**3.2 Bone protein expression**

The expression of the bone proteins sclerostin and osteocalcin in the mandibles of mice from the STS-135 and Bion-M1 missions was studied by immunohistochemistry (IHC) [48] (**Figure 11A**–**C**). Sclerostin is secreted by osteocytes and other cells embedded in mineralized tissues; it inhibits osteoblast proliferation and differentiation and induces osteoblast apoptosis [65, 66]. No differences in the intensity of sclerostin staining of osteocytes were observed between the flight mice and their respective controls in either mission. The percentage of reactive osteocytes, however, varied between the 2 missions and among the 3 groups and locations within the mandible. Overall, the percentage of reactive osteocytes was greater in the STS-135 mice than in the Bion-M1 mice, and greater in flight mice than in control mice. The alveolar bone anterior to the first molar and the interradicular bone of the first molar showed the greatest percentage of reactive osteocytes, whereas the septal bone between the first and second molars had the lowest percentage.

Sclerostin synthesis is increased by mechanical unloading (i.e., rodent HU, human bed rest) [67, 68]. However, the percentage of sclerostin-positive osteocytes was unaltered in male mouse tibiae after 3 or 7 days of HU [67]. Sclerostin-positive

#### **Figure 11.**

*Immunohistochemistry of mandibular bone and incisor teeth. (A) Sagittal section of a hemimandible from a Bion-M1 habitat control mouse, immunostained for osteocalcin. Bone (B), incisor (I), molars (M1-3), pulp (P). Note staining of odontoblasts lining the pulp chamber. (B) Sclerostin staining of osteocytes (arrows) in interseptal bone of a Bion-M1 flight mouse. A few osteocytes are unstained (arrowheads). Cementocytes (CC) in the cementum covering a molar root also stain for sclerostin. Periodontal ligament (PDL). (C) Osteocalcin staining of osteoblasts (arrows) of a Bion-M1 vivarium control mouse. (D) Immunostaining of dentin sialoprotein in odontoblasts (O) of a STS-135 flight mouse. Odontoblast processes (arrows) in predentin (PD) and dentin (D) are stained. Predentin and dentin stain less intensely. (E) Amelogenin staining of secretory ameloblasts (A) of a STS-135 vivarium control mouse. Tomes' processes of ameloblasts (arrows) are stained intensely. Enamel matrix (EM).*

**67**

*Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728*

pared to vivarium and habitat control mice [74].

food prevents significant bone resorption.

stay on the ISS [47].

Bion-M1 missions [48].

different flight lengths.

osteocytes increased in male rat femurs after 28 days of HU [69], and partial weight bearing (1/6G) for 21 days also increased their percentage in female mouse femurs [70]. No significant changes in serum sclerostin levels of astronauts and cosmonauts were observed after 4–6 months on the ISS [71, 72]. In the NASA Twins Study, a trend toward increased serum sclerostin levels was seen throughout the 340-day

Spaceflight also resulted in increases in the size of osteocyte lacunae, expression of matrix metalloproteinase (MMP) activity, and the percentage of osteocytes expressing tartrate-resistant acid phosphatase (TRAP) in pelvic bones of STS-131 flight mice, indicating osteocytic osteolysis [73]. Empty lacunae indicating osteocyte death doubled in number in the femoral cortex of Bion-M1 flight mice com-

Osteocalcin is a non-collagenous bone matrix protein produced by osteoblasts

Osteocalcin synthesis in rat long bones and vertebrae decreased after short-term spaceflight (4–14 days) [79–82]. Serum and urinary osteocalcin levels increased in cosmonauts during 1 month and 6 months spaceflight [83–85]. A trend toward increased serum osteocalcin levels was seen in astronauts and cosmonauts after 4-6 months on the ISS [72], and during a 340-day flight on the ISS [47]. These findings are consistent with decreased bone formation and increased bone resorption. The lack of apparent change in osteocalcin staining in the mandible, along with the lack of or small changes in bone volume, suggests that the continued mastication of

Osteocalcin gene and protein expression also have been studied in cultured osteoblasts or osteoblast-like cells flown in space. Production of osteocalcin message after 9 days of microgravity by human osteosarcoma cells stimulated with vitamin D3 and transforming growth factor beta was only 19% of that of similarly treated cells at unit gravity [86]. The steady-state levels of osteocalcin mRNA in human fetal osteoblast cells after 17 days of microgravity did not differ from controls [87]. Osteocalcin protein expression was slightly but significantly increased in cultured human osteoblasts flown for 11 days [88]. The different results for osteocalcin expression may be due to the different cell types, different culture conditions, and

The percentage of alveolar bone surface occupied by TRAP-positive osteoclasts (Oc.S./B.S. %) was increased in the mandibles of Bion-M1 flight and habitat control mice [48], indicating increased bone resorption. This correlates with the decreased BV observed in these mice compared to vivarium controls. Osteoclasts were present mainly along the mesial (anterior) surfaces of the alveolar bone, consistent with the normal physiological distal (posterior) drift of rodent molars. No differences were seen among the STS-135 mice groups, although the overall Oc.S./B.S. % was greater in these mice than in the Bion-M1 mice. This likely was due to the younger age of the STS-135 mice, as well as the increased proportion of sclerostin-positive osteocytes in these mice, as sclerostin promotes increased osteoclast formation and size [66]. In rats flown for 18.5 days on Cosmos 1129, there was a slight but non-significant decrease in alveolar bone resorption immediately after flight [57]. However, after 6 and 29 days of post-flight recovery, a significant decrease in bone formation and resorption was

observed, indicating a slowing of the normal distal drift of the molars.

(and odontoblasts). It binds calcium and has long been thought to regulate mineralization [75, 76]. More recently osteocalcin has been recognized as a major bone hormone that participates in the regulation of energy metabolism, brain development and cognition, and male fertility [77, 78]. No differences were observed in the IHC staining intensity of osteoblasts, bone matrix or osteoid between the flight mice and their respective controls from the STS-135 and

### *Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728*

*Beyond LEO - Human Health Issues for Deep Space Exploration*

The expression of the bone proteins sclerostin and osteocalcin in the mandibles of mice from the STS-135 and Bion-M1 missions was studied by immunohistochemistry (IHC) [48] (**Figure 11A**–**C**). Sclerostin is secreted by osteocytes and other cells embedded in mineralized tissues; it inhibits osteoblast proliferation and differentiation and induces osteoblast apoptosis [65, 66]. No differences in the intensity of sclerostin staining of osteocytes were observed between the flight mice and their respective controls in either mission. The percentage of reactive osteocytes, however, varied between the 2 missions and among the 3 groups and locations within the mandible. Overall, the percentage of reactive osteocytes was greater in the STS-135 mice than in the Bion-M1 mice, and greater in flight mice than in control mice. The alveolar bone anterior to the first molar and the interradicular bone of the first molar showed the greatest percentage of reactive osteocytes, whereas the septal

bone between the first and second molars had the lowest percentage.

Sclerostin synthesis is increased by mechanical unloading (i.e., rodent HU, human bed rest) [67, 68]. However, the percentage of sclerostin-positive osteocytes was unaltered in male mouse tibiae after 3 or 7 days of HU [67]. Sclerostin-positive

**3.2 Bone protein expression**

**66**

*intensely. Enamel matrix (EM).*

**Figure 11.**

*Immunohistochemistry of mandibular bone and incisor teeth. (A) Sagittal section of a hemimandible from a Bion-M1 habitat control mouse, immunostained for osteocalcin. Bone (B), incisor (I), molars (M1-3), pulp (P). Note staining of odontoblasts lining the pulp chamber. (B) Sclerostin staining of osteocytes (arrows) in interseptal bone of a Bion-M1 flight mouse. A few osteocytes are unstained (arrowheads). Cementocytes (CC) in the cementum covering a molar root also stain for sclerostin. Periodontal ligament (PDL). (C) Osteocalcin staining of osteoblasts (arrows) of a Bion-M1 vivarium control mouse. (D) Immunostaining of dentin sialoprotein in odontoblasts (O) of a STS-135 flight mouse. Odontoblast processes (arrows) in predentin (PD) and dentin (D) are stained. Predentin and dentin stain less intensely. (E) Amelogenin staining of secretory ameloblasts (A) of a STS-135 vivarium control mouse. Tomes' processes of ameloblasts (arrows) are stained* 

osteocytes increased in male rat femurs after 28 days of HU [69], and partial weight bearing (1/6G) for 21 days also increased their percentage in female mouse femurs [70]. No significant changes in serum sclerostin levels of astronauts and cosmonauts were observed after 4–6 months on the ISS [71, 72]. In the NASA Twins Study, a trend toward increased serum sclerostin levels was seen throughout the 340-day stay on the ISS [47].

Spaceflight also resulted in increases in the size of osteocyte lacunae, expression of matrix metalloproteinase (MMP) activity, and the percentage of osteocytes expressing tartrate-resistant acid phosphatase (TRAP) in pelvic bones of STS-131 flight mice, indicating osteocytic osteolysis [73]. Empty lacunae indicating osteocyte death doubled in number in the femoral cortex of Bion-M1 flight mice compared to vivarium and habitat control mice [74].

Osteocalcin is a non-collagenous bone matrix protein produced by osteoblasts (and odontoblasts). It binds calcium and has long been thought to regulate mineralization [75, 76]. More recently osteocalcin has been recognized as a major bone hormone that participates in the regulation of energy metabolism, brain development and cognition, and male fertility [77, 78]. No differences were observed in the IHC staining intensity of osteoblasts, bone matrix or osteoid between the flight mice and their respective controls from the STS-135 and Bion-M1 missions [48].

Osteocalcin synthesis in rat long bones and vertebrae decreased after short-term spaceflight (4–14 days) [79–82]. Serum and urinary osteocalcin levels increased in cosmonauts during 1 month and 6 months spaceflight [83–85]. A trend toward increased serum osteocalcin levels was seen in astronauts and cosmonauts after 4-6 months on the ISS [72], and during a 340-day flight on the ISS [47]. These findings are consistent with decreased bone formation and increased bone resorption. The lack of apparent change in osteocalcin staining in the mandible, along with the lack of or small changes in bone volume, suggests that the continued mastication of food prevents significant bone resorption.

Osteocalcin gene and protein expression also have been studied in cultured osteoblasts or osteoblast-like cells flown in space. Production of osteocalcin message after 9 days of microgravity by human osteosarcoma cells stimulated with vitamin D3 and transforming growth factor beta was only 19% of that of similarly treated cells at unit gravity [86]. The steady-state levels of osteocalcin mRNA in human fetal osteoblast cells after 17 days of microgravity did not differ from controls [87]. Osteocalcin protein expression was slightly but significantly increased in cultured human osteoblasts flown for 11 days [88]. The different results for osteocalcin expression may be due to the different cell types, different culture conditions, and different flight lengths.

The percentage of alveolar bone surface occupied by TRAP-positive osteoclasts (Oc.S./B.S. %) was increased in the mandibles of Bion-M1 flight and habitat control mice [48], indicating increased bone resorption. This correlates with the decreased BV observed in these mice compared to vivarium controls. Osteoclasts were present mainly along the mesial (anterior) surfaces of the alveolar bone, consistent with the normal physiological distal (posterior) drift of rodent molars. No differences were seen among the STS-135 mice groups, although the overall Oc.S./B.S. % was greater in these mice than in the Bion-M1 mice. This likely was due to the younger age of the STS-135 mice, as well as the increased proportion of sclerostin-positive osteocytes in these mice, as sclerostin promotes increased osteoclast formation and size [66]. In rats flown for 18.5 days on Cosmos 1129, there was a slight but non-significant decrease in alveolar bone resorption immediately after flight [57]. However, after 6 and 29 days of post-flight recovery, a significant decrease in bone formation and resorption was observed, indicating a slowing of the normal distal drift of the molars.
