Tissue Engineering and Regenerative Medicine Summer@Brown 2012

Thoughts? What products in biomedical engineering have you encountered? What products in biomedical engineering will be produced in the future?

Tissue Engineering Tissue Engineering is the in vitro development (growth) of tissues or organs to replace or support the function of defective or injured body parts, or the directed management of the repair of tissues within the body (in vivo). Research is presently being conducted on several different types of tissues and organs, including: Skin Cartilage Blood Vessels Bone Muscle Nerves Liver Kidney etc. etc. etc.

Tissue Engineering is comprised of… Bioengineers Material Scientists Cell and molecular biologists Immunologists Policy Makers Chemical Engineers Electrical Engineers Surgeons

“all tissues are comprised of several levels of structural hierarchy” Tissue Organization Before a tissue can be developed in vitro (outside of the body), first we must understand how tissues are organized in our body. “all tissues are comprised of several levels of structural hierarchy” These structural levels exist from the macroscopic level (centimeter range) all the way down the molecular level (nanometer range) there can be as many as 7-10 distinct levels of structural organization in some tissues or organs

Organization of the Tendon

Organization of the Kidney

How can we build the functional subunit outside of the body Functional Subunits The smallest level at which the basic function of the tissue/organ is provided is called a “functional subunit”: functional subunits are in the order of ~100 mm (whereas cells are of the order of ~10 mm) each organ is comprised between 10-100 x 106 functional subunits each functional subunit is comprised of a mixture of different cell types and extracellular matrix (ECM) molecules Separation of the functional subunit into individual cohorts (i.e. cells and ECM) leads to a loss of tissue function. For this reason, this is the scale that tissue-engineering tries to reconstruct. How can we build the functional subunit outside of the body (in vitro)?

Cellular Microenvironment Since cells are entirely responsible for synthesizing tissue constituents and assembly of the functional subunit, much attention is paid to the microenvironment surrounding the cell(s) of interest. The microenvironment, which can be very different depending on the type of cell, is typically characterized by the following: Different cell types Cell- Cell Communications Local Chemical Environment Local Geometry

Cellularity Packing Density: Cellular Communication: cell densities in tissues typically vary between 10 – 500 x 106 cells/cm3 relates to about 100 - 500 cells per microenvironment (100 mm)3 extreme cases, such as cartilage which has ~ 1 cell per (100 mm)3 thus its microenvironment is essentially 1 cell plus associated ECM Cellular Communication: Cells communicate in three principal ways: secretion of soluble signals cell-to-cell contact (geometry) cell- extracellular (ECM) interactions Cellular communication can affect all “cellular fate” processes (migration, replication, differentiation, cell proliferation and cell death) and the method(s) of communication used depends, in part, on how the cells are packed within the tissue.

Cellular Communications Soluble Signals: includes small proteins such as growth factors and cytokines (15-20 kDa), steroids, hormones bind to membrane receptors usually with high affinity (low binding constants: 10-100 pM) S. Waldman/B. Amsden

Cellular Communications Cell-to-Cell Contact: serve to create junctions between adjacent cells allowing for direct cytoplasmic communication gap junctions 1.5-2 nm diameter and only allow transport of small molecules ~1 kDa A gap junction or nexus is a specialized intercellular connection between a multitude of animal cell-types.[1][2][3] It directly connects the cytoplasm of two cells, which allows various molecules and ions to pass freely between cells.[4][5] S. Waldman/B. Amsden

Cellular Communications Cell-ECM Interactions: ECM is multifunctional and also provides a substrate that cells can communicate through since cells synthesize the ECM, they can modify the ECM to elicit specific cellular responses cells possess several specialized receptors that allow for cell-ECM interactions integrins, CD44, etc. also a mechanism by with cells respond to external stimuli (“mechanical transducers”) extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in animals. S. Waldman/B. Amsden

Local Geometry Geometry of the microenvironment depends on the individual tissue: needs to be re-created for proper tissue growth two-dimensional layers or sheets three-dimensional arrangements transport issues local geometry also affects how cells interact with the ECM the ECM serves as a substrate for cellular communications For these reasons, considerable effort has been geared at creating artificial ECM’s (aka scaffolds) to provide the appropriate substrate to guide in vitro tissue growth and development.

3D cultures Proliferation Gene expression Cell morphology Uniform, controllable size Metabolism Standardized Proliferation Low proliferation rate Gene expression Mimics native tissue

Tissue Engineering SJ Shieh and JP Vacanti Surgery 137 (2005) 1-7

Sources of Cells 2. Allograft 1. Autograft 4. Xenograft 3. Synograft Same-Species Autologous (donor back to donor) Allogenic (donor to recipient, same species) Syngeneic (genetically identical donor) Xenogenic (cross-species) 4. Xenograft 3. Synograft Patient Identical Twin Cross-Species

Culturing of Cells Types of Cell Culture monolayer (adherent cells) suspension (non-adherent cells) three-dimensional (scaffolds or templates) S. Waldman/B. Amsden

Culturing of Cells Sterilization Methods Growth Conditions ultraviolet light, 70% ethanol, steam autoclave, gamma irradiation, ethylene oxide gas Growth Conditions simulate physiological environment pH 7.4, 37°C, 5% CO2, 95% relative humidity culture (growth) media replenished periodically (depends on the cell type, cell concentrations that you are using) Culture (Growth) Media appropriate chemical environment pH, osmolality, ionic strength, buffering agents appropriate nutritional environment nutrients, amino acids, vitamins, minerals, growth factors, etc.

HOW?

Cells as Therapeutic Agents Blood transfusion RBCS into anemic patients to restore adequate O2 Platelet transfusion Platelets into patients with blood-clotting defects Bone-marrow transplantation Cancer patients undergoing high-dose of chemo or radiotherapies

More recent developments Encapsulated b-islet cells for diabetes Sheets of dermal fibroblasts for uclers and burns Chondrocytes for cartilage repair Liver and kidney cells grown in extracorporeal support devices

Cartilage – Carticel® C. A. B. D. http://www.carticel.com/patients/about/treatment.aspx

More recent developments Encapsulated b-islet cells for diabetes Sheets of dermal fibroblasts for uclers and burns Chondrocytes for cartilage repair Liver and kidney cells grown in extracorporeal support devices

Bioartificial Liver (BAL)

Tissue Engineering - Building Body Parts (video) Thinking question: What are the four steps that are used to engineer new tissue?

What are the four main steps used to engineer new tissue? Extract the cells from either the donor or the recipient Build a scaffold for the cells to grow on Supply nutrients and oxygen to the cells Grow cells on the scaffold until the scaffold dissolved and the tissue structure is robust enough for cells to continue to grow and attach

Cell-Scaffold Tissue Engineering Ohba, S. et al, IBMS BoneKEy, 2009, 405-419.

Cells are the functional units that make up tissues Cells are the functional units that make up tissues. Tissues then become the functional units that make up--- A. enzymes B. organs C. other cells D. DNA

Cells are the functional units that make up tissues Cells are the functional units that make up tissues. Tissues then become the functional units that make up--- A. enzymes B. organs C. other cells D. DNA

What is the tissue that researchers believe is the easiest to grow and why? Cartilage cells do not need a great deal of oxygen Cartilage cells receive nutrients by diffusion Cartilage do not require a large network of blood to survive

Tissue Engineering Scaffolds Scaffold Materials: synthetic polymers poly(lactide) ,poly(lactide-co-glycolide), poly(caprolactone)…. foams, hydrogels, fibres, thin films natural polymers collagen, elastin, fibrin, chitosan, alginate…. fibres, hydrogels ceramic calcium phosphate based for bone tissue engineering porous structures permanent versus resorbable degradation typically by hydrolysis (except for natural materials) must match degradation rate with tissue growth Chemical and Physical Modifications (synthetic materials): attachment of growth factors, binding sites for integrins, etc. nanoscale physical features

Tissue Engineering Scaffolds smooth muscle cells on unmodified poly(CL-LA) elastomer (L) and modified surface having bound peptide sequence (R) S. Waldman/B. Amsden

Emerging Medical Technologies Drug-Loaded Stents Guided Tissue Regeneration Resorbable Stents Transgenic Organ Generation Robotic Surgery Genomics/Proteomics Implantable Mini Blood Pump Glycomics Ventricle-Coronary Artery Shunt Protein Engineering Functional Electrical Stimulation Gene Therapy Nanotechnology Gene Expression Analysis (u-arrays) Tissue Augmentation Immunotherapy High-throughput Biomaterial Discovery Cell Multiplication & Transplantation Sustained, Local Drug Delivery Cell Function Regulation Scar Prevention/Reduction Stem cell Expression and Therapy Slide Courtesy of Art Coury

Medical “Holy Grails” Small diameter vascular prostheses Artificial trachea, esophagus, larynx Devices to prevent surgical adhesions Cartilage (articular, meniscal, disc) regeneration techniques Permanent cardiac assist implants Ligament replacement devices Fully functional artificial skin Bone healing devices Tissue adhesives, sealants Artificial blood Artificial vision, hearing devices Artificial cells Nerve regeneration devices Devices to reconstruct sections of the digestive tract Synthetic leaflet heart valves Devices to treat cancer Bioartificial internal organs (liver, pancreas, kidney, intestine) Cellular heart pacemaker Closed loop drug delivery devices Slide Courtesy of Art Coury