Graduate Student University of Toronto Mississauga, Ontario, Canada
Abstract Body : One of the more difficult tasks anthropologists and pathologists are tasked with is assessing the postmortem interval in early skeletonized remains. During this period, the assessed PMI can be rather wide. This study investigated additional modalities to aid in more precise PMI estimates by examining the degradation of osteopontin and osteocalcin.
Osteopontin is an acidic glycosylated phosphoprotein excreted by osteoclasts, while osteocalcin is a γ-carboxylated protein excreted by osteoblasts. Together they bind to form dilatational bands binding hydroxyapatite to collagen. Previous studies have indicated the stability of osteocalcin, yet limited work has been done to study the degradation of osteopontin. The present study examined the degradation of osteopontin, osteocalcin, and the change in their ratio.
Iliac bone samples were extracted from 25 individuals: 9 coming from biopsies representing a “perimortem” sample, 9 as a postmortem sample collected 48hrs postmortem, and 7 from embalmed cadaveric samples representing a 6-month sample. The bone samples from the 9 unembalmed postmortem samples were then divided and fixed in to 3 pieces at 7-day intervals to create an < 48hr-, 7-day, and 14-day postmortem time-groups. Samples were embedded in methyl methacrylate and sectioned to < 100μm. The 6-month sample group did not incorporate an osteocalcin component and only examined osteopontin degradation. Samples were labeled with either OC or OPN primary antibody and co-labeled with AlexaFluor 555. Imaging was conducted using a Carl Zeiss LSM800 confocal microscope at 400x magnification, with 50 optical slices, and a tile array of 447.23μm2. Analysis was conducted using BitPlane™ Imaris® v.9, examining the total protein count, mean pixel intensity, and mean standard deviation. Statistical analysis relied on a one-way ANOVA, Pearson correlation test, and logistic regression models on the OPN, OC, and the ratio of OC:OPN.
This study found that the OC remained relatively stable (p=0.198). The OPN count saw significant decrease with a strong correlation with these changes (p< 0.001, f=24.1; r=-0.832). The logistic regression likewise demonstrated a significant results (r2 = 0.723). When the 6-month sample is included the results of the ANOVA was p< 0.001 and the Pearson correlation being r=-0.848. The pixel intensity and standard deviations analyses found a significant variation in the standard deviations, while the mean intensity was not found to be significant (p=0.097). The results of the ratio analysis were also found to be significant at p=0.035. When taken all together, the results of this study have demonstrated that the degradation of OPN, and the ratio of OC:OPN can potentially be used as a means of assessing the PMI in bone from remains within a 6-month postmortem window.
