| With the development of voice surgery, lesions of the vocal folds can beremoved by voice surgeon under the microscope using laser therapy.But thelamina propria of the vocal fold can be easily damaged during the surgeryleading to scar which caused by composition changes of the extracellular matrixin the lamina propria. How to reduce the scar formation has troubled theresearchers for many years. Previously, researchers had injected different kindsof stem cells such as bone marrow mesenchymal stem cells, adipose derivedstem cells and embryonic stem cells into animal’s vocal folds in order to treatthe injured vocal folds. However, it is very painful when bone marrow wasobtained from the patient’s bones, and although adipose derived stem cells canbe easily isolated from fat tissue, there are large differences between the fattissue and the structure of vocal folds. The application of embryonic stem cellsstill have some ethical problems. These factors have limited their application in clinical.In recent years, researchers have successfully confirmed that there are manykinds of mesenchymal stem cells in higher animals’ bodies. Laryngeal mucosawith its wide range of tissue resource which is easy to harvest has the sameorigin with the vocal fold’ s structure, and has no ethical obstacles. So weassumed that there are mesenchymal stem cells existing in the laryngealmucosa. If such cells could be successfully isolated for the laryngeal defectsrepair, it will bring new hopes for the patients with vocal fold’s damage. Inprevious experiments, Liu Yang had successfully identified that there aremesenchymal stem cells existing in human laryngeal mucosa with rapidproliferative capacity and differentiation potential which are called laryngealmucosa mesenchymal stem cells (LM-MSCs). Therefore, the purposes ofpresent study are to trace the survival and differentiation of such stem cells(LM-MSCs)in an animal model and, to observe the morphological andhistological changes of wound repair of the vocal fold.1ObjectiveTo investigate wether there are laryngeal mucosa mesenchymal stem cells incanine laryngeal mucosa, then they will be isolated and identified for caninevocal folds injection. To observe the morphological and histological changes ofsuch stem cells in vocal fold wound repair.2Methods2.1Isolation, cultivation and identification of LM-MSCsCanine laryngeal mucosa mesenchymal stem cells were isolated fromlingual side of canine epiglottic mucosa by digestion culture method. Multipledifferentiation potentials were confirmed by adipogenic, chondrogenic and osteogenic differentiation. The biological characteristics of LM-MSCs wereevaluated by MTT assay and Colony Forming Cell Assay. The expression of cellsurface marker of LM-MSCs were tested by Flow cytometry.2.2Establishment of a canine vocal folds injury model by Laser5Chinese rural dogs, bilateral vocal folds of4dogs were exposed underlaryngoscope, a small amount of lidocaine was sprayed locally, the membranousvocal folds were damaged by semiconductor laser, one dog without any injurywas taken as control group. The general healing was observed4d,2w,4w and8w post-surgery, the vocal folds were obtained respectively. The left side ofvocal folds were embedded with paraffin, and sections of the injured part wereobserved by HE staining and Masson trichrome staining, EVG staining andAlcian blue staining to evaluate the arrangement and content changes ofcollagen fibers, elastic fibers, hyaluronic acid and inflammatory reaction. Whilethe right side of vocal folds were observed by frozen section with fibronectinimmunohistochemical staining.2.3Experimental study of laryngeal mucosa mesenchymal stem cells in theacute injured vocal folds2.3.1The impacts of laryngeal mucosa mesenchymal stem cells on themorphological changes of the injured vocal folds10Chinese rural dogs, bilateral vocal folds of10dogs were exposed underlaryngoscope, a small amount of lidocaine was sprayed locally, the membranouspart of vocal folds were damaged by semiconductor laser, to the depth of thethyroarytenoid muscle, vocal fold injection was carried out after injury,0.2mlmixture(about2×106cells) of LM-MSCs and collagen was injected into the leftvocal folds, while0.2ml collagen was injected into the right vocal folds only as the control. The vocal folds of6dogs were observed respectively,2w,4w and8w post-surgery under laryngoscope to access general healing.2.3.2The impacts of laryngeal mucosa mesenchymal stem cells on the laminapropria structure of the injured vocal foldsThe6dogs were sacrificed. ECM conposition changeds were observed by HEstaining, Masson trichrome staining, EVG staining, Alcian blue staining and toevaluate the distribution and content changes of collagen fibers, elastic fibersand HA. Fibronectin changes were accessed by immunohistochemistry offibronectin staining.2.3.3The survival of Laryngeal mucosa mesenchymal stem cells in the injuredvocal foldsThe survival and distribution of LM-MSCs were observed by frozen sections ofthe vocal folds under fluorescence microscope, Hochest fluorescent dye wasperformed for15min to observe the nuclei, then PBS rinsed the slices, theamount of implanted LM-MSCs were evaluated at different time points.2.3.4The differentiation of laryngeal mucosa mesenchymal stem cells in theinjured vocal folds4dogs were observed on4w and8w post-surgery respectively, and to trace thedifferentiation of the stem cells. Immunofluorescence staining of Vimentin andSmooth musle actin staining were performed to trace the capabilities of theinjected LM-MSCs differentiating into fibroblasts and myofibroblasts.3Results3.1Isolation, cultivation and identification of LM-MSCsLM-MSCs initially grew into a short spindle shape, then spreading in a longershape, like the shape of fibroblast, and had large nuclei. In vitro, LM-MSCs could differentiate into adipogenic, osteogenic and cartilage lineage respectively.The oil red O staining and alizarin red staining were positive. In cartilagedifferentiation positive signal of type II collagen by immunohistochemicalstaining could be observed. LM-MSCs showed high proliferation ability with nostagnation phase. The logarithmic phase of LM-MSCs began at3d, reached tothe growth peak at7d, and entered a growth plateau at8d. The colonyformation assay showed LM-MSCs had high colony formation ability about19.7%. Surface markers of LM-MSCs were analysed by flow cytometry, withpositive mesenchymal surface molecular markers (CD29-77.6%, CD44-75.5%,CD90-97.3%, CD105-38.3%), and negative hematopoiet surface molecularmarkers (CD34-1.8%, CD45-0.9%).3.2Establishment of a canine vocal folds injury model by Laser4d post-surgery, acute inflammation was observed in bilateral vocal folds, andthe lamina propria layer(LP) hadn’t been covered with epithelium.2wpost-surgery, acute inflammation was reduced and the LP had been covered withepithelium. The content of collagen was increased while the elastic fiber wasdecreased. The content of hyaluronic acid(HA) was decreased while thefibronectin was increased.4w post-surgery, the inflammation was disappeared,and scars were observed on the injured parts of the vocal folds. Collagen contentwas increased continually while the elastic fiber was decreased. The content ofHA was decreased while the fibronectin was increased.8w post-surgery, thescars and atrophy were observed. Collagen showed a thick bundle-like shapewhile the density of elastic fiber was stabilized. HA content was decreasedslightly while fibronectin was increased.3.3.1The impacts of Laryngeal mucosa mesenchymal stem cells on morphological changes of the injured vocal foldsUnder laryngoscope2w post-surgery, edema, congestion, irregular surface ofthe mucosa and granuloma could be observed on the bilateral vocal folds, andthe stem cell group had less granuloma and irregular surface than control group;4w post-surgery, edema and congestion could hardly be observed on thebilateral vocal folds, but irregular surface of the mucosa, granuloma and atrophycould be observed. The control group had larger atrophy than stem cell group;8w post-surgery, edema, congestion and newly formed granuloma couldn’t beobserved on the bilateral vocal folds, while scars could be seen on the injuredparts of the vocal folds, and the control group had larger scar and atrophy thanstem cell group.3.3.2The impacts of laryngeal mucosa mesenchymal stem cells on the laminapropria structure of the injured vocal foldsHistological study showed that2w post-surgery, the stem cell group had lessInflammatory cells invasion and fibrous tissue proliferation in lamina propriacompared with the control group. The contents of fibronectin in both groupswere increased, the content of fibronectin in the stem cell group was slightlylower than that of the control group; the content of collagen in the stem cellgroup was lower than that of control group, and stem cell group had moreorganized collagen fiber than control group; the contents of elastic fibers in bothgroup were reduced and disorganized, and the content in stem cell group washigher; the contents of HA in both group were decreased, while the content instem cell group was slightly higher than that of the control group.4wpost-surgery, both groups showed little Inflammatory cells invasion while moreproliferation of fibrous tissue in lamina propria than that on2w. The contents of fibronectin in both groups were increased continuously, the content offibronectin in the stem cell group was lower than that of control group; thecontent of collagen in the stem cell group was lower than that of control group,and control group had more disorganized collagen fiber than stem cell group; thecontent of elastic fiber in both group were reduced and disorganized, and thediameter of elastic fiber of stem cell group was bigger than the control; thecontents of HA in both group were decreased continuously, while the content instem cell group was higher than that of the control group.8w post-surgery, bothgroups showed no Inflammatory cells invasion with more fibrous tissues inlamina propria. The content of fibronectin in both groups were increased whilethe content of fibronectin in the stem cell group was lower; the content ofcollagen in the stem cell group was lower than that of control group, and controlgroup had more disorganized collagen fiber; the content of elastic fiber in bothgroup were stablized and disorganized, and content of elastic fiber in stem cellgroup was higher than control; the contents of HA in both group were decreased,while the content of the stem cell group was higher.3.3.3The survival of laryngeal mucosa mesenchymal stem cells in the injuredvocal folds2w,4w and8w post-surgery the implanted LM-MSCs could be observed in theLP under fluorescence microscope, a large number of red fluorescence labelledcells were observed on2w in lamina propria, suggesting that a large number oftransplanted cells were surviving at this time point and the amount of implantedLM-MSCs on4w was less than that on2w.8w post sugery, the implantedLM-MSCs could be still observed, while the amount of implanted LM-MSCswas decreased continuously. 3.3.4The differentiation of Laryngeal mucosa mesenchymal stem cells in theinjured vocal foldsImmunofluorescence staining of Vimentin showed that4w post-surgery the Dillabelled LM-MSCs emitted green fluorescence by the blue-ray excitation underfluorescence microscope, indicated the implanted LM-MSCs differentiated intofibroblasts. By the same method, the implanted LM-MSCs could differentiateinto fibroblasts on8w. Immunofluorescent stainings of SMA showed4wpost-surgery the Dil labelled LM-MSCs emitted green fluorescence by theblue-ray excitation under fluorescence microscope, indicated the implantedLM-MSCs differentiated into myofibroblasts, while the differentiated myofibroblasts by LM-MSCs couldn’t be observed on8w.4Conclusions4.1In this study, canine laryngeal mucosa mesenchymal stem cells weresuccessfully cultured by digestion culture mothed and identified, the adipogenic,chondrogenic and osteogenic differentiation of LM-MSCs were proved.LM-MSCs have rapid growth capacity and higher proliferation ability, and havehigh expression of mesenchymal stem cell surface markers.4.2The membranous portions of the canine vocal folds were injured bysemiconductor laser under laryngoscope. Under laryngoscopy congestion andedema were observed on bilateral vocal folds2w post-surgery, atrophy wereobserved on bilateral vocal folds4w post-surgery, scars were observed onbilateral vocal folds8w post-surgery. Histological study showed collagen wereincreased and disorganized, indicating scar tissue formation. The decrease ofelastic fiber indicated less elasticity of the vocal folds. The level of HA wasdecreased indicating worsen vibrating ability of vocal folds and inhibitory ability of collagen deposition. The increased fibronectin showed more fibrosisformation. These data showed that this experiment successfully established acanine vocal fold injury model, and provided a reliable animal model for thesubsequent experiments.4.3.1The implanted LM-MSCs could reduce congestion and edema in the LPcompared with control group. Stem cell group had more regular surface, lessatroph and scar formation than that of the control group.4.3.2The histological study showed LM-MSCs could not only reduceddeposition and disordered arrangement of collagen, but also increased elasticfiber and HA contents which ensure the effective vocal fold vibration. BesidesLM-MSCs could decrease fibronectin which can promote fibrosis of the laminapropria. The above results showed that LM-MSCs could change the microenvironment of the injured vocal folds by regulating the synthesis ofextracellular matrix components.4.3.3Under fluorescence microscope the implanted LM-MSCs could beobserved from2w to8w post-surgery, but as time went by，the number ofimplanted LM-MSCs reduced gradually.4.3.4The implanted LM-MSCs could differentiate into fibroblasts and myofibroblasts in injured vocal fold, suggesting that LM-MSCs could repair theinjured vocal folds through differentiating into the lamina propria cells directly. |
