Xu et al. Cell Biosci (2015) 5:54 DOI 10.1186/s13578-015-0045-7 RESEARCH Open Access The expansion of autologous adipose‑derived stem cells in vitro for the functional reconstruction of nasal mucosal tissue Xiao Xu, Liang Li, Cheng Wang, Yang Liu, Chong Chen, Junling Yan, Hong Ding and Su‑yang Tang* Abstract Background: It is established that adipose-derived stem cells (ADSCs) produce and secrete cytokines/growth fac‑ tors that antagonize mucosal injury. However, the exact molecular basis underlying the treatment effects exerted by ADSCs is ill understood, and whether ADSCs cooperate with adipose tissue particles to improve mucosal function in patients with empty nose syndrome (ENS) has not been explored. We investigated the impact of ADSCs on nasal mucosa, the associated mechanisms, and their use in the treatment of patients with ENS. Results: The nasal endoscope and mucociliary clearance assessments were significantly improved (P < 0.05) in patients with (n 28) and without (n 2) a rudimentary turbinate that received ADSCs combined with fat granules transplantation. =Patients experienced a= significant improvement in nasal obstruction and nasal mucociliary clear‑ ance after nasal turbinate angioplasty (P < 0.05). H&E staining, Masson’s staining, and AB-PAS staining confirmed that inflammation was significantly reduced, collagenous fibers became aligned, fewer deposits were observed, and the mucosal proteins generated from caliciform cells increased following treatment. After a 14-day incubation period, ADSCs developed a polygonal cobblestone shape characteristic of human epithelial cells. Furthermore, immunohisto‑ chemical analysis revealed the presence of epithelial markers such as cytokeratin-7, and cytokeratin-19. Western blot analysis showed the presence of specific epithelial cell markers including cytokeratin-7, cytokeratin-14 and cytokera‑ tin-19 in these epithelial like cells (ELC); these markers had low expression levels of ADSCs. Conclusions: The reconstruction of mucosal function by nasal turbinate angioplasty combined with ADSCs and autologous adipose tissue particle transplantation significantly improved the symptoms of patients with ENS. This is a new procedure that will improve mucosal restoration treatment options in patients with ENS. Furthermore, we under‑ took preliminary explorations of the underlying mechanisms involved, and found that transplantation of ADSCs could induce epithelial cells to improve mucosa function in patients with ENS in the micro-environment of injection areas. Keywords: Adipose derived stem cells, Empty nose syndrome, Turbinate reconstruction, Nasal mucosa Background engineering seed cells with several advantages compared In recent years, rapid developments in regenerative to other stem cells: they are abundant in source, easy to medicine have identified seed cells and applicable cell find and culture, not rejected, safe, will undergo trans- stroma for tissue engineering to restore tissue damage. layer multiple differentiation, and proliferate rapidly Adipose-derived stem cells (ADSCs) are excellent tissue [1–4]. Empty nose syndrome (ENS) is an iatrogenic dis- order most often recognized by the presence of paradoxi- *Correspondence: [email protected] cal nasal obstruction despite an objectively wide, patent Skin and Reconstructive Medicine Department, The General Hospital nasal fossa. The term ‘empty nose syndrome’ was initially of Chinese People’s Armed Police Forces, Beijing, People’s Republic used to describe certain symptoms associated with tissue of China © 2015 Xu et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. Cell Biosci (2015) 5:54 Page 2 of 9 loss and radiographic findings of a paucity of normal assessments (MCCs), nasal volumes (NVs), and in the anatomic structures within the nasal cavities. After the minimum cross-sectional areas (MCAs) of the nasal removal of the inferior nasal concha, 20 % of patients will cavities of patients at 3, 6, and 9 months postoperatively. develop ENS [5, 6]. The severity of the subjective sensa- The improvements in NAR and in MCAs were not sta- tion caused by ENS does not correlate with the findings tistically significant at 3 and 6 months after surgery, but of objective examinations and can cause conflict between where so, when compared to the initial visit, 9 months patients and doctors. There is no established treatment after surgery (*P < 0.05). The values of NV reduced after for the nasal symptoms experienced by patients with the implantation surgery and the change of NV average ENS; developing such treatments is an important clinical overall scores were statistically significant between the priority. initial visit versus those of 3 and 9 months after treat- ment (*P < 0.05). One patient has been observed over Results a period of 18 months. The symptoms of nasal crust- ADSC transplantation improved nasal mucosal clinical ing and difficulty with nasal breathing improved further symptoms (data not shown). The acoustic rhinometry and endo- Flow cytometric analysis demonstrated that ADSCs were scopic imaging findings were not significantly different positive for CD73, CD90, and CD105, and negative for 9 months after surgery. However, the saccharine clear- CD19, CD34, CD45, and HLA-DR (Fig. 1). The ADSCs ance time was greatly improved at 18 months compared were also capable of osteogenic and adipogenic differen- to that at 12 months after surgery (data not shown) tiation when cultured in the appropriate inducing media. (Fig. 4). Assessment of the degree of osteogenic and adipogenic diffentiation via Alizarin Red S and Oil Red O staining, ADSC transplantation treatment improved the histological respectively, revealed good results (Fig. 2). Following the appearance of the nasal mucosa of patients with ENS transplantation of ADSCs and fat particles into the areas Lymphocytes and neutrophils infiltrated the nasal of nasal damage to form turbinates, 30 ENS patients dis- mucosa prior to ADSCs injection (Fig. 5a). After played no signs of infection or allergy. Of these patients, ADSCs injection, the nasal mucosa section had fewer symptoms improved in 28, including increased moisten- infiltrated lymphocytes and neutrophils 30 days follow- ing of the nasal mucosa, decreased tension of the mucosa ing treatment (Fig. 5b). Deposits of thickened and dis- and of nasal cavity pain when breathing, and improved organized collagenous fibers were seen using Mason’s quality of sleep. Two patients experienced improvement staining between the glands in the nasal mucosa prior of nasal cavity dryness. However, excessive breathing to ADSC injection (Fig. 5c). Thirty days later, the colla- remained an issue. genous fibers were aligned in the nasal mucosa, and col- lagenous fiber deposition was decreased in the glands ADSCs transplantation improved the function of the nasal (Fig. 5d). As revealed by AB-PAS staining of mucosa mucosa in patients with ENS tissue, mucosal proteins generated from cyathiform Nasal endoscopy was conducted 3 months after treatment, cells were limited and the submucosal glands showed and the nasal cavities of patients were visibly more erythe- compensatory hyperplasia (Fig. 5e). Thirty days later matous, secreted more mucus and had fewer scabs (Fig. 3). the mucosal proteins generated from cyathiform cells were evenly distributed throughout the nasal mucosa Mucociliary clearance (MCC) assessments (Fig. 5f). Saccharine clearance times were 1526.23 ± 1000.43 s Differentiated human ADSCs expressed epithelial markers preoperatively, 1457.13 ± 1078.23 s at 3 months fol- low-up, 1232.03 ± 499.45 s at 6 months follow-up, and After 14 days of ADSC culture in a differentiating 1501.00 ± 1159.85 s at 9 months follow-up. Mucociliary medium containing 20–30 ng/ml EGF, 20–30 % of the clearance assessments showed improvement of saccha- cells acquired a rounded/polygonal shape. They prolifer- rine clearance times in the study group at 3 and 6 months ated, and formed an adherent monolayer, organized in postoperatively (Fig. 4). cobblestone-pattern clusters (Fig. 6). While pure popula- tions of ADSCs downregulated the expression of cytoker- Acoustic rhinometry assessments: ADSCs improved atin 19 (CK19), immunohistochemical analysis revealed nasal resistance and mucociliary clearance assessments, that the differentiated cells from the dark blue cobble- and reduced the nasal volumes and the cross‑sectional stone-pattern clusters were positive for CK19 (Fig. 6). areas of the nasal cavities of patients with ENS Microscopic analysis using Lucia software (Nikon Corpo- Acoustic rhinometry assessments showed improve- ration Co., Kanagawa, Japan), showed that these consti- ments in nasal resistance (NAR), mucociliary clearance tuted approximately 60 % of the total cells. Xu et al. Cell Biosci (2015) 5:54 Page 3 of 9 Fig. 1 Flow cytometric analysis. ADSCs are positive for CD73, CD90, and CD105, and negative for CD19, CD34, CD45, and HLA‑DR Sustained upregulation of CK7, CK14 and CK19 in ADSCs expressed in epithelial-like cells (ELC),