The effect of mechanical stresses on osteogenesis, the viability of osteocytes and their metabolic activity in organ culture of bones intermittently loaded "in vitro" are reported. Metatarsal bones, isolated from 12-day-old rats, were cultured in BGJb medium (with 10% foetal calf serum, 75 micrograms/ml of ascorbic acid, 100 U/ml of penicillin and 100 micrograms/ml of streptomycin), in humidified air enriched by 5% CO2 and 30% O2, and loaded in our original device for 1/2 an hour at 1 Hz. homotypic isolated and unloaded bones, cultured in the same medium, were taken as controls. The ALP (alkaline phophatase activity) increases in the media of loaded bones in comparison with the control bones. The percentage of viable osteocytes is significantly greater in loaded than in control bones. TEM observations demonstrate that in both loaded and control unloaded bones, osteocytes show well developed organelle machinery and several gap junctions with adjacent cellular processes. In the cells of loaded bones, however, a higher number of cytoplasmic organelles and gap junctions were found. In particular, RER increases twice, gap junctions three times. The induced osteogenesis and the TEM observations demonstrate the suitability of this experimental model and support the recent advanced hypothesis according to which the mechanical loading may exert a trophic function on osteocytes, stimulating both the proteic synthesis in the above-mentioned cells and the cell-to-cell communication. Furthermore, the loading is likely to exert a biological stimulus on osteoblasts via signalling molecules produced by osteocytes.

The trabeculae of the mastoid, the upper end of the femur, and the tibia were examined to ascertain whether they contain vascular channels according to a pattern similar to that observed in the calcaneus. The trabeculae were serially sectioned in transverse planes. Each section was microradiographed and photographed under ordinary and polarized light. On the photos of the individual sections (1) the number of the vascular channels, (2) the thickness of the trabecular segments with or without osteons, and (3) the maximum distance of the osteocytic lacunae from filtering surfaces (i.e., haversian canal walls or trabecular surfaces), were evaluated. About 80% of the vascular channels are haversian. Their frequency increases through the increase of the trabecular thickness and reaches 100% in those thicker than 428 microns. The distance of the deep-seated osteocytes from filtering surfaces appears almost the same in the thinner trabeculae, devoid of osteons, and in the thicker ones, containing osteons. Evidence is provided that osteons are present in numerous spongy trabeculae. Osteon formation is strictly related to the trabecular thickness so that the distance of the osteocytes from filtering surfaces does not exceed the critical value of 230 microns (in the mastoid). These findings are in agreement with those recorded in the calcaneus spongiosa. As the trabeculae studied in this research and those of the calcaneus are submitted to different mechanical loads, the main function of the endotrabecular osteons is conceivably to improve the deep-seated cell metabolism rather than the mechanical resistance of the trabeculae. On the other hand, the circumstance that most of the osteons are secondary indicates that they participate to the renewal of bone tissue.

This study analyzes the relationship between the function of femoral regions in the rat and the extent of collagen type I posttranslational modifications, to assess whether the different functional roles, i.e., mechanical or metabolic, of the bone tissues are related to the molecular structure of the matrix. For this purpose, 18 female, 100-day-old Sprague-Dawley rats were sacrificed, under anesthesia, and their femurs were removed and dissected free of adhering tissue. The spongy bone of the proximal metaphysis and the diaphysis were then selected as regions exerting prevalently a mechanical function, and the spongy bone of the distal metaphysis was selected as mainly related to metabolic function. Bone prepared from these regions was used to extract and purify the major component of the matrix, type I collagen. The content of hydroxyproline, hydroxylysine, glycosylated hydroxylysine, and pyridinium crosslinks was evaluated and the amount of each compound was expressed as a molar ratio to hydroxyproline. The amount of glycosylated hydroxylysine and pyridinium crosslinks in the distal metaphysis are significantly different from the amounts measured both in the diaphysis and the proximal metaphysis. On the contrary, the amounts of the same compounds in the diaphysis and the proximal metaphysis are statistically the same. The amount of free hydroxylysine, however, appears to be different in the proximal metaphysis and in the diaphysis. The conclusion is that matrix composition differs among different skeletal regions according to the main function they exert.

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