Eight of the twenty-eight samples were selected to report what can be visualized in virtual cross-sections in comparison to what was identified in the thin section micrographs as an introduction to how bioerosion presents at each level of the VHI. This subset consists of samples from the femur and mandible, and includes one faunal sample, a cremated sample, and a severely osteoporotic sample. Selected images are included in the text while the remaining ones can be found in the Supplementary Materials (S4–S28).
SB01/mandibular ramus (OHI 5/VHI 5) (Fig. 3)
OHI with LM
No MFD can be found in the entire thin section. There is reduced collagen content along the periosteal surface, but overall, collagen concentration is high. Enlarged lacunae and canaliculi, evidence of Wedl type 2 tunnelling, are visible throughout the entire thin section (Supplementary Material S4). The periosteal and endosteal surfaces are in good condition, and osteocyte lacunae are numerous. There is no damage to the microstructure. Sediment is visible along trabecular surfaces. A minority of the Haversian canals are filled with exogenous material.
The bone is in nearly perfect condition with no visible bioerosion. Grey values remain consistent within and between slices through the volume image. Lamellae around Haversian canals are defined as the cement lines that surround them. There are inclusions within a small number of canals and attached to trabeculae, as noted in the histology, which are not to be confused with bioerosion.
GE15/femur (OHI 4/VHI 4.5) (Fig. 4)
OHI with LM
The bacterial attack is arrested. The highest concentration of MFD is within the midline and endosteum; the trabeculae are affected and the periosteum mildly so. Microfissures follow the microanatomy along the periosteal surface. Collagen preservation is very good throughout the entire section. There is exogenous mineral precipitation within the Haversian systems, primarily along the endosteal surface with a milder manifestation along the midline.
Bioerosion is minimal. Grey values are mostly consistent throughout and between virtual slices with minor density loss (darker grey values), particularly around the midline and towards the endosteal surface. Bioerosion is concentrated around Haversian systems with numerous MFDs present as small clusters of perforations forming speckled areas of demineralization. While individual lamellae are visually indistinct, undifferentiated concentric lamellae surrounding the Haversian canals remain visible. Some cement lines can be distinguished in the filter contrast-enhanced figure (Supplementary Material S5) as confirmed by assessment of multiple virtual slices. The periosteal circumferential lamellae are eroded more so than the endosteal lamellae, which have undergone microbial attack, as have the surfaces of the trabeculae. Microfissures that move from the periosteum towards the midline along the anatomy of the Haversian systems appear to be post-depositional or post-excavation damage as bioerosion does not follow them. There are inclusions in canals near the endosteum.
S01/femur (OHI 2.75/VHI 3) (Fig. 5)
OHI with LM
Bioerosion is inhibited. MFD is equally dispersed throughout the entire section. Collagen preservation is mediocre though it is better within the unaffected lamellae. There are many enlarged canaliculi and lacunae, evidence of Wedl type 2 tunnelling. Most of the canals are filled with exogenous mineral precipitation. The endosteal surface is not eroded, though the periosteal surface is damaged.
A moderate level of bioerosion is present. Canals can be distinguished throughout much of the bone; when not visible, it is not due to bioerosion; the sample is an early adult and much of the endosteal lamellae remain where at this age, fewer secondary osteons have formed with cement lines. The periosteal lamellae are more heavily eroded. There are MFDs and patches of reduced density within the circumferential lamellae of the endosteal surface that connect with bioerosion surrounding Haversian systems. Lamellae of the Haversian canals are distinct within areas of relatively unaffected bone; the bioerosion, which presents as both MFD and patches of dark grey values, has primarily attacked the periosteum and the midline towards the periosteum. A large embedding artefact traverses the sample from the endosteum to the midline. Small patches of high density (white) material fill a few canals towards the endosteal surface and along the midline; the most external lamellae of the endosteum are also a higher density.
R03/femur (OHI 1/VHI 1.75) (Fig. 6)
OHI with LM
Bioerosion is present throughout the entire section. MFD is strongest along the midline towards the endosteum. A large area along the midline towards the endosteum remains mostly unaltered with unaffected lamellae still visible, though there are MFDs around the Haversian canals. Within this area, collagen content is very good and the lacunae remain a normal size. All canals and microfissures are filled with calcite. A large microfissure runs from the periosteum of the entire section through the histological anatomy, which may be the result of shrinkage as the bone dries in areas where there is no tunnelling. Microfissures following the microanatomy travel from the periosteum into the midline. Sediment is attached to the periosteal surface.
The bone has undergone bioerosion in large patches. Canals remain, particularly along the midline, but bioerosion is pervasive throughout and between virtual slices through the 3D image, particularly along the endosteum and towards the periosteum. Though bioerosion attacks the concentric lamellae, rings of denser bone separate erosion from the canals. Along the midline where bone remains better preserved, lamellae of Haversian systems are visible as are faint cement lines. Many canals are filled with dense (white) inclusions (calcite) in areas of heavy and light bioerosion. Circumferential lamellae, particularly along the endosteum, are heavily affected. The outer layer of the endosteum is also denser. Two large shrinkage-induced microfissures encircle the midline where bioerosion is arrested, as noted in the histology. The periosteal lamellae are eroded but are slightly denser than the endosteal lamellae.
GÖ03/femur (OHI 0/VHI 0) (Fig. 7)
OHI with SEM
The bacterial attack is extensive. MFDs are identifiable along the entire section with only Haversian canals remaining. A very small area of well-preserved bone remains along the endosteum; however, this surface is heavily eroded. Most of the canals are filled with an exogenous matrix, possibly bone fragments, soil, and/or calcite. The periosteum is also heavily eroded.
The bone has undergone extensive bioerosion with little unaltered bone remaining. Small Howship’s lacunae suggest incipient osteoporosis (Supplementary Material S6). Canals remain but bioerosion is extensive throughout and between virtual slices, and several are filled with a dense (white) material, particularly along the periosteum towards the midline. The periosteal circumferential lamellae have been sloughed; some remain along the endosteal surface, which is also encrusted with inclusions as are canals along this border. Although bioerosion affects the entirety of the sample, it is more prevalent (darker grey values) towards the periosteum where concentric lamellae can barely be distinguished, if at all, from the surrounding interstitial lamellae, and in a patch along the endosteum. Trails of lighter grey values where some visually undifferentiated lamellae of the Haversian systems can be seen are interspersed amongst heavy demineralization. In general, bioerosion is now so heavy that individual MFDs are indistinguishable.
S04/femur (OHI 0/VHI 0.25) (Fig. 8)
OHI with LM
Macroscopic examination revealed the individual to be osteoporotic; this was confirmed by histology. Bacterial attack is heavy; only Haversian canals are identifiable. There is no collagen preservation. There is exogenous mineral precipitation in most Haversian canals. Bioerosion is severe throughout the entire section. Exogenous sediment is attached to both the endosteal and periosteal surfaces with exogenous infilling along the endosteum.
The sample has undergone extensive bioerosion with little unaltered bone remaining. Canals remain distinct but bioerosion is extensive throughout and between virtual slices; however, the bone is also porous and demineralized in part due to an osteoporosis-induced reduction in bone mineral density (Marcus and Bouxsein, 2010). Many canals exhibit clear evidence of resorption, such as Howship’s lacunae and irregular borders. During senescence remodelling of Haversian systems, including smaller ones, results in the enlargement of canals and their coalescence (Seeman 2013) as found in this sample, and should not be confused with bioerosion. Patches of bioerosion (dark grey values) are present as are MFDs, which, upon closer inspection, appear to surround canals; however, because the concentric lamellae are indistinguishable, partly due to age, it is difficult to ascertain that MFD concentrates around canals. The circumferential lamellae of the periosteum and endosteum have been sloughed.
IN01/cremated femur (OHI 4.75/VHI 4.5) (Fig. 9)
OHI with LM
Macroscopic examination revealed the sample was cremated as confirmed by histology. It appears that there is no bioerosion, though discoloration makes the assessment difficult. Osteocyte lacunae are numerous and slightly enlarged, which is a common feature of cremated bone. Howship’s lacunae are also visible (Supplementary Material S7). There is exogenous mineral precipitation within the Haversian canals. The collagen signal is weak (Supplementary Material S7). Overall, the classic characteristics of concentric lamellae are lost as the matrix becomes homogeneous due to cremation (Hunger and Leopold 1978; Schultz 1986).
The consistently medium-dark grey grey-values of each virtual slice within the virtual stack (save for high density (bright white) exogenous mineral precipitation) confirm cremation; however, the evaluation was hindered by the inability to visualize certain anatomical features due to burning. The periosteal lamellae are missing, though some remain along the endosteum. Canals can be distinguished throughout, though they appear smaller than usual by visual inspection. However, a morphometric assessment is required to verify this. It is demonstrated that burning at medium-to-high/high temperatures results in shrinkage, cracking, and crystallisation (Hanson and Cain 2007; Boschin et al. 2015; Ellingham and Sandholzer 2020). Several long, narrow microfissures travel from both the periosteum and endosteum towards the midline; all are dense (whitish), which may be calcite. The visually undifferentiated lamellae surrounding Haversian canals are obscured by burning but are more clearly visible towards the endosteum and along the midline than the periosteum. That they remain visible may be due to the slightly denser cement lines that are visible around some Haversian systems.
R04/faunal long bone fragment (OHI 3.5/VHI 3) (Fig. 10)
OHI with LM
This bone was macroscopically identified as non-human. There is a clear pattern of bioerosion with MFD visible in patches throughout the entire section; however, bioerosion is inhibited. Under polarised light, a thick, high-density band can be seen along the periosteum and a thinner band along the endosteum. There is brownish staining along both surfaces. Haversian canals are visible in the histology, and the canaliculi are slightly enlarged (seen at higher magnifications, Supplementary Material S8). In areas without MFD, the lacunae are typical in size. Collagen preservation is poor even in the islands of bone that are free of MFD.
The bone has undergone moderate bioerosion. Some circumferential lamellae, individually visually undifferentiated, remain along the endosteum and periosteum; both surfaces have light grey, higher density bands as noted in the histology. Bioerosion manifests in patches relatively evenly throughout and between virtual slices, with a slightly higher concentration along the midline. Although MFDs have spread throughout the sample, they remain distinct and do not generally present as undifferentiated dark grey patches. There are several shrinkage artefacts that primarily travel longitudinally from the periosteum. There are few canals, as expected for plexiform animal bone, and the few Haversian lamellae that are visible are difficult to distinguish. There is potentially also osteon banding between two longitudinal microfissures below the transverse crack along the midline.
The following results (Table 3) show that exact inter-observer matching within each histological index is at least 54% but diminishes across all observations. However, it is useful to also assess approximate and general observational matches, as the histological indices are categorical across several criteria. This approach elucidates the rate of approximate agreement of minimal variation and minor variation. Assessing for these minimal disagreements (≤ 0.5) demonstrates a significant increase in observation agreement for both OHI and VHI ratings. Furthermore, a general matching with a threshold of ≤ 1.0 between observations demonstrates a significant increase of all categories, with a minimum of 93% agreement.
The results of our PCC analyses (Table 4) demonstrate a strong positive (0.94) correlation between the OHI as rated by two observers, and a stronger positive (0.96) correlation between the VHI as rated by two observers, with a strong correlation overall (0.97) between the mean values of OHI and VHI observations. To account for observational agreement by chance, LOA analysis was performed. As histological indices are based on multiple criteria with multiple descriptive indicators, the problem of equifinality arises. This approach helps address the chance to which observers agree and assesses agreement based on genuinely similar observations using the same rating scale. The LOA analysis (Table 5) demonstrates that for the OHI, observer 1 measures slightly biased (0.25), whereas for VHI observer 2 measures negligibly biased (− 0.02). These negligible differences indicate that both observers produce relatively similar results, with a trend of observer 1 rating marginally higher observations (4 s and 5 s) and observer 2 rating slightly lower observations (1 s and 2 s) (Fig. 11a, b).
A 95% confidence interval is used to determine the limits of agreement across observations (see Bland and Altman 1986). For the OHI, the systematic difference is greater than the VHI observations (Fig. 11a, b) as indicated by the upper and lower limit ranges. One observation (GÖ25) falls outside the LOA for the OHI assessment (Fig. 12a); however, this is the only difference where the observers rated a specimen significantly different. Similarly, VHI observations were consistently matching, and observer bias is distributed evenly with 1 observation falling outside the limits of agreement: GE08 where observer 1 rated the sample higher than observer 2 (Fig. 12b). Furthermore, the trend across observations is similar for both the OHI and VHI where observer 1 tends to rate more specimens higher and observer 2 tends to rate more specimens lower (Fig. 12c). However, the OHI illustrates a slightly more accentuated slope (y = 0.0639x + 0.0753) compared to the VHI (y = 0.0537x − 0.1671), demonstrating the minor differences in observations between the extreme ratings. This further shows that the observers are more prone to rate at either end of the scale and with minimal inherent bias in one observer compared to the other.