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United States Department of Agriculture

Agricultural Research Service

Title: Method for Preparation of Trabecular Bone Matrix for Histological Studies Using Scanning Electron Microscopy

item Crummy, Laurie - GF PUBLIC SCHOOLS
item Idso, Joseph
item Newman Jr, Samuel
item Hunt, Curtiss

Submitted to: North Dakota Academy of Science Proceedings
Publication Type: Other
Publication Acceptance Date: September 15, 1997
Publication Date: N/A

Interpretive Summary: The spongy' bone found in the middle of a long bone is formed and removed at a higher rate than is the compact bone found in the outside wall of the bone. Because many bone diseases affect the spongy bone more than compact bone, we need to find out more about how a poor diet changes the shape and therefore strength of spongy bone. To make measurements of the spongy bone, it was necessary to find a way of removing all the marrow and blood that fills all the 'holes' in the spongy bone but do it in such a way that the bone itself is not changed in any way. We took the lower leg bone from rats that were raised on diets known to damage bone structure. After the bones were cut lengthwise, they were put in an embalming solution and then cut again lengthwise to create two matching halves. Both halves were put in ether, then hydrogen peroxide to remove much of the fat. Finally, one half of each bone was digested in sodium hydroxide for 16 hours, the other half for 116 hours. A treatment period of 16 hours with sodium hydroxide did not clear the spongy bone of blood and marrow. In contrast, matched samples treated for 116 hours were clear of marrow such that spongy bone was visible in the depth of the sample. The overall structure of the spongy bone was not different between any of the matched samples, even when the rats received very poor magnesium nutrition. Also, the width of each 'icicle' of spongy bone did not vary discernibly between matched samples. The size of the pits that contained the bone cells prior to sodium hydroxide digestion were visible regardless of chemical treatment and their shape did not change with the longer treatment with sodium hydroxide. Our method is satisfactory for removing marrow and blood from bone without damaging the bone itself.

Technical Abstract: To improve the ability to assess the influence of various nutritional stressors on trabecular bone matrix morphology, we developed a chemical digestion technique to remove adjacent cellular material from trabecular bone samples prior to examination by scanning electron microscopy (SEM). To ensure that the clearing technique did not alter bone matrix morphology, we verified the efficacy of the technique by utilizing bone from control rats and from rats exposed to nutritional stressors that induce alterations in trabecular bone morphology. Proximal tibiae were isolated from rats fed 100 mg magnesium/kg diet (inadequate) and ~0.1 (low) or 2.0 (physiologic) mg boron/kg and 1000 IU Vit.D/kg throughout the experiment or between days 19 and 35 only. The proximal tibiae were halved in the frontal plane with a razor blade and placed in buffered formalin for 21 months. The halved proximal tibiae were cut again sagittally to create duplicate experimental bone samples. Both samples were immersed in ether, then 5% H2O2 then one of the matching halves was treated for 16 hours, the other for 116 hours, in 18% NaOH. After chemical digestion, matching halves were prepared for, and examined with SEM. Treatment for 116, but not 16 hours with NaOH cleared the trabecular bone of adjacent cellular material. Gross trabecular structure, width of individual trabeculae, or appearance of osteoblast lacunae or possible cannilicular channels or dense osteoid material were not different between any of the matched samples, even under conditions of extreme dietary stress. The described method is satisfactory for removing soft tissue from bone without risk of damaging the bone itself.

Last Modified: 4/18/2014
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