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

Abstract

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.

Plant-derived Cellulose Scaffolds for Bone Tissue Engineering

“…After the 4-week period in differentiation medium, both cell-seeded scaffold types displayed signs of ECM mineralization. Indeed, aggregates of minerals were visible on the scaffold constructs, specifically on the edges of the pores, which corroborate a previous study by Addison et al [22] using MC3T3-E1 extracellular matrix. These aggregates were not visible on the bare scaffolds without cells.…”

In vivovisualization of collagen fiber produced by cultured osteoblasts using sensitive second-harmonic-generation microscopy equipped with a 10-fs mode-locked Ti:sapphire laser

“…A series of these SHG image clearly visualized that the collagen fibers were first produced in the osteoblasts, were secerned outside of osteoblasts, and then formed the networks structure of thick collagen fibers. Such dynamics of the collagen fibers in the cultured osteoblasts will give significant knowledge in the bone tissue engineering [3]. …”

“…On the other hand, if the high-peak electric field in ultrashort pulse laser is interacted with the matter, the non-linear terms are included in the polarization wave in addition to the linear term. The resulting non-linear polarization P NL is given by the following equation (2) where χ (2) and χ (3) are the second-order and third-order non-linear optical susceptibilities. When the non-linear terms are included in the polarization wave, the temporal waveform of the polarization is distorted and then harmonic components of optical frequency ω of the incident light, namely 2 ω, 3 ω, •••••, appear.…”

Vibrational spectroscopy and imaging: applications for tissue engineering

“…For instance, MIR methods have been used to evaluate the bioactivity of acellular scaffolds by accessing the degree of apatite precipitation on them after immersion in physiological solutions, based on the presence and relative intensity of the ν 3 PO 4 band 108–111 . MIR spectroscopy has also been shown as a valuable analytical tool to reveal several properties of the mineralized matrix produced by osteogenic cells in cell culture systems 112–115 . This also includes the analyses of the mineral and matrix produced during treatment with osteogenic factors and drugs 116–119 .…”