Antiseptics Influence the Proliferation of Adipose-Derived Stem Cells
Cell proliferation was evaluated by the bromodeoxyuridine assay (Fig. 4, above, left). Because a concentration of 7.5% showed the most remarkable difference in adipose-derived stem cell viability between the tested antiseptics, we selected this specific concentration for further study, including the bromodeoxyuridine incorporation assay. Similar to the alamarBlue assay, cell proliferation was reduced by all antiseptics and saline. Although saline, Lavasept, and mafenide acetate led to a modest decrease in proliferation, Prontosan, Betaisodona, and Octenisept reduced adipose-derived stem cell proliferation by more than 70 percent.
Antiseptics Influence the Apoptosis and Necrosis of Adipose-Derived Stem Cells
We investigated the effect of antiseptics on cell death by the annexin V assay, which allows for the distinction between early/late apoptosis and necrosis (Fig. 4, above, right and Table 3). We compared the ratio between necrosis and apoptosis (sum of early and late apoptosis) and calculated the difference in necrosis between the antiseptics (Table 3). All antiseptics caused increased adipose-derived stem cell necrosis compared with growth medium. Octenisept and Betaisodona both led to cell death mainly by necrosis. Prontosan resulted in a higher degree of cell death compared with Lavasept, mafenide acetate, and saline, which all induced comparable rates of necrosis and apoptosis.
Antiseptics Influence the Expression of Stem Cell Markers of Adipose-Derived Stem Cells
CD29, CD34, CD73, CD90, and CD105 mRNA expression was examined by qualitative real-time polymerase chain reaction after treatment with 7.5% solutions of each antiseptic, saline, medium, staurosporine, hydrogen peroxide, and ultraviolet light (Fig. 5). Although the expression of none of the markers was changed by saline, Betaisodona caused a significant down-regulation of all five stem cell markers (CD34 was not detectable). Octenisept also led to a down-regulation of all markers except for CD105. Lavasept and Prontosan showed a distinctive down-regulation of CD34 and moderate down-regulation of CD29. Mafenide acetate was the only antiseptic that did not influence the expression of any stem cell markers significantly. Staurosporine reduced only CD105 expression, whereas hydrogen peroxide reduced all stem cell markers.
Antiseptics Influence the Adipogenic Differentiation of Adipose-Derived Stem Cells
The adipogenic differentiation potential of adipose-derived stem cells was examined by Oil Red staining (Fig. 4, below). The low concentration of 0.05% of antiseptics did not lead to increased cell death as confirmed by the alamarBlue assay. All of the tested antiseptics reduced the adipogenic differentiation of adipose-derived stem cells, whereas saline did not have a significant effect. Under Lavasept, mafenide acetate, and Prontosan treatment, adipogenic differentiation was reduced by almost 40 percent, whereas Betaisodona and Octenisept led to a reduction of almost 80 percent.
Adipose-derived stem cells are pluripotent mesenchymal stem cells located in the adipose tissue and are competent to self-renew, proliferate, and differentiate into multiple cell lines.17 Adipose-derived stem cells promote wound repair by differentiation into keratinocytes and fibroblasts, secretion of growth factors, and stimulation of angiogenesis.14,16 Because of high yield and easy, safe harvest procedures, adipose-derived stem cells represent a true alternative to bone marrow–derived stem cells.22 Improved wound repair by treatment with adipose-derived stem cells has been repeatedly reported in a multitude of clinical studies, and interest in the regenerative properties of adipose-derived stem cells continues to increase.2 In this study, we have examined the effect of different chemical antiseptics on the viability, proliferation, cell death, expression of stem cell markers, and adipogenic differentiation of adipose-derived stem cells in vitro.
Octenisept is a standard antiseptic used in Europe, and is composed of the antiseptic agents octenidine dihydrochloride and phenoxyethanol. However, serositis after peritoneal lavage, severe subcutaneous edema with fatty tissue necrosis in pediatric patients, and chronic soft-tissue inflammation combined with tissue necrosis of the hand have been reported with its use.23–27 As a result, the manufacturer issued a warning for the use of Octenisept for the irrigation of deep wounds with high force. In our experiments, Octenisept reduced the viability of adipose-derived stem cells even when diluted to a 1% solution, whereas the other tested antiseptics showed marginal to no reduction of viability. Adipose-derived stem cell viability is dependent on cell proliferation, which also was significantly reduced by Octenisept. In contrast to apoptosis, which is a well-controlled form of cell death that may be inhibited by certain measures, Octenisept induces necrosis, an irreversible and therefore more detrimental form of cell death.28,29 Necrosis is followed by an inflammatory response, which may further perpetuate the critical condition of wound repair disorders.30 Octenisept also reduced the expression of the stem cell markers CD29, CD34, CD90, and CD105. The International Society for Cellular Therapy defined the minimal criteria of cultured mesenchymal stem cells, among others, by the expression of CD73, CD90, and CD105.31 CD29 and CD34 are additional surface markers that are used consistently to characterize mesenchymal stem cells.32,33 Staurosporine is a well-investigated inducer of apoptosis,34 whereas high concentrations of hydrogen peroxide induce necrosis.35 As the pattern of stem cell marker reduction by Octenisept was similar to that of hydrogen peroxide, necrosis may be the most feasible explanation for Octenisept-related stem cell marker reduction. The capability of adipogenic differentiation, which is a hallmark of adipose-derived stem cells and an important contributor to tissue regeneration, was significantly reduced by Octenisept and may be another deleterious factor associated with its use.17,36
Povidone-iodine is the main antiseptic reagent of Betaisodona.37 For irrigation, cleansing, and bathing, the manufacturer recommends dilutions of 1:2 to 1:100. Betaisodona showed the second highest reduction of adipose-derived stem cell viability after Octenisept. Betaisodona further reduced adipose-derived stem cell proliferation and differentiation. Betaisodona led to a marked decrease in the expression of all five stem cell markers, which may primarily reflect necrosis of adipose-derived stem cells.
Prontosan and Lavasept are both antiseptic solutions containing the antimicrobial agent polyhexanide.38 Prontosan wound irrigation solution is a ready-to-use antiseptic, whereas Lavasept is distributed as a liquid concentrate and its use is recommended in a 0.2% solution. The cytotoxicity of Prontosan and Lavasept was significantly weaker than that of Octenisept and Betaisodona. The stronger reduction of adipose-derived stem cell viability and proliferation, and the increase of necrosis, by Prontosan compared with Lavasept may be explained by the higher initial concentration, which was 0.1% polyhexanide compared with 0.04% polyhexanide in Lavasept. Importantly, CD34+ adipose-derived stem cells are described as being more proliferative and possessing higher stemness.33 Both antiseptics reduced the expression of CD34. As neither staurosporine nor hydrogen peroxide led to a similar isolated CD34 reduction, this effect may present a polyhexanide-specific effect.
Mafenide acetate is a short-acting sulfonamide that is provided as a powder. It readily penetrates burn eschar, where it retains its antimicrobial activity even in an acidic environment. Mafenide acetate showed the mildest effect on the viability, proliferation, and cell death of adipose-derived stem cells in our study. Adipose-derived stem cells survived treatment with mafenide acetate even at a concentration of 10% and 25%. In addition, none of the stem cell markers were significantly altered by mafenide acetate. Since its first use in World War II, mafenide acetate’s primary indication has been the antibacterial control of burn-related wounds/mesh grafts. However, in some cases, off-label application of mafenide acetate for the treatment of wounds not related to burns is possible.39,40 Bennett et al. demonstrated that mafenide acetate was a highly effective antiseptic in a porcine wound model.41 Other studies support the beneficial effect of mafenide acetate on partial-thickness and full-thickness wounds.42,43 In chronic wounds reaching the subcutaneous tissue and particularly Pseudomonas aeruginosa–colonized wounds, mafenide acetate may represent a genuine alternative to other antiseptics. Drawbacks of mafenide acetate, however, are its high costs and a requirement for use within 48 hours once the powder is reconstituted.44
We also examined the effect of saline on adipose-derived stem cell viability. Plain saline is commonly used in clinical practice to irrigate wounds, either pure or as a diluent for antibiotics and antiseptics. We have shown that treatment of adipose-derived stem cells with pure saline led to a slight reduction of cell viability and proliferation, and a small increase of cell death, but did not influence stem cell marker expression and adipogenic differentiation. However, its general effectiveness in the prevention of infections is still the subject of debate.45 In the context of adipose tissue and adipose-derived stem cell harvest for aesthetic or reconstructive purposes, the use of saline (e.g., to rinse adipose tissue) may be regarded as rather uncritical.
Aside from the toxicity of antiseptics, the antimicrobial potency is pivotal. Interestingly, Müller and Kramer found that Octenisept had superior efficacy against Escherichia coli and Staphylococcus aureus compared with Lavasept, and Betaisodona showed the weakest antimicrobial efficiency.10 Hirsch et al. also identified Betaisodona as the least effective antiseptic against S. aureus, Enterobacter faecalis, P. aeruginosa, and E. coli compared with Octenisept, Prontosan, and Lavasept, all of which showed similar efficiency.6 Rode et al. studied the antimicrobial efficiency of mafenide acetate and Betaisodona ointment in rats infected with P. aeruginosa and found that mafenide acetate was superior to Betaisodona.46 Bennett et al. also reported higher efficiency of mafenide acetate than povidone-iodine–based antiseptics in a porcine burn model.41 Collectively, Betaisodona may be the least effective antiseptic, as it shows high adipose-derived stem cell toxicity and relatively low antimicrobial efficiency. Octenisept, by contrast, shows high adipose-derived stem cell toxicity but also high germicidal activity at low concentrations so that a higher dilution may be advocated.
Not only adipose-derived stem cells but also other cells such as keratinocytes and fibroblasts contribute to wound repair. In keratinocytes, Betaisodona and Octenisept showed high cytotoxicity and significant reduction of proliferation, whereas Prontosan and Lavasept exerted only minor effects.6 For fibroblasts, controversial results were reported. Müller and Kramer observed the highest cytotoxicity with Octenisept, followed by Lavasept and Betaisodona,10 wheras Hirsch et al. showed substantial cytotoxic and antiproliferative effects of Betaisodona and Octenisept but no such effects of Prontosan and Lavasept.6 Higher cytotoxic effects on chondrocytes were observed for povidone-iodine–based antiseptics compared with Lavasept.47 Consequently, Octenisept and povidone-iodine–based antiseptics appear to be more detrimental to cells than polyhexanide-based antiseptics.
We have to acknowledge some limitations of our study. We have to concede that our data were collected from in vitro experiments. In vitro experiments are surely a valuable approach and a starting point for unraveling underlying mechanisms. Nonetheless, the effects of an in vivo environment where surrounding cells, extracellular matrix, active perfusion, and additional physiologic processes may facilitate clearance of antiseptics and counterregulate toxic effects are neglected. To translate our results into clinical practice (i.e., the treatment of deep wounds), additional studies are needed. Different in vivo wound models48 may be used to examine the systemic influences. The harvest and analysis of adipose tissue from patients treated with the different antiseptics may deliver more applicable data and better reflect the in vivo effect of antiseptics on wounds and may therefore be considered in future studies.
Mafenide acetate exerted the mildest toxic effects on human adipose-derived stem cells. Lavasept and Prontosan showed moderate toxicity and may be regarded as an alternative to mafenide acetate. Octenisept and Betaisodona, in contrast, reduced adipose-derived stem cell viability, proliferation, and differentiation significantly and caused considerable adipose-derived stem cell necrosis.
Bong-Sung Kim, M.D., is supported by “START,” a program for young scientists of the Medical Faculty at the RWTH Aachen University (project number 691346, START 2013-2) and the Research Fellowship Program of the German Research Foundation (Deutsche Forschungsgemeinschaft GZ: KI 1973/1-1). Norbert Pallua, M.D., Ph.D., is supported by Deutsche Forschungsgemeinschaft grant PA 1271/5-1. Jürgen Bernhagen, Ph.D., is supported by Deutsche Forschungsgemeinschaft grants SFB1123/P03, SFB/TRR57-P07, and DFG BE1977/7-1, and by the Deutsche Forschungsgemeinschaft within the framework of the Munich Cluster for Systems Neurology (EXC 1010 SyNergy). Richard Bucala, M.D., Ph.D., is supported by National Institutes of Health grant R01 AI042310.
1. Braiman-Wiksman L, Solomonik I, Spira R, Tennenbaum TNovel insights into wound healing sequence of events.Toxicol Pathol200735767–779
2. Hassan WU, Greiser U, Wang WRole of adipose-derived stem cells in wound healing.Wound Repair Regen201422313–325
3. Frank C, Bayoumi I, Westendorp CApproach to infected skin ulcers.Can Fam Physician2005511352–1359
4. Bowler PG, Duerden BI, Armstrong DGWound microbiology and associated approaches to wound management.Clin Microbiol Rev200114244–269
5. Daeschlein GAntimicrobial and antiseptic strategies in wound management.Int Wound J201310Suppl 19–14
6. Hirsch T, Koerber A, Jacobsen F, et alEvaluation of toxic side effects of clinically used skin antiseptics in vitro.J Surg Res2010164344–350
7. Thomas GW, Rael LT, Bar-Or R, et alMechanisms of delayed wound healing by commonly used antiseptics.J Trauma20096682–90; discussion 90
8. Kolbenschlag J, Goertz O, Behr B, Daigeler A, Lehnhardt M, Hirsch TSkin antiseptics in plastic surgery (in German).Handchir Mikrochir Plast Chir201244254–258
9. Müller G, Kramer AComparative study of in vitro cytotoxicity of povidone-iodine in solution, in ointment or in a liposomal formulation (Repithel) and selected antiseptics.Dermatology2006212Suppl 191–93
10. Müller G, Kramer ABiocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity.J Antimicrob Chemother2008611281–1287
11. Sugihara H, Toda S, Yonemitsu N, Watanabe KEffects of fat cells on keratinocytes and fibroblasts in a reconstructed rat skin model using collagen gel matrix culture.Br J Dermatol2001144244–253
12. Aoki S, Toda S, Ando T, Sugihara HBone marrow stromal cells, preadipocytes, and dermal fibroblasts promote epidermal regeneration in their distinctive fashions.Mol Biol Cell2004154647–4657
13. Lee SH, Jin SY, Song JS, Seo KK, Cho KHParacrine effects of adipose-derived stem cells on keratinocytes and dermal fibroblasts.Ann Dermatol201224136–143
14. Moon KM, Park YH, Lee JS, et alThe effect of secretory factors of adipose-derived stem cells on human keratinocytes.Int J Mol Sci2012131239–1257
15. Campbell CA, Cairns BA, Meyer AA, Hultman CSAdipocytes constitutively release factors that accelerate keratinocyte proliferation in vitro.Ann Plast Surg201064327–332
16. Kim WS, Park BS, Sung JH, et alWound healing effect of adipose-derived stem cells: A critical role of secretory factors on human dermal fibroblasts.J Dermatol Sci20074815–24
17. Zuk PA, Zhu M, Mizuno H, et alMultilineage cells from human adipose tissue: Implications for cell-based therapies.Tissue Eng20017211–228
18. Toyserkani NM, Christensen ML, Sheikh SP, Sørensen JAAdipose-derived stem cells: New treatment for wound healing?Ann Plast Surg201575117–123
19. Coleman SRStructural fat grafts: The ideal filler?Clin Plast Surg200128111–119
20. Grasys J, Kim BS, Pallua NContent of soluble factors and characteristics of stromal vascular fraction cells in lipoaspirates from different subcutaneous adipose tissue depots.Aesthet Surg J201636831–841
21. Hemmrich K, Kappel BA, Paul NE, et alAntipsychotic drugs increase adipose stem cell differentiation: Implications for treatment with antipsychotic drugs.J Clin Psychopharmacol201131663–665
22. Kern S, Eichler H, Stoeve J, Klüter H, Bieback KComparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.Stem Cells2006241294–1301
23. Hupuczi P, Papp ZPostoperative ascites associated with intraperitoneal antiseptic lavage.Obstet Gynecol20051051267–1268
24. Parlakgumus A, Baykal A, Aran OAn unusual cause of chemical peritonitis.Acta Chir Belg2005105322–323
25. Hülsemann W, Habenicht RSevere side effects after Octenisept irrigation of penetrating wounds in children (in German).Handchir Mikrochir Plast Chir200941277–282
26. Schupp CJ, Holland-Cunz SPersistent subcutaneous oedema and aseptic fatty tissue necrosis after using octenisept.Eur J Pediatr Surg200919179–183
27. Franz T, Vögelin EAseptic tissue necrosis and chronic inflammation after irrigation of penetrating hand wounds using Octenisept.J Hand Surg Eur Vol20123761–64
28. Kanduc D, Mittelman A, Serpico R, et alCell death: Apoptosis versus necrosis (review).Int J Oncol200221165–170
29. Wang WZ, Fang XH, Williams SJ, et alAnalysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.Plast Reconstr Surg201313177e–85e
30. Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis NCrosstalk between apoptosis, necrosis and autophagy.Biochim Biophys Acta201318333448–3459
31. Dominici M, Le Blanc K, Mueller I, et alMinimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy20068315–317
32. Davies OG, Cooper PR, Shelton RM, Smith AJ, Scheven BAIsolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation.J Tissue Eng201562041731415592356
33. Suga H, Matsumoto D, Eto H, et alFunctional implications of CD34 expression in human adipose-derived stem/progenitor cells.Stem Cells Dev2009181201–1210
34. Belmokhtar CA, Hillion J, Ségal-Bendirdjian EStaurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms.Oncogene2001203354–3362
35. Saito Y, Nishio K, Ogawa Y, et alTurning point in apoptosis/necrosis induced by hydrogen peroxide.Free Radic Res200640619–630
36. Schmidt BA, Horsley VIntradermal adipocytes mediate fibroblast recruitment during skin wound healing.Development20131401517–1527
37. Durani P, Leaper DPovidone-iodine: Use in hand disinfection, skin preparation and antiseptic irrigation.Int Wound J20085376–387
38. Egli-Gany D, Brill FH, Hintzpeter M, Andrée S, Pavel VEvaluation of the antiseptic efficacy and local tolerability of a polihexanide-based antiseptic on resident skin flora.Adv Skin Wound Care201225404–408
39. Breton DWOff-label drug use in WOC nursing: Issues related to use of mafenide acetate to treat infected chronic wounds.J Wound Ostomy Continence Nurs200128253–258
40. Barillo DJUsing mafenide acetate in acute and chronic wounds.Ostomy Wound Manage2002Suppl5–10
41. Bennett LL, Rosenblum RS, Perlov C, Davidson JM, Barton RM, Nanney LBAn in vivo comparison of topical agents on wound repair.Plast Reconstr Surg2001108675–687
42. Argamaso RV, Garcia A, Freiman M, Lewin ML, Bharati SEffect of sulfamylon acetate on wound healing.Plast Reconstr Surg197046282–286
43. Kjolseth D, Frank JM, Barker JH, et alComparison of the effects of commonly used wound agents on epithelialization and neovascularization.J Am Coll Surg1994179305–312
44. Ibrahim A, Fagan S, Keaney T, et alA simple cost-saving measure: 2.5% mafenide acetate solution.J Burn Care Res201435349–353
45. Mueller TC, Loos M, Haller B, et alIntra-operative wound irrigation to reduce surgical site infections after abdominal surgery: A systematic review and meta-analysis.Langenbecks Arch Surg2015400167–181
46. Rode H, de Wet PM, Davies MR, Cywes SAn experimental evaluation of the germicidal efficacy of three topical antimicrobial agents in burns.Prog Pediatr Surg198114189–208
47. Schaumburger J, Beckmann J, Springorum HR, et alToxicity of antiseptics on chondrocytes in vitro (in German).Z Orthop Unfall201014839–43
48. Ansell DM, Campbell L, Thomason HA, Brass A, Hardman MJA statistical analysis of murine incisional and excisional acute wound models.Wound Repair Regen201422281–287
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