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.

Bone marrow stromal cells (MSCs) are a source of osteoblast precursors that can be recruited during bone remodeling or injury, both important processes in aging populations. With advancing age, alterations in bone structure and mineralization are often associated with an increase in osteoporosis and fracture risk. Changes in the number of osteoprogenitor cells and their osteogenic potential may occur with advancing age; however few studies have considered the influence of mechanical conditions. Here, we investigated the ability of bone MSCs from mature and aged rats to differentiate into osteoblasts and to respond to short and long periods of mechanical stimulation through signaling by ERK1/2, nitric oxide (NO), and prostaglandin E(2) (PGE(2)) during differentiation. Mineralization was delayed and reduced, but extracellular matrix production appeared less affected by increased age. Differentiating MSCs from aged animals had a decreased response to short and long periods of mechanical stimulation through ERK1/2 signaling, and to long periods of mechanical loading through NO signaling early and late during differentiation. Increases in relative PGE(2) signaling were higher in MSCs from aged animals, which could compensate for reduced ERK1/2 and NO signaling. The decreased mineralization may decrease the ability of cells from aged animals to respond to mechanical stimulation through ERK1/2 and NO signaling, with increased impairment over differentiation time. Decreasing the delay in mineralization of MSCs from aging animals might improve their ability to respond to mechanical stimulation during bone remodeling and injury, suggesting therapies for bone fragility diseases and tissue engineering treatments in elderly populations.

We report two immuoreactive species of thrombospondin-2 (TSP2), sized approximately 200 and 125 kDa, in the long bones of growing, but not skeletally mature, mice. In vitro osteoblasts secrete a 200-kDa species into the culture medium as early as day 3, and it appears in the cell-matrix layer by day 7. A 125-kDa species appears in the cell-matrix layer in parallel with mineralization; it is not detected in cell-conditioned medium. Unilateral tibial fracture induced a time-dependent upregulation of the 200-kDa species at the site of trauma. By contrast, relative levels of the 125-kDa species at the fracture site were lower than in bones from naive control animals. In the contralateral untouched control tibia, the 200-kDa species was rapidly and substantially reduced compared to bone harvested from naive control mice. Levels of the 125-kDa species in the untouched tibia declined gradually with time postfracture. TSP2 gene expression in uninjured control bone decreased modestly by 21 days postfracture. On the day of fracture, the osteoblast differentiation potential of MSCs harvested from uninjured bones decreased compared to those harvested from naive control animals. The presence of two isoforms suggests that TSP2 may undergo posttranscriptional or posttranslational processing in skeletal tissue. Our data also suggest that, in the context of trauma, the two TSP2 isforms are differentially modulated at injured and noninjured skeletal sites in an animal undergoing fracture healing.