Table 2.

Collagen type I physicochemical characterisation methods and expected results.

Characterisation	Method	Expectation	Reference
Biocompatibility
Biocompatibility and immunogenicity	MTT assay Live/dead assay Cell attachment Immunocytochemistry (ICC)	Cell proliferation and growth; SEM; more than 80% cell adhesion after 24 h; integrin-related protein expression and cell attachment	Addad et al., 2011 [99], Fauzi et al., 2016 [71], Fauzi et al., 2017 [104], Thievessen et al., 2015 [105]
Physical, Morphological, and Topographical (PMT) Characterisation for Three-dimensional stability
Weight	SDS-PAGE	Type I collagen is composed of β (250 kDa), α1 (130 kDa), and α2 (115 kDa)	Peng et al., 2010 [102], Parenteau-Bareil et al., 2010 [106], Fauzi et al., 2016 [71] Inanc et al., 2017 [107]
Native conformation	UV-circular dichroism (CD) spectroscopy	CD with a positive maximum absorption band at around 222 nm	Carvalho et al., 2018 [108]
Mechanical strength	Tensile strength and Young’s modulus	No gold standard, but fish and reptile should be more fragile than mammalian material	Amri et al. 2014 [109], Teramoto et al. 2012 [110]
Thermal stability	Thermogravimetry analysis (TGA) Differential scanning calorimetry (DSC)	No gold standard for what Td of biomaterial should be, but it must be stable to use normal temperatures or higher; in general, comparable or higher than native rat tail tendon collagen fibre Td of 65 °C; Td of extracted collagen in solution are 37–40 °C, 26- °C, and 6–20 °C, for mammalians, fish, and deep-sea animals, respectively	Miles and Bailey 1999 [111], Zhang et al., 2020 [112], Subhan et al., 2015 [113], Bozec and Odlyha, 2011 [114]
Porosity and pore size	SEM	Pore size > 80 µm for fibrogenic and <20 µm for chondrogenic growth; the average mean size for ovine-, bovine-, and porcine-derived scaffolds are 73.05 ± 10.79 µm, 85.84 ± 9.51 µm, and 87.32 ± 10.69 µm, respectively	Ghodbane and Dunn 2016 [115]
Biodegradation	Enzymatic biodegradation method	Depends on application example within 14 days for cutaneous wound healing	Mh Busra et al., 2019 [116] Salleh et al., 2022 [117]
Swelling ratio	Swelling ratio protocol	1000–2700% for biomaterials	Ghodbane and Dunn 2016 [115]
Water vapor transmission rate (WVTR)	WVTR method	Within range 2028.3  ±  237.8 g/m2/day to maintain a moist environment and enhance the normal healing phase	Xu et al., 2016 [118]
Surface and particle physicality	Contact angle zeta potential	Angle < 90° hydrophilic; isoelectric points (pIs) close to 6 at zero zeta potential	Chen et al., 2019 [119]
Chemical Characterisation
X-ray photoelectron spectroscopy (XPS)	XPS	Samples should show ≈0.1 atomic % as nominal sensitivity with an elemental sensitivity that may differ as much as ≈100; for the assessment of chemical components, a sample size larger than ≈10 μm will be convenient	Baer et al., 2019 [120]
Fourier transform infrared (FTIR)	FTIR	Collagen type I functional groups include amides I, II, and III; range of peak intensity between 1450 cm−1 and 1235 cm−1 commonly indicates the helical structure of collagen; amide A at the higher peak intensity of 3350 cm−1 can be attributed to collagen type I; At peak intensity of 1632 cm−1, this indicates the higher-order arrangement of the collagen structure, which refers to β-sheet and triple helix structure	Sasmal and Begam 2014 [121] Fauzi et al., 2016 [71]
Energy dispersive X-ray (EDX)	EDX	Major elements in collagen type 1 are oxygen, nitrogen, and carbon with a higher percentage of oxygen, followed by nitrogen and carbon	Fauzi et al., 2014 [122]
X-ray diffraction (XRD).	XRD	Collagen XRD generally consists of 2 clear peaks, where the first peak is sharper than the second peak; collagen type I from different sources of mammalian, avian, marine, fish, etc., via XRD has been proven closer to the amorphous phase rather than crystallinity	Zhang et al., 2011 [123], Fauzi et al., 2016 [71], León-Mancilla et al., 2016 [124]