Perivascular Stem Cells at the Tip of Mouse Incisors Regulate Tissue Regeneration.

Cells with in vitro properties similar to those of bone marrow stromal stem cells are present in tooth pulp as quiescent cells that are mobilized by damage. These dental pulp stem cells (DPSCs) respond to damage by stimulating proliferation and differentiation into odontoblast-like cells that form dentine to repair the damage. In continuously growing mouse incisors, tissue at the incisor tips is continuously being damaged by the shearing action between the upper and lower teeth acting to self-sharpen the tips. We investigated mouse incisor tips as a model for the role of DPSCs in a continuous natural repair/regeneration process. We show that the pulp at the incisor tip is composed of a disorganized mass of mineralized tissue produced by odontoblast-like cells. These cells become embedded into the mineralized tissue that is rapidly formed and then lost during feeding. Tetracycline labeling not only revealed the expected incorporation into newly synthesized dentine formation of the incisor but also a zone covering the pulp cavity at the tips of the incisors that is mineralized very rapidly. This tissue was dentine-like but had a significantly lower mineral content than dentine as determined by Raman spectroscopy. The mineral was more crystalline than dentine, indicative of small, defect-free mineral particles. To identify the origin of cells responsible for deposition of this mineralized tissue, we genetically labeled perivascular cells by crossing NG2(ERT2) Cre and Nestin Cre mice with reporter mice. A large number of pericyte-derived cells were visible in the pulp of incisor tips with some having elongated, odontoblast-like shapes. These results show that in mouse incisors, rapid, continuous mineralization occurs at the tip to seal off the pulp tissue from the external environment. The mineral is formed by perivascular-derived cells that differentiate into cells expressing dentin sialo-phosphoprotein (DSPP) and produce a dentine-like material in a process that functions as continuous natural tissue regeneration.

Probing differential optical and coverage behavior in nanotube-nanocrystal heterostructures synthesized by covalent versus non-covalent approaches.

Double-walled carbon nanotube (DWNT)-CdSe heterostructures with the individual nanoscale building blocks linked together by 4-aminothiophenol (4-ATP) have been successfully synthesized using two different and complementary routes, i.e. covalent attachment and non-covalent π-π stacking. Specifically, using a number of characterization methods, we have probed the effects of these differential synthetic coupling approaches on the resulting CdSe quantum dot (QD) coverage on the underlying nanotube template as well as the degree of charge transfer between the CdSe QDs and the DWNTs. In general, based on microscopy and spectroscopy data collectively, we noted that heterostructures generated by non-covalent π-π stacking interactions evinced not only higher QD coverage density but also possibly more efficient charge transfer behavior as compared with their counterparts produced using covalent linker-mediated protocols.

Characterization of porcine aortic valvular interstitial cell 'calcified' nodules.

Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-ß1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-ß1 (OST+TGF-ß1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-ß1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-ß1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that 'calcified' nodules formed from PAVICs grown in OST+TGF-ß1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.

Gene-expression analysis reveals that embryonic stem cells cultured under osteogenic conditions produce mineral non-specifically compared to marrow stromal cells or osteoblasts.

Pluripotent cells, such as embryonic stem cells (ESCs), divide indefinitely and can differentiate to form mineralised nodules in response to osteogenic supplements. This suggests that they may be used as a cell source for bone replacement strategies. Here, we related the expression of osteogenic and chondrogenic genes in cultures of murine ESCs, marrow stromal cells (MSCs) and calvarial osteoblasts (OBs) cultured under osteogenic conditions to the biochemical composition and quantity of mineral formed. Mineralisation, measured by calcium sequestration, was >2-fold greater in ESC cultures than in either MSCs or OBs. Micro-Raman spectroscopy and spectral mapping revealed a lower mineral-to-matrix ratio and confirmed a more diffuse pattern of mineralisation in ESCs compared to MSCs and OBs. Baseline expression of chondrogenic and osteogenic genes was between 1 and 4 orders of magnitude greater in MSCs and OBs than in ESCs. Osteogenic culture of MSCs and OBs was accompanied by increases in osteogenic gene expression by factors of ~100 compared to only ~10 in ESCs. Consequentially, peak expression of osteogenic and chondrogenic genes was greater in MSCs and OBs than ESCs by factors of 100-1000, despite the fact that mineralisation was more extensive in ESCs than either MSCs or OBs. We also observed significant cell death in ESC nodules. We conclude that the mineralised material observed in cultures of murine ESCs during osteogenic differentiation may accumulate non-specifically, perhaps in necrotic cell layers, and that thorough characterisation of the tissue formed by ESCs must be achieved before these cells can be considered as a cell source for clinical applications.