V11

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

„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).“

• 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, ...)

• 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

• In vitro studies (drug screening, tissue development, toxicity tests, ...)

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

• 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

• 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.

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

• 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

• Core protein + Glycoaminoglycan (GAG)

• GAG: long unbranched, negatively charged carbohydrate

• Tasks:

  • 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

• 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

• 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

• 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

• 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)

▪ 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