Fig 1.
Methods used to evaluate the two types of xenogenic bone particles compared in the present work.
The different methods and procedures performed in this work are summarized in a flow chart.
Fig 2.
Morphological characterization of both types of bone particles.
CP (collagen-based particles) and DP (deproteinized particles) were analyzed using a stereomicroscope (A, B, F, G), a scanning electron microscope (E, J), and a micro-CT scanner (C, D, H, I). Video images corresponding to panels D and I are available as S1 and S2 Files, respectively. Scale bar: 200µm.
Fig 3.
Morphological analysis of human cells cultured with CP and DP using phase-contrast microscopy.
Illustrative images are shown of human cells cultured for 24, 48 and 72h in direct contact (DC) or indirect contact (IC) with collagen-based particles (CP) or deproteinized particles (DP). Scale bars: 100 µm.
Fig 4.
Analysis of cell viability using Live & Dead assay (LD) and free DNA quantification (DNA).
The top panel corresponds to histograms representing the quantitative values obtained for each analysis method in each study group and controls, after 24, 48 and 72h of follow-up, shown as percentages of cell viability normalized to controls. The lower panel shows illustrative images of cells analyzed with LD. Live cells appear in green, whereas dead cells are stained in red. DC: direct contact; IC: indirect contact; CP: collagen-based particles; DP: deproteinized particles; CTR + : positive control of cells cultured without bone particles (live cells); CTR-: negative control of cells treated with 1% triton X-100 (dead cells). Statistical differences with the CTR+ group are labeled with asterisks in the histograms (*). Scale bars: 100 µm.
Fig 5.
CT scanning analysis of the craniofacial bones of animals included in the in vivo study.
A: CT images obtained in different planes (front view, left side, axial plane, and parasagittal plane). B: Size of the mandibular bone defect normalized to the negative control group (NEG), considered as 100% of the defect size. Results are shown as means and standard deviations, and significant differences with the negative control group are highlighted with asterisks (*). Native: positive control of non-operated animals; NEG: negative control group of animals subjected to bone defect devoid of any bone particles; CP: animals subjected to bone defect filled with collagen-based particles; DP: animals subjected to bone defect filled with deproteinized particles. The bone defect is labeled with white arrows in panel A.
Fig 6.
Histological analysis of animals grafted with the different types of bone particles and controls using hematoxylin and eosin staining (H&E).
Images obtained from each study group are shown at different magnifications, with images at higher magnifications corresponding to the square inserts in the images above. Native: positive control of non-operated animals; NEG: negative control group of animals subjected to bone defect devoid of any bone particles; CP: animals subjected to bone defect filled with collagen-based particles; DP: animals subjected to bone defect filled with deproteinized particles. White arrows point to illustrative areas of native mandibular bone; Yellow arrows label the dilated area of bone at the edges of the defect; Black arrows point to the critical bone defect; Giant cells are labeled with white arrowheads; M: mineralized structures. Scale bar: 200 µm for the top panel and 50 µm for the medium and lower panels.
Fig 7.
Histological analysis of animals grafted with different types of bone particles and controls using picrosirius red (PSR) and alcian blue (AB) histochemistry and osteocalcin (OCC) immunohistochemistry.
Native: positive control of non-operated animals; NEG: negative control group of animals subjected to bone defect devoid of any bone particles; CP: animals subjected to bone defect filled with collagen-based particles; DP: animals subjected to bone defect filled with deproteinized particles. M: Mineralized structures. Scale bar: 200 µm.
Fig 8.
Histological analysis of bone regeneration associated to the grafted particles in the CP and DP groups of animals.
Images correspond to osteocalcin immunohistochemistry analysis of defect areas close to the limits of the critical bone defect, after 2 months of follow-up. Grafted particles are highlighted with “M”, and asterisks (*) correspond to areas of native or regenerated bone tissue. Some cells showing positive osteocalcin staining signal that could correspond to differentiating osteoblasts are highlighted with blue arrows.