Altered bone development and turnover in transgenic mice over-expressing lipocalin-2 in bone

Lipocalin-2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over-expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed-down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate. In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate-resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional receptor activator of nuclear factor-κB ligand (RANKL) and of the IL-6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment

Bone Turnover in Wild Type and Pleiotrophin-Transgenic Mice Housed for Three Months in the International Space Station (ISS)

Bone is a complex dynamic tissue undergoing a continuous remodeling process. Gravity is a physical force playing a role in the remodeling and contributing to the maintenance of bone integrity. This article reports an investigation on the alterations of the bone microarchitecture that occurred in wild type (Wt) and pleiotrophin-transgenic (PTN-Tg) mice exposed to a near-zero gravity on the International Space Station (ISS) during the Mice Drawer System (MDS) mission, to date, the longest mice permanence (91 days) in space. The transgenic mouse strain over-expressing pleiotrophin (PTN) in bone was selected because of the PTN positive effects on bone turnover. Wt and PTN-Tg control animals were maintained on Earth either in a MDS payload or in a standard vivarium cage. This study revealed a bone loss during spaceflight in the weight-bearing bones of both strains. For both Tg and Wt a decrease of the trabecular number as well as an increase of the mean trabecular separation was observed after flight, whereas trabecular thickness did not show any significant change. Non weight-bearing bones were not affected. The PTN-Tg mice exposed to normal gravity presented a poorer trabecular organization than Wt mice, but interestingly, the expression of the PTN transgene during the flight resulted in some protection against microgravity’s negative effects. Moreover, osteocytes of the Wt mice, but not of Tg mice, acquired a round shape, thus showing for the first time osteocyte space-related morphological alterations in vivo. The analysis of specific bone formation and resorption marker expression suggested that the microgravity-induced bone loss was due to both an increased bone resorption and a decreased bone deposition. Apparently, the PTN transgene protection was the result of a higher osteoblast activity in the flight mice

Altered bone development and turnover in transgenic mice over-expressing lipocalin-2 in bone

Lipocalin-2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over-expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed-down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate. In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate-resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional receptor activator of nuclear factor-\u3baB ligand (RANKL) and of the IL-6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment

Effects of long time exposure to simulated micro- and hypergravity on skeletal architecture

This manuscript reports the structural alterations occurring in mice skeleton as a consequence of the longest-term exposition (90 days) to simulated microgravity (hindlimb unloading) and hypergravity (2g) ever tested. Bone microstructural features were investigated by means of standard Cone Beam X-ray micro-CT, Synchrotron Radiation micro-CT and histology. Morphometric analysis confirmed deleterious bone architectural changes in lack of mechanical loading with a decrease of bone volume and density, while bone structure alterations caused by hypergravity were less evident. In the femurs from hypergravityexposed mice, the head/neck cortical thickness increment was the main finding. In addition, in these mice the rate of larger trabeculae (60\u201375 \u3bcm) was significantly increased. Interestingly, the metaphyseal plate presented a significant adaptation to gravity changes. Mineralization of cartilage and bone deposition was increased in the 2 g mice, whereas an enlargement of the growth plate cartilage was observed in the hindlimb unloaded group. Indeed, the presented data confirm and reinforce the detrimental effects on bone observed in real space microgravity and reveal region-specific effects on long bones. Finally these data could represent the starting point for further long-term experimentations that can deeply investigate the bone adaptation mechanisms to different mechanical force environments

Quantification of trabecular thickness distribution in femurs.

<p>Wt (A-C) and PTN-Tg (D-F) flight experiment, ground and vivarium control mice. The value reported for the vivarium condition is the average of the three animals.</p

Non-weight-bearing bones analysis.

<p>On the left: quantification of bone thickness distribution in the calvaria of Wt2 (A) PTN-Tg1 (B) PTN-Tg2 (C) obtained from the <i>μ</i>CT analysis. On the right: parietal bone color maps of the Wt2 (D) PTN-Tg1 (E) and PTN-Tg2 (F) of flight, ground and a representative bone of the vivarium control.</p

Serum OPG level analysis.

<p>Luminex^® assay was performed on serum samples of flight and ground Wt2 and an average of flight and ground PTN-Tg1, PTN-Tg2 as well as an average of Wt1, Wt2, Wt3 and of PTN-Tg1, PTN-Tg2 and PTN-Tg3 vivarium mice in order to measure serum OPG level.</p

Histology on femurs on flight samples.

<p>Stevenel’s/Van Gieson staining was performed on the epiphyseal region of the same <i>μ</i>CT analyzed femurs. Flight Wt2 (A) ground Wt2 (B) vivarium Wt2 (C) flight PTN-Tg2 (D) ground PTN-Tg2 (E) and vivarium PTN-Tg2 (F) mice trabecular femur bone (B). Bm = bone marrow, Tb = trabecular bone, Gp = growth plate.</p

Osteocytes morphology.

<p>Stevenel’s/Van Gieson staining was performed on the diaphysial region of the same <i>μ</i>CT analyzed femurs. Flight Wt2 (A) ground Wt2 (B) flight PTN-Tg2 (C) and ground PTN-Tg2 (D) cortical bone, magnification 60x. Db = diaphyseal bone, O = osteocytes, N = nucleus.</p