Histological analysis of regenerated bone after implantation. (A) Hematoxylin and eosin (HE) and Azan Mallory staining of calvarial tissue sections was conducted 4 weeks after implantation. Newly regenerated bone in the blank, Matrigel™ alone, monolayer rat mesenchymal stem cells (MSCs)/Matrigel™, and MSC spheroids/Matrigel™ groups were evaluated histologically. The left and middle columns showed HE staining at low and high magnification, respectively. Solid lines indicate the edges of the host bone. Dotted lines indicate the defect filled with the connective tissue. In MSC spheroids/Matrigel™ group, the defect were filled with newly regenerated bone. In the right column showing Azan Mallory staining, the defects of the blank, Matrigel™ alone, monolayer rat mesenchymal stem cells (MSCs)/Matrigel™ groups were filled with mature collagenous fibers. Scale bar in the left column = 1 mm. Scale bars in the middle and right columns = 100 μm. (B) Percentages of newly regenerated bone in relation to the size of the original defect evaluated histomorphometricly 4 weeks after implantation. Replacement of newly bone in MSC spheroids/ Matrigel™ group was greater than that in other groups. *, significantly different at p < 0.05 compared with other groups. There are no significant difference in groups of gray columns jointed to horizontal bar.