Old Mice Have Less Transcriptional Activation But Similar Periosteal Cell Proliferation Compared to Young-Adult Mice in Response to in vivo Mechanical Loading

Mechanical loading is a potent strategy to induce bone formation, but with aging, the bone formation response to the same mechanical stimulus diminishes. Our main objectives were to (i) discover the potential transcriptional differences and (ii) compare the periosteal cell proliferation between tibias of young-adult and old mice in response to strain-matched mechanical loading. First, to discover potential age-related transcriptional differences, we performed RNA sequencing (RNA-seq) to compare the loading responses between tibias of young-adult (5-month) and old (22-month) C57BL/6N female mice following 1, 3, or 5 days of axial loading (loaded versus non-loaded). Compared to young-adult mice, old mice had less transcriptional activation following loading at each time point, as measured by the number of differentially expressed genes (DEGs) and the fold-changes of the DEGs. Old mice engaged fewer pathways and gene ontology (GO) processes, showing less activation of processes related to proliferation and differentiation. In tibias of young-adult mice, we observed prominent Wnt signaling, extracellular matrix (ECM), and neuronal responses, which were diminished with aging. Additionally, we identified several targets that may be effective in restoring the mechanoresponsiveness of aged bone, including nerve growth factor (NGF), Notum, prostaglandin signaling, Nell-1, and the AP-1 family. Second, to directly test the extent to which periosteal cell proliferation was diminished in old mice, we used bromodeoxyuridine (BrdU) in a separate cohort of mice to label cells that divided during the 5-day loading interval. Young-adult and old mice had an average of 15.5 and 16.7 BrdU+ surface cells/mm, respectively, suggesting that impaired proliferation in the first 5 days of loading does not explain the diminished bone formation response with aging. We conclude that old mice have diminished transcriptional activation following mechanical loading, but periosteal proliferation in the first 5 days of loading does not differ between tibias of young-adult and old mice. Female C57BL/6N mice were obtained at 5-months and 22-months were separated into groups (5 mice/day/age) and loaded for either one, three, or five bouts of daily loading, reflecting distinct phases of the loading response: early mechanosensation (day 1), activation of the bone formation cascade before matrix deposition (day 3), and active bone formation (day 5). Mice were anesthetized (3% isoflurane) and subjected to loading each morning for the specified number of bouts. With the mice prone, the right legs (tibias) were placed vertically in the loading fixture, with the knee positioned superiorly in a semispherical cup (10-mm-diameter) attached to the system actuator, and the foot held in a static fixture inferiorly (20 degrees of dorsiflexion). A preload (−0.5 N) was applied, and tibias were subjected to axial compression for 1200 cycles/day (4-Hz triangle waveform with a 0.1-s rest-insertion after each cycle) using the Electropulse 1000 materials testing. Based on prior strain gauging analyses, age-specific peak forces of −8 N and −7 N were selected for 5-month-old and 22-month-old mice, respectively, to engender average peak periosteal strains of −2200με at the cortical mid-shaft. After each loading bout, buprenorphine was delivered to mitigate pain from loading, and mice were returned to their cages to resume unrestricted activity. The left tibias served as non-loaded, contralateral controls. Left and right tibias were stripped of muscle, cut at the distal tibiofibular junction and 2 mm distal to the tibial plateau, centrifuged to remove the bone marrow, and snap frozen in liquid nitrogen. Samples were pulverized, and total RNA was extracted using TRIzol. Ribosomal RNA (rRNA) was removed using the Ribo-Zero rRNA Removal Kit, and all 60 samples (30 pairs of tibias) were subjected to RNA-seq with the HiSeq 3000 at 1 × 50 bp on 1 μg of RNA. The RNA-seq reads were aligned to the mouse genome (mm10), and gene counts were determined by the number of uniquely aligned, unambiguous reads.