Laryngotracheal stenosis (LTS) can be either congenital or acquired. Laryngeal stenosis is most often encountered after prolonged intubation. The mechanism for stenosis following intubation is believed to be hypertrophic scarring. Mesenchymal stem cells (MSCs) therapy has shown promising results in regenerative medicine. We aimed to systematically review the literature on MSC therapy for stenosis of the conductive airways.
PubMed, EMBASE, Google Scholar and the Cochrane Library were systematically searched from January 1980–January 2017 with the purpose of identifying all studies addressing the effect of MSC therapy on the airway. We assessed effect on inflammation, fibrosis, and MSC as a component in tissue engineering for treating defects in the airway.
We identified eleven studies (n = 256 animals) from eight countries evaluating the effect of MSCs as a regenerative therapy in the upper airways. The studies indicate that MSC therapy may lead to a more constructive inflammatory response as well as support tissue regeneration.
There may be a favorable effect of MSCs in inhibiting inflammation and as a component in tissue engineering. Given the heterogeneous nature of the included animal studies, any clear conclusion regarding the effect of tracheal stenosis in human subjects cannot be drawn. The included preclinical studies are however encouraging for further research.
Citation: Jakobsen KK, Grønhøj C, Jensen DH, Fischer-Nielsen A, Hjuler T, von Buchwald C (2017) Mesenchymal stem cell therapy for laryngotracheal stenosis: A systematic review of preclinical studies. PLoS ONE 12(9): e0185283. https://doi.org/10.1371/journal.pone.0185283
Editor: Gianpaolo Papaccio, Università degli Studi della Campania "Luigi Vanvitelli", ITALY
Received: May 18, 2017; Accepted: September 8, 2017; Published: September 21, 2017
Copyright: © 2017 Jakobsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This work was funded by The Danish Cancer Society (Grant number: R165-A10483-16-S7) and The University of Copenhagen (Grant number: A5090). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Characteristics of laryngotracheal stenosis
Laryngotracheal stenosis (LTS) is a broad term encompassing narrowing of the airway at the level of the larynx, subglottis, or trachea. Laryngotracheal stenosis (LTS) is a rare but severe condition. Besides the functional impairments as a result of airway obstruction, stenosis of the airways leads to considerable morbidity and mortality. [1,2] The main cause of LTS is intubation.  Current options for treating stenosis involve endoscopic dilation, laser surgery, laryngotracheal reconstruction, or life-long tracheostomy, but are often suboptimal as new scar tissue frequently develops leading to restenosis. [3,4] It has been suggested that LTS develops because of altered fibroblast responsiveness to anti-fibrotic signals during mucosal repair leading to excessive production of fibrosis.  Furthermore, there seems to be an altered inflammatory response leading to hypertrophic scars and a correlation between early inflammatory reaction to injury of the mucosa and the degree of scarring. 
The initial, preferred and less invasive method of treatment is endoscopic dilation, but this method has only proven success rates in paediatric patients of 64%; not incorporating the need for repetitive treatments due to re-stenosis and accompanying morbidity. [3,4] Alternatively, laryngotracheal reconstruction is required for severe stenosis. [3,7] This procedure holds high success rate but entails severe risks and subsequent morbidity. 
Mesenchymal stem cells
Mesenchymal stem cells (MSCs) are adult multipotent stem cells characterized by their adherence to plastic, their surface antigen expression, and their ability to differentiate into various connective tissue lineages including adipogenic, chondrogenic, myogenic and osteogenic cells. [8,9]
MSCs have during the last decades received extensive interest due to their potential therapeutic use as treatment for multiple diseases.  MSCs have shown anti-inflammatory and immunosuppressive properties, an ability to migrate to the exact site of injury, and a capacity to secret soluble factors crucial for cell survival and proliferation with minimal side effects. [10,11] Furthermore, MSCs currently indicate promising results in regenerative medicine.  In addition, MSCs are easily accessible for isolation most commonly from the bone marrow (BM-MSC) or adipose tissue (ASC) and have shown great expansion potential supporting the prospective of MSCs as a therapeutic agent. It has been shown that ASC increase wound healing with less scarring in skin ulcers  and the prospect of using ASC for therapeutic purpose in treating LTS is promising.
This study systematically evaluated the literature on the effects of MSCs on the conductive airway. We aimed to clarify the potential of MSCs in the conducting airways in regard to potentially treating stenosis and to evaluate their potential in a future human trial.
Materials and methods
This systematic review was conducted with reference to the Preferred Reporting Items for Systematic Reviews (PRISMA) statement. 
Systematic literature search and eligibility criteria
In January 2017, one author (KKJ) systematically searched the PubMed, EMBASE, Google Scholar, Cochrane Library, and clinicaltrials.org for articles in the English and Scandinavian languages. We included studies evaluating the effect of exogenous supplied MSC on the conductive airway in animals regardless of the publication date. Only studies concerning the conductive airways (trachea to the bronchioles) were included. Studies concerning the effector-mechanisms by which MSCs exert their properties were excluded as were studies evaluating the effect of MSCs on the airway in combination with other therapeutic regimes. Finally, studies concerning the effect of MSCs on the alveolar function, lung parenchyma and vascular bed were excluded. Due to conflict of interests the study done by Macchiarini, P. et al. on clinical transplantation of a tissue-engineered airway was excluded.
The following keywords (MeSH terms included in PubMed) were used: mesenchymal stromal cell, or MSC, or mesenchymal stem cell, adipose derived stromal cells, or adsc preadipocytes, or processed lipoaspirate cells, or stromal vascular fraction cells and Airways when searching PubMed and Embase. In Google Scholar, clinicaltrials.org and Cochrane Library the search were performed with the keywords “stem cell” and “airways”.
The following data were extracted from the included studies: study design, study participants, graft donor, source of graft, intervention, control groups, origin of stem cell, statistical tests.
The electronic searches identified 251 potentially eligible studies, of which eleven studies (256 animals) met the inclusion criteria (S1 Fig). All studies concerned the use of MSCs as a regenerative modality in the treatment of upper airway disease.
It was not possible to perform a meta-analysis due to the great variability in species, source of the stem cell, evaluation time, disease model, intervention and statistical tests across the studies.
The effect of MSCs on inflammation in the airway
Five studies (n = 153) evaluated the effect of MSCs on inflammation in the airways, (Table 1). [14–18] All studies used a preclinical model of induced asthma with either ovalbumin or toluene diisocyanate (TDI) as an inflammatory sensitizer (Table 1). [14–18]
Three of the studies (n = 100) administered MSCs after sensitization [15,17] while two studies (n = 53) [14,18] administered the MSCs before the sensitization.  All studies used BM-MSCs and administrated the MSCs via intravenous injection.
All studies could demonstrate a reduction in inflammation in the MSC treated group compared to the control group (Table 1). This was either measured as a significant decrease in the amount of inflammatory cells peribronchially [14,15,18] a reduction in the total white blood cell count, or a significant reduction in neutrophiles and eosinophils in blood samples [16,18]. Finally two studies demonstrated a significant reduction in the level of pro-inflammatory cytokines in the circulation in the MSC treated group compared to the control group. [14,17] In three of the studies the MSCs group was also demonstrated to have a significant reduction in goblet cell hyperplasia compared to the control group. [15,16,18]
The effect of MSCs on fibrosis in the airways
One study on induced asthma was not able to demonstrate a significant change in fibrosis after administration of MSCs, evaluated by the amount of collagen deposition.  Another study on induced asthma  demonstrated an increase in collagen deposition following the sensitizer. However, administering MSCs caused that the amount of collagen deposition, only reached the level in the sham group.
The use of MSCs in tissue repair
One study (n = 30) , investigated the participation of MSCs in the recovery of injured airway epithelium. Naphthalene was used to induce lung damage one month after MSC transplantation into the jugular vein. It was observed that BM-MSCs could adhere to the airways and form patches of epithelial lining in the conductive airways. 
Two studies (n = unknown) evaluated the repopulation of decellularized lung scaffold with MSCs for subsequent clinical transplantation. [21,22] Mendez JJ. et al.  showed attachment to the airways when using MSCs derived from adipose tissue. In regard to epithelization, Mendez JJ. et al. showed that MSCs from both bone marrow and adipose tissue underwent epithelial cell differentiation.
This contrasts to Daly AM. et al.  that repopulated their scaffolds with BM-MSCs but their cells did not differentiate into pseudostratified epithelia.
Three studies (n = 73) evaluated the repair of a tracheal defect by surgically replacing the damaged area with a tracheal graft or a scaffold. [7,19,23] Ott LM. et al.  and Gray FL. et al.  used patch-type decellularized scaffold to repair a tracheal defect and investigated if seeding of MSCs onto the scaffold would be beneficial for the tracheal repair, measured by survival rate, severity of the stenosis, the epithelialization, and histology of the scaffold. Go, T. et al. (n = 30)  investigated the engineering of a functional long-segment graft and replaced six cm of the trachea with a tracheal graft. The tracheal defects were made on the lamb fetus, and after the defect was repaired the fetus was returned to the uterine cavity.
Ott LM. et al. found that the survival rate was higher in the BM-MSC group compared to the scaffold-only group and had a higher level of immature cartilage which could indicate that BM-MSCs contribute to cartilage formation.
In regards to cross section area and preventing stenosis no clear results exist. Ott LM. et al. observed a larger cross section area in the scaffold-only group compared with the scaffold seeded with BM-MSC. Gray FL. et al. did not show a significant difference in cross section area between the two groups. Gray FL. et al. argued that the scaffolds were made from tracheal segments of rabbits and were thus not originally programmed to grow or dilate. Go T. et al. showed that MSC-derived chondrocytes seeded on the external surface resulted in less stenosis of the tracheal graft. Go T. et al.  continued showing the beneficial effect of epithelialization which help prevent bacterial/fungal contamination.
To the best of our knowledge, this study is the first systematic review to evaluate the effect of mesenchymal stem cells in the conducting airway and its therapeutic preclinical potential for treating laryngotracheal stenosis. As we identified no studies specifically evaluating the effect of MSCs in regards to treating stenosis by injecting stem cells into the airways, this review highlights the effects of MSCs on the airways in a general setting. The goal of the study was to investigate the effects of MSCs on the airway as a preclinical study to evaluate if it could be possible to treat stenosis by injection of stem cells to the airway. The result of our study highlighted three main areas respectively the impact on inflammation, on fibrosis and in tissue repair.
The studies included in this review differed in regards to origin of MSCs, the dose of MSCs, the study design, the animal model and the disease model. Collectively, both mice, rats, rabbits, pigs and lambs were used as experimental models. MSCs were derived from bone marrow, adipose tissue and amnion fluid and were extracted from different species including both human, mice, rats, rabbits, pigs, and lambs. Especially, the studies investigating MSCs in regards to tissue engineering differed considerably in methods and design and it is thus difficult to integrate the results. Furthermore, only two of the eleven studies included in this review reported randomization and only four studies reported blinding when analyses were performed. This is important to bear in mind in regards to potential biases. The studies also differed considerable in regards to the time MSCs were administered as compared to the intervention the animals were exposed to.
As stenosis of the airways is a process characterized by fibrotic wound healing  and altered inflammation  it is relevant to see if alteration of these factors using MSCs would adjust the development of stenosis. Inhibiting the healing process by inhibiting inflammation and fibrosis would possibly prevent scar tissue formation and prevent a reduced cross-sectional area of the airway. However, the consequence of impaired healing has not yet been investigated.
Another modality of treating severe stenosis with a significant narrowing of the airways, or a long-segment airway stenosis is by creating a graft to substitute the narrow area of the airway. A graft could be helpful in treating both acquired and congenital stenosis. The majority of the studies agreed upon the effect of MSCs in increasing epithelialization. Go, T. et al. showed the importance of epithelialization to avoid infections and to create a functional graft. When dealing with laryngotracheal stenosis it is essential to increase the cross-section area and to construction of a functional graft that can expand and widen the airway. The results on the effect of MSCs to aid in increasing the cross-section area were not conclusive.
Mendez JJ. et al.  concluded that MSCs derived from adipose tissue would be more beneficial than MSCs derived from BM when constructing a patch-type scaffold. Still a lot remains to be investigated such as the possible improved effects of MSCs derived from adipose tissue and if MSCs from adipose tissue is more beneficial in treating subglottic stenosis.
So far no studies on the effect of stem cells and their effect in the airways have been conducted in human trials. Animal studies are associated with a great deal of methodological problems that must be taken into account before the study can be transferred to humans. Methodological problems of animal experiments include difference and variation of metabolic pathways between disparate animal species. Furthermore, variations in dosing regime between human and animals exists as well as variability in methods of randomization, nuances in laboratory technique, and variability in the choice of comparison. [24,25]
In conclusion there are various effects of MSCs on the airways with consistent responses towards inflammation, whereas the effects on fibrosis and tissue repair were contradictory possibly related to methodological differences. Thus it is difficult to summarize the findings into a coherent conclusion. The presented results suggest that MSCs have potential in treating laryngotracheal stenosis but many issues remain to be investigated. Altogether, however, we find that the above-mentioned findings are encouraging for conducting a clinical study.
- 1. Morrissey MS, Bailey CM. Diagnosis and management of subglottic stenosis after neonatal ventilation. Arch Dis Child. 1990;65: 1103–4. Available: http://www.ncbi.nlm.nih.gov/pubmed/2248497 pmid:2248497
- 2. Holinger PH, Kutnick SL, Schild JA, Holinger LD. Subglottic stenosis in infants and children. Ann Otol Rhinol Laryngol. 1976;85: 591–9. pmid:791051
- 3. Gelbard A, Francis DO, Sandulache VC, Simmons JC, Donovan DT, Ongkasuwan J. Causes and consequences of adult laryngotracheal stenosis. Laryngoscope. 2015;125: 1137–43. pmid:25290987
- 4. Wentzel JL, Ahmad SM, Discolo CM, Gillespie MB, Dobbie AM, White DR. Balloon laryngoplasty for pediatric laryngeal stenosis: case series and systematic review. Laryngoscope. 2014;124: 1707–12. pmid:24222273
- 5. Singh T, Sandulache VC, Otteson TD, Barsic M, Klein EC, Dohar JE, et al. Subglottic stenosis examined as a fibrotic response to airway injury characterized by altered mucosal fibroblast activity. Arch Otolaryngol Head Neck Surg. 2010;136: 163–70. pmid:20157063
- 6. Sandulache VC, Chafin JB, Li-Korotky H-S, Otteson TD, Dohar JE, Hebda PA. Elucidating the role of interleukin 1beta and prostaglandin E2 in upper airway mucosal wound healing. Arch Otolaryngol Head Neck Surg. 2007;133: 365–74. pmid:17438251
- 7. Ott LM, Vu CH, Farris AL, Fox KD, Galbraith RA, Weiss ML, et al. Functional Reconstruction of Tracheal Defects by Protein-Loaded, Cell-Seeded, Fibrous Constructs in Rabbits. Tissue Eng Part A. 2015;21: 2390–403. pmid:26094554
- 8. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8: 315–7. pmid:16923606
- 9. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284: 143–7. Available: http://www.ncbi.nlm.nih.gov/pubmed/10102814 pmid:10102814
- 10. Squillaro T, Peluso G, Galderisi U. Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transplant. 2016;25: 829–48. pmid:26423725
- 11. Salem HK, Thiemermann C. Mesenchymal stromal cells: current understanding and clinical status. Stem Cells. 2010;28: 585–96. pmid:19967788
- 12. Mizuno H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch. 2009;76: 56–66. Available: http://www.ncbi.nlm.nih.gov/pubmed/19443990 pmid:19443990
- 13. Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. J Clin Epidemiol. 2009;62: 1006–1012. pmid:19631508
- 14. Sun Y-Q, Deng M-X, He J, Zeng Q-X, Wen W, Wong DSH, et al. Human pluripotent stem cell-derived mesenchymal stem cells prevent allergic airway inflammation in mice. Stem Cells. 2012;30: 2692–9. pmid:22987325
- 15. Ogulur I, Gurhan G, Aksoy A, Duruksu G, Inci C, Filinte D, et al. Suppressive effect of compact bone-derived mesenchymal stem cells on chronic airway remodeling in murine model of asthma. Int Immunopharmacol. 2014;20: 101–9. pmid:24613203
- 16. Mohammadian M, Boskabady MH, Kashani IR, Jahromi GP, Omidi A, Nejad AK, et al. Effect of bone marrow derived mesenchymal stem cells on lung pathology and inflammation in ovalbumin-induced asthma in mouse. Iran J Basic Med Sci. 2016;19: 55–63. Available: http://www.ncbi.nlm.nih.gov/pubmed/27096065 pmid:27096065
- 17. Firinci F, Karaman M, Baran Y, Bagriyanik A, Ayyildiz ZA, Kiray M, et al. Mesenchymal stem cells ameliorate the histopathological changes in a murine model of chronic asthma. Int Immunopharmacol. 2011;11: 1120–6. pmid:21439399
- 18. Lee S-H, Jang A-S, Kwon J-H, Park S-K, Won J-H, Park C-S. Mesenchymal stem cell transfer suppresses airway remodeling in a toluene diisocyanate-induced murine asthma model. Allergy Asthma Immunol Res. 2011;3: 205–11. pmid:21738887
- 19. Gray FL, Turner CG, Ahmed A, Calvert CE, Zurakowski D, Fauza DO. Prenatal tracheal reconstruction with a hybrid amniotic mesenchymal stem cells-engineered construct derived from decellularized airway. J Pediatr Surg. 2012;47: 1072–9. pmid:22703772
- 20. Serikov VB, Popov B, Mikhailov VM, Gupta N, Matthay MA. Evidence of temporary airway epithelial repopulation and rare clonal formation by BM-derived cells following naphthalene injury in mice. Anat Rec (Hoboken). 2007;290: 1033–45. pmid:17661377
- 21. Mendez JJ, Ghaedi M, Steinbacher D, Niklason LE. Epithelial Cell Differentiation of Human Mesenchymal Stromal Cells in Decellularized Lung Scaffolds. Tissue Eng Part A. 2014;20: 1735–1746. pmid:24393055
- 22. Daly AB, Wallis JM, Borg ZD, Bonvillain RW, Deng B, Ballif BA, et al. Initial Binding and Recellularization of Decellularized Mouse Lung Scaffolds with Bone Marrow-Derived Mesenchymal Stromal Cells. Tissue Eng Part A. 2012;18: 1–16. pmid:21756220
- 23. Go T, Jungebluth P, Baiguero S, Asnaghi A, Martorell J, Ostertag H, et al. Both epithelial cells and mesenchymal stem cell-derived chondrocytes contribute to the survival of tissue-engineered airway transplants in pigs. J Thorac Cardiovasc Surg. 2010;139: 437–43. pmid:19995663
- 24. Bracken MB. Why animal studies are often poor predictors of human reactions to exposure. J R Soc Med. 2009;102: 120–2. pmid:19297654
- 25. Pound P, Ebrahim S, Sandercock P, Bracken MB, Roberts I, Reviewing Animal Trials Systematically (RATS) Group. Where is the evidence that animal research benefits humans? BMJ. 2004;328: 514–7. pmid:14988196