Dental methods of post-mortem interval estimation. A pilot study on Feulgen reaction for DNA integrity on teeth pulp

Abstract

The estimation of late post-mortem intervals (PMIs) represents a challenge for the routine forensic pathologist's practice. Few studies have evaluated the reliability of teeth as stable and protected matrices rich in DNA. The pilot study aims to verify the feasibility of Feulgen reaction colorimetric measurement on odontoblastic cells from the dental pulp of extracted teeth as a useful tool to estimate late PMIs. Ten teeth obtained from healthy patients who underwent tooth avulsion for clinical reasons were included in the sample and crown pulps were extracted at five different PMIs (T0-baseline, 14 days, 1 month, 1.5 months, and 2 months). The PMI was assumed as the time elapsed from the tooth avulsion up to the pulp extraction. The Feulgen reaction and the colorimetric analyses with ImageJ software on odontoblastic cells of dental pulps were pursued in order to measure the residual dental DNA integrity according to the PMls. The study provides evidence for the persistence of odontoblasts up to 2 months post-mortem in a recognizable organization, hence useful in late PMIs. The method based on Feulgen reaction could be a reliable tool for estimating the late PMI, correctly distinguishing between PMI 0 and 14 days, but also between PMI 14 days and 2 months. To the best of our knowledge, the Feulgen reaction has never been applied to study the possible correlation of the dental pulp cells’ integrity and the PMI. Preliminary results suggest the feasibility of the proposed method for estimating the time elapsed since death, but further research is needed.

Keywords

Post-mortem interval DNA Feulgen reaction dental pulp forensic odontology histological analysis forensic biology

Introduction

The estimation of post-mortem intervals (PMIs), especially late PMIs, still represents a challenge for the routinely forensic pathologist's practice.

Late PMI is considered the period from the death when the tissues begin transformation and/or decomposition.¹ Several reliable methods have been validated to estimate early PMIs (from 3 to 72 h after death), and in particular the so-called compound method based on an algorithm combining the classical triad of post-mortem (PM) changes – rigour mortis, livor mortis, and algor mortis.^2–4

These PM parameters can only be applied within 48–72 h after death and the longer the time elapsed since death, the less reliable methods are available (e.g. the visual assessment of gross morphological/taphonomic changes of a corpse as well as the entomological study of insects), and the wider the error.¹^4–10 Many alternative methods have been experimented over the last few decades, some useful for archaeology and palaeontology (e.g. radioisotopes dating, or fluorescence techniques on bones).⁵ Other techniques analyse the thanatomicrobiome,¹¹ the PM proteomic profiling,^12,13 or the protein and nucleic acid degradation.^11,14–22

Most methods share the same limitation having been validated only for early PMIs, thereby they cannot be effectively used for PMIs exceeding 1 week after death.¹⁹ This limitation is partly due to the characteristic and tissue matrix selected for the study.^10,19–22 For late PMIs Vass et al.²³ studied nail samples exposed to different environmental conditions and detected RNA and DNA for up to 4 months. They concluded that nucleic acid showed time-wise degradation in nails and the amplification of some specific degraded fragments of nucleic acids over weeks and months can represent a useful marker for a long time span after death. On the contrary, RNA confirmed to be more susceptible to ribonuclease activity than DNA and, depending on the tissue matrix and storage temperatures, RNA can be either more or less stable with differential potential to accurately correlate with PMI.^13,22,24

In recent years, our research focused on the estimation of the late PMI using teeth as stable, resistant, and protected matrix²⁵ rich in DNA^26–28 and applying the next-generation sequencing analysis to study the post-mortal alterations of DNA occurring in the dental pulp.^29,30 Specific PM mutations of DNA resulted to correlate with the time after death up to at least 1 month. On the other hand, some authors analysed the PM histomorphology changes of the dental pulp obtaining promising results in terms of possible correlation with time elapsing after the death. Hence, these traditional histological methods should not leave the way to genetic analyses that are noticeably more expensive and require complex techniques and high standard labs.^31–40

The Feulgen reaction is a specific histochemical reaction used to quantify the amount of intact DNA and to localize vital cells in different tissues.⁴¹ Since the staining intensity is proportional to the DNA concentration, it is possible to measure the DNA integrity with image analysis and morphometrical approaches.⁴¹ Some studies applied this DNA-specific stain to investigate apoptotic cells and their evolution,^42,43 but at the best of our knowledge, it was never applied to study the possible correlation between the integrity of dental pulp cells and the PMI.

This pilot study aims to verify the feasibility of Feulgen reaction colorimetric measurement on odontoblastic cells of the dental pulp of extracted teeth as a possible tool to estimate late PMIs. The PMI is assumed as the time elapsed from tooth avulsion to pulp extraction for histological analysis^29,30,40 and the quantitative analysis of the DNA integrity is performed on samples extracted up to two months.

Materials and methods

Sample collection and storage

Teeth were extracted for clinical reasons from patients at the Dental Department of Careggi University Hospital in Florence, Italy, and the study obtained approval from the local Ethical Committee (no. 15208/2020). Informed consent for research and publication was obtained from each patient and all data collected were fully anonymized and processed in accordance with the European Union law (General Data Protection Regulation). Healthy subjects with indications for tooth extraction for clinical reasons (e.g. orthodontics, or impacted third molars) were included in the study. Only sound dental elements were included in the study according to the following criteria.

Inclusion criteria were permanent, complete, and healthy tooth (both mono or multiple rooted), absence of hard tissue pathologies (e.g. caries), periodontal pathologies, and severe wear, absence of dental treatments (e.g. fillings, endodontics, prostheses), and negative anamnestic history of the subject for systemic pathologies (e.g. diabetes). Exclusion criteria were deciduous teeth or permanent teeth with incomplete roots, presence of hard tissue pathologies (e.g. caries), periodontal pathologies, and severe wear, and presence of dental treatments (e.g. fillings, endodontics, prostheses).

A progressive numerical code was associated with each dental sample and only sex, date of birth, tooth position, avulsion date and time, pulp extraction date and time (PMI) were recorded on an Excel file for each patient.

The avulsion of the tooth was assumed as the moment of death since it interrupts pulp blood perfusion, as it happens at death.^29,30,40

Immediately after avulsion, teeth were rinsed with sterile saline solution and, when necessary, mechanically cleaned of external organic residues (e.g. periodontal tissue) to minimize potential influences of surface putrefactive processes on pulp degeneration. Teeth were then placed in sterilization bags, on which the sample data was recorded. Each bag was stored in the same room at standardized temperatures, with daily variations maintained within a range of 18 to 21 °C throughout all the established PMIs.

PMI set and pulp extraction

The sampling was divided into five groups based on the established PMI, as follows: group 1 representing PMI T0, that is, pulp extracted immediately after tooth avulsion (two teeth); group 2 representing PMI 14 days, that is, pulp extracted 14 days after tooth avulsion (two teeth); group 3 representing PMI 30 days, that is, pulp extracted 31 days after tooth avulsion (two teeth); group 4 representing PMI 45 days, that is, pulp extracted 45 days after tooth avulsion (two teeth); and group 5 representing PMI 60 days, that is, pulp extracted 60 days after tooth avulsion (two teeth).

At the respective PMI, each tooth was cut horizontally to separate the crown from the root with a flame diamond bur under water cooling, according to the pulp extraction protocol developed by Bianchi et al.⁴⁰ The cut was applied 2 mm below the fork for multi-rooted teeth, and 2 mm below the amelo–cemental junction in single-rooted teeth. After separating the crown from the roots, the mineralized tissue (dentine) of the pulp chamber floor was carefully smoothed with the bur under water cooling until the pulp was completely exposed. The pulp was separated from the walls using a thin probe and gently extracted with the help of tweezers. Once extracted, the pulp was immediately fixed by immersion in buffered 10% formaldehyde for histological analysis. The fixation time was consistent for each dental pulp sample.

Histological preparation

After 4-h fixation, the pulp samples were dehydrated in graded ethanol, cleared in Bio-Clear (Bio-Optica, Milan, Italy) and embedded in paraffin, orienting the major axis of the fragment parallel to the cut area of the paraffin block. Five micrometre thick sections were cut and left to adhere to glass slides at 40 °C overnight. Then, the sections were dewaxed, rehydrated, and stained with haematoxylin–eosin (H&E) and Feulgen reaction.⁴¹

With the H&E stain, the cell nuclei stained blue or dark purple with haematoxylin, which binds to nucleic acids, while the cytoplasm and extracellular components stained in various shades of pink or red-orange with eosin, which chiefly binds to proteins. This histological method is widespread for evaluating the morphology of organic components of dental pulp that are relevant for the qualitative assessment of PM degradation.^31–40

With the Feulgen reaction, the cell nuclei stained magenta red, the staining intensity being directly proportional to the amount of intact nuclear DNA. The cytoplasm and other structures not containing DNA remain colourless, ensuring a clear contrast that easily allows thresholding for morphometric analysis. The Feulgen reaction is a DNA-specific histochemical technique that enables the identification of viable cells and the quantitative evaluation of nuclear DNA integrity,⁴¹ enabling the objective analysis through an image-based and morphometric approach. Although no prior forensic application of this histochemical reaction for PMI estimation has been reported in the literature, the combined use of these two techniques allows for both qualitative and quantitative assessments of PM dental pulp degradation on the same sample. The two methodologies are based on the same preparatory process of the pulp sample and easily available stains as they are widely used for other purposes, offering advantages in terms of time and cost efficiency.

After staining, the slides were dehydrated in graded ethanol, cleared in Bio-Clear and mounted in a hydrophobic mounting medium.

Microscopy analyses

The H&E-stained slides were observed and photographed with a light microscope (Nikon) using 10×, 20×, and 40× objectives (10× eyepiece), while the Feulgen-stained ones were examined with 10×, 20×, 40×, and 100× (oil immersion) objectives. A digital camera connected to the microscope (Nikon) was used to take microphotographs at appropriate magnification as jpeg files for documentation and image analysis.

Qualitative analysis

This was aimed at the identification of the morphological structures of the dental pulp in the sections stained with H&E, that is, the presence and morphology of stromal cells, nerves, and blood vessels, as well as the appearance of collagen fibres and the absence/presence of extracellular matrix (ECM) vacuolization.

Quantitative analysis

This was performed on micrographs taken with the 100× oil immersion objective (final magnification: 1000×) to measure the intensity of the Feulgen reaction in the cell nuclei. Briefly, free-share ImageJ software (NIH, Bethesda, MD, USA) was used to select the nuclear profiles and measure the optical density of the magenta red Feulgen reaction, which directly correlates with the amount of intact DNA. The measurements are expressed as optical transmittance, the reciprocal of colour optical density; and given as arbitrary units in a scale comprised between 255 (white) and 0 (black).⁴¹

Statistical analyses

Statistical analysis of densitometry on the samples stained with the Feulgen reaction was performed using GraphPad Prism 5.0 software. Each individual tooth was considered as a statistical sampling unit, and for each tooth two microscopic areas (regions of interest – ROIs) at 100× magnification were analysed. In each ROI, 10 nuclei of odontoblasts were randomly chosen for densitometric analysis. For each PMI, two teeth from two different subjects were analysed and their values expressed as the mean ± SEM. The densitometric mean value derived from both samples corresponding to each PMI was used to minimize the risk of error from inter-individual variability. Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple comparison test. A p value ≤0.05 was considered statistically significant.

Results

Ten healthy teeth were collected from 10 healthy patients. The PMI was registered in hours (h), days (d), or months (m) and the sample information is reported in Table 1.

Table 1.

Sample dataset.

Group/ID code	Sex	Date of birth	Pathologies	Tooth	Tooth avulsion	Sample decontamination	Storage temperature (°C)	Pulp extraction	PMI	Fixation in buffered 10% formaldehyde (h)
G1/I	F	2004	X	38	20.05.24	Sterile saline solution and mechanical debridement	18–21	20.05.24	0	4
G1/L	M	2005	X	28	20.05.24	Sterile saline solution	18–21	20.05.24	0	4
G2/F	F	1991	X	48	04.03.24	Sterile saline solution and mechanical debridement	18–21	18.03.24	14 days	4
G2/G	F	1991	X	18	04.03.24	Sterile saline solution	18–21	18.03.24	14 days	4
G3/D	M	1959	X	38	15.02.24	Sterile saline solution	18–21	18.03.24	1 month	4
G3/E	F	1991	X	38	16.02.24	Sterile saline solution and mechanical debridement	18–21	18.03.24	1 month	4
G4/A	F	1974	X	28	06.02.24	Sterile saline solution	18–21	18.03.24	1.5 months	4
G4/H	F	1998	X	48	25.03.24	Sterile saline solution and mechanical debridement	18–21	09.05.24	1.5 months	4
G5/B	M	1947	X	18	08.02.24	Sterile saline solution	18–21	08.04.24	2 months	4
G5/C	M	1974	X	48	08.02.24	Sterile saline solution and mechanical debridement	18–21	08.04.24	2 months	4

PMI: post-mortem interval.

Figures 1 to 3 show the summary of the most significant histological characteristics of dental pulp at PMI T0 (baseline), PMI = 14 days, PMI = 1 month, PMI = 1.5 months, and PMI = 2 months obtained with the H&E stain. The pulp alterations in the two matched subjects were similar and consistent for T0, T 14 days, and T 1 month, therefore only one sample is shown and described in Figure 1. On the contrary, for T 1.5 months and T 2 months a certain degree of inter-sample variability emerged, thus both samples are reported and analysed (panels D/E in Figure 2 and panels F/G in Figure 3). Regardless of these slight individual differences, the alterations of the pulp (ECM, collagen fibres, stromal cells, blood vessels, and nerves), tended to increase with the time elapsed from death, especially from PMI = 1 month. In particular, a homogeneous distribution of ECM without vacuolization featured all samples up to PMI = 14 days. ECM vacuolization was detectable in all samples for PMI > 14 days, with an intra-PMI variability of diffuse or localized disaggregation at PMIs 1.5 and 2 months (Figures 2 and 3, samples A/C and H/B, respectively). Well-conserved stromal, endothelial, and nerve cells with roundish or elongated nuclei are detectable for PMIs up to 1 month. At PMI = 1.5 months, the samples showed intra-PMI different patterns of alteration with fairly preserved, still recognizable cells for sample A, but not visible blood vessels, nerves, and randomly distributed stromal cell nuclei with unclear morphology for sample H. Similar heterogeneous patterns were observed in the samples at PMI = 2 months (samples C and B, Figure 3). Collagen fibres become less defined for all PMIs > 1 month. For all PMIs, odontoblasts usually were clearly recognizable up to PMI > 14 days: they were distributed homogeneously at the inner dentinal surface and showed aligned nuclei and apical cell processes extending inside the dentinal tubules. On the other hand, a progressive disarrangement of the odontoblastic layer and disappearance of the odontoblastic processes inside the dentinal tubules was detected from PMIs > 1.5 months. However, several nuclei of stromal cells and odontoblasts were still recognizable up to 2 months after death.

Figure 1.

(Haematoxylin–eosin stain) magnification 10× (a) and 40× (b): histological characteristics of dental pulp at PMI T0 (baseline) (A), 14 days (B), 1 month (C). Panel A shows homogeneous distribution of extracellular matrix without vacuolization, well represented odontoblasts with homogeneous distribution and stratification of nuclei within the odontoblastic layer and odontoblastic processes inside dentinal tubules, well represented different cell types with circular, ovoid, and elongated-shaped nuclei, well conserved endothelial cells of blood vessels, well conserved nerve cells, and well defined collagen fibres. Panel B shows homogeneous distribution of extracellular matrix without vacuolization, well represented odontoblasts with homogeneous distribution and stratification of nuclei within the odontoblastic layer and odontoblastic processes inside dentinal tubules, well represented different cell types with circular, ovoid, and elongated-shaped nuclei, well conserved endothelial cells of blood vessels, well conserved nerve cells, and well defined collagen fibres. Panel C shows extracellular matrix with localized disaggregated and vacuolized areas, well represented odontoblasts with a reduced layer thickness and no visible odontoblastic processes inside dentinal tubules, well represented different cell types with circular, ovoid, and elongated-shaped nuclei, well conserved endothelial cells of blood vessels, well conserved nerve cells, and less defined collagen fibres. PMI: post-mortem interval.

Figure 2.

(Haematoxylin–eosin stain) magnification 10× (a) and 40× (b): histological characteristics of dental pulp from two different sample at PMI 1.5 months (panel D from sample A and panel E from sample H). The two samples show different stages of extracellular matrix degeneration. Panel D shows less defined collagen fibres, extracellular matrix with localized disaggregated and vacuolized areas, well represented odontoblasts but the layer thickness is reduced and no visible odontoblastic processes inside dentinal tubules, well conserved endothelial cells of blood vessels, and well conserved nerve cells. Panel E shows not defined collagen fibres, extracellular matrix with massive disaggregated and vacuolized areas, not visible blood vessels, nuclei still visible but distributed randomly among the pulp vacuoles, and unorganized odontoblastic layer. PMI: post-mortem interval.

Figure 3.

(Haematoxylin–eosin stain) magnification 10× (a) and 40× (b): histological characteristics of dental pulp from two different samples at PMI 2 months (panel F from sample B and panel G from sample C). The two samples show different stages of extracellular matrix degeneration. Panel F shows not defined collagen fibres, extracellular matrix with massive disaggregated and vacuolized areas, well represented odontoblasts but the layer thickness is reduced and no visible odontoblastic processes inside dentinal tubules, and no visible blood vessels. Panel G shows less defined collagen fibres, extracellular matrix with localized disaggregated and vacuolized areas, well represented odontoblasts but the layer thickness is reduced and no visible odontoblastic processes inside dentinal tubules, and well represented endothelial cells and blood vessels. PMI: post-mortem interval.

In Figure 4, representative images of the Feulgen staining at PMI = 2 months at different magnifications (10×, 40×, and 100×) are shown. These indicate that intact DNA can still be found in the 2-month PMI.

Figure 4.

Results of Feulgen reaction coloration of sample C at PMI of 2 months with magnifications 10× (a), 40× (b), and 100× (c). PMI: post-mortem interval.

Table 2 shows the levels of DNA integrity as can be argued by Feulgen reaction densitometry. The columns report the optical transmittance values for each sample at the different PMIs. The mean baseline transmittance was 216 ± 2 for PMI = T0. The transmittance values decreased to 178 ± 1.4, 179 ± 1, 180 ± 1.7, and 184 ± 1.8 in PMI = 14 days, PMI = 1 month, PMI = 1.5 months, and PMI = 2 months, respectively.

Table 2.

Colorimetric values by Feulgen reaction of DNA integrity obtained with ImageJ software at PMI T0 (baseline), 14 days, 1 month, 1.5 months, and 2 months.

Column statistics
Post-mortem interval	T0	T 14 days	T 1 month	T 1.5 months	T 2 months
Colorimetric values
Minimum	205	168	167	166	168
25% percentile	211	172	176	175	176
Median	218	177	179	178	186
75% percentile	220	184	181	186	190
Maximum	226	188	188	192	195
Mean	216	178	179	180	184
Std. deviation	6.2	6.4	4.6	7.6	8.3
Std. error	2.0	1.4	1.0	1.7	1.8
Lower 95% CI of mean	212	175	176	176	180
Upper 95% CI of mean	221	181	181	183	187
normality test
KS distance	0.15	0.12	0.12	0.11	0.14
P value	P > 0.10	P > 0.10	P > 0.10	P > 0.10	P > 0.10
Passed normality test (alpha = 0.05)?	Yes	Yes	Yes	Yes	Yes
P value summary	ns	ns	ns	ns	ns

PMI: post-mortem interval; CI: confidence interval; KS: Kolmogorov-Smirnov.

Table 3 and Figure 5 report the results of the statistical analysis (ANOVA and Newman–Keuls post-hoc test). Significant differences in DNA integrity were there between T0 (PMI baseline) and all the other PMI sets, from PMI = 14 days up to PMI = 2 months. Small, not-significant differences were detected among the PMIs longer than T0.

Figure 5.

Graphical representation of the results obtained by ANOVA and Newman–Keuls (NK) multiple comparison tests. ANOVA: analysis of variance.

Table 3.

Results obtained by ANOVA and Newman–Keuls (NK) multiple comparison tests.

One-way ANOVA
P value	P < 0.0001
P value summary	***
Are means signif. different? (P < 0.05)	Yes
Number of groups	5
F	66
R ²	0.76
Bartlett's test for equal variances
Bartlett's statistic (corrected)	6.9
P value	0.1431
P value summary	ns
Do the variances differ signif. (P < 0.05)	No
ANOVA table	SS	df	MS
Treatment (between columns)	12,000	4	3000
Residual (within columns)	3900	85	46
Total	16,000	89
Newman–Keuls multiple comparison test	Mean diff.	q	Significant? P < 0.05?	Summary
T14 days vs T0	−39	21	Yes	***
T14 days vs T 2 months	−6.0	3.9	Yes	*
T14 days vs T 1.5 months	−1.9	1.3	No	ns
T14 days vs T 1 month	−0.85	—	No	ns
T 1 month vs T0	−38	20	Yes	***
T 1 month vs T 2 months	−5.1	3.4	No	ns
T 1 month vs T 1.5 months	−1,1	—	No	ns
T 1.5 months vs T0	−37	20	Yes	***
T 1.5 months vs T 2 months	−4.0	—	No	ns
T 2 months vs T0	−33	17	Yes	***

ANOVA: analysis of variance.

Discussion

Teeth are known worldwide as a pillar for personal identification and age estimation,^{25,28,44–55} but few studies considered dental tissues as valuable source of data for instance as alternative matrix for PM toxicology^56,57 or for estimating the PMI.^{29,30,33–40} Previous literature reported promising results about the use of dental tissues for estimating the time elapsed since the death, but recent reviews stated the need for further studies and the validation of methods based on the PM degeneration of dental pulp and nucleic acids (RNA/DNA).^21,24,31,32

This study was based on 10 dental pulps obtained from healthy patients who underwent tooth avulsion for clinical reasons (impacted third molars). As suggested by previous studies,^{29,30,33–40} the time of avulsion was assumed as the time of death, because the dental extirpation causes the interruption of blood perfusion of the pulp as it occurs at death. Five different PMIs (T0-baseline, 14 days, 1 month, 1.5 months, and 2 months) were considered and two dental pulps for each PMI were analysed. To measure the residual DNA integrity of the dental pulp as a possible marker for estimating late PMIs, the Feulgen reaction and densitometric analysis on odontoblastic cells of the dental pulp were carried out.

In terms of tissue degeneration, the variability between the samples is limited for the baseline and PMI of 14 days and 1 month, with similar alterations of the ECM, blood vessels, collagen fibres, and different cells (stromal, vascular, nervous, and odontoblasts). On the contrary, we found a certain inter-sample variability of alteration pattern for PMI = 1.5 months and PMI = 2 months. The limited samples analysed here do not allow any definite conclusion about the factor that can cause this variability.

Despite the above-mentioned inter-sample variability, we observed an increasing pattern of degeneration of ECM, collagen fibres, blood vessels, and nerves in parallel with the increase in PMI, starting from 1 month after death. The most relevant findings are the persistence of various pulp cell types, in particular stromal cells and odontoblasts, up to 2 months. The odontoblastic layer appears to undergo a progressive decrease in density and alteration of its classic epithelial-like arrangement as the time elapses from death. At PMI = 14 days, odontoblasts are still well recognizable with homogeneously aligned nuclei and odontoblastic processes within the dentinal tubules. At PMI = 1 month, 1.5 months, and 2 months, the odontoblast layer disassembles and the odontoblastic processes inside dentinal tubules disappear, but scattered cells are still recognizable for all the considered PMIs (Figure 2). This finding is consistent with some previous studies. Bhuyan et al.³⁸ analysed 40 pulps of sound premolars extracted for orthodontic treatment at PMIs 24 h, 48 h, 72 h, 1 month, 3 months, 6 months, 1 year, and 2 years after avulsion (pulp death). Here, the nuclei of pulp cells underwent progressive autolytic changes over time, highlighting a substantial decrease in fibroblasts, immunologic and endothelial cells at PMI = 1 month. The odontoblastic layer remained intact in some peripheric areas up to PMI = 6 months and few odontoblastic nuclear debris were seen up to 1 year after pulp death. Bianchi et al.⁴⁰ analysed 27 dental pulps of 16 healthy subjects who underwent extraction of impacted third molars or premolars for orthodontic treatments. The tooth avulsion was assumed as the time of pulp death and different PMIs were analysed up to 2 weeks after death. Different histological changes are reported for blood vessels, collagen fibres, and ECM in PM dental pulp, even with a certain degree of inter-sample variability. Nonetheless, odontoblasts showed no evidence of cellular or nuclear lysis up to 14 days.

Different findings were reported by Caballín et al.,³³ who analysed 122 pulps extracted from impacted molars of living subjects within an early PMI of a week. They found that odontoblasts progressively detached from the dentin layer from 3 days up to 5–6 days after death with a complete disappearance at the beginning of the first week PM. Vavpotic et al.³⁵ obtained similar results on 32 teeth extracted from corpses of subjects aged 18–40 years old with a known time of death. They calculated a linear regression model of the reduction of nuclear density in the PM odontoblastic layer at two different storage temperatures, 23 °C and 4 °C, estimating the persistence of odontoblasts with 95% confidence interval of 110.03 ± 14.12 h (4.58 ± 0.59 days) at 23 °C temperature, and of 111.53 ± 9.35 h (4.64 ± 0.38 days) at 4 °C. The authors concluded that the density of odontoblasts and their histological disappearance could be a useful tool in estimating the early PMI up to 5 days after death, without significant differences among storage temperatures. A recent study conducted by Sato et al.⁵⁸ on 99 healthy third molars from 94 living subjects aged 18–40 years analysed at T0, 1 month, 3 months, and 6 months after death under different environmental conditions (controlled environment, drowned and buried teeth), highlighted that at PMI = 1 month the odontoblast nuclei disappeared completely regardless of the conservation conditions.

The difference obtained by these studies could be explained by the different techniques used to prepare the tooth and the pulp extraction. In fact, Caballín et al.,³³ Vavpotic et al.,³⁵ and Sato et al.⁵⁸ immersed the samples in a decalcifying substance for some months (formic acid or Ethylenediaminetetraacetic acid [EDTA], respectively) before cutting and preparing them histologically. Decalcification has conceivably induced cellular alteration and damage, then causing an ostensible cell lysis in the microscopic samples. On the contrary, Bhuyan et al.³⁸ and Bianchi et al.,⁴⁰ as well as the present study, have adopted a more conservative technique, since the pulp was extracted from teeth without previous decalcification which could have interfered with the histomorphology of the pulp cells and structures. Moreover, EDTA requires a long time to induce decalcification: hence, an insufficient incubation can result in cutting artefacts due to incomplete decalcification, whereas a prolonged incubation can result in artefacts of cell preservation or staining, especially on the peripheral odontoblast layer.⁵⁹ In turn, formic acid can induce even more pronounced artefacts to the odontoblastic layer, causing detachment of the pulp from the inner dentin surface.⁵⁹

The different preparations of the PM dental samples seem to be a discriminating factor on the possible detection of odontoblasts in PM dental pulps. Although the study sample is limited (10 teeth), the present finding of a well-preserved layer of odontoblasts with the typical epithelial-like structure, detectable up to 2 months after death, strengthens the conclusions of the previous studies suggesting the stability and persistence of these cells in PM dental pulps as an useful tool for estimating late PMIs.

Some previous studies³⁵ proposed a PMI estimation method based on the count of odontoblast cell density. However, in a recent research,⁴⁰ the authors found some difficulties in standardizing the cell counting procedure both manually and automatically, due to overlapping of cellular layers and especially cell fragmentation after death. On the other hand, Madsen⁶⁰ established that the most accurate method for the determination of the cellular content in cow's milk (e.g. leucocytes in cases of mastitis) is to estimate the integrity of the DNA via the Feulgen reaction rather than using the classical H&E staining. They developed a formula to calculate the cell amount from DNA content (1 µg of DNA representing about 145,000 cells), and assessed a 0.9 coefficient of correlation between the real cell content in the milk samples and the Feulgen reaction. In conclusion, they demonstrated that the accuracy of the Feulgen reaction in measuring the DNA content was as sensitive and specific as the direct microscopic cell count method, but it is endowed with higher reliability detecting even and especially damaged cells with residual DNA, not countable microscopically. Accordingly, we applied the Feulgen reaction⁴¹ to measure the PM amount of residual intact DNA in the odontoblasts, and we found that this decreased with the time from death. This analysis revealed the persistence of Feulgen-positive DNA in the odontoblastic layer up to PMI = 2 months, suggesting that the analysis of DNA rather than the mere alterations of cellular morphology may be a promising tool for the estimate of late PMIs. Indeed, the densitometric analysis of odontoblastic DNA at the different PMIs demonstrated a statistically significant decrease in DNA integrity between baseline = T0 and 14 days PMI, reaching a plateau up to 1.5 months and a further significant decrease at PMI = 2 months.

The noted PM stability of DNA in the dental pulp cells is partially consistent with previous literature which demonstrated a higher resistance of nucleic acids of dental tissues compared to other tissues, such as blood samples, splenic cells, and so on,^61,62 in which DNA integrity persisted up to maximum 72 h after death. In contrast, the DNA flow cytometric analysis conducted by Boy et al.⁶² on 57 human teeth, exhibited minimal DNA degradation up to 144 h post-extraction, without constant degradation, an useful finding to determine early PMIs. Similarly, Mansour et al.⁶³ studied 95 teeth from corpses conserved under different environmental conditions (fresh, burnt, buried, or skeletonized corpses, dry, indoor, or immersed in water) and found that the most reliable PM period for DNA analyses for identification purposes consisted in the first 10 days after death with a dramatic decrease in dental DNA integrity observed in the longer times. The peak of degradation of odontoblastic DNA was assessed between T0 and 2 weeks after death, a finding that is consistent with the present results obtained with densitometric analysis of the Feulgen reaction. Our study also demonstrates that another peak of DNA degradation occurs at 2 months after death. To the best of our knowledge, data on the progression of odontoblastic DNA degradation at PMI longer than 2 weeks has not been reported before,²¹ except for a preliminary note by Rubio et al.⁶⁴ Through quantitative real-time Polymerase Chain Reaction (PCR), they described a significant reduction of DNA concentration at 1 month post-extraction, a plateau between 1 and 12 months post-extraction, and a further decrease at 18 months post-extraction, accompanied by a significant reduction of the allelic dropout ratio, an index of DNA integrity. The use of real-time PCR suggests the impossibility to correlate a decrease in DNA integrity before a PMI = 18 months, while the odontoblastic DNA degradation studied by Feulgen reaction suggests the possibility to detect a correlation with the time of death of a subject in late PMIs, up to 2 months after death but earlier than 18 months.

Although the sample analysed is limited in the number (10 pulps) and in the PMIs considered (up to 2 months), the present study highlights that the Feulgen reaction applied on the odontoblastic DNA could be a reliable tool for estimating the late PMIs, correctly discriminating between PMI 0 and 14 days but still applicable to PMIs ranging between 14 days and 2 months. This new method has shown a discriminative capability inversely proportional with the time after death (2 weeks from baseline and 1.5 months up to 2 months PM), but it demonstrates a possible correlation between pulp cell DNA and PMI within PM ranges for which no other reliable method is available.^4–9

The main limitation of this preliminary study is the reduced number of samples and the relatively few PMIs considered, albeit the reported statistically significant differences endorse our method based on densitometric analysis of Feulgen reaction of odontoblastic DNA as a promising tool for PM analyses. In particular, preliminary results appear to discriminate between PMIs of 14 days and 2 months, where other methods often fail, but the present study cannot provide evidence regarding the potential of the Feulgen reaction to differentiate longer PMIs and the associated range of error, as the analysis was limited to this timeframe. Therefore, further research on a larger sample is needed to validate our findings and to better delineate the applicability of the method for both early and late PMIs. The potential influence of the inter-individual variability, as each tooth was obtained from a different subject, was not consider for this pilot study due to the limited sample size. This factor may represent a potential source of bias in the interpretation of the results, particularly in cases of later PMIs, where morphological manifestations are more irregular. Future studies involving comparative analyses of multiple teeth extracted from the same individuals are needed to assess the accuracy of DNA integrity, even in highly degraded samples, thereby enhancing the robustness of the preliminary findings. Another important limitation may concern the absence of the soft tissues normally surrounding teeth in the oral cavity (e.g. the periodontal ligament), which are themselves subject to PM changes and may influence the rate of pulp tissue degradation. The mechanisms underlying PM dental pulp degeneration, such as putrefaction with bacterial contamination, autolysis, or dehydration, are not clearly known. Since the primary aim of this pilot study was to assess the feasibility of a novel histological approach for evaluating DNA degradation in relation to the PMI, this variable was excluded from the experimental design. Further research investigating odontoblastic DNA in teeth surrounded by soft tissues is definitely needed to integrate this aspect.

Conclusions

Since the estimation of late PMI represents a challenge for the routine forensic pathologist's practice, this preliminary study proposes a novel method to estimate late PMIs up to 2 months based on the densitometric quantitation of Feulgen reaction, used as an index of odontoblastic DNA integrity, in PM dental pulp histological sections. The pilot study supports the feasibility of the method suggesting the Feulgen reaction potentially useful for estimating the late PMI. To the best of our knowledge, this method has never been applied for such purposes. Moreover, our results provide evidence that histologically recognizable odontoblasts persist up to PMI equal to 2 months, whereas other pulp components (ECM, collagen fibres, endothelial cells blood vessels, and nerves) undergo variable and unpredictable histological changes, especially from PMIs greater than month and a half, thus being unreliable markers for PMI estimation.

Albeit the promising preliminary results, further research improving the sample size and PMI ranges is needed to investigate the influence of the degeneration of soft tissues surrounding teeth on PM dental pulps and the inter-individual variability, to widen the late PMIs to better delineate the applicability of the method for both early and late PMIs, and to validate pilot findings.

Footnotes

ORCID iDs

Ilenia Bianchi

Emanuele Capasso

Ethical considerations

Ethics Committee of the Tuscany Region – Central Wide Area (CEAVC) (no. 15208/2020).

Consent to participate

Informed consent was obtained from all subjects involved in the study.

Consent for publication

Informed consent was obtained from all subjects involved in the study.

Author contributions

Ilenia Bianchi conceived of the study, participated in its design and coordination, contributed to the investigations and resources, conducted data curation, drafted the manuscript, and supervised the final version.

Martina Focardi contributed to the investigations and data curation.

Annalidia Seclì conducted the analyses and investigation, contributed to data curation, and drafting the manuscript.

Emanuele Capasso contributed to data curation.

Regina Rensi contributed to data curation.

Barbara Gualco contributed to resources and data curation.

Laura Calosi conducted to the analyses and investigation.

Daniele Bani conceived of the study, participated in its design and coordination, contributed to the investigations and resources, conducted data curation, and supervised the final version of the manuscript.

Vilma Pinchi conceived of the study, participated in its design and coordination, contributed to the investigations and resources, conducted data curation, drafted the manuscript, and supervised the final version of the manuscript.

All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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