Joris van Dongen

330 Chapter 14 reduced tenfold in comparison to the starting volume of lipoaspirate. 11,12 A reduction of volume is an indirect indication of the effectiveness of a mechanical isolation procedure because 90% of the volume of adipose tissue can be ascribed to adipocytes. The main difference between the FAT procedure and other mechanical isolation procedures is the centrifugation step prior to fractionation. Centrifugation is necessary to remove all infiltration fluid in harvested lipoaspirate. Infiltration fluid is used for local anesthetics and the fluid component has a protective effect on mature adipocytes during fractionation. The principle of fractionation is based on shearing of tissue and large cells; powerfully pushing adipose tissue through a small hole in a luer-to-luer hub. Adipocytes are flexible by changing their shape while being pushed through a small hole. Once adipocytes pass the small hole, reformation occurs into their natural shape. However, without a protective fluid around the adipocytes giving counter pressure, adipocytes will disintegrate. A comparable technique used in literature is the Nanofat procedure. 13 Nanofat procedure uses only decanted lipoaspirate and thus a large amount of protective infiltration fluid is still present. Consequently, a large number of adipocytes are still intact present in the final product. 14 A study of Mashiko et al. compared the Nanofat procedure with the FAT procedure and showed a larger number of adipocytes of 83.3% in the Nanofat fraction compared to 45.8% in the FAT procedure fraction. Moreover, the extracellular matrix portion was lower in Nanofat than in tSVF isolated by the FAT procedure (16.7% vs 54.2%). 15 These findings show that the Nanofat procedure is an emulsification and filtration procedure to increase injectability by mixing infiltration fluid with lipoaspirate rather than an mechanical isolation procedure of tSVF. In contrast to enzymatic isolation procedures, mechanical isolation procedures disrupt only adipocytes while maintaining all cell-cell communications including extracellular matrix. This difference might be of significant importance because individual cells in a single cell suspension of SVF tend to quickly migrate from the tissue, e.g. into the draining lymph nodes, directly after injection. The extracellularmatrix inmechanically- derived SVF, literally retains the cells on the site of injection resulting in a larger regenerative effect. Additionally, extracellular matrix itself has an important and often underestimated regenerative role because it functions as a natural instructive scaffold for both cells and growth factors. In this thesis, extracellular matrix from SVF obtained by the FAT procedure was isolated through an extensive decellularization protocol and using mild enzymatic proteolysis with pepsin converted into a self-assembling hydrogel. 16 This hydrogel was loaded with conditioned medium from cultured ASCs containing the complete secretome of ASCs in order to stimulate angiogenesis as well as fibroblast proliferation and migration in vitro . 16 These findings showed that