Force transducer

Penis samples were obtained from rats under anesthesia. The urethra, veins, and tunica albuginea were removed from penises. The remaining CCs were prepared in a chilled Krebs solution composed of 119 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl₂, 1.2 mM MgCl₂, 15 mM NaHCO₃, 1.2 mM NaH₂PO₄, and 11 mM glucose. The CCs were suspended in an organ bath, which contained control solution (37℃), aerated with 95% O₂ and 5% CO₂. One side of the prepared CC was clipped, and the other side was ligated and connected to a force transducer (ADInstruments Pty. Ltd). The force transducers were connected to a bridge amp and PowerLab 4/26 (ADInstruments), and tension was measured using LabChart8 software (ADInstruments). The CCs were allowed to equilibrate for at least 1 h at an optimal resting tension of 0.6 g. After equilibration, the contractile responses to high KCl (80 mM) Krebs solution were measured to determine the reference contraction. Next, to investigate contractile responses, cumulative dose curves for NA (10⁻¹⁰–10⁻⁴ M; Sigma‐Aldrich Co. LLC) were measured, and the results were expressed relative to the responses to high KCl Krebs solution. Dose–response curve was fitted to all the data by nonlinear regression based on the Boltzmann model using the Origin software (OriginLab Corp.).

The repaired samples with the hydrogel placement site covered by no more than 2 mm of surrounding muscle were retrieved eight weeks after implantation. A silk suture was used to attach the distal tissue to a force transducer (AD Instruments, Dunedin, New Zealand). Parallel platinum electrodes were positioned at the anastomoses near the implant site perpendicular to the direction of muscle contraction, which was determined by the nylon sutures. A model S48 stimulator was then used to apply electrical field stimulation (20 V at the electrodes) to the muscle (Grass Technologies, Middleton, WI, USA). Following a 10 min equilibration period, the optimal length was found based on the twitch response by varying the muscle’s length by rotating the micrometer head. A train of 0.2 ms square pulses lasting 1200 ms was used to measure the peak isometric contractile force at the optimal length over a range of frequencies (0–200 Hz). Power Lab/8sp (AD Instruments) was used to display and record force readings in real-time.

Vascular contractile and dilative function was expressed as the responsiveness of arteries to the norepinephrine (NE) or acetylcholine (Ach), as described previously (Zhu et al., 2013 (link)). The response of arteries to NE was measured by the PowerLab system via a force transducer (AD Instruments, Australia). SMA rings (diameter, 2–3 mm) were mounted on wire and suspended between a force transducer and a post attached to a micrometer, then immersed into an isolated organ chamber containing K-H solution. After equilibration for 2 h, the contractile responses of arterial rings to NE (1×10⁻¹⁰–1×10⁻⁴ mol/L) were measured. The dilative responses of arterial rings to Ach (1×10⁻¹⁰–1×10⁻⁴ mol/L) were measured subsequently.

Intact TA muscle‐tendon complexes were isolated from young and old mice and mounted vertically to a bath chamber containing carbogen‐(95% O₂/5% CO₂)‐saturated Krebs‐Ringer buffer (118 mM of NaCl, 4.75 mM of KCl, 24.8 mM of NaHCO₃, 1.18 mM of KH₂PO₄, 2.5 mM of CaCl₂ 2H₂O, 1.18 mM of MgSO₄, and 10 mM of glucose) at pH 7.4 and 25°C. The muscle was fixed to the bottom of the organ bath by a clamp, while the tendon was connected to a force transducer (AD Instruments, USA) by a string. The optimal muscle length (L₀) was determined as the length producing the highest twitch force at supramaximal voltage (100 V for 1 ms) using a modified previous protocol.⁴⁷, ⁴⁸ Twitch forces (mN) were determined using an electrical stimulator (AD Instruments, USA) at 100 V and 1 Hz. Tetanus forces (mN) were determined at 100 V from 10 to 200 Hz. The isometric forces were normalized by the weight of TA muscle. For analysis of resistance to muscle fatigue, the muscle was repeatedly stimulated every 30 s for 10 min. Then, the isometric forces were analysed as a percentage of the initial maximal contractile force. All experiments were performed at room temperature (25°C) and analysed using LabChart software (AD Instruments, USA).