The use of human samples was approved by the Institutional Review Board of Kurume University Hospital (No. 2456), and the animal study was approved by our Institutional Animal Care and Use Committee (No. 2014-189-2).

NTP was provided by the Nippon Zoki Pharmaceutical Co., Ltd. (Osaka, Japan). The biological activity of NTP was expressed in NTP units (U). Lidocaine was purchased from Aspen (Xylocaine Injection Polyamp 1%; Aspen, Tokyo, Japan).

Human rotator cuff tendons were obtained from 12 patients (six males and six females) who underwent arthroscopic rotator cuff repair for small or medium tear; the patients had a mean age of 63.4 years, and the approximate size of the specimens were 5 mm in width. The rotator cuff was repaired without excessive tension at the sides, and only trimmed and redundant tissues were harvested before or after the repair. Rat rotator cuff tendons were obtained from 12 adult Sprague-Dawley rats (20–25 weeks old).

Both human and rat rotator cuff tissues were rinsed twice with phosphate-buffered saline, cut into small pieces, and incubated in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37°C in a humidified atmosphere that contained 5% CO₂ in T-75 flasks. Twenty-four hours after incubation, tenocytes that were attached to the plastic bottom were maintained in the same condition, and tissues that were not attached to the plastic bottom were discarded. Semi-confluent tenocytes from passages 4 to 6 were used in all experiments.

Human and rat tenocytes from six donors were seeded in 96-well plates at 5,000 cells/well and incubated for 24 hours. The cells were exposed to lidocaine (0.01%, 0.05%, 0.1%, 0.2%), NTP (0.1, 0.25, 0.5, 1.0 U/mL), 0.1% lidocaine with NTP (0.1, 0.25, 0.5, 1.0 U), or control medium for the next 24 hours. Cell viability was examined using the WST-8 cell proliferation assay kit (Nakarai, Kyoto, Japan). Briefly, 20 μL of WST-8 reagent was added to each well, and the plates were incubated for 4 hours. The absorbance of each well, which represented its cell viability, was measured at 450 nm using a microplate reader (Bio-Rad Model 550; Bio-Rad Laboratories, Hercules, CA, USA).

Semi-confluent human and rat tenocytes from 10 donors were exposed to 0.1% lidocaine, 0.1 U/mL NTP, 0.1% lidocaine with 0.1 U/mL NTP, or a control medium in T-75 flasks for 24 hours. The tenocytes were trypsinized, collected by centrifugation, washed with phosphate-buffered saline, and labelled with annexin V-FITC and propidium iodide (PI) for 15 min according to the manufacturer’s protocol (ApoScreen Annexin V Apoptosis Kit; Southern Biotech, Birmingham, AL, USA). The cells were analyzed using a FACSDiva flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) to count apoptotic (annexin V-positive and PI-negative), necrotic (PI-positive), and viable (annexin V- and PI-negative) cells.

Rat tenocytes from two donors were exposed to 0.1% lidocaine, 0.1 U/mL NTP, or 0.1% lidocaine with 0.1 U/mL NTP for 24 hours, switched to normal medium, and incubated for an additional 72 hours. Whole cell extracts were lysed with radioimmunoprecipitation assay buffer containing Protease Inhibitor Cocktail (Nacalai, Kyoto, Japan) and a Halt Phosphatase Inhibitor Cocktail (Pierce Biotechnology, Waltham, MA, USA), separated using a 10% SDS-polyacrylamide gel, and transferred onto equilibrated polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% bovine serum albumin in tris-buffered saline-Tween (TBS-Tween) for 1 hour and incubated overnight at 4°C with primary antibodies specific for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000; AB_2536381; Invitrogen, Waltham, MA, USA) and BCL-2 (1:1000; ab196495; Abcam, Cambridge, UK). The membranes were incubated with secondary antibodies labelled with horseradish peroxidase, and positive signals were visualized using an image analyzer (LAS-4000; Fujifilm, Tokyo, Japan). GAPDH was used as an internal reference control. BCL-2 band intensities were measured with ImageJ software 1.52k (National Institutes of Health, Bethesda, MD, USA) and normalized to GAPDH band intensity.

Semi-confluent rat tenocytes that were incubated on glass cover slips were exposed to 0.1% lidocaine, 0.1 U/mL NTP, 0.1% lidocaine with 0.1 U/mL NTP, or a control medium for 24 hours and incubated with normal medium for an additional 72 hours. The cells were fixed with 4% formaldehyde at room temperature, rinsed with TBS, treated with 1% hydrogen peroxidase in methanol to deplete endogenous peroxidase activity, and blocked with 5% skim milk powder in TBS for 60 minutes. They were incubated overnight at 4°C with the primary antibody specific for cytochrome c (1: 200; 11940S; Cell Signaling Technology, Danvers, MA, USA), rinsed thrice with TBS, incubated with biotinylated anti-rabbit IgG (Histofine Simple Stain PO (R), Nichirei Corp., Tokyo, Japan) for 30 minutes, stained using a DAB kit, and counterstained with Mayer’s hematoxylin. Cytochrome c-positive cells were counted in 10 randomly selected high-power fields (HPF) in each condition, and a mean positive cell count per HPF was calculated.

Statistical analysis was performed using JMP ver. 12 (SAS Institute Inc., Cary, NC, USA). The Kruskal-Wallis test with the Wilcoxon test as a post-hoc analysis was used to evaluate statistical differences between groups. Values are shown as the mean±standard deviation. Differences with a p-value<0.05 were considered significant.

Cell viability was significantly decreased by 0.01%–0.1% lidocaine in both human and rat tenocytes compared to that in the control group cells. The values ranged from 88.6% in 0.01% lidocaine to 15.6% in 0.2% lidocaine in human tenocytes (all p<0.001) and from 89.5% in 0.01% lidocaine to 10.7% in 0.2% lidocaine in rat tenocytes which indicated a dose-dependent manner (all p<0.001). However, NTP alone did not affect cell viability at any dose in either human or rat tenocytes (Fig. 1A). In human tenocytes, the cell viability in the lidocaine alone group was 51.5%, and that in the lidocaine with 0.1–1 U/mL NTP group was 71.0%–74.1% (p=0.0253–p=0.05) (Fig. 1B). Similarly, in rat tenocytes, the cell viability in the lidocaine alone group was 58.7%, and that in the lidocaine with 0.1–1 U/mL NTP group was 88.5%–102% (p=0.0011–p=0.0017) (Fig. 1B). These results show that NTP significantly inhibited the cytotoxic effect of lidocaine in both human and rat tenocytes.

The flow cytometry results showed that, in human tenocytes, 0.1% lidocaine-induced significant apoptosis with the apoptotic cells, with the values increasing from 2.0%±0.77% in the control group to 11.7%±6.58% in lidocaine-treated cells (p<0.001). Similarly, in rat tenocytes, 0.1% lidocaine-induced significant apoptosis, with the apoptotic cell proportion increasing from 1.6%±0.94% in the control group to 12.7%±8.16% in the lidocaine-treated cells (p<0.001). The results indicated that 0.1 U/mL NTP/mL considerably inhibited this lidocaine-induced apoptotic effect when it was administered in combination with 0.1% lidocaine, reducing the apoptotic cell population to 4.51%±2.01% in human tenocytes and to 2.87%±2.05% in rat tenocytes (both p<0.001) (Figs. 2 and 3).

The Western blotting experiments demonstrated that exposure of rat tenocytes to 0.1% lidocaine decreased BCL-2 protein expression, and administration of 0.1 U/mL NTP inhibited this BCL-2 reduction from 41.0% in 0.1% lidocaine alone to 78.1% when used in combination with 0.1% lidocaine (Fig. 4).

The immunohistochemical staining of rat tenocytes showed that only a few cells (mean, 0.2 cells/HPF) in the control group were cytochrome c-positive. Cells treated with 0.1% lidocaine showed an increased number of cytochrome c-positive cells (mean, 0.9 cells/HPF) compared to that in the control group, and treatment with NTP resulted in a reduced number of cytochrome c-positive cells (mean, 0.3 cells/HPF) upon co-administration with lidocaine (Fig. 5).