**3. Effects of spaceflight on the mandible**

### **3.1 MicroCT analyses**

MicroCT was used to assess mandibular bone volume (BV) and bone mineral density (BMD) in mice from the STS-135 and Bion-M1 flights [48].

In the STS-135 mission no differences in BV or BMD were seen between the flight and habitat ground control mice. However, when compared to vivarium control mice, BV was greater in both flight mice and habitat ground control mice. Similarly, BMD was greater in both the flight and habitat control mice than the vivarium control mice, although only the habitat control values were statistically significant. The increased BV and BMD seen in the flight and habitat control mice may be due to the difference in composition and consistency of the NASA food bars [18] these mice consumed compared to the standard rodent chow eaten by the vivarium control mice. The food bars have about 20% less caloric value per gram than chow, requiring greater consumption and more masticatory effort for comparable nutrition.

**65**

tive measurement.

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

smaller in size [58].

occurred in BMD among these three groups, however.

In contrast, mandibular BV decreased in both flight and habitat control mice compared to vivarium control mice from the Bion-M1 mission [48]. No differences

These changes in flight and habitat control mice are likely due to the difference in diet between these two groups and the vivarium mice [49, 50]. The soft paste diet consumed by the Bion-M1 flight and habitat control mice required considerably less masticatory force than chow. Previous studies have shown that reduced mechanical loading results in decreased growth of the mandible [51, 52]. The different age and sex (9 week old females vs. 15–16 week old males, respectively) of the STS-135 and Bion-M1 mice also may have contributed to the different results for BV and BMD. The STS-135 mice were skeletally immature [53, 54], and female mice are physically more active than males [55] and have a different pattern of chewing and biting activity [56]. Earlier studies examined the effects of spaceflight and simulated weightlessness on mandibular bone. Periosteal osteogenesis was reduced in areas of the mandible not covered by muscle (molar region) in rats flown for 18.5 days on Cosmos 1129 [57]. Formation of alveolar bone was reduced on the modeling (mesial or anterior) side of the first molar, indicating a slowing of the normal distal (posterior) drift of rodent molars. There also was a decrease in alveolar bone mineral and collagen in fractions representing the most mature components, with a corresponding increase in the most immature fractions, suggesting a delay in maturation. The mandibles of rats flown for 12 days on Cosmos 1887 had relatively high Ca and Mg levels but otherwise a normal composition, although their hydroxyapatite crystals were

Studies of rodents subjected to hindlimb unloading (HU) for up to 4 weeks have been used as model of exposure to microgravity [5, 59, 60]. Although the effects on the spine and hindlimbs show similarities to changes seen in animals after spaceflight, effects on the mandible do not mimic those occurring after actual spaceflight. Ten to 14 days of HU did not alter maturation of mandibular bone matrix and mineral as seen in rats flown on Cosmos 1129 [61]. Similarly, 15 days of HU did not alter the total weight, ash weight, Ca content or Ca uptake of the mandible [62]. No significant effects on the mandible were seen after 28 days of HU [63]. An earlier study, however, found an increase in the dry and ash weights of the mandible after 3 weeks of HU [64]. In HU, the mandible still closes against gravity [60], thus spaceflight induced changes in the mandible are most likely a result of hypogravity [61]. Ghosh et al. [8] studied mandibles from mice flown on STS-131 and STS-135 (from the opposite side of the same STS-135 mice we studied [48]). They also found no difference in BV between the flight and habitat ground control mice from the 13-day STS-135 mission. However, mandibular BV was decreased in flight mice from the 15-day STS-131 mission. It is unlikely that the 2-day difference in flight length would result in a significant difference in BV. As noted by Ghosh et al., it is more likely that other factors are involved, such as the age difference and resulting skeletal maturation of the mice on the two flights (23 weeks vs. 9 weeks for STS-131 and STS-135, respectively), slight differences in food consumption, and/or the relatively low statistical power due to the small number of animals. They found no difference in BMD between the flight and habitat ground control mice from the STS-131 mission, but similar to our results, BMD was increased in the flight mice from the STS-135 mission. The factors mentioned above also could contribute to the differences in BMD seen between the mice on the two shuttle flights. The difference in BV between the Ghosh et al. study and our results for the STS-135 mice may be due to the different methods used to assess BV. Ghosh et al. measured BV in a single section (6 μm) from each mandible. In our study the results from 10 serial sections (120 μm span) per mandible were averaged, providing a more representa-

### *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*

using saliva to monitor astronaut physiology and health.

**3. Effects of spaceflight on the mandible**

**3.1 MicroCT analyses**

STS-135 flight mice, **Figure 8B**).

Interestingly, significant changes in gene expression occurred in the PG of habitat ground control mice compared to vivarium housed mice. In ground control female mice housed in the NASA Animal Enclosure Modules, the expression of 50 genes was significantly upregulated, and 33 genes were downregulated. For example, Western blotting of PG of STS-135 habitat control mice showed a decrease in the expression of α-amylase (**Figure 8B**). In ground control male mice housed in the Bion-M1 habitats, the expression of 42 genes was significantly upregulated, and 169 genes were downregulated. These results indicate that the effects of the habitats must be considered when assessing

Changes in the expression of secretory protein genes (**Figure 10**), for example, PKA-RII, largely paralleled the changes seen in protein expression as determined by immunogold labeling and Western blotting (e.g., PKA-RII expression in PG from

There have been a few previous studies of salivary glands of rodents following spaceflight, and saliva collected from astronauts and cosmonauts before and after spaceflight. No changes in PG morphology were seen by light microscopy in mice flown for 12.5 days on Apollo 17 [37]. The SMG of rats flown for 18.5 days on Cosmos 936 and Cosmos 1129 showed a reduced glycoprotein content as determined by light microscopic histochemical staining [38, 39]. The saliva of 18 astronauts making up the primary and backup crews for Skylab missions of 28-, 59- and 84-days was analyzed preflight and postflight for flow rate, electrolytes, protein, immunoglobulin A (IgA) and lysozyme [40]. Changes were minimal except for an increase in IgA and a decrease in lysozyme concentrations. α-Amylase in cosmonaut saliva was decreased following an 18-day flight (uncited statement in [41]). The use of saliva for monitoring drug absorption was proposed, and saliva levels of acetaminophen were determined in 1 astronaut [42]. Analyses of saliva of cosmonauts during long-term spaceflight showed levels of cortisol below baseline [43], whereas salivary cortisol biorhythms and concentrations were preserved in astronauts onboard Spacelab [44] and cosmonauts on the International Space Station (ISS) [45]. Transient increases in saliva urea and phosphate concentrations, monitored in 2 astronauts during 6 months on the ISS, were consistent with serum concentrations reported in earlier studies [46]. Although studied in astronaut plasma, not saliva, in the NASA Twin Study, EGF and NGF levels were altered during a 340-day flight and after landing [47]. Our studies and those cited above indicate the feasibility of

MicroCT was used to assess mandibular bone volume (BV) and bone mineral

In the STS-135 mission no differences in BV or BMD were seen between the flight and habitat ground control mice. However, when compared to vivarium control mice, BV was greater in both flight mice and habitat ground control mice. Similarly, BMD was greater in both the flight and habitat control mice than the vivarium control mice, although only the habitat control values were statistically significant. The increased BV and BMD seen in the flight and habitat control mice may be due to the difference in composition and consistency of the NASA food bars [18] these mice consumed compared to the standard rodent chow eaten by the vivarium control mice. The food bars have about 20% less caloric value per gram than chow, requiring greater consumption and more masticatory effort for compa-

density (BMD) in mice from the STS-135 and Bion-M1 flights [48].

the overall effects of spaceflight on animal (as well as human) physiology.

**64**

rable nutrition.

In contrast, mandibular BV decreased in both flight and habitat control mice compared to vivarium control mice from the Bion-M1 mission [48]. No differences occurred in BMD among these three groups, however.

These changes in flight and habitat control mice are likely due to the difference in diet between these two groups and the vivarium mice [49, 50]. The soft paste diet consumed by the Bion-M1 flight and habitat control mice required considerably less masticatory force than chow. Previous studies have shown that reduced mechanical loading results in decreased growth of the mandible [51, 52]. The different age and sex (9 week old females vs. 15–16 week old males, respectively) of the STS-135 and Bion-M1 mice also may have contributed to the different results for BV and BMD. The STS-135 mice were skeletally immature [53, 54], and female mice are physically more active than males [55] and have a different pattern of chewing and biting activity [56].

Earlier studies examined the effects of spaceflight and simulated weightlessness on mandibular bone. Periosteal osteogenesis was reduced in areas of the mandible not covered by muscle (molar region) in rats flown for 18.5 days on Cosmos 1129 [57]. Formation of alveolar bone was reduced on the modeling (mesial or anterior) side of the first molar, indicating a slowing of the normal distal (posterior) drift of rodent molars. There also was a decrease in alveolar bone mineral and collagen in fractions representing the most mature components, with a corresponding increase in the most immature fractions, suggesting a delay in maturation. The mandibles of rats flown for 12 days on Cosmos 1887 had relatively high Ca and Mg levels but otherwise a normal composition, although their hydroxyapatite crystals were smaller in size [58].

Studies of rodents subjected to hindlimb unloading (HU) for up to 4 weeks have been used as model of exposure to microgravity [5, 59, 60]. Although the effects on the spine and hindlimbs show similarities to changes seen in animals after spaceflight, effects on the mandible do not mimic those occurring after actual spaceflight. Ten to 14 days of HU did not alter maturation of mandibular bone matrix and mineral as seen in rats flown on Cosmos 1129 [61]. Similarly, 15 days of HU did not alter the total weight, ash weight, Ca content or Ca uptake of the mandible [62]. No significant effects on the mandible were seen after 28 days of HU [63]. An earlier study, however, found an increase in the dry and ash weights of the mandible after 3 weeks of HU [64]. In HU, the mandible still closes against gravity [60], thus spaceflight induced changes in the mandible are most likely a result of hypogravity [61].

Ghosh et al. [8] studied mandibles from mice flown on STS-131 and STS-135 (from the opposite side of the same STS-135 mice we studied [48]). They also found no difference in BV between the flight and habitat ground control mice from the 13-day STS-135 mission. However, mandibular BV was decreased in flight mice from the 15-day STS-131 mission. It is unlikely that the 2-day difference in flight length would result in a significant difference in BV. As noted by Ghosh et al., it is more likely that other factors are involved, such as the age difference and resulting skeletal maturation of the mice on the two flights (23 weeks vs. 9 weeks for STS-131 and STS-135, respectively), slight differences in food consumption, and/or the relatively low statistical power due to the small number of animals. They found no difference in BMD between the flight and habitat ground control mice from the STS-131 mission, but similar to our results, BMD was increased in the flight mice from the STS-135 mission. The factors mentioned above also could contribute to the differences in BMD seen between the mice on the two shuttle flights. The difference in BV between the Ghosh et al. study and our results for the STS-135 mice may be due to the different methods used to assess BV. Ghosh et al. measured BV in a single section (6 μm) from each mandible. In our study the results from 10 serial sections (120 μm span) per mandible were averaged, providing a more representative measurement.
