Extracellular Matrix

extracellular matrix (ECM)

• the living environment of the cells

• non-cellular component of tissues and organs; biological material composed of polymeric networks

• several types of macromolecules (proteins and polysaccharides)

  • locally secreted and assembled into organized mesh work in close association with the surface of the cell that produced them

  • smaller molecules (ions and water) are bound

Functions of the ECM

• survival

• proliferation

• migration

• adhesion

• functions as adhesive substrate

  • tracks to direct migratory cells

  • concentration gradients for haptotactic (directional movement or outgrowth of cells along a gradient of adhesive cues in the ECM) migration

• provides structure

  • defines tissue boundaries

  • provides integrity and elasticity to developing organs

  • degraded by invasive cells during development and disease

• presents growth factors to their receptors

  • controls spatial destruction of surface molecules in ECM

  • facilitates crosstalk between growth factor and ECM receptors

• sequesters and stores growth factors

  • allows for spatio-temporal (space and time) regulation of factor release

  • organizes morphogen gradients (signaling molecule that drives tissue development by forming a concentration gradient)

  • mediates release of factors in the presence of appropriate cell mediated forces or proteolytic degradation (enzymatic breakdown for proteins into peptides or amino acids)

• senses and transduces mechanical signals

  • defines mechanical properties permissive/instructive to cell differentiation

  • activates intercellular signaling the interaction with cell surface receptors

  • engages cytoskeletal machinery and works with growth factor signaling

Explain the ECM and cell interaction and how they influence each other

• ECM influences cell shape, fate, orientation, growth, division, adhesion, migration, and differentiation

• molecular signaling by cellular adhesion receptors (integrins)

• ECM is modified by cells as they proliferate, differentiate, and migrate

What are integrins?

• largest family of ECM molecule receptors

• linkers between the ECM and actin cytoskeleton

• transmembrane heterodimers (crucial mediators in cellular signaling and adhesion that span the cell membrane)

ECM Components

• water (about 65% of tissue weight)

• secreted by a variety of cells: fibroblasts, bone, and cartilage cells

• polymer-forming proteins

• structural proteins

  • elongated molecules usually insoluble

    • special mechanical properties from its unique structure

    • collagens and elastic fibers for strength and elasticity

• adhesive glycoproteins

• space filling molecules (GAGs and proteoglycans)

Collagen

• most abundant protein in mammals

• secreted by connective tissue cells as well as other cell types

• predominant structural component of ECM in bones, cartilage, tendons, and skin

• colleges superfamily has 28 members

Explain why collagen fibers are so strong

• long and highly stable molecules with with rigid structure that provides structural framework and strength of tissues

• triple helix secondary structure: three polypeptides twist around one another in rope like triple helix

• intra and inter molecular cross linked collagen fibers into strong fibrils

• self-assembly of collagen fibrils into collagen fibers due to hydrophobic, electrostatic, and cross-linking interactions

Collagen structure function relations

orientation pattern matches its mechanical function

collagen for biomedical applications

• tissue filler

• nerve guides

• wound healing

• drug delivery

• scaffold in tissue engineering

• bioinks

elastic fibers

• responsible for tissue elasticity

• in tissues that require reversible deformation or extension (lungs, skins, blood wall, elastic cartilage)

• inelastic collagen fibrils are interwoven with elastic fibers to limit stretching and prevent the tissue from tearing

Elastin mechanical behavior

• resist tension and allows reversible change to ECM by passive entropy-driven mechanism allow stretching and recoil

• ability to stretch 8x its resting molecular length and recoil without damage to the protein

Why does elastin allow many tissues in the body to resume their shape after stretching or contracting?

• during elastin synthesis: crosslinking of elastin precursor molecule (inter and intra molecularly)

• elastin is rich in hydrophobic amino acids that cluster to avoid water

Elastin functions for biomaterials

• scaffold

• hydrogel matrices

• enhance integration of scaffolds into the host

• provide biological cues for cell adhesion

Adhesive or linking glycoproteins: types and its purpose in biomaterials

• bind to both cells and ECM

  • laminin

  • fibronectin

• used for coatings and surface modification of scaffolds to improve host-biomaterial interaction

laminin

• adhesive or linking glycoprotein

• large ECM family of proteins with cross-like structure

• separate binding domains to cells and ECM molecules

• localized at the basement membrane: sheets of highly organized and specialized ECM beneath man types of cell sheets (especially epithelium and endothelium)

• function: organize ECM, regulate cell-matrix adhesion, regulate cell migration, and regulate cell shape

fibronectin

• adhesive or linking glycoprotein

• binds cell surfaces and different ECM molecules

• RGD amino acids sequence as the integrin-binding region

• functions: cell migration and proliferation, wound healing, maintenance of cell cytoskeleton

laminin/fibronectin based biomaterials

• used as coating and for surface modification of biomaterials

proteoglycans (PGs)

• core protein and GAGs (long chain polysaccharides with repeating disaccharide motif)

• large complexes that are highly diverse

• GAGs are highly negatively charged so they trap a great deal of water

• resist compression by water retention and occupying large volume

• form porous hydrated gels that fill most of the extracellular space

hyaluronic acid

• provides compression strength, lubrication, and hydration within the ECM and regulates cell functions

• great biocompatibility, biodegradability, bioactivity

• versatile in both drug delivery and tissue engineering applications

  • forming of coatings, nano particles, hydrogels

fibrinogen and fibrin

• fibrinogen: precursor of fibrin

• fibrin: provisional matrix during wound healing and also primary protein component of blood clots

  • rich in growth factors

• both promote cell adhesion and migration

• both are other ECM proteins

• used as hemostatic agent, sealant, scaffold, and bioink

Preclinical and clinical application of fibrin based scaffolds

Preclinical:

• skin humanized mouse model: grafting bioengineered human skin in immunodeficient mice

• study physiologic and pathologic processes

• preclinical platform to evaluate skin disease treatments

Clinical:

• skin tissue loss

• chronic skin ulcers

• rare skin diseases

Main use of ECM based biomaterials

• scaffolds for tissue engineering

  • biocompatible

  • biodegradable

  • bioactive

• temporary ECM to accommodate cells and support 3D tissue regeneration

• degraded in the body and replaced by natural ECM made by host

applications of ECM scaffolds

• tissue regeneration

• wound healing

• drug/growth factor delivery

• cellular models

Explain the concept of varying ECM compositions

• different cells have different ECM ratios

• varying composition of collagen, GAGs, and elastic fibers generate different mechanical properties

• Different types:

  • organ support: collagen, GAGs, elastic fibers

  • blood vessels: most collagen, GAGs, middle to most elastic fibers

  • cartilage: mid collagen, lots GAGs, lots elastic fibers

  • tendon: lots collagen

  • bone: lots collagen, lots mineral

Explain the composition of bone in regard to ECM composition

• small nano crystals of hydroxyapatite that are embedded with an organic matrix

what is the best scaffold for an engineered tissue?

• ECM of target tissue in its native state

• ECM derived scaffolds should mimic the architectural, biological, and mechanical features of the ECM:

  • porous and biodegradable

  • biocompatible and bioactive

  • recapitulate the architecture of the target tissue (chemical, physical, and mechanical parameters)

ECM derived biomaterial sources

• collagen from skin, tail tendon, fish scale

• fibronectin from plasma

• laminin from placenta and heart

• elastin from aorta

• from humans (autologous: directly from own body, or allogenic: from donor, or other species (xenogenic)

Types of ways to obtained ECM derived biomaterials: traditional purification method

traditional purification methods: tissue isolation and processing through mechanical disruption, enzymatic digestion and precipitation

• purification: ECM extraction and purification from animal tissues in the blood → bio polymer isolation (hyaluronic acid, elastin, fibrin, collagen, matrigel)

  • matrigel:

    • complex mixture of basement membrane proteins derived from mouse tumors

    • rich in ECM proteins: laminin, collagen, PGs

• liposuction → washing, homogenization, and centrifugation (high speed rotation to separate components of a mixture based on density, size, and molecular weight) → freeze drying and milling → human ECM powders derived from adipose tissue (body fat tissue)

Types of ways to obtained ECM derived EC derived biomaterials: recombinant technology

• production of ECM proteins in vitro (in petri dish) in bioreactors as combined proteins expressed in either bacteria, insect, or mammalian cell

• can tune, modify, and manipulate the expressed protein

types of ways to obtain ECM biomaterials: decellularization

• best scaffold for an engineered tissue is the ECM of the target tissue in its native state

• removing the cellular compartment of living tissues, creating an acellular (lacks cellular structure) ECM scaffold

• ECM structure and composition of native organ/ tissue is preserved

  • as well as large scale vascular networks

  • and specific functional and structural molecules

explain how biological, chemical, and physical decellularization methods work

biological:

• protease: essential enzyme that breaks down proteins into peptides or amino acids via hydrolysis

• trypsin: protease enzyme produced in the pancreas activated in the small intestine to break down dietary proteins into smaller peptides

• Dnase: enzyme that catalyzes hydrolysis on phosphodiester (strong covalent linkage connecting nucleotides in DNA and RNA) bonds in DNA

• Rnase: enzyme that catalyzes degradation of RNA into smaller components

chemical methods:

• detergents: cleansing method

• acids and bases: chemical agents for inducing hydrolytic degradation

• chelators: chemical agents that bind to metal ions to remove isolate or neutralize them by forming stable ring like structures

• latrunculin B: inhibitor of actin polymerization that leads to depolymerization of existing actin filaments and disruption of the cellular cytoskeleton

physical methods:

• freeze-thaw

• agitation

• perfusion: delivering oxygenated blood to body tissue

• scCO2: sterilizes tissue while decellularization it, physical solvent

• sonication: breaks intermolecular bonds between cells and ECM using high frequency ultrasonic waves

besides tissues and cell cultures, what else can decellularization break down?

organs

what happens after decellularization?

• recellularization of ECM scaffold with patient-derived cells

• transplantation of bioengineered organ

bio fabrication techniques for ECM derived biomaterials

• cell biology

• 3D prototyping

• material science

• micro fabrication

• regenerative medicine

example of purified component biomaterials in the lab

animal tissue → take blood → have genetically modified organism → create polymer isolation of hyaluronic acid, elastin, fibrin, collagen

example of decellularized tissue in the lab

animal/human cadavers tissue → decellularization → acellular matrix

example of in vitro generated biomaterials in the lab

donor cell seedling both 2D and 3D → ECM deposition from stimulus → 2D and 3D components decellularized physically, chemically, or enzymatically → cell-derived ECM for both 2D and 3D structures

What can you do after having the solubilized ECM?

• create hydrogels

• electrospinning (produces nano fibers woven from electrostatic force)

  • improves low mechanical properties of natural polymers and low biocompatibility of synthetic ones

• make bioink

acellular scaffolds in vivo function

• serve as vehicles for delivery of signals that augment the recruitment of endogenous cells (native cells)

cellular scaffolds function & in vivo

• bearing embedded cells to guide tissue formation

• in vivo: ^^ and serve as cell carriers

• cells with regenerative capacities

problems with autologous transplants and allogeneic transplants

autologous

• unhealthy cells

• insufficient number

allogeneic

• compatibility issues

when would you use acellular scaffolds

• extensive tissue loss

• aged patient

• huge number of commercials available decellularized scafolds

• limited immune reaction

• patient with genetic disease

• acute wounds

pros of acellular and cellular scaffolds

acellular

• huge number of commercials available decellularized scaffolds

• limited immune reaction

• low cost

• no regulatory and scientific challenges of cell based therapies

• no donor needed

• facilitates repair activity of native cells

cellular

• addition of cellular component with regenerative capacity

• increased biological activity over the material alone

• useful with patients with systemic pathologies like compromised native tissue repair responses like diabetes

types and applications of ECM based biomaterials coatings

• coatings to improve the host-device interaction

• host cells bind to ECM molecules through receptors

• laminin and fibronectin

• RGD sequence in laminin and fibronectin that binds cells through integrins

  • manipulate cell and tissue behavior through computer modeling of protein interactions

  • activate proper signaling pathways and interact with the cell surface receptors of their parent ECM molecule

• YIGSR sequence from laminin-derived cell binding involved in endothelial cell adhesion, neural crest migration, and cell signaling

  • other motifs related to laminin: IKVAV, PDSGR

cell adhesion photocontrolled spatio-temporally

• photo triggered cell adhesion by RGD caged peptides

challenges of ECM biomaterial therapeutic applications

• understanding the 3D architecture

• mimicking the unique biochemical and biophysical composition

• improving the manufacturing processes

• understanding and modulating changes that occur after in vivo placement and host remodeling