Beyond the Visible World. Bridging Macroscopic and Paleohistopathological Techniques in the Study of Periosteal new Bone Formation in Human Skeletal Remains

Abstract

Paleopathology, summarily defined as the study of past diseases, has on the differential diagnosis a major challenge. Taking into account the difficulties faced by paleopathologists on the study of ancient conditions, especially those of infectious origin involving periosteal new bone formation (PNBF), an investigation was conducted combining macroscopic and histological techniques. The purpose of this research was twofold: firstly, to analyze the macroscopic distribution of periosteal reactions by age and cause of death, anatomical location, laterality, symmetry, bone dispersion and type of new bone; and secondly, to microscopically examine and compare thin sections of periosteal new bone, in order to evaluate the existence (or not) of microstructural differences in individuals who died from: (1) tuberculosis infections-TB (Group 1, n=114); (2) non-TB infections (Group 2, n=89); and (3) other conditions (Group 3, n=50). For the macroscopic analysis an assemblage of 253 individuals from the Human Identified Skeletal Collection from the Bocage Museum (Lisbon, Portugal), 136 males and 117 females, comprising individuals with an age at death ranging from 2.5 months to 94 years old, was chosen. A total of 34 dry bone specimens: 26 belonging to 23 individuals from the Bocage Museum, and eight from eight individuals from archaeological contexts (14th-19th centuries) were prepared for histological examination under transmitted and polarized light. Analysis of the macroscopic results revealed a high frequency (71.2%) of PNBF in individuals younger than 45 years old ( =22.64 years old). Deposits of new bone were most commonly found in individuals who died from TB (Group 1=82.5%) in comparison with those who died from non-TB disorders (Group 2=42.7%) or from other conditions (Group 3=46%), and the results were highly significant. Only the PNBF located on ribs had a positive relationship with TB as the cause of death (Group 1). Individuals who died from TB infection were also those who showed a broader involvement of the rib cage. Here periosteal lesions were most frequently observed in the upper to middle segment (R1R6, 60.2% [405/673]), mainly in the left-side (41.2%). In the ribs, a predominance of diffuse lesions combining woven and lamellar foci was recurrently seen. In spite of the apparent relationship between periosteal rib lesions and TB, the presence of similar lesions in individuals who died from other diseases does not allow for the establishment of a definitive association. The frequency of PNBF in the appendicular bones was higher in the tibia (T) and fibula (Fi) of the individuals from Group 2 (T=36.7%; Fi=17.4%) and Group 3 (T=41.2%; Fi=17.1%), and in those older than 45 years of age (T=38.8%; Fi=17.5%). The analysis of the individuals with multiple periosteal bone involvement did not reveal significant differences among cause of death groups. The younger individuals from Group 1 were those with more deposits of new bone in the humerus, radius and ulna. With regard to the distribution of the periosteal lesions, symmetric foci were most often seen in the scapula and radius (n=5, respectively), whereas left-sided ones more common in the ulna (n=5), and right-sided ones in the humerus (n=4). In the lower limb bones, a predominance of symmetric lesions was observed in the tibia (n=37), femur (n=28) and fibula (n=15). Localized foci of new bone were commonly observed in the upper limb bones. In contrast, a high frequency of diffuse lesions was found on the shaft of the lower limb bones (e.g. tibia and fibula). Only the femur (n=28) showed a high prevalence of localized lesions. Differences in the type of new bones were also found between the upper (predominance of woven and woven/lamellar foci) and the lower limb bones (lamellar and woven/lamellar foci). In the appendicular skeleton, the lack of association between new bone formation, particularly in the tibia and fibula, and the underlying cause of death seems to corroborate the non-specificity of periosteal lesions, challenging their usefulness as an indicator of physiological stress. Furthermore, it may point out to the existence of inaccurate records of cause of death, or the coexistence of multiple conditions not identified in the obituary certificate. The value of the histological analysis on the study of bone lesions was observed at three main levels: (1) diagnosis of pathological conditions; (2) description of bone lesions; and (3) assessment to bone tissue quality. With regard to the diagnosis of pathological conditions, differences in the microstructure of PNBF were seen between Group 1 and Group 2 of cause of death and within groups. Multiple layers of “appositional bone” enclosing numerous primary vascular canals were the pattern most commonly observed (n=4) in the samples from Group 1. In contrast, three samples (one from Group 1, two from Group 2) presented a microstructure compatible with subperiosteal hematomas. These observations suggest that beyond pulmonary diseases other mechanisms may stimulate PNBF on the visceral surface of ribs. Histological analysis was also fundamental in the description and characterization of bone changes. For example, of the five samples with “consolidated” fracture callus, only two presented a truly mature and remodeled microstructure. This means that the outer surface of a bone lesion may not give a complete picture of the tissues response. In spite of the good preservation of some bone samples, massive diagenetic changes due to the action of bacteria and fungi were observed at microscopic level. This clearly suggests that gross inspection is not a good measure of the bone tissue quality. In contrast, microscopy is essential to differentiate between pseudopathology and physiological or pathological signs. This study demonstrated the difficulties in using periosteal lesions, especially those of the lower limb bones to ascertain the diagnosis of particular conditions. Furthermore, it also revealed the limitations and possible misinterpretations found in the macroscopic study of identified skeletal collections. In contrast, the histological analysis showed surprising results that reinforce the pertinence of applying histological techniques in the description and diagnosis of bone changes in human remains. Future studies based on well-documented collections with accurate medical records and/or samples retrieved from clinical cases will eventually solve some of the problems and limitations detected in this investigation. Increasing the sample size will also improve our understanding of the entire spectrum of new bone variation associated with a particular condition. It is possible that further research in the field of bone biochemistry, immunology and cell communication will shed light on the exact mechanism (or mechanisms) behind PNBF.