Bioreactor Strategy in Bone Tissue Engineering: Pre-Culture and Osteogenic Differentiation Under Two Flow Configurations

Abstract

Since robust osteogenic differentiation and mineralization are integral to the engineering of bone constructs, understanding the impact of the cellular microenvironments on human mesenchymal stem cell (hMSCs) osteogenic differentiation is crucial to optimize bioreactor strategy. Two perfusion flow conditions were utilized in order to understand the impact of the flow configuration on hMSC construct development during both preculture (PC) in growth media and its subsequent osteogenic induction (OI). The media in the in-house perfusion bioreactor was controlled to perfuse either around (termed parallel flow [PF]) the construct surfaces or penetrate through the construct (termed transverse flow [TF]) for 7 days of the PC followed by 7 days of the OI. The flow configuration during the PC not only changed growth kinetics but also influenced cell distribution and potency of osteogenic differentiation and mineralization during the subsequent OI. While shear stress resulted from the TF stimulated cell proliferation during PC, the convective removal of de novo extracellular matrix (ECM) proteins and growth factors (GFs) reduced cell proliferation on OI. In contrast, the effective retention of de novo ECM proteins and GFs in the PC constructs under the PF maintained cell proliferation under the OI but resulted in localized cell aggregations, which influenced their osteogenic differentiation. The results revealed the contrasting roles of the convective flow as a mechanical stimulus, the redistribution of the cells and macromolecules in 3D constructs, and their divergent impacts on cellular events, leading to bone construct formation. The results suggest that the modulation of the flow configuration in the perfusion bioreactor is an effective strategy that regulates the construct properties and maximizes the functional outcome.

Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells

“…While some groups consider short bioreactor culture durations (5 days) sufficient prior to implantation [21], others use moderate durations (10–21 days) [5, 26, 45, 54–57] that are pulsatile or intermittent [58, 59], or up to 5 weeks to generate more mature constructs [22–24]. To minimize delays between harvesting autologous cells and generating an osteogenic graft, we characterized MSC response under perfusion in vitro to select the shortest culture duration necessary for construct maturity.…”

The e-Incubator: A Magnetic Resonance Imaging-Compatible Mini Incubator

“…Perfusion flow and micromechanical ultrasound stress can both be used to transform the e-incubator to an MR compatible bioreactor. 26,27 The e-incubator can be integrated with multiple imaging modalities, thus providing different contrast mechanisms and spatial resolution. For example, the e-incubator has the potential to improve our understanding of tumorigenesis by providing continuous assessment of cellular to organ levels during this process.…”

The Potential Impact of Bone Tissue Engineering in the Clinic

“…However, these elements, in conjunction with bioreactors, may reveal a comprehensive restoration strategy. Recent research into bioreactor preculturing of bone implants has emphasized the study of co-culturing multiple cell types, staged growth factor dosing and the use of shear and compressive forces to drive fate [15]. However, as yet, there are no widespread bioreactor approaches used in the clinic to prepare bone repair implants (see section titled "Preculturing strategies: static versus perfusion bioreactor culture").…”

“…Static preculture involves the expansion of cells on a biomaterial matrix under static conditions of cell culture, whereas, dynamic preculturing occurs inside a bioreactor [98]. Dynamic preculture in bioreactors can mimic some aspects of an in vivo environment, including the incorporation of shear stress due to media circulation or direct mechanical stimulation [15,[99][100][101]. Unlike entirely in vivo therapies, the use of bioreactors makes it easy to prevent the risk of pleiotropic effects of high doses of bioactive molecules and to avoid attachment of competing, undesirable cell types (e.g., fibroblasts).…”

“…Three types of bioreactors may be used for BTE. They are: rotating wall, spinner flask and flow perfusion, among which, flow perfusion bioreactors have been found most suitable due to uniform cell seeding and the media flow coming in direct contact with the cell-seeded biomaterials causing predictable shear stress to the cells [15,[100][101]105].…”