Cartilage TE: from in vitro and in vivo models to the clinic!Module 3, Lecture 6!20.109 Spring 2010!2!Lecture 5 review!•! What are some advantages of ELISA as a protein assay?"•! What are some pros and cons of end-point RT-PCR as a transcript assay?"3!Topics for Lecture 6•! Imaging assays"•! Cartilage TE in vitro "•! Cartilage TE in vivo!•! Cartilage TE in the clinic"!Day 5-6: image analysis!•! Imaging data is often high throug–! 4D: time, x-y-z "–! requires computation, and!–! human design/interpretation"•! Many available analysis package–! some ~ $20-30K"–! NIH ImageJ = free"•! Your analyses"–! cDNA band intensities"–! automated cell counts"–! optional: explore other features"hput"s"© Nature Publishing Group. All rights reserved."This content is excluded from our Creative Commons license. For more information, see http://ocw.mit.edu/fairuse4!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/nature02238.5!Fluorescence microscopy!•! Light source"–! Epiuorescence: lamp (Hg, –! Confocal: laser (Ar, HeNe)"–! 2-photon: pulsed laser"•! Filter cube"–! Excitation"–! Dichroic mirror"–! Emission"–! Band-pass vs. long-pass"•! Detection"–! CCD camera"Xe)"Image from: Lichtman & Conchello, Nature Methods 2:910 (2005)"Field diaphragmAperture diaphragmFilter cubeArc lampRed fluorescence collected for viewing/imaging.Red fluorescence excited.Green light excites rhodamine.Image by MIT OpenCourseWare.6!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 athttp://www.microscopyu.com/articles/fluorescence/filtercubes/filterindex.html2) Spectrum profile graph for Nikon B-2A (Medium Band Blue Excitation) at http://www.microscopyu.com/articles/fluorescence/filtercubes/blue/b2a/b2aindex.html"7!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!8!Confocal uscopy permits 3D reconstruction!9!Polymer composite for cartilage TE!•! Porous PLA scaffold + ste•! Cells loaded in medium"–! elongated morphology"•! Cells loaded in alginate"–! round morphology"–! improved cell retention"m cells"PLA"PLA+alginate"Caterson et al., J Biomed Mater Res 57:394 (2001)"Copyright © 2001 John Wiley & Sons., Inc. Reprinted with permission. PLA"PLA/alg"10!Chondrogenesis in vitro!•" Porous PLA scaffold w/ or w/out alginate!•" Alginate alone somewhat chondrogenic!•" Alginate+TGF better than PLA+TGF!Day 7! Day 14!PLA+TGF!ALG+TGF!PLA!ALG!Caterson et al., J Biomed Mater Res 57:394 (2001)"Copyright © 2001 John Wiley & Sons., Inc. Reprinted with permission.1!Dynamic, 3 d!Dynamic, 3 w!•" Method: rotational culture of rabbit chondrocytes with no cytokines!•" Results!–" Mostly dynamic culture gave best resullow apoptosis, very rigid disc!–" Fresh ECM made: primarily CN II and –" Organized architecture, similar to in viv•" A scaffold-free method is inherentlybiocompatible!–" Any disadvantages?!–" Pros/cons of cell-free methods?!T. Nagai et al., Tissue Eng 14 (2008)!ts: PG !o! Scaffold-free in vitro cartilage TE!Static!Courtesy of Mary Ann Liebert, Inc. Used with permission.1Source: 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.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.13!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. 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"12 wks!4 wks!14!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 mont•! Matrix synthesis, implant integration with native tissue "•! Why use a large animal model (vs. small)?!hs"native!native!repair! repair!treated"untreated"Courtesy of Elsevier, Inc., http://www.sciencedirect.com.Used with permission.15!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"16!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"V.C. Mow, A. Ratcliffe, and S.LY. Woo, eds. Biomechanics of Diarthrodial Joints (Vol. I) Springer-Verlag New York Inc. 1990"!Aggrecan!Image © 2002 OPML. Courtesy of OPML.http://web.mit.edu/cortiz/www/AFMGallery/AFMGallery.html17!Treatments for cartilage damage!•! Strategy 1: enhance/provoke healin–! biologics: hyaluronic acid, TGF-#, etc."–! damage bone (stem cell effect)"g"•! 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)"S.W. O$Driscoll. J Bone Joint Surg 80:1795 (S. Poitras, et al. Arth Res Ther 9:R126 (2007C.M. Revell & K. A. Athanasiou. Tissue Eng P1998)")"t B-Rev 15:1 (2009)"Public domain image (Wikimedia commons)"Cutting edge of treatment!•! Cell-based