Conference 3D Printing: a new tool for making of tissue/L2

Question 1

What is the role of 3d printing with tissue replacements?

Answer 1

1. Patient=Heart attack or arthiritis
2. Using cells from the patient to restore lost function (if someone has had a heart attack part of their cardiac muscles have died-medicines cannot revive this)
3. You can introduce new cells to allow restoration of tissues
4. Can be from the patient themseves which are known as autologous cells (good due to know immune response)
5. Energetic cells (from same species but different person) which does have immune rejection.
6. Isolation of cells from bone marrow for example, and you need enough cell numbers to actually treat the patients which is why the cells are cultivated and multiplied in numbers.(cell proliferation)
7. Either inject cells straight back into the patient or put them in a scaffhold- this is like a base for the cells to attach to.(these scaffholds give a certain shape)
8. You can also have mechanical stimulus or enzyme growth factors to aid the cells, such as stem cells being able to differentiate into different cells.
9. Hopefully this therapy will treat the underlying cause which is arthritis or heart attack.

Question 2

3D printing scaffhold

Answer 2

This is when the geometry really plays a role within 3D printing when designing a scaffhold.

Can have certain geometry for different patients.

You can put the cells in the scaffhold which goes directly into the patient.

Question 3

Liver example.

Why do we need 3D printing in tissue engineering and regeneratve medicine?

Answer 3

• Made of milllions of structures called lobules.(green box on lecture) hexagonal shape.
• Cells arranged in very sepcific patterns when zoomed in, in order to work.
• Use 3D printing for these physiologically relevant structures.

Question 4

Organ transplantation and 3D printing

Answer 4

• Solid organs are all highly vascularised
• So when you are undergoing this transplantation you need to make sure that the highly vascularised network is still in place.
• Without the oxygen or nutrients the cells will die very quickly.
• 3D printing can pre fabricate those vasculature networks, which allows connection of that network to the blood allowing the cells to have oxygen and nutrients and not causing them to die.
• Sometimes it can be used immediately but sometimes has to mature (so cultivation outside the body)

Question 5

Cartilidge

Answer 5

No vasculature so you would think its easier.
But it is still very complex
Collagen fibres orients themsleves parallel to the surface at the top.
Towards the bottom surface the cells and the collagen are vertical
The reason for this is the mechanical properties.
Therefore 3D printing could play a role in this field.

Question 6

Subtractive Manufacturing vs 3D Printing (additive manufacturing.)

Answer 6

Advantages of additive:
* Complex structures with no additonal costs.
* Custom geometries
* Spatial control of multiple materials

Disadvantages
* Slow so not suitable for mass production
* Objects have lower quality(rough surface)
* Hard to get quality assurance.

Additive (3D) you end up having less waste material and you use material to start which doesnt need to be broken down.

Whereas subtractive you have a block of material which needs to be broken down then you get the 3d object and have more waste.

Question 7

A typical process for making 3D bioprinted tissues and organs

Answer 7

You have to select the imaging you are going to use for example X-ray.
Then how you are going to design it.
Material selectrion-Polymers?
Cell selection such as differentiated cells?
Bioprinting- inkjet?
Application- in vitro testing?

Question 8

Viscosity

Cell laden hydrogel

Answer 8

Green fluorescent cells, print lattice structures with hydrogel mixture, hydrogel is viscous enough to form filament which makes the 3D structure

Question 9

extrusion based

Bioprinted ear for microtia

Answer 9

3D printed a year in mice for 1 month.
Use the 3D printer by telling it what to print and what cells we need.
Cells are suspended in hydrogels, and a polymer was used caused a PCL-gives better overall shape of the ear.

Question 10

3D printing of vasculature like networks.

Answer 10

Sugar network can be embedded in a cell-containing hydrogel.
Sugars dissolve in water (WATER SOLUBLE), sugars will dissolve away and leave the channels within the 3D printed structure

Another way they achieved was a vascular like endothelial structure and a different cell type.
The potential for this tech was for breast resconstruction for people who have their breasts cut off.

Question 11

Bioprinting for engineered ovaries

Answer 11

Seed cells which will form follicles in ovary. The green aggregates is the follicles attaching to the 3d printed structure.
They put this in mice and the produced babies which were green due to the fluorescent labelled structures.

Question 12

Rabbit scenario

Bone/ cartlidge regeneration and Osteoarthritis

Answer 12

Uses to replace a whole lower jaw.

Osteoarthritis, can have wearing of the cartlidge. Thus 3D printed scaffholds with the shape of the humeral head can be used. This is because you can design the exact geometry.

TGFbeta3 is a GF which can encourgae stem cells in bone marrow to become a cell type called Chondrocytes.

Thus they needed to include that growth factor to promote the growth of chondrocytes in the cartlidge.

5-8 weeks post surgery you can see that the rabbit can now walk.

Question 13

3D bioprinting for alveolus

Answer 13

Produced air sac structure and a printed translucent structure to breath air.

Even have 3d printed valves which control the direction of blood.

Question 14

3D printed trachea splint (patient study)

Answer 14

Printed trcahea splint which keeps the collapsed trachea open.

Question 15

3D printed surgical guides

Answer 15

This helps the surgeons do their surgeries.
Drill is at pre designed locations
Then this allows for an easier dental implant

Question 16

3d printed tissue replacement for drug screening

Answer 16

Put 3 different cell types into the 3D structure, trying to recapisulate physiologcal relevance, but this is still quite far away from the real structure of the liver.

Question 17

Ethics and integrity

Answer 17

The scaffold of the cadevir trachea was seeded with the patients own cells (bronchial epithelial cells) and bone marrow stem cell derived chondrocytes.
Thye claimed success, however a few years later they developed their technology.
The manufactured a windpipe, which was a synthetic material which was biocompatible.

Lots of patients died, building up of mucus and lots of infections.

But in the original surgery they left a STENT in there, which is what was just keeping the trachea open, they never mentioned the STENT in the paper.

Question 18

Rheological property of inks

Answer 18

Viscosity is a measure of resistance to flow, inks need to be able to flow in the needle.
The viscosity can be meausred by a rheometer
Inks need to have a suitable viscosity profile to be used in extrusion based prointing
Inks viscosity constanty changes due to shear thinning.

The polymer chains in the needles are alligned straight, whereas in the cartridge and when exiting the polymers are coiled.

Measures force required to rotate, how far its rotated or how quickly

Question 19

Two types of rheometer flow curves and 3 sub types

Answer 19

zero shear viscosity, when stationary it can still flow (Shower gel), when reducing the shear rate the viscosity platos

Yield stress: Reducing shear raye the viscosity keeps increasing (handcream or toothpaste:have to apply force to make it flow)
Once out of the nozzle can form its own 3d shape.

Newtonian: Viscosity is constant

Shear thinning: Want this, due to be able to flow and with pressure the hydrogels are deformed thus the viscosity wil decrease and flow better in the cartridge.

Shear thickening: Starch on water, viscosity increases with shear rate

Question 20

Alginate(isolated from seaweed)

Hydrogels in extrusion based printing

Answer 20

• Colloidal or polymer networks that are physically crosslinked.
• Crosslinks means the physical or chemical links between indivdiual chains.
• When there is increased crosslinking the viscosity increases
  * Alginate
• Introduction of calcium ions can crosslink the individual polysacchardie chains to make a hydrogel network.(electrostatic interactions between carboxylate group and pulls the chains together)
• Hydrogel- now semi solid thus viscosity has increased.

PolyNiPAM
* Polymer chains polar groups when heated up form hydrogen bonding and exclude water molecules out which causes globule shape and a hydrogel.

Question 21

Extracellular matrix hydrogels

Answer 21

• Interstitial space with cells embedded in them with proteins, aminoglycan and GFs.
• Can use this material as the hydrogel to encapsulate the cells.
• 2014 research: Isolation of various tissues like heart and cartlidge (cells removed to prevent immunogenicity),
• Gets grounded to powder form and put in an ECM solution, at a high temp it forms a gel (more visocus and higher mechanical properties) but colder its a runny liquid.
• Mixed the cells in the ECM then 3d printed them with a polymer material to form a fake ECM.
  *

Question 22

Shear stress needs to be induced for a long period.

Shear stress induced death

Answer 22

Cell viability decreases with decreasing needle diameter

Have to control shear stress to a level that doesnt cause harm to the cells.

Smaller needles mean less cell viabaility so thus more cell deaths, bigger nozzle is the opposite.

Conical shape is better comapred to cyndrical needle.

Question 23

Hot melt extrusion printing

Answer 23

Thermoplastic polymers are heated which is a solid filament forms due to polymer cooling.

These are non crosslinked polymers.

Mechanically more robust than hydrogels

Question 24

look on lecture to clarify diagram

Two photon stereolithography

Answer 24

Ray of tiny mirrors which refracts light from individual mirrors which would be a higher resolution which allows you to cross link specific areas.

Focusing UV beam on the resin solidifies and crosslinks it.

The two photon system is so much better which focuses two photons on the same plane simultaneously to degrade into 2 radicals, thus exciatation volume is much smaller, thus much better resoultion.

Cells can then be seede in the scaffhold by this mechanism

Question 25

Uv irradiation cell death

Answer 25

High dosage can cause cell damage thus optimal UV needs to be used, which is a balance of cell damage and mechanical properties of the 3D printed constructs.

Question 26

Biocompataibility

Answer 26

Materials for implantation must be biocompatible

Must be in contact with tissues of the human body without causing harm.

ISO standards which can prove if a material intended for implanation is biocompatible.

Introduction of a new material have not been through these vigrous tests therefore people just use exsiting materials just to save money.

Question 27

ATMPs

Answer 27

Maybe on essay