Asked 24th Apr, 2020

Can pixel density of callus in fraturec union xrays can be used quqntify bone union ?

we take a XRay in a conservative treatement u see callus and decide its united
when u do plate osteisynthesis when there is no callus how to decide if there is a gap due to commuintion and whther the pixel densith in a x-ray can be used to decide there is caluus formation or bone formation or not?

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Would someone be able to provide assistance with alizarin red s/mineralization assay with SaOS-2 cells?
7 answers
  • David HughesDavid Hughes
I'm currently trying to induce and identify mineralization in my SaOS-2 cells.
The issue I'm having is that the whole well will stain red, and of course this intensity depends on how long the cells are left in the Alizarin Red S staining solution. I look at these cells over a time course of 0, 3, 6, 9 and 12 days. I grow these cells in DMEM F12 Glutamax media supplemented with 110mg/L sodium pyruvate and 1% penstrep. SaOS-2 cells were seeded at around passage 10 +/- a couple of passages.
My current culture protocol is as follows:
Seed SaOS-2 cells at 50,000 cells/cm2 in a 6-well plate and grow cells in DMEM F12 Glutamax supplemented with 110mg/L sodium pyruvate, 10% FCS and 1% Penstrep.
After the cells have reached confluence and I wash the day 0 in PBS and fix it with 4% PFA for 5 mins and store the cells at 4oC in 3ml PBS. and then add osteogenic media to the remaining wells.
Oestrogenic media = DMEM F12 glutamax supplemented with 110mg/L sodium pyruvate, 10% FCS, 1% penstrep, 50ug/ml ascorbic acid and 5mM Beta-glycerophosphate (I've also trialled 2mM B-GP and 10mM B-GP and did not notice any immediate differences).
I then culture cells for 3, 6, 9 and 12 days in this osteogenic media washing and fixing them at their respective time points.
Alizarin red protocol:
Once all time points have been fixed, I wash the cells twice with DI water and then add 1ml of 2% alizarin red (pH ~4.2) to the well and I've tried this staining for 5 minutes and up to 30 minutes, I then wash the wells 3 times with DI water.
I tend to see an overall staining of the well with small red dots showing in the wells. I am of course looking for nodules as other papers have indicated however I still see a bit of background and the nodules are not as intense nor as large as you'd see in publications (so it would seem). The contrast between the background and the specific dots is not as clear as it is shown in publications.
Upon inspection under EVOS XL Core microscope there are definitely nodules of cells where the stain is gathering in a more intense manner, suggesting the staining is specific, despite the overall culture well looking red.
Many publications have great images with clear nodules and minimal background whereas others have less defined clear staining and I'm unsure which I should use as a guideline.
While culturing the cells I definitely see cloudy areas which indicates to me a collagen matrix has been synthesised, before staining the cells I also seen black-ish looking deposits which I think may be mineral.
I am also detecting collagen 1 and alkaline phosphatase at the RNA and protein level (Col1 protein increasing over the time course) and osteocalcin RNA expression increasing over time suggesting something is definitely going on.
Any information or advice would be appreciated.

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