Nanovibrational stimulation inhibits osteoclastogenesis and enhances osteogenesis in co-cultures

Abstract

Models of bone remodelling could be useful in drug discovery, particularly if the model is one that replicates bone regeneration with reduction in osteoclast activity. Here we use nanovibrational stimulation to achieve this in a 3D co-culture of primary human osteoprogenitor and osteoclast progenitor cells. We show that 1000 Hz frequency, 40 nm amplitude vibration reduces osteoclast formation and activity in human mononuclear CD14⁺ blood cells. Additionally, this nanoscale vibration both enhances osteogenesis and reduces osteoclastogenesis in a co-culture of primary human bone marrow stromal cells and bone marrow hematopoietic cells. Further, we use metabolomics to identify Akt (protein kinase C) as a potential mediator. Akt is known to be involved in bone differentiation via transforming growth factor beta 1 (TGFβ1) and bone morphogenetic protein 2 (BMP2) and it has been implicated in reduced osteoclast activity via Guanine nucleotide-binding protein subunit α13 (Gα13). With further validation, our nanovibrational bioreactor could be used to help provide humanised 3D models for drug screening.

Figure 1

Nanovibrational stimulation setup and measurement. (a) Nanovibrational bioreactor and power supply. (b) 24 well cell culture plate attached to the bioreactor using a magnetic sheet. Reflective prismatic tape is placed in the wells (or on top of gels) so that the interferometer laser is reflected back to the detector. (c) Interferometer measuring bioreactor vibrations in the 24 well plate. (d) 2D nanovibrations in two 24 well plates. No cells were present during measurement. Measurements were taken in triplicate from each well, giving an average displacement of 40.6 nm at 1000 Hz frequency. (e) 3D (collagen gel) nanovibrations in a 24 well plate giving an average displacement of 44.4 nm at 1000 Hz frequency.

Figure 2

Osteoclast response to nanovibrational stimulation in 2D. (a) No detrimental effect on CD14⁺ cell viability was seen, as measured by Alamar blue (violin plots of individual data points, n = (d = 3, r = 3), statistics by t-test where *p < 0.05). However, (b) following 7 days of nanovibrational stimulation numbers of fused, multinucleate osteoclasts observed by TRAP staining were reduced (mean ± SD of individual data points, n = (d = 3, r = 3), statistics by t-test where *p < 0.05). Similarly, the mean osteoclast area (c) was also reduced with nanovibrational stimulation (violin plots of individual data points, n = (d = 3, r = 3), statistics by t-test where ***p < 0.001) after 7 days of culture; (d) typical TRAP staining of both control and 1000 Hz stimulated samples imaged at 10 × magnification. (e) SEM images at day 7 showing less osteoclasts were present following nanovibrational stimulation. (f) Resorption assay at day 7 showing less osteoclast activity following nanovibrational stimulation (mean ± SD of individual data points, n = (d = 3, r = 3), statistics by t-test where *p < 0.05). (g) qPCR for nanovibrated vs control CD14⁺ cells for transcripts related to osteoclastogenesis and inflammation. A trend towards repression of these genes in the nanovibrated cultures was observed (n = (d = 1–3, r = 3), statistics by t-test where *p < 0.05); full qPCR data is presented in Supplementary Fig. 2. (h) At the protein level, IL-6 was seen to be repressed (mean ± SD of individual data points, n = (d = 3, r = 4), statistics by t-test where *p < 0.05). Together, the data indicates a reduction in osteoclast forming activity of CD14⁺ blood mononuclear cells with nanovibrational stimulation.

Figure 3

BMSC and BMHC co-culture in 2D and 3D. (a) No detrimental effect on cell viability, as measured by Alamar blue, was seen (violin plots of individual data points, n = (d = 3, r = 3), statistics by t-test where **p < 0.01). Using TRAP stain to identify osteoclasts after 28 days of co-culture, (b) the number of osteoclasts was seen to reduce (mean ± SD of individual data points, n = (d = 3, r = 3), statistics by t-test where **p < 0.01) and (c) area of osteoclasts was decreased with nanovibrational stimulation (violin plots of individual data points, n = (d = 3, r = 3), statistics by t-test where *p < 0.05). (d) Typical TRAP stain of both control and 1000 Hz stimulated samples imaged at 10 × magnification. (e) Actin/DAPI immunofluorescence after 28 days of culture showed that osteoclast cells identified by multiple nuclei and by actin rings tended to have fewer nuclei following nanovibrational stimulation. Arrows indicate the multiple nuclei of each cell. (f) SEM images after 28 days of culture showed that while many osteoclasts could be seen in control co-cultures, fewer were observed, along with better spread BMSCs, following nanovibrational stimulation. Arrows indicate BMSCs; M = macrophage; OC = osteoclast. (g) Looking at osteogenesis after 28 days of culture using von Kossa staining in 2D BMSC monoculture, 2D co-culture and 3D co-culture, osteogenesis was enhanced in all conditions with nanovibrational stimulation (mean ± SD of individual data points, n = (d = 3, r = 4–5), statistics by t-test where *p < 0.05, ***p < 0.001); typical von-Kossa images from the co-cultures are shown below their corresponding graphs (2D = left, 3D = right). (h) qPCR for nanovibrated vs control 2D (top) and 3D (bottom) co-cultures for transcripts related to osteoclastogenesis, inflammation and osteogenesis showing a trend towards initial activation and then repression of osteoclast-related genes and activation of osteoblast related genes for nanovibrated cultures (n = (d = 1–4, r = 3–4), statistics by t-test where *p < 0.05, **p < 0.01 and ***p < 0.001). Together, the data indicates reduction in osteoclast forming activity and increase in osteoblast forming activity of the co-cultures in both 2D and 3D with nanovibrational stimulation. Full qPCR data is presented in Supplementary Figs. 4 and 5. (i) Untargeted metabolomic analysis for 2D and 3D co-culture. Lipid-based pathways were upregulated, particularly at day 14 in the 3D culture; steroid and cholesterol pathways are indicated by *. This suggests that cell growth and differentiation is more energetically demanding in 3D culture compared to 2D culture (n = 3).