Increased Bone Cell Research Articles

Comparative effects of calcitriol and calcimimetic on bone health in renal insufficiency

AbstractCalcitriol and calcimimetics are used to treat hyperparathyroidism secondary to chronic kidney disease (CKD). Calcitriol administration and the subsequent increase in serum calcium concentration decrease parathyroid hormone (PTH) levels, which should reduce bone remodeling. We have previously reported that, when maintaining a given concentration of PTH, the addition of calcimimetics is associated with an increased bone cell activity. Whether calcitriol administration affects bone cell activity while PTH is maintained constant should be evaluated in an animal model of renal osteodystrophy. The aim of the present study was to compare in CKD PTH‐clamped rats the bone effects of calcitriol and calcimimetic administration. The results show that the administration of calcitriol and calcimimetic at doses that induced a similar reduction in PTH secretion produced dissimilar effects on osteoblast activity in 5/6 nephrectomized (Nx) rats with secondary hyperparathyroidism and in Nx rats with clamped PTH. Remarkably, in both rat models, the administration of calcitriol decreased osteoblastic activity, whereas calcimimetic increased bone cell activity. In vitro, calcitriol supplementation inhibited nuclear translocation of β‐catenin and reduced proliferation, osteogenesis, and mineralization in mesenchymal stem cells differentiated into osteoblasts. In conclusion, besides the action of calcitriol and calcimimetics at parathyroid level, these treatments have specific effects on bone cells that are independent of the PTH level.

Zinc and Strontium-Substituted Bioactive Glass Nanoparticle/Alginate Composites Scaffold for Bone Regeneration.

The global population is growing older and entering an aging society. Aging results in severe tissue disorder and organ dysfunction. Bone-related injuries are particularly significant. The need for alternative bone replacement materials for human implants has grown over the past few decades. Alginate has the potential for use as a cell scaffold for bone tissue engineering due to its high bio-compatibility. To improve the bioactivity of alginate scaffolds, zinc- and strontium-containing sol-gel-derived bioactive glass nanoparticles (Zn-Sr-BGNPs) with sizes ranging from 100 to l40 nm were incorporated. Zn-Sr-BGNPs synthesized through the sol-gel process have a high sur-face-to-volume ratio, homogeneity, and purity, resulting in faster degradation. The therapeutic bivalent ions released from Zn-Sr-BGNPs strengthen the cell scaffold and improve the stimulation of the production and development of bone cells. Zn-Sr-BGNPs with different Zn to Si nominal ratios of 0, 1, and 1.5 were mixed with alginate in this research. The ratio of Zn in Zn-Sr-BGNPs and the ratio of Zn-Sr-BGNPs in scaffolds impact the pore size, swelling, and biological properties of synthesized composite scaffolds. The surface area and pore volume of a 1:1 1Zn-Sr-BGNP:Alg composite scaffold were 22.58 m2/g and 0.055 cm3/g, respectively. The incorporation of Zn-Sr-BGNPs improved the mechanical performance of the scaffolds up to 4.73 ± 0.48 MPa. The swelling rate decreased slightly from 2.12 (pure Alg) to 1.50 (1Zn-Sr-BGNP:Alg (1:1)). The 1Zn-Sr-BGNP:Alg (1:1) composite scaffold promoted bioactivity through apatite layer formation, increased bone cell proliferation via the dissolution products released from the scaffold, enhanced calcium deposition, and facilitated cell attachment. Thus, 1Zn-Sr-BGNP:Alg (1:1) composite scaffold is proposed as a possible artificial bone scaffold in bone tissue regeneration.

Feasibility evaluation of a new lattice for porous surface design in additive manufacturing medical implants under interfacial tensile bonded testing

Porous titanium implant surfaces fabricated with suitable porosity and size using additive manufacturing (AM) can enhance bone–implant stability. There is a critical need to understand the biomechanical performance of porous (lattice) type implants to improve bone ingrowth efficiency and interfacial bonded strength. This study proposes a new lattice type implant with appropriate/favorable physiological environment within the lattice for bone ingrowth and increasing bone-implant bonded strength. A unit (1mm3 cube) YM lattice with 65 % porosity and average pore size of 750 µm was designed as a 3D spherical structure to increase bone cell growth capacity with multiple-corners for accepting cell clustering and the transport of fluids, nutrients and the expelling of impurities between these interconnected through-holes. The finite element (FE) sub-modeling technique was used to investigate the implant-bone interface micro mechanical behavior, to include lattice pillars, bone within/around lattices for four different lattice types with YMU, YMR (YM lattice without deformation and with 30 % deformation), DU and DR (Diamond lattice without deformation and with 30 % deformation). The DU and YMR lattice implants (6 × 6 × 3 mm3 block) were selected and fabricated using a Ti6Al4V AM (each n = 5) and implanted into the distal right femurs of 10 rabbits for 8 weeks for observation. The interface tensile bonded strength test was performed after the rabbits were sacrificed. No lattice implant slipped in any of the rabbits after surgery. This confirms postoperative radiographs. The test results showed that both the bonded force and strength of the YMR lattice were found significantly higher than those of DU. The corresponding values were 113.14 ± 21.96 N and 5.39 ± 1.04 MPa for the YMR lattice and 41.41 ± 15.32 N and 1.97 ± 0.73 MPa for the DU lattice. This study proposed a YMR lattice that presented a suitable bone ingrowth environment and interfacial bond strength test for surface porous design for the AM medical implant.

Effects of silver/selenium/chitosan doped hydroxyapatite coatings on REX-734 alloy: Morphology, antibacterial activity, and cell viability

The goal of this study was to improve the antibacterial activity and cell viability of the Rex-734 alloy in biomedical applications. Various biocomposite coatings, such as single-layer hydroxyapatite (HAp), HAp/silver(Ag), and HAp/selenium(Se)/chitosan(Chit), were applied to Rex-734 alloy at various film thicknesses. The surface coatings were characterized by scanning electron microscopy, SEM, EDX, XRD and coating thickness and hardness of surface coatings were measured. The in vitro antibacterial activities of the coated and uncoated substrates were tested against Esherichia coli JM 103 and Staphylococcus aureus ATCC29293. In vitro cell–material interactions using Saos-2 osteoblast cells were characterized by the 3-(4,5-dimethylthiazol2-yl)− 2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. The post-coating sintering parameters were shown to have a significant effect on the surface morphology. When compared to single HAp and HAp/Ag coatings, Se and Chit additions to HAp powders revealed good crack-free coating surfaces. Crack-free coating morphologies were achieved with a sintering speed of 3 °C/min. While single-HA coatings had an antimicrobial efficiency of 83% against E. coli, this value increased to 94% with Ag additive and to 85% with Se additive. Similarly, Ag and Se additives improved HAp's antimicrobial efficiency against Staphylococcus aureus. Cell viability decreased in the HAp/Ag coating at 86.5% and 90.93%, but increased significantly (p < 0.05) in the HA/Se-Chit coating at 137,43% and 158.23% at the end of the first and seventh days of incubation, respectively. The addition of Ag/Se/Chit to HAp increased the viability of the Rex-734 cells. Furthermore, it was revealed that the HAp-doped Se/Chit duplex biocomposite coating groups increased bone cell viability at much higher rates than the single-layer HAp coatings.

A Mouse Model Suggests How an Industrialized Diet Alters Jaw Form

Food texture affects jaw form by creating biomechanical stress during chewing. Chewing prompts remodelling via increased bone cell activities, including cell signalling, differentiation, proliferation, and matrix secretion. Vitamin D may influence jaw form via regulation of these bone cell activities. Modern, post-Industrialized human diets are characterized by soft textured food with low levels of vitamin D, but how these two factors affect mandible shape and size is unclear. Via a mouse model and specialized diets, we tested the effects of dietary texture and vitamin D on mandibular form. We hypothesized that vitamin D intake is: 1) imperative for normal mandibular form, and; 2) a greater influence on mandibular form than diet texture. C57BL/6J mice weaned at 7 weeks were raised on a soft or hard diet with vitamin D (n=40; n=48), or on a soft diet without vitamin D (n=39). Post-sacrifice, adult mice were scanned with a Skyscan1172 micro-CT system and landmarked using Amira software. Landmark datasets underwent Generalized Procrustes Analysis followed by Principal Component Analysis (PCA) in Morphologika. Mice fed soft diets had shorter mandibles with diminutive alveolar bone, gentler masseteric crests, a wider angle between dentary bones, and larger bicondylar breadth. Diet groups occupied distinct but overlapping regions of shape space, with diet texture accounting for 30.8% (PC1) of total shape variance. Presence/absence of vitamin D accounted for 13.8% (PC2) of total shape variance between soft diet groups. Mice fed a soft diet with vitamin D had mandibles with more flaring angular processes, more robust masseteric crests, and elevated alveolar bone. Our results indicate that vitamin D influenced dentary bone shape less than diet texture did, and that diet had a greater role in mandibular bone remodelling, including increased variation at muscle attachment sites. Future work will explore whether vitamin D also influences muscle form and function.

Effect of laser cladded co-doped strontium fluorapatite nanopowder coating on the antibacterial and cell attachment of Ti-6Al-4V implants for bone applications

ABSTRACT In this study, doped strontium fluorapatite (Sr-FAP) nanopowders were synthesised by sol-gel and used as a coating to modify Ti-6Al-4V substrates through CO2 laser cladding. The Ca ions of FAP were substituted by a different amount of Sr ions to increase bone cell differentiation and antibacterial properties. Lattice parameters and crystal size values of different samples decrease with increasing the amount of Sr ion. According to the morphological results, the laser cladding Sr doped FAP nanopowders can create homogenously and crack-free coating on the Ti-6Al-4V substrates. The 0.6SrFAP nanopowder sample had a more hydrophilic nature in comparison to other Sr doped FAP nanopowders. This sample showed antibacterial properties and high cell adhesion, spreading, and viability while provided a bioactive and favourable surface for bone tissue regeneration. Therefore, 0.6SrFAP nanopowder laser cladding on the surface of Ti-6Al-4VV implants seems to be promising for further in-vivo investigations in bone applications.

Efficient fabrication of ultrafine-grained 316L stainless steel surfaces for orthopaedic applications

ABSTRACTCommonly used severe plastic deformation (SPD) methods are suitable for fabrication of bulk nano and ultrafine-grained metals. Drawbacks of these methods include durability of dies, geometrical restrictions and reduced ductility of the products. In this study, two common machining techniques used in manufacturing of orthopaedic components, turning and milling, were applied on 316L stainless steel as surface SPD to refine the surface microstructures of the workpiece. Machining with optimised parameters resulted in substantial grain refinement down to 98 nm on the surfaces. Biological experiments showed up to ∼70% and ∼280% increased bone cell density on milled and turned samples compared to conventionally machined 316L stainless steel at 5 days, which was correlated with nanocrystallisation and nanoroughness of the samples.

3-D printed Ti-6Al-4V scaffolds for supporting osteoblast and restricting bacterial functions without using drugs: Predictive equations and experiments

Conditions resulting from musculoskeletal deficiencies (MSDs) are wide-ranging and retain the likelihood for restricting motion or producing pain, especially in the lower back, neck, and upper limbs. Engineered scaffold devices are being produced to replace antiquated modalities that suffer from structural and mechanical deficiencies in the treatment of MSDs. Here, as-fabricated Ti-6Al-4V-based Hive™ interbody fusion scaffolds, commercialized by HD Lifesciences LLC, were assayed for their osteogenicity and antibacterial potential using a series of characterization and in vitro tests, as well as by quantitative analyses. A topographical assessment of the Hive™ meshes indicated that the elementally pure substrates are microscopically porous and rough, in addition to displaying structural heterogeneity. Roughness estimations and static contact angle measurements recommended the use of the as-fabricated Ti-6Al-4V substrates for supporting osteoblast attachment, especially, due to the improved surface roughness and wettability values of these scaffolds relative to the unembellished Ti-6Al-4V surfaces. Quantitative correlations relating the surface properties of roughness and energy were applied to predict cellular behaviors. Cell growth suppositions were experimentally corroborated. Critical in vitro data indicated the competencies of the Hive™ scaffolds for promoting the adhesion and proliferation of human fetal osteoblasts (hFOBs), accumulating substantial calcium deposition from metabolizing hFOBs, and restricting the attachment of bacteria. The model system that investigated the pre-adsorption of casein proteins along the Hive™ test substrates additionally furthered the notion that bacterial attachment may be restricted, with short-scale adhesion dynamics serving as the theoretical basis for this hypothesis. In this manner, this study showed that through predictive models and experiments, these novel 3D printed Ti-based scaffolds can increase bone cell while decreasing bacteria functions without using drugs. Statement of SignificanceSintered Ti-6Al-4V spinal fusion devices (Hive™) manufactured and marketed by HD Lifesciences LLC were assessed for their biocompatibility and antibacterial performance. A mixed methods approach was employed, whereby quantitative measures were used to predict the ability for Hive™ substrates to adsorb specialized proteins and to restrict bacterial surface colonization. In vitro tests that evaluated bone cell and bacterial adhesion, calcium deposition, and protein adsorption supported quantitative predictions. The data herein presented demonstrate the following: (1) surface energy is an important predictor of implant-cell interactions, (2) strong correlations exist between surface energy and surface roughness, (3) mathematical models can be used to improve and predict implant device perofrmance, and (4) porous, rough, 3D-printed materials perform well in terms of biocompatibility and antimicrobial efficacy.

Effects of Davallia formosana Hayata Water and Alcohol Extracts on Osteoblastic MC3T3-E1 Cells.

The Taiwanese native fern Davallia formosana Hayata (DFH) is used to treat bone diseases in classical Chinese medicine. We analyzed MC3T3E1 osteoblasts treated with different concentrations of water and ethanol extracts (10, 25, and 50 [both], and 100μg/mL [DFE only]) using cell viability, expression of osteoblast differentiation markers [bone morphogenetic protein 2 (BMP-2), collagen 1 (CoL-1), alkaline phosphatase (ALP), and Runt-related transcription factor 2 (Runx 2)], and mineralization. These were significantly increased by DFW or DFE after 24-h incubation compared with the untreated controls. Compared with other treatments, DFW 50 and DFE 100μg/mL significantly increased MC3T3E1 cell survival. DFW 25 and 50μg/mL increased bone BMP-2, CoL-1, ALP, and Runx2 protein expression, ALP activity, and mineralization more than DFE did. Repeated chromatographic separation of DFW yielded compound (-)-epicatechin-3-O-d-allipyranoside (ECAP), which was characterized using 1 H and 13 C nuclear magnetic resonance spectroscopy. (-)-Epicatechin-3-O-d-allipyranoside (0.01μg/mL) significantly increased cell survival (118.9%) and mineralization (218.7%) compared with that of the control treatment. We inferred that ECAP could mediate the main activity of DFW in bone formation, likely through BMP-2-induced Runx2 transcription, which increased bone cell differentiation factors ALP and CoL-1 and promoted mineralization. (-)-Epicatechin-3-O-d-allipyranoside could be an anti-osteoporotic agent. Copyright © 2017 John Wiley & Sons, Ltd.

Adult Paget's disease of bone: a review.

Adult PD of bone is the second commonest metabolic bone condition after osteoporosis. The condition is characterized by increased bone cell activity, with bone-resorbing osteoclasts often larger and containing more nuclei than normal, and osteoblasts producing increased amounts of disorganized bone. This leads to expanded bone of poor quality possessing both sclerotic and lytic areas. PD of bone has a strong genetic element, with a family history being noted in 10-20% of cases. A number of genetic defects have been found to be associated with the condition. The most common disease-associated variants identified affect the SQSTM1 gene, providing insights into disease aetiology, with the clinical value of knowledge of SQSTM1 mutation status currently under active investigation. The diagnosis may be suggested by an isolated raised total ALP without other identifiable causes. This can be confirmed on plain X-rays and the extent determined by isotope bone scan. The mainstays of treatment are the bisphosphonates, especially i.v. zoledronate, which results in long-term suppression of bone turnover. ALP is the usual means of monitoring the condition, although more specific bone turnover markers can be helpful, especially in coincident liver disease. Patients should be followed up to monitor for biochemical relapse or development of complications, which may require medical or surgical intervention.

Estrogen Withdrawal from Osteoblasts and Osteocytes Causes Increased Mineralization and Apoptosis

Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17β-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.

Understanding the wetting properties of nanostructured selenium coatings: the role of nanostructured surface roughness and air-pocket formation

Wetting properties of biomaterials, in particular nanomaterials, play an important role, as these influence interactions with biological elements, such as proteins, bacteria, and cells. In this study, the wetting phenomenon of titanium substrates coated with selenium nanoparticles was studied using experimental and mathematical modeling tools. Importantly, these selenium-coated titanium substrates were previously reported to increase select protein adsorption (such as vitronectin and fibronectin), to decrease bacteria growth, and increase bone cell growth. Increased selenium nanoparticle coating density resulted in higher contact angles but remained within the hydrophilic regime. This trend was found in disagreement with the Wenzel model, which is widely used to understand the wetting properties of rough surfaces. The trend also did not fit well with the Cassie–Baxter model, which was developed to understand the wetting properties of composite surfaces. A modified wetting model was thus proposed in this study, to understand the contributing factors of material properties to the hydrophilicity/hydrophobicity of these nanostructured selenium-coated surfaces. The analysis and model created in this study can be useful in designing and/or understanding the wetting behavior of numerous biomedical materials and in turn, biological events (such as protein adsorption as well as bacteria and mammalian cell functions).

Metabolic acidosis increases fibroblast growth factor 23 in neonatal mouse bone

Fibroblast growth factor 23 (FGF23) significantly increases with declining renal function, leading to reduced renal tubular phosphate reabsorption, decreased 1,25-dihydroxyvitamin D, and increased left ventricular hypertrophy. Elevated FGF23 is associated with increased mortality. FGF23 is synthesized in osteoblasts and osteocytes; however, the mechanisms by which it is regulated are not clear. Patients with chronic kidney disease have decreased renal acid excretion leading to metabolic acidosis, which has a direct effect on bone cell activity. We hypothesized that metabolic acidosis would directly increase bone cell FGF23 production. Using cultured neonatal mouse calvariae, we found that metabolic acidosis increased medium FGF23 protein levels as well as FGF23 RNA expression at 24 h and 48 h compared with incubation in neutral pH medium. To exclude that the increased FGF23 was secondary to metabolic acidosis-induced release of bone mineral phosphate, we cultured primary calvarial osteoblasts. In these cells, metabolic acidosis increased FGF23 RNA expression at 6 h compared with incubation in neutral pH medium. Thus metabolic acidosis directly increases FGF23 mRNA and protein in mouse bone. If these results are confirmed in humans with chronic kidney disease, therapeutic interventions to mitigate acidosis, such as bicarbonate administration, may also lower levels of FGF23, decrease left ventricular hypertrophy, and perhaps even decrease mortality.

Cigarette smoking and serum liver enzymes: the role of alcohol and inflammation

Smoking may affect the liver through inflammatory pathways and may aggravate the pathogenic effects of alcohol on the liver. We have examined the relationship between cigarette smoking and liver enzymes and the role of alcohol and C-reactive protein (CRP), a marker of inflammation. The subjects consisted of 4595 men aged 40-59 y with no history of coronary heart disease drawn from general practices in 24 British towns. Cigarette smoking was significantly associated with increased levels of gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) (P < 0.0001) and was inversely associated with increased aspartate aminotransferase (AST) after adjustment for alcohol intake, body mass index and physical activity. Compared with never smokers, heavy cigarette smokers (> or =40/day) were associated with increased odds of elevated GGT (> or =23 IU/L) (adjusted odds ratio [OR] 1.56 [1.08, 2.27]), which was abolished after adjustment for CRP (adjusted OR 1.27 [0.87, 1.86]). There was a significant interaction between smoking and alcohol on GGT. In the absence of heavy drinking, there was no association between smoking and GGT after adjustment for CRP. Among heavy drinkers, smoking was associated with increased levels of GGT independent of CRP. Smoking was associated with increased odds of elevated ALP (> or =11 IU/L) (adjusted OR 3.95 [2.77, 5.62]), which persisted after adjustment for CRP and white cell count (adjusted OR 2.90 [1.99-4.23]), possibly reflecting increased bone cell activity. The findings suggest that cigarette smoking does not cause liver injury, but may enhance the effects of alcohol on liver cell injury in heavy drinkers.

Titanium with Surface-Grafted Dextran and Immobilized Bone Morphogenetic Protein-2 for Inhibition of Bacterial Adhesion and Enhancement of Osteoblast Functions

Failure of bone and joint implants has been attributed mainly to poor bonding of the implant to bone tissue, and to bacterial infection. The probability of successful osseointegration or implant infection depends on the race for the surface between tissue cells and bacteria. One promising strategy to enhance tissue integration is to develop a selective biointeractive surface that increases bone cell (osteoblast) function while decreasing bacterial adhesion. In this in vitro study, the surface of titanium alloy substrates was first functionalized by covalently grafted oxidized dextran, which is known to have activity against bacterial adhesion. Bone morphogenetic protein-2 (BMP-2) was then covalently linked to dextran-grafted surfaces through a chemical conjugation process. The composition and properties of the surface were investigated by X-ray photoelectron spectroscopy and by measuring the surface density of BMP-2 using an enzyme-linked immunosorbent assay. Bacterial adhesion was assayed with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial adhesion on both the dextran and dextran-BMP-2-functionalized surfaces was significantly decreased compared to that on the pristine substrates. Further, the dextran-BMP-2 modified substrates with a surface protein density of >50 ng/cm(2) or higher significantly promoted osteoblast spreading, alkaline phosphatase activity, and calcium mineral deposition. Thus, the results from this study suggest that surface grafting of dextran in conjunction with the bone growth factor BMP-2 on metal surfaces can enhance tissue integration of implants through the dual functions of reducing bacterial adhesion and promoting osteoblast functions.

Chinese red yeast rice (Monascus purpureus-fermented rice) promotes bone formation

BackgroundStatin can induce the gene expression of bone morphogenetic protein-2. Red yeast rice (RYR, Hongqu), i.e. rice fermented with Monascus purpureus, contains a natural form of statin. This study demonstrates the effects of RYR extract on bone formation.MethodsBone defects were created in the parietal bones of two New Zealand white rabbits. In the test animal, two defects were grafted with collagen matrix mixed with RYR extract. In the control animal, two defects were grafted with collagen matrix alone. UMR 106 cell line was used to test RYR extract in vitro. In the control group, cells were cultured for three durations (24 hours, 48 hours and 72 hours) without any intervention. In the RYR group, cells were cultured for the same durations with various concentrations of RYR extract (0.001 g/ml, 0.005 g/ml and 0.01 g/ml). Bicinchoninic acid (BCA) assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and alkaline phosphatase (ALP) assay were performed to measure total protein, mitochondrial activity and bone cell formation respectively.ResultsThe test animal showed more formation of new bone in the defects than the control animal. RYR significantly increased the optical density in the MTT assay and ALP activity in vitro.ConclusionRYR extract stimulated new bone formation in bone defects in vivo and increased bone cell formation in vitro.

Effect of naringin on bone cells

Statin, a HMG-CoA reductase inhibitor, was shown to increase BMP-2 gene expression for bone formation, by blocking the mevalonate pathway in cholesterol production. We investigated the effect of naringin, a flavonoid available commonly in citrus fruits, which was also a HMG-CoA reductase inhibitor, in UMR 106 osteoblastic cell line in vitro. The control group consisted of cells cultured without any intervention for different time intervals (24 h, 48 h, and 72 h), whereas the experimental (naringin) group consisted of cells cultured with naringin of different concentrations (0.001 micromol/L, 0.01 micromol/L, and 0.1 micromol/L) for the same time intervals of the control. Colorimetric Tetrazolium (MTT) assay, total protein content assay, and alkaline phosphatase activity were used to measure the cellular activities. Results for the naringin group showed an increase in MTT assay compared with the control and the effect was dose dependent. At high concentration (0.1 micromol), the increases ranged from 60% to 80%. In the total protein content assay, naringin also showed an increase compared with control and the effect was also dose dependent. At high concentration (0.1 micromol), the increases ranged from 9% to 20%. In the alkaline phosphatase activity assay, naringin at high concentration (0.1 micromol) significantly increased the activity up to 20%. In conclusion, naringin significantly increased bone cell activities in vitro. This is the first study specifically attempted to investigate the effect of naringin on bone cell activities. Besides statin, this provided another example of mevalonate pathway blockage in the cholesterol production pathway by HMG-CoA reductase inhibition will increase the bone cell activities.

Fluid Shear Stress Synergizes with Insulin-like Growth Factor-I (IGF-I) on Osteoblast Proliferation through Integrin-dependent Activation of IGF-I Mitogenic Signaling Pathway

This study tested the hypothesis that shear stress interacts with the insulin-like growth factor-I (IGF-I) pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm(2) for 30 min followed by 24-h incubation with IGF-I (0-50 ng/ml). IGF-I increased proliferation dose-dependently (1.5-2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2-5.3-fold and that of IGF-I receptor (IGF-IR) by 2-4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12- and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test whether the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress +/- IGF-I was measured. Echistatin reduced basal proliferation by approximately 60% and the shear stress-induced mitogenic response by approximately 20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR.

Stimulation of bone cell differentiation by low-intensity ultrasound??a histomorphometric in vitro study

Several investigations have established a stimulatory effect of low-intensity ultrasound treatment on osteogenesis and fracture healing. The objective of this study was to examine whether the stimulatory effect of low-intensity ultrasound results in increased bone cell activity and/or proliferation. Twenty-four paired triplets of metatarsal bone rudiments of twelve 17-days-old fetal mice were dissected and divided into two groups. One group of bone rudiments was treated with pulsating low-intensity ultrasound (30 mW/cm2; 1.5 MHz) for 20 min/day for a period of 3 or 6 days. The other group served as controls. After culture, the metatarsal bone rudiments were prepared for computer aided light microscopy. The following histomorphometric parameters were determined: length, width and volume of the calcified cartilage and of the bone collar, and cell number. GLM analysis demonstrated that bone collar volume and calcified cartilage percentage were significantly higher in the ultrasound-stimulated rudiments compared to untreated controls. Further, the calcified cartilage volume bordering the hypertrophic zone was significantly higher than in the center of the bone rudiment. Ultrasound treatment did not change the number of the cells. These results suggest that the stimulatory effect of low-intensity ultrasound on endochondral ossification is likely due to stimulation of bone cell differentiation and calcified matrix production, but not to changed cell proliferation. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Stimulation of bone cell differentiation by low-intensity ultrasound––a histomorphometric in vitro study

Several investigations have established a stimulatory effect of low-intensity ultrasound treatment on osteogenesis and fracture healing. The objective of this study was to examine whether the stimulatory effect of low-intensity ultrasound results in increased bone cell activity and/or proliferation. Twenty-four paired triplets of metatarsal bone rudiments of twelve 17-days-old fetal mice were dissected and divided into two groups. One group of bone rudiments was treated with pulsating low-intensity ultrasound (30 mW/cm 2; 1.5 MHz) for 20 min/day for a period of 3 or 6 days. The other group served as controls. After culture, the metatarsal bone rudiments were prepared for computer aided light microscopy. The following histomorphometric parameters were determined: length, width and volume of the calcified cartilage and of the bone collar, and cell number. GLM analysis demonstrated that bone collar volume and calcified cartilage percentage were significantly higher in the ultrasound-stimulated rudiments compared to untreated controls. Further, the calcified cartilage volume bordering the hypertrophic zone was significantly higher than in the center of the bone rudiment. Ultrasound treatment did not change the number of the cells. These results suggest that the stimulatory effect of low-intensity ultrasound on endochondral ossification is likely due to stimulation of bone cell differentiation and calcified matrix production, but not to changed cell proliferation.