SI de Desarrollo del trabajo (DT)

To facilitate the counting of incremental lines in tooth cementum in an effort to provide accurate age at death estimates (see section 5 for details), it was necessary to remove teeth from the alveolar socket.

Preferably, one maxillary or mandibular premolar was carefully extracted from the alveolar bone. The tooth was embedded in Biodur E12 resin (Gunter von Hagens, Heidelberg) and left one week to harden in a desiccating box.

Twelve thin sections (70µm) of the cervical third portion of each sampled tooth root were made using a Leica 1600 Leitz Co. rotating microtome. The thin sections were fixated to glass slides with Eukitt and capped with cover slips. The slides were viewed using a Zeiss Axioskop 2 plus phase contrast light microscope under objective power first at 20x for scanning purposes and subsequently 40x for analysis. The microscopic image was digitalized using the AxioVs 3.0 and Photoshop 7.0 software. Incremental lines were manually counted directly on the monitor, starting with the first dark band immediately adjacent to wide light band marking the point of eruption (see Fig. 5.2) and tallying the number of successive dark bands outwards in the direction of the tooth root surface. Incremental lines were also counted digitally with the aid of a computer software program (Auto-TCA) specially developed by Czermak et al. (2006) at the Ludwig-Maximilians-University in Munich. The total number of counted lines was then added to the person’s estimated biological age in years at the point of eruption of that particular tooth.

An in depth specification to this method is provided in the section labeled TCA.

Sampling criterion

Tooth samples taken for this age estimation study had to meet two basic criteria in order to allow for a tooth cementum annulation (TCA) analysis. First, healthy intact teeth needed to be present. A number of individuals at Volders had either experienced total tooth loss during their lifetime or the cranial skeleton was not recovered. Others had incurred such extreme abrasive damage upon their teeth during their lifetime, that not only the crown, but also the upper portion of the tooth root dentine, from which the sections are normally taken, was missing.

Pathological conditions, such as periodontitis, caries or abscesses had deleterious effects rendering the diseased tooth inadequate for TCA analysis. Several important factors need to be taken into consideration with regards to dental pathology when sampling teeth for a cementum examination. Some researchers have shown that an unhealthy oral cavity environment can severely compromise the structural integrity and biochemical composition of the extracellular matrix, which plays a pivotal role in maintaining the homeostasis of cementum (Grzesik & Narayanan 2002). Others have shown that systemic diseases such as Type 2 diabetes or non-insulin-dependent-diabetes (NIDDM) can have a substantial influence upon tooth loss through their negative effects on cementum production and stimulation of alveolar bone resorption (Gokhan et al. 2004). In an effort to study the mechanisms of tooth loss, Gokhan et al. examined the teeth of 46 patients suffering from Type 2 diabetes, a disease which afflicts between 2-6% of the modern western population, and found significant differences in the thickness of cementum in 4 vertical areas of the tooth root spanning from the apex to the cervical portion. Paget’s disease, with a prevalence of approximately 3% in people over 40, has been shown to cause hypercementosis, leading to substantial formation of cementum, and conditions such as hypophosphotasia result in minimal cementum formation and accompanying tooth exfoliation (Kaplan et al. 1994).

Since histometric changes in tooth cementum are proven to be influenced by not only local factors and systemic disease but can also result from idiopathic conditions, the question then arises if teeth should be sampled at all from a skeleton if evidence exists for periodontal disease or extensive tooth loss resulting from a suspected or documented systemic disease that might in turn skew conclusions based on cementum structure. Foster (1994) indicates that more than 40% of Pima Indians in the United States have Type 2 diabetes, evidence that the prevalence of this disease can be culture specific. Certainly awareness of these facts would be vital before sampling such a population for this particular examination. In 1996, Houck and his colleagues gave the cautionary reminder that “biology does not always correlate with expected outcomes, particularly in such multifaceted traits as age”.

Another frequent problem encountered, which potentially altered tooth cementum structure and influenced the selection of tooth samples, was the destruction to teeth caused by diagenetic factors such as the acidic nature of the earth that some of the skeletons were buried in or the mechanically erosive hydrokinetic effects of the ground water some skeletons were subjected to. These sufficed to virtually dissolve some skeletons completely, leaving only the remnants of tooth crown enamel (Fig. 8.1).

In addition, a past construction project next to the cemetery resulted in the dumping of large quantities of mortar and lime in a shallow ditch made directly above a cluster of burials that was apparently utilized as a waste pit for discarded building materials. The skeletal remains, including the teeth, under this ditch were badly damaged by the chemical effects of the lime and unusable for this analysis. As with diseased teeth, it was important to exercise selectivity in choosing the samples so as not to include teeth that may have been exposed to corrosives and undergone postmortem structural changes.

Fig. 8.1. Photomicrograph showing diagenetically altered tooth root dentine and cementum layers. (Premolar, 75µm thick, 20x, Volders burial 27).

Second, in addition to selecting healthy tooth specimens, primarily teeth from adults were sampled. It is generally not necessary to apply the method to individuals younger than approximately 20 years of age, because various characteristics intrinsic to skeletal and dental development provide indicators more than sufficiently accurate to make an age at death assessment, however, several exceptions were made during examination of the Volders material.

Tooth sections

A point of concern related to the TCA method following sampling and sectioning, was where on the thin section to perform the microscopic scan. Anomalous tooth root structure can inadvertently lead to exaggerated or reduced line totals. It was therefore imperative to locate an area of tooth root that visually appeared to be normal prior to both the automated and manual scans. Areas displaying cementum fissures, cracks, indentations, wave-like projections, tears produced by the mechanical removal of the tooth and diagenetically altered or destroyed portions were all strictly avoided (Fig. 8.2).

Figure 8.2. Photomicrograph showing irregularly organized cementum layers. (3rd _{molar, 75µm thick, 40x,}

Volders burial 37).