Human macrophages and osteoclasts resorb ß-tricalcium phosphate in vitro but not mouse macrophages.

ß-TCP is a resorbable bony biomaterial but its biodegradation mechanisms in vivo remains unclear. Osteoclast can resorb ß-TCP but a role for macrophages has also been suggested by in vivo studies. However no in vitro study has clearly evidenced the action of macrophages in the resorption process. We prepared flat ß-TCP tablets with a smooth surface to investigate the in vitro capability of murine (RAW 264.7) and human macrophage cells (PBMCs) to resorb the biomaterial. In parallel, these cells were differentiated into multinucleated osteoclasts with M-CSF and RANK-L. The action of these cells was evaluated by scanning electron microscopy and Raman microspectroscopy after a 21 day culture on the tablets. Human macrophages and osteoclasts derived from PBMCs appeared able to resorb ß-TCP by forming resorption pits at the surface of the flat tablets. RAW macrophages were unable to resorb ß-TCP but they exhibited this possibility when they have been differentiated into osteoclasts. These cells can engulf ß-TCP grains in their cytoplasm as evidenced by light and TEM microscopy with production of carbonic anhydrase (revealed by the immunogold technique in TEM). The resorbed areas were characterized by severe degradation of the grains showing speckled and stick-like aspects indicating a chemical corrosion. The effect was maximal at the grain boundaries which have a slightly different chemical composition. Changes in the Raman spectrum were observed between the resorbed and un-resorbed ß-TCP suggesting crystal modifications. In contrast, un-differentiated murine macrophages were not able to chemically attack ß-TCP and no resorption pit was observed. RAW cell is not a representative model of the macrophage-biomaterial interactions that occur in human. This in vitro study evidences that both human osteoclasts and macrophages represent active cell populations capable to resorb ß-TCP.

Biomaterial granules used for filling bone defects constitute 3D scaffolds: porosity, microarchitecture and molecular composition analyzed by microCT and Raman microspectroscopy.

Biomaterials are used in the granular form to fill small bone defects. Granules can be prepared with a grinder from trabecular bone samples or provided as synthetic biomaterials by industry. Granules occupy the 3D-space and create a macroporosity allowing invasion of vascular and bone cells when the inter-granular pores are larger than 300 µm. We compared the 3D-porosity of granule stacks obtained or prepared with nine biomaterials Osteopure® , Lubboc® , Bio-Oss® , CopiOs® , TCP Dental® , TCP Dental HP® , KeraOs® , and TCH® in comparison with that of human trabecular bone. For each biomaterial, two sizes of granules were analyzed: 250-1000 and 1000-2000 µm. Microcomputed tomography determined porosity and microarchitectural characteristics of granular stacks and Raman microspectroscopy was used to analyze their composition. Stacks of 250-1000 µm granules had a much lower porosity than 1000-2000 µm granules and the maximum frequency of pores was always centered at 200-250 µm. One biomaterial contained substantial amount of cortical bone (Bio-Oss® ). The highest porosity and pore size was obtained with TCP Dental HP. Raman spectroscopy found differences in biomaterials of the same composition. Stacks of granules represent 3D scaffolds resembling trabecular bone with an interconnected porous microarchitecture. Small granules have created pores <300 µm in diameter; this can interfere with vascular colonization. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 415-423, 2019.

Maxillary Sinus Lift with Beta-Tricalcium Phosphate (ß-TCP) in Edentulous Patients: A Nanotomographic and Raman Study.

Sinus lift elevation restores bone mass at the maxilla in edentulate patients before the placement of dental implants. It consists of opening the lateral side of the sinus and grafting beta-tricalcium phosphate granules (ß-TCP) under the olfactory membrane. Bone biopsies were obtained in five patients after 60 weeks. They were embedded undecalcified in poly(methyl methacrylate) (pMMA); blocks were analyzed by nanocomputed tomography (nanoCT); specific areas were studied by Raman microspectroscopy. Remnants of ß-TCP were osseointegrated and covered with mineralized bone; osteoid tissue was also filling the inner porosity. Macrophages having engulfed numerous ß-TCP grains were observed in marrow spaces. ß-TCP was identified by nanoCT as osseointegrated particles and as granules in the cytoplasm of macrophages. Raman microspectroscopy permitted to compare the spectra of ß-TCP and bone in different areas. The ratio of the ~820 cm-1 band of pMMA (-CH2 groups) on the ν1 phosphate band at 960 cm-1 reflected tissue hydration because water was substituted by MMA during histological processing. In bone, the ratio of the ~960 cm-1 phosphate to the amide 1 band and the ratio ν2 phosphate band by the 1240-1250 amide III band reflect the mineralization degree. Specific bands of ß-TCP were found in osseointegrated ß-TCP granules and in the grains phagocytized by the macrophages. The hydration degree was maximal for ß-TCP phagocytized by macrophages. Raman microspectroscopy associated with nanoCT is a powerful tool in the analysis of the biomaterial degradation and osseointegration.