Table 5.

The effect of in vitro glycation on the mechanical properties of bone. Studies reporting no effect of glycation on a mechanical property shaded in blue grey.

Species (age)	Incubation protocol (buffer, pH, anti-microbial)	Effect on AGEs	Effect on mechanical properties a	Ref
Cortical	bone
Human tibia (34-to 85-yr.)	0.6 M ribose (HBSS with 30 mM HEPES, pH 7.2-7.4, PI) for 7 days at 37 °C	104% higher fAGEs	No significant difference in longitudinal crack initiation toughness	[88]
			35% lower longitudinal crack growth toughness (compact tension)
			42% reduction in failure strain, 72% lower creep rate, and 40% lower residual strain for stress levels > 100 MPa in multicyclic creep tests
Human tibia (57-to 97-yr.)	100 mM glucose (HBSS with 10 mM HEPES, pH 7.2-7.6, PI & 100 μg/mL streptomycin) for 3 and 7 days at 50 °C	175% higher fAGEs after 7 days of incubation	~40% higher indentation distance increase (IDI) after 3 days	[151]
			~18% higher IDI after 7 days
			No other cyclic reference point indentation (cRPI) properties reported
Human tibia (57-to 86-yr.)	0.6 M ribose (HBSS with 30 mM HEPES, pH 7.2-7.6, PI); 10 days at 37 °C	136% higher fAGEs	56% lower transverse crack initiation toughness (single edge notched beam)	[183]
			55% lower microindentation modulus
			No differences in cRPI properties including IDI
Bovine tibia (20-mo.)	0.67 M ribose (HBSS pH 7.3-7.6, 100 mL/L of toluene & chloroform & 0.5 mg/mL of gentamicin) for 15 days at 37 °C	Whitish compared to brownish color	No significant differences in elastic modulus and yield stress, but higher secant stiffness	[184]
			Lower post-yield strain and damage fraction
			No significant difference in J-integral but lower plastic component of J-int with quasi-static loading rate
			No significant differences in the elastic and plastic component of the J-int under fall-like loading rate
Bovine tibia (1.5- to 2-yr.) irradiated at 33 kGy	1.8 M ribose (PBS, pH 7.4, NS) for 24 h at 60 °C	No effect due to irradiation 100-fold increase in PEN	Relative to irradiated control, no significant difference in modulus	[185]
			10% and 20% higher yield and ultimate stress
			4% and 39% higher yield and failure strain
			73% higher work-to-fracture (three-point bending tests of machined specimens)
Bovine femur /tibia (18-mo.)	0.67 M ribose (HBSS b NS c, 10 mL/L of toluene & chloroform & 0.5 mg/mL of gentamicin) for 3, 8, 11, 17, 29, 38 days at 37 °C	Whitish compared to dark brownish color fAGE = 1416 × [1– e−time/12]	No significant difference in compressive & tensile modulus, regardless of time	[64]
			Tensile properties after 29-days
			4.2% higher yield strain
			9.3% higher yield stress
			12.3% lower DF d
			no difference in post-yield strain
Bovine femur (3-mo.)	0.2 M ribose (PBS, pH neutral, 1% penicillin-streptomycin & 1% fungizone & PIf) for 15 days at 37 °C *	PEN not detected in control and ~250 mmol/mol in ribose	No significant differences in yield stress, yield strain, ultimate stress, ultimate strain, post-yield strain, energy to yield, and post-yield energy to fracture (three-point bending of machined specimens), and micro-hardness.	[186]
Bovine femur (18-mo.)	0.67 M ribose (HBSS, NS, 10 mL/L of toluene & chloroform & 0.5 mg/mL of gentamicin) for 21 days at 37 °C	NS	No significant difference in thermal stability of mineralized bone. (Significantly higher melting temperature in ribated bone after demineralization in 45% formic acid)	[187]
Bovine metatarsal (18-to 24-mo.)	0.6 M ribose (SBF g with HEPES h, pH 7.4, 10 mL/L of toluene & chloroform & 0.5 mg/mL gentamycin) for 14 days at 37 °C	88% higher fAGEs PEN not detected in control and ~16 mmol/mol in ribose	No significant differences in modulus, yield stress, ultimate stress, and yield strain.	[65]
			26% and 27% lower post-yield strain and failure strain
			21% and 29% lower toughness and post-yield toughness
			11.5% and 11% DF and post-yield strain/failure strain (3pt bending of machined specimens)
Rat femur/ tibia (4-mo.)	0.2 M glucose (PBS e, pH 7.4, 0.1% sodium azide) for 60 days at 29 °C *	Whitish compared to brownish color	No significant differences in ultimate force, stiffness, displacement, energy-to-yield, and energy-to-fracture (three-point bending of diaphysis).	[188]
Trabecular	bone
Human proximal femurs (42-to 97-yr.)	0.6 M ribose (HBSS with 30 mM HEPES, pH 7.3-7.6, PI) for 7 days at 37 °C	Whitish compared to brownish color; 90% higher fAGEs	No significant differences in apparent modulus, yield strain, yield stress, ultimate strain, and ultimate stress 92% lower post-yield strain energy and 38% lower DF (compression tests of cores)	[189]
Human tibial plateau (64-yr.)	0.6 M ribose (HBSS with 30 mM HEPES, pH 7.3-7.6, PI) for 7 days at 37 °C	NS	No significant differences in apparent modulus at either 0.6% strain or 1.1% strain.	[190]
			No difference in toughness at 0.6% strain
			30% lower toughness at 1.1% strain (compression tests of trabecular cores).
Porcine mandibular condyles	0.2 M ribose (PBS; pH 7.4; 0.1% sodium azide & PI) for 15 days at 37 °C *	200% higher PEN	No significant difference in tissue-level modulus (nanoindentation on dry trabeculae).	[191]

^aHigher or lower change with glycation reported as 100 × (sugar – control) / control

^bHanks’ balanced salt solution (HBSS) is typically Hanks buffer and 1.3 mM CaCI₂

^cNot specified in the paper (NS)

^dDamage fraction (DF) is 1 – secant modulus at failure / initial modulus

^ePhosphate buffered saline (PBS)

^fProtease inhibitor (PI) cocktail is typically either a tablet (e.g., Roche Diagnostics) or 25 mM ε-amino-η-caproic acid, 5 mM benzamide, and 10 mM N-ethylmalemide

^gSimulated body fluid (SBF)

^hHEPES is a zwitterionic buffer

^*changed incubation media every day, every 3 days, every 4 days (volume not always specified), 5 times per week