Microniche geometry modulates the mechanical properties and calcium signaling of chondrocytes.

In articular cartilage, the function of chondrocytes is strongly related to their zone-specific microniche geometry defined by pericellular matrix. Microniche geometry is critical for regulating the phenotype and function of the chondrocyte in native cartilage and tissue engineering constructs. However the role of microniche geometry in the mechanical properties and calcium signaling of chondrocytes remains unknown. To recapitulate microniche geometry at single-cell level, we engineered three basic physiological-related polydimethylsiloxane (PDMS) microniches geometries fabricated using soft lithography. We cultured chondrocytes in these microniche geometries and quantified cell mechanical properties using atomic force microscopy (AFM). Fluorescent calcium indicator was used to record and quantify cytosolic Ca2+ oscillation of chondrocytes in different geometries. Our work showed that microniche geometry modulated the mechanical behavior and calcium signaling of chondrocytes. The ellipsoidal microniches significantly enhanced the mechanical properties of chondrocytes compared to spheroidal microniche. Additionally, ellipsoidal microniches can markedly improved the amplitude but weakened the frequency of cytosolic Ca2+ oscillation in chondrocytes than spheroidal microniche. Our work might reveal a novel understanding of chondrocyte mechanotransduction and therefore be useful for designing cell-instructive scaffolds for functional cartilage tissue engineering.

Stiff substrates enhance cultured neuronal network activity.

The mechanical property of extracellular matrix and cell-supporting substrates is known to modulate neuronal growth, differentiation, extension and branching. Here we show that substrate stiffness is an important microenvironmental cue, to which mouse hippocampal neurons respond and integrate into synapse formation and transmission in cultured neuronal network. Hippocampal neurons were cultured on polydimethylsiloxane substrates fabricated to have similar surface properties but a 10-fold difference in Young's modulus. Voltage-gated Ca(2+) channel currents determined by patch-clamp recording were greater in neurons on stiff substrates than on soft substrates. Ca(2+) oscillations in cultured neuronal network monitored using time-lapse single cell imaging increased in both amplitude and frequency among neurons on stiff substrates. Consistently, synaptic connectivity recorded by paired recording was enhanced between neurons on stiff substrates. Furthermore, spontaneous excitatory postsynaptic activity became greater and more frequent in neurons on stiff substrates. Evoked excitatory transmitter release and excitatory postsynaptic currents also were heightened at synapses between neurons on stiff substrates. Taken together, our results provide compelling evidence to show that substrate stiffness is an important biophysical factor modulating synapse connectivity and transmission in cultured hippocampal neuronal network. Such information is useful in designing instructive scaffolds or supporting substrates for neural tissue engineering.

[Age-related biomechanical properties of chondrocytes in rabbit knee articular cartilage].

OBJECTIVE: To fully demonstrate the alterations about the viscoelastic properties of chondrocytes in rabbit knee articular cartilage. METHODS: Fifteen New Zealand white rabbits were divided into 3 age groups: young group (1 month), adult group (8 months) and old group (31 months). All rabbits were sacrificed and isolated from knee joint and digested into chondrocytes. The micropipette aspiration combined with Half-space model was used to quantify changes in viscoelastic properties of chondrocytes. RESULTS: Experimental studies have shown that the changes of aspiration length with time in old group were obviously different from young group and adult group. But similar variations were found between the latter two groups. The viscoelastic properties of chondrocytes in old group exhibited a significantly lower instantaneous modulus E0 (0.55 +/- 0.05 kPa), equilibrium modulus E infinity (0.28 +/- 0.04 kPa) and apparent viscosity micro (4.10 +/- 0.61 kPa x s) as compared with young group (0.67 +/- 0.10), (0.37 +/- 0.09), (6.29 +/- 0.92) kPa x s (P < 0.001) versus adult group: (0.65 +/- 0.07), (0.35 +/- 0.05), (6.01 +/- 0.89) kPa x s (P < 0.001). But no difference were found between the latter two groups (P > 0.05). CONCLUSION: In response to a step pressure, chondrocytes in each group exhibited the viscoelastic solid creep behavior. The viscoelastic properties of chondrocytes markedly decreased in old group.

[Mechanical properties of chondrocytes isolated from normal articular cartilage: experiment with rabbit knees].

OBJECTIVE: To measure the mechanical properties of chondrocytes of articular cartilage of knee in order to provide relevant technical parameters into the development of cartilage tissue engineering. METHODS: Eight NZW rabbits were killed, and their bilateral knee joints were taken out. The articular cartilage of the left knees were used for histological study, and articular cartilage of the right knees was digested with 0.4% pronase and 0.025% collagenase type II so as to make isolation of chondrocytes. The viability rate of the isolated chondrocytes was detected by trypan blue staining. The diameters of the chondrocytes in the histological sections and single cell suspension were measured respectively. The mechanical properties of the chondrocytes were determined using micropipette aspiration technique coupled with half-space model. RESULTS: In the normal articular cartilage 4 structural zones were differentiated: tangential, transitional, radial, and calcified zones. The viability rate of chondrocytes was 98.2% on average after isolation. The mean diameter of the chondrocytes in the histological sections was (14.2+/-2.9) microm, not significantly different from that in the single cell suspension [(14.9+/-2.2) microm, t=1.31, P=0.19]. In response to a prescribed pressure, the chondrocytes exhibited viscoelastic solid creep behavior characterized initially by a jump in displacement followed by a monotonically decreasing rate of deformation that generally reached an equilibrium displacement. The cells were observed to deform to a length of as much as three times the radius of the micropipette without completely entering the micropipette. The cells were then extruded from the micropipette and completely recovered. The Young's modulus of the normal chondrocytes was (0.57+/-0.43) kPa, and the k1, k2, and micro of the viscoelastic parameters were (0.37+/-0.07) kPa, (0.29+/-0.04) kPa, and (6.36+/-1.12) kPa-s respectively. The k1 is positively correlated to the cell diameter (r=0.4, P=0.031). CONCLUSION: Chondrocytes from normal articular cartilage behave as a viscoelastic solid. Micropipette aspiration technique is an efficient method for the study of chondrocyte biomechanics.