Comparison Of Two Kinds Of In Vitro Preservation Methods On Decellularized Laryngeal Scaffold In Dogs

Laryngeal carcinoma is the most common carcinoma in head and neck cancers, with an eventually increasing prevalence recently in current clinical diseases. Hemilaryngectomy and total laryngectomy is the two main treatments of laryngeal carcinoma, which possess long-term postoperative hoarseness or pronunciation and seriously worsening quality of life. Statistics indicate that there are 250,000 patients receiving total laryngectomy each year. Laryngeal transplantation is a better alternative treatment to total laryngectomy, but only two cases were reported. Organization organ transplantation made great advance recently in clinical, and decellularized scaffold in the tissue engineering were successfully used. We succeeded in completing the larynx perfusion of the decellularized scaffold in rabbits and dogs, and also in detecting histocompatibility, immunogenicity and biomechanics of the scaffold, which provided theoretical and experiment evidence for the decellularized allograft laryngeal scaffold. In order to spare time for the clinical receptor, decellularized allograft laryngeal scaffold must be preserved prior to laryngeal transplantation. Three organ preservation methods in clinical include: cryopreservation, tissue culture preservation and organic preservation solution preservation. Cryopreservation is mainly used to preserve solid organ, with a limitation of insufficient constraints period which leads to a difficult survival in preserve cartilage cells. Tissue culture preservation is mainly used to preserve and cultivate cell. Organic preservation solution preservation is mainly used to preserve solid organ, and no research about the preservation of decellularized allograft laryngeal scaffold was reported so far. How should decellularized allograft laryngeal scaffold be preserved? Could preservation be prolonged? Could laryngeal scaffold be supported and cartilage cells survive? In our study, two kinds of in vitro preservation methods on decellularized laryngeal scaffold in dogs were used, aiming to evaluate the effects of the two methods in RPMI-1640 medium and HCA organ preservation solution to determine the better methods and time of decellularized laryngeal scaffold. Objectives1.To prepare decellularized allograft laryngeal scaffold by perfusion, explore the morphological observation, microanatomy, chondrocyte activity, and biomechanical characteristics of the decellularized allograft laryngeal scaffold, and compare two kinds of in vitro preservation methods on the decellularized allograft laryngeal scaffold in dogs.2.To evaluate which kind of in vitro preservation methods on the decellularized allograft laryngeal scaffold in dogs is fit for preservation, which will set a theoretical foundation for laryngeal scaffold of laryngeal transplantation. Methods 1.Morphological research on the decellularized allograft laryngeal scaffolds in dogsPerfusion decellularized allograft laryngeal scaffolds were made by cricothyroid artery perfusion with detergent(SDS, TritonX-100, PBS), which were appraised by morphological observation, microanatomy examination, and chondrocyte activity detection. 2. In vitro preservation research on decellularized allograft laryngeal scaffolds in dogsThirty-three perfusion decellularized allograft laryngeal scaffolds in dogs were made through perfusion with detergents by cricothyroid artery. The twenty-four decellularized allograft laryngeal scaffolds were separately preserved in the medium RPMI-1640(Group A) and HCA organ preservation solution(Group B) in Week 1, Week 2, Week 4, and Week 6. The other nine decellularized allograft laryngeal scaffolds were determined as blank control group(Group C). At each time point, allograft laryngeal scaffolds were evaluated by morphological observation, microanatomy examination, chondrocyte activity detection, and biomechanical characteristics. Results 1.Morphological research on the decellularized allograft laryngeal scaffolds in dogsMacroscopic view displayed that the decellularized allograft laryngeal scaffolds were structural integrity, pale, and good flexibility. HE staining of the coronal section of the decellularized laryngeal scaffolds showed that muscle cells were completely removed and cartilage cells were survived in cartilage lacuna. PDA-PI bifluorescence staining of the freezing microtome section of the decellularized laryngeal scaffolds indicated that cartilage cells were stained out sharply as yellowish-green and the structure of cartilage lacuna was intact. 2.In vitro preservation research on decellularized allograft laryngeal scaffolds in dogsFollow-up examinations were performed at Week 1, Week 2, Week 4, and Week 6 in the two groups. In group A, the shape and hardness of the scaffolds showed obvious change obviously and the cartilage cells were inactivated after a 2-week preservation. In group B, this happened after a 6-week preservation. The differences of thyroid cartilage thickness, thyroid cartilage compression modulus between group A and group B in Week 2 and Week 4 were both statistically significant(P<0.01). Conclusions1.The whole decellularized laryngeal scaffolds were obtained through cricothyroid artery perfusion with detergent. Laryngeal mucosa cells and muscle cells were completely removed, while cartilage cells were survived in cartilage lacuna and extracellular matrix were retained. These decellularized laryngeal scaffolds had perfect biomechanical characteristics.2.HCA organ preservation solution was better than RPMI-1640 medium in the preservation of decellularized laryngeal scaffold. It could be perfect preserved for 4 weeks with HCA organ preservation solution which suggested a better choice for decellularized laryngeal scaffold preservation.