Cranial bone histology of Metoposaurus krasiejowensis (Amphibia, Temnospondyli) from the Late Triassic of Poland

In this study, 21 skull bones of Metoposaurus krasiejowensis from the Late Triassic of Poland were investigated histologically. Dermal bones show a diploë structure, with an ornamented external surface. The ridges consist of mostly well vascularized parallel-fibered bone; the valleys are built of an avascular layer of lamellar bone. The thick middle region consists of cancellous bone, with varying porosity. The thin and less vascularized internal cortex consists of parallel-fibered bone. The numerous Sharpey’s fibers and ISF are present in all bones. The cyclicity of growth is manifested as an alternation of thick, avascular annuli and high vascularized zones as well as a sequence of resting lines. The detailed histological framework of dermal bones varies even within a single bone; this seems to be related to the local biomechanical loading of the particular part of the skull. The dynamic processes observed during the ornamentation creation indicate that the positions of the ridges and grooves change during growth and could be a specific adaptation to changing biomechanical conditions and stress distribution during bone development. In the supratemporal, the cementing lines show that the remodeling process could be involved in the creations of sculpture. The common occurrence of ISF suggests that metaplastic ossification plays an important role during cranial development. Endochondral bones preserved the numerous remains of calcified cartilage. This indicates that ossification follows a pattern known for stereospondyl intercentra, with relatively slow ossification of the trabecular part and late development of the periosteal cortex. The large accumulation of Sharpey’s fibers in the occipital condyles indicates the presence of strong muscles and ligaments connecting the skull to the vertebral column.


Nasal
The thin section was prepared from the frontal part of the right nasal. Its external surface possesses distinct ornamentation. In cross section, the sculptured ridges are moderate high (Fig. 5). Bone thickness ranges from 1 mm to about 8 mm, and the E:M:I ratio is 1:1.2:0.4. External cortex -The external cortex consists of parallel-fibered bone, with an average thickness about 270 µm. Only in the area of the ridges the cortex becomes significantly thicker, to nearly 1900 µm. The external cortex is relatively well vascularized.
The simply vascular canals are present in its deeper part, and some of them may fragmentary penetrate into the middle region. Within the shallow and wide grooves, the elongated, simple vascular canals (500 µm in length) are arranged in regular rows, parallel to the bone surface.
The ridges are practically avascular. Sporadically, the primary and slightly larger secondary osteons can be observed also in the deeper parts of external cortex. The numerous, short Sharpey's fibers are visible, mainly in the sculptural ridges. The rounded osteocyte lacunae are very numerous. The growth marks are not visible.
Middle region -The middle region is very vast and spongy area with well-developed vascular network (Fig. 6A). The relatively large, approximately 400 µm in length and irregularly arranged, simple vascular canals are present next the borders with external and internal cortexes. They can partially penetrate into the internal cortex. In the central part of the middle region, the simple vascular canals are almost absent, whereas very numerous secondary osteons with diameters ranging from 90 µm to 170 µm can be observed (Fig. 6B).
The thickness of the lamellar bone inside the secondary osteons is 140 µm approximately. In the central part of the middle region, large and irregular erosion cavities occur, with length ranging from 300 µm to over 2500 µm (Fig. 6C). On the edges of erosion cavities, the lamellar bone of variable thickness is present. The elongate osteocyte lacunae with branching canaliculi often appear.
Internal cortex -The internal cortex consists of parallel-fibered bone, with a maximum thickness of about 490 µm. It is almost avascular, except for few simple vascular canals which partially penetrate from the middle region (Fig. 6D). The Sharpey's fibers are not visible in the internal cortex. The elliptical osteocyte lacunae (about 20 µm in length) with branched canaliculi are numerous. They typically extend in ordered rows, oriented parallel to the collagen fibers. The growth marks are visible as an alternation of lamellar layers and two resting lines.

Lacrimal
The histological thin section was prepared from the middle part of the right lacrimal, above the edge of the orbit (Fig. 7). The external surface of the lacrimal bears the irregular sculpture of medium high (0.5 mm), steep ridges and wide grooves. The average bone thickness in cross-section is 2.7 mm, and the ratio E:M:I is 1:1.6:0.7. External cortex -The variable in thickness external cortex consists of parallel-fibered bone (Fig. 8A). The cortex is the thickest within the ridges, up to 430 µm, while in the sculptural grooves the bone thickness is approximately 130 µm. The external cortex is well vascularized with numerous, anastomosing simple vascular canals. These canals are more numerous in ridges then in grooves (Fig. 8B & 8C). The thin (about 10 µm in diameter), well mineralized Sharpey's fibers are packed in bundles. They occur within sculptural ridges and on sutural edges. In the upper parts of ridges, the distinct growth marks manifested as a sequence of thin resting lines and layers of parallel-fibered bone are present. The irregularly arranged osteocyte lacunae possess branched canaliculi.
Middle region -In its central parts large, ranging from 320 µm to almost 2500 µm in diameter, erosion cavities are present (Fig. 8D). The edges of the erosion cavities are partially surrounded by the thin layer of lamellar bone. The degree of vascularization of the middle region is relatively high, the irregularly arranged simple vascular canals and secondary osteons, about 110 µm in diameter, are very numerous (Fig. 8E). In some parts of the middle region, the small secondary osteons occur in dense clusters (Fig. 8F). The elliptically elongated osteocyte lacunae, approximately 20 µm in length, are numerous.
Internal cortex -The internal cortex, with variable thickness, reaching up to 1000 µm, consists of parallel-fibered bone. It is relatively well vascularized. The simple vascular canals are arranged in several rows, orientated with its long axis at an angle of about 45 0 with respect to the cortex. The secondary osteons appear very rarely. Elliptical osteocyte lacunae are very numerous and irregularly arranged in the bone matrix.

Prefrontal
The investigated section of the left prefrontal was prepared from the middle part of this bone (Fig. 9). The external surface of the bone possesses distinct ornamentation -high and steep ridges and deep grooves. Sectional bone thickness is about 5 mm, and the ratio E:M:I is 1:1.7:0.8.

Jugal & Postorbital
The histological sample was made from the left part of the skull. The external surface of jugal and postorbital is sculptured by high (of about 1 mm) ridges and deep, wide grooves ( Fig. 11). The average thickness of the sectioned bones is 5.9 mm and the ratio E:M:I is 1:1.5:0.7. External cortex -The well vascularized external cortex with relatively high thickness consists of parallel-fibered bone. The very numerous, irregularly arranged simple vascular canals are grouped in some fragments of cortex. In the central parts of the ridges, the simple vascular canals are elongated and relatively small (about 80 µm), while deeper they are almost double in length (Fig. 12A, D). The round simple vascular canals are arranged in several regular rows in the ridges (Fig. 12B). In the outermost row the simple canals are very small, about 60 µm in diameter, while deeper they become more elongated to about 150 µm in length. The not numerous small primary osteons occur mainly in the deeper parts of the external cortex. A short, well mineralized and packed in bundles Sharpey's fibers, with diameter about 10 µm, are observed next to the surface and on the sutural edges (Fig. 12C).
Within the sculptural ridges of jugal, the growth marks, manifested as a sequence of thin resting lines and the layers of parallel-fibered bone, can be observed (Fig. 12E, F). In some places the alternation of valleys and ridges is preserved. The rounded and elongated osteocyte lacunae with branched canaliculi are very numerous in the entire external cortex. The large erosion cavities, with lengths up to 1800 µm occur in the deeper parts of the middle region (Fig. 12G). The thickness of irregularly arranged trabeculae varies from 160 µm to over 450 µm. The interiors of the erosion cavities are partially lined by discontinuous layer of the lamellar bone. The middle region is heavily cracked (Fig. 12H). The slightly elongated osteocyte lacunae are very numerous.
Internal cortex -The internal cortex with a relatively constant thickness consists of parallel-fibered bone. Only in jugal, the cortex becomes slightly thicker. The vascular network is poorly developed, but in some places the simple vascular canals can be observed. The long axes of the numerous and extending radially simple vascular canals (100 µm in length) are orientated an angle of about 80 0 relative to the middle region (Fig. 12I). The secondary osteons are not numerous. The Sharpey's fibers are not visible. The elongated osteocyte lacunae are very numerous. They are oriented parallel to the collagen fibers and possess branched canaliculi (Fig. 12J). The growth marks are visible in jugal as an alternation of lamellar layers and three resting lines.

Postfrontal
The histological sample was prepared from the left postfrontal, behind the posterior edge of the orbit (Fig. 13). The clear ornamentation of external surface consists of about 0.3 mm high ridges and shallow grooves. The bone thickness ranges from 3 mm to 5 mm and the ratio E:M:I is 1:1.7:0.6.

Frontal
The histological sample was prepared from the middle part of the right frontal ( Fig.   15). The dermal sculpture of frontal is composed of high ridges and wide grooves. The average bone thickness is about 4.7 mm, and the ratio E:M:I is 1:1.5:0.6. Internal cortex -The thin internal cortex consists of parallel-fibered bone (Fig. 16D).
The average thickness of cortex is 420 µm. The vascularization of the internal cortex is moderate. The longitudinal, simple vascular canals appear more frequently than the secondary osteons. The Sharpey's fibers cannot be observed. The elliptically elongated osteocyte lacunae with a diameter about 20 µm are as numerous as in external cortex. They are oriented in regular rows parallel to the cortex fibers.

Parietal
The investigated section of the right parietal was prepared from the middle part of this bone, approximately 1.5 cm above the pineal foramen. The external surface of the parietal bears a polygonal sculpture builds from ridges about 1 mm high, and narrow pits (Fig. 17).
The thickness of the bone is about 5 mm, and the ratio E:M:I is 1:1.6:0.6. Internal cortex -The thin internal cortex consists of parallel-fibered bone. Its thickness is usually less than 150 µm, but in some areas may be up to 250 µm. The internal cortex is avascular in the entire width (Fig. 18F). The Sharpey's fibers are not visible. The number of the elongated osteocyte lacunae (about 20 µm in length) is considerably lower. The growth marks are visible as an alternation of lamellar layers and three resting lines.

Supratemporal
The thin section was prepared from the anterior part of the right supratemporal (Fig.   19). The external surface of this dermal bone possesses the polygonal sculpture with high, about 1.5 mm ridges and deep grooves. The sectional bone thickness is from 3 mm to 5 mm, and the ratio E:M:I is 1:1.6:0.6.

Squamosal 1
The histological thin section was prepared from the front part of the right squamosal (Fig. 21). The external surface of the squamosal is characterized by a clear developed sculpture, consists of the high for 2 mm ridges and of the wide grooves. The bone thickness is about 5 mm, and the ratio E:M:I is 1:1.2:0.7. The rounded osteocyte lacunae with branched canaliculi appear in the moderate numbers. In some places the alternation of valleys and ridges is preserved.
Middle region -The middle region is created by the well vascularized bone. The huge erosion cavities, in excess of 900 µm in diameter, are partially surrounded by the layers of the lamellar bone (Fig. 22B, C). The very numerous simple vascular canals are irregularly arranged in the bone matrix (Fig. 22D). The most numerous are, variable in size, secondary osteons. The largest, reaching up to 200 µm in diameter, are located in the central part of the middle region (Fig. 22E). In some areas, next the border with the internal cortex, the arrangement of the osteons is more orderly. The rounded osteocyte lacunae with branched canaliculi are very numerous.
Internal cortex -The internal cortex consists of parallel-fibered bone and it is much thinner than the external cortex. Its average thickness is 250 µm. The internal cortex is avascular on the entire width (Fig. 22F). The Sharpey's fibers are not visible. The osteocyte lacunae occur sporadically. Their shape is elliptically elongated to about 20 µm, and they are arranged in the several, cyclic rows.

Squamosal 2
The histological thin section was prepared from the posterior (near the otic notch) part of the right squamosal. The external surface of squamosal 2 is characterized by a shallow sculpture (Fig. 23). The bone thickness is about 3 mm, and the ratio E:M:I is 1:0.3:0.5. It is the only bone in which the middle region is the thinnest part of the bone. External cortex -The external cortex with an average thickness of about 1100 µm, consists of parallel-fibered bone (Fig. 24A). Next to the surface the thin, avascular layer with thick collagen fibers is visible. Deeper part of the cortex is poor vascularized, mainly by the irregularly shaped vascular canals. The thin Sharpey's fibers occur in the deeper parts of the sculptural ridges (Fig. 24B). The rounded osteocyte lacunae with branched canaliculi appear in the moderate numbers (Fig. 24C). The degree of vascularization increases in the posterior part of section.
Middle region -The external cortex transfers gradually to the middle region. The middle region is poorly porous (Fig. 24D, E). Only the few erosion cavities with thick layer of lamellar bone are preserved. The entire part of middle region is highly remodeled and filled by the relatively compact secondary bone (Fig. 24F).
Internal cortex -The avascular on the entire width internal cortex consists of parallelfibered bone. The Sharpey's fibers are not visible. The osteocyte lacunae are present in very low amounts.

Postparietal
The sample was taken/prepared from the middle part of postparietal (Fig. 25). The external surface covers the ornament, about 1.5 mm high, builds up from steep ridges and polygonal pits. The sectional bone thickness is about 10 mm, and the ratio E:M:I is 1:1.9:0.5.

Tabular
The histological sample was prepared from the right tabular, from about the middle part of this bone (Fig. 27). The external surface of tabular bears the polygonal sculpture consists of high, about 1 mm ridges and wide pits. The bone thickness ranges from 10 mm to 12 mm, and the ratio E:M:I is 1:2.2:0.8. The numerous and well mineralized Sharpey's fibers, with a diameter about 20 µm, occur in the upper and deeper parts of the cortex (Fig. 28C). The distinct collagen fibers create a thick Interwoven Structural Fibers (IFS). Within the sculptural ridges, the cyclical growth marks, manifested as a sequence of thin resting lines and the layer of parallel-fibered bone are present (Fig. 28D). In some places the alternation of valleys and ridges is preserved. The osteocyte lacunae with branched canaliculi and rounded in shape occur numerously within the bone matrix.
Middle region -The middle region consists of coarse cancellous bone. The very large erosion cavities, with a diameter up to 1500 µm, occur only in the deeper parts of the middle region, partially in the border with the internal cortex. Their edges are fragmentary surrounded by a thin layer of the lamellar bone. The most part of the middle region is highly remodeled; however the porosity of this bone is low (Fig. 28E). The secondary osteons, with a diameter about 120 µm, are more numerous. The slightly elongated osteocyte lacunae, about 10 µm in length, and with branched canaliculi are common in the middle region.
Internal cortex -The internal cortex with constant thickness of about 1000 µm, consists of parallel-fibered bone. The irregularly in shapes, simple vascular canals, about 100 µm in length, are present in large amounts (Fig. 28F). In some areas they are arranged in several rows at angle of about 40 0 to the internal cortex. The secondary osteons rarely appear, and in some places they penetrate into the internal cortex from the middle region. The numerous Sharpey's fibers are packed in bundles. The elongated osteocyte lacunae, about 10 µm in length and with branched canaliculi are numerous.

Quadratojugal
The histological sample was prepared from the middle part of the left quadratojugal.
The external surface of the bone possesses distinct sculpture, which consists of about 1 mm high ridges and the deep grooves (Fig. 29). The average thickness of quadratojugal is 5 mm, and the ratio E:M:I is 1:1.7:0.6. External cortex -The external cortex consists of parallel-fibered bone of a variable thickness. The thickest bone, exceeding 1000 µm, is in the area of the sculptural grooves (Fig.   30A). Various in sizes and shape simple vascular canals are numerous, especially within the ridges (Fig. 30B). The small primary osteons are rare. The well mineralized Sharpey's fibers can be observed within the sculptural ridges and in the ridges (Fig. 30C). The cyclical growth marks, manifested as a sequence of thin resting lines and layers of parallel-fibered bone, are present within the sculptural ridges. In some places the alternation of valleys and ridges is preserved. The osteocyte lacunae, about 10 µm in diameter and with branched canaliculi are numerous. Internal cortex -The relatively thick, of about 700 µm, and well vascularized internal cortex consists of parallel-fibered bone. The irregular and highly elongated, up to 300 µm long, simple vascular canals are numerous. Sometimes they are arranged in several, regular rows, parallel to cortex fibers (Fig. 30F). The secondary osteons of the average size appear in a much lower amount. The Sharpey's fibers are not visible. The rounded osteocyte lacunae with preserved canaliculi are very numerous.

Parasphenoid
The thin section was made from the posterior part of processus cultriformis (Fig. 33).
The bone has the clearly ornamented external surface (located ventrally). The sectional bone thickness ranges from 2 mm to 5 mm, and the ratio E:M:I is 1:2:1.5. Middle region -The coarse cancellous middle region is well vascularized. The numerous erosion cavities have various sizes, ranging from 300 µm to over 2500 µm in length ( Fig. 34A & 34B). Their lateral edges are partially surrounded by thin layers of the lamellar bone. The middle region possess well-developed vascular network. The secondary osteons, with diameters ranging from 80 µm to nearly 200 µm, appear in a large amount, and they are lined with thick, up to 100 µm, layer of the lamellar bone (Fig. 34C). The simple vascular canals are not visible within the middle region. The osteocyte lacunae with a diameter 10 µm, and with branched canaliculi are numerous (Fig. 34D).
Internal cortex -The relatively thin and moderate vascularized internal cortex consists of parallel-fibered bone. The irregular in shape, small simple vascular canals and secondary osteons, about 60 µm in diameter, are not numerous. The Sharpey's fibers are absent. The numerous, rounded and elongated osteocyte lacunae have branched canaliculi.
The alternation of two thick well vascularized and two avascular layers is visible, represent the zones and annuli, respectively.

Pterygoid
The histological section was prepared from the posterior part of the palatine branch, of the right pterygoid, in a place where the bone is thicker and contacts with the parasphenoid (Fig. 35). The external part of pterygoid is poorly preserved, but the preserved areas show a lack of the ornamentation. The average bone thickness is approximately 8 mm, and the ratio E:M:I is 1:5.4:2.

Quadrate
The left quadrate was sectioned transversally in the central part of the bone (Fig. 37). The diameter of the quadrate is about 20 mm. The partially preserved and well vascularized cortex consists of parallel-fibered bone.
Its thickness is 200 µm. The simple vascular canals occur sporadically, but the secondary osteons, with diameters ranging from 60 µm to 170 µm, are more numerous (Fig. 38A). The lamellar bone inside the secondary osteons is relatively thick, up to 60 µm. The Sharpey's fibers are very short and they occur only in the subsurface parts of the cortex. The elongated osteocyte lacunae mainly are present within the lamellar bone. They not possess canaliculi.
The growth marks are not visible.
The central part of the bone is trabecular with irregular and variable in thickness and length bone trabeculae. The trabecular thickness is approximately 150 µm. The most numerous trabeculae are in the central parts of the bone, less frequent, shorter and thicker, on the border with cortex (Fig. 38B, C). On the border with cortex the secondary osteons, with a diameter of about 100 µm, are observed. The elongated osteocyte lacunae to about 20 µm, in the subsurface parts of the cancellous bone are rare, while in the deeper parts of the middle region their number rapidly increasing. In the central part of middle region, the relatively large accumulations of calcified cartilage remains are visible (Fig. 38D).

Exoccipital
The thin section was prepared from the right exoccipital. The bone was cut approximately in their central part (Fig. 39). The diameter of the bone is about 20 mm.