Aged male rats regenerate cortical bone with reduced osteocyte density and reduced secretion of nitric oxide after mechanical stimulation

Abstract

Mechanical loading is integral to the repair of bone damage. Osteocytes are mechanosensors in bone and participate in signaling through gap junction channels, which are primarily comprised of connexin 43 (Cx43). Nitric oxide (NO) and prostaglandin E2 (PGE2) have anabolic and catabolic effects on bone, and the secretion of these molecules occurs after mechanical stimulation. The effect of age on the repair of bone tissue after damage and on the ability of regenerated bone to transduce mechanical stimulation into a cellular response is unexplored. The goal of this study was to examine (1) osteocytes and their mineralized matrix within regenerated bone from aged and mature animals and (2) the ability of regenerated bone explants from aged and mature animals to transduce cyclic mechanical loading into a cellular response through NO and PGE2 secretion. Bilateral cortical defects were created in the diaphysis of aged (21-month-old) or mature (6-month-old) male rats, and new bone tissue was allowed to grow into a custom implant of controlled geometry. Mineralization and mineral-to-matrix ratio were significantly higher in regenerated bone from aged animals, while lacunar and osteocyte density and phosphorylated (pCx43) and total Cx43 protein were significantly lower, relative to mature animals. Regenerated bone from mature rats had increased pCx43 protein and PGE2 secretion with loading and greater NO secretion relative to aged animals. Reduced osteocyte density and Cx43 in regenerated bone in aged animals could limit the establishment of gap junctions as well as NO and PGE2 secretion after loading, thereby altering bone formation and resorption in vivo.

Fig. 1

a Implant hardware. Arrows point to the cover and channel plates. Dashed lines illustrate the open parallel channels. b Femoral cortical defect. c Postoperative radiograph of implanted hardware. Arrows point to implants. d Three-dimensional microCT reconstructed isosurface of regenerated explants from mature and aged rats. e Regenerated explant within the three-point bending device. Arrows point to loading supports

Fig. 2

a Regenerated explant mineral density expressed as mean ± SD for mature (n = 6) and aged (n = 8) rats; *p <0.05 between groups. b Regenerated explant mineral-to-matrix ratio expressed as mean ± SD for mature (n = 6) and aged (n = 8) rats; *p < 0.05 between groups. c Femoral cortical bone crystallinity expressed as mean ± SD for mature (n = 10) and aged (n = 10) rats; **p < 0.01 between groups. d Regenerated explant secreted osteopontin concentration expressed as mean ± SD for mature (n = 6) and aged (n = 8) rats; **p < 0.01 between groups

Fig. 3

a Histological sections of regenerated explants and femoral cortical bone from mature (n = 6, 10) and aged (n = 8, 10) rats. Arrows point to lacunae, some of which contain osteocytes. b Lacunar density of regenerated explant sections from mature (n = 6) and aged (n = 8) rats and of femoral cortical bone from mature (n = 10) and aged (n = 10) rats expressed as mean ± SD. c Osteocyte density of regenerated explant sections from mature (n = 6) and aged (n = 8) rats and of femoral cortical bone from mature (n = 10) and aged (n = 10) rats expressed as mean ± SD. d Area of cleaved caspase-3 stain normalized to femoral cortical bone area surrounding the defect (expressed as a percentage) for mature (n = 10) and aged (n = 10) rats expressed as mean ± SD. e Cleaved caspase-3 (red)- and DAPI (blue)-stained sections of femoral cortical bone surrounding the defect from mature (n = 10) and aged (n = 10) rats at ×20 and ×40. Arrows point to the edges of the rectangular defect

Fig. 4

For all panels, the numbers of regenerated explants tested were n = 11 sham, n = 11 loaded for mature rats and n = 9 sham, n = 9 loaded for aged rats. a Western blots of phosphorylated, total Cx43, and GAPDH for sham-treated and loaded regenerated explant specimens from mature and aged animals. b Phosphorylated Cx43 densitometry expressed as mean ± SD for sham-treated and loaded regenerated explant specimens from mature and aged rats. c Cx43 densitometry expressed as mean ± SD for regenerated explant specimens from mature and aged rats