Inflammatory Bone Resorption and Antiosteosarcoma Potentials of Zinc Ion Sustained Release ZnO Chips: Friend or Foe?

Multifunctional zinc oxide (ZnO) has been generated as nanoparticles or nanorods and applied to various medical purposes since it exhibits several biological actions including anticancer activity. Especially, due to antibacterial activity and effects on bone regeneration, ZnO is widely used in implants and scaffolds in the orthopedic and dental fields. However, concerns over side effects have been raised recently in the clinical use of ZnO, and it is necessary to assess the safety of ZnO regarding its inflammatory potential in the bone environment. This made us hypothesize that the inflammatory activity of zinc ions released from ZnO NPs could be harmful to induce bone resorption but that their cytotoxicity would be beneficial to kill osteosarcoma. To clarify this hypothesis, in the present work, the effects of ZnO on bone matrix and abnormal bone environments were investigated quantitatively using ZnO chips, filter paper, or glass slides coated with thin films of ZnO grown via atomic layer deposition (ALD). ALD-grown ZnO thin films exhibit thickness with atomic precision, which enables the quantitative analysis of the effects of ZnO. <i>In vivo</i> application of ZnO chips to mouse calvarial bone induced bone resorption, presumably due to the activation of osteoclasts by zinc ion-induced TNF-α release. However, <i>in vitro</i> application of ZnO chips to osteosarcoma cells induced caspase-dependent apoptosis and oxidative stress. Taken together, the results showed two sides of ZnO as our hypothesis. Therefore, careful design and multiple evaluations for the safety and efficacy of ZnO materials are necessary for its successful clinical application

Effect of KM11073 on the activation of p38.

<p>Cells (1 × 10⁵ cells/well) were cultured in a 6-well plate for 1 day and then incubated with DMEM containing 5% FBS in the presence or absence of BMP-2 and/or KM11073 (<i>A</i>). Inhibitory effects of p38 inhibitors (1, SB202190; 2, PD169316; 3, SB203580) on the activation of p38 by BMP-2 and KM11073. Western blot analysis was performed with protein samples prepared with cells treated with each inhibitor for 30 min and then incubated with BMP-2 and KM11073 for 30 min (<i>B</i>). The relative, normalized ratio between phosphorylated protein and the protein itself was presented.</p

Quinoline Compound KM11073 Enhances BMP-2-Dependent Osteogenic Differentiation of C2C12 Cells via Activation of p38 Signaling and Exhibits <i>In Vivo</i> Bone Forming Activity

<div><p>Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl) piperazino][1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl]methanone (KM11073) strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP), an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the <i>in vivo</i> osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.</p></div

Osteogenesis-related gene expression in zebrafish.

<p>At 5.0 dpf, larvae were treated with KM11073 (1 μM), and after 1 day the mRNA levels were evaluated by quantitative real-time PCR. Fold changes relative to each gene level in the control are presented as mean ± standard deviation.</p><p>* <i>p</i> < 0.05</p><p>** <i>p</i> < 0.01 (compared to the control).</p><p>Osteogenesis-related gene expression in zebrafish.</p

Evaluation of the <i>in vivo</i> osteogenic activity of KM11073 in zebrafish and mouse calvariae.

<p>Five days after fertilization, zebrafish were treated with KM11073 (1 μM) for 2 days and then fixed and stained with alizarin red S. The parasphenoid (ps), notochord (n), ceratobranchial 5 (cb5), otolith (ot), and vertebrae (vb) are indicated with arrows (<i>A</i>). Collagen sponges soaked in 5 μl of 2.5 or 5 mM KM11073 were placed onto mouse calvarial bones. After 3-week implantation, the mice were sacrificed. Calvarial bones were removed, fixed, decalcified, embedded in paraffin, and sectioned. Sections were stained with H&E and photographed at 200 × magnification. Arrows indicate the thickness of newly formed woven bones (<i>B</i>). The thickness of newly formed woven bones was quantified compared to the scale bar.</p

Involvement of p38 in the KM11073-mediated enhancement of BMP-2-stimulated ALP induction.

<p>In a 96-well plate, cells (4 × 10³ cells/well) were treated with each inhibitor for 2 h and then treated with BMP-2 and KM11073. After 3 days, the cells were treated with each inhibitor. On differentiation day 6, ALP staining (A) and its activity (B) were assayed. ^###<i>p</i> < 0.001 compared to the BMP-2-treated group; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 compared to the group treated with BMP-2 and KM11073.</p

KM11073 enhanced BMP-2-induced osteoblast differentiation in C2C12 cells.

<p>Cell viability was assayed 1 and 3 days after treatment with KM11073 (<i>A</i>). Effect of KM11073 on BMP-2-stimulated ALP induction. Cells (4 × 10³ cells/well) were cultured in a 96-well plate for 1 day and then the medium replaced with DMEM containing 5% FBS and KM11073 in the presence or absence of rhBMP-2 (100 ng/ml). The medium was changed every 3 days. On differentiation day 6, ALP staining and its activity were assayed <i>(B)</i>. Effect of noggin on KM11073-mediated enhancement of BMP-2-stimulated ALP induction. Osteogenesis was enhanced by KM11073 in the presence of BMP-2 on differentiation days 0 and 2, and then noggin was treated on differentiation day 4. On differentiation day 6, ALP staining and its activity were assayed (<i>C</i>). *** <i>p</i> < 0.001 compared to the BMP-2-treated group; ^##<i>p</i> < 0.01, ^###<i>p</i> < 0.001 compared to the group treated with BMP-2 and KM11073.</p

Involvement of p38 inhibitors in the KM11073-mediated enhancement of BMP-2-stimulated induction of osteogenic genes.

<p>Cells were treated with each inhibitor for 2 h and then incubated with BMP-2 (100 ng/ml) and KM11073 (10 μM) for 3 days. The mRNA expression levels were evaluated by quantitative real-time PCR. Fold changes relative to each gene level in the control are presented as mean ± standard deviation</p><p>* <i>p</i> < 0.05</p><p>** <i>p</i> < 0.01 (compared to the control)</p><p>^#<i>p</i> < 0.05</p><p>^##<i>p</i> < 0.01 (compared to the group treated with BMP-2)</p><p>^†<i>p</i> < 0.05</p><p>^††<i>p</i> < 0.01 (compared to the group treated with BMP-2 + KM11073).</p><p>Involvement of p38 inhibitors in the KM11073-mediated enhancement of BMP-2-stimulated induction of osteogenic genes.</p

Chemical structure of KM11073.

<p>Chemical structure of KM11073.</p

Praeruptorin A Inhibits <i>in Vitro</i> Migration of Preosteoclasts and <i>in Vivo</i> Bone Erosion, Possibly Due to Its Potential To Target Calmodulin

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i>. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. <i>In silico</i> binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca²⁺/calmodulin and inhibit its downstream signaling pathways, including the Ca²⁺/calmodulin-CaMKIV-CREB and Ca²⁺/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. <i>In vivo</i> application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i> by targeting calmodulin and inhibiting the Ca²⁺/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca²⁺/calmodulin-calcineurin-NFATc1 signaling axis