Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering.

Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

2,4,5-Trimethoxyldalbergiquinol promotes osteoblastic differentiation and mineralization via the BMP and Wnt/ß-catenin pathway.

Dalbergia odorifera has been traditionally used as a medicine to treat many diseases. However, the role of 2,4,5-trimethoxyldalbergiquinol (TMDQ) isolated and extracted from D. odorifera in osteoblast function and the underlying molecular mechanisms remain poorly understood. The aim of this study was to investigate the effects and possible underlying mechanisms of TMDQ on osteoblastic differentiation of primary cultures of mouse osteoblasts as an in vitro assay system. TMDQ stimulated osteoblastic differentiation, as assessed by the alkaline phosphatase (ALP) activity, ALP staining, mineralized nodule formation, and the levels of mRNAs encoding the bone differentiation markers, including ALP, bone sialoprotein (BSP), osteopontin, and osteocalcin. TMDQ upregulated the expression of Bmp2 and Bmp4 genes, and increased the protein level of phospho-Smad1/5/8. Furthermore, TMDQ treatment showed the increased mRNA expression of Wnt ligands, phosphorylation of GSK3, and the expression of ß-catenin protein. The TMDQ-induced osteogenic effects were abolished by Wnt inhibitor, Dickkopf-1 (DKK1), and bone morphogenetic protein (BMP) antagonist, noggin. TMDQ-induced runt-related transcription factor 2 (Runx2) expression was attenuatted by noggin and DKK1. These data suggest that TMDQ acts through the activation of BMP, Wnt/ß-catenin, and Runx2 signaling to promote osteoblast differentiation, and we demonstrate that TMDQ could be a potential agent for the treatment of bone loss-associated diseases such as osteoporosis.

PML overexpression inhibits proliferation and promotes the osteogenic differentiation of human mesenchymal stem cells.

The promyelocytic leukemia (PML) gene, as an important tumor-suppressor, has been proven to regulate stem cell function in multiple tissues; however its role in human mesenchymal stem cells (hMSCs) remains unclear. In the present study, the effect of PML on regulating the proliferation and osteogenic differentiation of hMSCs was explored. New downstream genes that may be responsible for the regulation of PML were found, and possible mechanisms were analyzed. The lentiviral vector which encodes full-length human PML cDNA or shRNA against PML was transfected into hMSCs. RT-PCR and western blotting were used to detect mRNA and protein expression. Flow cytometry was used to analyze apoptosis and the cell cycle distribution. Osteogenic differentiation of hMSCs was induced by osteo-inductive medium for 7 to 14 days. cDNA microarray was used to scan the gene expression profile and to identify significant changes in gene expression. In the present study, we found that PML was stably expressed in hMSCs, and the expression was increased time-dependently along with cell osteogenic differentiation. Overexpression of PML inhibited hMSC proliferation by inducing apoptosis and arresting the cell cycle. However, PML enhanced the osteoblast differentiation potential of hMSCs. PML-overexpressing hMSCs had a significant increase in mineralized matrix production and ALP activity on day 7 under osteogenic or non-osteogenic differentiation conditions. Upregulation of integrin-binding sialoprotein (IBSP, bone sialoprotein) induced by PML overexpression was found. Our data indicate that PML regulates hMSCs as an inhibitor of cell proliferation but a promoter of osteogenic differentiation.

High phosphate feeding induced arterial medial calcification in uremic rats: roles of Lanthanum carbonate on protecting vasculature.

AIMS: High cardiovascular mortality in patients with end-stage renal disease is closely associated with arterial medial calcification (AMC) caused by hyperphosphatemia, the mechanism of which associated hormones (FGF-23, klotho) and osteochondrogenic events is unclear. We examined the effect of Lanthanum carbonate on AMC via regulating the abnormalities in phosphorus metabolism of uremic rats. MAIN METHODS: 45 healthy SD rats were randomly divided into 3 groups: Normal group (n=15), CRF group (n=15), CRF diet supplemented with 2% La (n=15). AMC in great arteries were evaluated by VonKossa. Osteochondrogenic specific genes were analyzed by Immunohistochemistry and qRT-PCR. Serum FGF-23 and klotho levels were detected by ELISA kit. KEY FINDINGS: Serum phosphate was markedly increased in CRF group (6.94 ± 0.97 mmol/L) and 2%La group (5.12 ± 0.84 mmol/L) at week 4, while the latter became hypophosphatemic (2.92 ± 0.73 mmol/L vs CRF group, p<0.01) at week 10. Inhibitory effect of 2%La on development of AMC was reflected by downregulated Runx2, Osterix, BSP, Osteocalcin and collagenII and a reduction of FGF-23 at week 4(vs CRF group, p<0.01) but not week 10. SIGNIFICANCE: Beneficial effects of Lanthanum carbonate on progression of AMC in CRF could be mainly due to the decreased phosphate retention and FGF-23 in early stage and likewise a reduction of bone-associated proteins via osteochondrogenic pathway. Lanthanum carbonate has no effect on soluble klotho and serum FGF-23 in late stage of CRF.

Transcriptional regulation of bone sialoprotein gene by CO(2) laser irradiation.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Low-power laser irradiation has a stimulating effect on cells and tissues. Although the carbon dioxide (CO(2)) laser is a hard surgical laser, we have attempted to use it at low energy density to achieve biological alterations. To investigate the effects of CO(2) laser irradiation on BSP gene transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were increased at 12 h after irradiation with the CO(2) laser (2 W, 20 s). Transient transfection assays using various sizes of the rat BSP gene promoter linked to the luciferase reporter gene showed that CO(2) laser irradiation induced luciferase activity of a -116 to +60 BSP promoter construct (pLUC3) at 12 h in the cells. Transcriptional stimulation by CO(2) laser irradiation was abrogated in the pLUC3 construct containing a 2-bp mutation in the fibroblast growth factor 2 response element (FRE). Gel shift analyses showed that CO(2) laser irradiation increased the binding of nuclear protein to FRE. These studies demonstrate that CO(2) laser irradiation increases BSP transcription via FRE in the rat BSP gene promoter.

Effects of low-level laser therapy on human osteoblastic cells grown on titanium.

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

Effects of low-level laser therapy on human osteoblastic cells grown on titanium

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumínio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possível sugerir possíveis benefícios do laser na osseointegração de implantes de Ti apesar do efeito deletério às células imediatamente após a irradiação.