Hrev_master Veins and Lymphatics 2017; volume 6:6814 [Veins and Lymphatics 2017; 6:6814] [page 51] Technique for intraoperatory harvesting of adipose derived stem cells: towards cell treatment of recalcitrant ulcers Ilaria Zollino,1 Maria Grazia Sibilla,2 Sergio Gianesini,1,2 Erica Menegatti,1,2 Mirko Tessari,1,2 Anna Maria Malagoni2 1Department of Morphology, Surgery and Experimental Medicine, Section of Translational of Medicine and Surgery, University of Ferrara; 2Sant’Anna University Hospital, Unit of Translational Surgery and Vascular Diseases Center, Ferrara, Italy Abstract Successful wound and ulcer repair remains a major biomedical challenge in the 21st Century. Innovative and alternative treatment options have been investigated over the last decade and stem cells applica- tion has been suggested as a possible novel therapy for regenerative medicine. In partic- ular, stem cells derived from adipose tissue have been attracting a lot of attention in recent years as an alternative to the use of cells derived from bone marrow. This tech- nical note describes the procedure intro- duced by Coleman for intraoperatory har- vesting of adipose derived-stem-cells and proposes a rationale for using it in difficult wound healing and recalcitrant ulcers. Introduction Stem cell technology is increasingly being employed to treat tissue and organs in patients. Surgeons are keen to transfer these treatment methods to other applications such as ulcer and wound healing. The use of adipose-derived-stem cells (ADSCs) appeals to surgeons due to the relative ease of locating sites for harvesting, the harvest- ing process and the amount of material available for use. ADSCs also offer several desirable factors including their osteogenic and chondrogenic potential, enhancement of angiogenesis, limitation of apoptosis and immunomodulatory function.1 Due to these advantageous characteris- tics, ADSCs can be explored as a promising tool to repair recalcitrant ulcers and difficult wounds in those cases where conventional treatments have failed. Although numerous promising stem cell approaches are advanc- ing in clinical trials, intraoperative stem cell therapies using ADSCs offer more immedi- ate benefits, by integrating an autologous cell source with a well-established surgical intervention in a single, safe procedure. Coleman’s technique ADSCs are generally extracted from adipose tissue using a multiple stepwise procedure. Coleman’s technique2 is both the first and the most popular method for tissue regeneration:3,4 it provides aspiration, cen- trifugation and the subsequent re-injection of autologous fat. First, Kleine’s solution made up of 250 mL normal saline, 20 mL of 1% carbocaine, 1 mL adrenaline and 2 ml bicarbonate is injected into the fat donor area.5 After diffu- sion of the solution the harvesting proce- dure is undertaken, using a two-hole blunt cannula [Byron Medical (a division of Mentor Corporation), Tucson, AZ, USA] fitted directly to a 10 mL Luer Lock syringe (BD Syringe Luer-Lok tip; Becton Dickinson, Franklin Lakes, NJ, USA), which helps to reduce the pressure generat- ed during the harvesting procedure and pre- serves the fat parcels.6 Following the fat harvesting, the lipoaspirate is processed via centrifugation of the lipoaspirate at 3000 rpm (rotor size: 16 cm; g force: 580) for 3 min in 10 mL syringes. This separates the fat into 3 layers. The upper layer and lower layer are discarded, as they are composed of oil from destroyed fat and blood respective- ly, leaving the middle layer, which contains a high concentration of stem cells7,8 and the stromal vascular fraction. Once the middle layer has been extract- ed, the micro-fat graft is transferred into 1ml syringes prior to injection into the tis- sue to be grafted (Figure 1). A multilayer technique is used to implant the aliquots of fat, with very small amounts of fatty tissue released into the recipient area in order to optimize the successful implantation of the graft. A blunt Coleman microcannula is used to deposit the micro-fat graft via a number of sub dermal and hypodermal tun- nels through numerous tissue planes. This technique of minimising the amount of micro-fat graft released with each introduc- tion of the cannula increases the surface area between the grafted fat and the tissue receiving it. In this way there is a reduction in fat damage and adipocyte necrosis, with an improvement in graft vascularization and three-dimensional fat distribution. The recently grafted fat has a readily available blood supply, which facilitates its survival and reduces the possibility of fat necrosis or calcification.9 Chronic venous leg ulceration and the potential role of adipose derived-stem-cells Chronic venous leg ulceration (CVU) occurs in about 10% of cases of chronic venous disease (CVD).10 Although mainly caused due to superficial venous insuffi- ciency, CVU becomes more prevalent when associated with other health issues such as diabetes, malnutrition, rheumatoid arthritis, chronic anaemia or second stage peripheral arterial disease.11,12 CVU can be treated through surgery or through a combination of compression, wound-care and debridement. Two ran- domised control trials have evaluated the healing rate and risk of ulcer recurrence fol- lowing superficial venous surgery. The long-term randomized study conducted by Zamboni et al.13 corroborates the effective- ness of minimally invasive surgical haemo- dynamic correction of reflux (CHIVA) for leg ulceration secondary to superficial venous reflux. Follow-up after a 3 year peri- od revealed a 100% healing rate at 31 days and a recurrence rate of 9% in those patients who received surgery, compared to the compression only group with a healing rate of 96% after 31 days and a recurrence rate of 38%. The ESCHAR study conducted by Correspondence: Ilaria Zollino, Department of Morphology, Surgery and Experimental Medicine, Section of Translational of Medicine and Surgery, University of Ferrara, via Aldo Moro 8, Ferrara, Italy. E-mail: zlll- ri@unife.it Key words: Coleman’s technique; adipose stem cells; recalcitrant ulcers. Contributions: IZ, manuscript design, con- ceiving and preparation; MGS, manuscript design, pictures collection; SG, manuscript design; EM, manuscript drafting; MT, manu- script drafting; AMM, manuscript drafting and conceiving. Conflict of interest: the authors declare no potential conflict of interest. Received for publication: 24 May 2017. Revision received: 15 June 2017. Accepted for publication: 17 June 2017. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright I. Zollino et al., 2017 Licensee PAGEPress, Italy Veins and Lymphatics 2017; 6:6814 doi:10.4081/vl.2017.6814 No n c om me rci al us e o nly How I do it Barwell et al.14 also advocates simple venous surgery as a benefit to patients with chronic venous ulceration. It assessed the ulcer recurrence rate as significantly reduced in its compression and superficial venous surgery group at 12%, compared to 28% recurrence in the compression alone group. However, a significant proportion of recalcitrant ulcers respond to treatment but do not heal.15-18 In these patients, advanced dressings and skin grafts are generally used to achieve healing21 and new regenerative approaches including the use of ADSCs have been proposed.19 The positive effect of ADSCs is due to an increase in vascularization, which is fun- damental to the healing process.20,21 ADSCs are able to release angiogenic factors, and have shown increased angiogenesis in wound healing when injected or delivered via a scaffold.22 Treatment is customised for each patient and requires a clean and healthy wound bed with no devitalised tis- sue and carefully managed protease levels.23 Debridement and application of adipose derived-stem-cells in the wound bed There are few reports about the treat- ment of venous leg ulcers (VLUs) using ADSCs. However, ADSCs have shown a positive impact on wounds healing in pre- clinical and clinical studies. Recent ADSCs applications in vitro and in vivo have demonstrated that they are attracted to the wound site and affect regeneration process- es by means of paracrine mechanisms in addition to fusion and differentiation, for instance, into keratinocytes or dermal fibrobasts.24-27 Promising applications in wounds and ulcers healing have been reported, although thus far these are small studies with a total of only 98 patients.28-32 Bartsich and Morrison33 discussed the long- term treatment of chronic sickle cell ulcers and the possible use of a skin graft and fat grafting to achieve healing. Current treat- ment using skin grafting and local wound care is often unsuccessful long-term, as wounds that have healed break down again. So, treatment involving permanent alter- ation of the wound bed, with recruitment of a new cell population and subsequent fat grafting, was applied. Our group was attracted by first reports on application of cell therapy in unhealed ulcers, and performed preliminary applica- tion in selected cases. In our procedure, we first perform a detailed wound bed prepara- tion following this sequence. Prior to the Figure 1. Coleman’s technique for harvesting adipose derived-stem-cells (ADSCs). In this particular case the harvesting is performed in the periumbilical area after tumescent local anaesthesia infiltration (A); the adipose tissue is centrifuged in order to separate the dif- ferent components (B); the samples are separated into: an upper layer of yellow oil, a mid- dle layer composed of adipose tissue with high concentration of stem cells and a bottom layer of blood (C); after oil (D) and blood (E) elimination the ADSCs are aspirated by Luer-Lock syringe technique (F). [page 52] [Veins and Lymphatics 2017; 6:6814] No n c om me rci al us e o nly How I do it procedure, the wound area is sanitized using Chlorexidine, then debridement of ulcers is performed by the means of Versajet device (VERSAJET™, Versajet Hydrosurgery System, Smith and Nephew, Hull, UK), a debridement tool able to cut and remove necrotic tissue and fibrin based on a high pressure jet of water which produces the Venturi effect.34 We emphasize here that wound bed preparation needs to be meticu- lous before implantation: the microenviron- ment for wound regeneration mainly depends on interactions between stem cell progenitors and their niche, and it provides proliferation and differentiation of the cells.35 After this stringent preparation of the wound bed, it is possible to apply Coleman’s Technique, as described above. The suspension of ADSCs is injected into the wound bed and the wound margins; a scaffold of commercial hyaluronic acid is positioned on the wound bed in order to cover the implanted cells. Finally, advanced foam dressing protects the healing area before the application of compression. Centrifugation according to Coleman’s procedure creates a graded density of fat with varying characteristics that influence lipoaspirate persistence, properties, and quality.36 Compared with other fat-non pro- cessing methods, centrifugation clears the fat from most blood remnants and shows the highest concentration of mesenchymal stem cells.37 It is safe and feasible, does not impair cell viability and can augment ADSCs content.38 The inoculation of these non manipulated cells present in the lower centrifuged high-density layer (HDL) could accelerate the healing of recalcitrant venous and mixed leg ulcers and could be used for the regeneration of large defects: the HDL displays the highest expression of mes- enchymal stem cells and endothelial mark- ers, such as vascular endothelial growth fac- tor A (VEGFA), indicating the larger vascu- lar potential of the cells in this layer (Figures 2 and 3).36 Moreover, in presence of specific growth factors, stem cells pres- ent in the HDL can differentiate into several lineages, including osteogenic, adipogenic, condrogenic, neuronal, glial, and endothe- lial lineages. Conclusions As a treatment which is based on a widely recognised, safe procedure, intraop- erative ADSCs therapy using Coleman’s technique provides a new alternative and potentially regenerative approach to the clinical challenges of recalcitrant ulcers and difficult wound healing. This sur- Figure 2. Pre-operative Martorell’s ulcer (A); intraoperative injection of adipose derived- stem-cells (B) post-operative leg ulcer after 4 weeks of treatment (C) and after 8 weeks of treatment (D). Figure 3. Pre-operative venous leg ulcer over the medial malleolus (A) and over the lateral malleolus (B); post-operative leg ulcer after 14 weeks of treatment (C-D). [Veins and Lymphatics 2017; 6:6814] [page 53] A B C D No n c om me rci al us e o nly How I do it [page 54] [Veins and Lymphatics 2017; 6:6814] gical intervention in one single, safe proce- dure could become an alternative treat- ment in selected patients, with promising results and future potential. However, at present, there are neither standardised pro- tocols for the clinical application of ADSCs in VLUs nor a consensus on the number of cells (present in the HDL) necessary for dif- ferent therapeutic options. Therefore, stan- dardised protocols and larger randomised controlled trials are indispensable to ensure the safety and effectiveness of ADSCs in VLUs application. References 1. Griffin M, Kalaskar DM, Butler PE, Seifalian AM. The use of adipose stem cells in cranial facial surgery. Stem Cell Rev 2014;10:671-85. 2. Pu LL, Coleman SR, Cui X, et al. Autologous fat grafts harvested and refined by the Coleman technique: a comparative study. Plast Reconstr Surg 2008;122:932-7. 3. Coleman SR. Facial recontouring with lipostructure. Clin Plast Surg 1997;24:347-67. 4. Philips BJ, Marra KG, Rubin JP. Healing of grafted adipose tissue: cur- rent clinical applications of adipose- derived stem cells for breast and face reconstruction. Wound Repair Regen 2014;1:11-3. 5. Wang G, Cao WG, Li SL. Safe exten- sive tumescent liposuction with seg- mental infiltration of lower concentra- tion lidocaine under monitored anesthe- sia care. Ann Plast Surg 2015;74:6-11. 6. Mojallal A, Auxenfans C, Lequeux C, et al. Influence of negative pressure when harvesting adipose tissue on cell yield of the stromal-vascular fraction. Biomed Mater Eng 2008;18:193-7. 7. Clauser L, Ferroni L, Gardin C, et al. Selective augmentation of stem cell populations in structural fat grafts for maxillofacial surgery. PLoS One 2014;9:e110796. 8. Gardin C, Bressan E, Ferroni L, et al. In vitro concurrent endothelial and osteogenic commitment of adipose- derived stem cells and their genomical analyses through comparative genomic hybridization array: novel strategies to increase the successful engraftment of tissue-engineered bone grafts. Stem Cells and Dev 2012;21:767-77. 9. Clauser LC, Consorti G, Tieghi R et al. Three-dimensional volumetric restora- tion by structural fat grafting. Craniomaxillofac Trauma Reconstr 2014;7:63-70. 10. Kelechi TJ, Johnson JJ, Yates S. Chronic venous disease and venous leg ulcers: An evidence-based update. J Vasc Nurs 2015;33:36-46. 11. Nüllen H, Noppeney T. [Diagnosis and treatment of varicose veins. Part 1: def- inition, epidemiology, etiology, classifi- cation, clinical aspects, diagnostic and indications]. Chirurg 2010;81:1035-44. 12. Gemmati D, Federici F, Catozzi L, et al. DNA-array of gene variants in venous leg ulcers: detection of prognostic indi- cators. J Vasc Surg 2009;50:1444-51. 13. Zamboni P, Cisno C, Marchetti F, et al. Minimally invasive surgical manage- ment of primary venous ulcers vs. com- pression treatment: a long-term ran- domized study. Eur J Vasc Endovasc Surg 2003;25:313-8. 14. Barwell JR, Davies CE, Deacon J, et al. Comparison of surgery and compres- sion with compression alone in chronic venous ulceration (ESCHAR study): randomised controlled trial. Lancet 2004;363:1854-9. 15. Thomas CA, Holdstock JM, Harrison CC, et al. Healing rates following venous surgery for chronic venous leg ulcers in an independent specialist vein unit. Phlebology 2013;28:132-9. 16. Marston WA, Crowner J, Kouri A, et al. Incidence of venous leg ulcer healing and recurrence after treatment with endovenous laser ablation. J Vasc Surg May 2017 [Epub ahead of print]. 17. van Gent WB, Hop WC, van Praag MC, et al. Conservative versus surgical treat- ment of venous leg ulcers: a prospec- tive, randomized, multicenter trial. J Vasc Surg 2006;44:563-71. 18. Mauck KF, Asi N, Undavalli C, et al. Systematic review and meta-analysis of surgical interventions versus conserva- tive therapy for venous ulcers. J Vasc Surg 2014;60:60S-70S.e1-2. 19. Valle MF, Maruthur NM, Wilson LM, et al. Comparative effectiveness of advanced wound dressings for patients with chronic venous leg ulcers: a sys- tematic review. Wound Repair Regen 2014;22:193-204. 20. Yolanda MM, Maria AV, Amaia FG, et al. Adult stem cell therapy in chronic wound healing. J Stem Cell Res Ther 2014;4:162. 21. Fraser JK, Wulur I, Alfonso Z, Hedrick MH. Fat tissue: an underappreciated source of stem cells for biotechnology. Trends Biotechnol 2006;24:150-4. 22. King A, Balaji S, Keswani SG, Crombleholme TM. The role of stem cells in wound angiogenesis. Adv Wound Care (New Rochelle) 2014;3:614-25. 23. Senet P. [Cellular therapy and leg ulcers: Future approaches]. Ann Dermatol Venereol 2015;142:519-22. 24. Nambu M, Kishimoto S, Nakamura S, et al. Accelerated wound healing in healing-impaired db/db mice by autolo- gous adipose tissue-derived stromal cells combined with atelocollagen matrix. Ann Plast Surg 2009;62:317-21. 25. Ebrahimian TG, Pouzoulet F, Squiban C, et al. Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing. Arterioscler Thromb Vasc Biol 2009;29:503-10. 26. Cho HH, Kyoung KM, Seo MJ, et al. Overexpression of CXCR4 increases migration and proliferation of human adipose tissue stromal cells. Stem Cells Dev 2006;15:853-64. 27. Zollino I, Zuolo M, Gianesini S, et al. Autologous adipose-derived stem cells: basic science, technique, and rationale for application in ulcer and wound heal- ing. Phlebology 2017;32:160-71. 28. Cervelli V, De Angelis B, Lucarini L, et al. Tissue regeneration in loss of sub- stance on the lower limbs through use of platelet-rich plasma, stem cells from adipose tissue, and hyaluronic acid. Adv Skin Wound Care 2010;23:262-72. 29. Cervelli V, Gentile P, De Angelis B, et al. Application of enhanced stromal vascular fraction and fat grafting mixed with PRP in post-traumatic lower extremity ulcers. Stem cell research 2011;6:103-11. 30. Marino G, Moraci M, Armenia E, et al. Therapy with autologous adipose- derived regenerative cells for the care of chronic ulcer of lower limbs in patients with peripheral arterial disease. J Surg Res 2013;185:36-44. 31. Bura A, Planat-Benard V, Bourin P, et al. Phase I trial: the use of autologous cultured adipose-derived stroma/stem cells to treat patients with non-revascu- larizable critical limb ischemia. Cytotherapy 2014;16:245-57. 32. Lee HC, An SG, Lee HW, et al. Safety and effect of adipose tissue-derived stem cell implantation in patients with critical limb ischemia: a pilot study. Circ J 2012;76:1750-60. 33. Bartsich S, Morrison N. Composite fat and skin grafting for the management of chronic sickle cell ulcers. Wounds 2012;24:51-4. 34. Fraccalvieri M, Serra R, Ruka E, et al. Surgical debridement with VERSAJET: an analysis of bacteria load of the wound bed pre- and post-treatment and skin graft taken. A preliminary pilot No n c om me rci al us e o nly How I do it [Veins and Lymphatics 2017; 6:6814] [page 55] study. Int Wound J 2011;8:155-61. 35. Wong VW, Levi B, Rajadas J, et al. Stem cell niches for skin regeneration. Int J Biomater 2012:926059. 36. Clauser L, Ferroni L, Gardin C, et al. Selective augmentation of stem cell populations in structural fat grafts for maxillofacial surgery. PLoS One 2014;9:e110796. 37. Condé-Green A, de Amorim NGF, Pitanguy I. Influence of decantation, washing, and centrifugation of adipocyte and mesenchymal stem cell content of aspirated adipose tissue: a comparative study. J Plast Reconstr Aesthet Surg 2010;63:1375-81. 38. Ibatici A, Caviggioli F, Valeriano V, et al. Comparison of cell number, viabili- ty, phenotypic profile, clonogenic, and proliferative potential of adipose- derived stem cell populations between centrifuged and noncentrifuged fat. Aesthetic Plast Surg 2014;38:985-93. No n c om me rci al us e o nly