Regulation of osteoblast activity by Pyk2-targeted approaches

Indiana University-Purdue University Indianapolis (IUPUI)The hormonal and cellular mechanisms controlling bone formation are not completely understood. The proline-rich tyrosine kinase 2 (Pyk2) is important for osteoblast (OB) activity and bone formation. However, female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone volume/total volume. Previously, our laboratory found ovariectomized Pyk2-KO mice supplemented with 17β-estradiol (E2) exhibited a greater increase in bone volume than WT mice treated with E2. The overall hypotheses of our studies are that Pyk2 regulates OB activity by modulating the E2-signaling cascade and that a Pyk2-inhibitor will promote OB activity and be suitable for bone regeneration applications. In Aim1, we determined the mechanism of action of Pyk2 and E2 in OBs. Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In the presence of E2 or raloxifene, a selective estrogen receptor (ER) modulator, both matrix formation and mineralization were further increased in Pyk2-KO OBs, but not WT OBs. Consistent with a role of Pyk2 in E2 signaling, Pyk2-depletion led to the proteasome-mediated degradation of ERα, but not ERβ. Finally, we found Pyk2-depletion and E2 have an additive effect on ERK phosphorylation, known to increase cell differentiation and survival. In Aim2, we developed a Pyk2-inhibitor loaded hydrogel and evaluated its viscosity, gelation time, swelling, degradation, and release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin exhibited viscosity and shear-thinning behavior suitable for use as an injectable-carrier. Importantly, the Pyk2-inhibitor-hydrogel was cytocompatible, retained its inhibitory activity against Pyk2 leading to an increase in OB activity. In conclusion, therapeutic strategies targeting Pyk2 may improve systemic bone formation, while Pyk2-inhibitor loaded hydrogels may be suitable for targeted bone regeneration in craniofacial and/or the other skeletal defects

Effect of Hydrofluoric Acid Etching Followed by Unfilled Resin Application on the Biaxial Flexural Strength of a Glass-based Ceramic

Indiana University-Purdue University Indianapolis (IUPUI)Background: Numerous studies have reported the use of hydrofluoric (HF) acid as one of the most effective methods for the achievement of a durable bond between glass-based ceramics and resin cements. Nevertheless, there is little information available regarding the potential deleterious effect on the ceramic mechanical strength. Objectives: (1) to investigate the effect of HF acid etching regimens on the biaxial flexural strength of a low-fusing nanofluorapatite glass-ceramic (IPS e.max ZirPress, Ivoclar Vivadent), (2) to study the ability of an unfilled resin (UR) to restore the initial (i.e., before etching) mechanical strength, and (3) to evaluate the effect of HF acid etching on the ceramic surface morphology before and after UR treatment via scanning electron microscopy (SEM). Methods: One hundred and forty-four disc-shaped (15 ± 1 mm in diameter and 0.8 ± 0.1 mm in thickness) IPS e.max ZirPress specimens were allocated into 12 groups, as follows: G1-control (no etching), G2-30 s, G3-60 s, G4-90 s, G5-120 s, G6- 60 + 60 s. Meanwhile, groups (G7- G12) were treated in the same fashion as G1-G6, but followed by silane and UR applications. Surface morphology evaluation of non-etched and etched IPS e.max ZirPress (G1-G12) was carried out by scanning electron microscopy (SEM). The flexural strength was determined by biaxial testing as described in ISO 6872. Statistics were performed using two-way ANOVA and the Sidak multiple comparisons (α = 0.05). In addition, the Weibull statistics were estimated. Results: A significant effect of etching time (p=0.0290) on biaxial flexural strength was observed. Indeed, G4 led to a significantly (p=0.0392) higher flexural strength than G1. Correspondingly, G10 revealed a considerably higher flexural strength than G7 (p=0.0392). Furthermore, biaxial flexural strength was significantly higher for G7 – G12 than for G1 – G6 (p<0.0001). For G1 – G6, G4 showed the highest Weibull characteristic strength while the lowest Weibull characteristic strength was seen in G6. In G7 – G12, the highest Weibull characteristic strength was presented in G10 whereas G7 had the lowest. Finally, the SEM data revealed that the HF acid etching affected the surface of IPS e.max ZirPress by generating pores and irregularities and more importantly that the UR was able to penetrate into the ceramic microstructure. Conclusion: Within the limitations of this study, HF acid etching time did not show a damaging effect on the biaxial flexural strength of the IPS e.max ZirPress ceramic. Moreover, the ceramic biaxial flexural strength could be enhanced after UR treatment

A Pyk2 Inhibitor Incorporated into a PEGDA-Gelatin Hydrogel Promotes Osteoblast Activity and Mineral Deposition

Pyk2 is a non-receptor tyrosine kinase that belongs to the family of focal adhesion kinases. Studies from our laboratory and others demonstrated that mice lacking the Pyk2 gene (Ptk2B) have high bone mass, which was due to increased osteoblast activity, as well as decreased osteoclast activity. It was previously reported that a chemical inhibitor that targets both Pyk2 and its homolog FAK, led to increased bone formation in ovariectomized rats. In the current study, we developed a hydrogel containing poly(ethylene glycol) diacrylate (PEGDA) and gelatin which was curable by visible-light and was suitable for the delivery of small molecules, including a Pyk2-targeted chemical inhibitor. We characterized several critical properties of the hydrogel, including viscosity, gelation time, swelling, degradation, and drug release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin (P1000:G10) exhibited Bingham fluid behavior that can resist free flowing before in situ polymerization, making it suitable for use as an injectable carrier in open wound applications. The P1000:G10 hydrogel was cytocompatible and displayed a more delayed drug release behavior than other hydrogels we tested. Importantly, the Pyk2-inhibitor-hydrogel retained its inhibitory activity against the Pyk2 tyrosine kinase, and promoted osteoblast activity and mineral deposition in vitro. Overall, our findings suggest that a Pyk2-inhibitor based hydrogel may be suitable for the treatment of craniofacial and appendicular skeletal defects and targeted bone regeneration

Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene

Bone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17β-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor β (ERβ). As a consequence, E2 signaling via ERβ was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERβ signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases

Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms

Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases

Can Cleansing Regimens Effectively Eliminate Saliva Contamination from Lithium Disilicate Ceramic Surface?

This study evaluated the effect of cleaning protocols on the bond strength of resin cement to glass-ceramic. Ceramic specimens (N=120, n=12 per group) were etched with hydrofluoric acid and rinsed with water. After saliva contamination, specimens were cleaned as follows: water, 37% H3PO4, cleaning-paste (Ivoclean), or isopropanol. Non-contaminated specimens acted as the control. Resin cement was bonded to the specimens, and tested either after 24 h or x5000 thermocycling. Both the cleaning method (p=0.001) and the storage conditions (p=0.005) significantly affected the bond strength results. In dry conditions, the groups PA and IV showed no significant difference, being also similar to the non-saliva contaminated control group (p⟩0.05). In dry conditions, no significant difference was observed between the mean DW and IS being significantly lower than those of other groups (p⟨0.05). Except for the group IV, thermocycling decreased the results significantly in all groups (p⟨0.05). Predominantly mixed failure type was observed in both dry and aged conditions. SEM micrographs of ceramic surfaces after cleaning agents showed no major differences but on the specimens from the IV group, small, rounded-zirconia particles were observed. In case of saliva contamination of acid-etched glass-ceramics, mechanical cleaning can restore adhesion to the baseline situation

The impact of hydrofluoric acid etching followed by unfilled resin on the biaxial strength of a glass-ceramic

Objectives: To evaluate the null hypotheses that hydrofluoric (HF) acid etching time would neither decrease the biaxial flexural strength of a glass-based veneering ceramic nor enhance it after silane and unfilled resin (UR) applications. Methods: Disc-shaped IPS e.max ZirPress specimens were allocated into 12 groups: G1-control (no-etching), G2-30 s, G3-60 s, G4-90 s, G5-120 s, G6-60 s + 60 s. Groups (G7-G12) were treated in the same fashion as G1-G6, but followed by silane and UR applications. Surface morphology and roughness (Ra and Rq) of the ceramics were assessed by means of scanning electron microscopy (SEM) and profilometry, respectively. Flexural strength was determined by biaxial testing. Data were analyzed by two-way ANOVA and the Sidak test (α = 0.05). Weibull statistics were estimated and finite element analysis (FEA) was carried out to verify the stress concentration end areas of fracture. Results: The interaction (etching time vs. surface treatment) was significant for Ra (p = 0.008) and Rq (0.0075). Resin-treated groups presented significantly lower Ra and Rq than non-treated groups, except for the 60 s group (p < 0.005). SEM revealed that etching affected the ceramic microstructure and that the UR was able to penetrate into the irregularities. A significant effect of etching time (p = 0.029) on flexural strength was seen. G7-G12 presented higher strength than G1-G6 (p < 0.0001). None of experimental groups failed to show 95% confidence intervals of σ 0 and m overlapped. FEA showed lower stress concentration after resin treatment. Significance: HF acid etching time did not show a damaging effect on the ceramic flexural strength. Moreover, the flexural strength could be enhanced after UR treatment. © 2013 Academy of Dental Materials