Bone idol

Uncovering the secrets of the mineralised osteocyte remnant

As ‘fossil’ cells embedded in living animals, mineralised osteocyte remnants have some fascinating biological stories to tell.

Inside the bone of humans and animals is a cell that can survive for thousands of years. "The mineralised osteocyte remnant is the only cell that survives," explains Lynne Bell, a Bioarchaeology Fellow at the Natural History Museum. "Other cells in the skeleton just die and leave empty lacunae and vascular systems in the bone matrix." Frozen at a specific moment in time, the mineralised osteocyte remnant is a biological relic that may tell us much about how cells have evolved over the past millennia and may even shed light on the lives of the individuals in which they lived and died.

With Wellcome Trust funding, Dr Bell is investigating the survival and biological significance of the osteocyte remnant. "I’m aiming to find out as much as I can about this cell: its morphology, its structure, where it occurs, whether it just occurs in humans or whether it occurs in other mammals, other species, and how far into time we can find these cells."

Osteocytes are what some bone-forming cells (osteoblasts) turn into during bone formation. They are no longer involved in bone formation – their principal role is thought to be in cell-to-cell signalling. The ‘mineralised osteocyte remnant’ is formed by a process akin to fossilisation as the cell dies. The process is usually triggered by age, although osteocytes can survive for up to 18 years – a ripe old age for a cell. "What makes the mineralised osteocyte remnant unique," says Dr Bell, "is that, in addition to its longevity, it’s the only fossil cell I know that is embedded in the skeleton of a living mammal."

Until recently, microscopists believed that, when osteocytes died, the spaces left in the bone (lacunae) simply infilled with mineral. Under the light microscope, all they could see was an amorphous mass of mineral. However, higher-power imaging – scanning electron microscopy and back-scattered electron imaging – revealed that the mineral in these lacunae was in fact intricately structured. "Imaging confirmed that we were actually seeing very small organised three-dimensional structures," says Dr Bell.

The next stage was to find out whether any remnants of the cell existed beneath these mineral structures. "What I wanted to know next was, OK, that’s the mineral. But is there anything organic inside it?" To find out, Dr Bell contacted Mike Kayser and Professor Joseph Ali at Stanmore Hospital. Together, they stripped away the mineral using a gradual decalcification process and examined the mineralised osteocytes under very high-power transmission electron microscopy.

The team were hardly able to believe what they saw. "The organisation that we saw with the mineral on was reproduced when the mineral was removed. The organic is giving structure to the mineral." For Dr Bell, it was an extraordinary discovery. "It just surpassed any kind of expectation that I had. I think all of us were shocked at the degree of survival of the organic. For us to really see it there was magical. That was a very special moment."

It appears that the structure of the cell itself was mediating the mineralisation process, acting as a kind of template. "So the cell’s appearance, when you strip off the mineral, is of a cell that’s in the process of dying. If it had died completely, there would just be empty space, but because it was mineralised while it was dying, the death process was arrested. It’s almost like you’ve frozen a moment. This is really interesting for anyone wanting to look at programmed cell death."

She intends to sample bone material spanning 200 million years – looking at reptilian and dinosaur bone as well as mammalian bone – to search for ancient mineralised osteocyte remnants. Recently, again in collaboration with colleagues at Stanmore, she attempted to decalcify a remnant found in a five-million-year-old fossil.

Unfortunately, after decalcification the delicate cell structures that remained went into solution. "It’s not in such good shape as a modern cell; there’s been a massive amount of loss, partly because there’s no surrounding collagen to give it structural support," explains Dr Bell. "But there is something there that’s organic, even in this fossil." A 200 000-year-old specimen looks more promising: "We’re hoping this will be a better candidate for study because the collagen is still there."

The cell components found in the remnant include various kinds of substructure of the cystoskeleton and remnant organelles. Fragments of DNA may also survive, and researchers at the University of Oxford are extracting and amplifying short stretches of DNA from Dr Bell’s mineralised remnants. However, she hopes DNA doesn’t grab all the attention. "The real value of the cell is not just its potential for DNA to survive – it’s a chance to consider the evolution of a cell over thousands, even millions, of years, to find out whether it changed over time, and if so, how."

Horses’ teeth and Roman princesses
Physical examination of skeletal remains can give many clues to the life and times of the deceased. As well as high power microscopical analysis, Dr Bell is using other tools to gather information about the ancient past. One such tool is oxygen isotope analysis, which can help to pinpoint the geographic origins of a skeleton.
She is concentrating on ‘delta-[O]18’, the ratio of two naturally occurring oxygen isotopes ([O]16 and [O]18) compared against a standard. Oxygen isotopes from drinking water are incorporated into the mineral part of the skeleton, where they become ‘fixed’. The delta-[O]18 value in the skeleton thus depends on the levels in the original drinking water. "delta-[O]18 has traditionally been used to reconstruct palaeotemperature, or past climates," explains Dr Bell. "But I’ve poached this method and applied it to locate ‘place’ instead. I can do that because the oxygen value of drinking water changes depending on where you live. As you move northwards from the equator to the North Pole, delta-[O]18 becomes progressively more negative – in other words, it becomes ‘lighter’."
By examining human enamel, which forms during childhood, Dr Bell has established delta-[O]18 cut-off values for Britain. "If the oxygen is outside these values, that indicates the person came from a more southerly or more northerly place," she says. "So you can see if someone was a native or an immigrant and trace their movements to some extent. Although I can’t say yet exactly where they’ve come from, I can say no, they weren’t born in Britain, they lived somewhere else before they came here. That’s amazing – we’ve never been able to do that before."
Finding a modern baseline for delta-[O]18 data proved problematic, since drinking water for humans today comes from isotopically mixed water held in reservoirs and hence gives no indication of provenance. Dr Bell came up with the idea of using wild horses instead. She looked at enamel from the teeth of horses living in wild conditions from Iceland all the way down to Sudan, and determined their isotope values. A clear correlation with latitude appeared, with heavier delta-[O]18 values becoming more common towards the south and, conversely, lighter values towards the north.
Last year, the team responsible for the Meet the Ancestors BBC television series got to hear of her work. She was invited to help reconstruct the movements of the Roman Princess whose skeleton was found in a stone sarcophagus at Spitalfields. The results suggested she was definitely not a local: "She was way outside of the range for Britain. The data suggested she was Mediterranean."

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