Dynamic Elastic Modulus of Porcine Articular Cartilage Determined at Two Different Levels of Tissue Organization by Indentation-Type Atomic Force Microscopy

Abstract

Cartilage stiffness was measured ex vivo at the micrometer and nanometer scales to explore structure-mechanical property relationships at smaller scales than has been done previously. A method was developed to measure the dynamic elastic modulus, |E(*)|, in compression by indentation-type atomic force microscopy (IT AFM). Spherical indenter tips (radius = approximately 2.5 microm) and sharp pyramidal tips (radius = approximately 20 nm) were employed to probe micrometer-scale and nanometer-scale response, respectively. |E(*)| values were obtained at 3 Hz from 1024 unloading response curves recorded at a given location on subsurface cartilage from porcine femoral condyles. With the microsphere tips, the average modulus was approximately 2.6 MPa, in agreement with available millimeter-scale data, whereas with the sharp pyramidal tips, it was typically 100-fold lower. In contrast to cartilage, measurements made on agarose gels, a much more molecularly amorphous biomaterial, resulted in the same average modulus for both indentation tips. From results of AFM imaging of cartilage, the micrometer-scale spherical tips resolved no fine structure except some chondrocytes, whereas the nanometer-scale pyramidal tips resolved individual collagen fibers and their 67-nm axial repeat distance. These results suggest that the spherical AFM tip is large enough to measure the aggregate dynamic elastic modulus of cartilage, whereas the sharp AFM tip depicts the elastic properties of its fine structure. Additional measurements of cartilage stiffness following enzyme action revealed that elastase digestion of the collagen moiety lowered the modulus at the micrometer scale. In contrast, digestion of the proteoglycans moiety by cathepsin D had little effect on |E(*)| at the micrometer scale, but yielded a clear stiffening at the nanometer scale. Thus, cartilage compressive stiffness is different at the nanometer scale compared to the overall structural stiffness measured at the micrometer and larger scales because of the fine nanometer-scale structure, and enzyme-induced structural changes can affect this scale-dependent stiffness differently.

Keratin network modifications lead to the mechanical stiffening of the hair follicle fiber

“…The related elastic moduli are calculated using the Hertz model (SI Text) (23,24). Note that the determined absolute moduli values depend on the tip shape and the used fitting model; sharp tips are expected to rather probe the local mechanics of the underlying fibrous network (SI Text) (25)(26)(27). The deduced elastic moduli recorded along the follicle axis are shown as boxplots in Fig.…”

Articular cartilage repair by genetically modified bone marrow aspirate in sheep

“…However, this is insufficient to detect local mechanical property variations of the examined tissue that reflect differences in cartilage structural organization at the molecular level. 41,42 To overcome these limitations, Stolz et al 22 proposed a novel, AFM-based approach they termed IT AFM. Their protocol enabled absolute measurements of the dynamic elastic modulus |E*| at two different length scales of tissue organization-micrometer and nanometer.…”

“…A sharp AFM tip has nominal radius of 20 nm that is smaller than an individual collagen fibril, Gene therapy in ovine articular cartilage repair A Ivkovic et al which typically measures around 50 nm. 22 Although microspherical tip is too big to detect subtle differences in orientation and amount of collagen fibrils, sharp pyramidal tip can discriminate such differences, resulting in higher stiffness values.…”

“…Mechanical properties (that is, stiffness) of articular cartilage and repair tissue were determined by measurements of |E*|, the dynamic elastic modulus of articular cartilage, at two different length scales of tissue organization: micrometer (|E*| micro ) and nanometer (|E*| nano ). Preparation of the cartilage samples, data acquisition and processing was done as described by Stolz et al 22 Briefly, spherical tips with radius of 7.5 mm (SPI Supplies, West Chester, PA, USA) were mounted onto the end of rectangular tipples silicon nitride cantilevers having nominal spring constants of 0.35 N m À1 (MicroMasch, San Jose, CA, USA) and used for micrometer-scale experiments. For nanometer-scale experiments, square-based pyramidal silicon-nitride tips with a nominal tip radius of 20 nm were used on V-shaped 200-mm-long silicon nitride cantilevers with a nominal spring constant of 0.06 N m À1 (Veeco Instruments Inc., Plainview, NJ, USA).…”

Dynamic patterns of mechanical stimulation co-localise with growth and cell proliferation during morphogenesis in the avian embryonic knee joint

“…No studies of the mechanical properties of the interzone were found in the literature by these authors. Atomic force microscopy (AFM) has previously been used as a type of nanoindenter in order to analyse the mechanical properties of biological specimens including growth plate cartilage (Allen and Mao, 2004;Radhakrishnan et al, 2004), articular cartilage (Stolz et al, 2004) and single cells (Rotsch et al, 1997).…”