Introduction
It has long been recognized that systemic acidosis causes depletion of the skeleton – an effect initially assumed to be the result of physicochemical dissolution processes. However more recent 2D in vitro studies revealed that protons directly activate osteoclasts to resorb mineralized tissue. The existence of this stimulatory effect has never been validated using human cells combined with a 3D culturing approach, believed to more closely mimic the in vivo situation.
Materials and Methods
A protocol was designed to create 3D scaffolds from trabecular bone samples, which were then used to study the effect of acidosis in 3D. Different protocols were checked on the following criteria: 1) extracellular matrix (ECM) preservation, 2) sterility and 3) cell removal, and were tested for their capabilities to generate scaffolds that allowed 4) monocyte attachment, 5) infiltration, 6) osteoclastic differentiation and 7) resorption. Microcomputed tomography (μCT) was used to assess ECM preservation and resorption, scaffold culturing was performed to evaluate sterility, a MTT and DNA assay were used to provide information on human monocyte attachment and infiltration, histological analyses gave insights into cell removal efficiency and osteoclastic differentiation, and quantitative real time polymerase chain reactions (qPCR) were performed to additionally evaluate osteoclastic differentiation and resorption activity. Lastly, μCT was used to investigate the influence of acidosis on osteoclastic bone resorption in 3D.
Results
Following a stepwise elimination procedure, the final protocol was shown to preserve the ECM, ensure sterility, efficiently remove host cells and create scaffolds that enabled attachment and infiltration of human monocytes. The ability of the 3D scaffolds to allow differentiation of monocytes towards bone resorbing osteoclasts could not be proven. Also, the resorption-promoting effect of low pH could not be confirmed in 3D.
Discussion and Conclusion
The inabilities to demonstrate differentiation, resorption and the resorption-stimulatory effect of acidosis are anticipated to be (partly) attributable to analysis technique being lacking. Multiple promising adjustments and additional analysis methods were suggested and are expected to be helpful in overcoming presently-encountered issues concerning (the detection of) differentiation and resorption. Confirmation of the promoting effect of acidosis on osteoclast activity in 3D would provide further rationale to base drug development for acidosis-related bone loss disorders on shifting the acid-base balance towards the alkaline direction.