Fe3+和Fe2+对原代培养的成骨细胞增殖、分化和矿化功能的影响

采用噻唑蓝(MTT)法、碱性磷酸酶(ALP)比活性测定、油红O染色和茜素红染色及定量分析,研究了不同浓度的Fe3+和Fe2+对原代培养的成骨细胞增殖、分化及矿化功能的影响.结果表明:浓度为1×10-9～1×10-4 mol·L-1的Fe3+和Fe2+促进成骨细胞增殖,但是在较高浓度1×10-3 mol·L-1时,它们则抑制成骨细胞增殖.与成骨细胞作用48 h,浓度为1×10-8～1×10-4 mol·L-1的Fe3+和Fe2+抑制其分化,但在较低的浓度1×10-9 mol·L-1时则对其分化没有影响:进一步延长作用时间为72 h,Fe3+对成骨细胞分化没有影响,除1×10-6mol·L-1浓度的Fe2+促进成骨细胞分化外,其他浓度的Fe2+则抑制其分化;测试浓度下的Fe3+对成骨细胞向脂肪细胞的横向分化表现为抑制或没有影响,而Fe2+的影响则依赖于浓度和作用时间.在1×10-8～1×10-5mol·L-1浓度范围内,Fe3+和Fe2+对矿化结节的影响表现出相反的效应.在较高浓度(1×10-4mol·L-1)下,它们促进矿化节结的形成,而在较低浓度(1×10-9mol·L-1)下,Fe3+抑制矿化节结的形成,Fe2+则没有影响.结果提示:浓度.作用时间和铁离子的价态都是影响Fe3+和Fe2+生物效应(从毒性到活性,从损伤到保护,从上调到下调)转变的关键因素.     

硝酸锶对原代培养的小鼠成骨细胞增殖、分化和矿化功能的影响

采用噻唑蓝(MTT)法、碱性磷酸酶(ALP)比活性测定、油红O染色、Ⅰ型胶原测定以及矿化结节染色及定量分析等方法,研究了不同浓度的硝酸锶对原代培养的成骨细胞增殖、分化、矿化功能以及横向分化为脂肪细胞的影响。结果表明:硝酸锶对成骨细胞增殖、分化、矿化功能以及横向分化为脂肪细胞的影响与作用浓度和时间密切相关,但没有呈现出剂量依赖性。结果提示,硝酸锶对骨代谢的影响是复杂的,其具有保护还是损害作用取决于作用浓度和时间,而且它们是影响硝酸锶生物效应(从损伤到保护)转变的关键因素。     

Vitamin D and bone: — How does vitamin D regulate bone formation and resorption? —

Vitamin D was discovered as an anti-rachitic agent, but even at present, there is no direct evidence to support the concept that vitamin D directly stimulates osteoblastic bone formation and mineralization. It appears to be paradoxical, but vitamin D functions in the process of osteoclastic bone resorption. Osteoclasts, the only cells responsible for bone resorption, develop from hematopoietic cells of the monocyte-macrophage lineage. In 1992, we hypothesized that a membrane-bound factor, designated as “osteoclast differentiation factor (ODF)”, is expressed on the plasma membrane of osteoblasts/stromal cells in response to osteotropic factors including the active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. Recently, four research groups including ours independently identified three key molecules (RANKL, RANK, and OPG) responsible for osteoclastogenesis. A long-sought-after ligand, ODF, was identical to RANKL. RANKL was a member of the membrane-associated TNF ligand family, which induced differentiation of spleen cells (osteoclast progenitors) into osteoclasts in the presence of M-CSF. RANK, a member of the TNF receptor family, was a signaling receptor essential for the RANKL-mediated osteoclastogenesis. OPG, a secreted member of the TNF receptor family, was a decoy receptor for RANKL. The discovery of RANKL, RANK and OPG opens a new era in the study of bone biology and the therapy of several metabolic bone diseases such as osteoporosis, rheumatoid arthritis, and periodontal diseases.     

Osteoblast cell membrane chromatography coupled with liquid chromatography and time‐of‐flight mass spectrometry for screening specific active components from traditional Chinese medicines

A method using osteoblast membrane chromatography coupled with liquid chromatography and time‐of‐flight mass spectrometry was developed to recognize and identify the specific active components from traditional Chinese medicines. Primary rat osteoblasts were used for the preparation of the stationary phase in the cell chromatography method. Retention components from the cell chromatography were collected and analyzed by liquid chromatography with time‐of‐flight mass spectrometry. This method was applied in screening active components from extracts of four traditional Chinese medicines. In total, 24 potentially active components with different structures were retained by osteoblast cell chromatography. There were five phenolic glucosides and one triterpenoid saponin from Curculigo orchioides Gaertn, two organic acids and ten flavonoids from Epimedium sagittatum Maxim, one phthalide compound and one organic acid from Angelica sinensis Diels, and two flavonoids and two saponins from Anemarrhena asphodeloides Bunge. Among those, four components (icariin, curculigoside, ferulaic acid, and timosaponin BII) were used for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation, alkaline phosphatase activity, levels of bone gla protein and collagen type 1, and promoted mineralized nodule formation. The developed method was an effective screening method for finding active components from complex medicines that act on bone diseases.     

Biogenic Polyelectrolyte Multilayers on Poly(L-lactide) Films for Control of Osteoblast Adhesion

Cell adhesion and spreading are important events during cell-biomaterial interaction, which control survival, growth and differentiation of cells. Layer-by-layer technique was used to generate multilayer coatings for regulating adhesion of primary osteoblasts on biomaterials. Polyelectrolyte multilayers (PEM) were based on poly (ethylene imine) as primary polycation layer. PEM were then prepared from chitosan (CHI) as polycation and heparin (HEP), or sulfated HA (sHA) as polyanions. It was observed that attachment and spreading of primary osteoblasts (pOB) was highly dependent on the composition of multilayers, as well as pH values of polyelectrolyte solutions. Results presented in this paper may pave the way for application of PEM surface coatings for bone-contacting implant materials.     

Msx2 mediates the inhibitory action of TNF-�� on osteoblast differentiation

TNF-α, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-α signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-α-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-α down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2^-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-α induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-α. TNF-α activated the NF-κB and the JNK pathways. Inhibition of NF-κB or JNK activation reduced the inhibitory effect of TNF-α on ALP expression, whereas TNF-α-induced Msx2 expression was only suppressed by the inhibition of the NF-κB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-α on BMP2-regulated osteoblast differentiation and that the TNF-α-activated NF-κB pathway is responsible for Msx2 induction.     

In vitro evaluation of biodegradable poly(butylene succinate) as a novel biomaterial

Poly(butylene succinate) (PBSU) can be easily synthesized by condensation polymerization of the starting materials of succinic acid and butan-1,4-diol. It has good degradability and possesses excellent processability. Due to these advantages, PBSU was first evaluated in the present study for its potential application as a novel biomaterial. The in vitro biocompatibility of the PBSU was evaluated by monitoring proliferation and differentiation of osteoblasts cultured on the PBSU film substrates for different periods. The results showed that the PBSU was biocompatible as the osteoblasts could proliferate and differentiate on the PBSU plates. In addition, the hydrolytic degradation behavior of the PBSU films in the phosphate-buffered saline (PBS) was also investigated and the results suggested that the PBSU degraded in the PBS solution with the same behavior as that of the degradable poly(alpha-hydroxyesters). In addition to the biocompatibility and hydrolytic degradation, some physical properties, including hydrophilicity, and mechanical and thermal properties of the PBSU substrates, were also determined and the results revealed that the PBSU was hydrophilic and ductile with excellent processability. The biocompatibility of the PBSU, together with the advantages of hydrolytic degradability, hydrophilicity, and excellent processability, indicated that PBSU has the potential to be used as a biomaterial for tissue repair. [Diagram: see text] Alkaline phosphate activity of osteoblasts cultured on PBSU and TCPS substrates for different time periods.     

In vitro response of hFOB cells to pamidronate modified sodium silicate coated cellulose scaffolds

The aim of the present study was to evaluate the suitability of cellulose-based scaffolds coated with pure sodium silicate gel and sodium silicate gels accumulated with different concentrations of the bisphosphonate pamidronate as scaffolds for attachment, proliferation and differentiation of human fetal osteoblasts (hFOB 1.19). Human osteoblasts were cultured in vitro for a period up to 14 days on different cellulose scaffolds. Unmodified and sodium silicate coated cellulose scaffolds were used as control. Two surface-coated modifications of cellulose were applied. The scaffolds were coated in a modified two-step dip coating process with pure sodium silicate gel and pamidronate enriched sodium silicate gel, respectively. In order to investigate the influence of the pamidronate, concentrations of 0.667 mg Na-pamidronate/ml sodium silicate solution, 0.333 mg Na-pamidronate/ml sodium silicate solution and 3.33 x 10(-3) mg Na-pamidronate/ml sodium silicate solution were used for the coating process. Cell proliferation, vitality and attachment were examined by means of cell counting, WST-1 test, fluorescence and scanning electron microscopy. The relative grade of differentiation of hFOB cells was examined by using quantitative real-time polymerase chain reaction (qRT-PCR) analysis for the gene expression of alkaline phosphatase and osteocalcin. Proliferation and differentiation of human osteoblasts was enhanced by the sodium silicate coatings accumulated with pamidronate compared to pure sodium silicate coatings. There was a reciprocal correlation of vitality with the concentration of pamidronate. The highest vitality was found on surfaces with the lowest pamidronate accumulation. Alkaline phosphatase, an early differentiation marker, was overexpressed after 7 days in cells on all pamidronate-containing surfaces (up to 350% compared to untreated cellulose). Osteocalcin, a late differentiation marker, was overexpressed after 14 days in cells on all coated surfaces (up to 300,000% compared to untreated cellulose). The results indicate that due to the modified coating procedure a homogeneous coating and thus, an enhancement of cell attachment and subsequent cellular functions can be achieved. Low concentrations of pamidronate seem to have a relevant effect on cell proliferation and vitality and, therefore, can be recommended for the improvement of the properties of a biomaterial.