Quantification of osteoclastic resorption of 3D biomaterials: towards better in vitro models for biomaterial degradation in vivo

摘要

Introduction: Resorbable bone graft materials have become an important alternative for autologous bone tissue for the regeneration of bone defects. The balance between new bone formation and resorption of the materials is crucial for successful remodeling. Cell-based resorption assays of biomaterials are performed using osteoclasts and osteoclast-like cell lines however the quantification of resorption pits requires planar substrates with relatively smooth surfaces (roughness below 1 μm). Therefore quantification of resorption activity through biochemical markers is desirable to evaluate the osteoclast-mediated degradation of 3D biomaterials. The focus of the present study was to explore possible correlations between the activity of osteoclast-specific enzymes tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase Ⅱ (CAⅡ) and cathepsin K (CSK) and osteoclast formation and activity on different 2D and 3D biomaterials. Materials and Methods: Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of more than 10 donors and were cultivated under stimulation with M-CSF and RANKL on cell culture polystyrene, membranes of mineralized collagen, calcium phosphate bone cements, collagen gels, bone chips and dentin. Osteoclast formation was studied microscopically (confocal laser scanning microscopy) and resorption pits, if appropriate, were analyzed using SEM. Comprehensive data sets were obtained for the activity of osteoclast-specific enzymes TRAP, CAⅡ and CSK at different time points of cultivation on the different materials. Results: The formation of multinucleated osteodasts was observed on all examined materials. However, number and size of osteodasts differed considerably on the materials, and there were also substantial differences between cells of different donors. TRAP activity did not always correlate with the osteoclast number and resorption area since also non-multinudeated cell species can be TRAP positive. Enzyme activity of CAⅡ and CSK showed a better correlation to osteoclast number and resorption area. Discussion: The quantification of osteodastic enzyme activities is commonly restricted to measurement of TRAP activity, while other osteodastic markers are only detected on gene expression level. This is the first study involving quantitative measurement of three different osteodastic enzyme activities of osteodasts on biomaterials. Our results indicate that measurement of TRAP activity is not sufficient to assess resorptive activity of osteodasts. CA Ⅱ activity gives better correlation to osteoclast formation than TRAP activity, however this assay might be disturbed by non-spedfic enzyme activities. CSK activity turned out to be a reliable marker for biomaterial resorption. More investigations involving other 3D biomaterial spedes like granules and foams are needed to consistently correlate CSK activity to osteodastic resorption. Conclusion: Quantitative measurement of osteoclast-specific enzyme activities, especially CAⅡ and CSK is an alternative method to assess osteodastic resorption of biomaterials. Enzyme activity quantification is advantageous in comparison to microscopic analyses since it is less time-consuming and also irregularly shaped materials can be examined.