Modeling nucleus pulposus regeneration in vitro: mesenchymal stem cells, alginate beads, hypoxia, bone morphogenetic protein-2, and synthetic peptide B2A.

STUDY DESIGN: Using a low cell density, hypoxic, alginate-bead culture system, the effects of bone morphogenetic protein-2 (BMP-2) and synthetic peptide B2A on cell proliferation and extracellular matrix (ECM) synthesis were assessed at days 0, 3, 5, and 7, using nucleus pulposus (NP)-like differentiated mesenchymal stem cells (MSCs). OBJECTIVE: This is a preliminary investigation into B2A's potential adjunctive role with MSCs and BMP-2, in NP regeneration. SUMMARY OF BACKGROUND DATA: B2A analogs, alone and in combination with BMP-2, have been shown to promote proliferation and ECM production in chondrocytes and MSCs. Articular chondrocytes and NP cells often respond in a similar manner to growth factor treatments, thus suggesting a potential role for B2A in treating disc degeneration by NP regeneration. METHODS: Using the NP regeneration in vitro model (low cell density, hypoxic, alginate bead culture), B2A and BMP-2 were evaluated alone and in combination, to determine effects on proliferation and ECM synthesis in the presence of transforming growth factor-beta 3 on NP-like differentiated MSCs. RESULTS: B2A administration induced mild proliferation of NP-like differentiated MSCs and diminished an initial wave of low-dose BMP-2-prompted apoptosis. Individually and in combination, B2A and BMP-2 were found to inhibit transforming growth factor-beta 3-permitted collagen accumulation; levels remained similar in their presence. Both collagen I (Col I) and collagen II (Col II) were found in almost all specimens, but increased B2A levels favored Col II unlike BMP-2, which favored Col I. BMP-2 resulted in a minor reduction in aggrecan synthesis, which was unchanged by B2A. CONCLUSION: Using this in vitro model, B2A induced proliferation, continuous aggrecan synthesis, and stabilized collagen accumulation favoring Col II. These characteristics are consistent with cells of the young, healthy NP, indicating potential use of the peptide early in an MSC-based NP-regeneration therapy; whereas, BMP-2 induced apoptosis, Col I accumulation, and aggrecan production hindrance, and was found untherapeutic.

Platelets undergo phosphorylation of Syk at Y525/526 and Y352 in response to pathophysiological shear stress.

Atherosclerotic plaques can lead to partial vascular occlusions that produce abnormally high levels of arterial wall shear stress. Such pathophysiological shear stress can promote shear-induced platelet aggregation (SIPA), which has been linked to acute myocardial infarction, unstable angina, and stroke. This study investigated the role of the tyrosine kinase Syk in shear-induced human platelet signaling. The extent of Syk tyrosine phosphorylation induced by pathophysiological levels of shear stress (100 dyn/cm(2)) was significantly greater than that resulting from physiological shear stress (10 dyn/cm(2)). With the use of phospho-Syk specific antibodies, these data are the first to show that key regulatory sites of Syk at tyrosines 525/526 (Y525/526) and tyrosine 352 (Y352) were phosphorylated in response to pathophysiological shear stress. Increased phosphorylation at both sites was attenuated by pharmacological inhibition of Syk using two different Syk inhibitors, piceatannol and 3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (OXSI-2), and by inhibition of upstream Src-family kinases (SFKs). Shear-induced response at the Syk 525/526 site was ADP dependent but not contingent on glycoprotein (GP) IIb-IIIa ligation or the generation of thromboxane (Tx) A(2). Pretreatment with Syk inhibitors not only reduced SIPA and Syk phosphorylation in isolated platelets, but also diminished, up to 50%, the platelet-mediated thrombus formation when whole blood was perfused over type-III collagen. In summary, this study demonstrated that Syk is a key molecule in both SIPA and thrombus formation under flow. Pharmacological regulation of Syk may prove efficacious in treating occlusive vascular disease.

Cyclic mechanical strain alters tissue-factor activity in rat osteosarcoma cells cultured on a titanium substrate.

Tissue factor (TF), a transmembrane glycoprotein, plays a role in the initiation of blood coagulation at sites of vascular injury. Activated products of coagulation may then enhance inflammatory responses. The present investigation assesses the ability of rat osteosarcoma (UMR-106) cells cultured on titanium alloy (Ti6Al4V) to express differential surface TF activity in response to cyclic mechanical strain. Strains ranged from -2000 micro-strain to +2000 micro-strain, and durations from 5, 10, and 20 min per day over 5 days to 24 h continuous stimulation. ROS cells exhibited significant TF activity as demonstrated by the conversion of Factor X to Factor Xa. Strains of +2000 micro-strain with 5-20-min duration exhibited decreased TF activity with duration from 1.4E-04 nM/cell to 8.7E-05 nM/cell. Additionally, ROS cells stimulated with calcium ionophore (A23187) exhibited at least twice the activity of nonstimulated cells. Strains of +1340 micro-strain with 5-20-min duration exhibited an increasing trend with 4.15E-05 nM/cell to 7.38E-05 nM/cell. Strain direction had no significant effect on TF activity. Thus, both mechanical and chemical stimuli induce differential expression of TF activity by ROS cells cultured on Ti6Al4V, a phenomenon that may potentiate or regulate the inflammatory responses associated with the implantation of orthopedic biomaterials.

Comparison of shear stress-induced platelet microparticle formation and phosphatidylserine expression in presence of alphaIIbbeta3 antagonists.

The use of platelet glycoprotein IIb-IIIa (alphaIIbbeta3) antagonists is an accepted practice in the treatment of acute coronary syndromes. Recent studies have demonstrated that alpha beta receptor antagonists are effective in inhibiting the procoagulant activity of platelets under static conditions. No investigation, however, has compared the ability of these platelet antagonists to inhibit platelet procoagulant activity, defined as an increase in phosphatidylserine (PS) expression, under conditions of shear stress. Thus, the goal of this study was to quantify the amount of microparticle formation and PS expression of platelets exposed to physiologic and pathophysiologic levels of shear stress in the absence and presence of three clinically approved parenteral alpha beta antagonists (abciximab, eptifibatide, and tirofiban). Flow cytometric results demonstrated that although microparticle formation was significantly inhibited by all three antagonists, PS expression by sheared platelets was affected differently depending on the antagonist present. Specifically, abciximab suppressed PS expression compared with the saline control; both abciximab and eptifibatide significantly reduced PS expression compared with tirofiban; and tirofiban potentiated PS expression relative to the saline control at the highest shear stress. This is the first demonstration of differential regulation of platelet PS expression and, by inference, procoagulant activity in the presence of alpha receptor antagonists under shear stress. The current results may have future importance in improving the design of platelet antagonists as well as defining the general role of fluid shear stress in platelet thrombus formation.

Inhibition of shear-stress-induced platelet aggregation and phosphotyrosine signaling by GPIIb-IIIa antagonists.

Shear-stress-mediated platelet thrombus formation has been implicated in the pathophysiology of cardiovascular diseases such as acute myocardial infarction and unstable angina. Although previous studies have established that fluid shear forces cause platelet aggregation, a direct comparison of GPIIb-IIIa antagonists used in the treatment of acute coronary syndromes on shear-induced platelet activation has not been reported. Therefore, the objective of the present study was to characterize the effects of the platelet antagonists abciximab, eptifibatide, and tirofiban on shear-mediated platelet activation and aggregation using flow cytometric and Western blotting techniques. Flow cytometric analyses indicated that all three platelet antagonists, when used at concentrations that saturated all GPIIb-IIIa receptors, significantly inhibited platelet aggregate formation and expression of the platelet activation marker p-selectin. None of the antagonists caused increased expression of GPIbalpha or GPIIb-IIIa on the platelet surface compared to untreated controls. Additionally, Western blotting demonstrated that a 72 kDa protein tentatively identified as Syk became phosphorylated in response to shear stress and that its phosphorylation was inhibited by each antagonist. The findings of this study indicate that abciximab, eptifibatide, and tirofiban, though possessing distinct biochemical and pharmacological properties, effectively and equivalently inhibit platelet aggregation, p-selectin expression, and intracellular tyrosine phosphorylation events induced by fluid shear stress.