Table 5.

In vitro and in vivo evaluation of IGTEOS.

Evaluation		Evaluation index		Evaluation methods
In vitro	Interfacial bonding strength	Sufficient bonding strength		Shear testing and peel testing
	Cell compatibility	Cell adhesion and viability		SEM and live/dead staining
	Chondrogenic differentiation	Chondrogenic markers	SOX9	Immunofluorescence staining
			Col-II
			Aggrecan (ACAN)
		Chondrogenic gene expression		Real time-PCR (RT-PCR) procedure
		Production of glycosaminoglycan (GAG)		Toluidine blue and sarfranin O staining
	Osteogenic differentiation	Osteogenic markers	RUNX2	Immunofluorescence staining Alkaline phosphatase (ALP) staining
			Osteocalcin (OCN)
			Alkaline phosphatase (ALP)
		Osteogenic gene expression		Real time-PCR (RT-PCR) procedure
		Calcium deposition		Alizarin RedS (ARS) staining/von Kossa
In vivo (animal model)	Macroscopic assessment	Gross morphology assessment scores using modified Wayne's grading scale		Based on the degree of defect repair, degree of integration and macroscopic appearance
	Microcomputed tomography (micro CT)	Percentage bone volume over total volume (% BV/TV)		Scanco Medical 40 Micro CT system
	Histological analysis	Repair tissue morphology, composition and arrangement, cell ECM production and scaffold degradation		Hematoxylin and Eosin (H&E) staining
		The presence of proteoglycans		Toluidine blue staining
		Production of Glycosaminoglycans		Safranin-O/Fast green staining
		Collagen		Masson's trichrome staining
		Fibrin and collagen fibres		Movat's pentachrome/Sirius red staining(in vitro)
	Immunohistochemistry analysis	Formation of type II collagen		Collagen were incubated with specific antibody
	Biomechanical	Modulus, Permeability, Poisson's ratio		Custom-designed indentation apparatus