The Reconstruction And Transplantation Of Compound Tissue-engineered Tubal Trachea Consisting Of Tracheal Epithelium Mucosae And Cartilage

It is often necessary to resect laryngotracheal stenosis and to reconstruct theresulting defects for patients who have various types of tracheal disease liketrauma or tumor that cause stenosis.Circumferentially resected tracheas are conventionallyreconstructed by endtoendanastomosis,but the general limits of saferesection are about half of the tracheal length in adults and probably one third insmall childen.Tracheal resection longer than these limits requires replacement.Using autogenous tissue such as costal cartilage,nasal septal cartilage to patch thedefects takes risk of adding wounds and the source of grafts is lmited.Allograftsand artificial materials are also limited because of infection and longtermrejection.Therefore,effective production of grafts for tracheal reconstruction is very important.As tissue enginnering technique develops quickly,tissueengineeredtrachea have been thought highy of increasingly.The key to success fortracheal reconstruction is to reconstruct cartilage stent,reepithelization andrevascularization.Now,the cartilage stent has been made in nude mousesuccessfully.But functional tracheal epithelium and good vascularization are stillbig problems.Choosing cell stent materials is also very substaintial.Collagen is importantnot only for supporting tissue construction,but also for many kinds of cells beingcultured in vitro. Collagenous gel is also suitable for differentiation and formationof epithelial cells.With this method,culture of tracheal epithelial cells of severalspecies,such as rats,guinea pigs,rabbits,dogs,and humans has been reported [13].SIS is a nature,biocompatible,acellular,collagenbasedmatrix derived fromthe porcine small intestine submocosa. For the past few years,SIS has beenwidely used for the repair of skeletal muscle, abdominal wall, dura matermembrane,bladder,and vessels [4] .Also,some researcher fabricated the frameworkof a biodegradable artificial esophagus with the epithelial cells and the myoblastcells seeded on the small intestinal submucosa [5] .PGA,PLA and the copolymer ofthe both are main synthetic materials of cartilage tissueengineeringstents. In ourprevious studies,we demonstrated that human nasoseptal chondrocytesPGAconstruct could develop into a new cartilage in predetermined shapes in athymicmice.In this study,we would seeded primary rabbit tracheal epithelial cells andcartilage cells onto the surface of SIS covered by collagen and a nonwoven meshof polyglycolic acid(PGA) respectively to form epitheliumSISand chondrocytesPGAconstructs.Then the two layers were stratified to make tubershaped structure,which was implanted into the donor of athymic mice to grow. By investigatingthe compound grafts'growth in vivo and animals'survival time,we cansupply theoretical and experimental evidence for the construction of compoundtracheal framework.Objective1. To investigate an appropriate model of rabbit tracheal primary epithelial cellculture.To find suitable way to make SIS as scaffold for tissue engineering.Toinvestigate the biocompatibility of SIS with epithelial cells and to explore thepossibility to construct tissue engineering epithelium with SIS as the scaffold andepithelial cells as the seed cells.2. To evaluate the biocompatibility of PGA with cartilage cells and thedegradation of the two stents of PGA and SIS in vivo.3. To investigate compound grafts'variation in athymic mice and animals'survival time,and identify the grafts'growth and formation of blood vessels.Materials and methods1. Model of tracheal epithelial cells primary culture and selfmadeSISpreparation and coculturewith epithelial cellsRabbit tracheal ciliated epithelial cells were obtained by an explantoutgrowth culture system,cultured with serumfreemedium supplementedhormones and growth factors in vitro,observed by photocontrast microscopy.Antikaratin monoclonal antibody immunohistochemical stain and scanningelectron microscope(SEM) were employed to verify the characteristics ofepithelial cells.Derived from rabbit jejunum,SIS was dealt with steps chemicaldecellulaand examined by histological stain.Then the SIS taken from rabbits was mixed with epithelial cells taken from homogeneous rabbits for culture invitro to form epitheliumSISconstruct.Histological observation was done underphase contrast microscope.2. Coculture of primary chondrocytes with PGA and construction of tubershapedof tracheaCartilage cells obtained from rabbit joint cartilage by enzyme digestion werecultured in vitro.After 1 week,the cells were characterized by photocontrastmicroscopy and also examined with hematoxylineosinstaining and AB/PASstaining.Then the cells were seeded onto a nonwoven mesh of PGA to form acellPGAconstruct. Then epitheliumSISlayer was stratified onto chondrocytesPGAlayer to form a compound, and the compoumd was wrapped around adiameter of 4 millimeter glass rod, and bound by absorbable suture.3. Compound tracheal construction cultured in vivoThe compound grafts were implanted into forelimb oxter of 9 athymicmice separately.The grafts'growth in vivo and animals'survival time wereobserved. The specimens were harvested 4 ,8,12 weeks after implantation andsubjected to gross morphologic and histologic analysis.Results1. Model of tracheal epithelial cells primary culture and selfmadeSISpreparation and coculturewith epithelial cellsMany tracheal epithelium cells with good proliferation property were foundin the culture system after 1 week. Epithelial cells distributed widely andconfluented at 10th day.During that time ciliary beating was active,and theverification tests presented positive. HE staining showed the two layers of theepithelium and SIS.Collagen was not evident. 2. Coculture of primary chondrocytes with PGA and construction of tubershapeof tracheaPrimary cartilage cells was very active and with good proliferationproperty.The cells confluenced in about 1 week,and the histological stainingverified cartilage cell characteristics.Then the cells were seeded onto a nonwovenmesh of PGA to form a cellPGAconstruct. The construct with epitheliumSISlayer above could make tubershapein vitro by wrapping around a diameter of4 millimeter and lenghth of 7 millimeter glass rod.3. Compound tracheal construction cultured in vivoIn vivo study,no evident inflammatory infection and rejection of the constructsafter implantation were found.After animals were killed in batch, the glassrods were taken out of the specimens. The gross specimens of 4 weeks revealedapproximately the same shapes as original predetermined shapes.At 8 and 12weeks newlyformedcartilage had fair elasticity and support ability,and luminaswere almost integrated. Histological observation demonstrated that new cartilagecells and tracheal epithelial layer were not typical yet in 4 weeks. In 8 weeks,thecolumnar ciliated epithelium was observed towards the face of the lumina but notintegrated,and the PGA were almost degradated.Until in 12 weeks,the SIS of thespecimens were nearly absorbed.At the time,there was an epithelium mucosaeabove the cartilage layer,but the structure of blood vessels could not be foundunder the tracheal mucosa in the whole study.Conclusion1. The explant outgrowth culture system may be useful in establishing a culturesystem for ciliated cells. In this way, using cultured ciliated epithelial cells asseeds for tracheal tissue engineering was potentially.Steps chemical decellular method was a good way made SIS as scaffold for tissue engineering because ofits security,easy to conservation and remake,and operability.SelfmadeSIS can beused as the scaffold to construct tissue engineering epithelium as it has goodbiocompatibility with tracheal epithelial cells.2. PGA also has good biocompatibility with cartilage cells without disturbing thecells form or inhibiting the growth and function of cartilage cells,so it can be agood kind of tissueengineeringcartilage scaffold.PGA and SIS can be degradatedin 8 and 12 weeks in vivo.3. After the grafts were embedded into athymic mice in 8 and 12 weeks,trachealepithelium and cartilage could be observed in histological staining but nostructure of blood vessels could be found under the tracheal mucosa.Therevascularization of the constructs should be further studied for longtermapplication.