MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP). In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with beta-GP. These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.
"At first, biologists analyzed osteoblast activities of cells seeded on glass or plastic, easy and convenient two-dimensional cell culture models –. These experiments allowed to study the osteoblast phenotype through the production of alkaline phosphatase, bone Gla protein (osteocalcin), various acid-rich SCPPs, and mineral deposition. "
[Show abstract][Hide abstract] ABSTRACT: Among persisting questions on bone calcification, a major one is the link between protein expression and mineral deposition. A cell culture system is here proposed opening new integrative studies on biomineralization, improving our knowledge on the role played by non-collagenous proteins in bone. This experimental model consisted in human primary osteoblasts cultured for 60 days at the surface of a 3D collagen scaffold mimicking an osteoid matrix. Various techniques were used to analyze the results at the cellular and molecular level (adhesion and viability tests, histology and electron microscopy, RT- and qPCR) and to characterize the mineral phase (histological staining, EDX, ATG, SAED and RMN). On long term cultures human bone cells seeded on the osteoid-like matrix displayed a clear osteoblast phenotype as revealed by the osteoblast-like morphology, expression of specific protein such as alkaline phosphatase and expression of eight genes classically considered as osteoblast markers, including , and . Von Kossa and alizarine red allowed us to identify divalent calcium ions at the surface of the matrix, EDX revealed the correct Ca/P ratio, and SAED showed the apatite crystal diffraction pattern. In addition RMN led to the conclusion that contaminant phases were absent and that the hydration state of the mineral was similar to fresh bone. A temporal correlation was established between quantified gene expression of DMP1 and IBSP, and the presence of hydroxyapatite, confirming the contribution of these proteins to the mineralization process. In parallel a difference was observed in the expression pattern of and which questioned their attributed role in the literature. The present model opens new experimental possibilities to study spatio-temporal relations between bone cells, dense collagen scaffolds, NCPs and hydroxyapatite mineral deposition. It also emphasizes the importance of high collagen density environment in bone cell physiology.
PLoS ONE 02/2013; 8(2):e57344. DOI:10.1371/journal.pone.0057344 · 3.23 Impact Factor
"The cells were seeded on surfaces without FCS during 12 h that to allow the BMP mimetic peptides act on receptors without hassle of serum proteins. For evaluation of matrix mineralization, we put dexamethasone (10 À8 M), ascorbic acid (50 mg/ml), and b-glycerolphosphate (8 mM) in the media for 4 days  "
[Show abstract][Hide abstract] ABSTRACT: The bone morphogenetic proteins (BMPs) are cytokines of the transforming growth factor beta family. Some BMPs such as BMP-2, BMP-7 and BMP-9 play a major role in the bone and cartilage formation. The BMP peptides corresponding to residues 73-92, 89-117, and 68-87 of BMP-2, BMP-7 and BMP-9 respectively as well as adhesion peptides (GRGDSPC) were grafted onto polyethylene terephthatalate (PET) surfaces. We evaluated the state of differentiation of pre-osteoblastic cells. The behavior of these cells on various functionalized surfaces highlighted the activity of the mimetic peptides immobilized on surfaces. The induced cells (observed in the case of surfaces grafted with BMP-2, 7 or 9 mimetic peptides and GRGDSPC peptides) were characterized on several levels. First of all, we focused on the evaluation of the osteoblastic markers such as the transcriptional factor Runx2, which is a critical regulator of osteoblastic differentiation. Secondly, the results obtained showed that these induced cells take a different morphology compared to the cells in a state of proliferation or in a state of extracellular matrix production. Induced cells were characterized by an increased thickness compared to non-induced cells. Thus, our studies prove a direct correlation between cell morphology and state of induction. Thereafter, we focused on characterizing the extracellular matrix formed by the cells on various surfaces. The extracellular matrix thickness was more significant in the case of surfaces grafted with mimetic peptides of the BMP-2, 7 or 9 and GRGDSPC peptides which once again proves their activity when immobilized on material surface. These results demonstrate that GRGDSPC and BMPs peptides, grafted to PET surface, act to enhance osteogenic differentiation and mineralization of pre-osteoblastic cells. These findings are potentially useful in developing engineered biomaterials for bone regeneration.
[Show abstract][Hide abstract] ABSTRACT: Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2004. Includes bibliographical references. (cont.) implementation of this scintigraphic method for quantitative studies of osteoblast-mediated mineralization in vitro. A 2-D truss finite element model is used to study the remodeling of trabecular bone. Using strain energy density (SED) as the optimization object and the trabecular width as the optimization variable, an optimal structure with minimum SED was achieved. This structure is similar to real bone in the dense outside, porous inside, and orientation of the trabeculae. The bone density distribution pattern also matched with previous result by other people. Different implants were introduced to simulate the replacement for the femoral head. It has been proved that the difference in Young's modulus between bone and implant materials is the main reason for the long-term bone loss (stress screening). This problem can be alleviated by proper implant design and resurfacing instead of replacing the whole femoral head. Initial fixation with bone and the long term bone loss are two main problems associated with total hip replacement (THR), which are studied by electron microscope and computer simulation in this thesis. Bare Titanium-6 wt% Aluminum-4 wt% Vanadium (Ti64) implants, Ti64 implants with plasma-sprayed hydroxyapatite (PSHA), and Ti64 implants with electrochemically-deposited hydroxyapatite (EDHA) were implanted into canine trabecular bone for 6 hours, 7 days, and 14 days to study the initial bone formation on these implant materials. Scanning electron microscope (SEM) results showed that at 7 days PSHA had a higher bone apposition ratio than Ti64 and EDHA samples; however, at 14 days, the bone apposition on EDHA increased to be similar to PSHA, much higher than that on Ti64. By transmission electron microscope (TEM), a layer of new bone tissue was observed on PSHA coating surface; in contrast, no much bone was found on EDHA surface. At 14 days, substantial bone was found on both EDHA and EDHA coating surface. Technetium-99m-methylene-diphosphonate (Tc-99m-MDP) labeling was used to quantify mineralization of cultures of MC3T3 osteoblast-like cells in vitro on tissue culture polystyrene (TCPS). The gamma signal from labeled samples was imaged with a gamma camera and compared with the calcium content in the same samples determined by inductively coupled plasma. The high correlation (0.88) between these two values validated that radiotracer uptake method as a quantitative analytical tool for certain mineralization studies in vitro. There was an association between mineralization and radionuclide uptake in the MC3T3 cultures on titanium alloy, but the attenuation of the gamma photons by the metal resulted in a less robust correlation. The results warrant by Hao Wang. Ph.D.
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