Immunofluorescence-guided atomic force microscopy to measure the micromechanical properties of the pericellular matrix of porcine articular cartilage

Wilusz, Rebecca E.; DeFrate, Louis E.; Guilak, Farshid

doi:10.1098/rsif.2012.0314

Cited by 58 publications

(63 citation statements)

References 69 publications

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“…The elastic moduli correlated to collagen alignment, with the stiffer outer region of the pellets showing a higher degree of collagen alignment compared with the central region. This observation is similar to zonal alterations seen in the microscale mechanical properties of native cartilage, with the superficial layer showing a high modulus due to an increased ratio of aligned collagen molecules to GAGs in this zone (42). These results indicate that the engineered cartilage has some of the features of native cartilage architecture, which may be critical to success in defect repair and developing in vitro models to study cartilage biology and mechanics.…”

Section: Discussionsupporting

confidence: 63%

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

Diekman

Christoforou

Willard

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP− cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilagespecific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twicepassaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.chondrocyte | bone morphogenetic proteins | transforming growth factor-beta | cartilage micromechanics | cartilage regeneration

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Section: Discussionsupporting

confidence: 63%

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

Diekman

Christoforou

Willard

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

232

250

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“…Direct quantification of PCM properties in situ with minimal disruption of native matrix integration between the PCM and ECM has recently been reported through applications of atomic force microscopy (AFM)-based microindentation (Figure 2b) (Allen and Mao, 2004; Darling et al, 2010; McLeod et al, 2013; Wilusz et al, 2012a, b; Wilusz and Guilak, 2014; Wilusz et al, 2013). Using a force spectroscopy technique known as stiffness or force-volume mapping (Radmacher et al, 1992), AFM can be used to collect arrays of indentation curves and map spatial variations in elastic modulus in the micromechanical environment of the chondrocyte.…”

Section: Introductionmentioning

confidence: 99%

The structure and function of the pericellular matrix of articular cartilage

2014

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Chondrocytes in articular cartilage are surrounded by a narrow pericellular matrix (PCM) that is both biochemically and biomechanically distinct from the extracellular matrix (ECM) of the tissue. While the PCM was first observed nearly a century ago, its role is still under investigation. In support of early hypotheses regarding its function, increasing evidence indicates that the PCM serves as a transducer of biochemical and biomechanical signals to the chondrocyte. Work over the past two decades has established that the PCM in adult tissue is defined biochemically by several molecular components, including type VI collagen and perlecan. On the other hand, the biomechanical properties of this structure have only recently been measured. Techniques such as micropipette aspiration, in situ imaging, computational modeling, and atomic force microscopy have determined that the PCM exhibits distinct mechanical properties as compared to the ECM, and that these properties are influenced by specific PCM components as well as disease state. Importantly, the unique relationships among the mechanical properties of the chondrocyte, PCM, and ECM in different zones of cartilage suggest that this region significantly influences the stress-strain environment of the chondrocyte. In this review, we discuss recent advances in the measurement of PCM mechanical properties and structure that further increase our understanding of PCM function. Taken together, these studies suggest that the PCM plays a critical role in controlling the mechanical environment and mechanobiology of cells in cartilage and other cartilaginous tissues, such as the meniscus or intervertebral disc.

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“…Experimental data suggests that the PCM stiffness is in the region of 15 -100 kPa [29,31,78,79]. Hence, the stiffness of the PCM is considerably lower than the ECM, but is significantly higher than the effective stiffness of the active cell.…”

Section: Discussionmentioning

confidence: 98%

Computational investigation of in situ chondrocyte deformation and actin cytoskeleton remodelling under physiological loading

2013

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Immunofluorescence-guided atomic force microscopy to measure the micromechanical properties of the pericellular matrix of porcine articular cartilage

Cited by 58 publications

References 69 publications

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

The structure and function of the pericellular matrix of articular cartilage

Computational investigation of in situ chondrocyte deformation and actin cytoskeleton remodelling under physiological loading

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