The Study of Bone: Electron Microscopy, X-Ray Diffraction, & Optical Birefringence 



235 



cized the conclusions of Engstrom and Finean, be- 

 cause observations with the electron microscope do 

 not support the view that the inorganic particles are 

 rod-shaped. 



On the other hand North, Cowan and Randall 

 <14) have been able to show that the collagen gives a 

 low-angle scatter not dissimilar to that obtained by 

 Engstrom and Finean. 



The array of results referred to show such diver- 

 gent points that they deserve a deeper critical exa- 

 mination. In the hope of contributing to the solLitit>n 

 of the problem, we have taken up Finean and 

 Engstrom's researches on human and cow bone, 

 integrating them with further observations with the 

 polarizing and electron microscope. 



A low-angle scatter apparatus somewhat similar to 

 that employed by Finean (9) was used. The scatter was 

 lecorded using Ni-filtered CuA'a radiation (A ^ 1.54 A). 

 We did not think it advisable to use monochromatic 

 radiation since with a system of the type existing in 

 ihe bone tissue, the use of Ni-filtered radiation (CuA'a) 

 does not entail more substantial errors than the use of a 

 monochromatic radiation. The intensity variation of the 

 low-angle scatter was measured using a Leeds and North- 

 rup automatic recording microphotometer. 



The electron microscope preparations were obtained 

 employing both the mechanical dissociation in a Waring 

 blendor and the pseudo-replica technique. The detailed 

 description of these techniques has been reported in 

 previous papers (2, 3). 



X-ray, polarizing microscope and electron microscope 

 investigations were performed on samples of cow's and 

 human bone. This last pertained to foetus, adult and 

 old man. The samples comprehended untreated bone, 

 decalcified bone and bone from which ossein had been 

 removed by trypsin digestion or according Gabriel's 

 method. 



In agreement with the results obtained by Eng- 

 strom and Finean, the low-angle scatter of x-rays 

 from sections of bone suggests that the scattering 

 elements are well aligned and symmetrical around 

 their long axes. Therefore the same elements appear 

 as if they were the same as ellipsoids of revolution, 

 the long axes being aligned in the direction of the 

 longitudinal axis of the bone. The short diameter of 

 the ellipsoids is of about 70 A and the long one of 

 about 200 A. 



Still in agreement with the results of Engstrom and 

 Finean, the low-angle scatter from the longitudinal 

 sections of bone from which ossein has been removed 

 according to Gabriel's method, suggests that the 

 shape and the dimensions of the scattering elements 

 are changed. In fact the short diameter of the ellip- 

 soids is about 80 A, while the long diameter is re- 

 duced to about 130 A. In addition, the intensity of 

 diffraction seems greatly increased by the removal of 

 ossein. 



These results provide arguments for the following 

 discussion. 



The low-angle x-ray scatter might be interpreted 

 either in terms of the holes irregularly placed inside 

 a homogeneous body or in terms of a particle scatter, 

 when inter-particle interference is not taken into 



consideration. In the general equation adopted by 

 Guinier (12) in treating the scatter from such 

 systems, the intensity of the scattered radiation 

 appears as a function of the square of the difTerence 

 of the electron densities pertaining to the single 

 components responsible for the scatter (particles and 

 surrounding medium or homogeneous body and the 

 holes). Such a condition does not enable it to be 

 established a priori to which of the two aforesaid 

 systems a low-angle scatter is related. In this respect 

 the choice of the system is suggested by evidence 

 deriving from the data of other investigations. 



The identity in the low-angle x-ray diffraction 

 pattern pertaining, respectively, to the bone, collagen 

 tissue ( 14) and calcified collagen ( 10) does not permit 

 the acceptance of the theory that the scattering units 

 pertain to inorganic particles, these latter being 

 completely lacking in collagen tissue. Therefore the 

 scattering units must be considered as related to 

 ellipsoidal entities (200 70 A) pertaining to the col- 

 lagen and oriented in parallel. In bone and calcified 

 collagen the calcium salts, enclosing the organic el- 

 lipsoids, increase the difTerence in electronic density 

 between the same organic particles of collagen and 

 surrounding medium. 



This interpretation of the low-angle x-ray scatter 

 from bone tissue in terms of holes (containing the 

 organic particles) irregularly placed inside a homo- 

 geneous body (the inorganic bone fraction) is also 

 supported by the following arguments. 



{a) The form birefringence of the bone tissue from 



0.009 



008 



007 



005 



-0003 



Fig. 1. Graph of the form birefringence obtained from bone 

 deprived of ossein according to Gabriel's method. 



