Lectures final

Question 1

What is the ECM and whats it made of

Answer 1

• The extracellular matrix (ECM) is a dynamic structure that is constantly remodelled to control tissue homeostasis
• The ECM in mammals is composed of around 300 proteins, known as the core matrisome

Question 2

what are ECM characteristics and roles

Answer 2

Different tissues have unique and specialized extracellular matrix (ECM) components and organization, which enables each ECM to carry out tissue-specific roles, including structural support, the transmission of forces and macromolecular filtration.

The molecules that are associated with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled determine the structure and the organization of the resultant ECM.

Question 3

What is ECM made of

Answer 3

The fibrous (collagens and elastin) and glycoprotein (fibronectin, proteoglycans and laminins) macromolecules that constitute the ECM have evolved structures and chemical properties that are particularly suited to their specific biological functions in their respective tissues

The architecture of the ECM is highly organized, which partly arises from the innate properties of its constituent molecules and their interactions and partly from the activities of the resident cells

Each class of ECM molecule is designed to interact with another class to produce unique physical and signalling properties that support tissue structure, growth and function.

Question 4

Uses of ECM

Answer 4

Small intestinal submucosa (SIS) is a native collagen-based extracellular matrix (ECM) of submucosal layer of intestinal wall

Decellularized ECM as a vascular graft
Stem cell growth support

Question 5

What are ECM vascular graphs used for, limitations, requirements

Answer 5

Vascular prostheses (grafts) are widely used for hemodialysis blood access, trauma repair, aneurism repair, and cardiovascular reconstruction.

Smaller-diameter (≤4 mm) grafts that would be valuable for many reconstructions have not been achieved to date, although hundreds of papers on small-diameter vascular grafts have been published.

General requirements
- not thrombose (clog)
not trigger hyperplasia (excessive growth of cells around it)
- maintain mechanical integrity
- meet surgeon handling requirements (e.g., suturability).

Question 6

current situation on synthetic graphs (standard, what uses, issues)

Answer 6

• Vascular grafts used in surgery today are composed primarily of expanded Teflon (ePTFE) or Dacron fabric (polyethylene terephthalate (PET)).
• ≤4 mm grafts could be used for limb blood vessel replacement, possibly saving many of the roughly 1 million limbs amputated each year worldwide.

Arterio-venous grafts used for dialysis access also have a high failure rate at 1 to 2 years, leading to expensive reoperations. (fail due to hyperplasia -> thrombosis)

Nylon grafts fail rapidly due to degradation—the amide backbone chemistry of nylon is identical to the amide backbone of proteins and the body has highly efficient enzymatic mechanisms for breaking down unwanted polyamides.

Burst strength of the grafts can be problematic

Autologous vessels still outperform decellularized vessel grafts and are the optimal standard of care

Question 7

Duration of implantation of vascular graphs

Answer 7

around 1 month animal studies
(Sometimes 3 or 6 months)

in humans: if Degradation and aneurism -> in the 5- to 10-year implant period.

Question 8

scCO2 grafts vs detergent decell grafts

Answer 8

vascular grafts produced by detergent methods may result in defective vessel wall structures, detergent residues, and the development of aneurysms after grafting.

ScCO2:
- higher biocompatibility,

• enhanced chemotactic migration of endothelial progenitor cells (chemically attracts blood stem cells),
• lower risk of vasculopathy,
• lower inflammatory and splenic immune responses
• better physiological-like tension responses after xenotransplantation (XTP)

Question 9

Whole organ engineering

Answer 9

• decellularization of the organ, (creates an acellular scaffold consisting of structural proteins such as collagen and laminins, as well as proteoglycans and polysaccharides)
  maintains the organ’s composition, architecture, and mechanical properties.
• scaffold seeded with progenitor cells and cultured in a bioreactor to mimic the natural heart functions.
• A bioreactor supports and protects the engineered construct, providing nutrients and a sterile environment.

Question 10

How to test an engineered the heart as a bioengineer

Answer 10

Tissue function test
Sterility
mechanical properties
cell distribution/viability
electrophysiology

Question 11

methods of decell

Answer 11

(a) Chemical – detergents: Sodium Dodecyl Sulfate (SDS), Triton X100, CHAPS, acids/bases, hypotonic/hypertonic solutions, alcohols, solvents.

(b)Biological – enzymes and nucleases: Deoxyribonucleases (DNases), Endonucleases, Trypsin, chelating agents.

(c)Physical – temperature, pressure, electroporation, and impact/force: mechanical agitation, slicing, mincing.

Question 12

scCO2 advantages

what does it solvate

Answer 12

• sterilization
• inert, non-toxic

can extract undamaged ECM components maintaining the original structures of polymers and proteins from native tissues

low reactivity to polar components (protein, polysaccharide chain) hinder biomolecule denaturation during the tissue extraction process

scCO2 -> liquid-like solute solubility and gas-like diffusion ability, which increases the CO2 tissue penetration ability and solubility of nonpolar molecules in the tissues

->increase DNA elimination and the tissue extraction efficiency compared to chemical solution-based decellularization methods

Question 13

Applications of decellularized tissues : 3D scaffold fabrication strategies

Answer 13

A) Process of decellularized tissue derived hydrogel fabrication.
B) flow tunable decelluarization method for whole human limb.
C) Decelluarized liver tissue cube fabrication for angiogenesis.
D) 3D printer technologies using bio ink formulation.
E) endodermal (gastric) organoid using decellularized ECM, epithelial (zonula occludens-1, epithelial cadherin and actin) and gastric (ezrin and mucin-5AC) markers

Question 14

How can we engineer macroscopic designs for hydrogel solute transport

Answer 14

• The spacing between polymer molecules in the network (that is, the mesh size) is tuneable from around 5 to around 100 nm
• At the molecular (or atomistic) scale, drugs can interact with the polymer chains via a range of mechanisms like covalent linkage to a polymer chain

Question 15

Requirements for hydrogel drug delivery systems

Answer 15

maintain the drug bioactivity, and through packaging, transport and storage, both the drug and hydrogel must be chemically and physically stable.

hydrogel should degrade on its own or be refillable,

Question 16

How can hydrogels be delivered for drug delivery and engineering controls

Answer 16

surgical implantation, local needle injection or systemic delivery via intravenous infusion.

controls
delivery method dep on application

delivery release profile (dt ct) and speed dep on application (perhaps synced with the tissue regen process)

(example skin needs to be durable, adhesive, and flexible)

deformability,shape and surface chemistry are other factors to consider in designing drug delivery systems.

Question 17

The multiscale properties of hydrogels

Answer 17

Macroscopic scale
architectural factors (such as hydrogel size and porous structure).

Mesh scale
drug diffusion is regulated by the mesh size and its temporal or stimuli- responsive evolution.

Molecular and atomistic scale.
affinity or binding between the drugs and the polymer chains
(covalent conjugation, electrostatic interactions and hydrophobic interactions)

Question 18

How does hydrogel size affect drug delivery method

Answer 18

Size:
Microgels smaller than 5 μm -> oral or pulmonary delivery, (not suitable for intravascular injection -> rapid circulation clearance)

Nanogels (10-100 nm) -> systemic drug delivery bc can go through small blood vessels through fenestrations in the endothelial lining, allowing for extravasation into tissues.

Hydrogels below 10 nm -> kidney filtration clear

0.5-10 micrometers can be phagocytized by macrophages.

Question 19

Mesh size of hydrogels (meaning, size, depend on)

Answer 19

Hydrogels consist of a cross-linked polymer network, and open spaces (that is, meshes) between polymer chains; the meshes allow for liquid and small solute diffusion.

typical 5 -100 nm

The mesh size depends on polymer and cross-linker concentrations, as well as external stimuli such as temperature and pH.

Question 20

Mesh size effect on drug delivery

Answer 20

if molecule small, diffuse right through
If molecule big, wait for polymer to degrade for it to come out

Question 21

Types of controlled drug delivery

what holds and releases molecules

Answer 21

Reservoir (membrane based delivery)
Mesh controlled
Tortuosity controlled
Degradation
Erosion
Deformation

Question 22

how can islet cells be implanted for insulin delivery

Answer 22

In hydrogel, safe from immune response, nutrients can pass through and insulin too

Question 23

What are the types of protein purification (goals differ)

Answer 23

• Preparative Purification – produce large scale (insulin, enzyme production)
• Analytical Purification – small scale and/or
  detection
  for a variety of research or analytical purposes, including identification, quantification, and studies of the protein’s post- translational modifications and
  function.
  can also be used for protein structural analysis

Question 24

Exploiting which characteristics can help purify proteins from complex mixtures?

Answer 24

• Size
• Charge
• Affinity

Question 25

Protein extraction methods and particular steps

Answer 25

Accessing the cytoplasm:
1. freeze-thawing cycles,
2. sonication
3. use of high pressure
4. Grinding
5. detergents.

During this process, proteases that are normally contained in specialized compartments within the cells will be released.

It is also necessary to inactivate proteases within the extract, and to keep the mixture cold to preserve the shape and biological activity of the proteins.

Question 26

Protein separation methods

Answer 26

• Precipitation and Differential Solubilization
• Dialysis
• Centrifugation
• Chromatography

Question 27

How does dialysis work and what type of technique is it

Answer 27

protein separation method
- separates dissolved molecules by size.
- biological sample placed inside a closed membrane, protein of interest is too large to pass through, but smaller ions can easily pass.
- As the solution comes to equilibrium, ions evenly distributed, protein remains concentrated in the membrane.
-> This reduces the overall salt concentration of the suspension

Question 28

Salt in salt out method description and type of method?

Answer 28

protein separation

Salting In: At low concentrations, adding salt can actually increase the solubility of proteins in water. The salt ions help to shield the charged areas on proteins, which reduces the electrostatic repulsion between proteins, allowing them to dissolve better in water.

Salting Out: As you add more salt, the salt ions start to “out-compete” the protein molecules for water molecules. Salt ions attract the water, making it less available for the proteins. Eventually, there isn’t enough water for the proteins to stay dissolved, and they begin to come out of solution, or precipitate. This is the “salting out” process.

Question 29

How does centrifugation work and what is it for

Answer 29

a solute that won’t damage the cellular components is used to create a layered density gradient during the centrifugation.

Then the protein sample is placed on top and the denser proteins will travel faster to the bottom because of centrifugal force until they reach the sucrose that is of their exact density (sinking through small ones until they float). The smaller ones will travel less far bc they “float” on top of less dense particles

Collect fractions

Question 30

What is chromatography for and what types do we have

Answer 30

Chromatography -> protein separation
second step in the purification pathway after a salting out or centrifugal step

• Size Exclusion
• Ion Exchange
• Hydrophobic Interaction Column(HIC)
• Affinity

Question 31

What is size exclusion chromatography (or Gel Filtration Chromatography)

Answer 31

Column with beads with tiny pores of a precise size.

• Molecules with sizes larger than the exclusion limit do not enter the pores and pass through the column relatively quickly by making their way between the beads.
• Smaller molecules, which can enter the pores, do so, and thus, have a longer path that they take in passing through the column.

Question 32

Hydrophobic interactions chromatography

Answer 32

• HIC media is amphipathic, allowing for separation of proteins based on their surface hydrophobicity.
• The column matrix has a hydrophobic ligand covalently attached.
• In high salt conditions, proteins will bind to the matrix with differing affinity, with more hydrophobic proteins binding more tightly than more hydrophilic proteins
• When the salt concentration is decreased, proteins that are more hydrophilic will be released first, followed more hydrophobic proteins.

Question 33

Ion exchange chromatography

Answer 33

Ion exchange chromatography separates molecules by ionic charge.

Anion exchange resins are positively charged, binding negatively charged compounds.

Cation exchange resins are negatively charged, binding positively charged compounds.

Proteins bind to the resins based on their charge and are eluted with a competing salt solution.

Collect and test all fractions if the protein’s charge is unknown.

Question 34

Affinity chromatography

Answer 34

Affinity chromatography is useful if you know that your protein of interest binds with a specific small molecule or ligand.