New insights into lidocaine and adrenaline effects on human adipose stem cells.

BACKGROUND: Adipose stem cells have gained great interest in plastic and reconstructive surgery with their ability to improve engraftment after fat transfer for soft tissue filling. It is therefore essential to know the effect of the drugs commonly used during the lipoaspiration procedure, such as lidocaine and adrenaline. Indeed, these drugs are infiltrated at the fat donor site for local anesthesia and for reduction of bleeding. This study analyzed the effects of these drugs on the viability of adipose-derived stem cells and on their inflammatory status. METHODS: Adipose-derived stem cells from lipoaspirates were grown in culture before being treated with different clinical doses of lidocaine at different times of exposure (1-24 h), and with adrenaline (1 µg/mL). Cytotoxicity was measured by lactate dehydrogenase assay and by flow cytometry with annexin V/propidium iodide staining. In parallel, the secretion of the proinflammatory cytokines tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) was tested by enzyme-linked immunoassay. RESULTS: Lidocaine affected cell viability after 24 h, even when the cells were exposed for only 1 or 2 h. Apoptosis was not involved in lidocaine cytotoxicity. Regarding inflammation, no TNFα was produced, and lidocaine decreased the levels of IL-6 and MCP-1 in a dose-dependent manner. In contrast, adrenaline did not influence cell viability or cytokine secretions. CONCLUSIONS: Adipose tissue should be handled appropriately to remove lidocaine and adrenaline, with such procedures as washing and centrifugation. This study provides new insights into the use of lidocaine and adrenaline for fat transfer or stem cell isolation from lipoaspirates. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Effect of Washes and Centrifugation on the Efficacy of Lipofilling With or Without Local Anesthetic.

BACKGROUND: Among the different parameters that influence fat graft survival and lipofilling success, the use of local anesthetic and the way to process the fat before injection have often been pointed out. Likewise, we evaluated different techniques for processing adipose tissue before its injection and analyzed the quality of the grafts. METHODS: Adipose tissue from the same patient was gently harvested from one side of the abdomen after infiltration of a tumescent solution without lidocaine and from the other side of the abdomen using a tumescent solution containing lidocaine 2%. Harvested tissue was prepared with different protocols, from simple decantation to advanced protocols including single or multiple washes and centrifugations. Each type of processed adipose tissue was then injected subcutaneously into immunodeficient mice. Adipose grafts were collected after 1 month and analyzed by histology with a detailed scoring method. RESULTS: After lidocaine use, decantation protocol led to adipose grafts of poor quality with high resorption rate and oil vacuole formation. Larger grafts were obtained after centrifugation, but centrifugation alone resulted in increased fibrosis and necrosis, with or without the use of lidocaine. Finally, multiple washes and centrifugations greatly improved the quality of the lipografts. CONCLUSIONS: Centrifugation alone is not sufficient and must be associated with multiple washes to improve graft quality. This article aims to provide further evidence of lidocaine and washing/centrifugation effects in fat grafting to provide easy tips aimed at ensuring graft efficiency with a long-term clinical outcome.

Effect of centrifugation and washing on adipose graft viability: a new method to improve graft efficiency.

BACKGROUND: Adipose tissue grafting is a promising method in the field of surgical filling. We studied the effect of centrifugation on fat grafts, and we propose an optimised protocol for the improvement of adipose tissue viability. METHODS: Adipose tissue was subjected to different centrifugations, and the volumes of interstitial liquid and oil released were measured to choose the optimal condition. Tissue from this condition was then compared to tissue obtained from two traditional techniques: strong centrifugation (commonly 3 min at 3000 rpm/900 g), and decantation, by injecting into immunodeficient mice. The cytokine interleukin-6 (IL-6) and chemokine monocyte chemotactic protein-1 (MCP-1) were assayed 24 h post-injection, and after 1 month of grafting the state of the lipografts was evaluated through macroscopic and histological analysis, with oil gap area measurement. RESULTS: Strong centrifugation (900 g, 1800 g) is deleterious for adipose tissue because it leads to until threefold more adipocyte death compared to low centrifugation (100 g, 400 g). In addition, mice injected with strong centrifuged and non-centrifuged adipose tissue have higher rates of blood IL-6 and MCP-1, compared to those grafted with soft centrifuged fat. Moreover, extensive lipid vacuoles were detectable on histological sections of the non-centrifuged lipografts, whereas lipografts from soft centrifugation contain a higher amount of connective tissue containing collagen fibres. CONCLUSION: It is necessary to wash and centrifuge adipose tissue before reinjection in order to remove infiltration liquid and associated toxic molecules, which in the long term are deleterious for the graft. However, strong centrifugation is not recommended since it leads very quickly to greater adipocyte death. Thus, soft centrifugation (400 g/1 min), preceded by washings, seems to be the most appropriate protocol for the reinjection of adipose tissue.

Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

BACKGROUND: The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation. METHODS: Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays. RESULTS: WE SHOW FOR THE FIRST TIME THAT THE PRODUCTION OF TNFALPHA IN MATURE HUMAN ADIPOCYTES IS MAINLY DEPENDENT UPON TWO PATHWAYS: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages. CONCLUSION: This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

Effect of PEA on LPS inflammatory action in human adipocytes.

N-Palmitoylethanolamide (PEA) is an endogenous lipid secreted by human adipocytes that possesses numerous anti-inflammatory properties. Human adipose tissue can be subjected to modulation of its inflammatory state by lipopolysaccharide (LPS). Here we demonstrate that LPS increases the secretion of interleukin-6 (IL-6) by human mature adipocytes via activation of the NFkappaB pathway. This effect is not inhibited by PEA. Inversely, LPS strongly inhibits adipose cell leptin release, with PEA acting as a potentiator of this inhibitory effect. These actions are not linked to a reduction in leptin gene transcription. Thus, PEA does not have an anti-inflammatory role in the secretion of IL-6 via NFkappaB at the adipocyte level, but instead seems to act at the heart of the LPS-stimulated pathway, which, independently of NFkappaB, inhibits the secretion of leptin.