Often upon the discovery of skeletal remains, one of the most important steps in producing a biological profile of the deceased is to estimate a probable age-at-death range, the sex, ethnicity and stature of the individual (Franklin 2010). There are many different methods of doing this, depending on whether the skeleton is juvenile or adult. When aging a juvenile skeleton, there are many methods that can be used such as the length of long bones or dental eruption (Garvin et al, 2012).
These can be used with a certain degree of reliability, in particular the development of the dentition is reported to be the least effected by environmental factors, and therefore considered the most preferable method of age determination for a juvenile skeleton (Scheur and Black, 2000). Markers of adulthood include the full fusion of the epiphysis in long bones, and the eruption of the third molars (Garvin et al, 2012). At this stage any further changes to the skeleton are considered degenerative, rather than developmental, and adult skeletal aging methods must be used (Garvin et al, 2012).
Adult skeletal aging methods are most commonly focused around four key areas. These are the pubic symphysis, sternal rib ends, cranial sutures and the auricular surface of the ilium (Garvin et al, 2012). Of these the pubic symphysis in particular is considered reliable (Wink, 2014). This is suggested by Berg (2008) that this is because the pubic symphysis appears to progress through a number of defined and unchangeable steps. Todd began documenting the changes that occur in the pubic bones relating to age (1920), describing 10 morphological phases that have an age range associated with them.
Since then adjustments and recommendations have been suggested to improve on Todd’s (1921) method of scoring the pubic symphysis, such as the proposal by Katz and Suchey (1986) to reduce to 10 phases used in Todd method to six. In an effort to improve the morphological descriptions of the six phases, Brooks and Suchey (1990) added 273 female pubic symphyses to the original sample of 739 male pubic symphyses. The Brook-Suchey method is considered the most popular method of aging with the pubic symphysis used (Garvin and Passalacqua, 2012).
Despite this, many methods of age determination through the pubis symphysis, including the Suchey-Brooks (1990) and Todd (1921) suffer from a loss of precision in age estimation above the age of ~40 years (Berg, 2008). Micro-Computed Tomography Micro-Computed Tomography (micro-CT) was first introduced by Feldkamp et al (1989) and can be used to investigate the internal structures of bone in a non-destructive manner (Ruegsegger et al, 1996). As this type of investigation becomes more common it is often used in medical research into areas such as osteoporosis (Bruker micro-CT).
By using a micro-CT, it is possible to investigate the micro-architecture of trabecular bone (Ruegsegger et al, 1996). Trabecular bone can be assessed by calculating morphometric indices which includes, but is not limited to, the bone volume fraction (BV/TV), the trabecular number (Tb. N), and the trabecular thickness (Tb. Th) (Bouxsein et al, 2010). Bouxsein et al (2010) define these terms as follows:The BV/TV can be obtained by calculating the ratio of segmented bone volume to the total volume of bone within the region of interest; The Tb.
N is a measure of the mean number of trabeculae per each unit length, usually 1/mm; Trabecular thickness refers to the mean thickness of trabeculae within the area of interest. The aim of this study would be to ascertain whether micro-CT could be utilised as a useful tool in the aging of bones, specifically whether measuring the bone volume fraction of a scan, the trabecular number, and trabecular thickness of the bone underlying the pubic symphysis could be used to ascertain the age of a bone. Our hypothesis are as follows: All three parameters (BV/TV, Tb. Th, and Tb. N) will decrease as age increases.
It is expected that while this will be seen in both sexes, the level of decrease will be greater in females that in males for all three parameters. Methods and Materials Subjects As it takes approximately 1 hour to scan and reconstruct a sample, five samples will be scanned daily for five days a week across a 12-week period. Therefore 300 samples will be scanned in total, which was confirmed to be an appropriate sample size through using a sample size calculation, as described by Kadam and Bhalerao (2010). To the best of our ability, there should be an even number of samples examined for each sex, and a reasonably ven distribution of ages between the two sexes. This will ensure that our hypotheses can be appropriately tested. This study will use a skeletal collection, and permission to access the samples required is already granted. Individuals should be 21 years old at the time of death, or older, to be included in this study, as individuals below this age would not have reached skeletal maturity, and therefore skeletal aging methods that are based on developmental markers would be more accurate, as previously discussed (Garvin et al, 2012).
No upper age limit is required for this study. Previous work by Yeni et al (2011) has suggested that race could have a statistically significant effect of the changes in trabecular micro-architecture in the vertebrae. While this has effect has not been specifically said to effect the bone beneath the pubic symphysis, individuals that were white Caucasian shall be used in this study to ensure that if race does have a significant effect on the trabecular bone underlying the pubic symphysis, it will not skew the results obtained in this study.
Any individual that suffered from a bone degenerative disease, such as osteoporosis, will automatically be excluded as a condition of this nature would cause an increased degeneration of the micro-architecture of the trabecular bone, and therefore would cause the bones to appear older than reality (Parfitt et al, 1983). Equally, Parfitt et al (1983) found that if an individual had suffered from a vertebral or hip fracture, there would be an effect on the level of trabecular bone, so any individual reported to have suffered from either a vertebral or hip fracture will be excluded.
Any bones that are found to have damage to the area of interest would not be used. It should be said that we are investigating the bone directly beneath the pubic symphysis as this is an area that is often used in age determination through macroscopic examination (Garvin et al, 2012), and previous studies have indicated that there is a relationship between bone degeneration and age in this region (Parfitt et al 1983). It is possible that there are other areas of the pubis that would be more suited to the utilisation of this method, and this should be investigated further.
Method Samples of bone shall be selected from the area found directly below the pubic symphysis. These samples will be scanned using a micro-CT scanner. To ensure that there is no movement of the samples during the scanning process, the samples will be fixed to the rotating stage using plasticine, or another similar substance (Bruker micro-CT, http://umanitoba. ca/faculties /health_sciences/medicine/units/cacs/sam/media/MN001_ Bone_microCT_analysis_general. pdf, no date).
As suggested by Peyrin et al (1998), a voxel size of 14x14x14µm3 would be used, as this resolution has been shown to ensure the images obtained are of a nigh enough quality to analyse the trabecular microstructure. The Skyscan CT- analyser software, from Bruker, would be used to reconstruct the scan images using the Feldkamp algorithm (Bruker micro-CT,http://umanitoba. ca/faculties/ health_sciences/medicine/units/cacs/sam/media/MN001_Bone_microCT_analysis_general. pdf, no date), and then visualise the 3D scans.
The Skyscan software feature region and volume of interest selection tools, which would allow the accurate selection of an appropriate area to analyse. Skyscan software can also be used to calculate the BV/TV, Tb. N and Tb. Th of the selected area (Bruker micro-CT, http://brukermicroct. com/products/ ctan. htm, no date). These three measurements would be taken for each sample, and recorded. Once this is completed the will be statistically analysed to ascertain whether there is a relationship between any of the three parameters and the age-at-death of the individual.
Statistical Analysis The results for each parameter would be analysed individually. The data for each sex will be plotted separately in scatterplots. The correlation will be tested, and it is expected that a negative correlation will be seen. Using the Pearson’s product-moment correlation, we will test whether the correlations are significant. An ANOVA test will be used to ascertain whether the sex of the individual has a significant impact on any of the results for each parameter.
The results will be considered statistically significant if a p-value of less the 0. 05 is achieved. Potential outcomes/Further Research Assuming that our results allow us to accept our hypotheses, this research could open the door to devising a new method of estimating age-at-death. This could follow a similar idea as the radiographic atlas produced by Greulich and Pyle (1959), which shows hand and wrist x-rays at various stages of development, to which an x-ray of an individual of unknown age could be compared to.
In this case, a standardised table would be formed, allowing the Forensic Anthropologist or researcher to ascertain the BV/TV, Tb. N and/or Tb. Th and compare this to the values in the table, giving an age range that the individual likely falls into. Before this could be done, further research would need to be undertaken into factors such as race and environment, which could have an effect on these figures. (Yeni et al, 2011). Should this be possible, it would provide a relatively quick and non-destructive method of estimating age.
Any method used to estimate the age of an adult skeleton has its limitations (Rissech et al, 2012. Merrit, 2013) and therefore the use of multiple methods should be encouraged (Matrille et al, 2007). This method would have the advantage of being centred on a similar area to another popular method, the Suchey-Brooks (1990) method, which means that should a partial skeleton be recovered, if the pelvis is present then there are at least two methods of age determination that could be employed.
There would be limitations to this method, primarily that micro-CT analysis of the trabecular micro-architecture could not be used on an individual that suffered from osteoporosis (Parfitt et al, 1983). Osteoporosis is a highly prevalent condition, that is estimated to effect more than 200 million people worldwide, and is more likely to affect woman than men (Reginster and Burlet, 2005).
