A Tissue-engineered Laryngeal Reconstruction: Using Perfusion-decellularized Natural Platform

The laryngeal functional reconstruction after total laryngectomy remains one of the most challenging aspects.The routine trentment in clinical medicine is pharynx-to-esophagus anastomosis and laryngeal stoma to keep respiration which brings enormous burdens to both patients and society.It was counted by International Non-larynx Individual Association that every year about 136 thousand individuals worldwide were diagnosed as laryngocancinoma and many of them faced a total laryngectomy.The ideal laryngeal functional reconstruction should achive the following four points:①To restart phonation by near normal breath and vocal cord ;②To set up a near normal swallowing function and prevent accidental aspiration ;③To maintain the upper respiratory tract and upper gastrointestinal tract function through oropharynx and nasal cavity ;④To satisfy the aesthetic demands.The ideal treatment for laryngeal functional reconstruction is still laryngeal transplantation, however, once a larynx is transplanted, individuals must undergo the lifelong immunosuppression and often trade for tumor recurrence, metastasis and multi-infection [2].It is all known that larynx is not the necessary organ for life.It is still a controversy that whether it is worth for patients undergoing the laryngeal transplantation at the risk of their lives or not. The creation of a low immune bioartificial larynx could theoretically solve those problems. Rencently the rapid development on tissue engineering brings the new solution for those problems.The biological scaffolds derived from decellularized organs or tissues retain the extracellular matrixs (ECMs) which are mainly consisted by collagen, have nealy no immunogenicity, provide scaffolds for new tissue growth and induct cells'adherence, migration and proliferation.Mesenchymal stem cells (MSCs) possess high proliferative and multi-potent differentiation capacities in vitro and they can differentiate into every tissue of mesoderm such as chondrocytes, myocytes and vascular endotheliocytes. Consequently, they have attracted great attention as a promising tool for tissue engineering and regenerative medicine in the last two decades. MSCs can be inducted into skeletal myocytes or cadiocytes by 5-azacytidine .It is a classic pathway to induce MSCs into myoblast by 5-azacytidine.In our study, we attempted to decellularize posterior cricoarytenoid muscle, lateral cricoarytenoid muscle and thyroarytehoid muscle by common carotid arterious perfusion with detergents and reserve intact three-dimensional geometry of extracellular matrix. We then repopulated decellularized rabbit larynxes with autogenetic mesenchymal stem cells of receptor rabbits induced by 5-azacytidine in vitro.And then we transferred those constructs into receptor rabbit greater omentums for cell growth and vascularization. We investigated that whether this method had the potential to facilitate a tissue-engineered method for laryngeal reconstruction. Objectives1. To compare the difference of the deculluarized larynx scaffold between perfusion method and immersion method, and find better way made deculluarized larynx as scaffold for tissue engineering.2. To investigate the biological characteristics of MSCs in vitro and its feasibility to be inducted differentiation into myocytes.3. To prepare a decellularized whole laryngeal scaffold by utilizing a perfusion-decellularized technique, reseed cells on it, and then construct a recellularized whole larynx. To provide the laryngeal functional reconstruction after the total laryngectomy with a new solution.Methods1. A comparative study of preparing decellularized larynx scaffold between perfusion method and immersion methodAll the larynxes were excised in a sterile fashion. The deculluarized larynx scaffold was obtained by perfusion method and immersion method respectively, and then comparative examinations were performed by the macroscopic view, histological view, scanning electron microscope (SEM) and cartilage vitality assay.2. Myogenesis induction and amplification of BMSCs inducted by 5-azacytidineBone marrow was aspirated from chinchilla rabbits'femurs in a sterile fashion. At 80% confluence, primary cells were washed twice in PBS and then cultured with 5-azacytidine for 24 hours to differentiate them into skeletal myocytes. Inverted microscope observation, cell growth curve and immuocytochemistry were performed. 3. A perfusion-decellularized technique for tissue-engineered larynx reconstructionPerfusion decelluarized larynxes were obtained by common carotid arterious perfusion with detergents.And then decellularized laryngeal scaffold were reseeded with inducted mesenchymal stem cells (MSCs). Composites were transferred into greater omentums of rabbits after one day's adherence and harvested after 4 weeks and 8 weeks respectively. Macroscopic view, histological examination and immunohistochemistry were performed.Results1. A comparative study of preparing decellularized larynx scaffold between perfusion method and immersion methodMacroscopic view showed that the larynxes perfused by sodium dodecyl sulphate(SDS )became transparent after two hours'perfusion, but the larynxes immersed by SDS over 16 hours still appeared pink-white . Histology and SEM indicated that compared with immersion group , perfusion group showed better deculluarized effect, more ventages and collagen fibers were retained , no intact cell or nuclei remained in decelluarized matrix and chondrocytes were still survival . Chondrocyte vitality assay indicated that the chondrocyte vitality rate of perfusion group was higher than 85% while the chondrocyte vitality rate of immersion group was lower than 80%.The chondrocyte vitality of perfusion group was superior to that of immersion group (P<0.01) .2. Myogenesis induction and amplification of BMSCs inducted by 5-azacytidineThe primary BMSCs isolated by density gradient centrifugation became adherent after 48-to-72 hours and showed fusiform shape after culturing for 4 days.Cells were at 80% confluence after 7-to-10 days .Cell growth curve indicated that the proliferation speeds of cells from P1 to P7 were not obviously different. Cells inducted by 5-azacytidine enlarged with pseudopodia protrusion and kept fusiform after three weeks. Immuocytochemistry examination showed positive expression for sarcomericα-actin.3. A perfusion-decellularized technique for tissue-engineered larynx reconstructionAll animals survived before samples were harvested. After 4 or 8 weeks vascularization formed and integrated cartilage frameworks still remained. Histological examination could clearly show the presence of muscle bundles and vessels. Immunohistochemical examination indicated that sarcomeric-αactin expressed positively in corresponding areas.Conclusions1. Compared with immersion method, perfusion method is a better way to construct deculluarized larynx as scaffold because it can achieve better deculluarized effect and retain chondrocyte vitality at the greatest extent in the deculluarized larynx scaffold.2. BMSCs can be inducted to differentiate into myocytes in vitro and can be chosen as seed cells for recellulariztion of decellularized larynx.3. It is feasible to reseed MSCs into the decellularized laryngeal muscle matrix for constructing a tissue-engineered larynx. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct. This construct has the potential to facilitate a tissue-engineered construct for laryngeal reconstruction.