Fig. 1: Mineralized spherule propagation and its correlation with phosvitin at the avian tendon mineralization front.

a Scheme showing the preparation of turkey gastrocnemius tendons. b–d TEM images of longitudinal sections (i) and transverse sections (ii) of nonmineralized (b), partially mineralized (c) and highly mineralized (d) regions of avian tendons. Mineralized spherules are highlighted with red circles, with a typical spheroidal shape in the longitudinal section (c-i) and a circular profile in the transverse section (c–ii). In the highly mineralized region, heavily mineralized collagen could be identified (d). Scale bars: 300 nm. e, f Bright field and CLSM images of partially mineralized tendons (e) and nonmineralized tendons (f). In the partially mineralized tendon, Phov was deposited at the mineralization front in the longitudinal section (e–i) and transverse section (e–ii). Phov was virtually absent in the nonmineralized tendon both longitudinally (f–i) and transversely (f–ii). Blue, DAPI for nuclei; red, alizarin red S (ARS) for minerals; cyan, type I collagen; green, Phov; BF, bright field. Scale bars: 50 μm. g Semiquantitative analysis of calcium and Phov fluorescence intensities in longitudinal sections of partially mineralized and nonmineralized tendons. Data are presented as mean ± SD, n = 6 biologically independent samples, two-tailed unpaired t test (the P-values are shown). Differences with P-values < 0.05 are considered significant. h, i Immunogold-EM images showing Phov localization in the transverse sections of partially mineralized (h) and nonmineralized (i) tendons. Gold-labelled Phov (highlighted by red rectangles) were frequently found around and within the mineralized spherule. The yellow arrow indicates the intrafibrillar mineralization of collagen fibrils within the mineralized spherule (h). Gold particles and minerals were absent in the nonmineralized tendon (i). Scale bars: 200 nm. Experiments were repeated independently (h, i) three times with similar results.