Biomineralization of a Self-Assembled Extracellular Matrix for Bone Tissue Engineering

Abstract

Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the understanding of bone formation and the development of a successfully engineered bone tissue scaffold. It is still unclear how ECM mechanical properties affect protein–mineral interactions in early stages of bone mineralization. We investigated the longitudinal mineralization properties of MC3T3-E1 cells and the elastic modulus of their ECM using shear modulation force microscopy, synchrotron grazing incidence X-ray diffraction (GIXD), scanning electron microscopy, energy dispersive X-ray spectroscopy, and confocal laser scanning microscopy (CLSM). The elastic modulus of the ECM fibers underwent significant changes for the mineralizing cells, which were not observed in the nonmineralizing cells. On substrates conducive to ECM network production, the elastic modulus of mineralizing cells increased at time points corresponding to mineral production, whereas that of the nonmineralizing cells did not vary over time. The presence of hydroxyapatite in mineralizing cells and the absence thereof in the nonmineralizing ones were confirmed by GIXD, and CLSM showed that a restructuring of actin occurred only for mineral-producing cells. These results show that the correct and complete development of the ECM network is required for osteoblasts to mineralize. This in turn requires a suitably prepared synthetic substrate for bone development to succeed in vitro.

Complementary effects of multi-protein components on biomineralization in vitro

“…A 20 nm thick coating of Sulfonated Polystyrene (SPS) polymer on either a clean Si wafer (500 μm thick) or a Silicon-Nitride membrane window (100 nm-thick) provides a surface conducive to protein fiber formation (Subburaman et al, 2006; Meng et al, 2009; Pernodet et al, 2003). After 4 days of incubation at 37 °C, proteins from solution are adsorbed on the SPS-coated silicon surface.…”

“…1C) self-assemble into fiber networks, of slightly different dimension. The SPS coating is necessary: when bare silicon substrates are used, no fibrillar ECM network forms; only small amounts of protein globules, <300 nm in height, can be imaged (Meng et al, 2009). In Fig.…”

“…This is only a lower boundary, however, since further details of grain size and strain are not accessible due to the geometry of the GIXD method, which causes additional peak broadening (see supplemental Fig. 4 of Meng et al (2009)). The EL sample has no X-ray diffraction pattern even after day 28 (Fig.…”

“…In addition, synthetic Hydroxyapatite and Amorphous Calcium Phosphate powders (Sigma–Aldrich, Inc., St. Louis, MO) as controls were investigated for reference in both glass capillary tubes (1.0 mm diam.) and as compressed powders on a blank SPS substrate (Meng et al, 2009). …”

Restoration of Critical-Size Defects in the Rabbit Mandible Using Porous Nanohydroxyapatite-Polyamide Scaffolds

“…[23][24][25][26][27] Meng et al have reported that bone tissue engineering requires endogenous bone cells, extracellular matrix, and scaffold materials to be addressed holistically as a three-component system. 28 Endogenous bone cells, which mainly differentiate from BMSCs, play an important role in bone formation. The fact that many small bone defects can heal by themselves demonstrates the key role played by endogenic BMSCs in bone regeneration.…”