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 , T ousand 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 fi brillar bone tissue, osteocytes, and blood vessels. Concerns that these fi ndings relate to contamination or biofi lm formation have been refuted. Notwithstanding the contro- versial 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 decalcifi ed or otherwise destructively studied for the presence of soſt tissues because complete bone specimens are highly prized by paleontologists and collectors. Over the past 50 years, soſt blood vessels, collagen bands, intact cells, bone cells (osteocytes), fi lopodia with primary and secondary branching, cell-to-cell junctions, intracellular nuclei, and other soſt tissue details have been observed and illustrated from various diff erent species of dinosaurs including Tarbosaurus bataar , Tyrannosaurus rex , Brachylophosaurus canadensis , and Triceratops horridus . [ 1 – 6 ]. Initial criticisms, which labeled these soſt structures as biofi lms [ 6 ], have been resolved as incorrect [ 7 ].
In 2012 I collected a large Triceratops horridus supraor- bital horn from the Hell Creek Formation at Glendive, Montana. T e horn yielded soſt sheets of fi brillar bone ( Figure 1 ) and life-like cells. A Triceratops rib specimen from the same deposit contained soſt blood vessels and red blood cell-like (RBC) microstructures. Remarkable preser- vation of individual bone osteocytes encapsulated within the stretchy sheets of fi brillar horn bone was observed, as were osteocytes positioned upon sheets of fi brillar bone adhering to permineralized vessels within the decalcifi ed 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 . T e 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 fi xed in a 2.5% solution of glutaraldehyde, buff ered with 0.1 M sodium cacodylate buff er at 4°C for 5 days, rinsed in distilled water and buff er, and stored in phosphate buff ered saline (PBS). Pieces, roughly 20 mm 3 in size, were
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extracted from the inner bone core by pressure fracture and were processed through a decalcifi cation protocol as follows: bone pieces were rinsed in pure water aſt er fi xation and were incubated in a solution of 14% sodium ethylenediamine tetraacetic acid (EDTA) at room temperature. T e EDTA was exchanged every 2 to 4 days for a period of 4 weeks aſt er which bone fragments were processed for scanning electron microscopy (SEM). Other pieces were soaked for 4 months in EDTA. Even
aſt er this treatment signifi cant bone mineral/hardened material remained; therefore, it is unknown whether complete decalcifi - cation in EDTA would yield soſt and transparent, vessel-like tissues, such as previously reported [ 7 – 11 ]. A soak in hydrofl uoric acid (HF) was not attempted, but it might prove more successful in liberating any soſt vessels that remain. Rib specimens were similarly fi xed, washed, and pressure fractured to reveal inner surfaces of compact bone ( Figures 2 and 3 ). Light microscopy of cells . Aliquots of decalcifi cation solutions (post soak) were transferred by pipette into tied off chambers of Snakeskin dialysis tubing, (T ermo Scientifi c, Rockford, IL) and were submerged into vials of distilled water for 2 weeks. Water was exchanged every 2 days, and aſt er 2 weeks cells were transferred aſt er dialysis onto glass microscope slides for examination and imaging on a Jenaval light microscope (Carl Zeiss Jena) equipped with a Jenoptik ProgRes (Jena, Germany) C14 plus camera. SEM imaging of bone . Aſt er a 4-week soak in EDTA,
decalcifi ed bone was air-dried and affi xed to aluminum stubs. For Figures 2 – 4 , bone specimens were sputter-coated with
Figure 1 : Portion of soft, stretchy fi brillar bone from Triceratops horn. Note embedded osteocytes (black arrows). Scale bar = 30 µm.
doi: 10.1017/S1551929515001133
www.microscopy-today.com • 2016 January
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