An electron-microscopic study of the crystalline inorganic component of bone and its relationship to the organic matrix

Robinson, Robert A.

Affiliations

¹Division of Orthopaedic Surgery, Department of Surgery, University of Rochester School of Medicine and Dentistry, Rochester

Abstract

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.
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.
The crystals in newly formed and mature bone have the same general size and tabular form.
The crystals lie in the cement substance and not in the collagen fibers.
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.
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.
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.
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.
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.
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.

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.
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.
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.
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).

Links

DOI: 10.2106/00004623-195234020-00013

PubMed: 14917711

WoS: A1952UF14000013

Cited Works (2)

Year	Entry
1917	Koch JC. The laws of bone architecture. Am J Anat. March 1917;21(2):177-298.
1947	Ruth EB. Bone studies, I: fibrillar structure of adult human bone. Am J Anat. January 1947;80(1):35-53.

Cited By (38)

Year	Entry
1963	Frost HM. Bone Remodelling Dynamics. Springfield, IL: Charles C. Thomas; 1963.
2014	Reznikov N, Shahar R, Weiner S. Bone hierarchical structure in three dimensions. Acta Biomater. September 2014;10(8):3815-3826.
2010	Hamed E, Lee Y, Jasiuk I. Multiscale modeling of elastic properties of cortical bone. Acta Mech. August 2010;213(1):131-154.
1952	Robinson RA, Watson ML. Collagen‐crystal relationships in bone as seen in the electron microscope. Anat Rec. November 1952;114(3):383-409.
1996	Ziv V, Wagner HD, Weiner S. Microstructure-microhardness relations in parallel-fibered and lamellar bone. Bone. May 1996;18(5):417-428.
2003	Su X, Sun K, Cui FZ, Landis WJ. Organization of apatite crystals in human woven bone. Bone. February 2003;32(2):150-162.
2003	Rubin MA, Jasiuk I, Taylor J, Rubin J, Ganey T, Apkarian RP. TEM analysis of the nanostructure of normal and osteoporotic human trabecular bone. Bone. March 2003;32(3):270-282.
2020	Reznikov N, Hoac B, Buss DJ, Addison WN, Barros NMT, McKee MD. Biological stenciling of mineralization in the skeleton: local enzymatic removal of inhibitors in the extracellular matrix. Bone. September 2020;138:115447.
1986	Weiner S, Price PA. Disaggregation of bone into crystals. Calcif Tiss Int. November 1986;39(6):365-375.
1993	Grynpas M. Age and disease-related changes in the mineral of bone. Calcif Tiss Int. February 1993;53(suppl 1):S57-S64.
1978	Jackson SA, Cartwright AG, Lewis D. The morphology of bone mineral crystals. Calcif Tiss Res. August 1978;25(3):217-222.
2020	Maghsoudi-Ganjeh M, Wang X, Zeng X. Nanomechanics and ultrastructure of bone: a review. Comput Model Eng Sci. 2020;125(1):1-32.
1994	Ziv V, Weiner S. Bone crystal sizes: a comparison of transmission electron microscopic and x-ray diffraction line width broadening techniques. Connect Tissue Res. 1994;30(3):165-175.
1992	Weiner S, Traub W. Bone structure: from ȧngstroms to microns. FASEB J. February 1992;6(3):879-885.
1968	Bonfield W, Li CH. The temperature dependence of the deformation of bone. J Biomech. 1968;1(4):323-329.
1992	Wagner HD, Weiner S. On the relationship between the microstructure of bone and its mechanical stiffness. J Biomech. November 1992;25(11):1311-1320.
1966	Cooper RR, Milgram JW, Robinson RA. Morphology of the osteon: an electron microscopic study. J Bone Joint Surg. October 1966;48A(7):1239-1271.
1970	Cooper RR, Misol S. Tendon and ligament insertion: a light and electron microscopic study. J Bone Joint Surg. January 1970;52A(1):1-20, 170.
1960	Smith JW. The arrangement of collagen fibres in human secondary osteones. J Bone Joint Surg. August 1960;42B(3):588-605.
2012	Vaughan TJ, McCarthy CT, McNamara LM. A three-scale finite element investigation into the effects of tissue mineralisation and lamellar organisation in human cortical and trabecular bone. J Mech Behav Biomed Mater. August 2012;12:50-62.
2022	Gaziano P, Monaldo E, Falcinelli C, Vairo G. Elasto-damage mechanics of osteons: a bottom-up multiscale approach. J Mech Phys Solids. October 2022;167:104962.
2001	Eppell SJ, Tong W, Katz JL, Kuhn L, Glimcher MJ. Shape and size of isolated bone mineralites measured using atomic force microscopy. J Orthop Res. November 2001;19(6):1027-1034.
1993	Landis WJ, Song MJ, Leith A, McEwen L, McEwen BF. Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction. J Struct Biol. January 1993;110(1):39-54.
1996	Landis WJ, Hodgens KJ, Song MJ, Arena J, Kiyonaga S, Marko M, Owen C, McEwen BF. Mineralization of collagen may occur on fibril surfaces: evidence from conventional and high-voltage electron microscopy and three-dimensional imaging. J Struct Biol. July 1996;117(1):24-35.
1999	Weiner S, Traub W, Wagner HD. Lamellar bone: structure–function relations. J Struct Biol. June 1999;126(3):241-255.
2022	Buss DJ, Kröger R, McKee MD, Reznikov N. Hierarchical organization of bone in three dimensions: a twist of twists. J Struct Biol X. 2022;6:100057.
2023	Micheletti C. Multimodal and Multiscale Characterization of Bone and Bone Interfaces in Health and Disease [PhD thesis]. University of Gothenburg; 2023.
2012	Hamed E. Multiscale Modeling of Elastic Moduli and Strength of Bone [PhD thesis]. University of Illinois at Urbana-Champaign; 2012.
2015	Beck EC. Development of Chondroinductive Hydrogel Pastes from Naturally Derived Cartilage Matrix [PhD thesis]. Lawrence, KS: University of Kansas; 2015.
2002	Egerer AK. The Relationship Between the Osteonal Cement Line and Bone Strength [PhD thesis]. Loma Linda University; June 2002.
2023	Buss DJ. Mineralization of Biological Fiber Systems [PhD thesis]. McGill University; July 2023.
2022	Gupta SD. Mineralized Tissues At the Osteochondral Junction in Healthy and Osteoarthritic Joints: Advanced Microimaging and Mechanical Studies [PhD thesis]. University of Oulu; 2022.
2015	Chana NK. Development of a 3D Perfusion System to Monitor Response of Osteoblast-Like Cells Incubated on Synthetic Bone Graft Substitute Granules With Varied Hierarchical Porosities Under Dynamic Conditions [PhD thesis]. Queen Mary University of London; September 2015.
2009	Ruppel ME. Alterations in the Mineral and Collagen Matrix of Bisphosphonate-Treated Bone and Osteoblasts [PhD thesis]. Stony Brook, NY: Stony Brook University; May 2009.
2013	Wang Y-K. Multi-Level Micromechanical Modeling of Bone Tissues [PhD thesis]. Los Angeles, CA: Los Angeles, University of California; 2013.
2016	Vrahna C. Investigating the Effects of Downstream Parathyroid Hormone (PTH) Targets on Bone Strength and Quality [PhD thesis]. Melbourne, Australia: The University of Melbourne; December 2016.
2015	Xu H. The Coupling Effect of Water and GAGs on the in Situ Toughness of Bone Are Age-Dependent [Master's thesis]. San Antionio, TX: University of Texas at San Antonio; December 2015.
2014	Reznikov N. The Adaptation of Mechanical Properties of Lamellar Bone Tissue [PhD thesis]. Weizmann Institute of Science; July 2014.