Induced osteogenesis includes a program of microRNAs (miRs) to repress the translation of genes that act as inhibitors of bone formation. How expression of bone-related miRs is regulated remains a compelling question. Here we report that Runx2, a transcription factor essential for osteoblastogenesis, negatively regulates expression of the miR cluster 23a∼27a∼24-2. Overexpression, reporter, and chromatin immunoprecipitation assays established the presence of a functional Runx binding element that represses expression of these miRs. Consistent with this finding, exogenous expression of each of the miRs suppressed osteoblast differentiation, whereas antagomirs increased bone marker expression. The biological significance of Runx2 repression of this miR cluster is that each miR directly targets the 3′ UTR of SATB2, which is known to synergize with Runx2 to facilitate bone formation. The findings suggest Runx2-negative regulation of multiple miRs by a feedforward mechanism to cause derepression of SATB2 to promote differentiation. We find also that miR-23a represses Runx2 in the terminally differentiated osteocyte, representing a feedback mechanism to attenuate osteoblast maturation. We provide direct evidence for an interdependent relationship among transcriptional inhibition of the miR cluster by Runx2, translational repression of Runx2 and of SATB2 by the cluster miRs during progression of osteoblast differentiation. Furthermore, miR cluster gain of function (i.e., inhibition of osteogenesis) is rescued by the exogenous expression of SATB2. Taken together, we have established a regulatory network with a central role for the miR cluster 23a∼27a∼24-2 in both progression and maintenance of the osteocyte phenotype.bone formation by miRs | Runx2-regulation of microRNA cluster | phenotype attenuation by miRs | feed forward-feedback miR control | SATB2 targeted by cluster miRs N umerous regulatory pathways governing osteoblast differentiation involve transcription factors, signaling molecules, and chromatin modifiers (1-3). Recent evidence has shown that osteogenic induction and differentiation are also regulated by posttranscriptional mechanisms, most significantly by temporally expressed microRNAs (miRs) (4, 5).miRNAs are small noncoding RNAs that significantly regulate the translation of protein coding genes in higher organisms (6, 7). These small RNAs (approximately 22 nt) are involved in almost every biological process, including early development, lineage commitment, growth and differentiation, cell death, and metabolic control (4-7). The identification of miRs that characterize cancer and genetic and metabolic abnormalities provide new approaches for treatment of diseases (8-11).The recent discovery of miRNAs in regulating in vivo bone formation (12) and in vitro osteoblast differentiation has provided insights into the potent activity of miRs in the skeleton (4,5,(13)(14)(15). Studies show that the osteogenic BMP2 down-regulates a cohort of miRs that inhibit bone formation (4, 16). In an introductory miR profilin...

The surface characteristics of biomaterials can influence protein adsorption, cellular functions, and ultimately tissue formation. Controlled chemical oxidation of titanium-based surfaces with a mixture of H(2)SO(4)/H(2)O(2) creates a nanopatterned surface that has been shown to affect early osteogenic events. The objective of this study was to evaluate the effect over time of this nanopattern on various key parameters of osteogenesis, and determine whether these effects ultimately translate into more mineralized matrix production. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvaria and grown on treated and untreated titanium discs for periods of up to 14 days. Alkaline phosphatase activity peaked earlier and cell number was higher as of day 7 on the nanopatterned discs. Immunofluorescence showed that the treated surface favored early bone sialoprotein and osteopontin secretion, and fibronectin accumulation. Alizarin red staining revealed that, at days 10 and 14, there were significantly more mineralized nodules on treated than on untreated discs. These results demonstrate that simple chemical treatment of titanium with H(2)SO(4)/H(2)O(2) accelerates the in vitro osteogenic potential of calvaria-derived cells. They also suggest that this treatment may represent an advantageous approach for producing "intelligent surfaces" that stimulate bone formation and enhance bone-implant contact.

One of the strategies to improve the mechanical performance of bioactive glasses for load-bearing implant devices has been the development of glass-ceramic materials. The present study aimed to evaluate the effect of a highly bioactive, fully-crystallized glass-ceramic (Biosilicate) of the system P(2)O(5)-Na(2)O-CaO-SiO(2) on various key parameters of in vitro osteogenesis. Surface characterization was carried out by scanning electron microscopy and Fourier transform infrared spectroscopy. Osteogenic cells were obtained by enzymatic digestion of newborn rat calvarial bone and by growing on Biosilicate discs and on control bioactive glass surfaces (Biosilicate) parent glass and Bioglass(R) 45S5) for periods of up to 17 days. All materials developed an apatite layer in simulated body fluid for 24h. Additionally, as early as 12 h under culture conditions and in the absence of cells, all surfaces developed a layer of silica-gel that was gradually covered by amorphous calcium phosphate deposits, which remained amorphous up to 72 h. During the proliferative phase of osteogenic cultures, the majority of cells exhibited disassembly of the actin cytoskeleton, whereas reassembly of actin stress fibers took place only in areas of cell multilayering by day 5. Although no significant differences were detected in terms of total protein content and alkaline phosphatase activity at days 11 and 17, Biosilicate supported significantly larger areas of calcified matrix at day 17. The results indicate that full crystallization of bioactive glasses in a range of compositions of the system P(2)O(5)-Na(2)O-CaO-SiO(2) may promote enhancement of in vitro bone-like tissue formation in an osteogenic cell culture system.

The aim of our study was to investigate the osteoinductive potential of a titanium (Ti) surface with nanotopography, using mesenchymal stem cells (MSCs) and the mechanism involved in this phenomenon. Polished Ti discs were chemically treated with H2 SO4 /H2 O2 to yield nanotopography and rat MSCs were cultured under osteogenic and non-osteogenic conditions on both nanotopography and untreated polished (control) Ti surfaces. The nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of key bone markers of cells grown under both osteogenic and non-osteogenic conditions. Additionally, the gene expression of α1 and β1 integrins was higher in cells grown on Ti with nanotopography under non-osteogeneic condition compared with control Ti surface. The higher gene expression of bone markers and Alp activity induced by Ti with nanotopography was reduced by obtustatin, an α1β1 integrin inhibitor. These results indicate that α1β1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. This finding highlights a novel mechanism involved in nanosurface-mediated MSCs fate and may contribute to the development of new surface modifications aiming to accelerate and/or enhance the process of osseointegration.

There is general agreement that rough surfaces improve both biologic and biomechanical responses to titanium (Ti) implants. The aim of this investigation was to study the effect of Ti surface roughness on the response of human bone marrow cell culture evaluating: cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on commercially pure titanium (cpTi) discs with fourdifferent average roughnesses (Ra). For attachment evaluation, cells were cultured for 4 h. After 21 days, cell proliferation, total protein content, and ALP activity were evaluated. For bone-like nodule formation, cells were cultured for 28 days. Data were compared by ANOVA and Duncan's multiple range test. Cell attachment was not affected by surface roughness. For cells cultured on Ti with Ra ranging from 0.80 microm to 1.90 microm, proliferation was reduced while total protein content, and ALP activity were increased. There was a non-statistically significant increase of bone-like nodule formation on a surface with Ra near 0.80 microm. These results suggest that for Ti an Ra ranging from 0.80 microm to 1.90 microm would optimize both intermediary and final cellular responses but not affect the initial response, and a smoother surface would not favor any evaluated response.