Fig. 1: Divalent cations released from alginate induce new bone formation.

a Reconstructed micro-computed tomography (µCT) images (scale bars = 1 mm) showing the new bone formation in mouse femurs grafted with Mg²⁺-, Cu²⁺-, or Zn²⁺-releasing alginate. Pure alginate grafted mice serve as control. b Corresponding measurements of bone volume fraction (BV/TV), bone mineral density (BMD of TV), and cortical bone area (Ct.Ar). c Representative images of hematoxylin and eosin (H&E) staining showing periosteal new bone formation (n = 3). Lower images (scale bars = 100 µm) are high-resolution versions of the boxed regions in the upper images (scale bars = 500 µm). P, periosteum; NB, new bone; OB, old bone; M, marrow. d Representative immunofluorescent images showing the presence of osteocalcin⁺ (OCN⁺) osteoblasts on the cortical bone surface. Lower images (scale bars = 50 µm) are high-resolution versions of the boxed regions in the upper images (scale bars = 200 µm). P, periosteum; Ct, cortical bone. e Representative images of tartrate-resistant acid phosphatase (TRAP) staining showing the presence of TRAP⁺ osteoclasts on the cortical bone surface. Lower images (scale bars = 100 µm) are high-resolution versions of the boxed regions in the upper images (scale bars = 400 µm). f Histomorphological analysis of osteoblast (N.Ob/Ct.B.Pm) and osteoclast (N.Oc/Ct.B.Pm) numbers on the cortical bone surface. Representative images of calcein/xylenol labeling showing periosteal new bone formation (g, scale bars = 100 μm), quantitative analysis of fluorescence intensity of calcein and xylenol (h), as well as corresponding measurement of bone formation rate (i). Data are mean ± standard deviation (s.d.) *P < 0.05, **P < 0.01 by 1-way analysis of variance (ANOVA) with Tukey’s post hoc test. Source data are provided as a source data file.