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. 2016 Mar;16(3):3136-3145.
doi: 10.1166/jnn.2016.12564.

Nanomechanics of Engineered Articular Cartilage: Synergistic Influences of Transforming Growth Factor-β3 and Oscillating Pressure

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Nanomechanics of Engineered Articular Cartilage: Synergistic Influences of Transforming Growth Factor-β3 and Oscillating Pressure

Arshan Nazempour et al. J Nanosci Nanotechnol. .
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Abstract

Articular cartilage (AC), tissue with the lowest volumetric cellular density, is not supplied with blood and nerve tissue resulting in limited ability for self-repair upon injury. Because there is no treatment capable of fully restoring damaged AC, tissue engineering is being investigated. The emphasis of this field is to engineer functional tissues in vitro in bioreactors capable of mimicking in vivo envi- ronments required for appropriate cellular growth and differentiation. In a step towards engineering AC, human adipose-derived stem cells were differentiated in a unique centrifugal bioreactor under oscillating hydrostatic pressure (OHP) and supply of transforming growth factor beta 3 (TGF-β3) that mimic in vivo environments. Static micromass and pellet cultures were used as controls. Since withstanding and absorbing loads are among the main functions of an AC, mechanical properties of the engineered AC tissues were assayed using atomic force microscopy (AFM) under a controlled indentation depth of 100 nm. Young's moduli of elasticity were quantified by modeling AFM force-indentation data using the Hertz model of contact mechanics. We found exposure to OHP causes cartilage constructs to have 45-fold higher Young's moduli compared to static cultures. Addition of TGF-β3 further increases Young's moduli in bioreactor samples by 1.9-fold bringing it within 70.6% of the values estimated for native cartilage. Our results imply that OHP and TGF-β3 act synergistically to improve the mechanics of engineered tissues.

Figures

Figure 1
Figure 1
Experimental design. (A(i)) A 10 μl droplet of expanded hAMSCs at a density of 1.6 × 107 viable cells/ml was placed in the center of each of 15 wells in two 24-well culture plates, one serving as a negative control (NC) with base medium and one as a positive control (PC) with growth factor supplemented medium. (A(ii)) 5 × 105 hAMSCs in 500 μl of EM were centrifuged in each of two microcentrifuge tube sets at 600 g for 5 minutes, four NC and four PC. (A(iii)) 6 × 106 hAMSCs in 2.5 ml of EM were injected into each reactor with two replicates at each condition, NC with and without oscillating pressure (OP) and PC with and without OP. (B(i)) Conical-shape bioreactor housed in a polycarbonate casing and topped with a pressurizing piston. (B(ii)) Schematic of conical-shape centrifugal bioreactor/pressurizing system. (B(iii)) Schematic process flow diagram for the bioreactor process system.
Figure 2
Figure 2
(a) A colloidal probe approaching the sample surface, (b) A 16 × 16 force-volume image generated from indenting the sample at 256 points. The scale bar represents force in nN. Each pixel in the force-volume image consists of an approach and a retraction curve. The approach curves are converted into force-indentation profiles like that shown later in Figure 4. Retraction curves could be used for adhesion data, but this was not relevant to this paper.
Figure 3
Figure 3
(a–c) Distribution of Young's moduli for each treatment group and (d and e) the log-normal probability distribution function for each histogram. (f) The distribution of Young's moduli and log-normal probability distribution function for bovine native articular cartilage.
Figure 4
Figure 4
Example curves showing different force-indentation profiles for micromass, pellet, and bioreactor samples treated with TGF-β3. Solid lines represent the Hertz fits to the data.
Figure 5
Figure 5
Means of distributions of the Young's moduli for all 6 conditions. Samples listed as OP are from the CBR and samples listed as PC were positive control samples which are supplemented with TGF-β3. NC stands for negative control which are samples without TGF-β3 supplementation. Error bars represent the standard error of the mean. All treatment groups were statistically significant from each other within a 99.9% confidence limit as indicated by the *.

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