Osteocyte and osteoblast apoptosis and excessive bone deposition accompany failure of collagenase cleavage of collagen - PubMed (original) (raw)

Osteocyte and osteoblast apoptosis and excessive bone deposition accompany failure of collagenase cleavage of collagen

W Zhao et al. J Clin Invest. 2000 Oct.

Abstract

Mice carrying a targeted mutation (r) in Col1a1, encoding a collagenase-resistant form of type I collagen, have altered skeletal remodeling. In hematoxylin and eosin-stained paraffin sections, we detect empty lacunae in osteocytes in calvariae from Col1a1(r/r) mice at age 2 weeks, increasing through age 10-12 months. Empty lacunae appear to result from osteocyte apoptosis, since staining of osteocytes/periosteal osteoblasts with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling is increased in Col1a1(r/r) relative to wild-type bones. Osteocyte perilacunar matrices stained with Ab that recognizes collagenase collagen alpha1(I) chain cleavage ends in wild-type but not Col1a1(r/r) calvariae. Increased calvarial periosteal and tibial/femoral endosteal bone deposition was found in Col1a1(r/r) mice from ages 3-12 months. Calcein labeling of calvarial surfaces was increased in Col1a1(r/r) relative to wild-type mice. Daily injections of synthetic parathyroid hormone for 30 days increased calcein-surface labeling in wild-type but caused no further increase in the already high calcein staining of Col1a1(r/r) bones. Thus, failure of collagenase cleavage of type I collagen in Col1a1(r/r) mice is associated with osteocyte/osteoblast death but increases bone deposition in a manner that mimics the parathyroid hormone-induced bone surface activation seen in wild-type mice.

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Figures

Figure 1

Figure 1

Presence of empty osteocyte lacunae in calvariae from wild-type and Col1a1r/r mice. (a) Sections of calvariae from 4-week-old wild-type and Col1a1r/r mice stained with H&E. Note some of the empty osteocyte lacunae (arrows) in the sections from the Col1a1r/r mice; all osteocyte lacunae are filled with cells in the section shown from the wild-type mice. (b) Quantification of empty osteocyte lacunae in calvariae from 4- to 6-week-old wild-type and Col1a1r/r mice. The number of empty lacunae per mm2 standard area is shown in the left graph and the percentage of empty lacunae per total lacunae is shown in the right graph.

Figure 2

Figure 2

Fluorescence photomicrographs of sections of calvariae from 4-week-old wild-type and Col1a1r/r mice stained by the TUNEL method. (a) Presence of TUNEL-positive cells in calvariae from wild-type and Col1a1r/r mice. Open bars indicate outer (top) and inner (bottom) surfaces of the periosteum. (b) Quantification of TUNEL_-_positive cells in calvariae from wild-type and Col1a1r/r mice.

Figure 3

Figure 3

Photomicrographs of calvarial sections from 4-week-old wild-type and Col1a1r/r mice stained with 9A4 mAb to the collagenase cleavage epitope. Two separate representative areas of sections from different mice are shown. No counterstain was used.

Figure 4

Figure 4

The thickness of calvariae in Col1a1r/r mice increases with increasing age. (a) Histological sections of calvariae from wild-type and Col1a1r/r mice at 4 weeks and 12 months of age, cut perpendicularly to the sagittal suture and stained with H&E. The outer periosteal surfaces of each section are at the top and the inner periosteal surfaces are at the bottom. The thickness of the calvariae was similar in 4-week-old wild-type and Col1a1r/r mice, but the thickness in 12-month-old Col1a1r/r mice was markedly increased, compared with wild-type mice. (b) Quantification of calvarial bone area in ten wild-type and ten Col1a1r/r mice (4 weeks old) and five wild-type and seven Col1a1r/r mice (10 months old).

Figure 5

Figure 5

Calcein labeling of bone surfaces from 3-month-old wild-type and Col1a1r/r mice. (a) Frozen sections of calvariae, examined by fluorescence microscopy, from mice given vehicle alone or PTH daily and injected with single doses of calcein at 1 and 11 days before sacrifice (see Methods). The outer periosteal surface in the sample from the vehicle-treated wild-type mouse is indicated by the solid open bar and the inner surface by the dashed open bar. In this section of calvariae from the untreated wild-type mice, the fluorescent calcein label was limited to inside the bone marrow cavity; only scattered label was seen occasionally at the outer periosteal surface. In the Col1a1r/r mice, marked and extensive labeling was evident at both periosteal surfaces. Note that the increased calvarial thickness in the 3-month-old Col1a1r/r mice. In the PTH-treated wild-type mice, there was marked calcein labeling at both surfaces. The calcein labeling, already extensive in the untreated Col1a1r/r mice, was not further increased by PTH. (b) Quantification of calcein labeling of active calvarial surfaces. (c) Quantification of bone apposition rate on active calvarial surfaces.

Figure 6

Figure 6

Presence of excessive endosteal bone in femurs from Col1a1r/r mice. (a) Longitudinal histological sections stained with H&E of femurs from 10-month-old wild-type and Col1a1r/r mice. The location of the periosteal surface (black and white arrows) and endosteal surface (black arrows) is indicated. In wild-type mice the endosteal surface was smooth, and there was no evident endosteal new bone. In Col1a1r/r mice, in contrast, abundant endocortical new bone covered the endosteal surface. (b) Sections of femurs, embedded in methyl methacrylate and examined by fluorescence microscopy, from 10-month-old wild-type and Col1a1r/r mice injected twice with calcein, 30 days apart. Bo, bone; Ma, marrow area. The periosteal surface is indicated by the solid open bar and the endosteal surface by the dashed open bar. Note minimal calcein label at the endosteal surface of the femur from the wild-type mouse in contrast to the striking labeling of the new endosteal bone (a) in the femur from the Col1a1r/r mouse.

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