CELL EXTRACELLLULAR MATRIX INTERACTIONS
CELL EXTRACELLLULAR MATRIX INTERACTIONS BIOL 4750
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This 42 page Class Notes was uploaded by Arianna Veum on Monday October 19, 2015. The Class Notes belongs to BIOL 4750 at Rensselaer Polytechnic Institute taught by George Plopper in Fall. Since its upload, it has received 32 views. For similar materials see /class/224830/biol-4750-rensselaer-polytechnic-institute in Biology at Rensselaer Polytechnic Institute.
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Date Created: 10/19/15
Linear Elasticity examples 0 E force I M A cross section area 60113770111661 9 9 9 9 9 Gxx 0 0m 0 IDslTess a 0 0 6 0 Uyy 1 8 0 GCX 0 Iquot xx E 8 i 0 0 strain is 2D 8 v 1 0 a z E l 1 1 l 1 8 E039 V039yy va HHi slTess is 2D 1D strain gt U porous filter bathing soluliion rigd impermeable ony subslraie porous filter rigg gt U cartila e samp e d impermeable any subslrale bathing soluliion Effects of Cell Seeding and Cyclic Stretch on the Fiber Remodeling in an Extracellular MatrixDerived Bioscaffold Tan D Nguyen MD Rui Liang MD Sa vio L Y Woo PhD DJSC Hon Shawn D Burton BS Changfu Wu PhD Alejandro Almarza PhD Michael S Sacks PhD and Steven Abramowitch PhD Critically Presented By Ryan Koppesl Nate Schiellel Christian Wagnerl and Karen Mack2 1 Department of Biomedical Engineering ENTOF 2 Department of Biology Em Th e A u t h 0 rs University of Pittsburgh Tan D Nguyen MD Rui Liang MD Shawn D Burton 35 Changfu Wu PhD Alejandro Almarza PhDquot Steven Abra mowitch PhD MSRC COASSOCiate Director Savio L Y Woo PhD D S c Hon MSR C Director Michael S Sacks PhD ETMML Director The Big Hitters Savio LY Woo 39BS Mechanical Engineering Chico State College 1965 39MS Mechanical Engineering University of Washington 1966 39PhD Bioengineering University Q f Wa shinton 1971 39DSc Honorary California State Universityrat39Chico 1999 Steven Abra mowitch Assistant Professor De artmjient of Bioengineering BS Applied Mathematics University of Pittsburgh 1998 39PhD Bioengineering University off Pittsburgh 2002i Michael S Sacks William Kepler Whiteford Professor Department of Bioengineering 39PhD Biomedical Engineering University of Texas Southwestern Medical Center at Dallas 1992 39MS Applied Mechanics Michigan State University 1983 39BS Applied Mechanics Michigan State University 1981 Additional Contributors Alejandro Almarza Assistant Professor Department of Bioengineering 39BS Chemical Engineering Florida State University 39PhD Chemical Engineering Rice University Changfu Wu Associate Department of Bioengineering Rui Liang MSRC Fellow Department of Bioengineering 39MDMS Medicine Xi39an Medical University China 1995 39Fellowship Medical research Bioengineering University of Tokyo Japan 2002 Tan D Nguyen MSRC Fellow Department of Bioengineering Shawn D Burton Summer Undergraduate MSRC Department of Bioengineering Ligaments and Tendons paratenon blood vessels Jymphalic vessels menes endotenon banding sublibril tropocollagen broblasts crlmped fibrils cles endotennn bundles of fascicles epitenon and the entire tendon paratenbn Note that blood and lymphatic vessels and nerves are cut in the crosssection within the endolenon from Kastelic et 31 1978 with permission 39 Ligament Healing Why does this tissue need help Small Intestine Submucosa for Improved Healing in Animal Models Ledet et al 2002 Spinal Ligament Goat Karaoglu et al 2008 Patella Tendon Rabbit Shamaporaled slsueaied Nontreated 3 a g 3 a m Strain m Figure 9 Typical stress strain curves for sharmuperated control SIStreated and nontreated groups nod swlions shm B 39mIml andrCI small mun a inhnulcnsum mnlml levels an 12 SIS Fiber Orientation Suhmucosa and Mucosa Intestine Submucosa OI degrees F rema Whale SIS specimen Muscle Layer ten mounting t t with he SALE data SL819 39n idicatinn LGIL quot t l i n at full 1quot 01Lit139 L 0 L h uctui39e and binxial mechanical t Collagen Fiber Orientation w 0 idem in a 9x an 1 min iioii 1e whim mg 5 mil i30 from Intestine Longitudinal AXIs Fibroblasts Commonly Found in Connective Tissue Critical in Wound Healing Collagen Production Fibroblast Liehih 1993 Cells SIS Remodeling quotWell why didn39f you say you wan l39ed The office to be func onalquot a combinalinn of fibroblast seedian and cyclic stretch SIS Test Specimens 35 Dog bone Specimens 0 Grip Dimensions 2cm x 1 cm Cut Along Longitudinal Axis of Small Intestine Testing Groups Group I Cell Seeded and Cyclic Loading n14 Group II Cell Seeded No Load n13 0 Group III Cyclic Loading No Cell Seeding n8 Cell Seeding 0 Primary Fibroblast cultures from Adult Female New Zealand White Rabbits MCLs Passage 2 fibroblasts at a density of 15x105 cellscm2 In the presence of 50 ugmL ascorbic acid Customdesigned Cyclic Stretching Tissue Culture System TOP VIEW Small Intestinal L Self inear 39 I AMan Filler Cap Actuator Connector Tissue Clarnlil5 Culture Chamber In Mounted Base SIDE VIEW PH 1 t quot r em and B a schemati L single station From leftto right each station con nfa linear 39 a u quot c k V ue clampstn submerge the scaffold and ibmersibleload cell to monitor the 10 generated by the i n39e connected to the lin 1r actuator and inad cell with staini the wall of the stretthing chamber and link to selfaligning c lectms C0 1139 images available onhne at WWV iiEberipllbC01nEli Gilbert et al 2007 Loading Protocol Preloaded to 005 N Standard Starting Point Stretched 10 of Length at 5 Hz 2hrs ON 2hrs OFF 2hrs ON Followed by 18hrs OFF 5 Consecutive ays r li tuator clliure Chamber i 1933 7 Tissne Clampg l a D a l w w c licberlo nli no to m lquot lcn SmallAngle Light Scattering SALS A way of finding fiber orientation Sacks MS et al Ann Biomed Eng 199725678 689 Why a laser 0 Beam Diameter of N 600 pm 0 Wavelength 6328 nm What is the approximate diameter of a collagen fiber and why does this matter 5003000 nm How does it work Computes the 2 D Fourier Transform 0 Can someone describe what a Fourier Transform is used for SALS Intensity from Tissues 1IQndum Fibers diffract the i g I Ilwl lilk39h llt39ll J a laser beam and is shown as a W scattered light intensity distribution Sacks MS et al Ann Biomed Eng 199725678689 Angle Quantification 1000 scan points for an area of 08x125 cm o Orientation at each point was found with respect to the longitudinal axis then averaged over the 1000 scan points to get mean angle and dispersion Orientation Index Orientation ndexOangle containing 50 ofthe fibers at one scan point E V C m E 39U a E E E h 0 Z 60 90 120 150 180 D degrees Bowes LE et al Wound Rep Reg 19997179186 Single Scan Point Results 39 Plot ofOI I Smaller Angle Z PurpleRed B Gm LargerAngle BlueGreen 55 45 45 45 v 25 Group III OI Figure from Previous Study OIdegrees Show good local alignment but poor alignment with adjacent tissue Shows both local and global alignment Bowes LE et al Wound Rep Reg 19997179186 OI Analysis Critiques 0 The OI orientation index is only based on single scan point 0 Only shows local orientation Orientation wrt to the stretch direction is not shown Fiber Orientation Results TABLE 1 THE EFFECTS OF CELL SEEDING AND CYCLIC STRETCHING ON THE ORIENTATION OF COLLAGEN FIBERS IN THE ECM BIOSCAFFOLD AS QUANTIFIED BY SALS Mean nnqlt Angular dispt i siun L t Groups Before A cr b tjfurc39 After 1 seeded and 253 71 374 cyclically stretched ll seeded and 39 301 nonstretched lll nonseeded 39 128 and cyclically stretched aIndicate signi cant difference before and after stretching MMPS 0 A family of enzymes known to degrade matrix proteins Cleave fibrils of collagen Can be synthesized by fibroblasts What can cause upregulation of MMPs Cyclic loading Stressdeprivation Previous MMP Study Similar study by same gr0up No real change in MMP gene expression I E 0 n D lt lt 3 Q N a 0 n E 2 E E Gilbert TW et al Tissue Eng 20071313131323 Collagen Synthesis 0 Increased Collagen I gene expression I o n lt L9 o o Gilbert TW et al Tissue Eng 20071313131323 How does remodeled collagen align 0 Their argument Cells align in stretch direction Break down collagen not in stretch direction synthesized collagen aligns in stretch direction do to cell alignment Confocal Fluorescent Microscopy Look at particular Laser is used to excite fluorescence Focus emitted Stain with Moecular fluorescent light through pinhole aperture Scan Ta 5 g thIn samples mm to bOCk O Ut 3D imaging when layered reflectln g lIght and Mammal 39 De ec quot Principal Light background fluorescence get Confizgmcfgg my OutolFocus I Apgquott quotee Light Rays quotquot 5 chhromatk Laser Mirror Exclkatlon So rce lnFocus Light Rays Excltatlon high a s y ng hl Source Inhale Aperture Figure 1 4 Focal Planes I httpserccarletonedudetailsimages8280html spedmequot Figure 3 Groups and II lncubated to stain Seeded stretched and Rhodamine phalloidin not stretched for actin fillaments o Examine cell Sytox Green for nucleii morphologyorientation new collagen End of 5 days r1 L1 httpwwwfgscnethomepageimagescryptolStnjpg httpwww 39 39 y39 39 lJpg FIG 3 Fluorescent staining of the cells seed bE39e39ered u39n d39eir confocal uorescent microscope A Group I in which theaer Indlcat tth tchin g direction B grOup Actin bers were stained in red and nuclei were stained in gre39e UColorim age s39available online at wwwiebertonin39ecomten Transmission Electron Microscopy TEM Imaging Beam of electrons Resolution in nm range Thin specimen slice Specimen embedded in a substrate Stains can be used on tissue samples Figure 4 TEI Imaging indicated by ind ica cs l39ib Seeded 8 Nonstretched Future Work 0 Stretching protocol to be optimized Mechanical properties of modified SIS need to quan ed Implantation of modified SIS l into animal models for wound healing properties Article Critiques Only one loading protocol was implemented Crimped fiber orientation not addressed No geneprotein expression studies for MMPs or collagen in this study No mention of their previous similar studies No real quantification of fiber remodeling Bold conclusions made from representative images mmeltI
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