Microscopical and elemental FESEM and Phenom ProX-SEM-EDS analysis of osteocyte- and blood vessel-like microstructures obtained from fossil vertebrates of the Eocene Messel Pit, Germany

Fossil samples

Tiny fragments of bone (≤1 cm³ from the fossil specimens, which were left after mechanical preparation of the exposed surface and preserved without application of any resin, glue, or stabilizing additives, were sampled to explore preservation of OBvF-like microstructures (Fig. 1). All samples belong to specimens housed at the Messel vertebrate collection of the Senckenberg Naturmuseum (SMF ME) in Frankfurt am Main, Germany. A total of nine samples were taken, including three turtle specimens: Allaeochelys crassesculpta SMF ME 2449, Neochelys franzeni SMF ME 1091, Euroemys kehreri SMF ME 1807; one mammal: Eomanis krebsi SMF ME 1573; one amphibian: undetermined frog bone SMF ME 11432; one crocodile: Diplocynodon darwini SMF ME 898; one lizard: osteoderms of Eurheloderma sp.; one bird? Messelornis SMF ME uncatalogued specimen; and one fish: Atractosteus sp. SMF ME 2751. The taxonomic attribution of the specimens was taken from the Senckenberg Museum collections database, some of the samples belong to specimens published as followed: Al. crassesculpta SMF ME 2449 (Joyce et al., 2012), N. franzeni SMF ME 1091 (Cadena, 2015), and E. krebsi SMF ME 1573 (Storch & Martin, 1994).

OBvF-like microstructure isolation and transmitted light microscopy

Bone fragments were demineralized using isodium ethylenediaminetetraacetic acid (EDTA) (0.5 M, pH 8.0 filter-sterilized using a 0.22 µm filter) as previously described (Schweitzer et al., 2008) for a period of 5 days to 2 weeks, or until OBvF-like microstructures emerged. These OBvF-like microstructures were observed using a Zeiss Axioskop 2 plus biological-transmitted light microscope, 40x, and 100X (oil immersion lens) and imaged with a Nikon camera coupled to the microscope.

FESEM-BSE microscopy

OBvF-like microstructures emerging from bone after demineralization were collected into 1.5 ml microcentrifuge tubes. Samples were rinsed 3 times (at 1,000 rpm) in E-pure water to remove EDTA buffers. After centrifugation, samples were passed over a 0.4 micron Nylon cell strainer, then the strainer containing fossil remains (thin-small bone matrix remains and the OBvF-like microstructures) was mounted on individual SEM stubs, and affixed to the stub with silver polish. Samples were imaged using a Field Emission Scanning Electron Microscope (FESEM Sigma VP SEM, Carl Zeiss NTS) located at the Geosciences Department, Goethe Universität, Frankfurt, Germany. Samples were imaged without coating under 0.90 kV EHT (primary-beam energy) with a working distance of 3.0 mm. Elemental analyses were conducted using a Phenom ProX desktop scanning electron microscope (LOT-QuantumDesign) equipped with a thermionic CeB6 source and a high sensitivity multi-mode backscatter electron (BSE) detector, 0.15 kV EHT (primary-beam energy), also at the Geosciences Department, Goethe Universität, Frankfurt, Germany. At least 3 different points were analyzed for elemental composition for each sample studied.

OBvF-like microstructure recovery

Of the nine samples of different vertebrate groups (turtles, mammals, birds, crocodiles, lizards, amphibians, and fish) studied here, only four preserved abundant OBvF-like microstructures: two of the turtles (Allaeochelys crassesculpta and Neochelys franzeni), the crocodile and the mammal.

Morphological characterization of OBvF-like microstructures from Messel Pit

Among vertebrate cells, osteocytes present a unique morphology because during development they become entrapped in the matrix they secrete; to obtain nutrients and eliminate waste, their cell membranes form extensions known as filopodia (Bonewald, 2011; Lin & Xu, 2011). The morphology of the osteocytes and the bony cavities in which they are housed in life, known as osteocyte lacunae, have been previously described (Schweitzer et al., 2005; Schweitzer, Wittmeyer & Horner, 2007a; Schweitzer et al., 2009; Schweitzer et al., 2013; Cao et al., 2011; Cadena & Schweitzer, 2012, and references therein). These same morphological features are exhibited by the osteocytes-like of vertebrates from Messel Pit described herein. Osteocytes-like microstructures from the turtles Allaeochelys crassesculpta and Neochelys franzeni vary from semi-oval to elongated (between 20 and 40 µm) (Figs. 2A–2E), to very elongated (∾50 µm) (Figs. 2F–2H), almost all of them preserving second- and third-level ramifications of filopodia, and are yellow to orange in color. In contrast to turtles, the osteocytes-like microstructures from the crocodile Diplocynodon darwini (Figs. 3A–3B) and the mammal Eomanis krebsi (Figs. 3C–3E) are slightly smaller, particularly the semi-oval osteocyte-like microstructures (between 10 and 25 µm). Additionally, they are translucent, and no staining is evident. However, they also exhibit second- and third-level ramifications of filopodia. Broken or fragmented osteocyte-like microstructures were also commonly observed (Figs. 3D–3E).

Blood vessel-like and collagen fibril-like microstructures were also recovered after demineralization of bone, particularly in Allaeochelys crassesculpta (Fig. 4). The blood vessel-like microstructures are characterized by a thin wall with a microgranular texture (Fig. 4A), similar in both texture and diameter (∾10 µm average) to the blood vessel-like microstructures reported previously from a Paleocene bothremydid turtle from Colombia (Cadena & Schweitzer, 2014). Collagen fibril-like microstructures were also found forming stripes or groups of several fibrils (Figs. 4B–4C). The fibrils appear stained, exhibiting yellow to orange coloration, yet retained the characteristic plywood-like arrangement exhibited in almost all vertebrate bone, including human bone (see Varga et al., 2013). The collagen fibril-like microstructures were texturally distinct from both the blood vessels-like and the osteocytes-like recovered from the same bone samples. It is important to point out here that at present the definition of these blood vessel- and collagen fibril-like microstructures is based on their resemblance to the morphology of the same microstructures in extant turtles (see Cadena & Schweitzer, 2012).

FESEM analyses of the osteocyte-like microstructures from Messel, particularly for the two species of turtles, Neochelys franzeni (Figs. 5A–5C) and Allaeochelys crassesculpta (Figs. 5D–5F), show that they consist of a material exhibiting a mottled texture externally and internally (Fig. 5B) (see elemental characterization section, for elemental characterization of this material). Some of the osteocyte-like microstructures have in their filopodia or sometimes over the exterior surface spheres that under transmitted light microscopy exhibit the same coloration as the osteocyte-like microstructures (Fig. 5C), and under FESEM they are not only spherical, but also have a pentagonal net-like divisions (Fig. 5B). The elemental composition of these spheres is still unknown at this time, since none of those was recovered during the analysis with the Phenom ProX-SEM-EDS. Another notable feature of the external surface of these osteocyte-like microstructures is the occurrence of marks or troughs, most of them circular, others linear to irregular (Figs. 5E–5F). Linear branching troughs have been reported to occur in vascular canals attributed to microbial activity (Kaye, Gaugler & Sawlowicz, 2008). The external surface of some of the osteocyte-like microstructures also exhibits weak striations (Figs. 5E–5F).

As it was observed under transmitted light microscopy, some of the osteocyte-like microstructures show breakage, some being cleaved in two (Fig. 3E). Using FESEM the break edges were examined, shedding light on the physical properties of the material constituting these osteocyte-like microstructures. For example the osteocyte shown in Figs. 5A–5B, suffered breakage during the filtering-mounting processes, possibly due to desiccation, preserving its upper left portion next to the main body of the microstructures. This suggests a fragile rather than a ductile material, with clearly defined, relatively straight-uniform edges, without evidence of bending (flexible) or fibrous components, which is also the case of other broken regions of the osteocyte-like microstructures (see Fig. 5B, right-down region). A 3-dimensional visualization “in situ” of the internal bone microstructural elements of the Messel Pit vertebrates wait to be done using nano-computer tomography, something that could provide not only more information about their original morphology, but also about their intrabone variation, as well as their volumetric density, distribution, and network connectivity.

Phenom ProX-SEM-EDS analysis of OBvF-like microstructures from Messel Pit

The elemental analysis of OBvF-like microstructures in the two turtle species using FESEM-BSE is summarized in Figs. 6–7. These included three osteocyte-like microstructures for Allaeochelys crassesculpta, two of them shown in Figs. 6A–6B (complete results in Supplemental Information 1), and three osteocyte-like microstructures for Neochelys franzeni, one shown in Fig. 6C (complete results in Supplemental Information 2). All osteocyte-like microstructures from both species show occurrence of three major elements: oxygen (O), carbon (C) and iron (Fe), in all cases with values above 10%; other elements like phosphorus (P), calcium (Ca), nitrogen (N) and silicon (Si) are also present but in minimal percentages usually below 5% (see Supplemental Informations 1 and 2). In contrast to the elemental composition of osteocyte-like microstructures, the collagen fibril-like microstructures and the remains of the bone matrix lack a high concentration of Fe (less than 1%). Instead, the composition is dominated by O, Ca, and P (all these with percentages above 8%) (Figs. 7A–7B, and Supplemental Informations 1 and 2). Blood vessel-like microstructures, however, like the osteocytes are also rich in Fe with values about 15% (Fig. 7C).