Embryology   Biology 441   Spring 2012   Albert Harris




What is bone made of? One third collagen fibers, by weight; two thirds calcium phosphate crystals by weight.

The collagen very strongly resists tension, but doesn't resist pressure, because of the flexibility of collagen fibers. The calcium phosphate crystals resist compression; but by itself would be brittle and pulling tension would crack it. The combination of collagen fibers with calcium phosphate crystals, tightly interwoven with each other, strongly resist both tension and compression. Mechanically, this combination is analogous to fiber-glass, steel-reinforced concrete and other composite materials.

Bone is caused to form by cells of a special differentiated cell type, called osteocytes. Many scientists like to make a distinction between osteocytes versus osteoblasts (their precursors).

The collagen part of bone is synthesized by osteocytes, and they secrete collagen as fibers. Nobody knows how osteocytes cause calcium phosphate crystals to form. It is traditional to ignore this gap in knowledge by saying that osteocytes "secrete" calcium phosphate; but if that ever happened, it would be easy to see in electron microscope sections of osteocytes in the parts of the body where bone is being made. Collagen fibers can be clearly seen inside cells by electron microscopy.

Calcium phosphate crystals have never been seen inside osteocyte cells, but show up very clearly in sections of bones.

Various theories have been proposed to explain what osteocytes do that causes calcium phosphate crystals to form amongst the collagen fibers that osteocytes secrete. It is possible that osteocytes secrete large amounts of either calcium ions OR phosphate ions; but they can't secrete both at the same place, because then they would combine and precipitate inside the cell (which would be visible by electron microscopy). Maybe some osteocytes secrete phosphate and others secrete calcium ions! Nobody has proposed that, but several other theories have been reported with some evidence supporting them. One idea is that osteocytes release closed membrane vesicles, and that the membranes of these vesicles actively pump calcium and phosphate into their interior, where they combine and precipitate. Alternatively, ATP could be used to pump calcium ions, and the phosphates released by hydrolysis of the ATP could combine with the calcium ions.

This is a subject where you could achieve a major medical breakthrough! Just figure out what the mechanism might be, and then design experiments that will turn out differently depending on which theory is actually correct. That's how you do research: consider every possible explanation that you and others can invent; then design experiments that will give different results according to which theory is true.

Unless sequencing DNA, or doing PCR, or In Situ labeling, or Southern or northern blotting etc. will give you a different result depending on which possible mechanism osteocytes use to precipitate calcium phosphate, then those methods can't help answer this question.

One theory of ossification (sometimes stated as fact) is that osteocytes secrete some substance ("osteoid") that cause crystallization by any calcium and phosphate ions that happen to be diffusing randomly around in solution, even where the concentration of dissolved calcium ions multiplied by the concentration of phosphate ions is NOT large enough to cause them to precipitate.

Please try to invent some experiments that would give certain results if this Osteoid Theory were true.

For example, suppose that you could isolate the Osteoid, separate from other proteins and separate from calcium phosphate solution. Then you could buy pure calcium phosphate crystals, make a saturated solution of this salt in water... and do what? ...and predict what result?

Alternative experimental strategies include soaking bones in water solutions of EDTA until all the calcium has been chelated away...and then doing what? If you take a chicken embryo, at an age when many bones are rapidly ossifying, dissect out pieces of partly-formed bones, and kept these alive in tissue culture, then what could you do with them that would help solve the problem?

Suppose that you isolated osteocytes in tissue culture... what experiments could you do with them?
Hint: You can add particular salts to the tissue culture media.
Another hint: Radioactive phosphate is one of the isotopes most used by biologists.
EDTA and other chemicals can chelate calcium ions, sort of like decreasing their effective solubility.
ATP itself is a strong chelator of calcium.
Fluorescent dyes can be bought that fluoresce (emit light at a certain longer wave-length in response to illumination with light of a shorter wavelength) differently, depending on the calcium ion concentration at each location, thus "mapping" differences in this concentration, as observed by microscopy.

A quote from the Wikipedia article on ossification:

"The exact mechanisms by which bone development is triggered remains unclear, but it involves growth factors and cytokines in some way."

Yes, and the exact mechanism of life on other planets remains unclear, but it involves chemicals in some way.

Any protein that specifically stimulates or inhibits any activity by other cells is, by definition, a cytokine. What they mean is "it involves some of those growth-influencing proteins that have already been discovered."

This article includes a preposterous suggestion that the use of plaster of Paris (which is calcium sulfate) to help heal broken bones might involve transfer of calcium salts from the plaster of Paris to the calcium phosphate of the bone. That is Crazy Talk.

Plaster of Paris casts work by holding broken bones rigidly in position, never by any transfer of ions (unless maybe you chew on the cast). Also to be found on the web are dangerously wrong ideas that EDTA can be used to clear calcifications from blocked arteries. Don't believe them, even though chelators are sometimes used at small concentrations to treat poisoning by lead, mercury or ions of other heavy metals.

Ossification is a subject greatly in need of better researchers, with solid training in inorganic chemistry. If you go into medical research, either as am MD or a PhD, these are subjects where you could make big discoveries.

Bone is one of the most dynamic tissues of the body. It is constantly dissolved by special cells called "osteoclasts".

Osteoclasts have dozens (or hundreds?) of nuclei per cell.
They become multi-nucleate by fusion of uninucleate cells.
These uninucleate cells are so similar to macrophages that they are believed to BE macrophages, and not a special kind.

Even though macrophages are one of the smallest cell types in the body (despite the Greek meaning of "macro") osteoclasts are fairly large - large enough that they can (and do) seal off areas of bone surfaces and secrete protein-digesting enzymes and acid (i.e. hydrogen ions, please don't call them "protons") into the space 'tented-over' (a phrase I just invented, because they do look like rather flat tents) between the osteocyte cell and an area of bone surface. The enzymes digest the bone and the acid dissolved the calcium phosphate.

Bone is then re-deposited by osteocytes. There is a constant balance between bone making and bone destroying. Stronger-than-usual forces imposed on bones somehow shift this counter-balance, causing a stressed bone (and even a stressed PART of a bone) to become stronger, thicker and more dense.

X-ray photos of tennis players' arm bones are dramatically different in the right arm as compared with the left arm.

The mesh-work of narrow strands of bone in their marrow also change in response to stress. These are formed parallel to the direction of largest stress, and also exactly perpendicular to the direction of largest stress (please look at the diagram).


Lack of stress on bones causes them to get weaker ("demineralize" people say, although there is also less collagen). This occurs in bones of invalids, and is very severe in astronauts. (Much more severe than NASA tells the public, according to more than 10 NASA scientists who have discussed it with me.)

Osteoporosis is a huge medical problem, especially for older women.

What is not yet known on this subject:
* Whether weakening of bones is because less bone is being deposited by osteocytes, or because more bone is being destroyed by osteoclasts. (or both)
** How bone detects the amounts, locations and directions of stress being imposed on them.
On the latter question, a very popular theory was that bone might have the property of being piezo-electric. Many kinds of crystals (e.g. quartz) have the property of generating a small voltage when a force is imposed on them. (any crystal whose spatial arrangement of charged ions is not symmetrical for inversion through a point will be piezoelectric; this phenomenon was discovered by Pierre Curie, and gave him the idea for Curie's principle, that you already know about.) Sonar depends on piezo-electric crystals; so do "ceramic" cartridges for phonographs, if you know what they are, and so do "atomic force microscopes")

Calcium phosphate is not piezoelectric; but bone is supposed to be slightly piezoelectric, due to the collagen. Personally, I suspect researchers may be detecting electro-osmotic voltages, and misinterpreting them. Researchers on this subject tend not to realize that piezoelectric voltages only last for a tiny period of time in conductive surroundings (lymph, blood, saline, etc) and are necessarily followed by a brief voltage in the opposite direction when the stress is relaxed.

When pushed or pulled steadily in a given direction, the combination of osteocytes making bone and osteoclasts breaking it down will lead to a gradual change in the bone's shape. (Orthodontia wouldn't work, otherwise).

At least a dozen different bisphosphonate drugs are being sold for very high prices as treatments for osteoporosis, and actually can be effective. These drugs all have in common two phosphate groups covalently bound to a carbon atom, which is also covalently bound to a chain of 3 to 10 carbons, sometimes nitrogens, sulfurs, or even carbon rings.

These chemicals were invented as water softeners in the late 1800s. "Water softeners" reduce the precipitation of calcium salts. Somebody decided to try these chemicals on old ladies; maybe they will have the opposite effect and prevent the dissolving of calcium phosphate. Everyone assumed that the two covalently linked phosphates must be getting incorporated into the calcium phosphate of the bone, and making it stronger and/or inhibiting its destruction by osteoclasts. More recently, it was found that bisphosphonates work by inhibiting osteoclasts. It does this by stimulating programmed cell death of osteoclasts.

Unfortunately, these drugs also turn out to stimulate programmed cell death of quite a few different cell types, not just osteoclasts. That explains why so much damage was caused to other organs.

This field of research is a mess. I hope some of you students will choose it for a research career. All those millions of old people suffering from osteoporosis deserve dedicated wise researchers, which they are not getting now.



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