The mechanical microenvironment of high concentration agarose for applying deformation to primary chondrocytes

Abstract

Cartilage and chondrocytes experience loading that causes alterations in chondrocyte biological activity. In vivo chondrocytes are surrounded by a pericellular matrix with a stiffness of ∼25-200 kPa. Understanding the mechanical loading environment of the chondrocyte is of substantial interest for understanding chondrocyte mechanotransduction. The first objective of this study was to analyze the spatial variability of applied mechanical deformations in physiologically stiff agarose on cellular and sub-cellular length scales. Fluorescent microspheres were embedded in physiologically stiff agarose hydrogels. Microsphere positions were measured via confocal microscopy and used to calculate displacement and strain fields as a function of spatial position. The second objective was to assess the feasibility of encapsulating primary human chondrocytes in physiologically stiff agarose. The third objective was to determine if primary human chondrocytes could deform in high-stiffness agarose gels. Primary human chondrocyte viability was assessed using live-dead imaging following 24 and 72 hours in tissue culture. Chondrocyte shape was measured before and after application of 10% compression. These data indicate that (1) displacement and strain precision are ∼1% and 6.5% respectively, (2) high-stiffness agarose gels can maintain primary human chondrocyte viability of >95%, and (3) compression of chondrocytes in 4.5% agarose can induce shape changes indicative of cellular compression. Overall, these results demonstrate the feasibility of using high-concentration agarose for applying in vitro compression to chondrocytes as a model for understanding how chondrocytes respond to in vivo loading.

Mechanotransduction in primary human osteoarthritic chondrocytes is mediated by metabolism of energy, lipids, and amino acids

“…A short loading timescale was used because initial early-time responses can set the trajectory for longer-term behavior. The loading protocol followed previously optimized methods [22] in which homogeneous deformations [20] were applied to the cell-seeded gels using a custom built bioreactor emulating physiological loading conditions: frequency of 1.1 Hz and average sinusoidal compressive strains of 5% with an amplitude of 1.9% based on initial gel height (Supplementary Figure 1). The loading frequency was selected based on the preferred stride rate in humans [23], and the applied strain profile was selected to be in the range of deformations measured in human patients using MRI [24].…”

“…cellular metabolites smaller than ∼1000 Da). Building upon previous methodology to encapsulate chondrocytes in agarose similar in stiffness to the human PCM, we profiled the metabolomic responses of primary human OA chondrocytes in response to applied compression at physiological levels [20, 21]. …”

Metabolic responses induced by compression of chondrocytes in variable-stiffness microenvironments

“…24 hours after encapsulation, the gels were placed in fresh antibiotic-free Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and cyclically compressed using a custom built bioreactor (Jutila et al, 2014). This system provides relatively homogeneous deformations with displacement precision of 1% and strain precision of 6.5% (Zignego et al., 2014). Sinusoidal, compressive strains of 5±1.9% (based on initial 12.7mm ± 0.01mm gel height) were applied to the gels at a rate of 1.1 Hz to physiologically mimic the human gait cycle for 0, 15 and 30 minutes (Sutter et al, 2015; Umberger and Martin, 2007).…”

“…Therefore, in two adjacent materials, the one with the lower stiffness will deform more. This explains why cells encapsulated in higher stiffness hydrogels experience larger deformations (Zignego et al, 2014). Cellular force transmission induces conformational changes in the internal structure of the cell, leading to molecular changes that can protect the cells during the dynamic change in environmental conditions (Dowling et al, 2013).…”

Study of the Mechanical Environment of Chondrocytes in Articular Cartilage Defects Repaired Area under Cyclic Compressive Loading

“…The material parameters of cartilage anisotropy, Poisson's ratio ν and the elastic modulus E , are related in a depth-dependent manner as follows [21–23]: $\begin{array}{c}\nu =0.08+0.1\left(\frac{h-y}{h}\right),\end{array}$$\begin{array}{c}E\left(y\right)=\frac{3.66}{46.2e^{-6.53y}+2.84},\end{array}$where h and y are thickness and depth of cartilage, respectively.…”