Avanti j 26 xpi centrifuge

All experimental procedures were approved by the Institutional Ethics Review Committee of Southern University of Science and Technology and the volunteer who agreed to participate signed informed consent forms. The experiments were carried out in accordance with the approved relevant guidelines and regulations. The AC used in this study was from the disposal a patient (53 years-old, Nanfang Medical School, Guangzhou, China) after total knee arthroplasty. Only the portions of AC classified as the Outerbirdge Grade 1 were selected for collagen fibril extraction. The extraction protocol of type II collagen fibril was modified based on the previous studies32 (link)33 (link). The AC tissues were harvested with a round punch and were cut into pieces with about 0.5 mm in thickness. The harvested AC tissues were dispersed using a homogenizer in the physiological saline (10× of sample volume) on the ice. Afterwards, the sample was centrifuged (Avanti-J-26 XPI centrifuge, Beckman Coulter) at 4500 rpm for 20 min at 4 °C for three times. After each centrifugation step, the supernatant was collected for the next centrifugation. The low temperature used here is to prevent collagen fibril from possible thermal degradation. The final supernatant was treated with CaCl₂ (2, 3, 4, 5, 10, and 20 mM, respectively).

Mesoporous tetrasulfide-based silica nanoparticles were synthesized according to a previously reported procedure in the literature [30 (link)]. Briefly, 110 mL of ultrapure water, 10 mL of absolute ethanol, 220 mg of CTAB, and 900 µL of an aqueous solution of NaOH (2M) were mixed in a 250 mL round-bottomed flask at 80 °C and under reflux at a stirring rate of 500 rpm for 30 min. Then, the stirring rate was increased to 1400 rpm and a mixture of TEOS and BTESPT with the molar ratio of 6.6:1 was added dropwise to the solution. The reaction was left under stirring for 6 h. After synthesis, nanoparticles were precipitated and washed three times with ethanol by centrifugation at 18,000 rpm and 4 °C for 10 min (Avanti J-26 XPI Centrifuge (Beckman Coulter, CA, USA)) and maintained in alcohol. The nanoparticles were suspended in acidic ethanol (1 mL of HCl 36.5% in 30 mL of ethanol 100%) and kept under reflux at 80 °C and 1500 rpm for 6 h. Then, they were washed three times with water and freeze-dried.

Myofibril proteins were isolated in accordance with the method of Morzel et al. [26 (link)] with slight modification as outlined by Nakyinsige et al. [27 (link)]. Approximately 2.5 g of pulverized muscle tissue was homogenized for 30 s on ice in 25 ml of extraction buffer containing 25 mM KCl, 150 mM NaCl, 4 mM EDTA, and 3 mM MgCl₂ at pH 6.5 to which protease inhibitor (CALBIOCHEM®, Cat # 55140, EMD Bioscience, Inc. Germany) was added. After filtration, the homogenate was incubated at 4°C with stirring. This was followed by centrifugation at 2000 g for 15 min at 4°C using an Avanti^® J-26XPI centrifuge (BECKMAN COULTER®, USA). The pellets were washed twice with 25 ml of a 50 mM KCl solution at a pH of 6.4 and once with 25 ml of 20 mM phosphate buffer of pH 6. The pellets were finally re-suspended in the same phosphate buffer and the protein concentration of the samples was estimated in accordance to the Bradford method [28 (link)] with the aid of Protein Assay Kit II 500–0002 (Bio-Rad, USA) following the micro plate protocol for colorimetric procedure.

BL21(DE3) Escherichia coli cells were transformed with desired plasmids and cultured in LB media at 37 °C. At OD₆₀₀ 0.6 to 0.8, the culture temperature was lowered to 20 °C, IPTG (GoldBio) was added at 0.5 mM to induce protein expression and cultures further incubated at 20 °C overnight. The cells were harvested by centrifugation at 4000g for 20 min in an Avanti J-26 XPI centrifuge with a JLA 8.1000 rotor (Beckman Coulter).