Luer lok syringe

Distal epiphyses of femurs and proximal epiphyses of tibias were manually dislodged using dull scissors, and attached soft tissues and woven bones were carefully removed using a cuticle nipper. Cells were dissociated from dissected epiphyses using five serial rounds of collagenase digestion, incubating with 2 Wunsch units of Liberase TM (Roche, Basel, Switzerland) in 2 ml Ca²⁺, Mg²⁺-free Hank’s Balanced Salt Solution (HBSS, Sigma H6648) at 37°C for 30 min. each time on a shaking incubator (ThermomixerR, Eppendorf, Hamburg, Germany). Single--cell suspension was generated using an 18-gauge needle and a 1 ml Luer-Lok syringe (BD), and filtered through a 70 µm cell strainer (BD) into a 50-ml tube on ice.

For embryonic samples, hind limbs were harvested and incubated with 2 Wunsch units of Liberase TM (Sigma/Roche 5401127001) in 2 ml Ca²⁺, Mg²⁺-free Hank’s Balanced Salt Solution (HBSS, Sigma H6648) at 37 °C for 15 min on a shaking incubator (ThermomixerR, Eppendorf). For postnatal samples, soft tissues and epiphyses were carefully removed from dissected femurs. After removing distal epiphyseal growth plates and cutting off proximal ends, femurs were cut roughly and incubated with 2 Wunsch units of Liberase TM and 1 mg of Pronase (Sigma/Roche 10165921001) in 2 ml Ca²⁺, Mg²⁺-free HBSS at 37 °C for 60 min on a shaking incubator. After cell dissociation, cells were mechanically triturated using an 18-gauge needle with a 1 ml Luer-Lok syringe (BD) and a pestle with a mortar (Coors Tek), and subsequently filtered through a 70 µm cell strainer (BD) into a 50 ml tube on ice to prepare single-cell suspension. These steps were repeated for 5 times, and dissociated cells were collected in the same tube. Cells were pelleted and resuspended in an appropriate medium for subsequent purposes. For cell culture experiments, cells were resuspended in 10 ml culture medium and counted on a hemocytometer.

We started by explanting the lungs with standard autopsy methods. Surgical ink was applied to the pleura on each lobe to designate anterior, posterior, and mediastinal surfaces and the lateral aspect.
We prepared a mini‐BAL catheter (Ballard‐Avanos, Alpharetta, GA) and a wash basin filled with hot tap water. Our lungs specimens were refrigerated, which hastens gelatinization.
First, we incubated the stock solution in hot tap water until contents were liquefied. We then navigated the cannula to the intended bronchus until wedged (Figure S1). The stock solution was agitated to disperse the solids, drawn up into the desired aliquot volume with a Luer‐Lok syringe (BD, Franklin Lakes, NJ) and injected 3–5 mL/s and the cannula left in place—1 min with cold specimens, 3 min at room temperature. Finally, the lungs were inflated fully with 10% formalin using standard post‐mortem techniques and stored at room temperature.

A single bolus of 1 mL hypertonic saline (5.85% NaCl) was injected in the VL (the middle third of the muscle belly) of the right leg to induce muscle pain. The site was cleaned with an alcohol swab and then the saline was manually injected using a 3 mL Luer-Lok syringe (BD, New Jersey, USA) connected to a 1.5 inch 25-gauge hypodermic needle (SurGuard2, Terumo, Japan) over a 20 s window (5 s pause after the insertion, a 10 s infusion period, followed by 5 s pause before needle withdrawal). An identical injection protocol was performed with the isotonic saline (CTRL condition).

Soft tissues and epiphyses were carefully removed from dissected femurs. After removing distal epiphyseal growth plates and cutting off proximal ends, femurs were cut roughly and incubated with 2 Wunsch units of Liberase^TM (Sigma/Roche 5401127001) and 1 mg of Pronase (Sigma/Roche 10165921001) in 2 ml Ca²⁺, Mg²⁺-free Hank’s Balanced Salt Solution (HBSS, Sigma H6648) at 37 °C for 60 min. on a shaking incubator (Thermomixer^R, Eppendorf). After cell dissociation, cells were mechanically triturated using an 18-gauge needle with a 1 ml Luer-Lok syringe (BD) and a pestle with a mortar (Coors Tek), and subsequently filtered through a 70 µm cell strainer (BD) into a 50 ml tube on ice to prepare single cell suspension. These steps were repeated for five times, and dissociated cells were collected in the same tube. Cells were pelleted and resuspended in an appropriate medium for subsequent purposes. For cell culture experiments, cells were resuspended in 10 ml culture medium and counted on a hemocytometer.

For embryonic samples, hind limbs were harvested and incubated with 2 Wunsch units of Liberase TM (Sigma/Roche 5401127001) in 2 ml Ca²⁺, Mg²⁺-free Hank’s Balanced Salt Solution (HBSS, Sigma H6648) at 37 ^°C for 15 min on a shaking incubator (ThermomixerR, Eppendorf). For postnatal samples, soft tissues and epiphyses were carefully removed from dissected femurs. After removing distal epiphyseal growth plates and cutting off proximal ends, femurs were cut roughly and incubated with 2 Wunsch units of Liberase TM and 1 mg of Pronase (Sigma/Roche 10165921001) in 2 ml Ca²⁺, Mg²⁺-free HBSS at 37 ^°C for 60 min on a shaking incubator. After cell dissociation, cells were mechanically triturated using an 18-gauge needle with a 1 ml Luer-Lok syringe (BD) and a pestle with a mortar (Coors Tek), and subsequently filtered through a 70 μm cell strainer (BD) into a 50 ml tube on ice to prepare single cell suspension. These steps were repeated for five times, and dissociated cells were collected in the same tube. Cells were pelleted and resuspended in an appropriate medium for subsequent purposes. For cell culture experiments, cells were resuspended in 10 ml culture medium and counted on a hemocytometer.

Polycaprolactone (average 80,000 Mn, Sigma, UK) was dissolved in a specific solvent and then propelled by an electric field onto a collector where polymer fibers formed as the solvent evaporated. Adding both the silver-doped and undoped nHA powder to the solution at 20 wt.% allowed it to be incorporated into the electrospun scaffold. PCL (1.5 g) was dissolved into a solvent mixture (90% Dichloromethane [DCM], 10% Dimethylformamide [DMF]) (Sigma Aldrich, UK) and stirred using a magnetic stirrer bar (5 mm bar, Sigma Aldrich, UK) for 2 h. The nHA powders were incorporated at 20 wt.% of the PCL component and added into the dissolved PCL solution. The polymer-ceramic-solvent mixtures were placed in an ultrasonic bath for 30 min, before stirring using a magnetic stirrer bar for 30 min to ensure even distribution of powder in the solution. A 1-mL Luer-Lok syringe (BD, EU) was loaded with 1 mL of the PCL/nHA solution and this was electrospun using a custom electrospinning rig described previously [28 (link)]. In total, 2 mL of solution at 1 mL/hr was used to fabricate each scaffold at 17 kV with a spinning gap between the needle and collector of 20 cm.