工程化组织的构建.pptx

工程化组织的构建.pptx

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四川大学华西临床医学院干细胞与组织工程研究室秦廷武,Dec.22,2006,工程化组织的构建（Constructofengineered-tissues）,Theconceptoftissueengineering,ThetermtissueengineeringwasofficiallycoinedataNationalScienceFoundationworkshopin1988tomeantheapplicationofprinciplesandmethodsofengineeringandlifesciencestowardfundamentalunderstandingofstructure-functionrelationshipsinnormalandpathologicalmammaliantissuesandthedevelopmentofbiologicalsubstitutestorestore,maintainorimprovetissuefunction.,Purposesoftissueengineering,Todevelopbiologicalsubstitutesforimplantationintothebody;Tofostertissueregenerationandremodeling;Toreplace,repair,maintain,orenhancefunctionoftissues.,MedicalapplicationpurposeResearchpurpose,Testingnewdrugs;Simulatingdiseasestodevelopbettertreatments;Reducingtheuseofanimaltissuesandorgansinbiologicalresearch.,组织工程研究内容,组织工程研究过程,工程化组织,构建模式体内体外,细胞改造,材料设计加工检测,生物学及力学检测,植入技术修复技术,细胞接种应力与生长,生物反应器、力学诱导与信号的转导,种子细胞,干细胞工程,支架材料,工程化组织的保存,植入技术修复技术,Simpleculturetechniques,Ifspecificsignalsarenotproperlyreceivedorprocessed,thenthecellsdedifferentiate（i.e.,becomenonspecificcelltypes）,becomedisorganized,andeventuallydie.,Dynamicmicroenvironment,Cellcoloniesneedexternalcuesorsignalstogrowintofunctional3Dtissues.Cellsareconstantlybombardedwithmechanical,electrical,structural,andchemicalcuesthatsignalthecellsaboutwhattheyshouldbedoing.,工程化组织的理解,Specialculture,Engineeredtissue,Theengineeredtissue，orconstruct，usuallyconsistsofthree-dimensionalscaffoldsandlivingcellswiththedesiredstructureandfunctionality.,细胞材料有机结合体,细胞,材料,？

BioreactorsforTissueCulture,Bioreactorsarepurpose-designedtissueculturesystem；Providingthedesiredbiochemicalandmechanicalenvironmentforcelladhesion,proliferation,differentiationandfunction；Devisinganddevelopingnewtypesofbioreactors,particularlyaddressingmasstransferandmechanicalsignaling.,简单,复杂,低维,多维,On-linemonitoringinengineeredtissue,Monitoringofthecellviability,functionalityandtissueformation；Developingcontinuoussamplingandanalyticaltechnologiesusingmicro-ornano-probes；Enablingefficientcontrolofthetissueculturebioreactorandoptimizationoftheconstructdesignandbio-processingoperation.,Cryopreservationofengineeredtissues,Owingtothelongproductioncycle,forexamplethreeweeksforengineeredskingraftstoeightweeksforengineeredcartilage,preservationofengineeredtissuesiscriticaltoensuretheoff-the-shelfavailabilitytoclinicians.Preservationandstorageofliveengineeredtissues,orbio-constructs,wouldbeoneofthemajorobstaclesforthecommercialization.,Mathematicalmodelingintissueengineering,Developingmathematicalmodelsforcellgrowthandtissueformation；Devisingexperimentaltechniquestodeterminemodelparameterssuchasoxygenconsumptionrateandeffectivediffusivityinthetissue；Themodelscanbeappliedbothinvivoandinvitro.,工程化组织的构建方法,体外构建（工程化椎间盘）,体内构建（工程化耳软骨）,体外构建,静态:

细胞或支架相对静止；动态:

细胞或支架相对运动；存在的问题：

环境条件、营养条件（生物反应器）。

在体外模拟体内细胞的动态微环境，包括生物化学、生物物理环境。

提供组织特有的生物的、化学的和物理的刺激，产生相应的传导信号。

并提供细胞营养及代谢通路。

生物反应器系统,静态:

常规二氧化碳培养箱；动态:

旋转培养系统、拉伸/压缩/扭转培养系统、电磁培养系统、声学/光学培养系统。

PerfusionofCultureSystem,Improvemasstransportthroughoutthescaffoldbyperfusingmediathroughcellseededscaffoldsandstimulatethosecellsviaflow-inducedshearstress.,Constructsareperfusedusingaperistalticpumpatvaryingflowratestoenhancematrixmineralization.,体外构建人工皮,Engineeredskinconstructsinvitrokeratinocytesgrowingonaplasmapolymersurface,SEM,体外构建的耳软骨,Newlyformedinvitroauricularcartilageconstruct,Earcartilageconstructwiththechondrocytes,动态构建:

有软骨陷窝;有软骨基质分泌.静态构建:

无软骨陷窝;无软骨基质分泌.,静态、动态构建的差异（软骨）,本位：

功能位置；皮下：

浅表位置；肌内：

深度位置；骨内：

骨膜内。

体内构建,体内构建的皮肤,Reconstructedhumanskinonthedorsalofthenudemiceat4weeksaftertransplantation,体内构建气管软骨,皮下：

浅表位置,皮下构建气管软骨,体内、外构建的差异,Invitroconstructsformedandmaintainedat8weeks:

Yellowishglisteningsurface；Resemblingnativecartilage；Stablebutsoftinconsistency.,Invivoconstructsaftermaturationinmice：

Whitishwithglistening；Firminconsistency.,体内、外构建的差异（关节软骨）,Articularcartilageconstructsinvitro,Articularcartilageconstructsinvivo,体内、外构建的差异（皮肤）,Apligrafconsistsoflivingcellsandstructuralproteins:

Lowerdermallayercombinestype1collagenandfibroblasts（dermalcells）,whichproduceadditionalmatrixproteins;Upperepidermallayerisformedbypromotinghumankeratinocytes（epidermalcells）tomultiplyandtodifferentiatetoreplicatethearchitectureoftheepidermis.,Apligrafwithout:

Melanocytes;Macrophages;Lymphocytes;Bloodvessels;Hairfollicles;Sweatglands.,工程化组织的检测,检测对象：

细胞、支架、工程化组织及植入后新生组织;检测指标：

形态学、组织学、分子生物学及生物力学。

形态学检测,光学显微镜；电子显微镜；荧光显微镜；激光共聚焦显微镜；原子力显微镜。

组织学检测,固定、包埋；切片（软、硬、冰冻）；染色（免疫组织化学：

因不同组织而定）；荧光或同位素标记；测量（半定量、定量）。

生物力学检测,样品制作、保存；实验设计：

选择指标、样本量、数据处理及统计方法；静态实验（拉伸、压缩、剪切、弯曲、扭转）；动态实验（疲劳、磨损、寿命）。

生物力学检测指标,强度指标：

拉伸强度、压缩强度、弯曲强度、扭转强度、剪切强度和疲劳强度；刚度指标：

扬氏模量、剪切模量、弯曲模量、扭转模量等；塑性指标：

延伸率、最大应变、断面收缩率、粘性系数等。

工程化肌腱的静态构建,体外静态构建举例,种子细胞与支架材料,种子细胞：

第2-4代鸡腱细胞,支架材料：

用缝线复合的PGA网,细胞接种及培养,细胞接种:

接种密度1106/支架）,细胞-支架复合物;静态培养时间:

6天;培养条件：

37，5%CO2，饱和湿度。

37，5%CO2,细胞-支架复合物,细胞接种工程化肌腱,静态构建的工程化肌腱,腱细胞长入支架中,沿纤维方向平行排列,并分泌胶原基质,组织学检测（HEstaining）,2W4W,6W,8W,组织学检测（Mallorystaining）,2W4W,6W,8W,支架材料的降解,2week,4week,6week,8week,454035302520151050,PGA支架TET2周4周6周8周,材料重量（mg）,支架材料降解速率,称重法,植入前后抗拉强度,120100806040200,支架,TET,2周,4周,6周,8周正常肌腱,抗拉强度（MPa）,单轴拉伸实验：

每个时间点5个样本;加载速度:

20mm/min;应力下降80%停止加载。

植入后胶原生成量,1000,300200,400,2周,4周,6周,8周,正常肌腱,羟脯氨酸（ug/gDryWt.）,*,*,碱水解法测羟脯氨酸,静态构建的结论,静态培养,无体内肌腱细胞特有的力学刺激;支架降解过快;植入后力学刺激小,粘连形成多;胶原合成少,且排列方向无明显取向性;新生腱生物力学强度差.?

!

?

工程化肌腱的体外动态构建,3-DCultureofTendonCellsunderMechanicalStrain,细胞特性,生物学特性：

形态学：

正常贴壁依赖性成纤维样肌腱细胞；致瘤性：

不致瘤；遗传性状：

二倍体细胞；结构：

正常细胞结构；功能：

能分泌型胶原。

力学特性：

变形性；黏附性；,3-DScaffold,3-Dscaffold,2g/mlCNI,5g/mlFN,PLGA85/15,PVAfoam,支架材料形态,大体,表面微观（SEM）,支架材料的力学特性,0,2,4681012Strain（100%）,Stress（MPa）,PVA+PLGA21.510.50,CellseedingIn3-Dscaffold,3-DScaffold,Cell-ScaffoldComposite,CellSuspension（2105/ml）,Seedingtwice!

Strainculturedevice,中国发明专利号:

ZL98121829.6,Mechanicalstrainpattern,10%elongation;12cycles/min;15min/h48h;48hrs.,应变场生物反应器,Scanningelectronphotomicrographs,静态组,动态组,组织学检测,H.E.染色,静态组（48h,100）,动态组（48h,100）,Proliferationresponse,210,3,4,HETCs,THETCs,TendoncellProliferationisincreasedundermechanicalstrain,Cellnumber（106/sponge）,ControlStrain,*,DNAsynthesis,Whenmeasuringcontinuous3HthymidineincorporationintoDNAittook48htodemonstratestretch-mediatedenhancementofDNAsynthesis,*,*,32.521.510.50,HETCsTHETCsTendoncell,3H-TdRincorporation（cpm103/sponge）,Control,Strain,Collagensynthesis,0.40,0.8,1.61.2,HETCs,THETCs,肌腱细胞Collagensynthesisissignificantlydifferentinstretchedculture,comparedwithnonstretchedcontrolculture,3H-Proline掺入量（cpm104/支架）,ControlStrain,*,*,Cellsbecamemoreactive,andspreadtothedirectionofthestrain.Aftera48-hourexposuretothestrain,cellnumberDNAsynthesiswereincreasedcomparedwithcontrols.Thecyclicstrainalsocausedanincreaseincollagenmatrixsynthesisbytendoncells.Cyclicmechanicalstrainsacteddirectlytostimulatethegrowthoftendoncells.Theseresultsarecompatiblewithasignificantroleforstrainintheconstructofengineeredtendonsinvitro.,动态构建的结论,Manychallenges,Missinginformationonhowtodevelopuniversaldonorcellsthatcouldbegiventoanyrecipient；Howtostimulateregenerationofcomplexmulticellularstructuresinvivo；Howtodirectthefunctionofitscells；Howtovascularizetheengineeredtissues;Successintissueengineeringisgoingtorequireinterdisciplinaryparticipationanddetermination.,Multi-disciplinarycooperation,Biologistssourceandgrowthecellsthatwillbeusedtoconstructtheengineeredtissue/organ;Chemistsdevelopasuitablescaffoldonwhichthecellscanbegrown;Engineersfabricatethescaffoldandensureithastheappropriatephysicalcharacteristics;Biochemistsandphysiologiststestcellsgrownonthescaffoldtoensurethattheyfunctioncorrectly;Cliniciansimplantandtesttheorgan/tissueinpatients.,