Short communicationDynamic nanomechanics of individual bone marrow stromal cells and cell-matrix composites during chondrogenic differentiation
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.
Section snippets
Cell culture and isolation
Bovine BMSCs were isolated and seeded into 2% alginate hydrogel beads at 20 × 106 cells/mL density, similar to the culture of chondrocytes (Ng et al., 2007). The beads were placed in chondrogenic culture medium containing high-glucose DMEM supplemented with 1% Insulin-Transferrin-Selenium (Sigma-Aldrich, St. Louis, MO), 100 nM dexamethasone (Sigma-Aldrich) and 10 ng/mL recombinant human transforming growth factor-β1 (R&D System, Minneapolis, MN) (Connelly et al., 2008). Culture medium was
Results
For all BMSC-matrix composites measured, both ||E⁎|| and δ increased significantly with increasing frequency from 1–316 Hz (one-way ANOVA, p<0.0001, Fig. 1b,c). In this experiment, the static indentation depth (≈1 μm) is≈10% of the total diameter of the cell-matrix composite (≈16.4 μm). In addition, linear dependences of |E⁎| versus f1/2 were observed for the composites at all culture days (R2>0.81 through LSLR, Fig. 1d). Over the 21 days of culture, we did not observe significant trends in |E⁎|
Poro-viscoelasticity of BMSC-matrix composites
The significant frequency dependence of |E⁎| and δ (Fig. 1) suggested the dominance of time-dependent poro-viscoelasticity of BMSC-matrix composites. Since the static indentation depth is ≈25% of the neo-matrix thickness, this frequency dependence likely involves contributions from both the matrix and intracellular structures. Poroelasticity is thus originated from fluid flow through both the cytoskeletal filaments and organelles within the cell (Charras et al., 2005), and the negatively
Conclusions
In this study, we quantified the dynamic nanomechanical properties of individual BMSCs and their associated tissue-engineered matrices. The non-linear frequency dependence was mainly attributed to fluid-flow induced poroelastic energy dissipation. In addition, despite increases in collagen and GAG contents in the PCM during culture, there was a lack of culture duration dependence for both |E⁎| and δ, suggesting a delay in the formation of hierarchical structure of the matrix similar to native
Conflict of interest statement
The authors affirm that they have no financial affiliation or involvement with any commercial organization that has direct financial interest in any matter included in this manuscript.
Acknowledgments
We thank the Institute for Soldier Nanotechnologies at MIT, funded through the U.S. Army Research Office, for use of instruments. This work was supported by National Science Foundation (grant CMMI-0758651), the National Institutes of Health (grant AR060331), the National Security Science and Engineering Faculty Fellowship (grant N00244-09-1–0064) and the Faculty Start-up Grant at Drexel University (LH).
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