Comparison of the Anabolic Effects of Reported Osteogenic Compounds on Human Mesenchymal Progenitor-derived Osteoblasts

Abstract

There is variability in the reported effects of compounds on osteoblasts arising from differences in experimental design and choice of cell type/origin. This makes it difficult to discern a compound's action outside its original study and compare efficacy between compounds. Here, we investigated five compounds frequently reported as anabolic for osteoblasts (17β-estradiol (oestrogen), icariin, lactoferrin, lithium chloride, and menaquinone-4 (MK-4)) on human mesenchymal progenitors to assess their potential for bone tissue engineering with the aim of identifying a potential alternative to expensive recombinant growth factors such as bone morphogenetic protein 2 (BMP-2). Experiments were performed using the same culture conditions to allow direct comparison. The concentrations of compounds spanned two orders of magnitude to encompass the reported efficacious range and were applied continuously for 22 days. The effects on the proliferation (resazurin reduction and DNA quantification), osteogenic differentiation (alkaline phosphatase (ALP) activity), and mineralised matrix deposition (calcium and collagen quantification) were assessed. Of these compounds, only 10 µM MK-4 stimulated a significant anabolic response with 50% greater calcium deposition. Oestrogen and icariin had no significant effects, with the exception of 1 µM icariin, which increased the metabolic activity on days 8 and 22. 1000 µg/mL of lactoferrin and 10 mM lithium chloride both significantly reduced the mineralised matrix deposition in comparison to the vehicle control, despite the ALP activity being higher in lithium chloride-treated cells at day 15. This demonstrates that MK-4 is the most powerful stimulant of bone formation in hES-MPs of the compounds investigated, highlighting its potential in bone tissue engineering as a method of promoting bone formation, as well as its prospective use as an osteoporosis treatment.

Keywords: bone formation; bone tissue engineering; matrix mineralisation; menaquinone-4; mesenchymal stem cells; osteoblasts; osteoporosis; vitamin K.

Figure 1

The baseline response of hES-MPs to osteogenesis induction media (OIM) over 22 days. (A) The metabolic activity. Boxplots showing minimum, 1st quartile, mean, 3rd quartile and maximum for (B) the total DNA, (C) ALP activity, (D) normalised ALP activity, (E) calcium deposition, and (F) collagen deposition. (n = 48). Whole well images (diameter ~15 mm) and a high magnification phase contrast image (scale bar: 100 μm) show typical matrix deposition characteristics.

Figure 2

The effect of DMSO normalised to 0% (horizontal dashed line). (A) The metabolic activity normalised to 0% at each time point, (B) D15 total DNA, (C) D15 normalised ALP activity, (D) D22 ARS, (E) D22 DR80, and (F) representative whole well photographs of ARS and DR80 staining at each concentration (diameter ~15 mm). (n = 8). No significant effect on any parameter for 0.01% or 0.1%. At 1% DMSO, the metabolic activity was significantly reduced from D7 onwards, with ALP, DNA, mineral, and collagen also all being significantly lower.

Figure 3

The effect of each compound and concentration on the metabolic activity. No significant difference in the metabolic activity for hES-MPs cultured in (A) OIM vs. BM/DMSO vehicle controls (n = 40), and (B) the vehicle controls for each compound, normalised to day 1 (n = 8). (C–E) The effect of each compound at the (C) lowest, (D) middle, and (E) highest concentrations on the metabolic activity, normalised to their respective vehicle control at each time point (horizontal dashed line). (n = 8). Lactoferrin (1 mg/mL) and lithium chloride (10 mM) significantly reduced the metabolic activity from day 15 onwards (p < 0.0001). 1 µM icariin significantly increased the metabolic activity on days 8 and 22 (p < 0.05). No other condition significantly deviated from the vehicle control growth curve.

Figure 4

The effect of each compound and concentration on the total DNA per well at day 15, normalised to their respective vehicle control (horizontal dashed line). The treatment with lactoferrin (1 mg/mL) and lithium chloride (10 mM) significantly lowered the total DNA (p < 0.001 & P <0.01, respectively). No other condition significantly deviated from the vehicle control. (n = 8).

Figure 5

The effect of each compound and concentration on the normalised ALP activity at day 15, normalised to their respective vehicle control (horizontal dashed line). The treatment with lactoferrin (1 mg/mL) resulted in a significantly lower normalised ALP activity, whilst the treatment with lithium chloride (10 mM) elevated the normalised ALP activity by 100% (both p < 0.01). No other condition significantly differed from the vehicle control. (n = 8).

Figure 6

(Top) The effect of each compound and concentration on the calcium deposition at day 22, normalised to their respective vehicle control (horizontal dashed line). The treatment with lactoferrin (1 mg/mL) and lithium chloride (10 mM) resulted in a significantly lower calcium deposition in comparison to the vehicle control (p < 0.001 & p < 0.01, respectively), whilst the MK-4 (10 µM) treatment resulted in a 50% greater calcium deposition (both p < 0.05). No other condition significantly deviated from the vehicle control. (n = 8). (Bottom) Representative whole well images (diameter ~15 mm) and high magnification (40×) phase contrast images (diameter 200 μm) of ARS staining of each compound at the highest concentration applied.

Figure 7

(Top) The effect of each compound and concentration on the collagen deposition at day 22, normalised to their respective vehicle control (horizontal dashed line). Lactoferrin (1 mg/mL) and lithium chloride (10 mM) significantly diminished the collagen deposition (p < 0.01 & p < 0.05, respectively). No other condition significantly deviated from the vehicle control. (n = 8). (Bottom) Representative whole well images (diameter ~15 mm) and high magnification (40×) phase contrast images (diameter 200 μm) of DR80 staining of each compound at the highest concentration applied.