Osteoporosis

You would make enormous contributions to human health, and reduce great suffering, if you could solve these problems.

Vertebrate bones are made of a tight mesh of (protein) collagen fibers mixed with the mineral salt calcium phosphate.
Notice that calcium carbonate is the mineral component of the skeletons of echinoderms, coral, mollusks, crustaceans, brachiopods, coralline algae, egg shells, and those few sponges that don't have silica skeletons. Only vertebrates use calcium phosphate, as far as I know.

It is normal for human bones to be constantly renewed, by dissolving combined with formation.

The mechanism of bone dissolving is known. But the mechanism of bone formation is not known. The collagen is secreted, but the calcium phosphate precipitates outside cells.
#1) Our main unsolved problem, here, is what mechanism causes precipitation of calcium phosphate part of bone.
#2) Our secondary unsolved problem is that increased mechanical loading somehow causes more bone to form, and decreases in force exerted on bones somehow tips the balance against bone formation.
It is NOT yet known whether these effects of loading (and lack of loading) are to decrease dissolving, or to increase deposition. Osteoporosis could be caused by more dissolving, or by less deposition, or by some combination.

#3) Another set of medical problems is that bone sometimes forms inside tendons and in the walls of arteries.

#4) It is NOT known by what signal bones detect or "feel" amounts of mechanical load. Experiments show that the effect is localized. The most popular theory is that mechanical loads produce local voltages, which either stimulate bone deposition or inhibit dissolving. Although this theory is often stated as fact, the supporting evidence is not good. Naive researchers were so impressed by finding that voltages really are induced by compressing or stretching bone, that they accepted the whole theory.

The dissolving is done by a special kind of cell, called osteoclasts, which are large and multinucleate (formed by fusion). They dissolve bone by secreting hydrogen ions (acid) and proteolytic enzymes into spaces between themselves and bone surfaces. The enzymes digest the collagen, and the hydrogen ions cause calcium phosphate to dissolve. By spreading out flat onto bone surfaces, and sealing tight to the bone around their edges, the acid and enzymes are kept localized and concentrated in oblong areas of bone surface on the order of a hundreds of micrometers on a side, and tens of micrometers deep (that look very much like the effects of ice cream scoops on the surface of a container of ice cream!)
NOTE: I need better information on the sizes of these digested areas of bone.

#5) another unsolved problem is why osteocytes (and also skeletal muscle cells) become multi-nucleated (many nuclei in same cytoplasm) by means of fusing many uni-nucleate cells, instead of (what would seem to be simpler) having repeated mitosis without cytoplasmic separation.

Bone is (somehow!?) caused to form by a special differentiated cell type called an osteocyte. Many people like to call them osteoblasts, and make distinctions between those inside the bone and those surrounded by bone. In my opinion, different names shouldn't be given to cells until some fundamental difference has been discovered, and that making pointless distinctions is a major reason why the true mechanisms remain unsolved. There has been some excellent science in this area, and lots and lots of very bad, meaningless science.

Osteocytes secrete collagen fibers (so do many other cell types) and somehow cause precipitation of calcium phosphate. This precipitation occurs next to the outside surfaces of osteocytes, but not inside them. Transmission electron microscopy can "see" calcium phosphate crystals very clearly (analogous to x-rays "seeing" bone), but you never see the mineral part of bone inside osteocytes (and believe me, scientists have searched VERY hard for it!).

Let's consider some possible theories (and try to invent more; and also try to invent experiments that could disprove or prove them)

Some background facts and ideas:

If you dissolved as much calcium chloride (which is very solutble in water) as possible in one container of water; and dissolved as much potassium phosphate (also soluble) as will dissolve in the water in another container; then mixing these two containers of water will result in precipitation of lots of crystals of calcium phosphate. Therefore, if a cell had vacuoles with ion pumps in the membrane, some that pump calcium ions into the vacuole and others that pump phosphate ions into the vacuole, then that ought to be an effective mechanism for causing formation of the mineral part of bone. It must be some sort of clue that it doesn't work that way.

A nifty alternative mechanism would be to have a membrane calcium pump that gets its energy from ATP and that releases its phosphate ions (i.e. from ATP -> ADP + P) into the vacuole, where they would combine with calcium ions.

But again, nobody can find vacuoles that contain calcium phosphate crystals! Despite years of searching!

At least one researcher found extracellular spheres of membrane, with calcium phosphate crystals inside them. These were hypothesized to be creating bone by pumping ions into their inside space.

Another theory has been that the real question is what inhibits precipitation of calcium phosphate, everywhere but in bones!
Some researchers claim(ed) that body fluids normally contain such high concentrations of calcium ions and phosphate ions that they are super-saturated with respect to precipitation of calcium phosphate crystals. What an interesting possibility! It would help explain many kinds of kidney stones, calcification of artery walls, calcification of tendons, among other medical ailments. Anybody would think this would have been conclusively proven true, or disproven, long ago. That shows how messed-up research is in this field.

What is the relevance (if any) of the following facts?

* Calcium ion concentrations are often used for intracellular signaling (e.g. Muscle contraction, & backing-up by ciliates) * Phosphates are often used for energy transfer (e.g. ATP, creatinine phosphate) and also to destabilize molecules. (Maybe there there is some incompatibility? Or difficulty in pumping both to the same place? But I don't know what!

* In the chain of three phosphates in ATP, the chemical bonds between the phosphates must be covalent, in contrast to the ionic bonding between phosphates in calcium phosphate crystals (Right?) But how to think about this?

A different kind of theory: That a certain protein ("osteoid") causes calcium ions and phosphate ions to precipitate even at ion concentrations that would not be high enough to cause calcium phosphate to precipitate by themselves.

Notice that this would be somewhat the opposite to chelation of calcium by EDTA! EDTA, EGTA, and also ATP (incidentally) bind to calcium ions and prevent them from precipitating (increase solubility).

There are fluorescent chemicals whose fluorescence changes as a function of calcium concentrations, and these are used to map concentrations of calcium ions inside cells.

It is not difficult to dissect developing bones out of early chicken embryos, and keep their cells alive in tissue culture media.

Alkaline phosphatase enzyme detaches phosphate ions from ATP and other carbon polymers, and sometimes located where bone is forming.

Strontium ions, zirconium ions, radium, uranium and technecium and other "bone seeking ions" get made into bone in among calcium ions But magnesium ions (and nearly all ions) don't get incorporated into bone.

I don't know any examples of "bone seeking" anions (that get substituted for phosphate). Arsenate is a possibility. (Probably arsenate is so poisonous because it gets bound to ATP instead of phosphate.) Please don't experiment with any poisons however, unless you really, really know what you are doing.

Teeth are made of two materials, a thin layer of enamel on the surfaces and dentine in the interior. Dentine is a special kind of bone (2/3 calcium phosphate crystals + 1/3 type I collagen (protein). Enamel is 95% calcium phosphate crystals and 5% protein of a completely different kind. Neither has any osteoclasts (I heard this at a dental research conference) and would like to find a paper on it. Rarely, osteoclasts find teeth, and if this happens they will destroy all the dentine. Enamel is much more rigid (than dentine or bone); but that makes it more brittle.

If you measure (and graph) how much force is needed to distort a material by 0.1%, 0.2% etc. for many materials the graph is linear up to around 2%, but then curves toward less slope. The slope of this graph is called "Young's modulus", and is steeper for harder materials. Brittleness results from this slope not becoming smaller for large forces.

Often, I have heard and read that osteoporosis causes bone to be more brittle; but I don't know whether scientists saying this know what brittleness means; they may just think it is a synonym for weakness. Reducing the ratio of collagen per amount of calcium phosphate could make bone brittle, if it actually occurred. Experts in this subject seem very ignorant of engineering concepts.

Diphosphonate chemicals were synthesized for "water softening" in the early 1900s, and sold cheaply by the ton. Then they were patented for incorporation into bone, and are sold for > $ 50 per small pill, to reduce osteoporosis.

This effect was assumed to result from making calcium phosphate resist being dissolved; But the real cause is widely (but not universally) thought to be killing of osteoclasts.

This field of science is a disgraceful mess.

Experiments?: (Please notice whether having a theory makes it easier to invent experiments)

Would you expect that osteoporosis would be increased by calcium chelating chemicals (oxalic acid)?

Would any theory be proved or disproved if oxalic acid didn't cause increased osteoporosis?

Which (if any) of these theories would predict that more bone will form if a person has more calcium in their diet?

By what experiment could you prove that osteoporosis is caused by (or is NOT caused by) reduced deposition of bone?

Suppose you had a drug that caused leakage of acid and/or enzymes out of the space between osteoclasts and bone. What effect would you expect? How could you test for such leakage? What medical use might such a drug have?

Other questions to discuss:

Synthetic chemicals have been made that have a carbon covalently bound to two phosphorus atoms, and a chain of carbons also attached to the carbon between the phosphorus atoms. Although the idea was that these would inhibit bone breakdown by making the bone stronger, their effects are really to induce programmed cell death of osteoclasts (and, unfortunately also kill other kinds of cells). These drugs are sold for very high prices, justified by the superhuman intelligence required to invent them. If a drug turns out to work some completely different way than anybody expected, then who deserves the profits?

* The company that synthesized the chemical?
** The people on whom the chemical was tested?
*** The scientist who discovered how the drug really worked?
**** Whoever figured out how to prevent news media from reporting things like this.

What about the possibility that some enzyme catalyses precipitation? Remember that enzymes work by lowering the activation energy of a chemical reaction. Does precipitation of any salt, such as calcium phosphate have an activation energy? I don't know any examples, but this possibility might be worth thinking about (maybe?).

Some possible experiments: What would it tell us if we did such-and-such an experiment, and observed that so-and-so happens?

Take a piece of bone containing living osteocytes and soak it in tissue culture media containing EDTA. (note: Adding EDTA to media is a standard tissue culture method for detaching cells, so we know it won't kill cells)

The goal would be to chelate the calcium ions, so that they would go into solution, also resulting in dissolving of the phosphate. This would leave you with just the collagen and the "osteoid", if there is such a thing. (Also, the living cells)

What would we expect to happen if this de-mineralized bone were put back in normal tissue culture medium (no EDTA?
* New bone would form at the same locations?
? Or maybe calcium phosphate wouldn't precipitate? Would this prove something? Would it disprove anything?

Some variations on this experiment:
When dissecting out the piece of bone to be used, leave a fairly large piece of tendon attached (i.e. tightly packed collagen).
* Will calcium phosphate crystals not also precipitate among the collagen fibers of the tendon?
? Or will calcium phosphate also precipitate in what had been the tendon?
Would that prove/disprove anything?

Deliberately kill some of the osteocytes in the piece of bone, either all those at one end, or in stripes, or some other pattern.
* Maybe calcium phosphate will dissolve less completely, or more slowly, next to the living osteocytes? (meaning what?)

* When this piece of demineralized bone is put back in tissue culture media, will calcium phosphate crystals form faster (or sooner? or only?) in the areas nearest the living bone cells?
? What would this prove, or disprove?
[Hint: suppose there really is an "osteoid" protein, that itself causes/promotes/accelerates re-precipitation of calcium phosphate]

Add a calcium-sensitive fluorescent dye to tissue culture medium, and look for any patterns or unequal distribution of fluorescence around an embryonic piece of bone. Specifically, will calcium concentration be depleted next to where bone is forming? Will calcium concentration be depleted only near living osteocytes?

Add one of the diphosphonate drugs to tissue culture medium. Then soak a piece of embryonic bone in this medium, Is there any way to prove whether or not the drug gets incorporated into newly forming bone?
Whether it gets incorporated into already-existing bone?
Whether dissolving of the calcium phosphate of bone is in any way reduced, or slower, or less complete, or requires more EDTA when that bone had been exposed to diphosphonate drugs?
Or is there less dissolving of calcium phosphate only from bones that were "synthesized" (for lack of a better word!) in the presence of a diphosphonate drug?

By adding EDTA and proteolytic enzymes to saline, and soaking pieces of embryonic bone in it, probably one could isolate reasonably pure populations of living osteocytes, that could then be put in tissue culture media, kept alive, growing, dividing and making more bone.
? What experiments could you do with such tissue cultures of living, bone producing osteocytes?
? Suppose they cause new bone to form around all surfaces of osteocytes?
? Suppose the new bone only forms between osteocytes, that are close to each other?
? Suppose the new bone only forms under osteocytes, in the sense of between them and their glass or plastic substrata?
? Suppose that bone production were strongly different depending of properties of the substratum? (For example. suppose that some areas were made to be negatively charged, and more bone got formed there?
? Or suppose some areas of substratum were smoother, or some areas were more flexible?
[Note: all these properties have been discovered (somewhat unexpectedly) to change differentiation of certain other cells!]

? What if you cultured osteocytes in tissue culture media that contained enough EDTA to prevent formation of the calcium phosphate component of bone?
? Would you expect the collagen and osteoid components of bone would still be secreted?
? What if you cultured many osteocytes on plastic for a long time, in the presence of enough EDTA to prevent bone formation;
And what if you then replaced the medium with normal medium (no EDTA), what are some alternative possible results?
* Bone production at the normal rate?
* Rapid bone formation (using the already-secreted collagen and osteoid, and/or other non-mineral components?
* Bone formation in areas where osteocytes had been (during the time when EDTA medium was used), even where no osteocytes are now located there? (Possibly because you killed or removed some of the osteocytes; or because osteocytes had crawled over that part of the substratum.)

Think what could be proved or disproved by these alternative observations!

[Notice how much of the discussion above is the exact reverse of what many/most people think is the "Scientific Method."

# Do experiments, where something-or-other ought to happen.
## Look for consistent, reproducible results of experiments.
### Try to invent hypothetical phenomena that would have predicted these observed results.]

What do you think about the following quote?

"Observations should never be accepted until they have been checked for consistency with relevant theories."

(I owe this quote to my former collaborator Prof. Micah Dembo of Boston University. Thank you, Micah.)