Intro. to Tissue Ch. 1-5

Modern Tissue Engineer’s Toolkit

Biomaterials

Cells and Cell Biology

Bioactive Materials

Fabrication Biotechnologies

functional biological substitutes

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life science toward the development of __________

Cells, scaffold, signal

What are the 3 pillars of Tissue Engineering?

Limitations of Tissue Engineering

Innervation, vascularization, an active immune response, external response and monitoring

Differentiation

the process by which stem cells become more specialized and can perform new functions

Totipotent

Can differentiate into all cells in the body plus placenta

Pluripotent

Can differentiate into all cells in the body

Multipotent

can differentiate into a limited range of all cell types

Oligopotent

can differentiate into only a few closely related cell types

Unipotent

can produce only one cell type

Stem-cell system niche (Extracellular Matrix)

ligands, stiffness, and growth-factor presentation

Stem-cell system niche (Soluble Cues)

morphogens, cytokines, nutrients, and metabolites

Stem-cell system niche (Neighboring Cells)

contact-dependent and paracrine signals

Stem-cell system niche (Physical Environment)

oxygen, geometry, flow, and mechanical loading

embryo

Primal Source of Stem Cells

Placenta, amniotic fluid, umbilical chord

Sources of Stem Cells (Fetal Tissue)

Bone marrow, adipose tissue, dental pulp (synovium), skin

Sources of Stem Cells (Adult Tissue)

Transdifferentiation

Fully differentiated cells that switch phenotypes to another mature cell without having to become an induced pluripotent stem cell first

Immunohistochemistry

Flow cytometry

Common transcription factors

Characterizing Stem Cells (Protein Expression)

Characterizing Stem Cells (Gene Expression)

RT-PCR

Microarray

RNA-sequencing

NANOG, OCT4, SOX2

Common Factors in Stem Cells

Limitations of iPSC

low and variable reprogramming efficiency

heterogeneity

epigenetic memory

genomic instability and mutations

cost and technical complexity

Week 1-2 (embryonic development)

Blastocyst and inner cell mass

Week 2 (embryonic development)

Bilaminar embryonic disc

Week 3 (embryonic development)

Primitive streak

Gastrulation

Week 5-8 (embryonic development)

Neurulation and organogenesis

epiblast; hypoblast

The two layers that form the bilaminar embryonic disk consist of the _______ and _______

It defines the primitive dorsal ventral axis

Gastrulation

Formation of the three germ layers: endoderm, mesoderm, and ectoderm

neural plate; neural tube

Neurulation is the developmental process where the ________ folds and closes to form the _____ which develops into the brain and spinal chord

pluripotent

The cells at the inner cell mass of the blastocyst are _______

Wnt Signaling (Embryo)

Axis formation and lineage decisions

BMP Signaling (Embryo)

Patterning and bone or cartilage differentiation

Sonic Hedgehog Signaling

Neural tube, limb, and somite patterning

Retinoic Acid Signaling

anterior-posterior patterning

Vasculogenesis

endothelial precursors assemble de novo into tubes

Angiogenesis

endothelial cells sprout from existing vessels

pericytes; smooth muscle cells

_____ support capillaries

______ reinforce larger vessels

PDGF-B; mural; halts

Endothelial cells express ____ on PDGFR-expressing _____ cells. Those cells release _____ back to the endothelial cells, which _____ angiogenic activity

Osteoblast Maturation 1st Step; Wnt, BMP, Hedgehog

Mesenchymal stem cells —> Osteoprogenitor

Morphogens: _____, ______, _____

Osteoblast Maturation 2nd Step; Wnt, BMP

Osteoprogenitor —> Pre-osteoblast

Morphogens: ______, ______

Osteoblast Maturation 3rd Step; Wnt, BMP

Pre-osteoblast —> Mature osteoblast

Morphogens: ______, ______

Osteoblast Maturation 4th Step; osteocyte; quiescent

Mature osteoblast to either an _______ or a _______ bone-lining cell

Endochondral Ossification

Long bones form from cartilage

Intramembrous Ossification

Bone forms from mesenchymal cells

Mainly flat bones and part of the clavicle

VEGF, TGFB, and FGF

Factors needed for establishment of endothelial pathways

Phosphorylation; GTP-binding proteins

Main classes of intercellular switches

Tyrosine; threonine, and serine

Amino acids that phosphate group mainly link to

Secondary Messengers

Small molecules inside the cell that are downstream and get activated

They relay and amplify the signal from the cell surface

cAMP, Calcuim, IP3 and DAG

Common secondary messengers include: ______, _______, _______, ________

G-protein; secondary messenger

GPCR Pathway: Ligand→ GPCR →_______ → ________ → ________ → cell response

dimerization; autophosphorylation; TF

RTK Pathway: Ligand → RTK _______ → _______ → ______ → cell response

growth; blood vessel

VEGF receptor is a ______ receptor

It is a ______ factor that promotes ______ ______ growth

IP₃, DAG, calcium and NO

Secondary messengers for RTK receptors

Ras and PI₃K

Signaling proteins in RTK pathway

SMAD

Signaling protein in ser/thr kinase receptor pathway

Also acts as a TF

ser/thr kinase

TGF-B is a ________ receptor

Wnt is not present

When _____ B-catenin dies

Wnt is present

When ______ B-catenin lives and drives gene expression

Reasons for the Extracellular Matrix

Structural support, cell adhesion, cell signaling, tissue repair and barrier protection

structural proteins, protein-polysaccharide complexes, adhesive glycoprotein

3 classes of molcules the ECM contains

triple; alpha

Collagen I-IV are characterized by their _______ helix of _______ chain polypeptides

Collagen I

Major role consists of inclues tensile strength

Representative in skin, tendon, bone

Collagen II

Major role in cartilage

Collagen III

Major role is compliant reticular matrix

Abundant in submucosa and early repair tissue

Collagen IV

Major role in network forming basement membranes

hydroophobic

Elastin is a ______ crosslinked structure

Elastin; lysine

This component’s role is the stretch and elastic recoil. _______-rich region create crosslinks

Abundant in arteries, lungs and skin

Fibronectin; RGD

rod-like polymers

role: cell adhesion (_____ units)

found in interstitial matrix or basement membrane

Laminin

Trimeric cross-linked peptide

role: scaffolding and cell communication

found in basement membrane

Glycosaminoglycans

long unbranched polysaccharide

role: water retention and signal binding

lo: hydrated matrices throughout the tissue

M1; M2

ECM bioscaffolds should promote a transition from ______ inflammatory macrophage to ________ anti-inflammatory

Decellularization

Remove all cellular components from scaffold while maintain the native nature of the ECM

Freeze thawing, force, pressure

Mechanical agents of decellularization

Acids/Bases, Hypotonic/hypertonic solutions, Detergents, Alcohol

Chemical agents of decellularization

Trypsin, nucleases, chelating agents

Biological Agents if Decellularization

Xenogeneic Effect

ECM components can elicit host immune responses

Inadequate analysis of residual cellular debris