Figure 1.
Experimental design and scaffold morphology.
(A) Schematic of Experimental Design. Osteoprogenitors from the bone marrow were expanded in vitro before seeding to collagen-hydroxyapatite scaffolds and implanted in critical size calvarial defects. To label areas of active mineralization, calcein was injected intraperitoneally one-day prior to euthanization at three weeks post-implantation. (B) Electron micrograph of the Col-HA scaffold showing cellular morphology. Scale bar is 500 µm. (C) Enlarged inset from (A). Scale bar is 100 µm. (D) Electron micrograph of Healos scaffold. Scale bar is 500 µm. (E) Enlarged inset from (D). Scale bar is 100 µm.
Figure 2.
Osteoprogenitor cells following six days of in vitro expansion.
(A) Phase contrast showing near confluent cell culture, (B) TdTomato reporter driven by OsterixCRE and (C) Merged image of (A) and (B) indicating a large fraction of cultured cells are osteoprogenitors.
Figure 3.
Radiographs of cell-scaffold constructs after 3 weeks in vivo.
(A)–(M) H and C denote Healos and Col-HA scaffolds, respectively. Scale bars are 1 mm. (N) Quantitation of radiographs. Light red bars indicate 95% confidence intervals and blue bars indicate one standard deviation.
Figure 4.
Histological sections of defects following 3 weeks of in vivo implantation.
(Left column) Defect wide view of scaffolds and mineralized tissue (darkfield channel), +TdTomato donor cells (red), and the mineralization label (green, calcein). Scale bars are 500 µm. (Right column) Magnifications of left column. Blue arrows indicate +TdTomato donor cells embedded in bone. White arrows indicate +TdTomato donor cells overlying mineral label. Scale bars are 100 µm.
Figure 5.
Quantitative histomorphometry of darkfield, donor cell, and calcein channels.
(A) Quantification of the darkfield area fraction in the defect area. (B) Quantification of donor cell area fraction using TdTomato signal. (C) Quantification of mineralizing surface using the calcein mineralization label. Light red bars indicate 95% confidence intervals and blue bars indicate one standard deviation.
Figure 6.
Donor cell distribution in the scaffolds.
(A) Comparison of donor cell distribution from the outside edge of the implant for Healos and Col-HA. Error bars indicate one standard deviation. Plot above shows a comparison of the means for each sample according to scaffold type. (B) Comparison of the mean donor cell distance from the outside edge of the implant. Light red bars indicate 95% confidence intervals and blue bars indicate one standard deviation.