Dynamic nanomechanics of individual bone marrow stromal cells and cell-matrix composites during chondrogenic differentiation

Abstract

Dynamic nanomechanical properties of bovine bone marrow stromal cells (BMSCs) and their newly synthesized cartilage-like matrices were studied at nanometer scale deformation amplitudes. The increase in their dynamic modulus, |E^⁎| (e.g., 2.4±0.4 kPa at 1 Hz to 9.7±0.2 kPa at 316 Hz at day 21, mean±SEM), and phase angle, δ, (e.g., 15±2° at 1 Hz to 74±1° at 316 Hz at day 21) with increasing frequency were attributed to the fluid flow induced poroelasticity, governed by both the newly synthesized matrix and the intracellular structures. The absence of culture duration dependence suggested that chondrogenesis of BMSCs had not yet resulted in the formation of a well-organized matrix with a hierarchical structure similar to cartilage. BMSC-matrix composites demonstrated different poro-viscoelastic frequency-dependent mechanical behavior and energy dissipation compared to chondrocyte-matrix composites due to differences in matrix molecular constituents, structure and cell properties. This study provides important insights into the design of optimal protocols for tissue-engineered cartilage products using chondrocytes and BMSCs.

Introduction

During the past decade, various nanomechanical approaches have been used to understand the mechanical integrity of individual primary chondrocytes and chondrocytes associated with pericellular matrices, including compression (Koay et al., 2008), indentation (Darling et al., 2010, Darling et al., 2006, Sanchez-Adams et al., 2013, Wilusz et al., 2013), shear (Ofek et al., 2010) and tension (Trickey et al., 2004). These studies laid the background on how biomechanical signals play a major role in chondrocyte gene expression and biosynthesis. Besides chondrocytes, bone marrow stromal cells (BMSCs) were recently shown to be a promising alternative cell source for cartilage tissue repair (Mauck et al., 2006). When seeded within a variety of tissue engineering scaffolds and subjected to chondrogenic factors, BMSCs can undergo chondrogenesis within a few days, and produce PCMs mainly composed of types II and VI collagen, aggrecan and other macromolecules found in articular cartilage (Kopesky et al., 2010a). Aggrecan molecules synthesized in vitro by BMSCs harvested from immature and adult equines have significantly longer glycosaminoglycan (GAG) chains and higher nano-compressive stiffness than aggrecan synthesized by chondrocytes (Kopesky et al., 2010a, Lee et al., 2010b).

As previous studies have focused on the biochemical composition and elastic-like tissue-level mechanics of the BMSC-synthesized neo-cartilage matrices (Connelly et al., 2007, Kopesky et al., 2010b), little is known about the dynamic nanomechanical properties of individual cells. Similar to chondrocyte-associated matrix, the BMSC-associated matrix provides important functions in cell signaling and mechanotransduction (Millward-Sadler et al., 2000, Potier et al., 2010) in response to both static and dynamic loadings. Knowledge of the dynamic nanomechanical properties of the BMSC-matrix composites will provide a critical measure of the potential success of BMSC-based cartilage tissue engineering (Han et al., 2011b). Here, we adapted the atomic force microscopy (AFM)-based dynamic oscillatory nanoindentation (Han et al., 2011a) to assess the dynamic mechanical properties of individual BMSC-matrix composites. The dependence on dynamic frequency and culture duration was investigated to study the poro-viscoelastic properties of this composite, and its culture time-dependent evolution. The results were compared to our previous study of chondrocyte-matrix composites (Lee et al., 2010a) to highlight the differences in engineered products from these two cell sources.