Motivation behind tissue engineering
Tissue engineering (TE)
Restoration of damaged tissue
Enhancement of natural tissue
Reducing the need for animal models in drug development
This dream is as old as the human imagination
What is tissue engineering?
„An interdisciplinary field that applies the principle of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function or whole organs (Science 1993).“
Importance of tissue engineering
The three pillars of tissue engineering
Primary cells
Are isolated by biopsy directly from human tissue
Mature cells
Limited potential for differentiation
Limited potential for proliferation
Change morphological / functional properties as they age
Only early passages should be used
Demonstrate the exact biological function as in vivo tissues, implantable
In vitro studies (drug screening, tissue development, toxicity tests, ...)
Cell lines
Mutated or gene modified primary cells
Easier to handle
Proliferate indefinitely
Avoid batch-to-batch variations
Same phenotypes and genotypes
In some cases do not replicate the target biological function
Preliminary studies
HeLa cell line
Oldest and most commonly used human cell line
Derived from cervical cancer from Henrietta Lacks (1951)
George Otto Grey discovered the immortality of HeLa cells
Stem cells
Induced pluripotent cells
Derived from adult somatic cells
genetically reprogrammed to an embryonic stem cell
Shinya Yamanaka introduced four specific genes (Myc, Ocr3/4, Sox2, Klf4) encoding transcription factors to somatic cells rendering them pluripotent stem cells
Allowed the clinical use of pluripotent stem cells without damaging a pre-implantation stage embryo
Why do cells need an extracellular matrix?
All normal human tissue-derived cells are anchorage-dependent cells and therefore need a surface for cell attachment and normal proliferation.
In vivo: The extracellular matrix (ECM) provides a suitable substrate for cell adherence.
The ECM is a dynamic, fibrillar, tissue type-specific 3D network of extracellular macromolecules linked together to form a structurally stable composite.
The ECM is mainly secreted by fibroblast cells.
Cell adhesion observed by a microscope
Components of the ECM - Collagen
Components of the ECM - Elastin
Elastin
Tropoelastin is produced in fibroblasts, smooth muscle cells, endothelial cells
Formation of elastin by coacervation and crosslinking of tropoelastin
Formation of elastic fibers by deposition of elastin on fibrillin
Present in vasculature, skin, lung, connective tissue
Provides tissue elasticity by being 1000x more flexible than collagen
Components of the ECM – Fibronectin & Laminin
Glycoprotein of high molecular weight (400 – 900 kDa)
Amino acid sequences for:
Binding to cellular integrin receptors (RGD-motif)
Binding to other ECM components such as collagen or proteoglycans
Tasks:
Linkage of cells to ECM molecules
Cell adhesion, migration, differentiation
Components of the ECM – Proteoglycans
Core protein + Glycoaminoglycan (GAG)
GAG: long unbranched, negatively charged carbohydrate
Binding of water to tissues
Providing viscoelastic properties to tissues
Providing compressive resistance to tissues
Regulation of mass transport by the formation of a diffusion and convection barrier
Storage and release of growth factors (GFs)
Organization of collagen fibers
Cell adhesion by the formation of focal adhesions
Transcription and presentation of integrins based on extracellular cues (e.g. ECM, substrate, ...)
Activation of integrin binding by the displacement of integrin inhibitors by talin (inside-out signaling)
Activation of integrin binding by linkage of ECM molecules and force application (outside-in signaling)
Focal adhesions formation by integrin clustering and a trans-plasmamembrane linkage (ECM-Integrin-actin)
Trans-plasmamembrane linkages allows for force transmission ▪ Downstream signaling influences adhesion, migration, survival, proliferation, differentiation
The ECM - summary
Acts as tissue type-specific mechanical support
Provides tissue-specific mechanical properties (Stiffness, viscoelasticity,...)
Controls tissue shape and organization
Regulates cell adhesion by providing binding sites
RGD amino acid sequences
Controls mass transport by providing a diffusion & convection barrier
Regulation of cellular behavior by mechanical cues and growth factors
Homeostasis
Proliferation
Migration / chemotaxis
Differentiation
Tissue development
Decellularized allogeneic or xenogeneic ECM
Remove all cellular and nuclear materials
Maintain composition, mechanical properties and biological activity
Combination of mechanical, physical and enzymatic processes
Mechanical delamination of certain layers
Physical: sonication, freezing and thawing
Enzymatic treatment: Trypsin
Chemical Treatment: Detergents or ionic solutions
Decellularization is critical why?
Cellular antigens can trigger inflammatory response or tissue rejection
Decellularized allogeneic/xenogeneic ECM
Well tolerated by human hosts
ECM components are well conserved over different species
Decellularization and recellularization of tissue
Commercially available decellularized products
Ease of processing makes decellularized ECM scaffolds a possible off-the-shelf product
Implantation with subsequenty cell migration onto the scaffold
Preseeding of autologous cells before the implant (patient specific medicine)
Extraction of specific ECM components
Requirements for synthetic and hybrid scaffolds
Biomaterial overview
Current state of the art
Controlling cell infiltration by scaffold pore size
Mimicking the 3D ECM architecture by foam-scaffolds
Influence of scaffold stiffness on cell behaviour
PEDOT:PSS microcarriers as injectable 3D tissue scaffold
Influence of particle crystallinity on cell behavior
▪ PEDOT:PSS particle properties can be tailored towards the needs of the desired cell / tissue type
▪ PEDOT:PSS conductivity allows for external stimulation or detection of electrical cell signaling
▪ Possible applications: Injectable 3D scaffold or production of whole cells and pharmaceuticals
Imitating the fibrous ECM structure by electrospun fiber scaffolds
Electrospun membranes as alveolar capillary mimics
Control of cell alignment & migration by scaffold topography
Single silk nano-fibers as scaffold for cell alignment
Neural cell alignment by injectable magnetic microgels
Scaffolds for cell actuation
Last changeda year ago