BACKGROUND: The molecular pathways of how endocrine disruptors affect bone mineral density (BMD) and bone remodeling are still unclear. The purpose of this experimental study is to determine the effects of di(2-ethylhexyl)phthalate (DEHP) on bone metabolism in ovariectomized mice. METHODS: Twenty-six-month-old female CD-1 mice were divided into 4 groups: control, low-dose DEHP, high-dose DEHP, and estrogen groups (n=5, each group). All mice were subjected to ovariectomy for the induction of artificial menopause and then exposed to corn oil, DEHP, and estrogen for 2 months. Micro-computed tomography (Micro-CT) of the bone and analysis of blood samples for bone markers were performed to observe the changes in bone metabolism. RESULTS: Osteocalcin level was decreased in the control, low-dose and high-dose DEHP group, the reduction width was greater in the high-dose DEHP group (−0.219 ng/mL) than control group (−0.077 ng/mL, P<0.05). C-terminal telopeptide of type I collagen level was increased in the control, low-dose and high-dose DEHP group, the increase range of low-dose DEHP group (0.329 ng/mL) showed greater than control group (0.093 ng/mL, P<0.05). Micro-CT analysis revealed that the BMD was significantly lower in the high-dose DEHP group (19.8×10⁻² g/cm³) than control group (27.2×10⁻² g/cm³, P<0.05). The structure model index was significantly higher in the high-dose DEHP group (2.737) than low-dose DEHP group (2.648) and estrogen group (2.63, P<0.05). It means the progression of osteoporosis in the high-dose DEHP group. CONCLUSIONS: These results confirm the negative effects of DEHP on bone health in ovariectomized mice. Further continuous studies on genetic pathways and other endocrine disruptors will be necessary to validate these findings.
Animals ; Bone Density ; Bone Remodeling ; Collagen Type I ; Corn Oil ; Diethylhexyl Phthalate ; Endocrine Disruptors ; Estrogens ; Female ; Humans ; Menopause ; Metabolism ; Mice ; Osteocalcin ; Osteoporosis ; Ovariectomy ; X-Ray Microtomography

The aim of this study was to prepare inclusion nanocomplexes of hyaluronic acid-β-cyclodextrin and simvastatin (HA-β-CD/SIM) and evaluate in vitro anti-inflammation effects on lipopolysaccharide (LPS)-activated synoviocytes and chondrogenic differentiation effects on rat adipose-derived stem cells (rADSCs). The β-CD moieties in HA-β-CD could incorporate SIM to form HA-β-CD/SIM nanocomplexes with diameters of 297–350 nm. HA-β-CD/SIM resulted in long-term release of SIM from the nanocomplexes for up to 63 days in a sustained manner. In vitro studies revealed that HA-β-CD/SIM nanocomplexes were able to effectively and dose-dependently suppress the mRNA expression levels of proinflammatory markers such as matrix metallopeptidase-3 (MMP-3), MMP-13, cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in LPS-stimulated synoviocytes. HA-β-CD/SIM-treated rADSCs significantly and dose-dependently enhanced mRNA expressions of aggrecan, collagen type II (COL2A1), and collagen type X (COL10A1), implying that HA-β-CD/SIM greatly induced the chondrogenic differentiation of rADSCs. Conclusively, HA-β-CD/SIM nanocomplexes will be a promising therapeutic material to alleviate inflammation as well as promote chondrogenesis.
Aggrecans ; Animals ; Chondrogenesis ; Collagen Type II ; Collagen Type X ; Cyclooxygenase 2 ; In Vitro Techniques* ; Inflammation ; Interleukin-6 ; Rats ; RNA, Messenger ; Simvastatin* ; Stem Cells ; Thrombospondins ; Tumor Necrosis Factor-alpha

Surface characteristics and cellular response to titanium surfaces that had been implanted with calcium and magnesium ions using plasma immersion ion implantation and deposition (PIIID) were evaluated. Three different titanium surfaces were analyzed: a resorbable blast media (RBM) surface (blasted with hydroxyapatite grit), a calcium ionimplanted surface, and a magnesium ion-implanted surface. The surface characteristics were investigated by scanning electron microscopy (SEM), surface roughness testing, X-ray diffraction (XRD), and Auger electron spectroscopy (AES). Human bone marrow derived mesenchymal stem cells were cultured on the 3 different surfaces. Initial cell attachment was evaluated by SEM, and cell proliferation was determined using MTT assay. Real-time polymerase chain reaction (PCR) was used to quantify osteoblastic gene expression (i.e., genes encoding RUNX2, type I collagen, alkaline phosphatase, and osteocalcin). Surface analysis did not reveal any changes in surface topography after ion implantation. AES revealed that magnesium ions were present in deeper layers than calcium ions. The calcium ion- and magnesium ion-implanted surfaces showed greater initial cell attachment. Investigation of cell proliferation revealed no significant difference among the groups. After 6 days of cultivation, the expression of RUNX2 was higher in the magnesium ion-implanted surface and the expression of osteocalcin was lower in the calcium ion-implanted surface. In conclusion, ion implantation using the PIIID technique changed the surface chemistry without changing the topography. Calcium ion- and magnesium ion-implanted surfaces showed greater initial cellular attachment.
Alkaline Phosphatase ; Bone Marrow* ; Calcium* ; Cell Proliferation ; Chemistry ; Collagen Type I ; Durapatite ; Gene Expression ; Humans* ; Immersion ; Ions* ; Magnesium* ; Mesenchymal Stromal Cells* ; Microscopy, Electron, Scanning ; Osteoblasts ; Osteocalcin ; Osteogenesis ; Plasma ; Real-Time Polymerase Chain Reaction ; Spectrum Analysis ; Titanium* ; X-Ray Diffraction

SRT1720, a new discovered drug, was reported to activate silent information regulator 1 (SIRT1) and inhibit the chondrocyte apoptosis. However, the underlying mechanism remains elusive. In the present study, the chondrocytes were extracted from the cartilage tissues of New Zealand white rabbits, cultured in the presence of sodium nitroprusside (SNP) (2.5 mmol/L) and divided into five groups: 1, 5, 10, and 20 μmol/L SRT1720 groups and blank control group (0 μmol/L SRT1720). MTT assay was used to detect the chondrocyte viability and proliferation, and DAPI staining and flow cytometry to measure the chondrocyte apoptosis. The expression levels of SIRT1, p53, NF-κB/p65, Bax, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) were detected by Western blotting and the expression levels of SIRT1, type II collagen, and aggrecan mRNA by RT-PCR. The results showed that in the SRT1720-treated groups, the nuclei of chondrocytes were morphologically intact and had uniform chromatin. In the blank control group, nuclear rupture into debris was observed in chondrocytes. With the SRT1720 concentration increasing, the chondrocyte viability increased, the apoptosis rate decreased, the protein expression levels of SIRT1 and PGC-1α and the mRNA expression levels of type II collagen and aggrecan increased ({ptP}<0.05), and the expression levels of p53, NF-κB and bax decreased (P<0.05). It was suggested that SRT1720 inhibits chondrocyte apoptosis by activating the expression of SIRT1 via p53/bax and NF-κB/PGC-1α pathways.
Aggrecans ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Cartilage, Articular ; cytology ; drug effects ; metabolism ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Chondrocytes ; cytology ; drug effects ; metabolism ; Chromatin ; chemistry ; drug effects ; metabolism ; Collagen Type II ; genetics ; metabolism ; Gene Expression Regulation ; Heterocyclic Compounds, 4 or More Rings ; pharmacology ; Nitroprusside ; toxicity ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; genetics ; metabolism ; Primary Cell Culture ; Rabbits ; Signal Transduction ; drug effects ; genetics ; Sirtuin 1 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVETo investigate the effect of eletroacupuncture with close-to-bone needling treatment on expression of Sox9, vascular endothelial growth factor (VEGF) and type X collagen (ColX) in impaired cartilage of rabbits with knee osteoarthritis (KOA) and explore its possible mechanisms.

METHODSForty New Zealand rabbits were randomized equally into normal control group, KOA model group, eletroacupuncture with close-to-bone needling group (CN group), and normal thrust needing group (NTN group). In the latter 3 groups, KOA was induced by Hulth-Telhag treatment and evaluated with X-ray examination, and 6 weeks after the modeling, eletroacupuncture for 20 min was administered in CN and NTN groups at the acupoints "Zusanli", "Waixiyan", "Neixiyan", "Liangqiu" and "Yinlingquan" in the left knee joints once daily for 5 days as a treatment cycle. After 5 treatment cycles, the rabbits were examined for behavioral changes, cartilage morphology, and Mankin scores; The protein and mRNA expressions of S0x9, VEGF, and ColX were examined using Westen blotting, immunohistochemistry, and RT-PCR as appropriate.

RESULTSThe rabbits in the model, CN and NTN groups showed significant changes in behaviors and cartilage histomorphology after the modeling and after the treatments. HE staining showed that cartilage injury was repaired and tended to recovery in CN and NTN groups. The cartilage pathologies was severer in the model group than in the normal control, CN and NTN groups (P<0.01); Sox9 protein increased and VEGF mRNA level decreased in CN and NTN groups after treatment as compared with those in the model group (P<0.01).

CONCLUSIONEletroacupuncture with close-to-bone needling can effectively improve KOA in rabbits probably by enhancing Sox9 and reducing VEGF and ColX expressions in the cartilage to inhibit hypertrophic differentiation of the chondrocytes, maintain chondrogenic phenotype and repair cartilage cells.

Acupuncture Points ; Animals ; Cartilage, Articular ; metabolism ; pathology ; Cell Differentiation ; Chondrocytes ; cytology ; Chondrogenesis ; Collagen Type X ; metabolism ; Electroacupuncture ; Knee Joint ; physiopathology ; Osteoarthritis, Knee ; therapy ; Rabbits ; SOX9 Transcription Factor ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product.
Alkaline Phosphatase ; Apoptosis ; Cartilage ; Cell Size ; Chondrocytes* ; Chromatin ; Collagen Type X ; Cytoplasm ; DNA ; Endoplasmic Reticulum ; Endoplasmic Reticulum, Rough ; Extracellular Matrix ; Fibroblasts ; Hypertrophy* ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Phenotype ; Vacuoles

Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo.
Amino Acids ; Animals ; Bone Marrow ; Bone Regeneration ; Calcium ; Ceramics* ; Collagen Type I ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Mice ; Miners ; Osteocalcin ; Osteogenesis ; Photoelectron Spectroscopy ; Regeneration* ; X-Ray Microtomography

It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.
Aggrecans ; genetics ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; Cartilage ; cytology ; metabolism ; Cell Differentiation ; drug effects ; genetics ; Cell Hypoxia ; Cells, Cultured ; Chondrogenesis ; drug effects ; genetics ; Collagen Type II ; genetics ; metabolism ; Collagen Type X ; metabolism ; Female ; Gene Expression ; drug effects ; Glycosaminoglycans ; metabolism ; Growth Differentiation Factor 5 ; pharmacology ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Engineering ; methods

OBJECTIVE: The purpose of this study was to verify the appropriateness of ovariectomized rats as the osteoporosis animal model. METHODS: Twelve female Sprague-Dawley rats underwent a sham operation (the sham group) or bilateral ovariectomy [the ovariectomy (OVX) group]. Eight weeks after operations, serum biochemical markers of bone turnover were analyzed; osteocalcin and alkaline phosphatase, which are sensitive biochemical markers of bone formation, and C-terminal telopeptide fragment of type I collagen C-terminus (CTX), which is a sensitive biochemical marker of bone resorption. Bone histomorphometric parameters and microarchitectural properties of 4th lumbar vertebrae were determined by micro-computed tomographic (CT) scan. RESULTS: The OVX group showed on average 75.4% higher osteocalcin and 72.5% higher CTX levels than the sham group, indicating increased bone turnover. Micro-CT analysis showed significantly lower bone mineral density (BMD) (p=0.005) and cortical BMD (p=0.021) in the OVX group. Furthermore, the OVX group was found to have a significantly lower trabecular bone volume fraction (p=0.002). CONCLUSION: Our results showed that bone turnover was significantly increased and bone mass was significantly decreased 8 weeks after ovariectomy in rats. Thus, we propose that the ovariectomized rat model be considered a reproducible and reliable model of osteoporosis.
Alkaline Phosphatase ; Animals ; Biomarkers ; Bone Density ; Bone Resorption ; Collagen Type I ; Female ; Humans ; Lumbar Vertebrae ; Osteocalcin ; Osteogenesis ; Osteoporosis ; Ovariectomy ; Rats ; Rats, Sprague-Dawley ; Salicylamides ; X-Ray Microtomography

BACKGROUNDThere is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.

METHODSCartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.

RESULTSBoth the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable.

CONCLUSIONSExpression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.

ADAM Proteins ; ADAMTS5 Protein ; Cartilage ; pathology ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Chondrocytes ; metabolism ; Collagen Type II ; genetics ; Collagen Type X ; genetics ; Glycosaminoglycans ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; genetics ; Osteoarthritis ; genetics ; pathology