The Healing Effect of Conditioned Media and Bone Marrow-Derived Stem Cells in Laryngotracheal Stenosis: A Comparison in Experimental Dog Model

Abstract

Background: Differences in causes, severities, areas of stenosis, and the association with swallowing and phonation of larynx and trachea can result into Laryngotracheal stenosis (LTS).This study evaluated the healing effect of bone marrow stem cells (BMSCs) in experimentally induced LTS dog model.

Methods: Seven dogs were enrolled. BMSCs were isolated from proximal humerus and shoulder of a dog and cultured in media containing alpha minimal essential medium, fetal bovine serum, penicillin and streptomycin, and L-glutamine. BMSCs were characterized morphologically, by RT-PCR, and osteogenic induction. Karyotyping was undertaken for chromosomal stability. Mechanical trauma to laryngeal mucosa was identically conducted by Tru-cut punch forceps in right and left vocal folds. Two milliliter of conditioned media or BMSCs (2×10⁶) were injected in the right site of the tissue and the left side was considered as control after LTS induction. The larynx was visualized 2, 4 and 6 weeks after treatment. Six weeks post-treatment, the larynges were evaluated histologically.

Results: BMSCs were adhered to culture flasks, spindle shape and positive for mesenchymal marker and negative for hematopoietic markers. Osteogenic induction was verified by Alizarin red staining. Karyotype was normal. A complete epithelialization and minimal chronic inflammatory cell infiltration were noted in submucosa of both left (control) and right (cases) vocal folds. The healing effect of conditioned media and BMSCs in comparison to the control group was more prominent.

Conclusion: As thickness of fibrosis in cases were less than control group, conditioned media and BMSCs were shown to be good choices in healing of LTS.

Keywords: Bone marrow; Conditioned media; Dog; Healing; Laryngotracheal stenosis; Stem cell.

Fig. 1

BMSCs are seen as spindle shape morphology in passage 3 (×20

Fig. 2

RT-PCR of BMSCs were positive for CD90 and negative for CD34 and CD45 (M, 50 bp ladder; lane1, CD90; lane2, CD34; lane3, CD45

Fig. 3

Canine chromosomes at metaphase (left) and karyotyping (right) of BMSCs (2n=78) were shown

Fig. 4

Alizarin red staining denoted to calcification and osteogenic differentiation of BMSCs

Fig. 5

Complete epithelialization and minimal chronic inflammatory cell infiltration in left vocal fold (Control) sections with significant amount of submucosal fibrosis (A: H&E, B: Masson Trichrome, ×40

Fig. 6

Complete epithelialization and minimal chronic inflammatory cell infiltration and relatively less submucosal fibrosis in right vocal fold (Case) sections (A: H&E, B: Masson Trichrome, ×40