Mechanobiologically induced bone-like nodules: Matrix characterization from micro to nanoscale.

In bone tissue engineering, stem cells are known to form inhomogeneous bone-like nodules on a micrometric scale. Herein, micro- and nano-infrared (IR) micro-spectroscopies were used to decipher the chemical composition of the bone-like nodule. Histological and immunohistochemical analyses revealed a cohesive tissue with bone-markers positive cells surrounded by dense mineralized type-I collagen. Micro-IR gathered complementary information indicating a non-mature collagen at the top and periphery and a mature collagen within the nodule. Atomic force microscopy combined to IR (AFM-IR) analyses showed distinct spectra of "cell" and "collagen" rich areas. In contrast to the "cell" area, spectra of "collagen" area revealed the presence of carbohydrate moieties of collagen and/or the presence of glycoproteins. However, it was not possible to determine the collagen maturity, due to strong bands overlapping and/or possible protein orientation effects. Such findings could help developing protocols to allow a reliable characterization of in vitro generated complex bone tissues.

Retinoic Acid Excess Impairs Amelogenesis Inducing Enamel Defects.

Abnormalities of enamel matrix proteins deposition, mineralization, or degradation during tooth development are responsible for a spectrum of either genetic diseases termed Amelogenesis imperfecta or acquired enamel defects. To assess if environmental/nutritional factors can exacerbate enamel defects, we investigated the role of the active form of vitamin A, retinoic acid (RA). Robust expression of RA-degrading enzymes Cyp26b1 and Cyp26c1 in developing murine teeth suggested RA excess would reduce tooth hard tissue mineralization, adversely affecting enamel. We employed a protocol where RA was supplied to pregnant mice as a food supplement, at a concentration estimated to result in moderate elevations in serum RA levels. This supplementation led to severe enamel defects in adult mice born from pregnant dams, with most severe alterations observed for treatments from embryonic day (E)12.5 to E16.5. We identified the enamel matrix proteins enamelin (Enam), ameloblastin (Ambn), and odontogenic ameloblast-associated protein (Odam) as target genes affected by excess RA, exhibiting mRNA reductions of over 20-fold in lower incisors at E16.5. RA treatments also affected bone formation, reducing mineralization. Accordingly, craniofacial ossification was drastically reduced after 2 days of treatment (E14.5). Massive RNA-sequencing (RNA-seq) was performed on E14.5 and E16.5 lower incisors. Reductions in Runx2 (a key transcriptional regulator of bone and enamel differentiation) and its targets were observed at E14.5 in RA-exposed embryos. RNA-seq analysis further indicated that bone growth factors, extracellular matrix, and calcium homeostasis were perturbed. Genes mutated in human AI (ENAM, AMBN, AMELX, AMTN, KLK4) were reduced in expression at E16.5. Our observations support a model in which elevated RA signaling at fetal stages affects dental cell lineages. Thereafter enamel protein production is impaired, leading to permanent enamel alterations.

Nanoscale precipitation coating: the deposition of inorganic films through step-by-step spray-assembly.

Thin films and surface coatings play an important role in basic and applied research. Here we report on a new, versatile, and simple method ("precipitation coating") for the preparation of inorganic films, based on the alternate spraying of complementary inorganic salt solutions against a receiving surface on which the inorganic deposit forms. The method applies whenever the solubility of the deposited material is smaller than that of the salts in the solutions of the reactants. The film thickness is controlled from nanometers to hundreds of micrometers simply by varying the number of spraying steps; 200 spray cycles, corresponding to less than 15 min deposition time, yield films with thicknesses exceeding one micrometer and reaching tens of micrometers in some cases. The new solution-based process is also compatible with conventional layer-by-layer assembly and permits the fabrication of multimaterial sandwich-like coatings.