Design and validation of a novel bioreactor principle to combine online micro-computed tomography monitoring and mechanical loading in bone tissue engineering

Abstract

Mechanical loading plays an important role in bone remodeling in vivo and, therefore, has been suggested as a key parameter in stem cell-based engineering of bone-like tissue in vitro. However, the optimization of loading protocols during stem cell differentiation and subsequent bone-like tissue formation is challenged by multiple input factors, which are difficult to control and validate. These include the variable cellular performance of cells harvested from different patients, nonstandardized culture media components, the choice of the biomaterial forming the scaffold, and its morphology, impacting a broader validity of mechanical stimulation regimens. To standardize the cell culture of bone-like tissue constructs, we suggest the involvement of time-lapsed feedback loops. For this purpose we present a prototype bioreactor that combines online, nondestructive monitoring using micro-computed tomography and direct mechanical loading of three-dimensional tissue engineering constructs. Validation of this system showed displacement steps down to 1 microm and cyclic sinusoidal loadings of up to 10 Hz. Load detection resolution was 0.01 N, and micro-computed tomography data were of high quality. For the first time, the developed bioreactor links time-lapsed, nondestructive, and dynamic imaging with mechanical stimulation, designed for cell culture under sterile conditions. This system is believed to substantially improve today's experimental options to study and optimize osteogenic stem cell culture and differentiation at the interface with mechanical stimulation.

Three-Dimensional Characterization of Tissue-Engineered Constructs by Contrast-Enhanced Nanofocus Computed Tomography

“…According to specific study objectives, a customized strategy will be required to extract the necessary information in a robust way, ranging from (1) accurate and detailed (100s of mm scale) identification and quantification of different TE construct components limited to small TE construct samples and using phase-contrast imaging, 25 to (2) using standard desktop micro-or nano-CT as a routine 3D imaging technique for whole TE construct analysis (mm to cm scale) and quantification of mineralization after in vivo implantation. 10,13 The latter approach, without the use of a contrast agent, has also been used for static or bioreactor in vitro cultures, 16,18,19 showing distinct contrast differences between the mineralized matrix and scaffold. Phase-contrast imaging [24][25][26] or micro-CT combined with osmium tetroxide as a contrast agent 28 has shown its potential to assess nonmineralized ECM in an in vitro engineered TE construct.…”

“…Fluid flow is known to enhance matrix synthesis within TE constructs during perfusion culture. 19,40,50 Furthermore, differences in fibrillar collagen organization of the ECM, which are flow dependent, 51 could also explain the ECM differences that were observed by PTA staining as the ECM morphology could influence the binding mechanism of PTA to the ECM proteins. In Figure 5, it may also be observed that the difference in collagen content, as determined by Picrosirius Red staining, for the different flow rates is less pronounced than the one observed with the PTA staining.…”

“…6,[9][10][11][12][13][14] Furthermore, it has been employed for time-lapsed follow-up of mineralization inside scaffolds during in vitro static [15][16][17] or bioreactor cultures. 13,18,19 In most of these studies, polymeric, ceramic, collagen scaffolds, or composites were used, in which, the mineralized extracellular matrix (ECM) could be separated from the scaffold for the purpose of volume calculations and no significant material-dependent artifacts were present. However, when imaging the ECM or tissue growth in metallic scaffolds or around implants, additional caution has to be taken during image analysis as metal artifacts 20,21 can significantly influence the accuracy of the quantification of the newly formed ECM or tissue volume.…”

Development and evaluation of a device for simultaneous uniaxial compression and optical imaging of cartilage samples in vitro

“…To examine the accuracy of the entire device when exerting loads, the evaluation procedure employed by Hagenmüller et al 42 was adopted. Accordingly, displacement and force were recorded during the compression of commercially available springs (Gutekunst + Co. KG, Metzingen, Germany).…”

Effect of fetal bovine serum on mineralization in silk fibroin scaffolds

“…Highly and less ordered b-sheets are connected by amorphous network chains [11]. SF is a widely used scaffold material for bone tissue engineering (TE) applications [12][13][14][15][16], due to its excellent biocompatibility [17], controllable degradation [18] and favorable mechanical properties [19]. SF is an interesting scaffold material for bone TE consid- ering its ability to regulate the formation of HA nanocrystals when exposed to simulated body fluid (SBF).…”