Proterochersis porebensis, Szczygielski & Sulej, 2016, Szczygielski & Sulej, 2016

Szczygielski, Tomasz, Klein, Nicole, Słowiak-Morkovina, Justyna & Scheyer, Torsten M., 2023, Limb histology of the Triassic stem turtles Proterochersis porebensis Szczygielski & Sulej, 2016 and Proganochelys quenstedtii Baur, 1887 with insights into growth patterns of early turtles, Comptes Rendus Palevol 22 (32), pp. 635-665 : 640-644

LONG BONE MICROANATOMY AND BONE HISTOLOGY OF PROTEROCHERSIS POREBENSIS

All sectioned bones of Proterochersis porebensis present a well-developed, compact cortex and medullary area filled with cancellous bone ( Fig. 2 A-I). Thickness of the cortices (without erosion cavities and remodeling) is 2.2-3.4 mm, and 1.2-1.7 mm for the middle-sized and small humerus specimen and 0.7-2.4 mm and 0.7-1.3 mm for the large and small femora, respectively. The matrix of the cortex is composed predominantly of parallel-fibered bone ( Figs 2 A-I; 3A-D; 4A-F; 5; 6) that often shows high organization (approaching but not exactly the same as the lamellar bone in the same sections) and is even locally grading into lamellar bone in some samples, and in the middle-sized humerus ZPAL V. 39/433 ( Fig. 4B) and large femur ZPAL V. 39/500 ( Fig. 6D) it locally takes a highly organized appearance, approaching lamellar bone. The matrix shows annular (ring-like) lines of arrested growth (LAGs), which appear relatively even in the small femur ZPAL V. 39/499 ( Figs 2F; 5A) and in the deeper part of the cortex of the small humerus ZPAL V. 39/439 ( Figs 2A; 3C, E), but show more pronounced waviness in the external part of the cortex of ZPAL V. 39/439 ( Figs 2A; 3C) and in the larger ZPAL V. 39/433 ( Figs 2D; 4A, E) and ZPAL V. 39/500 ( Figs 2H; 6E). The vascularization in the cortex is generally poor, especially in the external layers, although some local variability is visible along the perimeter of the bones (e.g. the cortex of the humeri is clearly more vascularized ventrally than dorsally; Figs 2 A-E; 3A-D; 4A-F). The vascularization pattern is sub-longitudinal, arranged in circular patterns, with slight radial inclination visible in all specimens except ZPAL V. 39/499 (small femur). The vasculature in most sectioned bones is predominantly primary (primary canals without primary osteons and, locally, osteons). The incipient secondary remodeling (sparsely scattered secondary osteons, Fig. 4E) is present in the middle-sized humerus ZPAL V. 39/433. Only in the large femur ZPAL V. 39/500 is the secondary remodeling significant and, in some places, nearly reaches the external surface, although much of the primary tissue and some primary vascular canals are still locally present ( Fig. 6). The small femur ZPAL V. 39/499 retains mostly non-remodeled cortex, but evidence of active remodeling is visible at the border between the cortex and the medullary region, where two to three overlapping generations of lamellar bone are present ( Fig. 5). The large femur ZPAL V. 39/500 differs from the other sampled bones in having a clearly scalloped external surface due to a network of vascular grooves, which is visible also macroscopically ( Figs 1M, N; 2H, I; 6). These grooves seem to be mostly imprints of superficial vasculature, but numerous vascular openings are also present on the dorsoposterior surface of the distal expansion of that specimen.

The extent of the cancellous area appears to be smaller in the humeri than in the femora and more clearly defined in the small than in the middle-sized and large specimens ( Fig. 2 A-I). The trabeculae partly retain primary tissue in interstitial spaces, which are lined by endosteal lamellar bone, especially close to the cortex, and even in the smallest sampled specimens clearly incorporate periosteal tissue ( Figs 2 A-I; 3; 4C, D;5; 6). Especially in the large femur ZPAL V. 39/500, the boundary between the endosteal cancellous and periosteal cortical region is gradual, with large areas of the periosteal cortex remodeled into either intertrabecular spaces or irregularly meandering secondary osteons ( Figs 2H, I; 6). In the same specimen, trabeculae deeper inside the bone are comparatively very thin. The thickness and length of the trabeculae is, nonetheless, varied; they appear more slender and longer in the central and dorsal part of the medullary area ( Fig. 2 A-I), but some regions of increased thickness are locally evident, especially in the middle-sized humerus ZPAL V. 39/433 ( Figs 2D, E; 4G, H).

The CT data ( Fig. 7) illustrate a gradual ontogenetic progression of the trends observed in the histological sections and reveal the microstructural patterns along the bone length. Both the absolute and relative thickness of the cortices appears to increase with increasing bone size. In that regard, ZPAL V. 39/446 (cortex 1.6-2.16 mm) and ZPAL V. 39/156 (cortex 1.9- 2.3 mm), which are intermediate in size between the sectioned ZPAL V. 39/439 and ZPAL V. 39/433, are also intermediate structurally – their cortices are thicker than in ZPAL V. 39/439 (1.2-1.7 mm), but thinner than in ZPAL V. 39/433 (2.2- 3.4 mm; note that the cortical thickness in sectioned specimens is undervalued due to the slightly more distal plane of sectioning). The large humerus (ZPAL V. 39/50, cortex 2.8-3.4 mm) and femur (ZPAL V. 39/432, cortex 3.2-4 mm, compared to 0.7-1.3 mm in ZPAL V.39/499 and 2-2.4 mm in ZPAL V. 39.500) generally follow the trends of cortical thickening with increasing size suggested by the smaller specimens.

As indicated by previous studies, the centers of ossification are located slightly more proximally than the midshaft and are associated with the largest thickness of the cortices ( Fig. 7A, C, E, G). In small humeri, the cortices in that region are relatively compact (in ZPAL V. 39/446 more so than in ZPAL V. 39/156) and the trabecular bone in the bone center has a relatively uniform structure ( Fig. 7 A-D) probably indicating low remodeling (although potentially also dependent on stress distribution patterns and load). In ZPAL V. 39/50, however, the cortex is somewhat more vascularized (although still relatively poorly), but the interior trabecular region is much less defined and denser than in smaller specimens, indicating more pronounced remodeling ( Fig. 7E, F). There is no central marrow cavity in the studied humeri. Conversely, in the large femur ZPAL V. 39/432, the internal trabecular region is still distinct (even though it is denser than in the areas further away from the ossification center) and there is a small, irregular region devoid of trabeculae located around the level of the ossification center ( Fig. 7G, H). This cavity is connected with the bone exterior via two well-defined nutrient canals extending: 1) dorsoproximoanteriorly and eventually opening as a nutrient foramen at the anterodorsal base of the trochanter major; and 2) ventrodistally and opening as a nutrient foramen in the popliteal fossa, between the tibial and fibular condyle (see Appendices 1-16; and Morphosource).

Proximally and distally from the centers of ossification, the thickness of the cortex steadily decreases so in the areas of the proximal and distal articular surfaces the trabeculae nearly reach the bone exterior ( Fig. 7A, C, E, G). The cortex, however, retains a relatively constant thickness across the intertubercular fossa of the humeri ( Fig. 7A, C; Appendices 1-16; and Morphosource). In ZPAL V. 39/ 446 in that area it is perforated by numerous vascular canals roughly parallel to the long axis of the bone ( Fig. 7A).

Trabeculae are relatively long and thick in the small humerus ZPAL V. 39/446 ( Fig. 7A). In the slightly larger ZPAL V. 39/156 they retain a similar morphology close to the cortex (especially ventrally) and around the level of the center of ossification, but become finer and more densely packed in the middle of the more proximal and distal portions of the bone ( Fig. 7C). In the large humerus ZPAL V. 39/50, they are generally more irregular, with larger and smaller intertrabecular spaces and thinner and thicker trabeculae scattered without a clear pattern ( Fig. 7E). In the large femur ZPAL V. 39/432, the pattern of longer trabeculae closer to the cortex and finer trabeculae more interiorly is also noticeable, but less clear than in the humeri ( Fig. 7G).

Overall, based on the histological sections and CT images, the humeri appear denser than the femora of comparable ontogenetic stages.