Macroscopy analysis of the bone surface that delimits the fenestra on the right squamosal bone of Big John show alterations that could be consistent to inflammatory processes18. In anatomical region where the periosteum is close to the skin surface, localized ossifying periostitis, sometimes accompanied by a lytic response, can be observed secondary to trauma12.
Histological and chemical analyzes of the bone sample from the margin of the fenestra demonstrate that it consists of newly formed bone tissue that appears porous and disorganized, with numerous vascular canals and osteocytic lacunae (Figs. 2a, 3a), where layers of bone matrix that were undergoing mineralization tend to fill the opening (Fig. 3b). At higher magnification, the margin of the bone tissue facing the lumen of the fenestra shows resorption areas, with morphology and dimensions that correspond to the Howship lacunae that are characteristic of the remodeling of human bone tissue (Fig. 2b)19. All of these histological characteristics are compatible with previously metabolically active and remodeling bone.
The specific cellular and molecular processes that occur during bone remodeling in modern reptiles and dinosaurs are not known in detail. Therefore, the reference interpretive models here are the physiology and biochemistry of healing bone tissue in mammals (including humans). Bone remodeling begins with a phase of bone resorption by osteoclasts. Through a combination of acid dissolution of the mineralized matrix and digestion of the organic matrix by proteolytic enzymes, small areas of bone resorption are formed (the so-called Howship lacunae); these are concave in shape and a few microns in diameter18.19. The bone resorption is followed by a phase of apposition of osteoid substance by the osteoblasts, which undergoes mineralization through precipitation of calcium phosphate.
The main constituents of the mineralized bone matrix in new bone are Ca, O and P (which are typically present in calcium hydroxyapatite), and also S (Fig. 4). The phases of bone resorption and neoformation are separated by a transition phase, during which macrophage-like mononuclear cells deposit an initial layer of bone matrix that forms the cementing line that delimits the osteons20. This cementing line is rich in sulphated proteins21which explains the presence of S among the elements that constitute bone during the mineralization phase.
The distribution of S in repaired bone tissue might also be linked to glycosaminoglycan’s and glycoproteins in the osteoid substance, which is the preosseous substance found in ossification centers. Glycosaminoglycan’s contain sulphate and are essential for osteogenesis22,23,24. Baylink et al. demonstrated that the concentration of glycosaminoglycan’s and S are elevated in the osteoid substance, and then are reduced during the mineralization process, as a result of enzymatic digestion10. The results of the studies carried out by Baylink were confirmed by Takagi et al., Who showed a reduction in S in areas undergoing mineralization and in the calcified matrix25. The removal of glycosaminoglycan’s and their sulphate compounds might be the prerequisite to the start of the calcification process of the osteoid substance. The SEM images in the present study showed the mineralization front of the bone tissue in the remodeling phase, whereby the distribution map of the elements highlighted the presence of S in the areas that had still been occupied by the osteoid substance that had not yet calcified ( Fig. 4).
The bone that was more distant from the margins of the lesion was mainly made up of bone matrix with few vascular channels and reduced numbers of osteocytic lacunae. These are histological characteristics that are compatible with compact bone tissue that has completed or almost completed the remodeling phase. In this region, moreover, there were lower amounts of S, which confirms the hypothesis of a gradual reduction in S during deposition of hydroxyapatite in the osteoid substance.
The edge of the lesion thus appeared to be made up of remodeling bone tissue undergoing mineralization, as seen for both the light and dark areas of the histological preparations. Conversely, the innermost region that was more distant from the fenestra showed well mineralized bone or bone that was in an advanced state of mineralization.
The results of the histological and chemical analyzes show that the bone that circumscribed and partially filled the lesion had been made up of metabolically active and remodeling tissue (Figs. 2, 4). The fenestra is therefore of traumatic origin, and at the time of the death of Big John, the lesion was still healing.
The presence of newly formed bone and the histological characteristics of bone remodeling that was in progress excludes the possibilities that the lesion occurred post mortem (ie, that it is of a taphonomic nature) and that the fenestra represented an anatomical variant. This conclusion is supported by the lack of regular, sharp and blunt margins for the lesion. Furthermore, although congenital squamosal fenestrae have been described for the subfamily of Chamsmosaurinaethey have not been found in Triceratops or Anchiceratops to date9. Instead, the lesion was possibly caused by the horn of another Triceratops. The mediolateral diameter (equal to 5 cm) and shape of the caudal region of the fenestra coincide with those of the apex of the supraorbital horns. Furthermore, the Triceratops used their horns in intraspecific combat3.
Farke hypothesized three models of engagement in the intraspecific combat of Triceratops2. The “single horn contact position” defines the contact that occurs between only one postorbital horn of each combatant, where lesions of the squamosal bone are inflicted by the nasal horn. In the “oblique horn locking position”, the skulls come into contact when they are slightly rotated with respect to each other, and contact occurs between both postorbital horns of both individuals. This model predicts lesions inflicted by the nasal horn in the rostral portion of both the parietals and squamosal’s. The third model of engagement is defined as “full horn locking”, which consists of the collision between the skulls when tilted at about 45 ° with respect to the horizontal plane, and involves contact between both postorbital horns of the opponents. This combat mode will result in injuries in the region of the temporal fenestra. The location of the lesion analyzed here on the squamosal bone of Big John does not respect these predictive models of Farke for horn use in Triceratops2. It appears likely that the wound was instead inflicted from behind Big John (Supplementary Fig. S4), whereby the rival’s horn would have penetrated the frill and then slipped towards the rostrum, giving this lesion the shape of a keyhole. According to Aufderheide and Rodrìguez-Martìn26 pointed weapons could generate a perforating lesion with sharply-defined edges. The blow may also have caused comminution and loss of fragments and radiating fissures, that were hidden by bone remodeling in the months following the trauma. In describing his models on the trauma inflicted by the horns in the fighting between TriceratopsFarke did not exclude that there might also be other engagement dynamics besides those he hypothesized2.
Big John appears to have survived this trauma for some time. Two cases have been described in the literature where adult specimens of Centrosaurus survived extensive traumatic lesions to the skull9. However, it remains difficult to establish how long Big John survived following this trauma. Although the bone remodeling process can be first recognized in the third to fourth weeks after the trauma11, the healing times vary according to the species and to the extent of the trauma. Considering the healing times of traumatic injuries in modern reptiles, along with the size of the traumatic injury and the amount of bone repair, it is likely that the death of Big John occurred at least 6 months after this traumatic event27.28.
This study confirms the existence of intraspecific fighting in Triceratops. Furthermore, although the physiological and cellular mechanisms underlying the healing process in dinosaurs are still largely unknown, it would appear to be similar to those described in humans and mammals. Further histological and microanalytical investigations on fossil remains with traumatic lesions might shed light on the bone physiopathology of these reptiles.