Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

Mineralization of dense collagen hydrogel scaffolds by human pulp cells.

While advances in biomineralization have been made in recent years, unanswered questions persist on bone- and tooth-cell differentiation, on outside-in signaling from the extracellular matrix, and on the link between protein expression and mineral deposition. In the present study, we validate the use of a bioengineered three-dimensional (3D) dense collagen hydrogel scaffold as a cell-culture model to explore these questions. Dental pulp progenitor/stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into an extracellular matrix-like collagen gel whose fibrillar density was increased through plastic compression. SHED viability, morphology, and metabolic activity, as well as scaffold mineralization, were investigated over 24 days in culture. Additionally, measurements of alkaline phosphatase enzymatic activity, together with immunoblotting for mineralized tissue cell markers ALPL (tissue-non-specific alkaline phosphatase), DMP1 (dentin matrix protein 1), and OPN (osteopontin), demonstrated osteo/odontogenic cell differentiation in the dense collagen scaffolds coincident with mineralization. Analyses of the mineral phase by electron microscopy, including electron diffraction and energy-dispersive x-ray spectroscopy, combined with Fourier-transform infrared spectroscopy and biochemical analyses, were consistent with the formation of apatitic mineral that was frequently aligned along collagen fibrils. In conclusion, use of a 3D dense collagen scaffold promoted SHED osteo/odontogenic cell differentiation and mineralization.

Enhanced osteoblast adhesion on transglutaminase 2-crosslinked fibronectin.

Fibronectin (FN) is a cell adhesion protein that binds integrins in a process also involving the protein-crosslinking enzyme transglutaminase 2 (TG2) as a co-receptor. The cell-adhesive property of TG2 has been linked to a complex formation with FN and to its ability to crosslink and polymerize FN on the cell surface. We tested here the effects of extracellular FN, before and after in vitro crosslinking and polymerization by TG2, on MC3T3-E1 osteoblast adhesion. We show that TG2-mediated crosslinking creates large, compacted chain-like protein clusters that include both TG2 and FN molecules as analyzed by Western blotting and atomic force microscopy. Crosslinking of FN significantly promotes osteoblast adhesion as measured by crystal violet staining, and enhances beta(1)-integrin clustering on the cell surface as visualized by immunofluorescence microscopy. We hypothesize that TG2-mediated crosslinking enhances the cell-adhesive properties of FN by increasing the molecular rigidity of FN in the extracellular matrix.

Calcium phosphate thin film processing by pulsed laser deposition and in situ assisted ultraviolet pulsed laser deposition.

Calcium orthophosphates (CaP) and hydroxyapatite (HA) were intensively studied in order to design and develop a new generation of bioactive and osteoconductive bone prostheses. The main drawback now in the CaP and HA thin films processing persists in their poor mechanical characteristics, namely hardness, tensile and cohesive strength, and adherence to the metallic substrate. We report here a critical comparison between the microstructure and mechanical properties of HA and CaP thin films grown by two methods. The films were grown by KrF* pulsed laser deposition (PLD) or KrF* pulsed laser deposition assisted by in situ ultraviolet radiation emitted by a low pressure Hg lamp (UV-assisted PLD). The PLD films were deposited at room temperature, in vacuum on Ti-5Al-2.5Fe alloy substrate previously coated with a TiN buffer layer. After deposition the films were annealed in ambient air at 500-600 degrees C. The UV-assisted PLD films were grown in (10(-2)-10(-1) Pa) oxygen directly on Ti-5Al-2.5Fe substrates heated at 500-600 degrees C. The films grown by classical PLD are crystalline and stoichiometric. The films grown by UV-assisted PLD were crystalline and exhibit the best mechanical characteristics with values of hardness and Young modulus of 6-7 and 150-170 GPa, respectively, which are unusually high for the calcium phosphate ceramics. To the difference of PLD films, in the case of UV-assisted PLD, the GIXRD spectra show the decomposition of HA in Ca(2)P(2)O(7), Ca(2)P(2)O(9) and CaO. The UV lamp radiation enhanced the gas reactivity and atoms mobility during processing, increasing the tensile strength of the film, while the HA structure was destroyed.