Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice

Abstract

The presence of skeletal hypomineralization was confirmed in mice lacking the gene for bone alkaline phosphatase, ie, the tissue-non-specific isozyme of alkaline phosphatase (TNAP). In this study, a detailed characterization of the ultrastructural localization, the relative amount and ultrastructural morphology of bone mineral was carried out in tibial growth plates and in subjacent metaphyseal bone of 10-day-old TNAP knockout mice. Alizarin red staining, microcomputerized tomography (micro CT), and FTIR imaging spectroscopy (FT-IRIS) confirmed a significant overall decrease of mineral density in the cartilage and bone matrix of TNAP-deficient mice. Transmission electron microscopy (TEM) showed diminished mineral in growth plate cartilage and in newly formed bone matrix. High resolution TEM indicated that mineral crystals were initiated, as is normal, within matrix vesicles (MVs) of the growth plate and bone of TNAP-deficient mice. However, mineral crystal proliferation and growth was inhibited in the matrix surrounding MVs, as is the case in the hereditary human disease hypophosphatasia. These data suggest that hypomineralization in TNAP-deficient mice results primarily from an inability of initial mineral crystals within MVs to self-nucleate and to proliferate beyond the protective confines of the MV membrane. This failure of the second stage of mineral formation may be caused by an excess of the mineral inhibitor pyrophosphate (PPi) in the extracellular fluid around MVs. In normal circumstances, PPi is hydrolyzed by the TNAP of MVs' outer membrane yielding monophosphate ions (Pi) for incorporation into bone mineral. Thus, with TNAP deficiency a buildup of mineral-inhibiting PPi would be expected at the perimeter of MVs.

Figure 1

Micro-CT images of upper tibias from WT (A) and TNAP−/− (B) mice. Overall, mineral content was reduced in TNAP−/− mice, mineralized cortex was thinner, and epiphyses (Ep) were often not visible. Ep, epiphysis; GrPI, growth plate; Met, metaphysis.

Figure 2

Alizarin red calcium stains for mineral in TNAP WT growth plate (A and B), metaphysis (C) and cortex (D) versus TNAP-deficient growth plate (E and F), metaphysis (G) and cortex (H). TNAP-deficient tibias showed respectively a significant reduction in alizarin-stained mineral in growth plate, metaphysis (where in TNAP deficiency there was an excess of blue-staining osteoid bone matrix), and in cortex. A, magnification × 280; E, magnification × 354; B–D and F–H, magnification × 1100.

Figure 3

FT-IRIS images of TNAP WT versus TNAP-deficient growth plate and metaphysis. The mineral:matrix (A) was calculated from the ratio of the area of phosphate 900-1200 cm⁻¹ absorbance to the area of the protein amide I absorbance from 1590 to 1720 cm^[minus1. The distribution of the crystallinity (B) of the mineral phase was calculated by establishing the ratio of the intensity of the absorbance at 1030 cm⁻¹ to that at 1020 cm⁻¹. Means ± standard deviations are shown for each value.

Figure 4

Transmission electron micrographs of TNAP WT versus TNAP-deficient growth plates (A versus C) at the onset of mineralization. In the upper growth plate, (A versus C), needle-like crystallites of apatitic mineral were present in both WT and TNAP-deficient MVs, although mineral crystallites were more prominent in WT. At the junction between growth plate and metaphysis (B and D), the size and number of extravesicular mineral deposits in cartilage matrix was reduced in TNAP-deficient animals (D) versus WT (B), and often TNAP-deficient mineral appeared fragmented and more granular (D). Small, leaf-like, electron-dense proteoglycan granules are seen in the background cartilage matrix where they are attached to randomly arranged, faintly visible type II collagen fibrils. A and D, magnification × 115,000; B, magnification × 71,000; C, magnification × 150,000.

Figure 5

Normal uncalcified osteoid (Ost) layer (A) versus widened osteoid layer in TNAP-deficient tibial metaphyseal bone (B). A few intact matrix vesicles, containing apatite-like needles (indicated by arrows and shown at higher magnification in inserts, are present in the uncalcified osteoid of both TNAP wild-type and TNAP-deficient tibias. M, mineralized bone matrix; Obl, osteoblast; Ost, osteoid. A and B, magnification × 25,000; A inset, magnification × 61,000; B inset, magnification × 127,000.

Figure 6

Diagram outlining the metabolic sequence that occurs when ATP is hydrolyzed by nucleoside triphosphate pyrophosphohydrolase (NTPPase, also known as PC-1). Following ATP hydrolysis by NTPPase to adenosine monophosphate (AMP) plus inorganic pyrophosphate (PPi), both of these ester phosphates are further hydrolyzed to orthophosphate (PO₄³⁻) by alkaline phosphatase (TNAP). The resulting orthophosphate molecules are incorporated into calcium phosphate mineral. Abnormally high levels of non-hydrolyzed PPi block the formation of calcium phosphate mineral.