Non-Overlapping Functions for Pyk2 and FAK in Osteoblasts during Fluid Shear Stress-Induced Mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2+/+ and Pyk2−/− primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2+/+ and Pyk2−/− osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK−/− osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts

Influence of Sterilization Technologies on Electrospun Poly(ester urea)s for Soft Tissue Repair

Degradable poly­(ester urea)­s (PEU)­s were electrospun into nanofiber sheets and assessed for their potential to be used in soft tissue repair. The level of residual solvent was measured and the effects of ethylene oxide and electron beam sterilization techniques on molecular mass, mass distribution, and morphology were quantified. Two PEU compositions that formed stable nanofiber sheets were advanced into a pilot study <i>in vitro</i> and <i>in vivo</i> as candidate materials for hernia repair. Cell viability, spreading, proliferation, and migration were examined <i>in vitro</i>. Nanofiber sheets were implanted subcutaneously into mice and analyzed via microangiography and histology for tissue incorporation. Nanofiber sheets performed similarly to decellularized extracellular matrix (ECM) <i>in vitro</i>, but the lack of sufficient pore structure inhibited cellular infiltration after 14 days of culture. The lack of microporous features in nanofiber sheets also contributed to low levels of cellular infiltration, angiogenesis, and matrix deposition <i>in vivo</i>. A preliminary study to increase pore size in nanofibers was performed using coaxial electrospinning resulting in significant improvement in tissue infiltration <i>in vivo</i>

Analysis of OPN expression in osteoblasts exposed to FF.

<p>(A) Expression of OPN mRNA as determined by qRT-PCR analysis of Pyk2^+/+ and Pyk2^−/− osteoblasts exposed to either 2 hour of FF followed by a 20 hour post-flow incubation or maintained in static culture conditions. (B) Expression of OPN mRNA as determined by qRT-PCR analysis of FAK^+/+ and FAK^−/− osteoblasts exposed to either 2 hour of FF followed by a 20 hour post-flow incubation or maintained in static culture conditions. (C) Graphical representation of data in Fold Difference. Experiments were performed in triplicate, and n≥3 for each experimental group in each trial. Error bars represent standard error. ^aStatistically significant difference between static and FF (p<0.05). ^bStatistically significant difference between static conditions (p<0.05).</p

Analysis of COX-2 protein levels in response to FF.

<p>(A) Western blot analysis of COX-2 protein levels in Pyk2^+/+ and Pyk2^−/− osteoblasts exposed to either 1 hour of FF followed by a 3 hour post-flow incubation (F) or maintained in static culture conditions (S). (B) Western blot analysis of COX-2 protein levels in FAK^+/+ and FAK^−/− osteoblasts exposed to 2 hours of FF (F) or maintained in static culture conditions (S). Vinculin was analyzed as a loading control. Graphs represent quantification of c-Fos protein levels expressed as raw densitometry units. Experiments were performed in triplicate, and n≥3 for each experimental group in each trial. Error bars represent standard error. ^aStatistically significant difference between static and FF (p<0.05).</p

Analysis of c-Fos protein levels in response to Fluid Flow (FF).

<p>(A) Western blot analysis of c-Fos protein in Pyk2^+/+ and Pyk2^−/− osteoblasts exposed to 30 minutes of FF (F) or maintained in static culture conditions (S). (B) Western blot analysis of c-Fos protein in FAK^+/+ and FAK^−/− osteoblasts exposed to 30 minutes of FF (F) or maintained in static culture conditions (S). Vinculin was analyzed as a loading control. Graph represents quantification of c-Fos protein levels expressed as raw densitometry units. Experiments were performed in triplicate, and n≥3 for each experimental group in each trial. Error bars represent standard error. ^aStatistically significant difference between static and FF (p<0.05).</p