R. Steendijk et al.: Microradiographic and Histological Observations 175 



Microradiographic and Histological Observations in 

 Primary Vitamin D-resistant Rickets 



R. Steendijk, J. Jowsey, A. van den Hooff, H. K. L. Nielsen 



Departments of Paediatrics and Orthopaedic Surgery and Histological Laboratory, 



University of Amsterdam, The Netherlands; 



Section of Surgical Research, Mayo Clinic, Rochester, Minn., U. S. A. 



Impaired mineralisation of bone in primary vitamin D-resistant rickets (familial 

 or essential hypophosphataemia) is not limited to the presence of wide osteoid bands 

 covering the trabeculae of spongious bone or lining the central canals of growing 

 osteones. There also is a conspicuous lack of mineral around many osteocyte lacunae 

 and their canaliculi as shown by microradiography of undecalcified bone (Engfeldt 

 et al., 1956; Jowsey, 1964). Ordinary histological examination of decalcified bone 

 has hitherto failed to demonstrate any abnormality in the organic matrix at these 

 sites. No exhaustive studies have yet been published and a more detailed investigation 

 into the structural and histochemical characteristics of the perilacunar matrix was, 

 therefore, indicated. 



Material and methods 



Diaphyseal bone from the fibulae of 2 young adult patients with vitamin 

 D-resistant rickets was removed in the course of orthopaedic procedures. Appropriate 

 pieces were fixed in ZC/o ethanol for subsequent embedding in methyl-methacrylate 

 and microradiography. Similar pieces were fixed in lO^/o formalin, or in a mixture 

 of formalin, mercuric chloride and picric acid for subsequent decalcification in EDTA 

 or in nitric-picric acid and embedding in paraffin. From the latter material 6 jli 

 sections were cut and treated with various histological stains (see "Results"). Some of 

 the EDTA-decalcified material was cut in the frozen state at 20 // and used unstained 

 for phase-contrast microscopy, or stained with a modified Schmorl-method (O.OP/o 

 azure II in water at pH 9.0, followed by 20 ml saturated picric acid in 200 ml of 

 water). Sections from the same blocks were treated with Bodian's silver stain. 



Results 



The pattern of uptake of the azure Il-picric acid stain in decalcified bone from 

 the rachitic patients appeared disturbed in a specific way. In addition to its normal 

 distribution along the walls of the lacunae and canaliculi, the stain was concentrated 

 in the vicinity of many osteocyte lacunae, i. e. at the sites where microradiographs of 

 undecalcified sections revealed a subnormal degree of mineralisation. The localisation 

 was not strictly perilacunar, but rather extending from the lacunae toward the center 

 of the osteone, along the centripetal canaliculi (Fig. 1). Careful inspection of the 

 microradiographs disclosed that the lack of bone mineral was also predominant on 

 that side of the lacunae (Fig. 2). At high magnifications of the stained sections, it 

 appeared that neither lacunae nor canaliculi were enlarged and that the matrix had 

 taken up the stain in a globular fashion (Fig. 3). Similar pictures were obtained after 

 Bodian's silver impregnation technique. By phase-contrast microscopy of 20 // de- 

 calcified unstained sections, the abnormal pattern of the matrix was visible as a 

 conglomeration of small globules, interrupting the normal lamellar architecture of 



