Mineral Content and Extracellular Matrix Protein Expression in Mouse Growth Plates During Epiphyseal Fusion: An Observational Study

Abstract

In humans, the growth plate cartilage is completely replaced by bone in late puberty, resulting in epiphyseal fusion. However, in rats and mice, commonly used experimental model systems, the growth plate does not fuse completely even after sexual maturation, making it difficult to elucidate mechanisms involved in epiphyseal fusion. In this study, we investigated age-related changes in the mouse growth plate to better understand the process of epiphyseal fusion. We used scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM/EDS) to examine the distributions and concentrations of minerals in the growth plate. In SEM images, the hypertrophic zone was observed as a bright area and other zones as dark areas at 10 weeks of age (W10). The bright area was further expanded at W55 than at W10. EDS analysis showed that P and Ca concentrations were high in this area, while C and O concentrations were low, indicating that the growth plate had calcified during aging. Alcian blue histochemistry revealed that the glycosaminoglycans of aggrecan were distributed in the growth plate at both W10 and W55. Immunohistochemistry showed that aggrecan and type II collagen were expressed throughout the growth plate at W10, but sparsely at W55. Type I collagen was expressed weak at both W10 and W55. Type X collagen and MMP-13 expression were observed in the hypertrophic zone at W10 but not at W55. This study demonstrated that although the mouse growth plate calcifies with age, it remains calcified cartilage for an extended period without being replaced by bone.

Keywords: Aging; Calcified cartilage; Epiphyseal fusion; Extracellular matrix; Growth plate; Scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM/EDS).

Fig. 1

Three-dimensional micro-CT images of the knee and the proximal tibia. A The light blue line indicates the position of the coronal plane shown in B. B Coronal plane at W10. C The light blue line indicates the position of the coronal plane shown in D. D Sagittal plane at W55. Incomplete epiphyseal fusion is observed and several growth plate bridges are indicated by arrows. (A, anterior side; P, posterior side; R, right side; L, left side)

Fig. 2

BSE images of the proximal end of the tibia as visualized by SEM. A Proximal tibial growth plate at W10. B Enlargement of the boxed area shown in A. This shows dark areas of the resting zone and proliferative zone as well as a bright area of the hypertrophic zone within the growth plate at W10. C Proximal tibial growth plate at W55. D Enlargement of the boxed area shown in C. Some islands within the dark area (*) can be observed within the bright area

Fig. 3

Elemental maps of the proximal tibial growth plate obtained via EDS. A Elemental maps of C (blue), P (red), and Ca (green) at W10 and W55. B BSE images and overlaid images of C, P, and Ca. Co-localized dots of P and Ca are shown as yellow in the overlay image. Scattered spots of mineral deposits are indicated by arrowheads

Fig. 4

EDS line scans for C (blue) and O (yellow)—two main components of organic matter such as proteins, and P (red) and Ca (green), which are the main minerals in calcified tissues, along the light blue line on the BSE image. A Growth plate at W10. B Growth plate including the dark area at W55. C Growth plate excluding the dark area at W55. The concentrations of C, P, and Ca in the proximal tibial growth plate were measured via EDS point analysis. D The atomic percentage of C. E The atomic percentage of P. F The atomic percentage of Ca. (n = 6; Kruskal–Wallis tests with Dunn’s post-hoc tests; *P < 0.05)

Fig. 5

Sections of the proximal tibial growth plate stained with H&E or Alcian blue. A Growth plate stained with H&E at W10. B Growth plate stained with H&E at W55. C Growth plate stained with Alcian blue at W10. D Growth plate stained with Alcian blue at W55. GP growth plate

Fig. 6

Sections of the proximal tibial growth plate following immunohistochemistry experiments. A Immunoreactivity of aggrecan at W10. B Immunoreactivity of aggrecan at W55. C Immunoreactivity of type II collagen at W10. D Immunoreactivity of type II collagen at W55. E Immunoreactivity of type I collagen at W10. F Immunoreactivity of type I collagen at W55. (*, bone)

Fig. 7

Sections of the proximal tibial growth plate following immunohistochemistry experiments. A Immunoreactivity of MMP-13 at W10. Hypertrophic chondrocytes expressing MMP-13 are indicated by arrowheads. B Immunoreactivity of MMP-13 at W55. C Immunoreactivity of type X collagen at W10. D Immunoreactivity of type X collagen at W55