Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 g peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (−81%) and bone formation (−80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.

Three-dimensional (3D) cell-laden scaffolds are becoming more prevalent in bone tissue repair and regeneration. However, the influence of physical scaffold properties on cell behavior is still unclear. In this study, we fabricated four different alginate concentration (0.8, 1.3, 1.8 and 2.3%alg) composite cell-laden porous scaffolds using a 3D bioprinting technique. The aim was to investigate the changes of physical properties affected by the alginate concentration and the influences on cell behavior. The study showed that the different alginate concentration scaffolds had uniform macropores (500–600 μ m) with compressive moduli ranging from 1.5 kPa (0.8%alg) to 14.2 kPa (2.3%alg). Long-term structural integrity of the printed scaffolds was achieved when cultured in cell culture media, but not when cultured in phosphate buffered saline (PBS). Scaffold structure, swelling behavior, and compressive moduli decreased with culturing time and higher alginate concentration lead to more stable physical scaffold properties. Meanwhile, human mesenchymal stem cell (hMSCs) laden non-printed and bioprinted composite scaffolds were fabricated. Bioprinting did not affect cell viability, but alginate concentration had a significant influence on cell viability and cell morphology. Lower alginate concentration scaffolds (0.8%alg) showed higher cell viability (84% ± 0.7% versus 68% ± 1.3%) compared to higher alginate concentration scaffolds (2.3%alg) at day 14. Live cell image in the 0.8%alg scaffolds demonstrated the formation of a 3D interconnected cellular network, while cells in the 1.8 and 2.3%alg scaffolds formed spheroids. In conclusion, this study broadens the design space for alginate-based bioinks for 3D bioprinting. Higher alginate concentration preserved better scaffold fidelity and mechanical properties. Better cell viability and cell spreading morphology was achieved in lower alginate concentration scaffolds, which is relevant for potential applications in bone tissue engineering.

Low‐magnitude high‐frequency vibration (LMHFV) provokes anabolic effects in non‐fractured bone; however, in fracture healing, inconsistent results were reported and optimum vibration conditions remain unidentified. Here, we investigated frequency dependent effects of LMHFV on fracture healing. Twelve‐week‐old, female C57BL/6 mice received a femur osteotomy stabilized using an external fixator. The mice received whole‐body vibrations (20 min/day) with 0.3g peak‐to‐peak acceleration and a frequency of either 35 or 45 Hz. After 10 and 21 days, the osteotomized femurs and intact bones (contra‐lateral femurs, lumbar spine) were evaluated using bending‐testing, µ‐computed tomography, and histomorphometry. In non‐fractured trabecular bone, vibration with 35 Hz significantly increased the relative amount of bone (+28%) and the trabecular number (+29%), whereas cortical bone was not influenced. LMHFV with 45 Hz failed to provoke anabolic effects in trabecular or cortical bone. Fracture healing was not significantly influenced by whole‐body vibration with 35 Hz, whereas 45 Hz significantly reduced bone formation (−64%) and flexural rigidity (−34%) of the callus. Although the exact mechanisms remain open, our results suggest that small vibration setting changes could considerably influence LMHFV effects on bone formation in remodeling and repair, and even disrupt fracture healing, implicating caution when treating patients with impaired fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1006–1013, 2014.

In recent decades, the field of bone mechanobiology has sought experimental techniques to unravel the molecular mechanisms governing the phenomenon of mechanically regulated fracture healing. Each cell within a fracture site resides within different local microenvironments characterized by different levels of mechanical strain; thus, preserving the spatial location of each cell is critical in relating cellular responses to mechanical stimuli. Our spatial transcriptomics–based “mechanomics” platform facilitates spatially resolved analysis of the molecular profiles of cells with respect to their local in vivo mechanical environment by integrating time-lapsed in vivo micro–computed tomography, spatial transcriptomics, and micro–finite element analysis. We investigate the transcriptomic responses of cells as a function of the local strain magnitude by identifying the differential expression of genes in regions of high and low strain within a fracture site. Our platform thus has the potential to address fundamental open questions within the field and to discover mechano-responsive targets to enhance fracture healing.