Vascular Patches Tissue-Engineered with Autologous Bone Marrow

Vascular Patches Tissue-Engineered with Autologous Bone Marrow-Derived Cells and Decellularized Tissue Matrices Seung-Woo Cho, Hee Jung Park, Ju Hee Ryu, Soo Hyun Kim, Young Ha Kim, Cha Yong Choi, Min-Jae Lee, Jong-Sung Kim, In-Sung Jang, Dong-IK Kim, Byung-Soo Kim Biomaterials 26 (2005) 1915-1924 Presented by: Audra Bieg, Sugato De, Jose Gonzalez-Cobos, Monica Maestas April 13, 2007 Biomaterials  International Journal  “…all those materials used in medical devices in which contact with tissues of the patient is an important and guiding feature of their use and performance” Implantable Medical Devices Tissue Engineering Drug Delivery Systems Source: http://www.elsevierinternational.com/journals/biom/    Authors Seung-Woo Cho Department of Chemical Engineering, Hanyang University School of Chemical Engineering, Seoul National University Cha Yong Choi School of Chemical Engineering, Seoul National University Min-Jae Lee Division of Vascular Surgery, Samsung Medical Center Hee Jung Park Division of Vascular Surgery, Samsung Medical Center Jong-Sung Kim Division of Vascular Surgery, Samsung Medical Center Ju Hee Ryu Department of Chemical Engineering, Hanyang University School of Chemical Engineering, Seoul National University In-Sung Jang Division of Vascular Surgery, Samsung Medical Center Soo Hyun Kim Biomaterials Research Center, Korea Institute of Science and Technology Dong-Ik Kim Division of Vascular Surgery, Samsung Medical Center Young Ha Kim Biomaterials Research Center, Korea Institute of Science and Technology Seung-Woo Cho Department of Chemical Engineering, Hanyang University Background  Heart Disease Most common cause of death in western countries  At least 58 million Americans suffer from some form  Source: http://www.cdc.gov/DHDSP/library/imag es/fs_heart_disease.gif Ischaemic Heart Disease   Reduced blood flow to the heart Most common cause is atherosclerosis  Formation of multiple plaques  Caused by the rupturing of plaques  Heart attack Ischaemic Heart Disease  Results in: Temporary damage and pain  Loss of muscle activity  Permanent heart muscle damage  Structural damage to the heart valves  Source: http://www.womanshealth.net/images/heart_disease_fact.jp g Synthetic Vascular Patches   Used to reconstruct vascular conduits Common material types Polytetrafluoroethylene  Polyethylene terephthalate   Pros Good mechanical properties  In vivo durability  Source: http://en.wikipedia.org/wiki/Image:PTFE -3D-vdW.png Synthetic Vascular Patches  Problems  Thrombus formation  Blood clot Due to blood/tissue incompatibility  Calcification  Infection  Lack of growth capabilities  Source: http://fig.cox.miami.edu/~cmallery/150/p hysiol/c42x16blood-clot.jpg Tissue Engineered Vascular Patches  Autologous vascular cells  Taken from same specimen  Pros Natural tissue formation  Antithrombogenic  Biocompatible  Durable  Capable of growth and repair  Tissue Engineered Vascular Patches   Cells isolated from vascular biopsies Problems Highly invasive surgery  Risk of morbidity at biopsy site  Bone Marrow Derived Cells (BMCs)  Differentiate into: Endothelial cells  Smooth muscle cells   Pros Less invasive  Doesn’t use blood vessels  Source: http://www.meddean.luc.edu/lumen/Med Ed/orfpath/images/endo.jpg The Process  How do you make a tissue-engineered vascular patch?    Fabricate decellularized tissue matrix. Isolate bone marrow from primary source. Induce differentiation of bone marrow mononuclear cells.  Smooth Muscle Cells & Endothelial Cells   Seed cells onto tissue matrix. Implant into source.  Test for vascular tissue regeneration on patches. Decellularized Tissue Matrix     Canine inferior vena cavas explanted. Wash in phosphate buffered saline (PBS). Immerse in distilled water for one day. Decellularize using Triton X-100. (3 Days, Shaking)   How does this work? Triton X-100 is a non-ionic detergent.   Destroys cell membranes by attacking phospholipids. Solubilizes membrane proteins in their native state.  Wash again with distilled water. (3 Days, Shaking). http://www.usbio.net/ Decellularized Tissue Matrix  Preparing The Vascular Patch: Open decellularized IVCs in longitudinal direction.  Freeze dry under a vacuum.  Why?  (1) Creates porous texture to facilitate cell adhesion. (2) Kills ALL remaining cells.  Sterilize with ethylene oxide gas.  http://home.howstuffworks.com/freeze-drying.htm Decellularized Tissue Matrix   Figure 1 (A) Vascular patch matrix.  30 mm Long x 15 mm Wide What kind of staining?  (B) Canine IVC, no cells.  Decellularized Tissue Matrix     Figure 1 Shows native ECM. (C) Van Gieson’s Staining  Collagen is red, elastin is grayish. Collagen stains blue. Muscle cells would have been red. (D) Masson’s Trichrome Staining     What does the presence of the native extracellular matrix indicate about the vascular patches? It provides the microenvironments for adhesion and proliferation of the seeded cells. H&E Staining  H & E Staining: Haematoxylin & Eosin Staining   Widely used in medical diagnosis. Hematoxylin (Basic Dye)   Colors basophilic structures with blue-purple hue. Regions rich in nucleic acids (ribosomes, cell nucleus). Colors eosinophilic structures pink. Intracellular and extracellular proteins, most of cytoplasm. RBCs are intensely red.  Eosin Y (Alcohol Based Acidic Dye)    "T Cells Cause Lung Damage in Emphysema". PLoS Med 1(1): e25. H&E Staining "T Cells Cause Lung Damage in Emphysema". PLoS Med 1(1): e25. Patient In End-Stage Emphysema: •What color are the cell nuclei? What color are the RBCs? What color is the ECM? •Blue-Purple, Red & Pink. (Air spaces are white). Polychrome Staining  Three or more dyes applied sequentially.    Less porous tissues colored by the smallest dye molecule. Why? Large molecules penetrate at the expense of small ones. Weigert’s Hematoxylin stains the nuclei. Solution A (Plasma Stain) – Red Dyes (acid fushion, acetic acid, water). Solution B – Phosphomolybdic acid and distilled water. Solution C (Fibre Stain) – Stains collagen blue. Connective tissue (collagen is stained blue). Red smooth muscle cells, keratin fibers. Light pink or red cytoplasm. Black cell nuclei.  Masson’s Trichrome Staining         Van Gieson’s Staining   Mixture of picric acid and acid fuchsin. Simplest method for staining collagen.   Weigert’s Hematoxylin is used. Rinses in distilled water and ethyl alcohol. Collagen stains bright pink. Muscle, cytoplasm, RBC, fibrin stained yellow. Nuclei stained black.    Decellularized Tissue Matrix   Figure 1 (E) Cross section of matrix.    Porous, multi-layered structures. Cell-adhesion and cell seeding. 3-D Space for tissue formation. What does this image show? (What is absent from the surface?) No endothelium.  (F) Luminal surface of matrix.   Decellularized Tissue Matrix  Advantages as a scaffold?     Maintain environments for cell adhesion, proliferation. Similar mechanical properties as native vasculature. Low immunological response (antigens removed).  Prevents immune responses that lead to rejection. Potential of growing in-vivo.    ECM in decellularized matrix degrades is replaced in-vivo by autologous cells, SMCs and fibroblasts. Potential for growth and repair. Decellularized matrix biodegradation occurs at a slower rate than synthesis of autologous ECMs. BMC Isolation & Culture  Why use bone marrow cells?  They differentiate into ECs and SMCs.  Less invasive than harvest of vascular cells. http://stemcells.nih.gov/StaticResources/info/scireport/images BMC Isolation & Culture 1. 2. 3. Bone marrow aspirated from canine with syringe containing heparin. Why? Bone marrow mixed with PBS, centrifuged on Ficoll-Paque gradient. BMMNCs were isolated from layer between F-P reagent and blood plasma. (Approximately 1.0 x 107 cells obtained from 10 mL of aspirate.) http://www.biomedcentral.com/content/figures/1471-2342-6-7-2.jpg BMC Isolation & Culture  Diffentiation Into Smooth Muscle Cells:   Medium 199, Fetal Bovine Serum, Streptomyocin Done in humidified air with 5% carbon dioxide. Human fibronectin in EBM-2 medium. Human vascular endothelial growth factor (VEGF). Human basic fibroblast growth factor (bFGF). Human epidermal growth factor. Human insulin-like growth factor. Ascorbic Acid.  Diffentiation Into Endothelial Cells:         Why use human growth factors to induce differentiation? Why is only growth medium needed for SMC differentiation? BMC Characterization     Cells cultured for three weeks.  6.8 x 106 ECs, 7.2 x 107 SMCs. Characterized using immunohistochemical staining. Primary antibodies were used against cell-type indicator. EC Staining:  Von Willebrand Factor  CD31  What are these? http://www.crystal.chem.uu.nl/group-gros/complex-gain-both.jpg BMC Characterization  Figure 2 (A) Cobblestone morphology.  (B) vWF postive.  (C) CD31 positive.  (G) vWF negative.  BMC Characterization  SMC Staining:    (D) Smooth muscle morphology. (E) Smooth muscle α-actin positive. (F) Smooth muscle myosin heavy chain positive. Avidin-biotin immunoperoxidase kit. DAB subsrate solution kit.  Visualized Using:   Cell Seeding    Static Culture – scaffold placed in cell suspension to allow absorption of cells SMCs uniformly seeded onto decellularized tissue matrices After 2 hours ECs are seeded onto luminal sides of the matrices Cell Seeding  What are the problems related to static culture?  Cell Distribution with majority of the cells attached to surface  Possible Solutions Dynamic Culture  Seeding cells by injection or applying vacuum to ensure penetration of cell suspension  Vascular Patch Implantation    BMCs-seeded patches were implanted into inferior vena cava of BM donor dogs Animal Carefully Anesthetized Latero-posterior thoracotomy procedure followed Vascular Patch Implantation   Sections of the IVCs (15 x 30 mm) were resected and replaced with the tissue engineered vascular patches. No anticoagulants or antiplatelets were administered post-operatively Immunohistochemistry   Relies on specific reaction between applied antibody and the tissue constituent Labels include: enzymes, fluorescent dyes, colloidal metals and isotopes http://www.defenselink.mil/news/ Immunocytochemistry Immunohistochemistry  Why is a secondary antibody used instead of a labeled primary antibody? Signal Amplification  General technique can be applied to any antibody no matter which animal it was raised in simply by changing secondary antibody species  Histological and IHC Analyses Three weeks later…   Figure A – H & E staining shows vascular tissue generation Figure B – Masson’s Trichrome staining shows collagen regeneration Scale bar: 200 microns Once again!   Figure C – van Gieson’s staining indicates presence of elastin fibers (arrows) Figure D – cells on luminal sides stained positively for vWF (Von Willebrand Factor - ECs) Scale bar: 200 microns Once again!!!   Figure E – Patches stained positively for SM alpha actin showing SM tissue regeneration Figure F – Patches stained positively for SMMH showing SM tissue regeneration Scale bar: 200 microns Immunohistochemistry and BMCs   Prior Implantation BMCs were labeled using Cell Tracker fluorescent dye Figure A – cells observed mainly on the luminal sides of the matrices Scale Bar: 200 microns IHC and BMCs: Three weeks later…  Cells are found in luminal side of patches  They’re alive! Scale Bar: 200 microns IHC and BMCs: Three weeks later…  Cells are found in the medial sides of patches They’re alive! Why is the signal attenuated compared to prior implantation signal?  Migration of cells to media  Signal dilution of membrane incorporated dye by labeled cell division  Scale Bar: 200 microns SEM Physics NOT!! Scanning Electron Microscopy (SEM)   SEM provides 3D-like topographical information of bulk specimens at nano resolution Biological materials can be imaged but special sample preparation is required Why? http://mse.iastate.edu/images/microscopy/striped2.jpg Sample Preparation  Samples are prepared to withstand vacuum conditions Why vacuum? Carefully dehydrated to prevent wrinkles and imaging artifacts  Sample must conduct electricity How do we make a mosquito conductive?  Sputter Coater     Specimen is placed on a conductive film in an argon atmosphere Gold cathode ray tube High voltage ionizes argon molecules that strike the gold membrane Gold atoms cover biosample www.steve.gb.com/science http://www.ruhr-uni-bochum.de How SEM works?    Electron beam generated at e- gun Focus and intensified by condenser electro magnetic lenses Scanning coils move the focus beam in a raster scan manner http://www.mos.org/sln/SEM/tour15.html Secondary e- and Image Formation    Secondary e- knocked loose from the surface Detector counts the electrons and sends signal to amplifier Image is generated from intensity at any given spot http://www.mos.org/sln/SEM/tour15.html SEM Findings  SEM endothelium examinations a) b) Native canine IVCs endothelium Regenerated endothelium in the vascular patches - 3 weeks Scale bar: 30 microns Vascular Patch Formation Why is this Decellularization step important? It removes the cells that produce an immunogenic response. Vascular Patch Formation The Big Picture Describe what methods they used to show that the vascular patch was viable with in the body? Thrombosis Aneurysmal Dilation    Why would you want to prevent this from occurring?  Mechanical Properties   Immunogenic Response Calcification Final Conclusions  Based on the results of this experiment do you believe that the vascular patch is ready for use in humans? Yes…  No…  Suggested Future Studies  What future studies would you suggest? Mechanical  Small vessels  Arterial Studies  Long Term Studies  Calcification  Thrombus formation  Aneurysmal dilation  Term Thrombus Calcification Definition Blood Clot Calcium Deposition Importance Major cause of Vascular patch failure Atherosclerosis Factor No immunogenic responses Autologous Cells from the same source or donor Cells taken from the bone marrow which can be differentiated into endothelial and smooth muscle cells Bone Marrow Derived Cells Tidon x-100 H&E Staining Van Gieson’s Staining Masson Trichrome Staining Non-Ionic Detergent Staining method using Haematoxylin and Eosin Used to Decellularize the matrix Aides in structure identification Simplest stain for Collagen Stain using a mixture of picric acid and acid fuchsin Three Color Staining Protocol Term vWF CD 31 Smooth muscle Alpha Actin Definition Blood glycoprotein involved in coagulation Cell Adhesion Molecule Structure within Smooth Muscle Cells Importance Marker for endothelial cells Marker for endothelial cells Phenotypic marker for SMCs Phenotypic marker for SMCs Smooth Muscle Myosin Heavy Chain Dynamic Culture Structure within Smooth Muscle Cells Cell seeding technique Promotes uniform distribution of cells Provides 3D-like topographical information of bulk specimens at nano resolution Image Generation Identification and localization of tissue constituents SEM Scanning Electron Microscopy Secondary Electrons Emitted from sample surface Relies on specific reaction Immunohistochemistry between applied antibody and the tissue constituent