We obtained human tibial plateau osteochondral specimens from a skeletally mature male at the end of his second decade of life undergoing limb salvage for a distal femoral tumor not involving the regions of the specimens. After surgical excision, the specimen was stored in sterile lactated Ringer’s solution at room temperature until the experimental cartilage injury was created (approximately 4 hours after excision). To create the injury, we used a curette to make a single sagittal groove beneath the central portion of the lateral plateau penetrating through the cancellous bone into the subchondral bone. The specimen then was subjected to a three-point bending force across the weakened area until the cartilage and subchondral bone fractured. The osteochondral specimens then were placed in standard cell culture conditions with DMEM for 4 days at 37°C in the incubator with 5% CO2. The specimens then were fixed in zinc-buffered formalin (Z-Fix; Anatech Ltd, Battle Creek, MI) at 4°C overnight. Specimens were dehydrated, embedded in paraffin, sectioned at 7-μm thickness, and mounted on Superfrost® Plus slides (Fisher Scientific, Pittsburgh, PA). Sections were deparaffinized in xylene and rehydrated before subsequent analysis. A minimum of four near-adjacent sections were analyzed by each of the methods described subsequently.
Optimization of the signal-to-noise ratio involved adjusting the duration and dose of proteinase K digestion for all techniques, whereas TUNEL staining required dilution of TdT concentrations. Excessive proteinase K digestion created excess background noise. The absence of positive staining in areas of the growth plate where no apoptosis is believed to occur shows these techniques have sufficient specificity. The growth plate cartilage was obtained from one surgical specimen taken at the time of epiphyseodesis, washed in saline, and immediately fixed in Z-fix. After decalcification of these specimens, using 0.45 mol/L EDTA and 0.1 mol/L Tris buffer, the growth plates were dehydrated, embedded in paraffin, and sectioned at 7-μm thickness. A minimum of four sections then was analyzed using each of the same protocols.
Detection of double-stranded DNA breaks by TUNEL was performed using direct incorporation of fluorescein-labeled nucleotides with modification of the protocol provided with the ApopTag® Fluorescein Direct (Millipore Corp, Bedford, MA). After deparaffinization and rehydration, the samples were treated with 20 μg/mL proteinase K (Roche, Indianapolis, IN) for 15 minutes. After washing, the samples were incubated in Equilibrium Buffer for 30 seconds. Fifty-five microliters of TUNEL reaction mixture consisting of 12.4 μL terminal deoxynucleotidyl transferase enzyme, 28.9 μL reaction buffer, and 68.8 μL sterile phosphate-buffered saline (PBS) was added to the sections and incubated at 37°C in a humidified chamber for 1 hour. We found this dilution of the TdT reaction mixture was useful in reducing background noise from prior experiments. The reaction was stopped with stop/wash buffer and incubated for 10 minutes. Slides then were washed in PBS, counterstained with DAPI, 4′,6-diamidino-2-phenylindole (Vector Labs, Burlingame, CA), and observed under fluorescent microscopy.
Apoptosis detection using DNA denaturation analysis was performed following a protocol provided by Chemicon (Temecula, CA). After deparaffinization and rehydration, sections were permeabilized in 0.1 mg/mL saponin for 20 minutes and washed in PBS. Sections then were digested with 10 μg/mL proteinase K for 10 minutes and washed in distilled H2O. Slides were incubated in 50% formamide w/v in distilled H2O for 20 minutes at 56° to 60°C and then washed in ice-cold PBS for 5 minutes. Endogenous peroxide was quenched. Nonspecific binding was blocked with 3% nonfat dry milk. The primary antibody, anti-mouse F7-26 anti-ssDNA (Millipore) at 1:10 dilution, was applied for 30 minutes at room temperature in a humidified chamber and then washed in PBS. The secondary antibody, biotinylated anti-mouse IgM (Vector Labs) at 1:200 dilution, was applied for 30 minutes and washed in PBS. Streptavidin-peroxidase (Millipore) incubation for 30 minutes was followed by development in 3,3′-diaminobenzidine (DAB) (Millipore), washed in PBS and distilled H2O, and counterstained with DAPI (Vector Labs). Negative controls were run without primary antibody.
For the anti-activated caspase-3 method, sections were deparaffinized, rehydrated, and then incubated in 1 mg/mL hyaluronidase (Sigma-Aldrich Corp, St Louis, MO) in PBS for 60 minutes at 37°C and washed in PBS. Endogenous peroxide then was quenched. Nonspecific binding was blocked with 5% normal goat serum. Proteinase K at 10 μg/mL was applied for 10 minutes and sections were washed in PBS. Sections then were incubated in the primary antibody, 1:100 anti-active caspase-3 pAb rabbit IgG (Promega Corp, Madison, WI) overnight at 4°C, followed by anti-rabbit IgG biotinylated antibody (Vector Labs) at 1:100 dilution in PBS, and then streptavidin-peroxidase. Slides were developed in DAB, washed in PBS and distilled H2O, and counterstained with DAPI. Negative controls were run without primary antibody incubation.
Single-base 3′ overhangs and blunt ends in double-stranded DNA were detected using ISOL following the protocol provided in the ApopTag® ISOL kit (Millipore). After deparaffinization and rehydration, endogenous peroxide was quenched with 3% hydrogen, and the sections then were incubated in 10 μg/mL proteinase K for 10 minutes. Sixty microliters working-strength DNA ligase solution, 54 μL biotin-labeled oligonucleotide, and 6 μL T4 DNA ligase were placed on the sections, and the sections were incubated in a humidified chamber for 14 hours at 20°C. Oligonucleotides specific for single-base 3′ ends (Oligo A) and blunt ends (Oligo B) were used with single-base 3′ detection being more specific for apoptosis and blunt end detection being more sensitive. After incubation in T4 DNA ligase, the sections were washed in PBS, and streptavidin-peroxidase was applied for 30 minutes. The signal was developed in DAB, the reaction was stopped with PBS followed by distilled H2O washes, and the slides were counterstained in DAPI.
For histologic analysis, near-adjacent sections were stained with hematoxylin and eosin (Fisher Scientific, Fairlawn, NJ) and safranin O (Fisher Scientific) using standard methods to assess proteoglycan loss.
For the injured cartilage specimens, the injury zone was defined as the area of cartilage starting from the edge of the fractured cartilage and extending up to 1.3 mm away from the injury site. The control zone was defined as an area of cartilage approximately 10 mm from the cartilage fracture site. We intentionally chose areas that should be positive and negative. The deep layer provides a better control for true-negatives in testing detection methods as a result of processing and culture conditions that can affect the superficial layer of cartilage. This study was not designed to look at the model system of the injury, but rather the ability of these detection methods to distinguish between positive and negative cells. With superficial cartilage, there may be a baseline level of apoptosis resulting from storage. The deep cartilage should not be expected to have cells undergoing PCD.
Each zone of analysis measured 1.3 mm × 1.3 mm with the depth of visualization determined by the depth of field of the microscope objective. Images were captured digitally at one-megapixel resolution with a Zeiss AxioCam mounted on an Axioskop 2 microscope (Carl Zeiss, Inc, Thornwood, NY). Appropriate filters were used for fluorescence-based techniques. We (AD) performed semiautomated cell counting using Photoshop® (Adobe Systems Inc, San Jose, CA) and Scion Image (Scion Corp, Frederick, MD) software packages optimized to detect cells based on nuclear and/or cytoplasmic staining. The apoptotic index for each area was calculated by dividing the number of positive cells by the total number of cells in the area of analysis.
Results are presented as the apoptotic index in percentage ± standard error of the mean. We compared the apoptotic indices from the injury zone with those of the internal control of the zone of chondrocytes in the central area and also between detection methods in the injury zone, using unpaired two-tailed t tests. We performed all analyses using GraphPad Prism® (GraphPad Software, Inc, La Jolla, CA).