Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CMADSC) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CMADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CMADSC. Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell–derived approach to TM wound healing.
Get full access to this article
View all access options for this article.
References
1.
GurtnerGC, WernerS, BarrandonY and LongakerMT. (2008). Wound repair and regeneration. Nature, 453:314–321.
2.
MoleDR, BlancherC, CopleyRR, PollardPJ, GleadleJM, RagoussisJ and RatcliffePJ. (2009). Genome-wide association of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible transcripts. J Biol Chem, 284:16767–16775.
3.
AraganeY, YamadaH, SchwarzA, PoppelmannB, LugerTA, TezukaT and SchwarzT. (1996). Transforming growth factor-alpha induces interleukin-6 in the human keratinocyte cell line HaCaT mainly by transcriptional activation. J Invest Dermatol, 106:1192–1197.
4.
Santa MariaPL, AtlasMD and GhassemifarR. (2007). Chronic tympanic membrane perforation: a better animal model is needed. Wound Repair Regen, 15:450–458.
5.
Santa MariaPL, RedmondSL, AtlasMD and GhassemifarR. (2010). Histology of the healing tympanic membrane following perforation in rats. Laryngoscope, 120:2061–2070.
6.
MakinoK, AmatsuM, KinishiM and MohriM. (1990). Epithelial migration in the healing process of tympanic membrane perforations. Eur Arch Otorhinolaryngol, 247:352–355.
7.
KaftanH, HerzogM, MieheB and HosemannW. (2006). Topical application of transforming growth factor-beta1 in acute traumatic tympanic membrane perforations: an experimental study in rats. Wound Repair Regen, 14:453–456.
8.
RahmanA, OliviusP, DirckxJ, Von UngeM and HultcrantzM. (2008). Stem cells and enhanced healing of chronic tympanic membrane perforation. Acta Otolaryngol, 128:352–359.
9.
GoncalvesS, BasE, GoldsteinBJ and AngeliS. (2016). Effects of cell-based therapy for treating tympanic membrane perforations in mice. Otolaryngol Head Neck Surg, 154:1106–1114.
10.
KimWS, ParkBS, SungJH, YangJM, ParkSB, KwakSJ and ParkJS. (2007). Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci, 48:15–24.
11.
WeilBR, MarkelTA, HerrmannJL, AbarbanellAM and MeldrumDR. (2009). Mesenchymal stem cells enhance the viability and proliferation of human fetal intestinal epithelial cells following hypoxic injury via paracrine mechanisms. Surgery, 146:190–197.
12.
JavazonEH, KeswaniSG, BadilloAT, CrombleholmeTM, ZoltickPW, RaduAP, KozinED, BeggsK, MalikAA and FlakeAW. (2007). Enhanced epithelial gap closure and increased angiogenesis in wounds of diabetic mice treated with adult murine bone marrow stromal progenitor cells. Wound Repair Regen, 15:350–359.
13.
GnecchiM, ZhangZ, NiA and DzauVJ. (2008). Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res, 103:1204–1219.
14.
LauK, PausR, TiedeS, DayP and BayatA. (2009). Exploring the role of stem cells in cutaneous wound healing. Exp Dermatol, 18:921–933.
15.
HockingAM and GibranNS. (2010). Mesenchymal stem cells: paracrine signaling and differentiation during cutaneous wound repair. Exp Cell Res, 316:2213–2219.
16.
WalterMN, WrightKT, FullerHR., MacNeilS and JohnsonWE. (2010). Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell Res, 316:1271–1281.
17.
WuY, ChenL, ScottPG and TredgetEE. (2007). Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells, 25:2648–2659.
18.
MatsudaK, FalkenbergKJ, WoodsAA, ChoiYS, MorrisonWA and DilleyRJ. (2013). Adipose-derived stem cells promote angiogenesis and tissue formation for in vivo tissue engineering. Tissue Eng Part A, 19:1327–1335.
19.
ChenL, TredgetEE, WuPY and WuY. (2008). Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One, 3:e1886.
SpandowO, HellstromS and DahlstromM. (1996). Structural characterization of persistent tympanic membrane perforations in man. Laryngoscope, 106(Pt 1):346–352.
22.
RedmondSL, LevinB, HeelKA, AtlasMD and MaranoRJ. (2011). Phenotypic and genotypic profile of human tympanic membrane derived cultured cells. J Mol Histol, 42:15–25.
23.
ReimandJ, KullM, PetersonH, HansenJ and ViloJ. (2007). g:Profiler—a web-based toolset for functional profiling of gene lists from large-scale experiments. Nucleic Acids Res, 35:W193–W200.
24.
TehBM, RedmondSL, ShenY, AtlasMD, MaranoRJ and DilleyRJ. (2013). TGF-alpha/HA complex promotes tympanic membrane keratinocyte migration and proliferation via ErbB1 receptor. Exp Cell Res, 319:790–799.
25.
AndoY and JensenPJ. (1993). Epidermal growth factor and insulin-like growth factor I enhance keratinocyte migration. J Invest Dermatol, 100:633–639.
26.
ClymerMA, SchwaberMK and DavidsonJM. (1996). The effects of keratinocyte growth factor on healing of tympanic membrane perforations. Laryngoscope, 106(Pt 1):280–285.
27.
HaaseI, EvansR, PofahlR and WattFM. (2003). Regulation of keratinocyte shape, migration and wound epithelialization by IGF-1- and EGF-dependent signalling pathways. J Cell Sci, 116(Pt 15):3227–3238.
28.
TehBM, MaranoRJ, ShenY, FriedlandPL, DilleyRJ and AtlasMD. (2013). Tissue engineering of the tympanic membrane. Tissue Eng Part B Rev, 19:116–132.
29.
YuanF, LeiYH, FuXB, ShengZY, CaiS, and SunTZ. (2008). Promotive effect of adipose-derived stem cells on the wound model of human epidermal keratinocytes in vitro [in Chinese]. Zhonghua Wai Ke Za Zhi, 46:1575–1578.
30.
AltmanAM, YanY, MatthiasN, BaiX, RiosC, MathurAB, SongYH and AltEU. (2009). IFATS collection: human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. Stem Cells, 27:250–258.
31.
LeeSH, LeeJH and ChoKH. (2011). Effects of human adipose-derived stem cells on cutaneous wound healing in nude mice. Ann Dermatol, 23:150–155.
32.
HuangSP, HsuCC, ChangSC, WangCH, DengSC, DaiNT, ChenTM, ChanJY, ChenSG and HuangSM. (2012). Adipose-derived stem cells seeded on acellular dermal matrix grafts enhance wound healing in a murine model of a full-thickness defect. Ann Plast Surg, 69:656–662.
33.
LeeSH, JinSY, SongJS, SeoKK and ChoKH. (2012). Paracrine effects of adipose-derived stem cells on keratinocytes and dermal fibroblasts. Ann Dermatol, 24:136–143.
34.
KinnairdT, StabileE, BurnettMS, LeeCW, BarrS, FuchsS and EpsteinSE. (2004). Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res, 94:678–685.
35.
AndrikopoulouE, ZhangX, SebastianR, MartiG, LiuL, MilnerSM and HarmonJW. (2011). Current Insights into the role of HIF-1 in cutaneous wound healing. Curr Mol Med, 11:218–235.
36.
HongWX, HuMS, EsquivelM, LiangGY, RennertRC, McArdleA, PaikKJ, DuscherD, GurtnerGC, LorenzHP and LongakerMT. (2014). The role of hypoxia-inducible factor in wound healing. Adv Wound Care (New Rochelle), 3:390–399.
37.
AhluwaliaA and TarnawskiAS. (2012). Critical role of hypoxia sensor—HIF-1alpha in VEGF gene activation. Implications for angiogenesis and tissue injury healing. Curr Med Chem, 19:90–97.
38.
SemenzaGL, JiangBH, LeungSW, PassantinoR, ConcordetJP, MaireP and GiallongoA. (1996). Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol Chem, 271:32529–32537.
39.
MylotteLA, DuffyAM, MurphyM, O'BrienT, SamaliA, BarryF and SzegezdiE. (2008). Metabolic flexibility permits mesenchymal stem cell survival in an ischemic environment. Stem Cells, 26:1325–1336.
40.
RehmanJ, TraktuevD, LiJ, Merfeld-ClaussS, Temm-GroveCJ, BovenkerkJE, PellCL, JohnstoneBH, ConsidineRV and MarchKL. (2004). Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation, 109:1292–1298.
41.
HuLF, PanTT, NeoKL, YongQC and BianJS. (2008). Cyclooxygenase-2 mediates the delayed cardioprotection induced by hydrogen sulfide preconditioning in isolated rat cardiomyocytes. Pflugers Arch, 455:971–978.
42.
MirotsouM, JayawardenaTM, SchmeckpeperJ, GnecchiM and DzauVJ. (2011). Paracrine mechanisms of stem cell reparative and regenerative actions in the heart. J Mol Cell Cardiol, 50:280–289.
43.
HakubaN, TaniguchiM, ShimizuY, SugimotoA, ShinomoriY and GyoK. (2003). A new method for closing tympanic membrane perforations using basic fibroblast growth factor. Laryngoscope, 113:1352–1355.
44.
AlzahraniM and SalibaI. (2015). Hyaluronic acid fat graft myringoplasty vs fat patch fat graft myringoplasty. Eur Arch Otorhinolaryngol, 272:1873–1877.
45.
Santa MariaPL, RedmondSL, AtlasMD and GhassemifarR. (2011). Keratinocyte growth factor 1, fibroblast growth factor 2 and 10 in the healing tympanic membrane following perforation in rats. J Mol Histol, 42:47–58.
46.
KanemaruS, UmedaH, KitaniY, NakamuraT, HiranoS and ItoJ. (2011). Regenerative treatment for tympanic membrane perforation. Otol Neurotol, 32:1218–1223.
47.
LouZC and WangYBZ. (2013). Healing outcomes of large (>50%)traumatic membrane perforations with inverted edges following no intervention, edge approximation and fibroblast growth factor application; a sequential allocation, three-armed trial. Clin Otolaryngol, 37:446–451.
48.
HsiaoST, LokmicZ, PeshavariyaH, AbbertonKM, DustingGJ, LimSY and DilleyRJ. (2013). Hypoxic conditioning enhances the angiogenic paracrine activity of human adipose-derived stem cells. Stem Cells Dev, 22:1614–1623.
49.
ChoKS, LeeDG, ShinDH, ParkYD and ChonKM. (2010). The importance of vascular endothelial growth factor in the healing of acute tympanic membrane perforation. Am J Otolaryngol, 31:309–314.
50.
MaxsonS, LopezEA, YooD, Danilkovitch-MiagkovaA and LerouxMA. (2012). Concise review: role of mesenchymal stem cells in wound repair. Stem Cells Transl Med, 1:142–149.
51.
RosenbergerP, SchwabJM, MirakajV, MasekowskyE, MagerA, Morote-GarciaJC, UnertlK and EltzschigHK. (2009). Hypoxia-inducible factor-dependent induction of netrin-1 dampens inflammation caused by hypoxia. Nat Immunol, 10:195–202.
52.
Santa MariaPL, RedmondSL, McInnesRL, AtlasMD and GhassemifarR. (2011). Tympanic membrane wound healing in rats assessed by transcriptome profiling. Laryngoscope, 121:2199–2213.
53.
DursunE., DogruS, GungorA, CincikH, PoyrazogluE and OzdemirT. (2008). Comparison of paper-patch, fat, and perichondrium myringoplasty in repair of small tympanic membrane perforations. Otolaryngol Head Neck Surg, 138:353–356.
54.
SalibaI, AlzahraniM, ZhuT and ChemtobS. (2014). Growth factors expression in hyaluronic acid fat graft myringoplasty. Laryngoscope, 124:E224–E230.
55.
MadlenerM, ParksWC and WernerS. (1998). Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair. Exp Cell Res, 242:201–210.
56.
LegrandC, GillesC, ZahmJM, PoletteM, BuissonAC, KaplanH, BirembautP and TournierJM. (1999). Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling. J Cell Biol, 146:517–529.
57.
MakelaM, LarjavaH, PirilaE, MaisiP, SaloT, SorsaT and UittoVJ. (1999). Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes. Exp Cell Res, 251:67–78.
58.
MohanR, ChintalaSK, JungJC, VillarWV, McCabeF, RussoLA, LeeY, McCarthyBE, WollenbergKR, et al. (2002). Matrix metalloproteinase gelatinase B (MMP-9) coordinates and effects epithelial regeneration. J Biol Chem, 277:2065–2072.
59.
WernerS, BreedenM, HubnerG, GreenhalghDG and LongakerMT. (1994). Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse. J Invest Dermatol, 103:469–473.
60.
WilgusTA, MatthiesAM, RadekKA, DoviJV, BurnsAL, ShankarR and DiPietroLA. (2005). Novel function for vascular endothelial growth factor receptor-1 on epidermal keratinocytes. Am J Pathol, 167:1257–1266.
61.
BrownLF, YeoKT, BerseB, YeoTK, SengerDR, DvorakHF and van de WaterL. (1992). Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med, 176:1375–1379.
62.
DetmarM, BrownLF, BerseB, JackmanRW, ElickerBM, DvorakHF and ClaffeyKP. (1997). Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin. J Invest Dermatol, 108:263–268.
63.
SenCK, KhannaS, BabiorBM, HuntTK, EllisonEC and RoyS. (2002). Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing. J Biol Chem, 277:33284–33290.
64.
QiL, HigginsSP, LuQ, SamarakoonR, Wilkins-PortCE, YeQ, HigginsCE, Staiano-CoicoL and HigginsPJ. (2008). SERPINE1 (PAI-1) is a prominent member of the early G0—> G1 transition “wound repair” transcriptome in p53 mutant human keratinocytes. J Invest Dermatol, 128:749–753.
65.
Keski-OjaJ and KoliK. (1992). Enhanced production of plasminogen activator activity in human and murine keratinocytes by transforming growth factor-beta 1. J Invest Dermatol, 99:193–200.
66.
SimoneTM, LongmateWM, LawBK and HigginsPJ. (2015). Targeted inhibition of PAI-1 activity impairs epithelial migration and wound closure following cutaneous injury. Adv Wound Care (New Rochelle), 4:321–328.
67.
SimoneTM, HigginsCE, CzekayRP, LawBK, HigginsSP, ArchambeaultJ, KutzSM and HigginsPJ. (2014). SERPINE1: a molecular switch in the proliferation-migration dichotomy in wound-“activated” keratinocytes. Adv Wound Care (New Rochelle), 3:281–290.
68.
ProvidenceKM, HigginsSP, MullenA, BattistaA, SamarakoonR, HigginsCE, Wilkins-PortCE and HigginsPJ. (2008). SERPINE1 (PAI-1) is deposited into keratinocyte migration “trails” and required for optimal monolayer wound repair. Arch Dermatol Res, 300:303–310.
69.
GrossmanRM, KruegerJ, YourishD, Granelli-PipernoA, MurphyDP, MayLT, KupperTS, SehgalPB and GottliebAB. (1989). Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes. Proc Natl Acad Sci U S A, 86:6367–6371.
70.
YoshizakiK, NishimotoN, MatsumotoK, TagohH, TagaT, DeguchiY, KuritaniT, HiranoT, HashimotoK, et al. (1990). Interleukin 6 and expression of its receptor on epidermal keratinocytes. Cytokine, 2:381–387.
71.
GallucciRM, SimeonovaPP, MathesonJM, KommineniC, GurielJL, SugawaraT and LusterMI. (2000). Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice. FASEB J, 14:2525–2531.
72.
ScottKA, ArnottCH, RobinsonSC, MooreRJ, ThompsonRG, MarshallJF and BalkwillFR. (2004). TNF-alpha regulates epithelial expression of MMP-9 and integrin alphavbeta6 during tumour promotion. A role for TNF-alpha in keratinocyte migration?. Oncogene, 23:6954–6966.
73.
SongHY, JuSM, GohAR, KwonDJ, ChoiSY and ParkJ. (2011). Suppression of TNF-alpha-induced MMP-9 expression by a cell-permeable superoxide dismutase in keratinocytes. BMB Rep, 44:462–467.
74.
ShipleyGD, PittelkowMR, WilleJJJr, ScottRE and MosesHL. (1986). Reversible inhibition of normal human prokeratinocyte proliferation by type beta transforming growth factor-growth inhibitor in serum-free medium. Cancer Res, 46(Pt 2):2068–2071.
75.
HashimotoK. (2000). Regulation of keratinocyte function by growth factors. J Dermatol Sci, 24(Suppl 1):S46–S50.
76.
SeckerGA, ShorttAJ, SampsonE, SchwarzQP, SchultzGS and DanielsJT. (2008). TGFbeta stimulated re-epithelialisation is regulated by CTGF and Ras/MEK/ERK signalling. Exp Cell Res, 314:131–142.
77.
FinchPW and RubinJS. (2004). Keratinocyte growth factor/fibroblast growth factor 7, a homeostatic factor with therapeutic potential for epithelial protection and repair. Adv Cancer Res, 91:69–136.
78.
ShirakataY (2010). Regulation of epidermal keratinocytes by growth factors. J Dermatol Sci, 59:73–80.
79.
SogabeY, AbeM, YokoyamaY and IshikawaO. (2006). Basic fibroblast growth factor stimulates human keratinocyte motility by Rac activation. Wound Repair Regen, 14:457–462.
80.
WernerS. (1998). Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Rev, 9:153–165.
81.
FinchPW, RubinJS, MikiT, RonD and AaronsonSA. (1989). Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science, 245:752–755.
82.
TsuboiR, SatoC, KuritaY, RonD, RubinJS and OgawaH. (1993). Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes. J Invest Dermatol, 101:49–53.
83.
CoffeyRJJr, DerynckR, WilcoxJN, BringmanTS, GoustinAS, MosesHL and PittelkowMR. (1987). Production and auto-induction of transforming growth factor-alpha in human keratinocytes. Nature, 328:817–820.
84.
DorfleutnerA, HintermannE, TaruiT, TakadaY and RufW. (2004). Cross-talk of integrin alpha3beta1 and tissue factor in cell migration. Mol Biol Cell, 15:4416–4425.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
