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Organisationseinheit der BAM
Osteoclastic bioresorption of biomaterials: Two- and three-dimensional imaging and quantification
(2010)
Purpose: Bioresorbable materials have been developed in the hope that the body will replace them with newly formed tissue. The first step of this remodeling process in bone is the bioresorption of the material by osteoclasts. The aim of this study was to analyze osteoclastic resorption of biomaterials in vitro using the commonly used two-dimensional methods of light-microscopy (LM) and scanning electron microscopy (SEM) in comparison with infinite focus microscopy (IFM), a recently developed imaging method allowing for three-dimensional surface analysis. Methods: Human hematopoietic stem cells were cultivated in the presence of the cytokines M-CSF and RANK-L for 4 weeks directly on dentin and a calcium phosphate cement. Osteoclast development was surveyed with standard techniques. After removal of the cells, resorption was characterized and quantified by LM, SEM and IFM. Results: Osteoclast cultures on the biomaterials presented the typical osteoclast-specific markers. On dentin samples LM, SEM as well as IFM allowed for discrimination of resorption. Quantification of the resorbed area showed a linear correlation between the results (LM vs. SEM: r=0.996, p=0.004; SEM vs. IFM: r=0.989, p=0.011; IFM vs. LM: r=0.995). It was not possible to demarcate resorption pits on GB14 using LM or SEM. With IFM, resorption on GB14 could be visualized and quantified two- and three-dimensionally.
Commonly, to determine osteoclastic resorption of biomaterials only the resorbed area is measured. The depth of the resorption pit, however, may also be important for the performance of a material. To generate such data we used two calcium phosphate ceramics (Ca10 and Ca2). The solubility of the materials was determined according to DIN EN ISO 10993-14. They were scanned three-dimensionally using infinite focus microscopy and subsequently cultivated for 4 weeks in simulated body fluid without (control) or with human osteoclasts. After this cultivation period osteoclasts number was determined and surface changes were evaluated two- and three-dimensionally. Ca10 and Ca2 showed solubilities of 11.0 ± 0.5 and 23.0 ± 2.2 mg g-1, respectively. Both materials induced a significant increase in osteoclast number. While Ca10 did not show osteoclastic resorption, Ca2 showed an increased pit area and pit volume due to osteoclastic action. This was caused by an increased average pit depth and an increased number of pits, while the average area of single pits did not change significantly. The deduced volumetric osteoclastic resorption rate (vORR) of Ca2 (0.010.02 µm3 µm-2 day-1) was lower than the remodelling speed observed in vivo (0.08 µm3 µm-2 day-1), which is in line with the observation that implanted resorbable materials remain in the body longer than originally expected. Determination of volumetric indices of osteoclastic resorption might be valuable in obtaining additional information about cellular resorption of bone substitute materials. This may help facilitate the development of novel materials for bone substitution.