Determination of PMI is an important parameter in forensic evaluation. Considerable morphologic and histologic changes take place in the body after death attributed to the absence of circulation of blood and loss of regulatory mechanisms causing enzymatic dissolution of the injured cells leading to cellular and nuclear degeneration. Diverse histopathologic methods have been employed to identify the characteristic degenerative changes and determine the time elapsed after death in various soft tissues of the body (Zdravković et al. 2006; Pittner et al. 2016; Kushwaha et al. 2010) of which, we found only four studies on histological evaluation of PMI changes in human dental pulp (Gawande et al. 2012; Carrasco et al. 2017; Rajesh et al. 2016; Duffy et al. 1991).
Dental pulp is enclosed in hard tissue and hence it is well protected and preserved for a longer time. Preservation of dental pulp is seen long after damage to other tissues is evident. Thus, it is an ideal tissue for analysis of post-mortem changes. Rajesh et al. (2016) histologically noted the degenerative changes up to 36 h whereas Gawande et al. (2012) listed the findings up to 96 h only. A comparative study on the putrefaction rate of dental pulp in extracted teeth of humans, pigs, and unextracted teeth within the jaws of a pig was done by Duffy et al. in northwest coastal climate of Canada until 14 days. Carrasco et al. (2017) attempted to quantify these changes up to 6 months. Ours is a first of its kind study, where we analyzed the tissue morphologic changes, staining characteristics, and structural details in H & E-stained pulp tissue harvested at varying time intervals up to 2 years.
The body undergoes putrefaction which is majorly affected by the circumstances of death, external environmental factors, and internal factors of the deceased (Gawande et al. 2012). The relative humidity, the temperature, and the bacterial load of the environment also affect the desiccation of the soft tissue. In our study, to curtail the influence of external factors on pulp tissue, we blocked the apical foramen by a piece of modeling wax. We had kept the tooth at room temperature, the mean being 36 °C. An attempt has been made to estimate the histologic changes in the tooth pulp which is preserved within the hard enclosure of enamel and dentin up to 2 years from the time of extraction which is considered equivalent to the time of death.
The array of methods has been previously described to retrieve the pulp like sectioning the tooth with disk (Gawande et al. 2012), decalcification (Vavpotic et al. 2009), and using rotary instruments and files (Carrasco et al. 2017). These methods exposed pulp to excessive heat and/or chemicals which could alter the morphologic and histologic characteristics. In our study, we have used a less aggressive method in which the tooth is split with small sharp strokes applied to chisel by a mallet and whole pulp harvested without exposing to chemicals or heat (Fig. 1).
On morphological evaluation, the color of the pulp tissue gradually changed from pink at 24 h to dirty pink at 48 h followed by pale pink at 72 h. The consistency ranged from soft to firm to jelly up to 72 h and later became dehydrated, dried, desiccated, and friable by the end of 2 years (Fig. 2). A similar finding was found in only one study by Mehendiratta et al. (2015) who studied the time-related changes in dental pulp samples of porcine teeth. No evidence of any literature on the morphologic analysis of human pulp was found in post-mortem teeth.
Pulp is a soft, vascular connective tissue rich in fibroblasts and defense cells embedded in a soft gelatinous ECM lined by odontoblasts (Goldberg and Hirata 2017). In the present study, the light microscopic features of the degenerative changes in the constituents of pulp have been categorized at varying time intervals for up to 2 years. A rapid degeneration was observed until 72 h. The rate of decomposition had gradually slowed down beyond one month with relatively less histologic variation up to 2 years. This may be attributed to the fact that the desiccation of the pulp tissue after 1 month aids to the preservation of the tissue (Michaud and Foran 2011)
The ECM of pulp consists of a delicate network of collagen fibrils, fibronectin, tenascin, other non-collagenous proteins, and proteoglycans which imparts an eosinophilic background in H & E-stained sections of pulp (Goldberg and Hirata 2017). A decline in staining property of the ECM is seen with increasing time. This was in agreement with the study by Gawande et al. but in contrast with observations by Mehendiratta et al. (2015) and Duffy et al. (1991) who documented increased eosinophilic areas and condensation. Few small vacuolations were noted in group 1 samples which progressively increased in size and number in the subsequent groups nearly filling up the entire stroma by 2 years. Identical observations were reported by Mehendiratta et al. (2015), Rajesh et al. (2016), and Carrasco et al. (2017). The release of intracellular hydrolytic enzymes and anaerobic putrefying bacteria causes decomposition of these ECM components producing gas (Mehendiratta M et al. 2015) which might be reflected by these vacuolations.
The nuclear density of fibroblasts and defense cells progressively reduced with increasing time interval after which they were diminished to fragmented nuclear debris. These observations were consistent with the findings of the quantitative analysis done by Carrasco et al. (2017). A series of autolytic changes were observed in the nuclei of these cells which included karyorrhexis at 72 h, pyknosis at 1 month, and karyolysis by 6 months. The nuclei of the fibroblasts in our study were comprehendible up to 6 months which was similar to the study by Carrasco et al. (2017). On the other hand, rapid degeneration of the cells with nuclear stability was noted up to 144 h by Mehendiratta et al. (2015) and 2 weeks by Duffy et al. (1991). This may be attributed to the difference in environmental conditions where the temperature and humidity come into effect. Moreover, in these studies, the samples were buried in the soil which would have accelerated the rate of degeneration due to the favorable bacterial and fungal proliferation. In our study, a temperature of 36 °C and relatively lower humidity similar to the experiment by Carrasco et al. (2017) would have resulted in desiccation of the pulp tissue leading to longer nuclear stability.
Although our methodology permitted limited preservation of the odontoblastic layer, a layer of odontoblasts with palisading nuclei was seen in the periphery in some areas up to 6 months (Figs. 2 and 3). Nuclear stability of the odontoblasts was also noted till 6 months which slowly declined over time. Vavpotic et al. (2009) found a linear regression model for relative values of odontoblastic density until 120 h.
In the present study, the integrity of the blood vessels and RBCs were seen up to 72 h after which they were not discernable. Mehendiratta et al. (2015) in their study found faint outlines of blood vessels with few endothelial cells remaining in subsurface samples at 96 h. The blood vessel parameter can prove as a useful tool to estimate early PMI.
On the microbiological assessment of the tissue sections with Gram stain, Gram-positive Staphylococci and Streptococci were noticed in all groups of study. Hyde et al. (2013) have documented a shift in the nature of bacteria from aerobic like Staphylococcus and Enterobacteriacae to anaerobic like Clostridia and Bacteroides in cadavers. They further noticed Streptococcus among the top ten genera in the pre bloat swab of mouth samples in human remains. There are no studies to date on the microbiological flora of the post-mortem decomposing pulp.
This study was limited to a small sample size of 40 teeth and the study design being qualitative analysis. Since literature pertaining to the topic was sparse with no satisfactorily conclusive results, we conducted this observational study to analyze the exploratory possibilities with a small sample size and charted our findings. Further researches on a larger sample size can be done to quantitatively analyze and standardize these qualitative characteristics.
In the present study, the unfixed ante-mortem pulp tissue simulates the autolytic changes which take place in a post-mortem tissue. This study has its scope where the remains of the deceased could be studied at diverse circumstances and climatic conditions. It would also be interesting to carry out studies pertaining to the time to which successful DNA extraction can be done from the nuclei of the pulp in different environmental conditions. This can provide information on the identity of the individual which will be valuable in forensic investigation.