Low magnitude high frequency vibrations expedite the osteogenesis of bone marrow stem cells on paper based 3D scaffolds

Abstract

Anabolic effects of low magnitude high frequency (LMHF) vibrations on bone tissue were consistently shown in the literature in vivo, however in vitro efforts to elucidate underlying mechanisms are generally limited to 2D cell culture studies. Three dimensional cell culture platforms better mimic the natural microenvironment and biological processes usually differ in 3D compared to 2D culture. In this study, we used laboratory grade filter paper as a scaffold material for studying the effects of LHMF vibrations on osteogenesis of bone marrow mesenchymal stem cells in a 3D system. LMHF vibrations were applied 15 min/day at 0.1 g acceleration and 90 Hz frequency for 21 days to residing cells under quiescent and osteogenic conditions. mRNA expression analysis was performed for alkaline phosphatase (ALP) and osteocalcin (OCN) genes, Alizarin red S staining was performed for mineral nodule formation and infrared spectroscopy was performed for determination of extracellular matrix composition. The highest osteocalcin expression, mineral nodule formation and the phosphate bands arising from the inorganic phase was observed for the cells incubated in osteogenic induction medium with vibration. Our results showed that filter paper can be used as a model scaffold system for studying the effects of mechanical loads on cells, and LMHF vibrations induced the osteogenic differentiation of stem cells.

Keywords: Filter paper; Mesenchymal stem cells; Osteogenic differentiation; Scaffold; Vibration.

Fig. 1

The schematic representation a and photography b of the vibration platform utilized in the study

Fig. 2

The viability of D1 ORL UVA cells on filter paper scaffolds was determined via MTT assay. Cell viability was assessed under normal growth and ostogenic induction conditions for 10 days. *: p ≤ 0.05 between day 1 and day 10; §: p ≤ 0.05 between control and vibration at day 10; ¥: p ≤ 0.05 between growth and osteogenic condition at day 10; calculated by ANOVA followed by S-N-K post-hoc test. GC growth control, GV: growth vibration, OC: osteogenic control, OV osteogenic vibration

Fig. 3

Fluorescent microscope images of D1 ORL UVA-EGFP cells showing proliferation of cells on filter paper scaffolds; a day 1, b day 7, c day 14 and d day 21 after cell seeding. Scale bar: 200 µm. GC growth control, GV growth vibration, OC osteogenic control and OV osteogenic vibration

Fig. 4

a Phase contrast micrographs of D1 ORL UVA cells in tissue culture plates, stained with Alizarin red on day 14. Scale bar: 100 µm. Red color indicates calcium deposits. b Quantification of Alizarin red S (ARS) staining by CPC extraction normalized to total protein. a–c Differences in dissolved ARS dye concentration between groups calculated by ANOVA followed by S-N-K post hoc test for p ≤ 0.05. GC growth control, GV growth vibration, OC osteogenic control, OV osteogenic vibration

Fig. 5

a Stereomicroscope images of D1 ORL UVA cells seeded on paper scaffolds, incubated in regular growth medium or osteogenic induction medium and stained with Alizarin red on days 14 and 21. Red color indicates calcium deposits. b Quantification of Alizarin red S (ARS) staining by CPC extraction normalized to total protein. a, b, c: differences in dissolved ARS dye concentration between groups calculated by ANOVA followed by S-N-K post hoc test for p ≤ 0.05. GC growth control, GV growth vibration, OC osteogenic control, OV osteogenic vibration, EP empty paper

Fig. 6

Gene expression levels of D1 ORL UVA stem cells that were induced either with application of vibration or with osteogenic induction medium treatment after 14 days. a, b, c: differences in gene expression level between groups calculated by ANOVA followed by S-N-K post hoc test for p ≤ 0.05. GC growth control, OC osteogenic control, GV growth vibration, OV osteogenic vibration

Fig. 7

FTIR spectra of filter paper samples with D1 ORL UVA stem cells that were incubated in regular growth media or osteogenic media for 14 and 21 days with vibration or under static conditions. a Spectra of samples incubated in growth medium and the non-cell seeded empty paper, b spectra of samples incubated in osteogenic induction medium and empty paper c spectra of samples in growth condition after the spectrum of empty paper was subtracted from each and d spectra of samples in osteogenic condition after the spectrum of empty paper was subtracted from each. GC growth control, GV growth vibration, OC osteogenic control, OV osteogenic vibration, EP empty paper, d14: 14th day of cell culture, d21: 21st day of cell culture