**4. Effects of spaceflight on teeth**

## **4.1 Incisor growth and development**

The rodent mandible (and maxilla) contains, on each side, 1 incisor separated by a toothless region (diastema) from 3 molars (**Figure 11A**). The molars develop and erupt in a process similar to those of humans. The incisor, however, forms and erupts continuously, allowing the mandibular and maxillary incisors to maintain contact as the covering enamel and supporting dentin are worn away as the animal eats. The cells that form enamel, ameloblasts, originate from oral ectoderm; their stem cells are located in the cervical loop of the incisor, embedded within the bone of the mandible at the apical (posterior) end of the tooth. Ameloblasts initially deposit a partially mineralized matrix based mainly on the protein amelogenin. When the final thickness of the matrix is achieved, the ameloblasts undergo a morphologic and functional differentiation and begin to remove the matrix and add Ca and PO4 until the enamel is fully mineralized. The cells that form dentin, odontoblasts, are derived from craniofacial ectomesenchyme. Throughout the life of the tooth, odontoblasts continually form and mineralize dentin, which has a collagenbased extracellular matrix. Odontoblasts also synthesize several non-collagenous proteins, including dentin sialoprotein (DSP), dentin phosphoprotein, osteocalcin, bone sialoprotein, osteopontin and dentin matrix protein-1 [89].

### **4.2 MicroCT analyses**

Incisor teeth of flight and habitat ground control mice from the Bion-M1 mission had significantly greater enamel, dentin and overall volumes than the vivarium control mice (**Figure 12**). Enamel thickness in MicroCT sections at the 1st molar level was similar in all three mouse groups. In the flight and habitat control mice, however, the enamel had reached its full thickness at the 3rd molar level, and the thickness was greater in these 2 groups than in the vivarium control mice at both the 2nd and 3rd molar levels. Dentin thickness at all 3 molar levels in the flight and habitat control mice, as well as tissue density (mg hydroxyapatite/cm3 ), was greater than that of the vivarium control mice. Additionally, as visualized in 3D reconstruction images, incisor mineralization in the flight and habitat control mice began further posteriorly than in vivarium control mice (**Figure 12G**–**H**). These observations are consistent with a decreased incisor eruption rate in the flight and habitat control mice, most likely due to the soft paste diet consumed by these two groups [90–92]. In the STS-135 mice, there was a trend toward an increased incisor volume in the flight mice compared to the vivarium control mice, but no differences in enamel and dentin volumes, thicknesses or tissue densities.

Previous studies of spaceflight effects on tooth development have produced differing/conflicting results. In some investigations there were no differences between flight and control animals in Ca and P content of incisor dentin [58, 93, 94]. In other studies Ca and P concentrations were increased [57, 95, 96], or decreased [97]. These varying results are likely due to differences in age and sex of the animals, diet, length of the flight, the region of the tooth examined, as well as the methods used in these investigations.

#### **4.3 Tooth protein expression**

The expression of osteocalcin, DSP, amelogenin and PKA-RII was evaluated by IHC in the tissues and cells of the teeth [48]. Odontoblasts and some cementocytes of molar roots expressed strong reactivity for osteocalcin (**Figure 11A**); weaker

**69**

**Figure 12.**

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

reactivity was seen in predentin and dentin. Semiquantitative analysis revealed no differences in staining intensities among any of the flight and control groups. Immunostaining for DSP was strong in odontoblasts (**Figure 11D**) and some cementocytes; weaker reactivity was observed in predentin, dentin, molar cementum, enamel matrix, osteoblasts and bone. Significant differences in DSP staining intensities were observed among the Bion-M1 mouse groups. Predentin and cellular cementum (and bone) stained more intensely in flight mice than in vivarium controls. Secretory ameloblasts (**Figure 11E**) and enamel matrix (especially during enamel maturation) stained positively for amelogenin. Reactivity was significantly decreased in enamel matrix of Bion-M1 habitat control mice. Both odontoblasts and ameloblasts were immunoreactive for PKA-RII. Staining intensity was decreased in odontoblasts of Bion-M1 flight and habitat control mice compared to vivarium controls, and decreased in secretory ameloblasts of STS-135 flight and habitat control mice. In addition to effects due to microgravity, the observation of decreased amelogenin and PKA-RII reactivities in the habitat control mice suggest that the

*MicroCT images of mandibles of flight (A, C, E, and G) and vivarium control (B, D, F, and H) mice from the Bion-M1 mission. Sections at the level of the 1st (A and B), 2nd (C and D) and 3rd (E and F) molars. Incisor dentin (D) and enamel (E), molar (M), pulp chamber (P). Note the earlier appearance of mineralized enamel (images E vs. F), thicker dentin and smaller central pulp chambers in the incisors of the flight mice. Three-dimensional reconstructions of the mandibles (G and H) show that incisor mineralization begins (arrows) further posterior in flight mice than in vivarium control mice. Habitat ground control mice showed* 

habitat environment may play a role in cellular and tissue responses.

There are few reported studies of the effects of spaceflight on oral tissues other than bone and teeth. Histologic examination of periodontal tissues and oral mucosa

**5. Effects of spaceflight on other oral tissues**

*the same features as flight mice. Scale bars = 1 mm.*

#### **Figure 12.**

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

bone sialoprotein, osteopontin and dentin matrix protein-1 [89].

in enamel and dentin volumes, thicknesses or tissue densities.

The rodent mandible (and maxilla) contains, on each side, 1 incisor separated by a toothless region (diastema) from 3 molars (**Figure 11A**). The molars develop and erupt in a process similar to those of humans. The incisor, however, forms and erupts continuously, allowing the mandibular and maxillary incisors to maintain contact as the covering enamel and supporting dentin are worn away as the animal eats. The cells that form enamel, ameloblasts, originate from oral ectoderm; their stem cells are located in the cervical loop of the incisor, embedded within the bone of the mandible at the apical (posterior) end of the tooth. Ameloblasts initially deposit a partially mineralized matrix based mainly on the protein amelogenin. When the final thickness of the matrix is achieved, the ameloblasts undergo a morphologic and functional differentiation and begin to remove the matrix and add Ca and PO4 until the enamel is fully mineralized. The cells that form dentin, odontoblasts, are derived from craniofacial ectomesenchyme. Throughout the life of the tooth, odontoblasts continually form and mineralize dentin, which has a collagenbased extracellular matrix. Odontoblasts also synthesize several non-collagenous proteins, including dentin sialoprotein (DSP), dentin phosphoprotein, osteocalcin,

Incisor teeth of flight and habitat ground control mice from the Bion-M1 mission

), was

had significantly greater enamel, dentin and overall volumes than the vivarium control mice (**Figure 12**). Enamel thickness in MicroCT sections at the 1st molar level was similar in all three mouse groups. In the flight and habitat control mice, however, the enamel had reached its full thickness at the 3rd molar level, and the thickness was greater in these 2 groups than in the vivarium control mice at both the 2nd and 3rd molar levels. Dentin thickness at all 3 molar levels in the flight and habitat control mice, as well as tissue density (mg hydroxyapatite/cm3

greater than that of the vivarium control mice. Additionally, as visualized in 3D reconstruction images, incisor mineralization in the flight and habitat control mice began further posteriorly than in vivarium control mice (**Figure 12G**–**H**). These observations are consistent with a decreased incisor eruption rate in the flight and habitat control mice, most likely due to the soft paste diet consumed by these two groups [90–92]. In the STS-135 mice, there was a trend toward an increased incisor volume in the flight mice compared to the vivarium control mice, but no differences

Previous studies of spaceflight effects on tooth development have produced differing/conflicting results. In some investigations there were no differences between flight and control animals in Ca and P content of incisor dentin [58, 93, 94]. In other studies Ca and P concentrations were increased [57, 95, 96], or decreased [97]. These varying results are likely due to differences in age and sex of the animals, diet, length of the flight, the region of the tooth examined, as well as the methods

The expression of osteocalcin, DSP, amelogenin and PKA-RII was evaluated by IHC in the tissues and cells of the teeth [48]. Odontoblasts and some cementocytes of molar roots expressed strong reactivity for osteocalcin (**Figure 11A**); weaker

**4. Effects of spaceflight on teeth**

**4.1 Incisor growth and development**

**4.2 MicroCT analyses**

used in these investigations.

**4.3 Tooth protein expression**

**68**

*MicroCT images of mandibles of flight (A, C, E, and G) and vivarium control (B, D, F, and H) mice from the Bion-M1 mission. Sections at the level of the 1st (A and B), 2nd (C and D) and 3rd (E and F) molars. Incisor dentin (D) and enamel (E), molar (M), pulp chamber (P). Note the earlier appearance of mineralized enamel (images E vs. F), thicker dentin and smaller central pulp chambers in the incisors of the flight mice. Three-dimensional reconstructions of the mandibles (G and H) show that incisor mineralization begins (arrows) further posterior in flight mice than in vivarium control mice. Habitat ground control mice showed the same features as flight mice. Scale bars = 1 mm.*

reactivity was seen in predentin and dentin. Semiquantitative analysis revealed no differences in staining intensities among any of the flight and control groups. Immunostaining for DSP was strong in odontoblasts (**Figure 11D**) and some cementocytes; weaker reactivity was observed in predentin, dentin, molar cementum, enamel matrix, osteoblasts and bone. Significant differences in DSP staining intensities were observed among the Bion-M1 mouse groups. Predentin and cellular cementum (and bone) stained more intensely in flight mice than in vivarium controls. Secretory ameloblasts (**Figure 11E**) and enamel matrix (especially during enamel maturation) stained positively for amelogenin. Reactivity was significantly decreased in enamel matrix of Bion-M1 habitat control mice. Both odontoblasts and ameloblasts were immunoreactive for PKA-RII. Staining intensity was decreased in odontoblasts of Bion-M1 flight and habitat control mice compared to vivarium controls, and decreased in secretory ameloblasts of STS-135 flight and habitat control mice. In addition to effects due to microgravity, the observation of decreased amelogenin and PKA-RII reactivities in the habitat control mice suggest that the habitat environment may play a role in cellular and tissue responses.
