histology. Cheng et al. also claim that Coleman’s article2 disagreed with our data. After reviewing this article, we have found no such statement. This article describes Coleman’s method for fat grafting and does describe gentle negative pressure using a syringe, but it never states that this is the optimal method. In fact, Coleman advocates for further research and states, “in vivo studies are still needed to determine actual survival rates.” The authors also present a series of pictures of syringe suction fat using low and high suction pressures, before and after centrifugation. We have concern over some of the inconsistencies regarding these pictures. First, why are the precentrifugation and postcentrifugation samples in different-size syringes? If the authors wanted to compare the effect of fat/triglyceride/tumescent fractions, they should have used the same syringes before and after centrifugation without introducing error from transferring a sample to a different syringe. In addition, there is a discrepancy between the volume in the precentrifugation and postcentrifugation samples. The “low suction” (their Fig. 1, above, left, and below, left) goes from 12 cc before centrifugation to 5 cc after centrifugation. Even in our highest-speed centrifugation studies using 25,000 g for 3 minutes, we did not see a reduction in volume by this amount. Furthermore, the authors claim that there is a difference in free lipid between the two harvest groups. Unfortunately, the authors fail to quantify the difference of triglyceride fraction to see whether they were statistically significant. Regardless, we cannot infer long-term survival data from in vitro experiments such as those performed by Cheng et al. We have published an in vivo study investigating the roles of pressure and shear in autologous fat grafting using a well-described small animal model. Our endpoints, weight and histology, provide useful information and do not attempt to infer adipocyte viability from surrogates such as metabolic activity. Furthermore, investigators using assays for metabolic activity in a small animal model should be cautious, as nonadipocyte cell lines such as neutrophils and fibroblasts can erroneously increase metabolic activity in severely injured fat grafts. DISCLOSURE The authors have no financial interest to declare in relation to the content of this communication. Jeffrey H. Lee, M.D. Michael C. McCormack, M.B.A. William Gerald Austen, Jr., M.D. Massachusetts General Hospital, Boston, Mass.