| Laryngeal transplantation is considered to be ideal method of laryngeal functionreconstruction. Now there are two clinical successful cases of laryngeal transplantation.But they have to use immunosuppressants for life, which will increase the risk of thetumor recurrence, opportunistic infections and metabolic disease. Biological materialsof low immunogenicity maked by cell technology, has been applied in the part oftissue repair. The cell biological material of early were limited to some tissue, such asskin, bladder, small intestine, thin layer of tissue or organs and the pericardium. Ottmade the first extracellular matrix scaffold of mice heart by perfusing coronary arteryin2008, which opened a new era of acellular bioscaffold of complex organ. We hadmade successfully the acellular bioscaffold of canine larynx and rabbit with Ott，smethodandcarried the immunology and mechanical testing, which confirmed that thebiomechanical properties of the scaffold did not be changedandwhich has thecharacteristics of low immunogenicity. But the bioscaffold whether can survive for along time when implanted into the host was not yet clear. We transplanted the saffoldinto pectoralis major of host canineandmade sure the activity through grossobservation, histological examination andcartilage cell survival rate, further estimatedthe character of the caffold. To explore new methods for whole laryngeal functionreconstruction. Objective:1. To make Complete canine dog，s larynx scaffold of acellular by perfusion method,make sure whether there are change on the morphology, organization structure,immunogenicity and the cartilage cell activity of the dog，s laryngeal.2. To make sure the larynx scaffold of acellular whether can survival for along-term in pectoralis major of reservoir dogs by animal preparetions experimentin vivoandprovides the experimental basis and theoretical guidance for the nextlaryngeal transplantation.Method:1. To make the canine larynx scaffold of acellular and finish its related inspection.The canine larynx in experimental group retain cricothyroid arteries on both sides,infused continuously with SDS, TritonX-100, and remove all cell componentsin withstrong immunogenicity in laryngeal mucous membrane, muscle and ligament, retainthe extracellular matrix of immunogenicity and cartilage cells of low immunogenicity,obtain the canine larynx scaffold of acellular with low immunogenicity, conduct theinspection by general observation, histological examination, immunological test andcartilage cell survival rate.2. Preparetions experiment in vivoThe canine larynx scaffold of acellular and laryngeal specimens were implantedinto the right side pectoralis majorto of host dog of experimental group and controlgroup respectively. The host dogs were executed at2nd weeks,1st month, and2ndmonth separately postoperation, take out the buried plantsandconduct and finished theinspection by general observation, histological examination and cartilage cell survivalrate.Result；1. To make the canine larynx scaffold of acellular and finish its related inspection. The Color white, the form was not change, surface did not collapse, there was nostatistical significance on left-right diameter size of lamina of cricoid cartilage beforeand after the infusion of the canine larynx scaffold of acellular. Epithelial cells,muscle cells, gland and blood vessels were disappeared, perichondrium slightlydiscrete, cartilage lacunae was filled with the cartilage cell, which were seen on larynxscaffold of acellular by HE dyeing. There was statistically significant on DNA contentof cricothyroid muscle before and after the infusion. But DNA content of cricoidcartilage, thyroid cartilage and epiglottis cartilage did not show statistically significantbefore and after the infusion. Acellular tissue matrix of cricothyroid muscle, posteriorcricoarytenoid muscle and transverse arytenoids did not express housekeeping genesβ-actin. There was no statistically significant on cartilage cell survival rate before andafter the infusion.2. Preparetions experiment in vivoAll host dogs live to end point of observation. General observation ofexperimental group：the scaffold redden after two weeks；the surface adhere redmembrane material；The red is more significant, soft tissue on the surface thickening,volume Smaller at1st month；we can see blood vessels in surface of soft tissue of thescaffold after two month. General observation of control group：the specmin whiten,cartilage thinned, joint disconnected, there are inflammatory secretions around partiallaryngeal, there are not adhesion with the surrounding muscles after two weeks；Therewere Small visible cartilage remnants, which attached connective tissue after1month.Cricoid cartilage left-right diameter comparison： there was statistically significant(P <0.05) at each time point postoperation experimental group compared with controlgroup；there was not statistically significant (P>0.05) that2nd weeks postoperationcompared with pre-operation and2nd month postoperation compared with1st monthpostoperation in experimental group；there was statistically significant (P <0.05) that1st month postoperation compared with2nd week postoperation；there was statistically significant (P <0.05) that2nd weeks postoperation compared with pre-operation incontrol group. HE staining show：the scaffold was infiltrated by leukocyte, butcartilage cells and perichondrium has no obvious change at2nd week postoperation；loose connective tissue was seen on the surface of the scaffold, fibroblast cells andblood vessels were visible by high-power microscope at1st month postoperation；loose connective tissue thicken, fibroblast cells and blood vessels grown in number orquantity, perichondrium thinned, Cartilage cells were filled in cartilage lacuna at2ndpostoperation；the laryngeal specimens of control group were infiltrated with a largenumber of lymphocytes and perichondrium were complete after2weeks, connectivetissue proliferated, perichondrium fractured, lymphoid cells came into cartilage andcartilage cells were absorbed largely at1st month. There were no statisticalsignificance(P>0.05) about Cartilage cell survival rate that at2nd week postoperationcompared with pre-operation,1st month postoperation compared with2nd weekpostoperation,2nd month postoperation compared with1st month in experimentalgroup. There were statistical significance(P<0.05) about Cartilage cell survival ratethat at2nd week postoperation compared with pre-operation and1st monthpostoperation compared with2nd week postoperation in control group.Conclution:1. We could get the complete canine larynx scaffold of acellular by cricothyroidartery perfusing ion agent, which dislodged cells components with strongimmunogenicity of muscle and mucous membrane and retained extracellularmatrix of no immunogenicity and chondrocytes of low immunogenicity. The form,size and volume of the scaffold did not obvious change and biomechanicalproperties did not change. The scaffold could be used as a biological engineeringmaterials for next embedded experiment in vivo.2. Normal laryngeal induced acute rejection after embedded in vivo, larynx scaffoldwas damaged gradually and disappear. 3. The canine larynx scaffold of acellular did not take place severe immune rejectionafter embedded in vivo. the form was normal, volume diminished slightly, thesurface was covered by loose connective tissue and cartilage cells keeped highersurvival rate of the scaffold at2nd month postoperation, which could providesupport for all laryngeal transplantation of next step. |
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