18 doi:10.1017/S1551929515001133 www.microscopy-today.com • 2016 January Preservation of Triceratops horridus Tissue Cells from the Hell Creek Formation, MT Mark H. Armitage Electron Microscopy Laboratory, Micro Specialist, 587e North Ventu Park Road, STE 304, Thousand Oaks, CA 91320 micromark@juno.com



Abstract: Dinosaur soft tissues are shown to be remarkably preserved to the sub-micron level of ultrastructure despite environmental and biological factors associated with burial for millions of years. Light microscopy and scanning electron microscopy (SEM) reveals soft tissue features such as fibrillar bone tissue, osteocytes, and blood vessels. Concerns that these findings relate to contamination or biofilm formation have been refuted. Notwithstanding the controversial nature of these discoveries, soft dinosaur tissues should be systematically searched for and thoroughly characterized in other dinosaur remains. Introduction Remarkably preserved cells and tissues from dinosaurs have been reported since the mid 1960s [1], however until recently, dinosaur bone specimens usually have not been decalcified or otherwise destructively studied for the presence of soft tissues because complete bone specimens are highly prized by paleontologists and collectors. Over the past 50 years, soft blood vessels, collagen bands, intact cells, bone cells (osteocytes), filopodia with primary and secondary branching, cell-to-cell junctions, intracellular nuclei, and other soft tissue details have been observed and illustrated from various different species of dinosaurs including Tarbosaurus bataar, Tyrannosaurus rex, Brachylophosaurus canadensis, and Triceratops horridus. [1–6]. Initial criticisms, which labeled these soft structures as biofilms [6], have been resolved as incorrect [7]. In 2012 I collected a large Triceratops horridus supraorbital horn from the Hell Creek Formation at Glendive, Montana. The horn yielded soft sheets of fibrillar bone (Figure 1) and life-like cells. A Triceratops rib specimen from the same deposit contained soft blood vessels and red blood cell-like (RBC) microstructures. Remarkable preservation of individual bone osteocytes encapsulated within the stretchy sheets of fibrillar horn bone was observed, as were osteocytes positioned upon sheets of fibrillar bone adhering to permineralized vessels within the decalcified horn bone [6]. Variable-pressure scanning electron microscopy (VPSEM) of uncoated specimens was not attempted at that time, nor were individual cells isolated from the specimen for further analysis. In this article I describe VPSEM and cell isolation results from the Triceratops horn. Materials and Methods Specimen preparation. The hand-sized pieces of the horn, somewhat “pie-slice” in shape and extending from the exterior horn surface to the inner trabecular (cancellous) bone (core), were fixed in a 2.5% solution of glutaraldehyde, buffered with 0.1 M sodium cacodylate buffer at 4°C for 5 days, rinsed in distilled water and buffer, and stored in phosphate buffered saline (PBS). Pieces, roughly 20 mm3 in size, were extracted from the inner bone core by pressure fracture and were processed through a decalcification protocol as follows: bone pieces were rinsed in pure water after fixation and were incubated in a solution of 14% sodium ethylenediamine tetraacetic acid (EDTA) at room temperature. The EDTA was exchanged every 2 to 4 days for a period of 4 weeks after which bone fragments were processed for scanning electron microscopy (SEM). Other pieces were soaked for 4 months in EDTA. Even after this treatment significant bone mineral/hardened material remained; therefore, it is unknown whether complete decalcification in EDTA would yield soft and transparent, vessel-like tissues, such as previously reported [7–11]. A soak in hydrofluoric acid (HF) was not attempted, but it might prove more successful in liberating any soft vessels that remain. Rib specimens were similarly fixed, washed, and pressure fractured to reveal inner surfaces of compact bone (Figures 2 and 3). Light microscopy of cells. Aliquots of decalcification solutions (post soak) were transferred by pipette into tied off chambers of Snakeskin dialysis tubing, (Thermo Scientific, Rockford, IL) and were submerged into vials of distilled water for 2 weeks. Water was exchanged every 2 days, and after 2 weeks cells were transferred after dialysis onto glass microscope slides for examination and imaging on a Jenaval light microscope (Carl Zeiss Jena) equipped with a Jenoptik ProgRes (Jena, Germany) C14plus camera. SEM imaging of bone. After a 4-week soak in EDTA, decalcified bone was air-dried and affixed to aluminum stubs. For Figures 2–4, bone specimens were sputter-coated with Figure 1: Portion of soft, stretchy fibrillar bone from Triceratops horn. Note embedded osteocytes (black arrows). Scale bar = 30µm.

lded osteocytes with a higher degree of ultrastructural preservation than previously reported [3,6,9,12]. Uncoated bone surfaces show lacunae depressions (Figures 5–7), extensive filopodia (Figures 6–10), collagen aggregates (Figure 7), and cell surfaces displaying the indented impressions of overlying and compressing bone (Figures 6 and 8). Figures 9 and 10 show cells that were successfully isolated from fibrillar bone. In future work, it is hoped that individual cells such as these can be examined using immunohistochemistry for the presence of endogenous proteins. Conclusion Claims of contamination and biofilm replication have been dismissed [7,12], and identification of intra-cellular and intra-nuclear proteins have been verified showing that these are endogenous dinosaur tissues [12]. Therefore claims that these are not original dinosaur tissues appear to be questionable.