A three-dimensional osteochondral composite scaffold for articular cartilage repair.

There is a recognized and urgent need for improved treatment of articular cartilage defects. Tissue engineering of cartilage using a cell-scaffold approach has demonstrated potential to offer an alternative and effective method for treating articular defects. We have developed a unique, heterogeneous, osteochondral scaffold using the TheriForm three-dimensional printing process. The material composition, porosity, macroarchitecture, and mechanical properties varied throughout the scaffold structure. The upper, cartilage region was 90% porous and composed of D,L-PLGA/L-PLA, with macroscopic staggered channels to facilitate homogenous cell seeding. The lower, cloverleaf-shaped bone portion was 55% porous and consisted of a L-PLGA/TCP composite, designed to maximize bone ingrowth while maintaining critical mechanical properties. The transition region between these two sections contained a gradient of materials and porosity to prevent delamination. Chondrocytes preferentially attached to the cartilage portion of the device, and biochemical and histological analyses showed that cartilage formed during a 6-week in vitro culture period. The tensile strength of the bone region was similar in magnitude to fresh cancellous human bone, suggesting that these scaffolds have desirable mechanical properties for in vivo applications, including full joint replacement.

Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes.

Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K(+) current (I(K(ACh))) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by approximately 25% (vs. untreated controls) in field-stimulated myocytes. S1P(1) was shown to be involved by using the S1P(1)-selective agonist SEW2871 on myocytes isolated from S1P(3)-null mice. However, in these myocytes, S1P(3) can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P(1) antagonist VPC23019. Since S1P(1) activates G(i) exclusively, whereas S1P(3) activates both G(i) and G(q), these results strongly implicate the involvement of mainly G(i). Additional experiments using the I(K(ACh)) blocker tertiapin demonstrated that I(K(ACh)) can contribute to the negative inotropy following S1P activation of S1P(1) (perhaps through G(ibetagamma) subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I(K(ACh))) can reduce L-type calcium current and thus can decrease the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G(i) to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca(2+)](i); and 2) a second pathway that acts via G(i) to activate I(K(ACh)) and reduce APD. This decrease in APD is expected to decrease Ca(2+) influx and reduce [Ca(2+)](i) and myocyte contractility.

Sphingosine-1-phosphate receptor expression in cardiac fibroblasts is modulated by in vitro culture conditions.

The bioactive molecule sphingosine-1-phosphate (S1P) binds with high affinity to five recognized receptors (S1P(1-5)) to affect various tissues, including cellular responses of cardiac fibroblasts (CFbs) and myocytes. CFbs are essential components of myocardium, and detailed study of their cell signaling and physiology is required for a number of emerging disciplines. Meaningful studies on CFbs, however, necessitate methods for selective, reproducible cell isolations. Macrophages reside within normal cardiac tissues and often are isolated with CFbs. A protocol was therefore developed that significantly reduces macrophage levels and utilizes more CFb-specific markers (discoidin domain receptor-2) instead of, or in addition to, more commonly used cytoskeletal markers. Our results demonstrate that primary isolated, purified CFbs express predominantly S1P(1-3); however, the relative levels of these receptor subtypes are modulated with time and by culture conditions. In coculture experiments, macrophages altered CFb S1P receptor levels relative to controls. Further investigations using known macrophage-secreted factors showed that S1P and H(2)O(2) had minimal effects on CFb S1P(1-3) expression, whereas transforming growth factor-beta1, TNF-alpha, and PDGF-BB significantly altered all S1P receptor subtypes. Lowering FBS concentrations from 10% to 0.1% increased S1P(2), whereas supplementation with either PDGF-BB or Rho-associated protein kinase inhibitor Y-27632 significantly elevated S1P(3) levels. S1P(2) and S1P(3) receptor levels are known to regulate cell migration. Using cells isolated from either normal or S1P(3)-null mice, we demonstrate that S1P(3) is important and necessary for CFb migration. These results highlight the importance of demonstrating CFb culture purity in functional studies of S1P and also identify conditions that modulate S1P receptor expression in CFbs.

An analysis of the effects of stretch on IGF-I secretion from rat ventricular fibroblasts.

Mechanical force can induce a number of fundamental short- and long-term responses in myocardium. These include alterations in ECM, activation of cell-signaling pathways, altered gene regulation, changes in cell proliferation and growth, and secretion of a number of peptides and growth factors. It is now known that a number of these autocrine/paracrine factors are secreted from both cardiomyocytes and ventricular cardiac fibroblasts (CFb) in response to stretch. One such substance is IGF-I. IGF-I is an important autocrine/paracrine factor that can regulate physiological or pathophysiological responses, such as hypertrophy. In this study, we addressed the possible effects of mechanical perturbation, biaxial strain, on IGF-I secretion from adult rat CFb. CFb were subjected to either static stretch (3-10%) or cyclic stretch (10%; 0.1-1 Hz) over a 24-h period. IGF-1 secretion from CFb in response to selected stretch paradigms was examined using ELISA to measure IGF-I concentrations in conditioned media. Static stretch did not result in any measurable modulation of IGF-I secretion from CFb. However, cyclic stretch significantly increased IGF-I secretion from CFb in a frequency- and time-dependent manner compared with nonstretched controls. This stretch-induced increase in secretion was relatively insensitive to changes in extracellular [Ca(2+)] or to block of L-type Ca(2+) channels. In contrast, thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, remarkably decreased stretch-induced IGF-I secretion from CFb. We further show that IGF-I can upregulate mRNA expression of atrial natriuretic peptide in myocytes. In summary, cyclic stretch can significantly increase IGF-I secretion from CFb, and this effect is dependent on a thapsigargin-sensitive pool of intracellular [Ca(2+)].