Infrastructural and Crystallographic Study of Experimental Calcifications 163 



Materials and methods 



Nephrocalcinosis was produced in rats (Wistar or Long Evans) according to 

 FouRMAN (1959) by daily intraperitoneal injections for 6 to 8 days of 1.5 ml of a 

 10 per cent solution of calcium gluconate. The animals were killed directly after the 

 last injection or 10 days or so later; the deposits did not appear to be different in 

 either case. Fragments taken from the kidneys were fixed in P/o osmium tetroxide 

 mixture according to Sjostrand and embedded in methacrylate or Epon. At the same 

 time control examinations were made using the light microscope in particular after 

 staining according to von Kossa and microincineration. 



In another experiment, surgical fibrin sponges impregnated with calcium gluconate 

 were introduced into the peritoneal cavity of Wistar rats. After 5 days fragments 

 were taken as above for light and electron microscopic examination. 



Crystallographic examinations were made by electron diffraction (Triib-Taiiber 

 diffractograph and Siemens Elmiskop electron microscope), using thin sections 

 prepared as for the electron microscope. Residues from organs prepared by the 

 method of Gabriel (1894) were also analysed by X-ray diffraction. 



Results 



1. Renal deposits 



In the kidneys two kinds of deposits are observed. Some, mostly intracellular, of 

 small extent, consist of clusters of needle-shaped crystals 170 A long and 70 A wide. 

 They are very similar to the apatite crystals of bone. Others, in large number, appear 

 as very fine granular masses of about 50 A diameter. They are extracellular and 

 generally found within the basement membrane of the proximal convoluted tubules, 

 which is thickened and modified. The grains are often arranged in concentric layers, 

 like LiESEGANG rings. In exceptional cases small clusters of needle-shaped deposits are 

 found among them. A detailed description was previously reported (Policard et al., 

 1960, 1961). 



Occasionally small, dense granulations are seen in mitochondria near the basement 

 membrane. They are similar to those already described in the mature osteocyte (Baud 

 and DuPONT, 1965). 



Electron diffraction studies of selected areas of the intracytoplasmic needle- 

 shaped deposits show patterns consistent with apatite (d = 3.41, 3.09, 2.79, 2.60, 2.26, 

 1.92, 1.81, 1.71, 1.44, 1.245, 1.160). 



The total calcareous residue of the kidney prepared by the method of Gabriel 

 should correspond to the granular deposits which are by far in the majority. With 

 X-ray diffraction, a pattern similar to that of whitlockite or /] tricalcium phosphate 

 is obtained. It may be, however, that the residue was modified by the method of 

 preparation used. Extensive areas in ultrathin sections give an electron diffraction 

 pattern with 10 equidistances (d = 4.05, 3.63, 2.92, 2.43, 2.34, 2.19, 2.04, 1.72, 1.54, 

 1.49); some of them might correspond to whitlockite. 



2. The granulome surrounding the sponges 

 A direct electron microscopic examination of the granulome shows numerous 

 needle-shaped deposits similar to the intracytoplasmic deposits of the kidneys. The 

 needles, often arranged in small clusters, are found in the residues of the sponge or in 



