The Effect of Mechanical Stimulation on Mineralization in Differentiating Osteoblasts in Collagen-I Scaffolds

Abstract

Developing a viable and functional bone scaffold in vitro that is capable of surviving and bearing mechanical load in vivo requires an understanding of the cell biology of osteoprogenitor cells, particularly how they are influenced by mechanical stimulation during cell differentiation and maturation. In this study, mechanical load was applied using a modified FlexCell plate to impart confined compression to collagen-I scaffolds seeded with undifferentiated murine embryonic stem cells. The activity, presence, and expression of osteoblast-cadherin (OB-Cad) and connexin-43, as well as various pluripotent and osteogenic markers were examined at 5-30 days of differentiation as cells were stimulated to differentiate to osteoblasts with and without applied mechanical load. Fluorescence recovery after photobleaching, immunofluorescence, viability, von Kossa, and real-time polymerase chain reaction assessments revealed that mechanical prestimulation of this cell-seeded scaffold altered the expression of OB-Cad and connexin-43 and resulted in significant differences in the structure and organization of mineralization present in the collagen matrix. Specifically, cells in gels that were loaded for 40 h after 5 days of differentiation and then left to fully differentiate for 30 days produced a highly structured honeycomb-shaped mineralization in the matrix; an outcome that was previously shown to be indicative of late osteoblast/early osteocyte activity. This study highlights the potential of mechanical load to accelerate differentiation and enhance osteoblast communication and function during the differentiation process, and highlights a time point of cell differentiation within this scaffold to apply load in order to most effectively transduce a mechanical signal.

The Role of Gap Junctions and Mechanical Loading on Mineral Formation in a Collagen-I Scaffold Seeded with Osteoprogenitor Cells

“…In a previous study performed by our group, FRAP showed that there was a significant difference between average maximum percent recovery in early (Day 5) and late-differentiated (Days 15-30) gels in the presence and absence of loading (Figure 2A, a previously published result) (16). In this study, gels treated with octanol and AGA showed a significant reduction in fluorescence recovery values ( Figure 2B).…”

“…Specifically, confined compression of early-differentiated cells produced a honeycomb structure to the mineralization present, a quality associated with late osteoblast/early osteocyte activity (16). Prestimulated cells also upregulated early and late osteoblast markers (16). In this current study, gap This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.…”

“…Our group has previously shown that mechanical pre-stimulation of day 5 cells within this scaffold produced a drastic increase in the amount and organization of mineralization present (16). Specifically, confined compression of early-differentiated cells produced a honeycomb structure to the mineralization present, a quality associated with late osteoblast/early osteocyte activity (16). Prestimulated cells also upregulated early and late osteoblast markers (16).…”

“…When mechanical pre-stimulation in the form of confined compression is applied to these cells at a Day 5 differentiated state, we have found this increases the amount and organization of mineralization present in the matrix, and significantly increases connexin-43 (Cx-43) expression (16).…”

ClC‐3 Promotes Osteogenic Differentiation in MC3T3‐E1 Cell After Dynamic Compression

“…The effects of mechanical stimuli on osteoblastic cells have been studied in 2-D culture by applying cyclic tensile strain or compression [Ziros et al, 2002;Visconti et al, 2004;Koike et al, 2005]. Furthermore, some studies observed the effects of biomechanical forces on osteoblastic cells in 3-D culture systems [Duty et al, 2007;Sanuki et al, 2007;Rath et al, 2008;Dumas et al, 2009;Chatterjee et al, 2010;Damaraju et al, 2014Damaraju et al, , 2015Tanaka and Tachibana, 2015]. Other studies observed cell growth in 3-D cultures in the absence of any mechanical stimuli [Cao et al, 2003].…”