Immunological Study On Decellularized Whole Laryngeal Scaffold

Laryngeal transplantation is an ideal way to restore laryngeal function aftertotal laryngectomy. The first real problem faced by laryngeal transplantation isthe immune rejection and the use of immunosuppressive agent. Rapid andsevere immune rejection is a major obstacle to successful transplantation. If wecan construct a low immunogenicity bio-artificial larynx ,this problem may besolved. Rencently ,with the rapid development of tissue engineering , acellularscaffold brings hope to construct a low immunity larynx . Acellular scaffold isobtained by chemical detergent, which removed cells and soluble protein of thetissues or organs, retaining only the organizational structure of the insolublematrix components. The extracellular matrixs (ECMs) mainly contain collagen.The collagen provide scaffolds for cell adherence, cell migration,proliferationand tissue growth, . However, there is no research about the immunogenicity ofthe decellularized laryngeal scaffold. In our study, we attempted to construct adecellularized laryngeal collagen scaffold which reserved three-dimensionalgeometry extracellular matrix by utilizing a perfusion-decellularized technique and evaluate the immunogenicity of the decellularized laryngeal scaffold .Objectives1. To prepare an extracellular matrix laryngeal scaffold by utilizing a perfused,decellularized technique and investigate its feasibility to be athree-dimensional geometry extracellular matrix whole laryngeal collagenscaffold. Morphological observation was conducted on the decellularizedwhole laryngeal scaffold.2. To appraise the decellularized whole laryngeal scaffold's immunogenicity byimplanted the scaffold in para-laryngeal muscles in rabbits, which will set atheoretical foundation for the application in the tissue engineering.Methods1. Morphological observation on the decellularized whole laryngeal collagenscaffoldPerfusion decelluarized whole laryngeal collagen scaffolds were obtainedby carotid arterious perfusion with detergents. They were evaluated bymacroscopic view, scanning electron microscope (SEM) and histologicalexamination.2. Immunological study on decellularized whole laryngeal scaffoldTwelve perfusion decelluarized laryngeal scaffolds were obtained fromrabbits through perfusion detergents by common carotid arterious. The twelvedecellularized laryngeal scaffolds and the twelve fresh larynxes were separatelyimplanted in para-laryngeal muscles of rabbits and harvested after two weeks,four weeks, twelve weeks and twenty-four weeks, respectively. Then, performedmacroscopic view, histological examination and lymphocyte infiltration test onthese samples. Results1. Morphological observation on the decellularized whole laryngeal scaffoldMacroscopic view showed that the decellularized whole laryngeal collagenscaffold became transparent after two hours perfusion by detergents SDS.Perfusion decellularized laryngeal collagen scaffold can preserve the laryngealextracellular matrix and produce an acellular whole laryngeal collagenarchitecture. Histology and SEM indicated that the perfused laryngeal scaffoldhad a better deculluarized effect. The ventages and collagen fibers were retainedperfectly. HE staining of the coronal section of decellularized larynx indicatedthat the structure of laryngeal vocal cord, ventricular band, elastic cone wasintact. Muscle cells was completely removed.The fiber-like structure and thedirection of the extracellular matrix were remained.2. Immunological study on decellularized whole laryngeal scaffoldThe decelluarized larynx did not show obvious immunological rejectionafter implanted in the para-laryngeal muscles of the reeipient rabbits. Thevolume of implanted larynx became smaller but retain cartilage scaffold.Larynxes in the control group presented the serious immunological rejection andthe majority tissues of the larynxes were disintegrated and substituted by thefibrous connective tissues after four weeks. The peripheral tissues weredamaged and necrotic at different degrees. The quantity of the lymphocyteinfiltration in the control group is higher than that in the experiment group andthe result had the statistical significance (P<0.01).Conclusions1. Perfused, decellularized technique can achieve deculluarized effect. Theepithelial cells and muscle cells were removed, while extracellular matrix and cartilage were retained. The perfused technique can preserve thelaryngeal extracellular matrix and cartilage framework and construct anacellular whole laryngeal collagen scaffold.2. Decellularized larynx scaffold shows a low immunity laryngeal which couldbe a satisfactory material for laryngeal repair.