| Patients with advanced laryngeal or hypopharyngeal cancer sometimes need totallaryngectomy which impaired their speech and swallowing function. Patients with totallaryngectomy cann’t talk with other people often feel self-esteem, depression and anxiety.It was report that about45%patients with total laryngectomy have depressive symptoms,which are much higher than the patients with other cancer. With improvement of the headand neck surgery technology, the5-year survival rate has increased. Laryngeal cancertreatment should focus on to improve the patient’s quality of life from the simple improvethe survival rate. We shoule focue on recover and reconstruct in patient’s laryngealfunction.Reconstruction of the laryngeal function became the most fervent clinicaltreatment need for patients with advanced laryngeal carcinoma and severe throat injury.The larynx is a complex organ composed of cartilage, muscle, mucous membranes, and glands. Currently, there is no pratical way to reconstruct the comples structure of thelarynx by conventional surgery. Laryngeal transplantation is the only effectivereconstruction method for total laryngectomy and patients with severe laryngeal injury.The transplanted larynx can make the patient a near-normal voice and adequate swallowand respiration function. It has been reported that nearly75%laryngectomy patient willaccept a larynx transplant if offered. However, the first case of laryngeal transplantation ispatient with severe laryngeal injury, in order to avoid immune rejection aftertransplantation, long-term use of immunosuppressive agents in the following12years postoperation. Immunosuppressive agents which often lead to infection, tumor recurrence ormetastasis, greatly limited the wide spread use for patient with laryngeal cancer. Due tothe special nature of laryngeal anatomical structure, there is no natural or syntheticsubstitute that can mimic the anatomical structure of the larynx, recent advances in tissueengineering may offer a promising alternative to organ replacement. Decellularization ofallogeneic or xenogeneic donor organs such as the heart, liver, and lung provide anacellular, natural, three-dimensional (3D) biological scaffold material that can be seededwith selected cell populations. Preliminary studies in animal models have shown that a3D,decellularized matrix scaffold with seed cells can result in the formation of functionaltissue. A human decellularized laryngeal scaffold has also been made. However, studies onthe compliance of a decellularized trachea have shown that decellularization techniques,which have be used in decellularized tracheas and larynxes, can increase the complianceof the trachea, which will likely be at a high risk for tracheomalacia and potentially evenpostoperative stenosis when implanted in a recipient.Maintenance of the laryngeal framework depends mainly on the cartilage matrix. Aschondrocytes play important roles in synthesizing and secreting cartilage matrices andcollagen fibers, viable chondrocytes are vital for maintaining the3D structure of thelaryngeal framework. Cartilage is an avascular structure, in which chondrocytes receivenutrition by diffusion through the matrix. The absence of direct vascularization andpresence of a dense proteoglycan-collagen matrix will insulate chondrocytes from hostantigens. Therefore, perfusion of decellularizing agents through the laryngeal vasculature might remove the higher immunogenic tissue of the larynx and preserve chondrocyticviability.It is widely known that recipients of allografts tend to reject grafts without the use ofimmunosuppressive agents post-implantation. Immunogenicity depends largely on theexpression of major histocompatibility complex (MHC) antigens that can initiate organtransplantation rejection. Studies on the distribution of MHC-II antigens in the humanlarynx have shown that they were primarily found in mucosal surface epithelium,submucosal glands, and perichondrium, but not in thyroid chondrocytes or cartilagematrix, suggesting that the cartilage are not the major antigenic structures of the larynxand thus responsible for transplant rejectionWe constructed a low-immunogenic, whole-laryngeal scaffold in with the viabilityof laryngeal cartilage of the scaffold were preserved, while the cellular elements of othertissue were decellularized using perfusion technology by pump. The general observation,histological observation, microstructure observation, DNA content analysis, chondrocyteactivity analysis shown that structural integrity, non-immunogenic laryngeal cartilagescaffolds can be constructed; the acellular laryngeal cartilage implants to receptoromentum is detected the biocompatibility and immunogenicity of decellularzied laryngealcartilage scaffold. The bone marrow mesenchymal stem cells in vitro were amplificatedand seeded on the decellularized laryngeal cartilage scaffold. The cells could growth onthe scaffold. It could prove the toxicity and cell compatibility of the scaffold. Cell markedbone marrow mesenchymal stem cells could grow well on the scaffold, which couldprovide a theoretical basis for tissue engineering.Part one: In vitro study of the decellularized cartilage laryngeal scaffold[Objective] Constructe decellularized cartilage laryngeal scaffold by perfusion thedetergent through the Target-controlled infusion pump and in vitro studies scaffold, toexplore the feasibility of the preparation of tissue engineering laryngeal scaffold.[Methods] Male, SD rats, weighing200g, were randomly divided into experimental group and control group.The experimental group, separate and ligate the carotid arterybranches, then perfuse the detergent via the carotid artery to the superior thyroid arteryand the inferior thyroid artery by target-controlled infusion pump. The perfusion sequenseis heparin physiological solution,1%SDS,1%Triton X-100, PBS; The control group, thelaryngeal tissue removed from the body throat, without any treatment, just dipping in aPBS solution containing streptomycin and penicillin.The persistence time is as well as theperfusion time. Both the experment Specimens and control group specimens wereobserved by gross observation, histological observation, scanning electron microscopy,DNA content molecules, cartilage viability testing, and immunological detection.[Results] The images of the decellularized larynxes confirmed the efficacy of thedecellularization process in removing the majority of the cellular element. After14h ofperfusion with SDS, the larynxes appeared to be translucent and dilated, and retained theirshapes and stiff consistency. The treatment for decellularizing removed most of cellularcomponents from the mucosal and laryngeal muscle layers, and relatively preserved theextracellular matrix and laryngeal cartilage. The3D architecture of the larynx was notaltered. SEM demonstrated that all cellular components were removed, but the wholeorgan ECM network was preserved. After decellularzation, collagen bundles and ECMwith irregular luminal can be seen on the surface of laryngeal scaffolds (Epiglottis, falsevocal cord, vocal cord, cricoids and thyroid cartilage DNA analysis showed that thehousekeeping gene in the decellularized, cartilage-free, laryngeal sample were notamplified, but the GAPDH was amplified in the decellularized laryngeal samples withpreserved cartilage. The average DNA content of the decellularized laryngeal sampleswith preserved cartilages was less than that in the fresh laryngeal samples. Thechondrocyte viability of thyroid cartilage and cricoid cartilage were not changed greatly.Viable chondrocytes were found in all decellularized laryngeal cartilage layers. There wasno significant difference in the survival rate between the decellularized laryngeal cartilageand the fresh laryngeal cartilage.The decellularized larynx did not show the presence ofthe markers of MHC-I and MHC-II on the decellularized matrix. [Coclusions] The perfusion of decellularization detergents can construct adecellularized, whole-laryngeal scaffold while preserving the cartilage. The other cellularcomponents in soft tissue were mostly removed during this process. The decellularizedwhole-laryngeal scaffold had an excellent anatomical structure and might be a good sourceof tissue engineering for scaffolds or frameworks for laryngeal reconstruction and offers apromising reconstruction material for patients.Part two: In vivo study of the decellularized cartilage laryngeal scaffold[Objectives] Implanted the decellularized laryngeal scaffold into the allograftrecipient animals’ abdominal cavity and observed the response of the receptors on thedecellularized laryngeal scaffold. Explore the immunogenicity and biocompatibility of thescaffold.[Methods] The experiment group: put the BN rat perfution decellularized laryngealscaffold which was made as the part one described, on the receptor SD rats’ greateromentum. The control group: put the BN rat’s fresh larynx on the the receptor SD rats’greater omentum without any treatment. The experimental and control groups after2weeks,4weeks,8weeks,12weeks implantation, explanted the specimens for grossobservation, HE staining, CD3+T cells and CD8+T cell immunohistochemistry.[Results] Both the control group animals and experimental group animals allsurvived to each observation time, the animals are generally in good condition, goodwound healing, and no obvious wound dehiscence, bleeding, pus overflow situation. All ofthe implanted fresh larynx samples showed strong immunological rejection. The freshlarynxes were surrounded by fibrous capsules and greater omentum. The greater omentumwhich encapsulated the fresh larynxes became thicker weeks after implantation. Thecontrol group had shown that the lymphocytic infiltration in the external and internal ofthe implanted larynxes. Mucosa, glands, muscles, cartilage tissue were damaged bydifferent degree. Compared to the implanted fresh larynxes, the implanted, decellularizedlarynxes were covered by a thinner greater omentum and there were no signs of acute orchronic rejection. Neovascularization and fibroblaset-like cells were observed on the decellularized laryngeal scaffold4weeks post-implantation.[Conclusions] The perfusion decellularized laryngeal scaffold implanted in thereceport’s peritoneal cavity did not elicit strong immune rejection.At the same time therewere neovascularization and fibroblaset-like cells on the decellularized laryngeal scaffoldpost-implantation. This indicated that the decellulatized laryngeal scaffold with good cellbiocompatibility could promote the tissue and organ regeneration.Part three: Recellularization of the decellularized cartilage laryngeal scaffold[Objective] To investigate the methods of isolation, culture and identification of ratbone marrow mesenchymal stem cells (BMMSCs). Seeded the BMMSCs on thedecellularized cartilage laryngeal scaffold, observe the growth of the BMMSCs. Explorethe feasibility of the decellularized cartilage laryngeal scaffolds’recellularization.[Methods] BMMSCs were isolated from bone marrow of SD rats by desity gradientcentrifugation and differential ashesion methods. Cells were identificated by immunecytochemistry methods. The leach liquor of the decellularized laryngeal scaffold was usedto culture cells to observe the state of the cells and understand the toxicity of the scaffold.The BMMSc labeled by CM-Dil were seed on decellularized matrix of cricothyroid,posterior crico-arytenoid muscle and voval cord by the density1×106.The scaffold wasculture in37℃，5%CO2incubator for3days,5days7days. The histological, fluorescencemicroscopy, scanning electron microscopy were used to observe cell growth on thescaffold.[Results] The third generations BMMCs were growing fast. The morphology of thepassaged cell is relatively uniform.Most of them are spindle-shape, whirlpool growth,maintain a good growth state.Immuncytochemistry results showed that CD29and CD44positive. The positive rate is more than90%. The results is in coincidence with thestandards of BMMSc.The BMMSc have a good growth rate in the leaching liguor. AfterDM-Dil markers, bone marrow mesenchymal stem cells positive mark rate almost canreach100%.The CM-Dil-labeled BMMSc seeded on decellurized scaffold dispersed onthe surface of the stent.The seeded cells on the decellulatied scaffold can be observed growth on the outside and inside of the scaffold by histology study.The cells were in goodstate and evenly distributed.The scanning electron microscope showed that BMMSc wereshown that a small amount of cells were grown and extended between cells after3dayspost seeded.After5days seeded, the cells were distributed like a sheet on the surface ofthe scaffold. After7days, the cells on the surface of the scaffold is formed a layer offilm-like structure on the scaffold.[Conclusions] The pured BMMSc can be acquired by desity gradient centrifugationand differential ashesion method. The BMMSc seeded on the decellularized scaffold couldhave a good growth rate and generation. This could demonstrate that the decellularizedscaffold is non-toxic and does not cause cell death or mutation. There is a good affinitybetween the cells and the scaffold. The decellularized scaffold has a good biocompatibility.BMMSc can be used as seed cells for tissue engineering larynx. |
PDF Full Text Request