AN ELECTRON-MICROSCOPIC STUDY OF THE CRYSTALLINE INORGANIC COMPONENT OF BONE AND ITS RELATIONSHIP TO THE ORGANIC MATRIX
ROBERT A. ROBINSON

Abstract

1. The inorganic crystalline component of bone occurs as crystals having average dimensions of 500 Angström units in length by 250 Angström units in width by 100 Angström units in thickness.

2. The crystals have a size range which they do not apparently exceed in normal bone. This range is from 1,500 Angström units in length to about 20 Angström units ±. The crystals are tabular in form, and in mature cortical bone some of the crystals have a regular hexagonal outline.

3. The crystals in newly formed and mature bone have the same general size and tabular form.

4. The crystals lie in the cement substance and not in the collagen fibers.

5. The pattern of atomic spatial arrangement characteristic of the hydroxyapatite crystal lattice may develop in matrices having considerable physical and chemical variation Within this wide range of variation lies the physiological range of calcifying intrasomatic matrices. The fact that intrasomatic calcifications in general are hydroxyapatite is interpreted as follows: The hydroxyapatite crystal lattice is more stable under physiological conditions than alternate spatial arrangements of oxygen, hydrogen, calcium, and phosphorus atoms in other theoretically possible crystal lattices. At the periphery of a crystal lattice certain heterogenous substitutions may occur. This peripheral layer appears to be in contact with the cement substance of the bone matrix.

6. The collagen fibers in bone are fairly uniform in transverse measurement, between 500 and 1200 Angström units according to our micrographs. No visible difference is observed in these electron micrographs between these fibers and those of other connective tissues.

7. The collagen fiebers in bone apparently swell on mild heating in water and the interfibrillar cement substance swells when it comes in contact with hyaluronidase.

8. Some of the crystals and cement substance closely ensheathe the collagen fibers. In the crystal layer most closely applied to the fiber the long axes of the crystals parallel the axis of the fibers and form a periodic pattern of rings around the fibers, the intervals between the rings being about. 640 Angström units.

9. By the use of the blending method of bone-sample preparation, it was possible to detect this parallel orientation of the inorganic crystals and the collagen fibers only in those layers of crystals most adjacent to the fibers. In the interfibrillar spaces no such orientation could be definitely shown. However, in any group of crystals combining in large masses (in the interfibrillar spaces) they assumed a laminated structure. This tendency to lie with their broader, flat surfaces opposing may be a function of their tabular shape, and the added factor of packing is due to mechanical compression.

10. Calculations based on the dimensions of the inorganic crystals of bone observed in the electron microscope showed that:

A crystal 500 Angström units by 250 Angström units by 100 Angström units has (a) a surface area per gram of crystals of 106 m.2 and (b) the outer atomic layer in proportion to all atoms in the hydroxyapatite crystal was about 12 to 14 per cent.

A crystal 500 Angström units by 250 Angström units by 150 Angström units has (a) a surface area per gram of crystals of 84 m.2 and (b) an outer atomic layer in which the proportion to all atoms in the hydroxyapatite crystals was about 9 to 10 per cent.

On the basis of x-ray diffraction of samples used for electron micrography, it would appear that the inorganic crystals seen in the electron microscope are hydroxyapatite. The crystals were considered to be rectangular solids of tabular form with a specific gravity of 3.0, a calcium-phosphorus ratio of 2.16, and unit cell dimensions of A axis = 9,47 Angstöm units and C axis = 6.88 Angström units, for the purpose of these calculations.

11. The calculations of size and surface area based on dimensions of the inorganic crystals of bone observed in the electron microscope correlate well with x-ray diffraction, gas adsorption, radio-active calcium and phosphsorus exchange data, and the CO3 "space" of bone presented by other investigators. This correlation of data lends full support to the author's conclusion that the crystals seen in these electron micrographs of bone are not artifacts of the preparation methods but pictures of the crystals as they occur in vivo.

12. The relationship of the inorganic crystals to the organic matrix is discussed. The structural characteristics of bone as skeletal material depend on this relationship. The site of crystal development and their size and shape appear to depend on this relationship Others have shown that bone crystals form only in the matrix after the matrix has "matured" as shown by collagen fiber development. The theory is proposed that the collagen fibers may act as a nidus for crystal formation in the bone matrix.

Certain special staining characteristics and chemical analyses suggest that the cement substance of calcifying tissue is different from that in non-calcifying fibrous tissues. By electron micrographs it is shown that, although hydroxyapatite crystals may form in other normal or pathological tissues as well as in bone, the sizes or shapes of such crystals are often different from those found its bone. A special specificity of bone matrix as compared with other calcifying matrices is thus suggested by the characteristic size and shape of the crystals which it produces.

13. The cells as the organizing and sustaining members of the bone matrix play an essential role in this organic-inorganic relationship. It is suggested that, by slight alteration of the cement substance, thought to be due to cellular activity, the equilibrium between the calcium and phosphorus atoms of bone crystals, the calcium and phosphorus atoms of the cement substance, the calcium and phosphorus atoms of the extracellular fluid about the bone matrix, and finally the calcium and phosphorus atoms in the blood plasma, are controlled. The osteocytes responoding to variations in the circulating hormone concentrations may thus alter the cement substance, making more or less calcium available to the other extracellular fluids from the bone crystals.

14. The inorganic crystals of bone observed in the electron microscope do not contribute osteogenic potency to a bone graft or bone transplant (outside of their function as a "honeycomb" that holds and possibly stabilizes the osteogenic material of the organic matrix of fresh bone).