Osteoinductivity of calcium phosphate mediated by connexin 43.

Recent reports have alluded to the osteoinductive properties of calcium phosphate, yet the cellular processes behind this are not well understood. To gain insight into the molecular mechanisms of this phenomenon, we have conducted a series of in vitro and in vivo experiments using a scaffoldless three dimensional (3D) dental pulp cell (DPC) construct as a physiologically relevant model. We demonstrate that amorphous calcium phosphate (ACP) alters cellular functions and 3D spatial tissue differentiation patterns by increasing local calcium concentration, which modulates connexin 43 (Cx43)-mediated gap junctions. These observations indicate a chemical mechanism for osteoinductivity of calcium phosphates. These results provide new insights for possible roles of mineral phases in bone formation and remodeling. This study also emphasizes the strong effect of scaffold materials on cellular functions and is expected to advance the design of future tissue engineering materials.

Anisotropy of chemical bonds in collagen molecules studied by X-ray absorption near-edge structure (XANES) spectroscopy.

Collagen type I fibrils are the major building blocks of connective tissues. Collagen fibrils are anisotropic supramolecular structures, and their orientation can be revealed by polarized light microscopy and vibrational microspectroscopy. We hypothesized that the anisotropy of chemical bonds in the collagen molecules, and hence their orientation, might also be detected by X-ray photoemission electron spectromicroscopy (X-PEEM) and X-ray absorption near-edge structure (XANES) spectroscopy, which use linearly polarized synchrotron light. To test this hypothesis, we analyzed sections of rat-tail tendon, composed of parallel arrays of collagen fibrils. The results clearly indicate that XANES-PEEM is sensitive to collagen fibril orientation and, more specifically, to the orientations of carbonyl and amide bonds in collagen molecules. These data suggest that XANES-PEEM is a promising technique for characterizing the chemical composition and structural organization at the nanoscale of collagen-based connective tissues, including tendons, cartilage, and bone.

Relationships between dentin and enamel mineral at the dentino-enamel boundary: electron tomography and high-resolution transmission electron microscopy study.

To better understand the nature of the relationships between mineral phases at the dentino-enamel boundary (DEB), we performed electron tomography (ET) and high-resolution transmission electron microscopy (HR-TEM) of the apical portions of rat incisors. The ET studies of the DEB at the secretory stage of amelogenesis revealed that nascent enamel crystals are co-aligned and closely associated with dentin crystallites in the mineralized von Korff fibers, with the distances between dentin and enamel crystals in the nanometer range. We have further studied the relationships between dentin and enamel crystals using HR-TEM lattice imaging of the DEB. Among dozens of high-resolution micrographs taken from the DEB we were able to identify only one case of lattice continuity between dentin and enamel crystals, indicating direct epitaxy. In other cases, although there was no direct continuity between the crystalline lattices, power spectra analysis of lattice images revealed a very high level of co-alignment between dentin and enamel crystals. Hence, we propose here that the high degree of alignment and integration between dentin and enamel mineral can be established either by epitaxy or without direct interactions between crystalline lattices, probably via regulation of mineral formation and organization by integrated organic matrices of dentin and enamel at the DEB.

Transient amorphous calcium phosphate in forming enamel.

Enamel, the hardest tissue in the body, begins as a three-dimensional network of nanometer size mineral particles, suspended in a protein gel. This mineral network serves as a template for mature enamel formation. To further understand the mechanisms of enamel formation we characterized the forming enamel mineral at an early secretory stage using X-ray absorption near-edge structure (XANES) spectromicroscopy, transmission electron microscopy (TEM), FTIR microspectroscopy and polarized light microscopy. We show that the newly formed enamel mineral is amorphous calcium phosphate (ACP), which eventually transforms into apatitic crystals. Interestingly, the size, shape and spatial organization of these amorphous mineral particles and older crystals are essentially the same, indicating that the mineral morphology and organization in enamel is determined prior to its crystallization. Mineralization via transient amorphous phases has been previously reported in chiton teeth, mollusk shells, echinoderm spicules and spines, and recent reports strongly suggest the presence of transient amorphous mineral in forming vertebrate bones. The present finding of transient ACP in murine tooth enamel suggests that this strategy might be universal.