Mechanobiological Assessment of TMJ Disc Surfaces: Nanoindentation and Transmission Electron Microscopy

Objectives: Temporomandibular disc is a mechanically robust fibrocartilage tissue exhibiting highly elastic compressive, shear, and tensile moduli with structurally dense extracellular matrix that supports functional loading of the joint. The aim of this study was to illustrate structural complexities of the superior and inferior disc surfaces, to demonstrate the robust mechanical ability of the disc as a whole may be due to depth-dependent regional/layered variation, and also to provide characterization data imperative for future tissue engineering efforts focused on restoring function to the joint. Material and Methods: Nanoindentation was used to assess tissue zones in conjunction with detailed Transmission Electron Microscopy to define structural attributes that influence the temporomandibular disc function. Results: The disc architecture adjacent to the superior surface was shown to have three distinct regional segments within the interface layer: 1-a surface peripheral layer; 2-subsurface region; and 3-a layer of helical matrix bundles. The inferior surface displayed an interface layer (20 µm) that showed limited cell populations with little depth-dependent structural variation, a stiffer elastic modulus and reduced energy dissipation compared to the superior surface. These data indicate that the primary function of the inferior surface is resistance to compression rather than load distribution during joint motion. Conclusions: These are the first works that demonstrate that the superior central surface of the he temporomandibular disc is structured in depth-dependent isometric layers, each of which provides different mechanical function supporting the bulk tissue’s properties. From a clinical perspective these data have potential to define regions susceptible to fatigue that may translate to diagnostic criteria to better define the stages of dysfunction

Adaptation of Endothelial Cells to Physiologically-Modeled, Variable Shear Stress

<div><p>Endothelial cell (EC) function is mediated by variable hemodynamic shear stress patterns at the vascular wall, where complex shear stress profiles directly correlate with blood flow conditions that vary temporally based on metabolic demand. The interactions of these more complex and variable shear fields with EC have not been represented in hemodynamic flow models. We hypothesized that EC exposed to pulsatile shear stress that changes in magnitude and duration, modeled directly from real-time physiological variations in heart rate, would elicit phenotypic changes as relevant to their critical roles in thrombosis, hemostasis, and inflammation. Here we designed a physiological flow (PF) model based on short-term temporal changes in blood flow observed <em>in vivo</em> and compared it to static culture and steady flow (SF) at a fixed pulse frequency of 1.3 Hz. Results show significant changes in gene regulation as a function of temporally variable flow, indicating a reduced wound phenotype more representative of quiescence. EC cultured under PF exhibited significantly higher endothelial nitric oxide synthase (eNOS) activity (PF: 176.0±11.9 nmol/10⁵ EC; SF: 115.0±12.5 nmol/10⁵ EC, p = 0.002) and lower TNF-a-induced HL-60 leukocyte adhesion (PF: 37±6 HL-60 cells/mm²; SF: 111±18 HL-60/mm², p = 0.003) than cells cultured under SF which is consistent with a more quiescent anti-inflammatory and anti-thrombotic phenotype. <em>In vitro</em> models have become increasingly adept at mimicking natural physiology and in doing so have clarified the importance of both chemical and physical cues that drive cell function. These data illustrate that the variability in metabolic demand and subsequent changes in perfusion resulting in constantly variable shear stress plays a key role in EC function that has not previously been described.</p> </div

Parallel plate culture system.

<p>Endothelial cell monolayers were grown to confluence on glass coverslips, then affixed to parallel plate flow chambers (A) using a vacuum pump. Peristaltic pumps (B) are used to impose pulsatile flow of culture media (C) over the endothelial monolayers. The rotational speed of the pumps is controlled by an external computer (D) via RS-232 linkage.</p

Inflammation-associated gene expression in flow-conditioned endothelial cells.

<p>Shown is fold mRNA expression (with respect to static-cultured endothelial cells) of cell adhesion molecules (A) and genes with roles in recruitment of inflammatory cells (B). Results are presented as mean±SEM. An embedded asterisk indicates a significant difference with respect to static controls; an asterisk over a bracket indicates a significant difference between flow groups. Abbreviations: <i>PSGL-1</i>: P-selectin glycoprotein ligand-1; <i>ICAM-1</i>: intercellular adhesion molecule-1; <i>VCAM-1</i>: vascular cell adhesion molecule-1; <i>PECAM-1</i>: platelet-endothelial cell adhesion molecule-1; <i>ALOX5</i>: arachidonate 5-lipoxygenase; <i>PLA2G4C</i>: cytosolic phospholipase A2 gamma; <i>ADAM17</i>: ADAM metallopeptidase domain 17; <i>MCP-1</i>: monocyte chemoattractant protein-1; <i>CX3CL1</i>: fractalkine.</p

Cardio-protective gene expression in flow-conditioned endothelial cells.

<p>Shown is fold mRNA expression (with respect to static-cultured EC) of genes that promote or inhibit cardiovascular disease progression. Results are presented as mean±SEM. An embedded asterisk indicates a significant difference with respect to static controls; an asterisk over a bracket indicates a significant difference between flow groups. Abbreviations: <i>EDN1</i>: endothelin-1; <i>PTGIS</i>: prostacyclin synthase; <i>SOD1</i>: superoxide dismutase-1.</p

HL-60 cell adhesion to flow-conditioned endothelial cells.

<p>Endothelial cell monolayers were grown to confluence and cultured under steady flow (A,C,E,G) or physiological flow (B,D,F,H) for 24 hours. During the last four hours, endothelial cells were activated with 1 U TNF-a to stimulate adhesion molecule expression. At hour 24, monolayers were removed from flow chambers and incubated for 10 minutes with a bolus of GFP+ HL-60 cells (1000 cells/mm²) and stained as described (A,B: DAPI; C,D: F-actin; E,F: GFP+ HL-60 cells; G,H: overlay). Shown are representative images (40x) from each condition. Scale bar: 20 microns. (I): HL-60 cell adhesion in 15 predetermined locations per monolayer was quantified. Results are displayed as mean±SEM (n = 5–6). Asterisk denotes significant difference in means between groups as determined by Student’s t-test.</p