Preclinical models for in vitro mechanical loading of bone-derived cells

Abstract

It is well established that bone responds to mechanical stimuli whereby physical forces are translated into chemical signals between cells, via mechanotransduction. It is difficult however to study the precise cellular and molecular responses using in vivo systems. In vitro loading models, which aim to replicate forces found within the bone microenvironment, make the underlying processes of mechanotransduction accessible to the researcher. Direct measurements in vivo and predictive modeling have been used to define these forces in normal physiological and pathological states. The types of mechanical stimuli present in the bone include vibration, fluid shear, substrate deformation and compressive loading, which can all be applied in vitro to monolayer and three-dimensional (3D) cultures. In monolayer, vibration can be readily applied to cultures via a low-magnitude, high-frequency loading rig. Fluid shear can be applied to cultures in multiwell plates via a simple rocking platform to engender gravitational fluid movement or via a pump to cells attached to a slide within a parallel-plate flow chamber, which may be micropatterned for use with osteocytes. Substrate strain can be applied via the vacuum-driven FlexCell system or via a four-point loading jig. 3D cultures better replicate the bone microenvironment and can also be subjected to the same forms of mechanical stimuli as monolayer, including vibration, fluid shear via perfusion flow, strain or compression. 3D cocultures that more closely replicate the bone microenvironment can be used to study the collective response of several cell types to loading. This technical review summarizes the methods for applying mechanical stimuli to bone cells in vitro.

Figure 1

Vibration in monolayer. (a) Cells are seeded into a multiwell plate and cultured to 80% confluence upon which the plate is clamped into a rig (b) for vibration at 0.3 g and up to 100 Hz, which is sufficient to engender shearing forces without creating fluid shear.

Figure 2

Rocking ‘see-saw' platform with media-filled six-well culture plates and shear stress profile. (a) Cells are seeded into multiwell plates and cultured to 80% confluence. (b) Platform tilts at a predetermined angle (indicated by double-headed arrow) and fluid flow-induced shear stress (calculated from Equation (2)) that is oscillatory in nature (c) is produced across the well bottom (x/L is a point in space along the well bottom, where x/L=0.5 is the center) at different points in the cycle. Matrix production is enhanced by ‘see-saw' rocking in osteogenic progenitor cells. (d) Second harmonic generation imaging shows increased collagen deposition/organization by human dermal fibroblasts (hDFs) and human embryonic stem mesenchymal progenitors (hES-MPs) subjected to OFSS versus static (no-flow) conditions. (e) Mineral deposition is increased by OFSS stimulation compared with static culture, but only in osteoinduction groups. BM, basal media; OIM, osteoinduction media; OM, osteogenic media.

Figure 3

Schematic of parallel-plate fluid flow system and chamber setup. (a) Cells are seeded onto a slide and allowed to attach. Typically, a silicon gasket is sandwiched between a quartz glass slide (with cells attached) and a polycarbonate distributor held together by vacuum or stainless-steel and Teflon plates screwed together. Inlet and outlet slots on the distributor or plates allow fluid flow from a media reservoir into the chamber driven by a pulsatile pump. (b) Oscillatory fluid flow is provided by attaching an oscillatory pump system. (c) Commercially available Ibidi pump system with parallel-plate chamber, the pump can produce static, pulsatile and oscillatory flow profiles. Inset shows disposable single and multichannel chambers.

Figure 4

Schematic of the FlexCell system and an in vitro four-point bending model. (a) For the FlexCell system, osteoblasts are seeded as a monolayer onto FlexCell compatible bases and plates. A vacuum underneath the well draws down the rubber seal beneath the FlexCell and in doing so causes stretch to occur beneath the cells. For the in vitro four-point bending jig, (b) osteoblasts were seeded as a monolayer onto the custom-made plastic slides and bathed in media in the jig apparatus housed in a 37 °C incubator with 5% CO₂. (c) The cells experience a strain of 3400 μɛ over a period of 10 min during which the slide is deformed 600 times.

Figure 5

Setting up and loading 3D cell cultures. (a) Cell suspension is added dropwise on to scaffolds and incubated for 1–3 h for cell attachment, medium topped up and scaffolds incubated for cell growth. Scaffolds can be loaded from as early as 24 h after seeding. Loading may be applied by (b) vibration, (c) fluid flow, (d) compression or (e) strain.

Figure 6

Coculture preparation and compressive loading. (a) Collagen mix is prepared and added into wells. MLO-Y4 cell suspension is mixed with collagen that is allowed to polymerize with incubation. MC3T3-E1 cell suspension is added to the surface of the gel followed by incubation for up to 7 days. (b) Cocultures are then loaded via a 16-well silicone loading plate of the same dimensions (10 mm diameter) as a standard 48-well tissue culture plate but with a 150-μm-thick base and with holes for its attachment to a BOSE loading instrument. The collagen gels are contained in the wells of the silicone plate, and the entire plate is stretched to apply a strain to the gels.