Effect of Pulse Frequency on the Osteogenic Differentiation of Mesenchymal Stem Cells in a Pulsatile Perfusion Bioreactor

Engineered bone tissues are promising materials for the repair of large tissue deficits, but to be clinically effective they must be biologically active and capable of stimulating the normal bone remodeling processes upon implantation (e.g., integration, vascular infiltration, and new bone formation). One strategy for creating such materials is to culture mesenchymal stem cells (MSCs) [1–3] within porous three-dimensional scaffolds, as these cells are capable of synthesizing a bone-like extracellular matrix (ECM) containing bioactive growth and differentiation factors (e.g., bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) [4]) that can enhance osteoblastic differentiation of MSCs in vitro [5]. However, a recent in vivo study showed that bone-like ECM alone was not able to demonstrate a significant osteogenic response [6], underscoring the need for developing new strategies to enhance formation of a bone-like ECM. Medium perfusion may be an important component for forming a bone-like ECM in vitro as it serves for two complementary purposes. First, perfusion delivers oxygen and nutrients to the cells deep within large (> 1 cm) three-dimensional scaffolds [7–9]. This overcomes diffusional mass transport limitations, which otherwise restricts cell viability and ECM deposition to the outer surfaces of biomaterial scaffolds [7]. Second, perfusion enhances several phenotypic markers of osteoblastic differentiation, including alkaline phosphatase (ALP) activity [7,10], synthesis of type I collagen [11], osteocalcin (OC) [11], and osteopontin (OPN) [11], and mineral deposition [10,12]). Further, this biological response is sensitive to the flow conditions. For example, increasing the perfusion rate [12] or the fluid viscosity [13] has been shown to increase mineral deposition. However, perfusion has been shown to decrease cell density [14], which suggests that higher flow rates or medium viscosities might reduce the quality of bone-like ECM formed. Alternatively, dynamic perfusion regimens (e.g., oscillatory and pulsatile flow) may improve engineered bone tissue quality. To date only a hand-full of studies have examined the effect dynamic perfusion in porous three-dimensional scaffolds. A few short term studies (≤ 49 h) involving MC3T3-E1 model osteoprogenitor cells in three-dimensional porous scaffolds have demonstrated an increase in synthesis of prostaglandin E2 (PGE2) [14,15] and cyclooxygenase-2 (COX-2) [16] relative to steady flow. In addition, one long term study (14 days) in perfused scaffolds showed an increase in OPN mRNA expression with dynamic flow for MC3T3-E1 cells [17]. In contrast, dynamic flow regimens have been studied extensively in two-dimensional culture, and have demonstrated that osteoblastic cells are more responsive to dynamic flow conditions. In particular, dynamic flow has been shown to enhance mRNA expression of osteopontin, BMP-2, BMP-7 [18], and VEGF-A [18,19], synthesis of PGE2, and activation of the mitogen-activated protein kinases ERK and p38 [15,19–21]. In addition, evidence suggests that cell response is sensitive to the frequency of pulsatile flow [20,22,23], although varying trends have been reported. Jacobs et al. [20]—who measured intracellular calcium release in response to both oscillatory and pulsatile flow at 0.5 to 2 Hz—reported a decrease in cell response with increasing frequency. In contrast, Nauman et al. [23] reported that PGE2 production increased with increasing frequency, while Mullander et al. [22] reported no change in PGE2 production but an increase in nitric oxide release with increasing frequency (over the range from 1 to 9 Hz). To date the effect of dynamic perfusion on osteoblastic differentiation of MSC in three-dimensional porous scaffolds has not been described. Therefore, the goal of this study was to determine the effect of pulsatile perfusion on osteogenic differentiation of MSCs in three-dimensional culture. To accomplish this MSCs—expanded from bone marrow explants—were seeded into poly(D,L-lactic-co-glycolic acid) (PLGA) foam scaffolds and subjected to pulsatile flow at frequencies of 0.083, 0.050, and 0.018 Hz for 15 days. These very low frequencies were chosen to ensure that the shape of the pulsatile waveforms would not depend on frequency, and previous evidence indicates that these low frequencies stimulate late markers of osteoblastic differentiation in two-dimensional cultures [18]. Cell density, ALP activity, mRNA expression of OPN and OC mRNA expression, and accumulation of PGE2 and OPN in conditioned medium were measured, and compared with cells cultured statically or under steady perfusion.