Osteocalcin mRNA Levels Research Articles

Role of TNF alpha and PLF in bone remodeling in a rat model of repetitive reaching and grasping

We have previously developed a voluntary rat model of highly repetitive reaching that provides an opportunity to study effects of non-weight bearing muscular loads on bone and mechanisms of naturally occurring inflammation on upper limb tissues in vivo. In this study, we investigated the relationship between inflammatory cytokines and matricellular proteins (Periostin-like-factor, PLF, and connective tissue growth factor, CTGF) using our model. We also examined the relationship between inflammatory cytokines, PLF and bone formation processes. Rats underwent initial training for 5 weeks, and then performed a high repetition high force (HRHF) task (12 reaches/min, 60% maximum grip force, 2 h/day, 3 days/week) for 6 weeks. We then examined the effect of training or task performance with or without treatment with a rat specific TNFalpha antibody on inflammatory cytokines, osteocalcin (a bone formation marker), PLF, CTGF, and behavioral indicators of pain or discomfort. The HRHF task decreased grip strength and induced forepaw mechanical hypersensitivity in both trained control and 6-week HRHF animals. Two weeks of anti-TNFalpha treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNFalpha treatment attenuated task-induced increases in inflammatory cytokines (TNFalpha, IL-1alpha, and MIP2 in serum; TNFalpha in forelimb bone and muscles) and serum osteocalcin in 6-week HRHF animals. PLF levels in forelimb bones and flexor digitorum muscles increased significantly in 6-week HRHF animals, increases attenuated by anti-TNFalpha treatment. CTGF levels were unaffected by task performance or anti-TNFalpha treatment in 6-week HRHF muscles. In primary osteoblast cultures, TNFalpha, MIP2 and MIP3a treatment increased PLF levels in a dose dependent manner. Also in primary osteoblast cultures, increased PLF promoted proliferation and differentiation, the latter assessed by measuring Runx2, alkaline phosphatase (ALP) and osteocalcin mRNA levels; ALP activity; as well as calcium deposition and mineralization. Increased PLF also promoted cell adhesion in MC3T3-E1 osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNFalpha, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro.

The role of pyrophosphate/phosphate homeostasis in terminal differentiation and apoptosis of growth plate chondrocytes

Extracellular inorganic phosphate (P i) concentrations are the highest in the growth plate just before the onset of mineralization. The study reported here demonstrates that P i not only is required for hydroxyapatite mineral formation but also modulates terminal differentiation and apoptosis of growth plate chondrocytes. Extracellular P i stimulated terminal differentiation marker gene expression, including the progressive ankylosis gene ( ank), alkaline phosphatase (APase), matrix metalloproteinase-13 (MMP-13), osteocalcin, and runx2, mineralization, and apoptosis of growth plate chondrocytes. The stimulatory effect of extracellular P i on terminal differentiation and apoptosis events of growth plate chondrocytes was dependent on the concentration, the expression levels of type III Na +/P i cotransporters, and ultimately P i uptake. A high extracellular P i concentration was required for the stimulation of apoptosis, whereas lower P i concentrations were required for the most effective stimulation of terminal differentiation events, including terminal differentiation marker gene expression and mineralization. Suppression of Pit-1 was sufficient to inhibit the stimulatory effects of extracellular P i on terminal differentiation events. On the other hand, increasing the local extracellular P i concentration by overexpressing ANK, a protein transporting intracellular PP i to the extracellular milieu where it is hydrolyzed to P i in the presence of APase, resulted in marked increases of hypertrophic and early terminal differentiation marker mRNA levels, including APase, runx2 and type X collagen, and slight increase of MMP-13 mRNA levels, but decreased osteocalcin mRNA level, a late terminal differentiation markers. In the presence of levamisole, a specific APase inhibitor to prevent hydrolysis of extracellular PP i to P i, ANK overexpression of growth plate chondrocytes resulted in decreased mRNA levels of hypertrophic and terminal differentiation markers but increased MMP-13 mRNA levels. In conclusion, with extracellular PP i inhibiting and extracellular P i stimulating hypertrophic and terminal differentiation events, a precise regulation of PP i/P i homeostasis is required for the spatial and temporal control of terminal differentiation events of growth plate chondrocytes.

Drynaria fortunei J. Sm. promotes osteoblast maturation by inducing differentiation-related gene expression and protecting against oxidative stress-induced apoptotic insults

Aim of the study Drynaria fortunei J. Sm. is one variety of the traditional Chinese medical herb Gusuibu. This study was aimed to evaluate the effects of water extracts of Kunze on regulation of osteoblast maturation and its possible mechanisms. Materials and methods Primary osteoblasts prepared from neonatal rat calvarias were exposed to the water extracts of Kunze (WEK), and the cytotoxicity was assayed. Osteoblast maturation was evaluated by analyzing cell mineralization. RT-PCR was executed to determine the effects of WEK on regulation of osteoblast differentiation-related gene expression. Nitrosative stress and apoptotic cells were quantified using flow cytometry. Results Exposure of rat calvarial osteoblasts to WEK did not affect cell viability, but significantly promoted osteoblast mineralization. WEK induced osteoprogenitor proliferation-related insulin-like growth factor-1 mRNA, but did not affect collagen type 1 mRNA expression. Treatment with WEK likewise induced the expression of matrix maturation-related bone morphogenetic protein (BMP)-2 and BMP-6 mRNA. Consequently, WEK enhanced the levels of mineralization-related alkaline phosphatase, ostepontin, and osteocalcin mRNA in osteoblasts. In addition, exposure of osteoblasts to WEK alleviated nitrosative stress-caused apoptotic insults. Conclusions This study shows that WEK can promote osteoblast maturation by regulating bone differentiation-related gene expression and defending against nitrosative stress-induced apoptotic insults.

Tensile Loading Modulates Bone Marrow Stromal Cell Differentiation and the Development of Engineered Fibrocartilage Constructs

Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

Osteoinduction in Umbilical Cord- and Palate Periosteum-Derived Mesenchymal Stem Cells

Adult abdominoplasty (AA) fat is an ideal source for mesenchymal stem cells (MSCs) because it is discarded after surgery, abundant, and easy to harvest. Children however, do not have the same abundant quantities of fat as adults, nor are they likely to undergo a procedure during which fat is routinely discarded. Hence, finding an alternate source for MSCs in children is a reasonable strategy. Two such sources are the palate periosteum (PP) and the umbilical cord (UC). Advantages for PP as a source of MSCs are accessibility during palate repair, ease of harvest, and minimal risk to the patient. The UC, like AA, is a discarded tissue, with a theoretically unlimited supply, which can be harvested in children with craniofacial bone abnormalities in advance of reconstructive procedures. Our objective in this study is to characterize MSCs from 3 sources (AA, PP, and UC) by surface marker prevalence, and to assess osteoinductive capability. Institutional review board approval was obtained for harvest of AA, PP, and UC. The presence of MSCs was determined using immunostaining and flow cytometry for cell surface markers CD73, CD90, CD105, and SSEA-4. Osteogenesis was induced using osteogenic medium. Osteoinduction was evaluated using Alizarin red staining, and real-time polymerase chain reaction for bone morphogenetic protein-2, alkaline phosphatase, and osteocalcin at 7, 14, and 21 days. MSCs from AA, PP, and UC all stained positive for CD73, CD90, CD105, and SSEA-4. Flow cytometry demonstrated significant differences in expression of CD90 and SSEA-4 but similar values for CD73 and CD105. Following osteoinduction, MSCs from all sources stained positive for calcium deposition. In UC MSCs, reverse transcriptase-polymerase chain reaction demonstrated greater elevation in bone morphogenetic protein-2 and alkaline phosphatase mRNA beginning at day 7 and extending to day 21. Osteocalcin mRNA levels were comparable for all 3 sources of MSCs. For children with craniofacial bone defects, UC-derived MSCs may be ideal for tissue engineered bone: temporally, the UC can be harvested in advance of surgical timing for the need for bone, is readily available, easy to harvest, and leads to osteoinduction that is more robust than either AA or PP.

Lymphocyte enhancer-binding factor 1: an essential factor in odontoblastic differentiation of dental pulp cells enzymatically isolated from rat incisors

Lymphocyte enhancer-binding factor 1 (Lef1), a HMG-domain protein, is thought to play important roles in inductive tissue interaction during tooth development. Lef1 knockdown in mice causes arrest at the bud stage in tooth development. As this gene participates in the regulation of a large and diverse set of peptide growth factors in ectomesenchymal cell differentiation of dental papilla, Lef1 appears to be a key factor in odontoblast differentiation. However, the relationship between Lef1 and odontoblast differentiation is still unclear. To analyze the biological roles of Lef1 in regulating odontoblast differentiation, we transiently overexpressed or suppressed Lef1 in cultured dental pulp cells. Lef1-overexpressing cells expressed higher levels of dentin sialoprotein (DSPP), osteocalcin and alkaline phosphatase (ALP) mRNA and formed larger numbers of mineralized nodules compared to control cells. However, Msx-1 expression or cell proliferation was unaffected by overexpression of Lef1. To further examine the role of Lef1 in dental pulp cells, we knocked down Lef1 expression in dental pulp cells using short interfering RNA (siRNA). Transient expression of siRNA against Lef1 markedly reduced Lef1 mRNA levels, and Lef1-suppressed cells expressed lower levels of DSPP, osteocalcin and ALP mRNA compared to control cells. Furthermore, the formation of mineralized nodules was inhibited by siRNA against Lef1; however, neither Msx-1 expression or cell proliferation was inhibited by siRNA against Lef1. These results outline the role of Lef1 in accelerating odontoblast differentiation by regulating DSP and osteocalcin mRNA expression in dental pulp cells, confirming that Lef1 is a key factor for odontoblast differentiation.

Role of zinc in cellular zinc trafficking and mineralization in a murine osteoblast-like cell line

Zinc (Zn) supplementation stimulates bone growth in Zn-deficient humans and animals. A biphasic pattern of mineralization has been observed in cultured osteoblasts; an initiation phase and a progression phase. We used MC3T3-E1, a murine osteoblastic cell line, to elucidate the physiological role of Zn in osteoblast mineralization and cellular Zn trafficking during the mineralization event. Cells were cultured in media containing Chelex-treated fetal bovine serum and 1, 4, 10 and 20 μM Zn as ZnSO 4 for 14 days (early phase of mineralization) or 21 days (mid-to-late phase of mineralization). During the early phase of mineralization, Alizarin Red staining indicated that mineralization was increased by Zn in a dose-dependent manner. Although Zn exposure did not affect monolayer Zn concentration, metallothionein (MT) mRNA expression increased dose-dependently as assessed by real-time PCR. During the late phase of mineralization, mineralization was maximal at 1 μM Zn and monolayer Zn concentration reflected Zn exposure. The increase in MT mRNA expression during the late phase was similar to that during the early phase, but the difference in expression between culture Zn concentrations tended to be smaller. ZnT-2 mRNA expression decreased significantly with increasing zinc concentrations in the culture medium during the early phase, but increased significantly during the late phase. Osteocalcin mRNA levels were positively correlated to Zn exposure at both time points. Taken together, we propose that Zn may play an important role in osteoblast mineralization through Zn trafficking involving Zn storage proteins and Zn transporters.

The effect of dietary phosphatidylcholine/phosphatidylinositol ratio on malformation in larvae and juvenile gilthead sea bream ( Sparus aurata)

Malformation in commercially raised fish, such as cranial, vertebral and gill cover deformities is a major factor reducing their market value. Although these deformities are most apparent in the juvenile and adult stages they may originate from suboptimal nutrition during the critical larval rearing stage. Previous research hypothesized that dietary phosphatidylinostol (PI) was more effective in reducing deformities than the main membrane phospholipid, phosphatidylcholine (PC). Consequently, the aim of this study was to test the effect of different dietary ratios of PC and PI fed to the gilthead sea bream ( Sparus aurata) larvae, on developmental performances in juvenile fish in terms of survival, growth and malformation rate. Four microdiet (MD) treatments, that differed in their PC/PI ratio and replaced 75% of the Artemia ration (wt/wt), were fed to 20–34 dph (days post hatching) sea bream larvae. In addition to the high PC/PI or low PI containing MD control, a commercial reference treatment (100% Artemia ration) was also given. At 40 dph, the larvae were graded in all treatments into small (< 1.3 mg dry wt larva − 1) and large (> 2.9 mg dry wt larva − 1) larvae, in order to test if growth rate influenced treatment effect throughout development to 141 dph. There was no marked ( P > 0.05) treatment effect on growth rate in 40 dph larvae. On the other hand in later juvenile development (67 dph), decreasing dietary PC/PI ratio contributed to significantly ( P < 0.05) better growth and ( P > 0.05) higher survival. Moreover, reducing dietary PI markedly ( P < 0.05) increased jaw (cranial) deformity in both size groups at 67 dph which may have adversely affected juvenile feeding on a dry hard starter feed. Conversely, increasing dietary PI (reducing PC/PI ratio) showed a non-significant trend of increased skeletal deformity which was markedly ( P < 0.05) higher in faster growing larvae in all MD treatments. Although there was no clear effect of PC/PI ratio on gill cover deformity rate, there was a size dependent susceptibility to this deformity where smaller larvae showed the highest incidence of this malformation. Osteocalcin (BGP) mRNA levels correlated well ( R 2 = 0.964) with development in the faster growing fry fed the high PI diet. Higher production of BGP may have reduced ( P < 0.05) the jaw deformity while tending to cause over-mineralization and deformity of the skeleton. The results suggest an effective dietary PC/PI ratio of 1.28 for sea bream larvae during culture.

Alendronate Induces Mineralization in Mouse Osteoblastic MC3T3-E1 Cells: Regulation of Mineralization-Related Genes

Alendronate, an aminobisphosphonate, is an effective reagent to reduce fracture risk in osteoporotic patients. Although several studies suggest that bisphosphonates affect osteoblast differentiation, how they affect the genes relating to the mineralization step remains unknown. The present study was performed to clarify the effects of alendronate on mineralization and its related genes in mouse osteoblastic MC3T3-E1 cells. Alendronate at 10 (-8) and 10 (-7) M induced mineralization in MC3T3-E1 cells. As for the genes that suppress mineralization, alendronate enhanced the level of PC-1 mRNA in a dose-dependent manner in 7-day cultures in semiquantitative RT-PCR, although it reduced the levels of PC-1 mRNA in 21-day cultures. On the other hand, alendronate did not affect the levels of ANK, osteopontin and matrix Gla protein mRNA in both 7- and 21-day cultures. Moreover, alendronate reduced the level of osteocalcin mRNA at 10 (-7) and 10 (-6) M in 14-day cultures of these cells. As for the expression of alkaline phosphatase (ALP), an important positive regulator of mineralization in osteoblasts, alendronate enhanced the levels of ALP mRNA and protein at 10 (-7)-10 (-5) M. In conclusion, low-dose alendronate induced mineralization in mouse osteoblastic cells. The regulation of PC-1, osteocalcin and ALP by alendronate might play some role in these effects.

Bone turnover and its related molecular mechanism in STZ-induced diabetic rats

Objective To study the bone turnover and its related molecular mechanism in STZ-induced diabetic rats. Methods Of 30 male SD rats studied, 15 were induced diabetics by intravenous injection of streptozotocin (50 mg/kg)and fed for 8 weeks. After the sacrifice of both the diabetic and control groups, serum Ca, P, alkaline phosphatase (ALP), and osteocalcin were determined, and 24 h urinary Ca and urinary cross-linked N-telopeptide of type Ⅰ collagen (NTx)and creatinine (Cr)ratio were also determined. The left tibia was dissected for bone histomorphometry analysis. Right femur and lumbar vertebrae (L1-L4) were reserved for bone mineral density (BMD) determination. The right tibia was separated for the study of bone tissue RANKL/osteoprotegerin, Core binding factor 1 (Cbfa1) ,osterix and osteocalcin mRNA level which was performed by real-time quantitative reverse transcription polymerase chain reaction assay. Results No significant difference was found in serum Ca, P, and ALP levels between 2 groups of rats. ST-Z-induced diabetic rats were characterized by extreme hyperglycemia, marked weight loss, polyuria, and hypercalciuria. A low-turnover osteopenia was evidenced in diabetic rats by decreased BMD in both femur [(0. 099±0.013) vs (0. 139 ± 0.013 g/cm~3) , P < 0.01] and lumbar vertebrae [(0. 107±0.011)vs (0. 149±0.009) g/cm~3, P<0.01] , reduced serum osteoealcin level [a marker of formation, (3.03±0.52) vs (6. 18±0.71) ng/ml ,P<0. 01]) ,decreased urine NTx/Cr ratio [(5. 67±0.86) vs (5.23±0.98) nmol/g Cr, P<0. 05], decreased trabeeular volume and thickness, and reduced bone label surface and bone formation rate [(0. 44±0. 11) vs (0. 78±0. 14) μm/d,P<0. 01] by bone dynamic study. The RANKL/ osteoprotegerin [(0.57±0.11)vs (0.89±0.13) ,P<0.01] ,osterix [(1.93×10~(-4)±0.65×10(~-4))vs (4.19×10~(-4)± 0.71×10~(-4)) ,P<0.01] ,Cbfa1 [(26.68×10~(-4)±6.53×10~(-4))vs (37.21×10~(-4)±7.14×10~(-4)) ,P<0.01] ,and osteocalcin [(2.25×10~(-4)±1.19×10~(-4))vs (3.43×10~(-4)±1.63×10~(-4)) ,P<0.01] mRNA expressions were declined in the bone tissue of the tibia in the ST-Z-induced diabetic rats, as compared with the control. Conclusion A low-turnover osteopenia is evidenced in STZ-induced diabetic rats by significant decrease of both osteoclastic marker(RANKL/ osteoprotegerin)and osteoblastic marker (osterix ,Cbfa1 ,osteocalcin)mRNA levels in tibia. Key words: Diabetic mellitas, experimental; Bone histomorpbometry; Real-time quantitative polymerase reaction; RANK ligand; Osteoprotegerin; Core binding factor 1; Osterix; Osteocalcin; Cross-linked N-telopeptide of typelcollagen

Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold

This study examined the osteogenic differentiation of cultured human periosteal-derived cells grown in a three dimensional collagen-based scaffold. Periosteal explants with the appropriate dimensions were harvested from the mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the cells were divided into two groups and cultured for 28 days. In one group, the cells were cultured in two-dimensional culture dishes with osteogenic inductive medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In the other group, the cells were seeded onto a three-dimensional collagen scaffold and cultured under the same conditions. We examined the bioactivity of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and measurements of the calcium content in the periosteal-derived cells of two groups. Periosteal-derived cells were successfully differentiated into osteoblasts in the collagen-based scaffold. The ALP activity in the periosteal-derived cells was appreciably higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The calcium level in the periosteal-derived cells seeded onto three-dimensional collagen scaffolds showed a 5.92-fold increase on day 7, 3.28-fold increase on day 14, 4.15-fold increase on day 21, and 2.91-fold increase on day 28, respectively, compared with that observed in two-dimensional culture dishes. These results suggest that periosteal-derived cells have good osteogenic capacity in a three-dimensional collagen scaffold, which provides a suitable environment for the osteoblastic differentiation of these cells.

Inositol hexakisphosphate inhibits mineralization of MC3T3-E1 osteoblast cultures

Inositol hexakisphosphate (IP6, phytic acid) is an endogenous compound present in mammalian cells and tissues. Differentially phosphorylated forms of inositol are well-documented to have important roles in signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit soft tissue calcification (specifically renal and vascular calcification) by binding extracellularly to calcium oxalate and calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent inhibition of mineralization without affecting cell viability, proliferation or collagen deposition. Osteoblast differentiation markers, including tissue-nonspecific alkaline phosphatase activity, bone sialoprotein and osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly increased protein and mRNA levels of osteopontin, a potent inhibitor of crystal growth and matrix mineralization. Inositol alone (without phosphate), as well as inositol hexakis-sulphate, a compound with a high negative charge similar to IP6, had no effect on mineralization or osteopontin induction. Binding of IP6 to mineral crystals from the osteoblast cultures, as well as to synthetic hydroxyapatite crystals, was confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by binding to growing crystals through negatively charged phosphate groups and by induction of inhibitory osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of osteopontin gene expression.

Conditional Disruption of Pkd1 in Osteoblasts Results in Osteopenia Due to Direct Impairment of Bone Formation

PKD1 (polycystin-1), the disease-causing gene for ADPKD, is widely expressed in various cell types, including osteoblasts, where its function is unknown. Although global inactivation of Pkd1 in mice results in abnormal skeletal development, the presence of polycystic kidneys and perinatal lethality confound ascertaining the direct osteoblastic functions of PKD1 in adult bone. To determine the role of PKD1 in osteoblasts, we conditionally inactivated Pkd1 in postnatal mature osteoblasts by crossing Oc (osteocalcin)-Cre mice with floxed Pkd1 (Pkd1(flox/m1Bei)) mice to generate conditional heterozygous (Oc-Cre;Pkd1(flox/+)) and homozygous (Oc-Cre;Pkd1(flox/m1Bei)) Pkd1-deficient mice. Cre-mediated recombination (Pkd1(Delta flox)) occurred exclusively in bone. Compared with control mice, the conditional deletion of Pkd1 from osteoblasts resulted in a gene dose-dependent reduction in bone mineral density, trabecular bone volume, and cortical thickness. In addition, mineral apposition rates and osteoblast-related gene expression, including Runx2-II (Runt-related transcription factor 2), osteocalcin, osteopontin, and bone sialoprotein, were reduced proportionate to the reduction of Pkd1 gene dose in bone of Oc-Cre;Pkd1(flox/+) and Oc-Cre;Pkd1(flox/m1Bei) mice. Primary osteoblasts derived from Oc-Cre;Pkd1(flox/m1Bei) displayed impaired differentiation and suppressed activity of the phosphatidylinositol 3-kinase-Akt-GSK3beta-beta-catenin signaling pathways. The conditional deletion of Pkd1 also resulted in increased adipogenesis in bone marrow and in osteoblast cultures. Thus, PKD1 directly functions in osteoblasts to regulate bone formation.

Ectopic expression of telomerase enhances osteopontin and osteocalcin expression during osteogenic differentiation of human mesenchymal stem cells from elder donors

Age related bone loss is one of the most prevalent diseases in the elder population. The osteoblasts are the effectors cells of bone formation and regeneration. With the aging the osteoblasts become senescent reducing their ability to produce bone. Cellular replicative senescence is triggered by telomers shortening. Telomerase elongate the telomers length and maintain the cell proliferative capacity. Here, we demonstrated that the expression of human telomerase reverse transcriptase mediated by an adenovirus vector increases the levels of osteopontin and osteocalcin mRNA during the in vitro osteogenic differentiation of elderly human mesenchymal stem cells. Bone marrow human mesenchymal stem cells were obtained from old donors (>65 years) and induced to differentiate into osteoblasts for 14 days. The levels of mRNA of human telomerase reverse transcriptase, osteopontin and osteocalcin during the differentiation were assessed by semi-quantitative PCR before and during the differentiation on days 7 and 14. Infected cells showed 1.5 fold increase in telomerase expression. Also telomerized cells exhibit 1.5 fold increase in osteopontin and 0.5 fold increase in osteocalcin expression compared to primary osteoblasts isolated from the same donors. The transformed cells were not able to form tumours in NUDE mice.

The notch‐responsive transcription factor Hes‐1 attenuates osteocalcin promoter activity in osteoblastic cells

Notch signaling plays a key role in osteoblast differentiation. A major transcriptional downstream regulator of this pathway is the helix-loop-helix (HLH) transcription factor Hairy/Enhancer of Split 1 (Hes-1). Here we investigated the function of Hes-1 in osteoblastic cells. Endogenous Hes-1 gene expression decreases during progression of bone cell phenotype development in MC3T3-E1 osteoblasts suggesting that it is a negative regulator of osteoblast differentiation. Forced expression of Hes-1 inhibits osteocalcin (OC) mRNA levels, and luciferase assays indicate that Hes-1 directly represses OC promoter activity. In vitro and in vivo protein/DNA interaction assays reveal that recombinant Hes-1 binds specifically to an E-box in the proximal promoter of the OC gene. Deletion of the Hes-1 WRPW domain (MHes-1) that recruits the co-repressor Groucho abrogates repression of OC promoter activity by Hes-1, but also blocks Hes-1 binding to the promoter. The latter result suggests that exogenous Hes-1 may be recruited to the OC promoter by both protein/DNA and protein/protein interactions. We conclude that the Notch-responsive Hes-1 protein is capable of repressing OC gene transcription in osteoblastic cells through an E-box in the proximal promoter. Hes-1 may contribute to osteoblast growth and differentiation by controlling basal bone-specific transcription directly through interactions with transcriptional regulators that are known to bind to the OC gene promoter.

Chondroprotective effect of the bioactive peptide prolyl-hydroxyproline in mouse articular cartilage in vitro and in vivo

To investigate the direct effect of prolyl-hydroxyproline (Pro-Hyp) on chondrocytes under in vivo and in vitro conditions in an attempt to identify Pro-Hyp as the bioactive peptide in collagen hydrolysate (CH). The in vivo effects of CH and Pro-Hyp intake on articular cartilage were studied by microscopic examination of sections of dissected articular cartilage from treated C57BL/6J mice. In this study, mice that were fed diets containing excess phosphorus were used as an in vivo model. This mouse line showed loss of chondrocytes and reduced thickness of articular cartilage, with abnormality of the subchondral bone. The in vitro effects of CH, Pro-Hyp, amino acids and other peptides on proliferation, differentiation, glycosaminoglycan content and mineralization of chondrocytes were determined by MTT activity and staining with alkaline phosphatase, alcian blue and alizarin red. Expression of chondrogenesis-specific genes in ATDC5 cells was determined by semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). In vivo, CH and Pro-Hyp inhibited the loss of chondrocytes and thinning of the articular cartilage layer caused by phosphorus-induced degradation. In the in vitro study, CH and Pro-Hyp did not affect chondrocyte proliferation but inhibited their differentiation into mineralized chondrocytes. A combination of amino acids such as proline, hydroxyproline and prolyl-hydroxyprolyl-glycine did not affect chondrocyte proliferation or differentiation. Moreover, CH and Pro-Hyp caused two and threefold increases, respectively, in the staining area of glycosaminoglycan in the extracellular matrix of ATDC5 cells. RT-PCR indicated that Pro-Hyp increased the aggrecan mRNA level approximately twofold and decreased the Runx1 and osteocalcin mRNA levels by two-thirds and one-tenth, respectively. Pro-Hyp is the first bioactive edible peptide derived from CH to be shown to affect chondrocyte differentiation under pathological conditions.

Hesperetin stimulates differentiation of primary rat osteoblasts involving the BMP signalling pathway

Hesperidin found in citrus fruits has been reported to be a promising bioactive compound for maintaining an optimal bone status in ovariectomized rodent models. In this study, we examined the capacity of hesperetin (Hp) to affect the proliferation, differentiation and mineralization of rodent primary osteoblasts. Then, the impact of Hp on signalling pathways known to be implicated in bone formation was explored. We exposed osteoblasts to physiological concentrations of 1 μM Hp (Hp1) and 10 μM Hp (Hp10). Neither proliferation nor mineralization was affected by Hp at either dose during 19 days of exposure. Hp at both doses enhanced differentiation by significantly increasing alkaline phosphatase (ALP) activity from Day 14 of exposure (Day 19: Hp1: +9%, Hp10: +14.8% vs. control; P<.05). However, Hp did not induce an obvious formation of calcium nodules. The effect of Hp10 on ALP was inhibited by addition of noggin protein, suggesting a possible action of this flavanone through the bone morphogenetic protein (BMP) pathway. Indeed, Hp10 significantly induced (1.2- to 1.4-fold) mRNA expression of genes involved in this signalling pathway (i.e., BMP2, BMP4, Runx2 and Osterix) after 48 h of exposure. This was strengthened by enhanced phosphorylation of the complex Smad1/5/8. Osteocalcin mRNA level was up-regulated by Hp only at 10 μM (2.2 fold vs. control). The same dose of Hp significantly decreased osteopontin (OPN) protein level (50% vs. control) after 14 days of culture. Our findings suggest that Hp may regulate osteoblast differentiation through BMP signalling and may influence the mineralization process by modulating OPN expression.

Enhanced osteoblast response to an equal channel angular pressing-processed pure titanium substrate with microrough surface topography

This study investigated the surface characteristics and in vitro biocompatibility of ultrafine-grain pure titanium substrates produced by equal channel angular pressing (ECAP) using MC3T3-E1 pre-osteoblast cells, compared with those of conventional coarse-grain pure titanium (CP) and Ti–6Al–4V (Ti64) substrates. All Ti surfaces were grit-blasted with hydroxyapatite particles to produce microrough surfaces. The surface characteristics were evaluated by electron back-scattered diffractometry, scanning electron microscopy, contact angle and surface energy measurements, and optical profilometry. The morphology of spread cells, cell attachment, viability, alkaline phosphatase (ALP) activity, quantitative analysis of osteoblastic gene expression and mineralization nodule formation on different surfaces were evaluated. ECAP-processed substrates showed a significantly lower water contact angle and higher surface energy compared with coarse-grain CP and Ti64 substrates ( p < 0.05). They also showed enhanced cell spreading, attachment, viability and ALP activity compared with the CP and Ti64 surfaces ( p < 0.05). Real-time polymerase chain reaction analysis showed notably higher ALP, osteopontin and osteocalcin mRNA levels in cells grown on the ECAP surfaces than on the CP and Ti64 surfaces, and the ECAP surfaces showed significantly greater mineralization nodule formation compared with the CP and Ti64 substrates ( p < 0.05). These results demonstrate the superior osteoblast cell compatibility of microroughened Ti surface made of ECAP-processed ultrafine-grain pure Ti substrates over coarse-grain pure Ti and Ti64 substrates.

Development of a Hybrid Baculoviral Vector for Sustained Transgene Expression

Baculovirus is a promising gene delivery vector but its widespread application is impeded as it only mediates transient transgene expression in mammalian cells. To prolong the expression, we developed a dual baculovirus system whereby one baculovirus expressed FLP recombinase while the other harbored an Frt-flanking cassette encompassing the transgene and oriP/EBNA1 derived from Epstein-Barr virus. After cotransduction of cells, the expressed FLP cleaved the Frt-flanking cassette off the baculovirus genome and catalyzed circular episome formation, then oriP/EBNA1 within the cassette enabled the self-replication of episomes. The excision/recombination efficiency was remarkably enhanced by sodium butyrate, reaching 75% in human embryonic kidney-293 (HEK293) cells, 85% in baby-hamster kidney (BHK) cells, 77% in primary chondrocytes, and 48% in mesenchymal stem cells (MSCs). The hybrid baculovirus substantially prolonged the transgene expression to approximately 48 days without selection and >63 days with selection, thanks to the maintenance of replicons and transgene transcription. In contrast to the replicating episomes, the baculovirus genome was rapidly degraded. Furthermore, an osteoinductive growth factor gene was efficiently delivered into MSCs using this system, which not only prolonged the growth factor expression but also potentiated the osteogenesis of MSCs. These data collectively implicate the potential of this hybrid baculovirus system in gene therapy applications necessitating sustained transgene expression.

Dioxins interfere with differentiation of osteoblasts and osteoclasts

We have previously shown that the environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) affects bone growth, modelling and mechanical strength in vivo. In this study, we utilized differentiation of bone marrow stem cells to osteoblasts and osteoclasts as a model system to study the effects of TCDD on bones. Stem cells were isolated from bone marrow of femurs and tibias of rats and mice. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells using quantitative RT-PCR. TCDD significantly and dose-dependently decreased the mRNA levels of RUNX2, alkaline phosphatase and osteocalcin. Also the activity of alkaline phosphatase was significantly inhibited in both rat and mice cells. In the case of osteoclasts, TCDD decreased the number of TRACP+ multinucleated cells, with corresponding decreases in the number of F-actin rings and the area of resorption. Studies in AHR-knockout mice indicated that TCDD has no effect on the expression of osteoblastic differentiation markers suggesting that TCDD mediates its effects by AHR. Both osteoblastic and osteoclastic effects took place at very low doses of TCDD, as in most cases 100 fM TCDD was enough to significantly affect the differentiation markers. Therefore, differentiation of osteoblasts and osteoclasts from bone marrow stem cells seems to be a very sensitive target for TCDD. Disrupting effects in osteoblastic cells, in addition to disturbed osteoclastogenesis, may thus play a role in adverse effects on bone quality in TCDD exposed animals.