Guive Balooch

Glucocorticoid-treated mice have localized changes in trabecular bone material properties and osteocyte lacunar size that are not observed in placebo-treated or estrogen-deficient mice.

This study compares changes in bone microstructure in 6‐month‐old male GC‐treated and female ovariectomized mice to their respective controls. In addition to a reduction in trabecular bone volume, GC treatment reduced bone mineral and elastic modulus of bone adjacent to osteocytes that was not observed in control mice nor estrogen‐deficient mice. These microstructural changes in combination with the macrostructural changes could amplify the bone fragility in this metabolic bone disease.

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.

An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors.

Rodent incisors grow throughout adult life, but are prevented from becoming excessively long by constant abrasion, which is facilitated by the absence of enamel on one side of the incisor. Here we report that loss-of-function of sprouty genes, which encode antagonists of receptor tyrosine kinase signaling, leads to bilateral enamel deposition, thus impeding incisor abrasion and resulting in unchecked tooth elongation. We demonstrate that sprouty genes function to ensure that enamel-producing ameloblasts are generated on only one side of the tooth by inhibiting the formation of ectopic ameloblasts from self-renewing stem cells, and that they do so by preventing the establishment of an epithelial-mesenchymal FGF signaling loop. Interestingly, although inactivation of Spry4 alone initiates ectopic ameloblast formation in the embryo, the dosage of another sprouty gene must also be reduced to sustain it after birth. These data reveal that the generation of differentiated progeny from a particular stem cell population can be differently regulated in the embryo and adult.

Nanomechanical Properties of Hydrated Carious Human Dentin

Most restorative materials are bonded to caries-affected dentin that has altered structure. We tested the hypothesis that hydrated dentin of the transparent zone did not have increased hardness or elastic modulus. Nanoindentation by modified AFM was used to determine site-specific elastic modulus and hardness for components of hydrated dentin from 8 carious and non-carious human teeth. Indentations in intertubular dentin were made at intervals from pulp through the affected layers (subtransparent, transparent, and discolored zones). The values of intertubular dentin increased slightly from near the pulp into the transparent zone, then remained constant or decreased slightly through transparent dentin (E, 18.3 GPa; H, 0.8 GPa; confirming the hypothesis), and decreased markedly through the discolored region. Peritubular dentin values were unaltered in transparent dentin, and intratubular mineral had values between those of normal peritubular and intertubular dentin. Superficial areas contained distorted tubules without peritubular dentin or intratubular mineral.

Pharmacologic Inhibition of the TGF-β Type I Receptor Kinase Has Anabolic and Anti-Catabolic Effects on Bone

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-β has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-β signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-β signaling on bone remain unclear. To examine the role of TGF-β in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-β type I receptor (TβRI) kinase on bone mass, architecture and material properties. Inhibition of TβRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TβRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TβRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TβRI inhibitors may be effective in treating conditions of skeletal fragility.

The dentin-enamel junction: a natural, multilevel interface

Abstract Teeth contain two major calcified tissues, enamel and dentin, that are joined by an interface known as the dentin–enamel junction (DEJ). Enamel is the hard and brittle outer portion of the tooth that cuts and grinds food and dentin is composed of a tougher biological composite, that can absorb and distribute stresses. The DEJ is a complex and critical structure uniting these two dissimilar calcified tissues and acts to prevent the propagation of cracks from enamel into dentin. The DEJ has a three-level structure, 25–100 μm scallops with their convexities directed toward the dentin and concavities toward the enamel; 2–5 μm microscallops; and a smaller scale structure. Mechanical properties measurements, chemical differences and imaging have been used to determine the functional width of the DEJ. AFM based nanoindentation gave values of 11.8 μm, microRaman yielded a width of 7.0 μm, while the smaller probe used for AFM nanoscratching yielded 2.0 μm, and values from dynamic modulus mapping were less than 1 μm. The unique architecture of the DEJ may account for this variation based on enamel–dentin phase intermixing. The ultimate goal is to use the DEJ as a biomimetic model for other interfaces joining dissimilar materials.

Local mechanical and optical properties of normal and transparent root dentin

The mechanical and optical properties of healthy and transparent root dentin are compared using atomic force microscopy (AFM), micro-Raman and emission spectroscopies and fluorescence microscopy. The elastic modulus and hardness of intertubular and peritubular transparent and healthy dentin did not differ appreciably. The tubule filling material in the transparent zone, however, exhibited values between peritubular and intertubular dentin. Raman spectroscopy revealed a shift in the 1066 cm−1 band to 1072 cm−1 from normal to transparent intertubular dentin. The material filling the tubule lumen in transparent dentin showed an increase in frequency of the band near 1070 cm−1 as well. The emission spectral characteristics under 351 nm photoexcitation indicate differences between normal and transparent intertubular dentin. A transition region of about 300 μm between normal and transparent dentin was identified. In this region the intertubular emission properties were the same as for normal dentin, but tubules were filled. The filling material had emission characteristics closer to the normal intertubular than to transparent intertubular dentin.

On the Increasing Fragility of Human Teeth With Age: A Deep-UV Resonance Raman Study†

UV resonance Raman spectroscopy (UVRRS) using 244‐nm excitation was used to study the impact of aging on human dentin. The intensity of a spectroscopic feature from the peptide bonds in the collagen increases with tissue age, similar to a finding reported previously for human cortical bone.

Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-β and Runx2 in bone is required for hearing

Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue‐specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue‐specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage‐specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor‐β (TGFβ)‐responsive pathway that controls osteoblast differentiation. Deregulated TGFβ or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/− mice, inhibition of TGFβ signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.

Coupled Nanomechanical and Raman Microspectroscopic Investigation of Human Third Molar DEJ

The dentino-enamel junction (DEJ) connects enamel, that covers the outer surface of a tooth, to a thicker underlying dentin. The DEJ is a critical interface that permits joining these materials that have widely dissimilar mechanical properties. AFM-based nanoindentation and Raman microspectroscopy were used to define the width and composition of human molar DEJ. Indentation elastic modulus and hardness of enamel, dentin, and DEJ were determined along lines of indents made at 2 μm intervals across the DEJ. Indents made at maximum loads at each end of the indent lines were used to make visible markers allowing Raman microspectroscopy at 1 μm intervals across the DEJ, while using the nanoindent markers for orientation and location. Functional DEJ width estimates were made based on results from nanoindentation and Raman microspectroscopy. DEJ width estimates ranged from 4.7 (±1.2) μm to 6.1 (±1.9) μm based on hardness and 4.9 (±1.1) μm to 6.9 (±1.9) μm based on modulus. DEJ width based on Raman peak intensity variations were 8.0 (±3.2) μm to 8.5 (±3.1) μm based on the phosphate peak, and 7.6 (±3.2) μm to 8.0 (±2.6) μm for C–H stretching mode. These estimates are in the range of DEJ width estimates reported using nanoindentation.