Micro-CT, bone histomorphometry, and real-time RT-PCR analyses after unloading at 4 months of age.

<p>(A, B) Micro-CT analysis. Tail suspension was performed for 2 weeks using male wild-type mice [control group, 13 mice; unloaded group, 9 mice] and <i>BCL2</i> transgenic mice [control group, 8 mice; unloaded group, 8 mice] at 4 months of age. A, Micro-CT images of femurs. Scale bars = 0.5 mm. B, Trabecular bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were evaluated by micro-CT. (C) Bone histomorphometrical analysis of trabecular bone. The trabecular bone volume (BV/TV), osteoid thickness (O.Th), number of osteoblasts (N.Ob/B.Pm), number of osteoclasts (N.Oc/B.Pm), eroded surface (ES/BS), mineral apposition rate (MAR), double-labeled surface (dLS/BS), and bone formation rate (BFR/BS) were measured on distal femoral metaphysis in wild-type mice [control group, 8 mice; unloaded group, 11 mice] and <i>BCL2</i> transgenic mice [control group, 8 mice; unloaded group, 6 mice] at 4 months of age. (D) <i>Ctsk</i> expression. Tail suspension was performed for 3 days and <i>Ctsk</i> expression was examined by real-time RT-PCR analysis using osteoblast-enriched samples from wild-type mice [control group, 9 mice; unloaded group, 11 mice] and <i>BCL2</i> transgenic mice [control group, 6 mice; unloaded group, 5 mice] at 4 months of age. The values of the control groups were defined as 1, and relative levels are shown. In B–D, data are presented as the mean ± S.D. *vs. control. *, ♯ P<0.05; **, ♯♯ P<0.01.</p

Expression of bone matrix protein genes in Bcl2^−/− mice.

<p>(A) Real-time RT-PCR analysis of <i>Bcl2</i>, <i>Runx2</i>, <i>Osterix</i>, <i>Col1a1</i>, <i>osteopontin</i>, and <i>osteocalcin</i>. RNA was directly extracted from newborn calvariae of wild-type (wt) and Bcl2^−/− mice. The values of wild-type mice were defined as 1, and relative levels are shown. wild-type mice, n = 6; Bcl2^−/− mice, n = 15. *vs. wild-type mice. *P<0.05, **P<0.01, ***P<0.001. (B–U) In situ hybridization analysis of <i>Col1a1</i>, <i>osteopontin</i>, and <i>osteocalcin</i>. The sections of femurs from Bcl2^+/− mice (B, D, F, H, J), Bcl2^−/− mice (C, E, G, I, K, M, O, Q, S, U), and wild-type mice (L, N, P, R, T) at birth (B–K) and at 2 weeks of age (L–U) were stained with H–E (B, C, L, M) or hybridized with <i>Col1a1</i> (D, E, N, O), <i>osteopontin</i> (F, G, P, Q), and <i>osteocalcin</i> (H–K, R–U) probes. Boxed regions in H, I, R, and S are magnified in J, K, T, and U, respectively. Arrows in I indicate the appearance of <i>osteocalcin</i>-expressing cells in the bone collar. Similar results were obtained in two newborn mice and three 2-week-old mice in each genotype and representative data are shown. In situ hybridization using the sense probes showed no significant signals (data not shown). Bars: 100 µm (B–I, L–S); 50 µm (J, K, T, U).</p

Bone Regeneration Using Dentin Matrix Depends on the Degree of Demineralization and Particle Size

<div><p>Objectives</p><p>This study aimed to examine the influence of particle size and extent of demineralization of dentin matrix on bone regeneration.</p><p>Materials and Methods</p><p>Extracted human teeth were pulverized and divided into 3 groups according to particle size; 200, 500, and 1000 μm. Each group was divided into 3 groups depending on the extent of demineralization; undemineralized dentin (UDD), partially demineralized dentin matrix (PDDM), and completely demineralized dentin matrix (CDDM). The dentin sample was implanted into rat calvarial bone defects. After 4 and 8 weeks, the bone regeneration was evaluated with micro-CT images, histomorphometric and immunohistochemical analyses. Osteoblasts were cultured on UDD and DDM to evaluate the cell attachment using electron microscope.</p><p>Results</p><p>Micro-CT images and histological observation revealed that CDDM had largely resorbed but UDD had not, and both of them induced little bone formation, whereas all particle sizes of PDDM induced more new bone, especially the 1000 μm. Electron microscopic observation showed osteoblasts attached to DDM but not to UDD.</p><p>Conclusions</p><p>PDDM with larger particle size induced prominent bone regeneration, probably because PDDM possessed a suitable surface for cell attachment. There might be an exquisite balance between its resorption and bone formation on it. PDDM could be considered as a potential bone substitute.</p></div

Immunohistochemical analysis of Sost after unloading.

<p>Immunohistochemistry using anti-Sost antibody in tibial sections of control (A, B, E, F) and unloaded (C, D, G–J) groups in wild-type (A, C, E, G, I) and <i>BCL2</i> transgenic (B, D, F, H, J) mice at 4 months of age. The boxed regions with asterisks in A–D are magnified in E–G, respectively. The boxed regions in G and H are magnified in I and J, respectively. In F and H, closed arrows indicate Sost-positive osteocytes and open arrows indicate Sost-negative osteocytes. The lacunae with cellular debris in <i>BCL2</i> transgenic mice were non-specifically stained with Sost antibody (F, H). The sections were counterstained with methylgreen. Note that Sost is distributed through canaliculi throughout bone in wild-type mice but not in <i>BCL2</i> transgenic mice (I, J). Scale bars = 0.5 mm (A–D); 50 µm (E–H); 10 µm (I, J). (K–N) Frequency of Sost-positive cells in cortical bone. Sost-positive cells were counted in the anterior (K, M) and posterior (L, N) sides of cortical bone at the metaphysis (K, L) and mid-diaphysis (M, N) of tibiae. Tail suspension was performed for 14 days using male wild-type mice [control group, 7 mice; unloaded group, 9 mice] and <i>BCL2</i> transgenic mice [control group, 9 mice; unloaded group, 8 mice] at 4 months of age. The number of Sost-positive osteocytes was presented as a percentage of the total number of osteocytes. Only the cells with a nucleus were counted. Data are presented as the mean ± S.D. *vs. control. **P<0.01. (O) Western blot analysis using anti-β-catenin antibody. Proteins were extracted from osteoblast fractions from wild-type and <i>BCL2</i> transgenic mice at 4 months of age. β-actin was used as an internal control.</p

Micro-CT and bone histomorphometric analyses of cortical bone (A, B) Micro-CT analysis.

<p>Micro-CT images of mid-diaphyses of femurs (A) and cortical thickness, total tissue volume, and bone marrow volume (B) in male wild-type mice (wt) and <i>BCL2</i> transgenic mice (tg) at 10 weeks [wt, 17 mice; tg, 7 mice], 4 months [wt, 14 mice; tg, 10 mice], and 6 months [wt, 6 mice; tg, 6 mice] of age. Data are presented as the mean ± S.D. (C–G) Dynamic histomorphometric analysis of cortical bone at 4 months of age. C and D, Cross-sections from the mid-diaphyses of femurs of male wild-type mice (C) and <i>BCL2</i> transgenic mice (D), in which calcein had been injected twice. Scale bars = 0.5 mm. E–G, Mineral apposition rate (MAR) (E), double-labeled surface (dLS/BS) (F), and bone formation rate (BFR/BS) (G) in the endosteum (En) and periosteum (Pe) at the mid-diaphyses of femurs of wild-type mice (w, blue) and <i>BCL2</i> transgenic mice (t, red). Data are the mean ± S.D. of 10 mice. *vs. wild-type mice. *, ♯, $ P<0.05; **, ♯♯ P<0.01; ***, ♯♯♯ P<0.001. (H) Comparison of the serum osteocalcin level in male four wild-type mice and five <i>BCL2</i> transgenic mice at 4 months of age. Data are presented as the mean ± S.D. *vs. wild-type mice. **P<0.01.</p

A model of osteocyte functions.

<p>(A) In the loaded (physiological) condition, the osteocyte network inhibits osteoblast function, enhances osteoclastogenesis, and negatively regulates bone mass. (B) In the unloaded condition, the effect of osteocyte network on osteoblast function is augmented through the induction of Sost in osteocytes and that on osteoclastogenesis is augmented through the induction of Rankl in osteoblasts, resulting in reduced bone mass. The thickness of the lines and arrows in A and B reflects the strength of the effects.</p

Bone morphometric analysis, BrdU and TUNEL staining, and real-time RT-PCR analysis of apoptosis-related genes in Bcl2^−/− mice.

<p>(A) Bone histomorphometric analysis. The trabecular bone volume (bone volume/tissue volume, BV/TV), number of osteoblasts (N.Ob/B.Pm), and number of osteoclasts (N.Oc/B.Pm) were compared in femurs between 6 wild-type and 4 Bcl2^−/− mice at 2 weeks of age. B.Pm, bone perimeter. (B–H) BrdU labeling (B, C) and TUNEL staining (D, E) of sections of femurs from wild-type mice (B, D) and Bcl2^−/− mice (C, E). Bars  = 50 µm. BrdU-positive osteoblastic cells (F), TUNEL-positive osteoblastic cells (G), and TUNEL-positive osteocytes (H) were counted and shown as a percentage of the number of osteoblastic cells or osteocytes. wild-type mice, n = 7; Bcl2^−/− mice, n = 5 in F. wild-type mice, n = 8; Bcl2^−/− mice, n = 5 in G and H. (I) Real-time RT-PCR analysis of apoptosis-related genes. RNA was directly extracted from newborn calvariae of wild-type and Bcl2^−/− mice. wild-type mice, n = 6; Bcl2^−/− mice, n = 15. *vs. wild-type mice. *P<0.05, **P<0.01.</p

Increase of osteoid in <i>BCL2</i> transgenic mice at 4 months of age.

<p>Cortical bone (A–D) and trabecular bone (E–H) of femurs in wild-type (A, C, E, G) and <i>BCL2</i> transgenic (B, D, F, H) mice at 4 months of age. The boxed regions in A, B, E, and F are magnified in C, D, G, and H, respectively. Osteoid was visualized by Goland-Yoshilki method. Scale bars = 50 µm (A, B, E, F); 10 µm (C, D, G, H). (I) Osteoid thickness. Data are presented as the mean ± S.D. *vs. wild-type mice. *P<0.05, **P<0.01. wt, 4 mice; tg, 5 mice.</p

Transgene expression, osteocyte number, and the frequencies of TUNEL-positive lacunae.

<p>(A, B) Real-time RT-PCR analyses of the expression of transgene (A) and <i>Col1a1</i> (B). The expression levels of the transgene and <i>Col1a1</i> were examined using RNA that had been extracted from the whole femurs at 2 weeks of age [wt, 8 mice; tg, 7 mice] and osteoblast-enriched samples at 5–6 weeks [wt, 13 mice; tg, 11 mice], 10 weeks [wt, 8 mice; tg, 5 mice], and 4 [wt, 3 mice; tg, 9 mice] and 6 [wt, 5 mice; tg, 8 mice] months of age. The values of wild-type mice were defined as 1, and relative levels are shown in A. The value at 4 months of age was defined as 1, and relative levels are shown in B. (C–J) Immunohistochemical analysis. Sections of wild-type mice at 2 weeks of age (C) and <i>BCL2</i> transgenic mice at 2 weeks (E), 6 weeks (G), and 4 months (I) of age were reacted with anti-BCL2 antibody. Boxed regions in C, E, G, and I are magnified in D, F, H, and J, respectively. The arrows in H indicate immature osteocytes, which expressed the transgene. The lacunae, which were TUNEL-positive and contained cellular debris of dead osteocytes, were non-specifically reacted with anti-BCL2 antibody in <i>BCL2</i> transgenic mice. Scale bars  = 100 µm (C, E, G, I); 10 µm (D, F, H, J). (K) The number of osteocytes in cortical bone. The number of osteocytes was counted in the cortical bone of femurs at 10 weeks [wt, 9 mice; tg, 12 mice] and 4 months [wt, 14 mice; tg, 13 mice] of age. (L and M) Frequencies of TUNEL-positive lacunae in cortical bone (L) and trabecular bone (M). TUNEL-positive lacunae were counted in femurs at 5–6 weeks [wt, 5 mice; tg, 6 mice], 10 weeks [wt, 7 mice; tg, 9 mice], and 4 [wt, 4 mice; tg, 4 mice] and 6 [wt, 4 mice; tg, 7 mice] months of age. The number of TUNEL-positive lacunae was presented as a percentage of the total number of lacunae. In A, B, and K–M, data are presented as the mean ± S.D. *vs. wild-type mice. *P<0.05, **P<0.01, ***P<0.001, ^#P<0.05,^##P<0.01, ^$$$P<0.001.</p

Expression and activation of FoxOs in Bcl2−/− calvariae.

<p>(A) Real-time RT-PCR analysis of the expression of FoxOs. RNA was directly extracted from newborn calvariae of wild-type and Bcl2^−/− mice. wild-type mice, n = 6; Bcl2^−/− mice, n = 15. *vs. wild-type mice, **p<0.01, ***p<0.001. (B) Reporter assay of Gadd45a promoter using wild-type and Bcl2^−/− primary osteoblasts. Similar results were obtained in two independent experiments and representative data are shown. (C, D) Western blot analysis. Protein was extracted from newborn calvariae of wild-type and Bcl2^−/− mice. The intensities of the bands were normalized against each β-actin, the normalized values in wild-type mice were set as 1, and relative levels are shown. Similar results were obtained in three independent experiments and representative data are shown. (E) Real-time RT-PCR analysis. RNA was directly extracted from calvariae of wild-type and Bcl2^−/− newborn mice. wild-type mice, n = 6; Bcl2^−/− mice, n = 15. *vs. wild-type mice. *P<0.05, **P<0.01. (F) <i>p53</i>, <i>Pten</i>, and <i>Igfbp3</i> expression in primary osteoblasts. The cDNA in Fig. 3I was used for real-time PCR analysis. n = 10−12. *vs. wild-type primary osteoblasts. **P<0.01. (G) Induction of <i>Pten</i> by p53. p53^−/− osteoblasts were infected with p53-expressing retrovirus or empty retrovirus. Next day, the cells were plated at the concentration of 1.5×10⁵/well in 48 well plates (day 0). 50 µg/ml ascorbic acid and 10mM β-glycerophosphate were added at day 1, and mRNA was extracted at day 4. The expression of <i>p53</i>, <i>Pten</i>, and <i>Igfbp3</i> was examined by real-time RT-PCR. Similar results were obtained in two independent experiments and representative data are shown. n = 12−13. *vs. empty retrovirus. **P<0.01, ***p<0.001. (H) Schematic presentation of the signaling pathway for FoxO activation. p53 induces Pten mRNA and Igfbp3 mRNA. Pten and Igfbp3 inhibit Akt activation. Akt inhibits the activation of FoxOs. Activation of JNK and Mst1 activate FoxOs. p53 failed to induce Igfbp3 in vitro (G). Dotted arrows indicate that the activation did not occur in Bcl2^−/− mice (C).</p