An experimental model for studying the biomechanics of embryonic tendon: Evidence that the development of mechanical properties depends on the actinomyosin machinery

Abstract

Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe-linear-fail stress-strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live-cell imaging and (14)C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.

Fig. 1

Tendon construct formation and mechanical testing. (A) Schematic representation of the formation of a tendon construct. From left to right, during 7 days in culture, cells contract the fibrin gel, replace the fibrin with collagen fibrils, and form a mechanically robust linear construct. (B) Left to right, images of a T0 construct in the culture dish, mounted on a sand paper window frame, and being mechanically tested to failure.

Fig. 2

Typical stress–strain curves for T0, T7 and T10 tendon constructs shown in comparison with data from 13-day ECMT. All curves show the distinct regions (toe, linear and failure) characteristic of tendon. The tendon constructs show a progressive increase in both stiffness (gradient of linear region) and breaking stress with time from T0 to T10.

Fig. 3

Summary of data for the mechanical properties of tendon constructs (from T0 to T42) shown compared with the corresponding data on 13-day ECMT. (A) The ultimate tensile stress (UTS) of tendon constructs increased over the initial culture period from 0.87 ± 0.07 MPa at T0 to 2.10 ± 0.20 at T10 (p < 0.05). After T10 the UTS stabilized, reaching 1.52 ± 0.21 at T42 (p < 0.05). The UTS in the T10 construct was similar to that exhibited by 13-day ECMT, which had a value of 1.59 ± 0.19 MPa. (B) The elastic modulus of tendon constructs also increased in the initial culture period from 4.25 ± 0.32 MPa at T0 to 12.91 ± 1.60 MPa at T10 (p < 0.05). The elastic modulus stabilized after T10, reaching 10.82 ± 2.47 at T21 (p < 0.05). The elastic modulus at T10 was similar to that exhibited by 13-day ECMT, which had a value of 11.47 ± 0.82. (C) Failure strain was greater for the tendon constructs than for ECMT up to T21, after which the constructs showed a similar failure strain to that exhibited by 13-day ECMT. ^⁎p < 0.05, ^†p > 0.2.

Fig. 4

Transmission electron microscopy of tendon constructs and embryonic chick metatarsal tendon (ECMT). Typical images of transverse sections of tendon constructs at T0 (A), T7 (B) and T42 (C) and 13-day ECMT are shown. Plots of mean fibril diameter and mean fibril volume fraction as a function of time in tendon constructs and 13-day ECMT are shown.

Fig. 5

Comparison of construct transverse area, cell volume fraction and cell number. (A) Plots of transverse area and cell volume fraction for the tendon construct as a function of days in culture. The constructs initially maintain a constant transverse area from T0 to T7. Prolonged culture results in a 43% decrease in transverse area over the next 14 days. In contrast, the cell volume fraction showed an initial 39% decrease from T0 to T7 before stabilizing at a value of ~ 0.3 by T21. (B) Quantification of the live-cell population of tendon constructs over time in culture. Bracketed numbers at the head of the bars are the percentage of cells in the S-phase. Red bars correspond to pre-contraction of the tendon construct, black bars to post-contraction.

Fig. 6

Triton X-100 treatment of T0 tendon constructs arrests development of mechanical properties. (A) Ultimate tensile stress (UTS) of the control tendon constructs increased 2-fold during T0 to T7. In contrast, the Triton-treated constructs showed no significant increase in UTS. (B) Elastic modulus of the control tendon constructs increased 2.2-fold during T0 to T7. The Triton-treated constructs, however, showed no significant change in the elastic modulus. ^⁎p < 0.05, ^†p > 0.2.

Fig. 7

Expression of non-muscle MYH9 and MYH10 by cells in tendon constructs. (A) PCR gel electrophoresis analysis of MYH9 and MYH10 and col1a1 gene expression. (B) Western blot for NMMHC-IIB protein. (C, D, E) Real-time PCR expression of MYH9 (C), MYH10 (D) and col1a1 (E) in tendon constructs between T0 and T21. Expression of MYH9 and MYH10 was significantly reduced in tendon constructs compared to monolayer, whereas col1a1 was significantly increased. ^⁎p < 0.05.

Fig. 8

The effect of cytochalasin and blebbistatin treatment on the mechanical properties of tendon constructs at T0. Two-hour incubation with either cytochalasin or blebbistatin was immediately followed by mechanical testing. No significant differences in elastic modulus or UTS were seen between inhibitor treated constructs and control.

Fig. 9

The effects of cytochalasin and blebbistatin on the development of mechanical properties of tendon constructs. T0 tendon constructs were incubated separately with cytochalasin and blebbistatin for 24 h and the constructs were incubated for a further 48 h in an inhibitor-free culture medium. Control samples contained 0.1% DMSO for the first 24 h. ^⁎p < 0.05, ^†p > 0.2.

Fig. 10

Demonstration of live cells post-treatment with blebbistatin. After 24-hour incubation with blebbistatin constructs were stained with calcein-AM. (A) A transmitted light image of a construct. (B) Confocal microscope image of the boxed area in (A). Live cells appear green.

Fig. 11

Continuous labeling with ¹⁴C-proline. (A) Tendon constructs (at T0 or at T21) were treated with either blebbistatin or 0.1% DMSO for 4 h and then ¹⁴C-proline was added for a further 1 h. Proteins were extracted as previously described (Canty et al., 2004). (B) Sequential salt extractions (S1, S4) for extracellular proteins followed by an NP-40 detergent extraction (N) for intracellular proteins demonstrated that blebbistatin did not affect the ability of the cells to secrete procollagen and to convert procollagen to collagen.

Fig. 12

Live-cell imaging of cells in tendon constructs. Cells seeded in fibrin gels at T6 were treated with either DMSO, blebbistatin, or cytochalasin and imaged for 12 h at 5-min intervals. (A) Schematic showing the fibrin gel (shaded grey) contracting around the fixed-position pins. The box indicates the area imaged. (B) Cell contraction was highlighted with colored lines tracking the position of individual cells over time. Cell contraction was abolished by treatment with blebbistatin or with cytochalasin. Scale bar, 100 μm.