Experimental Study On Adipose-derived Stem Cells Combined With Acellular Dermal Matrix Particles For The Acute Injuried Vocal Fold

The extracellular matrix of lamina propria in the normal vocal fold is basic of vocal fold vibration.Vocal folds after they are injuried will result in vocal fold scaring, leading to dysphonia, affecting seriously the quality of the patients'life. To improve or restore the sounds by vocal fold scaring, there is no effective clinical treatment. Currently, it is hot or difficulty topic about the prevention and treatment of vocal fold scarring. Over the past decades, with the development and application of tissue engineering, researchers found some favorable results for scarred vocal folds treated with stem cells compounding the scaffold material. Therefore, the ideal seed cells and scaffold material selection is the major works in now. It may promote the injured vocal fols healing.In this study, adipose derived stem cells of rabbit (rabbit adipose-derived stem cells, rASCs) combined acellular dermal matrix particles (micronized acellular dermal matrix, MADM) were injected into the injured vocal folds and observing the results that rASCs-MADM restore the injured vocal folds.1 Objective:To exploring the feasibility on rASCs-MADM for vocal folds injection and Studing the effect about the rASCs-MADM repairing injured vocal folds.2. Method2.1 Rabbit inguinal adipose tissue was removed, digested with collagenase, the rASCs were fractional cultured. Cell morphology was observed under inverted microscope. Cell growth pattern was detected using the MTT, the cellular multi-directional differentiation was detected with orientation induced culture, and cells were frozen-thawed.2.2 No epidemic of fresh fetal bovine skin was removed layer of epidermis and dermal papilla with mechanical method, the reticular layer of dermis were used and made of acellular dermal matrix with trypsin digestion-NaOH digestion- freeze-thaw method, and were cut into particles using physical methods. The rASCs labeled with DiI and MADM were composite cultured. The cell adhesion materials were observed with fluorescence microscope and scanning electron microscope,the rASCs proliferation after combined MADM was detected using MTS colorimetric assay. The complexes cultured for 3 days, intramuscular injection of normal rabbit vocal mixed amount of collagen. The vocal gross morphology was observed with endoscopes after 2, 4, and 8w, and then individually drawn, section was frozen, the survival and distribution of rASCs in the vocal cords were observed with fluorescence microscope, the inflammation rejection of materials to vocal cord tissue and material degradation were observed with HE staining.2.3 Six adult male New Zealand white rabbits, weighing 2.7-3.2kg, were randomly divided into 2 groups. Laryngeal split to reveal bilateral vocal folds undergoing anesthesia, semiconductor laser (power: 8W) damaged the1/2 tissue after bilateral vocal folds membranous to the thyroarytenoid muscle, the general form of bilateral vocal cord was observed with endoscopes after 2, 4w, and then individually drawn, the lamina tissue changes in the damaged site, the lamina collagen arrangement was observed Van Gieson staining.2.4 Eighteen New Zealand white rabbits, weighing about 2.7-3.2kg, were divided intoⅠ,Ⅱ,Ⅲgroup.Ⅰgroup were injected rASCs-MADM in left vocal folds,Ⅱgroup were injected rASCs in left vocal folds,Ⅲgroup were injected MADM in left vocal folds, the right vocal folds of every group is only just injury, and three rabbits vocal folds was control group. The repair was observed with endoscopes after 2, 4, and 8w, and then drawn after 8w, the vocal fold lamina structure was observed HE staining, and the collagen in vocal folds lamina arrangement was observed Van Gieson staining.3 Results:3.1 It is easy to get the rASCs by isolated culture in vitro. The rASCs getted by this way have strong reproductive activity and can differentiate for adipose cells, sclerotomal cells and sarcoblasts in different microenvironment. The capacity of reproductive activity is still strong after cryopreservation and recovery.3.2 The collogens holded in MADM have fine successions and form stereo-network structure with each other. The pore in it is about 20-50μm. The MADM can be incised into corpuscles and the diameter is about 200-450μm. rASCs grew well after sticking on the corpuscles and can proliferate unceasingly. The capacity of proliferation has significant deviation compared with general culture. rASCs-MADM have slight immunologic rejection after jinjecting into vocal folds of rabbits. We observed the survival cells and portion of MADM in 8weeks after injection, which demonstrate that rASCs-MADM have good biological compatibility, suitable degradation, and is a good support materials.3.3 The laryngeal cleft surgery to set up the acute injuried model of vocal folds has limited damage and no complication. The goodness of this model is the procedure is simple, the location and degree is identified, and no obvious adherence formed. We observed that the vocal folds epithelium mucosae have finished coverage and a bulk of collogens in proper layer in 2weeks after injury; and we also found that the surface of vocal folds was irregular, scar was formed, and more collogens in proper layer in 4 weeks after injury compared with in 2 weeks after injury.3.4 After the acute injury and injection of vocal folds, we observed the result by endoscope in 2, 4, and 8 weeks. The injured site in vocal folds of rASCs-MADM, rASCs injection groups were smooth and less granulation tissue formed, whiles the injured site in vocal folds of Injection MADM, injured groups were irregular and more granulation tissue formed. HE staining and Van Gieson staining showed that the collagen in vocal folds lamina of rASCs-MADM, rASCs injection group arranged orderly, while the collagen in MADM injection group were bent bundle and arranged close-up. Lots of collagen deposited in injured group. The collagen was fracture and arranged disorderly4 Conclusions:4.1 ASCs are abundance, easily harvest, isolated, cultured in vitro.ASCs have both strong reproductive activity and multi-lineage potential.SO it has wide applicate prospects.4.2 MADM is easily made. It has a good 3D structure, biocompatibility and appropriate degradation. MADM is good scaffold material of cells and filling material for injection of vocal folds. 4.3 It is feasible that we seted up the acute injuried model of vocal folds of rabbit by the laryngeal cleft surgery, which has limited damage and is easily works.4.4 ASCs can adhere on MADM and continue growth, MADM significantly promote cell proliferation. They format rASCs-MADM complex, which can inject into vocal folds. After we injected the rASCs-MADM complex for injuring vocal folds, we observed that it not only had compatibilization but also decreased the collagen deposition in lamina propria. To some extent, the injection of complex can prevent the injured vocal folds atrophy and scar formation. It has a better restored effect than only injection of ASCs and MADM.