Comparison of Common Maceration Techniques to Prepare Porcine Bone for Fluorescence Analysis Using Alternative Light Sources (ALS)

Abstract

Objectives
Investigating the impact of three common maceration techniques on the collagen content and autofluorescence of porcine bone, to ascertain the most suitable preparation method for bone undergoing ALS analysis.

Materials and Methods
Hot water (80°C), biological washing powder (55°C), and enzymatic (55°C) maceration were used to prepare thirty porcine ribs (Sus scrofa domesticus) (n=10). Ribs were photographed before and after maceration using blue light (Crime-Lite 2, 450nm), coupled with an orange camera filter. Thermogravimetric analysis was used to quantify collagen content, and a bespoke computer program: The Osteo-Fluorescence Calculator (OFC) was used to quantify bone fluorescence.

Results
Ribs macerated in hot water exhibited homogenous fluorescence and produced a 5.5% average increase in fluorescence levels (n=10, s.d.=9.36, p=0.012) alongside a 11.2% loss in collagen content (n=10, s.d.=0.09, p=0.023). Biological washing powder was destructive to bone surfaces and produced an average collagen loss of 22.9% (n=10, s.d.=0.05, p= <0.001), while fluorescence was augmented (54.49%) and inconsistent (n=10, s.d.=27.46, p=0.180). Enzymatic maceration produced an average increase in fluorescence of 23.2% (n=10, s.d.=23.72, p=0.180), with a mostly consistent appearance except for some dark patches, and experienced a 19.5% loss in collagen content (n=10, s.d.=0.09, p=0.001).

Conclusions
Hot water maceration produced fluorescence results comparable to fresh bone with little impact on bone collagen and provides a suitable preparation technique for osseous ALS examination. Biological washing powder was destructive to bone collagen and produced exaggerated, inconsistent fluorescence and therefore should be avoided. Enzymatic maceration was the fastest method but requires an optimised formulation.