ANHB3323 (19) - regenerative therapies, stem cells 2

3 factors in tissue engineering

1. Cells

2. Growth factors

3. scaffolds

Steps after tissue assembly in Tissue engineering

1. Conditioning

2. Implantation

3. Vascularisation

Ear disease - regenerative therapy

middle: Tympanic membrane tissue engineering

Inner: Cochlear hair cell regeneration and tissue engineering:

- pluripotent stem cell models

Tympanic Membrane

Eardrum (middle ear)

Layers:

- epithelial

- fibrous

- mucousal

Acute middle ear perforation

damaged eardrum

28 days to fully heal

Tympanic Membrane and stem cells

Tympanic membrane has stem cell niche in middle

stem cells (keratinocytes) proliferate, daughter cells growth outwards towards periphery

Keratinocyte biology assays

Assess cell:scaffold interactions

Examples:

- scratch wound assay

- MTT assay

Scratch wound assay

Keratinocytes on top of scaffold

Scratch the keratinocytes

View and analyse migration of keratinocytes (as they fill the gap)

MTT assay (what does it assess)

Assesses cell adhesion and proliferation

Silk scaffolds for chronic Tympanic membrane perforation

Creating a tympanic membrane scaffold from silk:

- 3d print

- electrospin

Laser doppler vibrometer

measures vibrations with laser

can see how eardrum vibrates, to be able to replicate it

Optical Coherence Tomography Imaging

Imaging shape and prototyping tympanic membrane

can view scaffold material as it interacts with different things

Bioink development (requirements for eardrum scaffold printing)

During printing:

- must be viscous, but able to flow

- must have rapid coagulation

After printing:

- low stiffness (for proliferation and migration)

- high enough stiffness to maintain integrity

Bottom up Silk processing (3 steps)

1. degumming

2. dissolving (dissolve silk)

3. dialysis (wash away chemicals)

[used to cast molds for tympanic membrane scaffolds]

Secretome stem cell

wound healing via paracrine factor secretion

paracrine factor = stimulates keratinocyte growth

(applicable to tympanic membrane)