Flexure-Based Device for Cyclic Strain-Mediated Osteogenic Differentiation

Abstract

Biophysical strain has been applied widely for bone regeneration. However, application of low-magnitude strains to cells on small-thickness scaffolds is problematic, especially in rodent calvarial defect models, because general translation systems have limitations in terms of generating low-magnitude smooth signals. To overcome these limitations, we developed an in vitro biophysical-stimulation platform for stimulation of cells on small-thickness scaffolds for rodent calvarial bone defects. The customized flexure-based translational nanoactuator enables generation of low-magnitude smooth signals at the subnano- to micrometer-scale. This nanoactuator, which is equipped with a piezoelectric actuator, is suitable for biological applications because it can generate friction-free motion with a high resolution. Moreover, its operation without wear or deterioration eliminates contamination factors in cell culture environments. The developed in vitro biophysical-stimulation platform using these nanoactuators showed predictable operational characteristics. Also, a few-micrometer sinusoidal signal was generated successfully without any distortion. Three-dimensional scaffolds fitting the critical-size rat calvarial defect model were fabricated using poly(caprolactone), poly(lactic-co-glycolic acid), and tricalcium phosphate. Runt-related transcription factor 2 expression was increased upon stimulation of human adipose-derived stem cells (ASCs) on these scaffolds were stimulated in the in vitro biophysical-stimulation platform. Additionally, the use of this platform resulted in up-regulation of alkaline phosphate, osteopontin, and osterix expression compared to the non-stimulated group. These preliminary in vitro results suggest that the biophysical environment provided by the in vitro biophysical-stimulation platform influences the osteogenic differentiation of ASCs.

Load‐induced osteogenic differentiation of mesenchymal stromal cells is caused by mechano‐regulated autocrine signaling

“…In our study, the expression of RUNX2 in hBMSCs was found to be downregulated upon cyclic compressive loading (Figure ). This observation stands in contrast to previous reports in which comparable experimental setups were used (Jagodzinski et al, ;Michalopoulos et al, ; Shin Kang et al, ; Sittichokechaiwut et al, ). It is known that RUNX2 regulates the expression of osteoblast‐specific genes such as collagen type 1, bone sialoprotein, osteocalcin, and RUNX2 itself (Franceschi & Xiao, ).…”

“…An accumulation of attracted bone marrow MSCs at locations of increased mechanical strain could represent a conceivable physiological mechanism in healing of bone fractures and bone defects. Previous in vivo and in vitro studies reported that mechanical loading induces the differentiation of osteoprogenitors into osteoblasts (Jagodzinski et al, ; Michalopoulos et al, ; Shin Kang et al, ; Sittichokechaiwut et al, ; Turner, Owan, Alvey, Hulman, & Hock, ). Concerning our finding that hBMSC migration is reduced in response to cyclic compression, this led to the assumption that a reduction in migration is caused or at least accompanied by an onset of osteogenic differentiation.…”

“…In comparison with others, we can exclude previously reported indirect effects of mechanical loading on oxygen concentration and nutrient supply inside the biomaterial due to the chosen macroporous architecture (Witt et al, ). A further major difference is the low total cell number in relation to the volume of cell culture medium in the bioreactor (1.5 × 10 5 cells/27 ml medium) compared with the cultivation of 3D cell seeded constructs in well plates with low medium volume (Michalopoulos et al, ; Shin Kang et al, ; Sittichokechaiwut et al, ). This, in combination with the small but decisive fluid flow in the bioreactor, leads to a strong dilution of signaling proteins secreted by the cells and hinder autocrine biochemical self‐stimulation.…”

“…Following this motivation, various test systems for the in vitro application of mechanical stimulation have been developed, including force application in cell culture well plates (Michalopoulos et al, ; Shin Kang et al, ; Sittichokechaiwut, Edwards, Scutt, & Reilly, 2010) or bioreactors (Jagodzinski et al, ; Kopf, Petersen, Duda, & Knaus, ; Ode et al, ; Petersen, Joly, Bergmann, Korus, & Duda, ). Diverse effects of mechanical stimulation on cell migration were reported.…”

“…However, studies that worked without additional osteogenic triggers reported on an induction of osteogenic differentiation by cyclic bulk material deformation ( f = 1 Hz), reflected by an increased expression of Runt‐related transcription factor 2 (RUNX2; Michalopoulos et al, ), Alkaline phosphatase (ALP), and osteopontin (Shin Kang et al, ) in human MSCs. Interestingly, mechanical loading was previously shown to induce the expression of bone morphogenetic protein type 2 (BMP2) in MSCs (Wang et al, ).…”