ALP Gene Expression Research Articles

The Effect of Vibration Treatments Combined with Teriparatide or Strontium Ranelate on Bone Healing and Muscle in Ovariectomized Rats.

The aim of the present study was to study the effect of combined therapy of teriparatide (PTH) or strontium ranelate (SR) with whole-body vibration (WBV) on bone healing and muscle properties in an osteopenic rat model. Seventy-two rats (3months old) were bilaterally ovariectomized (Ovx), and 12 rats were left intact (Non-Ovx). After 8weeks, bilateral transverse osteotomy was performed at the tibia metaphysis in all rats. Thereafter, Ovx rats were divided into six groups (n=12): (1) Ovx-no treatment, (2) Ovx+vibration (Vib), (3) SR, (4) SR+Vib, (5) PTH, and (6) PTH+Vib. PTH (40μg/kg BW sc. 5×/week) and SR (613mg/kg BW in food daily) were applied on the day of ovariectomy, vibration treatments 5days later (vertical, 70Hz, 0.5mm, 2×/day for 15min) for up to 6weeks. In the WBV+SR group, the callus density, trabecular number, and Alp and Oc gene expression were decreased compared to SR alone. In the WBV+PTH group, the cortical and callus widths, biomechanical properties, Opg gene expression, and Opg/Rankl ratio were increased; the cortical and callus densities were decreased compared to PTH alone. A case of non-bridging was found in both vibrated groups. Vibration alone did not change the bone parameters; PTH possessed a stronger effect than SR therapy. In muscles, combined therapies improved the fiber size of Ovx rats. WBV could be applied alone or in combination with anti-osteoporosis drug therapy to improve muscle tissue. However, in patients with fractures, anti-osteoporosis treatments and the application of vibration could have an adverse effect on bone healing.

Reactive oxygen species regulatory mechanisms associated with rapid response of MC3T3-E1 cells for vibration stress

Although many previous studies have shown that refractory period-dependent memory effect of vibration stress is anabolic for skeletal homeostasis, little is known about the rapid response of osteoblasts simply derived from vibration itself. In view of the potential role of reactive oxygen species (ROS) in mediating differentiated activity of osteoblasts, whether and how ROS regulates the rapid effect of vibration deserve to be demonstrated. Our findings indicated that MC3T3-E1 cells underwent decreased gene expression of Runx2, Col-I and ALP and impaired ALP activity accompanied by increased mitochondrial fission immediately after vibration loading. Moreover, we also revealed the involvement of ERK-Drp1 signal transduction in ROS regulatory mechanisms responsible for the rapid effect of vibration stress.

Injectable photocrosslinkable hyaluronic acid hydrogels incorporated with platelet lysate enhance the dentinogenic differentiation of human dental pulp stem cells

Injectable photocrosslinkable hyaluronic acid hydrogels incorporated with platelet lysate enhance the dentinogenic differentiation of human dental pulp stem cells

Cross-Talk Between VEGF and BMP-6 Pathways Accelerates Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

Deficiency in vascular endothelial growth factor (VEGF) or bone morphogenetic proteins (BMPs) results in fracture non-unions. Therefore, it is indispensable to comprehend the combined effect of VEGF and BMPs on the osteogenic differentiation of osteoprogenitor mesenchymal stem cells (MSCs) that are either naturally occurring at the fracture repair site or exogenously added to enhance the bone repair. We found that the combination of VEGF and BMP-6 enhanced COL1A2 expression, which correlated with upregulated expression of osterix, Dlx5, and Msx2 in human adipose-derived stem cells (hADSCs). Cross-talk between VEGF and BMP-6 pathways upregulated activation of p38 mitogen-activated kinase (p38 MAPK) and inhibited activation of protein kinase B (PKB, also known as Akt), whereas phosphorylation of "mothers against decapentaplegic" homologs 1/5/8 (Smads 1/5/8) and extracellular signal-regulated kinases 1 and 2 (ERK 1/2) was not affected. Consistent with these findings, p38 inhibitor SB203580, or siRNA knockdown of osterix, abrogated crosstalk between the VEGF and BMP-6 pathways and significantly reduced the observed upregulation of COL1A2. Nuclear translocation of the phosphorylated form of osterix was also inhibited by SB203580. Although crosstalk between the VEGF-BMP-6 pathways did not show an effect on the extent of mineralization, inhibition of any one of the three components that were upregulated through the cross-talk, i.e., osterix, Dlx5, and p38 activation, led to a complete inhibition of mineralization. Inhibition of PKB/Akt activation, which is attenuated through the cross-talk, significantly enhanced ALP gene expression. These observations imply that crosstalk between the VEGF and BMP-6 signaling pathways enhances osteogenic differentiation of MSCs.

Proliferation and osteogenic response of MC3T3‐E1 pre‐osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria

Dense zirconia ceramics are used in bone applications due to their mechanical strength and biocompatibility, but lack osseointegration. A porous interface in contact with bone tissue may lead to better bone bonding but the biological properties of porous zirconia are not widely explored. The present study focuses on the manufacturing of an yttria- (YSZ) and a magnesia-stabilized (MgSZ) porous zirconia, and on their in vitro biological investigation. The sintered ceramics had similar characteristics of porosity, pore size and interconnectivity. Their elastic moduli and compressive strength values were within the range of the values of human cortical bone. MC3T3-E1 pre-osteoblasts were used to investigate the proliferation, alkaline phosphatase (ALP) activity, collagen deposition and expression profile of four genes involved in bone metabolism of cells on porous ceramics. Scanning electron and fluorescence microscopy were employed to visualize cell morphology and growth. Pre-osteoblasts adhered well on both ceramics but cell numbers on YSZ were higher. Cells exhibited an increase in ALP activity and collagen deposition after 14 days on both MgSZ and YSZ, with higher levels on YSZ. Real-time quantitative polymerase chain reaction (qPCR) showed that the expression of bone sialoprotein (Bsp) and collagen type I (col1aI) were significantly higher on YSZ. No significant differences were found in their ability to regulate the early gene expression of Runx2 and Alp. Nevertheless, the biomineralized calcium content was similar on both ceramics after 21 days, indicating that despite chemical differences, both scaffolds direct the pre-osteoblasts toward a mature state capable of mineralizing the extracellular matrix.

Effects of non-thermal atmospheric pressure air plasma on gene expression at SLAtreated titanium surfaces

Dental implants manufacturers have developed high hydrophilic and rough implant surfaces which in turn exhibited better osseointegration than conventional ones. Based on in vitro studies, sand blast, large grit, acid etching(SLA)-treated surfaces with microstructural characteristics may promote the process of osseointegration. But when the SLA surface is kept dry in the air, it becomes hydrophobic. Appropriate non-thermal atmospheric pressure air plasma jet(air-NTAPPJ) processes render surfaces hydrophilic. The purpose of this research is to air-NTAPPJ on SLA surface treatment to improve the osteogenic functionality. The experiment was carried out through the a cytotoxicity assay and real-time reverse transcriptase-polymerase chain reaction(RT-PCR). The results were that the cell viability and gene expression of ALP, OCN, OPN, and Runx-2 of rMSC enhanced by air-NTAPPJ treated. Thus, air-NTAPPJ application can lead to an improved osseointegration.

Multi-walled carbon nanotubes promote cementoblast differentiation and mineralization through the TGF-β/Smad signaling pathway.

Excretion of cementum by cementoblasts on the root surface is a process indispensable for the formation of a functional periodontal ligament. This study investigated whether carboxyl group-functionalized multi-walled carbon nanotubes (MWCNT-COOH) could enhance differentiation and mineralization of mammalian cementoblasts (OCCM-30) and the possible signaling pathway involved in this process. Cementoblasts were incubated with various doses of MWCNT-COOH suspension. Cell viability was detected, and a scanning electron microscopy (SEM) observed both the nanomaterials and the growth of cells cultured with the materials. Alizarin red staining was used to investigate the formation of calcium deposits. Real-time PCR and western blot were used to detect cementoblast differentiation and the underlying mechanisms through the expression of the osteogenic genes and the downstream effectors of the TGF-β/Smad signaling. The results showed that 5 µg/mL MWCNT-COOH had the most obvious effects on promoting differentiation without significant toxicity. Alp, Ocn, Bsp, Opn, Col1 and Runx2 gene expression was up-regulated. Smad2 and Smad3 mRNA was up-regulated, while Smad7 was first down-regulated on Day 3 and later up-regulated on Day 7. The elevated levels of phospho-Smad2/3 were also confirmed by western blot. In sum, the MWCNT-COOH promoted cementoblast differentiation and mineralization, at least partially, through interactions with the TGF-β/Smad pathway.

Alendronate-Eluting Biphasic Calcium Phosphate (BCP) Scaffolds Stimulate Osteogenic Differentiation.

Biphasic calcium phosphate (BCP) scaffolds have been widely used in orthopedic and dental fields as osteoconductive bone substitutes. However, BCP scaffolds are not satisfactory for the stimulation of osteogenic differentiation and maturation. To enhance osteogenic differentiation, we prepared alendronate- (ALN-) eluting BCP scaffolds. The coating of ALN on BCP scaffolds was confirmed by scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDS), and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An in vitro release study showed that release of ALN from ALN-eluting BCP scaffolds was sustained for up to 28 days. In vitro results revealed that MG-63 cells grown on ALN-eluting BCP scaffolds exhibited increased ALP activity and calcium deposition and upregulated gene expression of Runx2, ALP, OCN, and OPN compared with the BCP scaffold alone. Therefore, this study suggests that ALN-eluting BCP scaffolds have the potential to effectively stimulate osteogenic differentiation.

Zoledronic Acid Reverses Acute Bone Impairment in a Mouse Model for Sickle Cell Disease

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder, characterized by the synthesis of pathological hemoglobin S (HbS). Up to now, limited studies in SCD patients have proposed a bone loss related to recurrent acute vaso-occlusive events (VOCs). Here, we designed a study in a humanized mouse model for SCD to evaluate (i) bone structure turnover and micro-architecture by histomorphometric approach; (ii) osteoblastic differentiation by RT-PCR analysis of bone runx2, sparc and alp gene expression. We used humanized healthy control (Hbatm1(HBA)TowHbbtm3(HBG1,HBB)Tow) and SCD (Hbatm1(HBA)TowHbbtm2(HBG1,HBB*)Tow) mice. Mice were divided into different groups as detailed in the enclosed flow-chart. In steady state, we first evaluated bone-marrow erythropoiesis by FACS analysis based on CD44-TER119 strategy. No signs of ineffective erythropoiesis were detectable in SCD mice compared to controls. Bone analysis was performed by histomorphometric approach after double labelling with demeclocycline followed by calcein to evaluate the dynamic parameters of bone turnover and analyzed by a specific software (bone3.1, Explora Nova, Larochelle, France). The histomorphometric bone analysis revealed (i) reduced bone volume with associated decreased trabecular number and increased trabecular separation; (ii) increased bone-turnover measured as bone formation rate (BFR) and activation frequency (AcF); (iii) decreased number of nodes (NdN) and node termini ratio; (iv) increased marrow star volume (MSV) and fractal dimension (D) compared to healthy mice. The bone molecular analysis revealed a significant down-regulation of runx2, sparc and alp gene expression in SCD mice compared to healthy ones. These data indicate a severe osteoporosis with bone fragility associated with alteration of osteogenic differentiation in SCD mice. Zoledronic acid (Zol) is the most powerful available bisphosphonate used to reduce bone turnover and bone fragility. Since VOCs are life-threatening complication of SCD and mainly involved skeletal system, requiring multidiscliplinary approach, we exposed SCD and control mice to hypoxia/reoxygenation (H/R) stress to mimic acute VOCs (see flow-chart). Mice were exposed to hypoxia (8% oxygen, 10 hours) followed by reoxygenation (21% oxygen) for either 18 hours to carry out molecular analysis or 10 days to carry out histomorphometric analysis. In H/R exposed vehicle treated SCD mice, we found (i) a significant increase in AcF (bone turnover), without differences in bone structure and microarchitecture compared to SCD mice under normoxia; (ii) a further reduction in osteogenic differentiation molecular markers. Zol (100 μg/ Kg in a single ip injection) was administrated either 7 days before (Zol-pre) or immediately after (Zol-post) H/R stress. In SCD mice, both Zol-pre and Zol-post significantly prevented the H/R induced increased bone turnover, while no differences were detectable in bone structure and microarchitecture compared to vehicle treated H/R SCD mice. The molecular analysis showed that Zol increased the osteogenic related gene expression, indicating a positive induction of osteogenic differentiation. Our data suggest that murine SCD is characterized by a significant bone impairment related to the unbalance between osteoblastic/osteoclastic activity in favor of osteoclastic one in presence of a down-regulation of osteogenic differentiation. In SCD, Zol plays a pivotal role decreasing osteoclastic activity and promoting osteogenic differentiation, representing a powerful new therapeutic strategy to limit bone disease in SCD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro.

Clinical trials using bone morphogenetic protein-2 (BMP2) for bone reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect of an alternative treatment strategy in which human bone marrow–derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured in osteogenic medium. The effects of preconditioning were analyzed with regard to proliferation and expression of osteogenic markers at both gene and protein level. The results were compared to those from cultures with continuous BMP2 stimulation. A significant increase in proliferation was seen with both precondition and continuous stimulation with BMP2, with no difference between the treatments. Preconditioning with BMP2 significantly increased gene expression of RUNX2, COLI, ALP, and OC, and protein levels of COLI and ALP. This was not found with continuous stimulation. The role of preconditioning with BMP2 in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of hMSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided.

Expression of bone markers and micro-CT analysis of alveolar bone during orthodontic relapse.

To investigate biological changes in alveolar bone occurring during orthodontic relapse. Rat maxillary first molars were moved mesially for 10 days. After orthodontic tooth movement (OTM), appliances were removed, and the molars were allowed to relapse for one, three, five, seven, 14 or 21 days. Changes in 3D morphometric parameters of bone located mesial to the first molars were evaluated by micro-CT. Total RNA was isolated from the same bone site, and real-time RT-PCR was used to measure the expression of bone formation and resorption markers. One day after appliance removal, the molars relapsed to a mean 73% of the achieved OTM and then steadily relapsed to 93% at 21 days. Tissue mineral density and per cent bone volume increased over the experimental period. Inversely, there was a decrease in total porosity. Gene expression of OCN, Coll-I and ALP decreased during OTM, whilst as the molars relapsed showed tended to increase. Gene expression of RANKL and TRAP increased during OTM. Changes in mRNA expression of H(+)-ATPase were minor. By 21 days post-appliance removal, the remodelling process in rats appeared to have returned to control levels. Bone tissue reactions on a molecular level are similar during OTM and orthodontic relapse. These findings validate the importance of immediate retention following active OTM.

Low-level laser therapy for osteonecrotic lesions: effects on osteoblasts treated with zoledronic acid.

Clinical studies have shown that low-level laser therapy (LLLT) can improve local tissue healing of bisphosphonate-induced osteonecrosis of the jaw. However, the effects of laser irradiation on bisphosphonate-treated osteoblasts have not been completely elucidated. Human osteoblasts were cultured in plain culture medium (DMEM). After 48 h, plain DMEM was replaced by DMEM with no fetal bovine serum, for a 24-h incubation followed by addition of zoledronic acid (5 μM) for additional 48 h. Cells were subjected to LLLT (InGaAsP; 780 ± 3 nm; 0.025 W) at 0.5, 1.5, 3, 5, and 7 J/cm(2), three times every 24 h. Cell viability, total protein production, alkaline phosphatase activity (ALP), mineral nodule formation, gene expression of collagen type I and ALP, and cell morphology were evaluated. LLLT at 0.5 J/cm(2) increased cell viability of cultured osteoblasts. ALP activity and gene expression, in addition to mineral nodule formation and Col-I gene expression, were not increased by LLLT. LLLT applied to ZA-treated cells increased Col-I expression at 0.5, 1.5, and 3 J/cm(2) but did not improve any other cell activity assessed. LLLT showed limited effects on bisphosphonate-treated osteoblasts.

The pH in the Microenvironment of Human Mesenchymal Stem Cells Is a Critical Factor for Optimal Osteogenesis in Tissue-Engineered Constructs

The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue-engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human Bone marrow-derived mesenchymal stem cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, that is, 0.5 U more alkaline than that in two other cell-containing materials (hydroxyapatite/tricalcium phosphate [HA/TCP] and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics, which consistently promoted the formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, which displayed a pH of around 7.8, promoted 20-30-fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared with the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, the alkaline phosphatase [ALP] activity and gene expression of RUNX2, ALP, and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. The formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (>7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected, but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells.

Different BGMs associated to MSC: Can tissue engineering help GBR?

Purpose: Bone augmentation in oral surgery relies on osteogenic andosteoconductive properties of bone graftmaterial (BGM). Several types of BGMs with different chemical compositions andmicrostructures are today available, including natural derived or artificially modified heterologous bone, and synthetically synthetized bioceramics. A further improvement is represented by the tissue engineering approach, where autologous mesenchymal stem cells (MSCs), expanded in vitro, are seeded on BGM prior implantation in the bone defect. In this study the effect of different bone substitutes on MSC differentiation when they were adopted as cell culture substrates has been evaluated. Methods andmaterials:HumanMSCwere cultured in vitro in the presence of BGMs of different origins (natural bone derived, synthetic and mixed). Cellular adhesion on BGMs were analyzed with SEM. Alkaline phosphatase (ALP – early marker of the osteogenic differentiation) gene expression was evaluated by real time PCR andALP enzyme activitywas determined at different culture time points (7, 14 and 28 days). In vivo osteogenic differentiation of implanted tissue constructs (BGM+MSC) was investigated taking advantage of an ectopic bone formation in an immunodeficient mouse model and analyzed by histology. Results: A good cell adhesion to all BGMs was observed already after 6h from plating and cells were successfully cultured for 28 days. ALP gene expression and enzymatic activity was detectable after 14 day culture. The ALP gene expression analysis indicated that the MSC cultured with BGM derived from natural bone (BioGen, Bio-Oss) presented a steady increase of the ALP expression reaching a significantly higher level at 28th day of osteoinduction compared to the other BGMs. Instead, cells cultured in the presence of BGMs of synthetic origin (Bonit, Skelite), presented themaximum level of ALP gene expression at day 14 with a subsequent stabilization in the expression at day 28. Interestingly the cells cultured in the presence of mixed origin (Pepgen P-15) BGM presented a peak in the gene expression at day 7. The in vivo osteogenic test showed a good bone formation at the periphery of all BGMsparticles seededwith BMSCs. Neo-formed bone progressively filled the gap between the particles bridging them after 8 weeks, maintaining a peripheral vasculature. Conclusion: All tested BGMs implemented bone formation in guided bone regeneration. BGMs of animal origin showed greater ALP expression at 28 days.

Bond strength evaluation of adhesive systems containing natural antimicrobial components

Purpose: Bone augmentation in oral surgery relies on osteogenic andosteoconductive properties of bone graftmaterial (BGM). Several types of BGMs with different chemical compositions andmicrostructures are today available, including natural derived or artificially modified heterologous bone, and synthetically synthetized bioceramics. A further improvement is represented by the tissue engineering approach, where autologous mesenchymal stem cells (MSCs), expanded in vitro, are seeded on BGM prior implantation in the bone defect. In this study the effect of different bone substitutes on MSC differentiation when they were adopted as cell culture substrates has been evaluated. Methods andmaterials:HumanMSCwere cultured in vitro in the presence of BGMs of different origins (natural bone derived, synthetic and mixed). Cellular adhesion on BGMs were analyzed with SEM. Alkaline phosphatase (ALP – early marker of the osteogenic differentiation) gene expression was evaluated by real time PCR andALP enzyme activitywas determined at different culture time points (7, 14 and 28 days). In vivo osteogenic differentiation of implanted tissue constructs (BGM+MSC) was investigated taking advantage of an ectopic bone formation in an immunodeficient mouse model and analyzed by histology. Results: A good cell adhesion to all BGMs was observed already after 6h from plating and cells were successfully cultured for 28 days. ALP gene expression and enzymatic activity was detectable after 14 day culture. The ALP gene expression analysis indicated that the MSC cultured with BGM derived from natural bone (BioGen, Bio-Oss) presented a steady increase of the ALP expression reaching a significantly higher level at 28th day of osteoinduction compared to the other BGMs. Instead, cells cultured in the presence of BGMs of synthetic origin (Bonit, Skelite), presented themaximum level of ALP gene expression at day 14 with a subsequent stabilization in the expression at day 28. Interestingly the cells cultured in the presence of mixed origin (Pepgen P-15) BGM presented a peak in the gene expression at day 7. The in vivo osteogenic test showed a good bone formation at the periphery of all BGMsparticles seededwith BMSCs. Neo-formed bone progressively filled the gap between the particles bridging them after 8 weeks, maintaining a peripheral vasculature. Conclusion: All tested BGMs implemented bone formation in guided bone regeneration. BGMs of animal origin showed greater ALP expression at 28 days.

Quercetin protects osteoblast development in the presence of oxidative stress in fetal rat calvaria cells

Studies examining quercetin (QRC) effects on bone health are limited. The purpose of this study was to examine the extent that QRC impacts osteoblast cell development in the presence of oxidative stress, using osteoblast‐like cells isolated from fetal rat calvaria. At confluence, cells were pre‐treated for 12 hours with QRC aglycone (0 or 20 μM). Cells were then incubated for 4 days with QRC aglycone continued at the same dose as pre‐treatment (0, 20 μM) with or without hydrogen peroxide (H2O2; 0, 300 μM), a known inducer of oxidative stress. There were four groups: untreated Control, QRC alone, H2O2 alone, and QRC/H2O2. Expression of genes related to osteoblast phenotype development (runt‐related transcription factor 2, Runx2; alkaline phosphatase, ALP; type I collagen, COLL; bone sialoprotein, BSP; and osteocalcin, OCN) were analyzed using real‐time quantitative PCR. Additionally, alkaline phosphatase staining was used as an indicator of cells with osteoblastic potential. Results for Controls and QRC treated cells were similar. H2O2 alone suppressed ALP, BSP, and OCN gene expression (p < 0.05) and alkaline phosphatase staining. QRC, in the presence of H2O2, partially prevented the H2O2‐ induced suppression (p < 0.05). These results suggest that QRC is partially protective of osteoblast development in the presence of oxidative stress.Grant Funding Source: USDA Agricultural Research Services

Study on Osteoblast like Behavior of Umbilical Cord Blood Cells on Various Combinations of PLGA Scaffolds Prepared by Salt Fusion

The osteogenic potential of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) on porous poly lactide-co-glycolide (PLGA) scaffolds have been reported to differentially support osteogenic differentiation based on polymer composition (80:20, 75:25 and 70:30 percent of PLA: PGA, respectively). Along with polymer composition; fused NaCl crystal matrix prior to solvent casting improves the porosity and pore interconnectivity in 3D scaffolds, which has significant impact on cell proliferation. FTIR and XRD studies of PLGA scaffolds also verified the intermolecular interactions, phase distribution and crystallinity in scaffolds. Among three scaffold combinations, sample B (75:25) has showed maximum porosity with optimum water uptake/retention abilities. Impact of polymer composition and porosity on cell proliferation was investigated through MTT assay, where sample B was observed to be supporting better cell proliferation,due to its internal structure. The above results were further confirmed by ALP and Col-I gene expression studies using RT-PCR. Immuno fluorescent studies also revealed the extracellular filamentous actin organization over the scaffolds, where cell adhesion and proliferation was found to be higher with increase in PGA content, which is a hydrophilic polymer.

Identification of a Vibration Regime Favorable for Bone Healing and Muscle in Estrogen-Deficient Rats

Numerous whole-body vibration (WBV) devices of various forces are available on the market, although their influence on the musculoskeletal system is not yet understood. The effect of different WBVs on bone healing and muscle function was evaluated in rats ovariectomized at 3 months of age. 2 months after ovariectomy, bilateral metaphyseal tibia osteotomy and T-plate osteosynthesis were performed. Rats were divided into groups: intact, OVX, and OVX exposed to vertical WBVs of 35, 50, 70, or 90 Hz (experiment 1) or horizontal WBVs of 30, 50, 70, or 90 Hz (experiment 2) 5 days after osteotomy (0.5 mm, 15 min/day for 30 days). The tibia and gastrocnemius and soleus muscles were collected. Vertical vibrations (>35 Hz) improved cortical and callus densities, enlarged callus area and width, suppressed the tartrate-resistant acid phosphatase gene, enhanced citrate synthase activity, accelerated osteotomy bridging (35 and 50 Hz), upregulated the osteocalcin (Oc) gene (70 Hz), and increased relative muscle weight (50 Hz). Horizontal vibrations reduced cortical width (<90 Hz) and callus density (30 Hz), enhanced alkaline phosphatase (Alp) gene expression (50 Hz), decreased the size of oxidative fibers (35 and 70 Hz), and increased capillary density (70, 90 Hz). Biomechanical data; serum Oc, Alp, and creatine kinase activities; body weight; and food intake did not change after WBVs. Vertical WBVs of 35 and 50 Hz produced more favorable results than the higher frequencies. Horizontal WBV showed no positive or negative effects. Further studies are needed to elucidate the effects of WBV on different physiological systems, and precautions must be taken when implementing WBV in the treatment of patients.

Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus

Hypertrophy-like changes are often observed in chondrocytes during the development of osteoarthritis (OA). These changes play a crucial part in the OA-associated cartilage degradation and osteophyte formation. However, the pathogenesis leading to such changes is still unknown. In this study, we investigated the mechanism by which these hypertrophy-like changes are induced from the viewpoint of impaired glycolytic metabolism. The effect of sodium fluoride (NaF) on glycolytic metabolism of cultured chondrocytes was confirmed by measurement of intracellular adenosine triphosphate (ATP) production. Translocation of phosphorylated Smad1/5/8 to the nucleus was evaluated by subcellular fractionation and Western blotting. Chondrocyte hypertrophy-like changes were investigated by real-time RT-PCR and Western blot analysis of differentiation markers. ATP production was dose-dependently decreased by NaF in the human chondrocytic cell line HCS-2/8. In addition, both chondrocyte proliferation and differentiation were inhibited, whereas cell death was promoted by treatment with NaF. Interestingly, combinational treatment with NaF and lactate enhanced translocation of phospho-Smad1/5/8 to the nucleus, as well as gene expression of ALP, VEGF, COL10a1, and matrix metalloproteinase13 (MMP13), which were the markers of late mature and hypertrophic chondrocytes. Furthermore, the production of type X collagen and activation of MMP9 were also promoted under the same conditions. These findings suggest that decreased ATP production by NaF promotes hypertrophy-like changes via activation of phospho-Smad1/5/8 in the presence of lactate. Novel metabolic aspects of OA pathogenesis are indicated herein.

Osteopromotive Activity of a Novel Pyrazole Carboxamide Derivative

To investigate the effect of 1-(4-(tert-butyl)benzyl)-N-(4-methoxyphenyl)-3-phenyl-1H-pyrazole-5-carboxamide (Pyr-C) on the proliferation and osteogenic differentiation of MC3T3-E1 cells. MTT and BrdU incorporation assay were used to determine cell survival and proliferation. The gene expression levels of osteogenic markers were determined using real-time PCR and ALP activity was detected. Western-blot analysis was used to determine the protein expression of BSP and OPN. The long-term effect of Pyr-C on mineralization deposition was measured by Alizarin Red Staining. Pyr-C inhibited cell proliferation and increased ALP activity. Gene expression of ALP, BSP, OCN, Runx2, and Osterix was up-regulated in Pyr-C-induced group. Pyr-C increased the protein expression of BSP at day 7, 14 and 21, and OPN at day 14, 21 and 28. Meanwhile, Pyr-C enhanced the mineral deposition. Pyr-C inhibits proliferation and stimulates osteogenic differentiation of MC3T3-E1 cells.