ch7_in_vitro

ch7_in_vitro - Harvard-MIT Division of Health Sciences and...

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Unformatted text preview: Harvard-MIT Division of Health Sciences and Technology HST.523J: Cell-Matrix Mechanics Prof. Myron Spector Massachusetts Institute of Technology Harvard Medical School Brigham and Women's Hospital VA Boston Healthcare System 2.785j/3.97J/BEH.411/HST523J IN VITRO MODELS FOR CELL CONTRACTION M. Spector, Ph.D. SKIN WOUND HEALING IN VIVO METHODOLOGY Non-regenerating, well-defined wound Evaluate the result measure contraction as the decrease in the area of the wound scar or regeneration: density and organization of collagen Summary: methodolgy of experiments in vivo The approach of the skin experiments was to let the experimental animal answer the question as to whether our implants can induce regeneration. The studies were performed in both the guinea pig and the swine. It is important to use a non-regenerating wound model so that there is noambiguity in interpreting the outcome as regeneration or intact tissue left in the wound bed. In the skin, the full-thickness removal of epidermis and dermis is a non-regenerating wound. An ECM analog with a particular combination of characteristics is grafted into the wound, and, after sufficient time for healing, the tissue in the wound is assessed for contraction and to determine if this tissue is scar or regenerated tissue. We then modify the matrix characteristics (chemical comp, pore size/orient, and degradation) and repeat the process. IV Yannas, et al. SKIN WOUND CONTRACTION Decrease in wound area with time: A = A0e-kt A, A0, t, k, area initial area time "specific rate of contracture"; amount by which a unit area of wound decreases per day; uniform for rectangular and triangular wounds and lower for circular wounds Billingham and Russel, 1956 SKIN WOUND CONTRACTION A = A0e-kt A0 Area k circular Inc. k rectangular t, time Billingham and Russel, 1956 IN VITRO MODELS FOR CELL CONTRACTION Cells seeded into a preformed collagenGAG sponge Measure dimensional change Cell force monitor Cells wrinkling a flexible (silicone) membrane Measure % cells wrinkling the membrane Cells cast in a collagen gel (lattice) Measure the dimensional change COLLAGEN-GAG MATRICES Analogs of Extracellular Matrix Type I (bovine tendon) Type II (porcine) Chondroitin 6-sulfate Photo removed for copyright reasons. Photo removed for copyright reasons. Freeze-dried Dehydrothermally cross-linked IV Yannas, et al. PNAS, 1989 Canine articular chondrocytes in a collagen-GAG matrix synthesize type II collagen (red) but they also contract the matrix 15 Days in Culture Photo removed for copyright reasons. Type II collagen (red) immunohistochemistry Our laboratory has investigated the use of porous type I and type II collagen matrices for in vitro and in vivo articular cartilage tissue engineering for several years. To briefly summarize some previous findings, it has been found that .... As shown in this matrix seeded with third passage canine chondrocytes and cultured for 15 days and stained for type II collagen. However, it is also known that the passaged chondrocytes also display .... Additionally, we have seen that levels of biosynthetic activity... CR Lee, et al, Biomat. 2001;22:3145 Cross-link Density 0.35 0.3 Mean SEM; n=4 Swelling Ratio* 0.25 0.2 0.15 0.1 0.05 0 DHT UV GTA EDAC *Swelling ratio ~ cross-link density [Weadock, et al., 1983] All groups significantly different; p<0.05 Adult canine articular chondrocytes (passage 3) contract a type I collagen-GAG matrix, reflected in the decrease in diameter Highly x-link., high modulus Graph removed for copyright reasons. Little x-link., low modulus CR Lee, et al., Biomat. (In press) Chondrocyte Proliferation Correlation with Contraction? 60 50 DNA (g) 40 30 20 10 0 0 10 20 30 DHT UV GTA EDAC Day Chondrocyte Biosynthesis Correlation with Contraction? Proline Incorporation nmol/hour/g DNA 0.15 0.03 Sulfate Incorporation 0.1 0.02 0.05 0.01 0 2 7 15 29 0 2 7 15 29 Day DHT UV GTA Day EDC Osteoblastic cells (MC3T3-E1) contracting a collagen-GAG matrix Photo removed for copyright reasons. Pores compressed as specimens decrease in size (no evident dissolution) 1 wk Loss of SMA Photo removed for copyright reasons. Photo removed for copyright reasons. 2 wk 4 wk C. Menard, et al., Biomat. 2000;21:1867 MC3T3-E1 Cells in a Collagen-GAG Matrix, 4 wks Photo removed for copyright reasons. C. Menard, et al., Biomat. 2000;21:1867 LINEAGE OF THE ARTICULAR CHONDROCYTE Progenitor Cells* Bone marrow stromal cell (mesenchymal stem cell) Pericyte ? Smooth muscle cell ? Synovial cell ? * All express SMA Marrow Stromal Cells in Monolayer Culture (7 days) SMA IH (>90% SMA+) Neg. control Photos removed for copyright reasons. Lapine, p2 D. Cai, et al. Tiss. Engr. 7;829-841(2001) Canine, p1 Mesenchymal Stem Cell-Seeded Matrices Graph and two photos removed for copyright reasons. D. Cai, et al. Tiss. Engr. 7;829-841(2001) CORRELATION OF CONTRACTION WITH SMA CONTENT Regulation of SMA Expression Time in culture Selected growth factors TGF-1 increases SMA expression Western blot evaluation of the SMA content of human articular chondrocytes with time in culture Diagram and graph removed for copyright reasons. B. Kinner and M. Spector JOR 2001;19:233-24 Does contraction increase with increasing SMA? Passage 7 cells that contain more SMA cause 40% more contraction of the collagen-GAG matrix Graph removed for copyright reasons. Subtract contraction of non-seeded matrix and normalize to cell number B. Kinner and M. Spector JOR 2001;19:233-24 Graph removed for copyright reasons. SMA Western Blots Articular Cartilage Photo removed for copyright reasons. Meniscus Photo removed for copyright reasons. Zaleskas, et al., ECR 270:21(2001) SMA WESTERN BLOT RESULTS Human Articular Chondrocytes Graph removed for copyright reasons. Zaleskas, et al., ECR 270:21(2001) Cell Force Monitor Diagram removed for copyright reasons. See Freyman, T.M., I.V. Yannas, R. Yokoo, and L.J. Gibson. "Fibroblast contraction of a collagen-GAG matrix." Biomaterials 22 (2001) 2883-2891. Adult Canine Articular Chondrocytes Cell Force Monitor Graph removed for copyright reasons. Zaleskas, et al., ECR 270:21(2001) 0.6 Cell Force Monitor Results Human Articular Chondrocytes 0.5 0.4 Force per Cell [nN] PDGF PDGF PDGF Control Control Control TGF-B TGF-B 0.3 0.2 0.1 0 0 5 10 Time [h] 15 20 Zaleskas, et al., ECR 270:21(2001) IN VITRO MODELS FOR CELL CONTRACTION Cells seeded into a preformed collagenGAG sponge Measure dimensional change Cell force monitor Cells wrinkling a flexible (silicone) membrane Measure % cells wrinkling the membrane Cells cast in a collagen gel (lattice) Measure the dimensional change Fibroblasts on Silicone Membranes SMA IH L. Suh, et al. Fibroblast Wrinkling of a Silicone Membrane E, elongated S, stellate CC, cell-cell L. Suh, et al. Lung Fibroblasts on a Silicone Membrane SMA (red) Photos removed for copyright reasons. See Figure 2 in Hinz, B. et al. "Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity." Mol. Biol. Cell 12:2730-2741 (2001). F-actin Overlay SMA yellow Wrinkling of a Silicone Membrane by Fibroblasts Correlates with SMA Graphs removed for copyright reasons. See Figure 1 in Hinz, B. et al. "Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity." Mol. Biol. Cell 12:2730-2741 (2001). Graph removed for copyright reasons. See Figure 3 in Hinz, B. et al. "Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity." Mol. Biol. Cell 12:2730-2741 (2001). IN VITRO MODELS FOR CELL CONTRACTION Cells seeded into a preformed collagenGAG sponge Measure dimensional change Cell force monitor Cells wrinkling a flexible (silicone) membrane Measure % cells wrinkling the membrane Cells cast in a collagen gel (lattice) Measure the dimensional change COLLAGEN GEL CONTRACTION ASSAY Cells cast in gelatin. Gel can be fixed to the culture dish or freefloating. In one assay, the gel is fixed to the dish for 5 days and then released and the dimensional change recorded. Fibroblasts from Dupuytren's Contracture in a Collagen Gel Graphs removed for copyright reasons. MB Vaughn, et al., ECR 257:180 (2000) Fibroblasts Cast in a Collagen Gel (Lattice) Figure removed for copyright reasons. See Figure 4 in Hinz, B. et al. "Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity." Mol. Biol. Cell 12:2730-2741 (2001). Fibroblasts in Collagen Gels for 5 days: IH SMA, red; F-actin, green in A, B, D, E; ED-A FN, green C, F; nuclei, blue Photo removed for copyright reasons. See Figure 5 in Hinz, B. et al. "Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity." Mol. Biol. Cell 12:2730-2741 (2001). IN VITRO MODELS FOR CELL CONTRACTION Cells seeded into a preformed collagenGAG sponge Measure dimensional change Cell force monitor Cells wrinkling a flexible (silicone) membrane Measure % cells wrinkling the membrane Cells cast in a collagen gel (lattice) Measure the dimensional change CONTRACTILE CONNECTIVE TISSUE CELLS Express SMA in vivo Capable of contracting collagen-GAG matrices in vitro SMA-positive cells retain differentiated phenotype SMA trait derived from the stem cell Amount of contraction correlated with the SMA content SMA and contraction up-regulated by TGF-1 Roles have yet to be determined, but may be both positive and negative ...
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This note was uploaded on 11/11/2011 for the course BIO 20.410j taught by Professor Rogerd.kamm during the Spring '03 term at MIT.

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