Cartilage TE from in vitro and in vivo models to the clinic Module 3 Lecture 6 20 109 Spring 2010 Lecture 5 review What are some advantages of ELISA as a protein assay What are some pros and cons of endpoint RT PCR as a transcript assay 2 Topics for Lecture 6 Imaging assays Cartilage TE in vitro Cartilage TE in vivo Cartilage TE in the clinic 3 Day 5 6 image analysis Imaging data is often high throughput 4D time x y z requires computation and human design interpretation Many available analysis packages some 20 30K NIH ImageJ free Your analyses cDNA band intensities automated cell counts optional explore other features Nature Publishing Group All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse 4 Source Mempel T R et al T cell Priming by Dendriticcells in Lymph Nodes Occurs in Three Distinct Phases Nature 427 8 January 2004 154 159 doi 10 1038 nature02238 Fluorescence microscopy Light source Epi uorescence lamp Hg Xe Confocal laser Ar HeNe 2 photon pulsed laser Red fluorescence collected for viewing imaging Filter cube Excitation Dichroic mirror Emission Band pass vs long pass Aperture diaphragm Field diaphragm Filter cube Green light excites rhodamine Arc lamp Red fluorescence excited Detection CCD camera Image by MIT OpenCourseWare Image from Lichtman Conchello Nature Methods 2 910 2005 5 Speci cations for Day 3 imaging Live Dead Dyes Green 490 ex 520 em Red 490 ex 620 em Excitation 450 490 nm Dichroic 500 nm Emission 515 nm Images removed due to copyright restrictions 1 Schematic of filter cube optical block Figure 1a at http www microscopyu com articles fluorescence filtercubes filterindex html 2 Spectrum profile graph for Nikon B 2A Medium Band Blue Excitation at http www microscopyu com articles fluorescence filtercubes blue b2a b2aindex html 6 Types of microscopy Epi uorescence noisy due to out of plane light Confocal pinhole rids out of plane light 2 photon femtoliter volume excited in plane Epi uorescence Confocal 7 Confocal uscopy permits 3D reconstruction 8 Polymer composite for cartilage TE Porous PLA scaffold stem cells Cells loaded in medium PLA elongated morphology Cells loaded in alginate round morphology improved cell retention PLA PLA alg PLA alginate Caterson et al J Biomed Mater Res 57 394 2001 Copyright 2001 John Wiley Sons Inc Reprinted with permission 9 Chondrogenesis in vitro Porous PLA scaffold w or w out alginate Alginate alone somewhat chondrogenic Alginate TGF better than PLA TGF PLA TGF ALG TGF PLA Day 7 Day 14 ALG Caterson et al J Biomed Mater Res 57 394 2001 Copyright 2001 John Wiley Sons Inc Reprinted with permission 10 Scaffold free in vitro cartilage TE Method rotational culture of rabbit chondrocytes with no cytokines Results Mostly dynamic culture gave best results low apoptosis very rigid disc Fresh ECM made primarily CN II and PG Organized architecture similar to in vivo Static Dynamic 3 d A scaffold free method is inherently biocompatible Any disadvantages Pros cons of cell free methods Dynamic 3 w T Nagai et al Tissue Eng 14 2008 Courtesy of Mary Ann Liebert Inc Used with permission Source Nagai T et al Characteristics of a Scaffold Free Articular Chondrocyte Plate Grown in Rotational Culture Tissue Engineering Part A 14 no 7 July 2008 1183 1193 11 Interlude What TE topics would you like to hear more about list on board tree kangaroo cutest animal ever scienti c proof Photo of young tree kangaroo removed due to copyright restrictions Cells and scaffolds in vivo Y Liu et al Tissue Eng 12 3405 2006 Stem cells and or injectable natural matrix gelatin HA in rabbit knee defects Matrix and cells both contributed synergy 4 wks 12 wks Courtesy of Mary Ann Liebert Inc Used with permission Source Liu Y et al Osteochondral Defect Repair with Autologous Bone Marrow Derived Mesenchymal Stem Cells in an Injectable in Situ Cross Linked Synthetic Extracellular Matrix Tissue Engineering 12 no 12 December 2006 3405 3416 doi 10 1089 ten 2006 12 3405 13 Large animal in vivo model D Barnewitz et al Biomaterials 27 2882 2006 Biodegradable scaffold with autologous cells Examined horses and dissected joints after 6 12 months Matrix synthesis implant integration with native tissue Why use a large animal model vs small native repair treated native untreated repair Courtesy of Elsevier Inc http www sciencedirect com Used with permission 14 Advantages of working in vivo Ability to mimic human disease state Ability to mimic therapy surgery applied to humans especially true for large animal models Can compare results to gold standard treatment The construct interfaces with an actual wound the immune system etc more realistic environment Toxicity studies more meaningful 15 Cartilage pathology Cartilage has little regeneration capacity why Early damage can promote later disease Osteoarthritis pathology PG and collagen loss PG size water content strength chondrocyte death Symptoms loss of mobility pain Aggrecan Image 2002 OPML Courtesy of OPML http web mit edu cortiz www AFMGallery AFMGallery html V C Mow A Ratcliffe and S LY Woo eds Biomechanics of Diarthrodial Joints Vol I Springer Verlag New York Inc 1990 16 Treatments for cartilage damage Strategy 1 enhance provoke healing biologics hyaluronic acid TGF etc damage bone stem cell effect Strategy 2 replace tissue joint replacement synthetic or donated tissue invasive or ber optic partial cell and or scaffold implantation immature therapy Other supplemental mechanical electrical stimulation debridement rid debris Public domain image Wikimedia commons S W O Driscoll J Bone Joint Surg 80 1795 1998 S Poitras et al Arth Res Ther 9 R126 2007 C M Revell K A Athanasiou Tissue Eng Pt B Rev 15 1 2009 17 Cutting edge of treatment Cell based therapies on the market e g Carticel Scaffold based approaches in trials e g NeoCart INSTRUCT The Center for Orthopaedics and Sports Medicine All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse Histogenics Corporation All rights reserved This content is excluded from our Creative Commons license For more information see http ocw mit edu fairuse 18 Many clinical trials are ongoing FGF MSCs Screenshot from www clinicaltrials gov May 2010 19 Lecture 6 conclusions Both in vitro and in vivo models of cartilage repair can reveal valuable insights but have different strengths Cell based therapies have come to market for cartilage TE and scaffold based