The simple filtration by means of the MilliGraft® Kit of a disaggregated lipoaspirate allows to extract the class of cells defined as progentors with characteristics of

adult stem cells present in the nanofat exclusively on the basis of their dimensions. It also allows the elimination of the fibrous branches and cell membranes destroyed by the emulsion phase and obtain a population of cells deprived of the inflammatory component. This method was used in regenerative and aeshetic medicine treatments with excellent and lasting clinical results in the follow-up phase

Svolacchia, Fabiano

Svolacchia, Lorenzo

English

The simple filtration by means of the MilliGraft® Kit of a disaggregated lipoaspirate allows to extract the class of cells defined as progentors with characteristics of
adult stem cells present in the nanofat exclusively on the basis of their dimensions. It also allows the elimination of the fibrous branches and cell membranes destroyed by the emulsion phase and obtain a population of cells deprived of the inflammatory component. This method was used in regenerative and aeshetic medicine treatments with excellent and lasting clinical results in the follow-up phase

Fabiano Svolacchia

Lorenzo Svolacchia

Archivio della ricerca- Università di Roma La Sapienza

 Svolacchia and Svolacchia. Scr Med 2019;50(3):117-21.ORIGINAL ARTICLEDOI:10.5937/scriptamed50-21881Dermal Regeneration with MilliGraft® Kit of Nanofat: the Micrograft of Adipose Tissue. A Clinical Assessment StudyFabiano Svolacchia1, Lorenzo Svolacchia2Department of Anatomical, Histologi-cal, Medical and Legal Sciences and the Locomotor Equipment, Section of Human Anatomy, Laboratory of Exper-imental Morphology, University of Rome "La Sapienza", Rome, Italy.Faculty of Medicine and Surgery,  Uni-versity of Rome "La Sapienza", Rome, Italy.Correspondence:FABIANO SVOLACCHIAE: fabiano.svolacchia@uniroma1.itThis article should be cited as follows: Svolacchia F, Svolacchia L. Dermal regeneration with MilliGraft® kit of nanofat: the micrograft of adipose tissue. A clinical assessment study. Scr Med 2019;50(3):117-21.Received: 29 April 2019Revision received: 30 May 2019Accepted: 3 June 2019ARTICLE INFO(1)(2)Copyright © 2019 Svolacchia and Svolacchia. This is an open access article distributed under the Creative Commons Attribution License (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.ABSTRACTThe simple filtration by means of the MilliGraft® Kit of a disaggregated lipoaspi-rate allows to extract the class of cells defined as progentors with characteristics of adult stem cells present in the nanofat exclusively on the basis of their dimensions. It also allows the elimination of the fibrous branches and cell membranes de-stroyed by the emulsion phase and obtain a population of cells deprived of the in-flammatory component. This method was used in regenerative and aeshetic med-icine treatments with excellent and lasting clinical results in the follow-up phase. Key words: dermal regeneration, nanofat, mesenchymal stromal progenitor cells, microfiltrate, MilliGraft®. INTRODUCTIONThe physiological turnover of adult tissues loses efficiency after the first third of life through chro-no-aging and photo-aging due to the progressive loss of the normal cellular turnover guaranteed by adult stem cells contained in the niches of differentiated tissues. The niches are made up of specialised cells protecting the mesenchymal stromal progenitor cells (MSCs) that are contin-ually attacked by oxygen free radicals and ultra-violet B (UVB) radiation.1 Through the influence of these highly reactive chemical species, adult stem cells will be carried out in a phase of quies-cence that will force them to remain in the G0/G1 phase, influencing and facilitating their tran-sition in the subsequent phase of senescence.2 The senescence that adult stem cells will meet will prevent the physiological regeneration of tissues of the tissue progenitor population. Pre-vious studies have been conducted on tissue pro-genitors from the dermis.3In the present clinical study, it was assumed that, in order to improve photo-aging and chrono-ag-ing damage, the dermis could be replenished with a vital micrograft from adipose tissue that contains the progenitors with adult staminal markers and those that best embody the char-acteristics of adult stem cells. The adipose tis-sue was chosen because of the simplicity of ex-traction and its abundance. It was processed and disaggregated through the MilliGraft® kit that al-lowed to deprive the tissue of cellular debris and fibrous shoots.4 Such a suspension was injected into the dermis. It was also hypothesised that the triglycerides derived from the fragmentation of adipocytes increased the potential of engraft-ment5- 7 of the side population that remains sus-pended in triglycerides after filtration.8It was also assumed that the adult stem cells de-rived from adipose tissue and their secretomes were able to allow for normalisation of the ex-tracellular matrix with neocollagenogenesis and neovasculogenesis in the dermis without being influenced by hypoxia of the recipient tissue typical for aged tissue.4 The method adopted is 118the MilliGraft® kit, a method based exclusive-ly on the dimensions of the vital grafts and the triglyceride vehicle5-7 in which the micrograft is suspended.9 With the MilliGraft® kit it was able to prepare a sample of adipose tissue according to the technique of cytometry. By means of this method the tissue is disaggregated by special devices and filtered according to the necessary measures. The flow cytometer showed then that the side population, which is the one that best embodies the values of the progenitor, is small-er than the differentiated cells, that they have a greater cytoplasmic complexity and a greater ex-pression of the stem markers. The task of these cells is to precisely maintain the equilibrium, the tissue homeostasis and the regenerative capacity of adult tissues in an incredibly flexible process.4The choice of adipose tissue was also dictated by the biological reasons for the wealth of side population contained in it even after disaggrega-tion.5,6 In fact, it is possible to isolate 5x103 cells from a gram of it, about 500 times more than from the bone marrow9 with the surface expres-sion of CD 105, 90, 73 and 44 as markers8 typical for adult stem cells10. Mesenchymal stromal pro-genitor cells (MSCs) are a population of non-hae-matopoietic and multipotent cells and are able to self-renew and differentiate into mesodermal cell lines. Under the normal conditions of a cell culture, they adhere to plastics and differentiate into osteoblasts, chondroblasts and adipocytes.11 MSCs isolated from adipose tissue can function as a source of cells for the repair and dermal re-generation affected by chrono- and photo-ag-ing.12 These cells have shown not to lose their vitality during the various cultivation steps up to the passage number eight and without showing typical senescence characteristics of differentiat-ed tissues.13 They possess a greater proliferative capacity than the bone marrow MSCs.14Therefore, the preparation of adipose tissue ob-tained by means of the MilliGraft® kit allowed to obtain the side population and the vascular stro-mal fraction exclusively according to the princi-ple of cell size.3 The dimensional characteristics of the MilliGraft® kit allowed for the exclusion of the inflammatory fibrous shoots and cellular debris in a reproducible way without the use of enzymes.12 Despite of the fact that a part of these cells can be lost during the disaggregation,15 the filtration has shown that more colonies can be obtained in culture than in the unfiltered nano-fat.16 The vascular stromal fraction is rich in pericytes and active mesenchymal cells that are able to accelerate the process of tissue regener-ation1.7, 18The disaggregation of adipose tissue allows for the release of lipoprotein lipase and adiponectin. The function of lipoprotein lipase is to hydrolyse triglycerides in fatty acids and glycerol and is a key enzyme for the metabolism of lipoproteins and triglycerides. Lipoprotein lipase induces an increase in retention and absorption of all classes of lipoproteins.19,20 Consequence of this induction is an increase in energy production in cells and greater protein synthesis. Adiponectin enables the catabolism of fatty acids and inhi- bition of inflammatory processes by improving metabolic energy in tissues.6 Through the me-sotherapeutic supplementation of adipose-de-rived MSCs the formation of inflammasomes is inhibited and the regulation of the population of macrophages M1 is activated through the pros-taglandin E.27It should be considered that fibroblasts are the most represented cells in the dermis. They are able to accumulate triglycerides21 and to degrade them together with cholesterol esters in two dif-ferent catabolic ways.22 Injection of a microfil-tered adipose tissue through MilliGraft® kit in the dermis also enables the regulation of EGFR and ERBB3 and to normalization of sebaceous lipogenesis. In fact, triglycerides are important compounds for the skin, produced by the seba-ceous glands. It was also hypothesised that the triglycerides that carry the MSCs decrease the re-lease of LDH and reactive oxygen species (ROS) generation.23 According to this hypothesis, there is a greater cellular vitality, a higher metabolic activity of the cells and a higher level of ATP.24Triglycerides as a vehicle for suspended MSCs also cause vasodilation and increased local blood flow.25 For this reason, it was also hypothesised that a temporary increase in blood flow in the dermis with consequent physiological normal-ization of the tissue increases the replication and the plastic potential of MSCs.26 Triglycerides used as a vehicle for MSCs promote cell mem-brane phospholipids in human fibroblasts.27The study was aimed at evaluating the clinical results of the improvement of the skin condition and to clinically verify the therapeutic response by using a microfiltrate of the population of the adipose tissue.3Svolacchia and Svolacchia. Scr Med 2019;50(3):117-21.119METHODS Figure 1: Tissue estractionFigure 2: Decantation of fatFigure 4: Clinical Mean score of treatmentFigure 3: MicrofiltrateA total of 124 female patients were studied and their age ranged between 28 and 72 (average age 48). They signed informed consent for the use of lipoaspirate for experimental procedures. The study was performed following the stan-dards of the local ethics committee and in ac-cordance with the Helsinki Declaration (2000). None of the patients presented any specific pa-thology other than the dermal-epidermal aging process. Once the donor area was identified, the Klein solution was injected to anaesthetise the collection site. The adipose tissue was extracted under a tumescent anesthesia that allows the extraction of a greater number of viable grafts than a local anesthesia.28 The adipocytes were extracted through a 10 ml syringe with Luer lock and a 16-gauge needle (Figure 1).876543210time 0 after 30 days after 90 daysfoldsMean scoreSvolacchia and Svolacchia. Scr Med 2019;50(3):117-21.An aliquot of 6/7 ml of tissue that has been de-canted to eliminate anesthesia fluids was ex-tracted (Figure 2) to obtain 5 ml of adipose tissue that was disaggregated through the MilliGraft® kit and filtered to 50 microns in order to obtain the vital side population3,9 (Figure 3) and Micro-Graft® according to the principles of cytometry. In this way the fibrous shoots and cellular debris were eliminated.4,16 The microfiltrate was used for face, neck and décolleté treatments with 1 ml Luer lock syringes and 30 gauge needles. All the patients have shown an improvement with the treatment they have undergone and they have expressed satisfaction with the improvements achieved. Inspection visits and evaluations were made on the first day (D1, baseline) and after 30 (D30) days of treatment, with a follow-up visit after three months (Figure 4). 120REFERENCESDISCUSION CONCLUSION1. Pontieri GM, Russo MA, Frati L, editors. General pa-thology. 4th edition. Padua: Piccin Editore, 2011. Ital-ian.2. Dicarlo M, Teti G, Iezzi I, Cerqueni G, Manzotti S, Falconi M, Mattioli-Belmonte M. Detecting senescent fate in mesenchymal stem cells: a combined cytofluo-rimetric and ultrastructural approach. Biogerontology 2018;19(5):401-14.3. Svolacchia F, De Francesco F, Trovato L, Graziano A, Ferraro GA. An innovative regenerative treatment of scars with dermal micrografts. J Cosmet Dermatol 2016;15(3):245-53.4. Svolacchia F, Svolacchia L. A protocol of a new regen-erative treatment of chrono-aging and photo-aging with progenitor cells from adipose micrograft obtained from MilliGraft® Kit. Acta Sci Med Sci 2019;3(3):30-5. Hilaire N, Salvayre R, Thiers JC, Bonnafé MJ, Nègre-Salvayre A. The turnover of cytoplasmic triacyl-glycerols in human fibroblasts involves two separate acyl chain length-dependent degradation pathways. J Biol Chem 1995;270(45):27027-34. 6. Díez JJ, Iglesias P. The role of the novel adipocyte-de-rived hormone adiponectin in human disease. Eur J Endocrinol 2003;148(3):293-300. 7. Park HJ, Kim J, Saima FT, Rhee KJ, Hwang S, Kim MY, et al. Adipose-derived stem cells ameliorate coli-tis by suppression of inflammasome formation and regulation of M1-macrophage population through prostaglandin E2. Biochem Biophys Res Commun 2018;498(4):988-95.8. Svolacchia F. A simple regenerative treatment of chro-no-aging and photo-aging with adult mesenchymal stem cells from adipose tissue extracted. Research-Gate DOI:10.13140/RG.2.2.30935.52649 Septem-ber 2018. Available at https://www.researchgate.net/publication/327381774_A_simple_regenera-tive_treatment_of_chrono-aging_and_photo-aging_with_adult_mesenchymal_stem_cells_from_adi-pose_tissue_extracted 9. Hass R, Kasper C, Böhm S, Jacobs R. Different pop-ulations and sources of human mesenchymal stem This in vivo study of the MilliGraft® kit tech-nique enabled the evaluation of a new method of dermal regeneration inspired by the cytometry method applied to a nanofat on our patients. The cytometry counts the cells in a suspension and identifies them through the markers. Because MSCs are the same as a classic lipoaspirate in the disaggregated adipose tissue, it was separat-ed from the fibrous shoots and cell membranes of the destroyed adipocytes to make sure that a less polluted side population is achieved. The presence of MSCs is provided by the size of the filter used and the size of tissue progenitors.3,4 In vivo results we obtained with the MilliGraft® kit show that tissue progenitors can be isolat-ed from the examined samples. This technique is suitable for preserving the side population thanks to the filtration measurement to which the nanofat is subjected. In this way, it was in-jected into the dermis of our patients through the mesotherapeutic technique, a considerable amount of viable MicroGraft deprived of the potentially inflammatory component. These Mi-croGrafts are able to normalise a physiological production of collagen type I and type III.29Through filtration we have overcome and atten-uated the biological events caused by the pro- duction of inflammatory cytokines produced by macrophages and dendritic cells present in the dermis to an increasing extent at the age1 and triggered by fibrous shoots and by the cell mem-branes of the destroyed adipocytes.The fibrous shoots and cell membranes that are injected through the nanofat technique are recognized by the TOLL-LIKE system to trig-ger, within that class of cells and other cells, the pro-inflammatory NF-Kb1 program interfering with the rejuvenation treatment.The initiation of the inflammatory phenome-non through the injection of nanofat has been demonstrated30,31 and the inflammatory state related to the dermis selects fibrotic fibroblasts with production of fibrotic collagen.32 Through this simple procedure it will be possible to inject a nanofat deprived of the inflammatory compo-nent.The treatment was well tolerated and the clini-cal results confirmed the working hypothesis on dermal-epidermal regeneration with the Milli-Graft® kit procedure.ACKNOWLEDGEMENTSCONFLICT OF INTERESTNone.None.Svolacchia and Svolacchia. Scr Med 2019;50(3):117-21.Svolacchia and Svolacchia. Scr Med 2019;50(3):117-21. 121cells (MSC): a comparison of adult and neonatal tis-sue-derived MSC. Cell Commun Signal 2011;9:12. doi: 10.1186/1478-811X-9-12. 10. Chan AK, Heathman TR, Coopman K, Hewitt CJ. Mul-tiparameter flow cytometry for the characterisation of extracellular markers on human mesenchymal stem cells. Biotechnol Lett 2014;36(4):731-41.  11. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for de-fining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8(4):315–7.12. Kalinina N, Kharlampieva D, Loguinova M, Butenko I, Pobeguts O, Efimenko A, et al. Characterization of se-cretomes provides evidence for adipose-derived mes-enchymal stromal cells subtypes. Stem Cell Res Ther 2015;6:221. doi: 10.1186/s13287-015-0209-8. 13. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294–1301.14. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294–301.15. Bi HS, Zhang C, Nie FF, Pan BL, Xiao E. Basic and clinical evidence of an alternative method to pro- duce vivo nanofat. Chin Med J (Engl) 2018;131(5):588-93.16. Lo Furno D, Tamburino S, Mannino G, Gili E, Lom-bardo G, Tarico MS, et al. Nanofat 2.0: experimental evidence for a fat grafting rich in mesenchymal stem cells. Physiol Res 2017;66(4):663-71.17. Bertheuil N, Chaput B, Ménard C, Varin A, Laloze J, Watier E, et al. Adipose mesenchymal stromal cells: definition, immunomodulatory properties, mechani-cal isolation and interest for plastic surgery. Ann Chir Plast Esthet 2018 64(1):1-10.18. Gentile P, Scioli MG, Bielli A, Orlandi A, Cervelli V. Comparing different nanofat procedures on scars: role of the stromal vascular fraction and its clinical im- pli-cations. Regen Med 2017;12(8):939-52. 19. Fernández-Borja M, Bellido D, Vilella E, Olivecrona G, Vilaró S. Lipoprotein lipase-mediated uptake of li- poprotein in human fibroblasts: evidence for an LDL receptor-independent internalization pathway. J Lipid Res 1996;37(3):464-81. 20. Hilaire N, Salvayre R, Thiers JC, Bonnafé MJ, Nègre-Salvayre A. The turnover of cytoplasmic triacyl- glycerols in human fibroblasts involves two separate acyl chain length-dependent degradation pathways. J Biol Chem 1995;270(45):27027-34. 21. Jeney F, Bazsó-Dombi E, Oravecz K, Szabó J, Nagy IZ. Cytochemical studies on the fibroblast-preadipocyte relationships in cultured fibroblast cell lines. Acta His-tochem 2000;102(4):381-9. 22. Hilaire N, Nègre-Salvayre A, Salvayre . Cytoplasmic triacylglycerols and cholesteryl esters are degraded in two separate catabolic pools in cultured human fibro-blasts. FEBS Lett 1993;328(3):230-4. 23. Jebali A, Kazemi B. Triglyceride-coated nanoparticles: skin toxicity and effect of UV/IR irradiation on them. Toxicol In Vitro 2013;27(6):1847-54.24. 24. Bayerle-Eder M, Polska E, Kopf A, Roden M, Waldhäusl W, Pleiner H, et al. Free fatty acids exert a greater effect on ocular and skin blood flow than triglycerides in healthy subjects. Eur J Clin Invest 2004;34(8):519-26. 25. Polak K, Schmetterer L, Luksch A, Gruber S, Pols-ka E, Peternell V, et al. Free fatty acids/triglycerides increase ocular and subcutaneous blood flow. Am J Physiol Regul Integr Comp Physiol 2001;280(1):R56-61. 26. Forsyth NR, Kay A, Hampson K, Downing A, Talbot R, McWhir J. Transcriptome alterations due to physio- logical normoxic (2% O2) culture of human embryonic stem cells. Regen Med 2008;3(6):817-33.27. Miller M, Motevalli M, Westphal D, Kwiterovich PO Jr. Incorporation of oleic acid and eicosapentaenoic acid into glycerolipids of cultured normal human fi-broblasts. Lipids 1993;28(1):1-5 28. Wang WZ, Fang XH, Williams SJ, Stephenson LL, Baynosa RC, Khiabani KT, et al. Lidocaine-induced ASC apoptosis (tumescent vs. local anesthesia). Aes-thetic Plast Surg 2014;38(5):1017-23.29. Jung JY, Shim JH, Choi H, Lee TR, Shin DW. Human dermal stem/progenitor cell-derived conditioned me-dium improves senescent human dermal fibroblasts. Int J Mol Sci 2015;16(8):19027-39.30. Janis JE, Harrison B. Wound healing: part I. Basic sci-ence. Plast Reconstr Surg 2014;133:199e-207e.31. Sezgin B, Ozmen S, Bulam H, Omeroglu S, Yuksel S, Cayci B, et al. Improving fat graft survival through pre-conditioning of the recipient site with microneedling. J Plast Reconstr Aesthet Surg 2014;67:712-20. 32. Jelaska A, Strehlow D, Korn JH. Fibroblast heteroge-neity in physiological conditions and fibrotic disease. Springer Semin Immunopathol 1999;21(4):385-95.

Dermal regeneration with MilliGraft® kit of nanofat: the micrograft of adipose tissue. A clinical assessment study

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Dermal regeneration with MilliGraft® kit of nanofat: the micrograft of adipose tissue. A clinical assessment study

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