Histological Method to Study the Effect of Shear Stress on Cell Proliferation and Tissue Morphology in a Bioreactor

Abstract

BACKGROUND: Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. METHODS: In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental setup using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. RESULTS: Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. CONCLUSION: We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.

Channeling Effect and Tissue Morphology in a Perfusion Bioreactor Imaged by X-Ray Microtomography

“…The samples were scanned by X-ray micro-tomography and the tridimensional arrangements of the beads and the tissue were obtained after image reconstruction. Preliminary experiments showed that tissue proliferation on glass and polyacetal bead stacks in static conditions (without flow) was negligible compared to dynamic conditions [39,40], producing too little tissue volume to be resolved by X-ray microtomography. Additionally, mass transport estimates in similar culture conditions and close flow rate indicate that oxygen and nutrient supply is in excess during most of the three weeks of culture [39].…”

“…The cylindrical growth chamber (13.8 mm diameter, 5 cm high, made of polycarbonate) is randomly filled by mono disperse polyacetal beads (2 mm diameter). Let us note that bead-based scaffolds have been identified as good candidates for tissue engineering and therefore have been investigated multiple times [4,32,39,40,[44][45][46][47]. The beads are immobilized by two polycarbonate grids (0.5 cm high), inserted in the growth chamber at the bioreactor inlet and outlet.…”

“…2 Materials and Methods Following previous tissue engineering studies using murine fibroblasts [38][39][40], these cells have been chosen for our model system because they belong to the family of connective tissue cells (like chondrocytes, osteoblasts, ...) [41]. Additionally, they respond to mechanical stimuli [42], which is relevant to our perfusion bioreactor model and have a rapid proliferation rate.…”

Assessment of the interplay between scaffold geometry, induced shear stresses, and cell proliferation within a packed bed perfusion bioreactor

“…The formation of multi‐layered structure require 3‐D environments (at the cell scale). Such sites actually exist in stackings of beads: they are typically located at the contact point between beads . On the contrary, stackings of cubes seem to have fewer sites able to induce 3‐D (multi‐layered) proliferation.…”

“…Methodology combining histological procedure and image analysis allows the description and quantification of cell proliferation within bioreactor. 53 However, this method can only generate a crude rather than comprehensive depiction of the 3D scaffold, primarily because its destructive nature leads to an incomplete representation of the 3D tissue. Over the last 10 years, nondestructive three-dimensional image-based technology has made steady progress (i.e., the ability to visualize micrometric objects).…”