Table 3.

Biomolecules used for tympanic membrane regeneration by tissue engineering.

Biomolecule	Properties	Model	Main findings
EGFl9,48-52	Mitogenic effect	Animal	Greater tympanic closure in less time Neomembrane thickness and histology similar to the native membrane; the thickness achieved in spontaneous closure is less than half the normal thickness of the TM Stimulated neovascularization and fibroblast number Stimulated proliferation (mainly of the squamous layer) Long-term application leads to re-perforation and cholesteatoma formation
		Human	No improvement of tympanic closure No toxicity
TGF-α53,54,60		In vitro	More effective than EGF in promoting colony dispersion and injuries healing Greater pro motility activity than EGF
		Animal	TGF-α was not observed in normal TM, but it was expressed after a perforation
TGF-β65	Chemotaxis induction Extracellular matrix production Angiogenesis stimulation Possible excess of scar tissue Thicker TM	Animal	Reduction of perforation closure time Need to repeated application to achieve the above beneficial effects Possible formation of a disorganized fibrous scar
PGF45,46,55-59	Stimulation of fibroblast, endothelial cells and keratinocytes proliferation and differentiation Stimulation of collagen fibrils growth Vasodilation promoting Stimulation of protease production	Animal	Epithelial and/or connective tissue hiperplasia Increased success of tympanic closure when applied directly to the perforation; if applied with Gelfoam®, it forms a voluminous scar that protrudes from the middle ear cavity and ossicles (use of Gelfoam® inadvisable)
		Human	Increased tympanic closure rate and reduced time of TM closure Enhanced hearing recovery Possible epithelial pearl formation Reduction of middle ear infections Hyperplasia of granulation tissue - which disappears in 5-7 days
KGF60,61	Reactive oxygen species detoxyfication Promotion of re-epithelialization Keratinocytes proliferation and migration	Animal	Enhanced epithelial migration and proliferation in the first steps No increase in tympanic closure rate More organized repair process
PDGF62,63	Fibroblast mitogen	Animal	Increased tympanic closure rate Reduction of perforation closure time More abundant connective fibrous tissue layer
		Human	No increase in tympanic closure
VEGF64	Fibroblast mitogen Angiogenesis stimulation Induction of collagen deposition Induction of epithelialization	Animal	VEGF is more specific and important than bFGF in acute perforation closure
Autologous serum from peripheral blood21	Promotion of wound healing Lack of antigenicity Large quantity of growth factors	Human	ASET does not require anesthesia, reduces or completely closes chronic perforations and implies a continuous supply of growth factors by the own patient Further studies are required to conclude whether the beneficial effect is due to the serum or to the scaffold used for the tympanic membrane closure
Human umbilical cord serum68	Large quantity of growth factors (greater concentration of EGF, NGF and TGF-αβ than in autologous serum from peripheral blood)	Animal	Applied with a 3D collagen scaffold, it enhances significantly chronic perforation closure and hearing capacity from early stages than with paper patches Neomembrane thickness similar to the native membrane Further studies are required to conclude whether the beneficial effect is due to the serum or to the scaffold used for the tympanic membrane closure
Hyaluronic acid19,69	Viscoelastic properties It can be used as a scaffold or as a biomolecule administered in drops Increased motility and phagocytic activity of polimorfonuclear leucocytes	Animal	Reduced time in tympanic perforation closure Increased closure success rate than natural closure Increased levels of FGF and VEGF
		Human	No increased tympanic closure success rate
Human insulin66,70	Neovascularization Increased fibroblast growth rate Increased keratinocytes proliferation, migration and differentiation from the perforation edges Activation of insulin receptor and IGF 1 receptor	Animal	Beneficial effect in perforation epithelialization Neovascularization Formation of finger-like projections Fibroblast activity Presence of inflammatory cells in the lamina propria
	Stimulation of keratyn migration	Human	Increased micro-vascularization from the remnants of tympanic membrane or graft towards the perforation, with the induction of inflammation and epithelialization from the perforation edges Halfen the perforation size after its forth or fifth application
Platelet-rich plasma67	α granules of platelets contain growth factors	Animal	Reduction of the average time of tympanic closure
Plasminogen71,72	Degradation of fibrin and extracellular matrix proteins	Animal	Reduced time of tympanic closure

EGF, epidermic growth factor; TM, tympanic membrane; TGF-α, -β, transformant growth factor type α, -β; FGF, fibroblast growth factor; KGF, keratinocyte growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; ASET, autologous serum eardrop therapy; NGF, nerve growth factor; IGF-1, insulin-like growth factor type I.