Biomechanical Study On Canine Decellularized Laryngeal Scaffold

Total laryngectomy used in the treatment of advanced laryngeal cancer has saved many patients, lives, but it causes permanent loss of laryngeal function, and seriously affects the quality of life of patients. There is still no ideal method of reconstruction. To solve the reconstruction of laryngeal function, doctors try to perform laryngeal transplantation. On the one hand, the human laryngeal transplantation of allogeneic donor source is inadequate; on the other hand, severe immune rejection limits the conduct of laryngeal transplantation. Lifelong immunosuppression can be partially reduced immune rejection, but lead to tumor recurrence, metastasis and infection. The development of tissue engineering makes repair and reconstruction of tissues and organs possible. In the revelation of the skin, cartilage and other tissues and organs, the researchers have tried to use tissue engineering to reconstruct whole larynx. Because of the fine structure and complexity of the whole larynx, research work is now still concentrated in the reconstruction of partial laryngectomy defect. Reconstruction of whole larynx after total laryngectomy needed for further innovation. In recent years, decellularized scaffolds provide a new method for reconstruction. Acellular Technology removes cells in the organs or tissue and retains the natural structure and micro-environment. Ideal tissue engineering scaffold materials not only maintain certain physical and chemical properties, but also need to have certain mechanical strength to maintain its structure. Hou et al[1]prepared a soft tissue decellularized laryngeal scaffold with intact laryngeal cartilages using a perfusion-decellularized method, reseeding muscle cells on it, and then reconstructed a recellularized larynx. Histological examination clearly showed the presence of vessels and muscle bundles. Immunohistochemical examination indicated that sarcomeric-? actin expressed positively. Ma et al[2] implanted the laryngeal scaffolds in para-laryngeal muscles of rabbits and harvested after several weeks. The decelluarized larynx did not show obvious immunological rejection in the recipient rabbits. This research revealed that the laryngeal scaffolds have good biocompatibility and low immunogenicity. However, whether the cartilage has good biomechanical properties after perfusion and or laryngeal scaffolds can meet the supporting role are still uncertain. In our study, we attempted to decellularize muscle through bilateral cranial artery antegrade infusion with detergents and retained intact three-dimensional geometry of extracellular matrix, effective removal of the larynx muscles and mucosal tissues which have strong immunogenicity. We detect biomechanical properties of the decelluarized scaffold before and after the infusion, and to provide a new method for laryngeal reconstruction.Objectives1. To prepare a decellularized canine laryngeal scaffold by using a perfusion-decellularized method .To clarify if preparation of canine laryngeal scaffold is feasible and to find the best reperfusion method.2. To evaluate mechanical properties of the deculluarized larynx scaffolds, and to provide theoretical and experimental basis for reconstruction of laryngeal functional after the total laryngectomy.Methods1. Preparation of canine decellularized laryngeal scaffold by using a perfusion-decellularized method. The acellular larynx scaffold was obtained from dogs through cranial thyroid arteries perfusion with detergents. They were evaluted by the macroscopic view, histological view (hematoxylin and eosin stain) and scanning electron microscope (SEM).2. Biomechanical study on decellularizaed canine laryngeal scaffoldComparative examinations were performed by histological view (Alcian blue stain and Masson stain), SEM and biomechanical properties between perfusion group and control group. IPP6.0 software detected the content of collagen and glycosaminoglycan on the basis of Alcian blue stain and Masson stain, and SEM were used to observe cartilage matrix. The compressive modulus and tensile strength were used to evaluate biomechanical properties.Results1. Preparation of decellularized canine laryngeal scaffold by using a perfusion-decellularized methodThe control group showed red and structural integrity; the reperfusion group showed that cricothyroid muscle can be transparent after 3 hours'perfusion by sodium dodecyl sulphate (SDS), and then after 20 hours'the muscle and mucosa completely transparent. The decellularized laryngeal scaffold appeared pale asphyxia at last. The structure does not complete collapse, in addition to color changes in external morphology compared with the control group, no significant change in larynx.HE staining: control group showed complete laryngeal muscle fibers, and chondrocytes were filled in cartilage lacunae; experimental laryngeal muscle cells have been removed, meanwhile the fiber structure still existed and retained the direction of the extracellular matrix. The structure of cartilage was integrity, and a large number of active cells were filled in the lacunae in the cartilage. Scanning electron microscope (SEM): the control group showed structural integrity and no pores of muscle. Muscle cells of perfusion group had been completely removed, the fiber network structure were not destroyed.2. Biomechanical study on canine decellularizaed laryngeal scaffoldAlcian blue stain and Masson stain: IPP software results showed that there were no obvious changes about lycosaminoglycan and collagen between perfusion group and control group. Scanning electron microscope: collagen fibers of cartilage matrix were no significant changes, and no obvious fracture after perfusion.The compressive modulus of thyroid cartilage was (1.0645±0.0733)Mpa in experimental groups and (1.1476±0.1078)MPa in control group ,showing no significant difference(t=1.424,P>0.05), neither in compressive modulus of annular cartilage {(1.6849±0.1093) Mpa in experimental groups and (1.6715±0.0947) Mpa in control group, t=0.627,P>0.05}.The tensile strength of thyroid cartilage between experimental(5.7358±0.8849) Mpa and control groups(6.1824±1.3259) MPa did not have the statistical significance(t=0.627,P>0.05)Conclusions1. The improved perfusion way can effectively remove cells of dog's muscles and laryngeal mucosa, and retained the spatial structure of the extracellular matrix.2. The biomechanical properties were no significant different between control group and perfusion group. These results indicate that perfusion method can construct a perfect biomechanical acellular larynx scaffold which could be a better selection for laryngeal reconstruction with tissue engineering method. Key words:Larynex; Stents; Perfusion, regional; Compressive strength; Tensile strength; Dogs...