Thermal energy is used in > 100,000 procedures annually to treat partial-thickness injuries to cartilage.a Although temperature- and power-controlled devices are available for chondroplasty procedures, the clinical effect is the result of heat. Rapid, visual macroscopic modification of the articular surface during arthroscopy appears to take place at approximately 70°C and is likely the result of the combined denaturation of matrix proteoglycans and collagen. This temperature is consistent with collagen denaturation.1–3 Microscopically, initial morphologic changes have been detected in 250-μm sections of cartilage treated at 56.5°C,4 and this is consistent with early morphologic changes that have been detected in joint capsular tissues.3,5–8 However, most RFE devices used for chondroplasty operate at temperatures > 100°C.
Injured articular cartilage has a poor healing response because of its lack of blood vessels and reparative cells.9–11 Chondrocytes have limited mitotic activity once mature and are also limited in their ability to increase matrix synthesis or migrate to the site of injury.12 Thus, there is a narrow margin of safety for procedures that cause cartilage damage. Limited repopulation of the cartilage matrix may be a result of allowing pluropotential cells from subchondral bone access through microfractures or may possibly be attributable to pluropotential cells in the synovium.13–15
Several methods can be used to assess cell viability. Light microscopy and many conventional stains can be used to differentiate intracellular morphologic characteristics. Confocal laser microscopy and viability (live-dead) staining with a red fluorescent compound and a green fluorescent cell marker has emerged as the premier method for assessing chondrocyte viability.16–20 The green fluorescent cell marker is a freely diffusible esterase compound that is hydrolyzed in the cytoplasm of viable cells and yields green fluorescence. The red fluorescent compound has a high affinity for nuclear DNA.21 Intact plasma membranes have an extremely low permeability for the red fluorescent marker; thus, red staining indicates that cells have damaged plasma membranes and are nonviable.
The purpose of the study reported here was to evaluate chondrocyte viability after subjecting osteochondral sections to increasing temperatures, to evaluate the response of chondrocytes over time after thermal insult, and to evaluate the response with regard to defined cartilage layers. We hypothesized that increasing temperatures would reduce chondrocyte viability and that the critical temperature would be approximately 55°C. In addition, we hypothesized that once the critical temperature was reached, loss of chondrocyte viability would be permanent and the chondrocytes would not recover viability over time in culture. On the basis of other preliminary cryopreservation studies22,23 and an understanding of the variability of chondrocyte function among matrix layers, we also hypothesized that viability would vary depending on the cartilage layer evaluated.
Materials and Methods
Sample population—Thirty humeral heads were collected from 15 mature female sheep immediately after the sheep were euthanized by an overdose of pentobarbital.b Full-thickness osteochondral explants were removed from each humeral head by use of a band saw. The cartilage was sectioned (by use of irrigation with physiologic saline [0.9% NaCl] solution) into uniform sections (1.5 mm × 1.5 cm × 1.0 cm). Sections were washed 3 times with sterile physiologic saline solution, and each section was then placed in a separate well of a 24-well tissue culture plate containing 1.5 mL of hybridoma cell culture mediumc that contained 50 μg of gentamicin/mL.d The working volume of each well was 2.0 mL. Samples were cultured at 37°C, 96% humidity, and 5% carbon dioxide. Medium was changed every 48 hours.
Cartilage explants (n = 318) were randomly assigned to temperature groups. Explants were placed in preheated (37°, 45°, 50°, 55°, 60°, or 65°C) sterile physiologic saline solution. Tissues were submerged at the assigned temperature for 5 minutes. After thermal treatment, samples were returned to tissue-culture medium and incubated at 37°C for up to 14 days by use of culture techniques described elsewhere.24 The day of thermal treatment and initiation of culture was designated as day 0.
Chondrocyte viability was assessed by use of confocal laser microscopy and viability staining on day 0 and days 1, 3, 7, and 14 after thermal treatment. Explants were stained by incubation at 23°C for 30 minutes in 1 mL of sterile physiologic saline solution that contained 0.4 μL of a green fluorescent dyee and 10 μL of a red fluorescent dye.f Imaging was accomplished by use of a confocal laser microscopeg equipped with a krypton-argon laser and the necessary filter systems for fluoreceinh and rhodaminei at an excitation wavelength of 495 nm. Images were collected as a 25-μm, single-plane image. The confocal laser microscope was calibrated by use of a micrometer measured through an objective lens (10X) used for the study.
Cartilage thickness was measured in each image by use of an imaging system.j Cartilage was characterized into 4 consecutive layers extending from the most superficial layer (layer 1) to the deepest layer (layer 4). Each of the 4 layers comprised 25% of the total cartilage thickness. Three 125- μm-wide samples of each layer were chosen randomly, and all stained cells within this portion of the layer were counted and designated as alive or dead. The volume of each sample was calculated by use of the 125-μm-wide sample, multiplied by the measured thickness of the sample, and then multiplied by 25 μm (ie, the depth of the sample). The percentage of live cells was determined by use of the following equation: (No. of green-labeled cells/[No. of red-labeled cells + No. of green-labeled cells]) × 100.
Data analysis—Mean and SEM for the number of live cells, number of dead cells, and percentage of live cells were determined for each temperature-time subgroup. Statistical analysis was performed with the general linear-models technique for an ANOVAk for temperature, duration of culture time, cartilage layer, and animal. When significance was detected, differences were identified by use of the Duncan multiple range test. Significance for all analyses was set at P < 0.05. When comparisons were not significant, the difference needed between populations to detect a significant difference at power = 0.8 and α = 0.05 was calculated.
Results
Chondrocyte viability within each temperature treatment, irrespective of the number of days in incubation or layer of cartilage, was summarized (Table 1). To evaluate the effect of depth of cartilage, the mean ± SEM percentage of viable cells on day 0 in cultures incubated at 37°C was as follows: layer 1, 76 ± 3.5%; layer 2, 79 ± 3.4%; layer 3, 62 ± 6.1%; and layer 4, 50 ± 6.2%. Percentage of viable cells decreased significantly for each increase in temperature higher than 45°C. The largest decrease in percentage of viability was detected when temperatures increased from 50° to 55°C.
Mean ± SEM values for chondrocytes incubated for up to 14 days after exposure for 5 minutes at various temperature treatments.
| Temperature (°C) | Dead cells (× 10−6μm3) | Live cells (× 10−6μm3) | Percentage of live cells |
|---|---|---|---|
| 37° | 69.0±3.6a | 125.3±4.6a | 62.9±1.4a |
| 45° | 75.8±3.7a | 125.5±5.0a | 60.7±1.3a |
| 50° | 85.3±3.0b | 86.0±3.6b | 43.4±1.3b |
| 55° | 73.3±2.6a | 16.1±1.1c | 18.9±1.2c |
| 60° | 109.9±3.0c | 12.1±1.1c | 7.8±0.6d |
| 65° | 97.2±3.0d | 1.92±0.3d | 1.5±0.2e |
Within a column, means with different superscript letters differ significantly (P<0.05).
Chondrocyte viability for each cartilage layer, irrespective of the number of days in incubation and treatment temperature, was summarized (Table 2). The percentage of viable cells was significantly lower for layer 4, compared with percentages for the other 3 layers. There was a decrease of 42% in the number of labeled cells (alive and dead) per unit volume between the most superficial and the deepest cartilage layer.
Mean ± SEM values for chondrocyte viability determined on the basis of cartilage layer.*
| Layer | Dead cells (× 10−6μm3) | Live cells (× 10−6μm3) | Percentage of live cells |
|---|---|---|---|
| 1 | 103.2±3.2a | 78.1±3.8a | 33.9±1.2a |
| 2 | 92.7±2.5b | 68.6±3.1b | 36.2±1.2a |
| 3 | 75.3±2.3c | 56.9±3.0c | 34.4±1.3a |
| 4 | 72.3±2.2c | 31.9±1.9d | 20.7±1.0b |
Cartilage thickness was measured in each image, and the cartilage was characterized into 4 consecutive layers (layer 1, most superficial; layer 4, deepest); each of the 4 layers comprised 25% of the total cartilage thickness.
See Table 1 for remainder of key.
Analysis of chondrocyte viability, based on treatment temperature and layer of cartilage, revealed a pattern for the chondrocytes in the deepest layer to have the lowest viability at each temperature (Table 3). Mean ± SEM values for the ratio of the percentage of live cells in the deepest layer to percentage of live cells in the most superficial layer were 0.71 ± 0.02 at 37°C, 0.65 ± 0.02 at 45°C, 0.55 ± 0.01 at 50°C, 0.56 ± 0.01 at 55°C, 0.59 ± 0.01 at 60°C, and 0.06 ± 0.01 at 65°C. Layers 1 through 3 had similar percentages for viable cells at each temperature, except at 45°C (power = 0.8 for a difference of 8.8%). In addition, chondrocyte viability was significantly less when the treatment temperature was increased from 45° to 50°C, and there was a larger percentage decrease in viability when the temperature was increased from 50° to 55°C (Table 4).
Mean ± SEM percentage of live cells for each cartilage layer* incubated for up to 14 days after exposure for 5 minutes at each treatment temperature.
| Layer | 37°C | 45°C | 50°C | 55°C | 60°C | 65°C |
|---|---|---|---|---|---|---|
| 1 | 65.6±2.6a,A | 61.6±2.2a,A | 48.9±2.3a,B | 20.1±2.3a,C | 8.9±1.3a,D | 1.7±0.4a,E |
| 2 | 70.3±2.6a,A | 69.4±2.3b,A | 51.2±2.5a,B | 21.6±2.2a,C | 8.7±1.2a,D | 2.1±0.4a,E |
| 3 | 68.2±2.8a,A | 70.7±2.3b,A | 45.5±2.6a,B | 21.3±2.6a,C | 8.1±1.3a,b,D | 1.8±0.4a,E |
| 4 | 46.7±2.8b,A | 40.2±2.7c,B | 27.2±2.2b,C | 11.4±2.0b,D | 5.3±1.1b,E | 0.1±0.1b,F |
Within a row, means with different superscript letters differ significantly (P<0.05).
See Tables 1 and 2 for remainder of key.
Mean ± SEM percentage of live cells at various time points during a 14-day culture period after exposure for 5 minutes at each treatment temperature.
| Day | 37°C | 45°C | 50°C | 55°C | 60°C | 65°C |
|---|---|---|---|---|---|---|
| 0 | 75.7±2.4a,A | 70.7±2.5a,A,B | 67.8±3.3a,B | 36.0±2.9a,C | 3.9±0.7a,D | 1.5±0.4a,D |
| 1 | 44.4±3.5b,A | 55.5±4.2b,B | 41.1±2.8b,A | 14.5±2.2b,C | 2.8±0.5a,D | 0.3±0.2a,D |
| 3 | 59.4±2.7c,A | 63.7±2.5a,A | 38.9±2.8b,B | 14.6±2.0b,C | 9.7±1.3b,C | 3.5±0.7b,D |
| 7 | 65.2±2.8c,A | 51.3±2.7b,B | 44.5±2.4b,C | 21.5±3.1c,D | 14.8±2.2c,E | 0.9±0.4a,F |
| 14 | 65.1±3.6c,A | 55.6±2.9b,B | 38.9±2.3b,C | 7±1.7d,D | 8.3±1.4b.D | 1.4±0.4A.e |
Day 0 = Day of 5-minute exposure to temperature treatment and initial day of culture.
See Tables 1 and 3 for remainder of key.
Interestingly, when chondrocyte viability was evaluated over time in culture, the results revealed that the percentage of live cells increased at 37°C from days 3 to 14, 45°C at day 3, 55°C at day 7, 60°C from days 3 to 14, and 65°C at day 3 (Table 4).
Discussion
Analysis of the results of the study reported here revealed an important relationship between increases in temperature and chondrocyte death. Temperatures of 50° to 55°C appear to be the critical temperature range that caused chondrocyte death after exposure for 5 minutes. In addition, this study revealed that there was some recovery of cell viability over time for cells exposed at the lower temperatures evaluated. However, after exposure to temperatures of 50°C or higher for 5 minutes, there was minimal recovery of cell viability after thermal insult.
The critical temperature range identified in the study reported here is supported by results of other studies25–28 in which investigators evaluated thermal injury in osteoblasts and chondrocytes. In another study,29 researchers used flow cytometry to evaluate thermal damage to nasal septal cartilage and determined chondrocyte viability of 28% after exposure to 56°C for as little as 15 seconds and cell viability of 3% after exposure to 65°C for 3 seconds. The response of chondrocytes exposed to 45°, 50°, or 55°C for exposure times ranging from 30 seconds to 3 minutes was evaluated, and cell viability was determined by use of methylthiotetrazole (3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyltetrazolium bromide; thiazolyl blue), which is dependent on mitochondrial dehydrogenases.27 In that study,27 cell viability was not affected by treatment at 45°C and the response to treatment at 50°C was unclear. However, the acute response of osteoarthritic cartilage to treatment at 55°C was a decrease in viability of 10% at 30 seconds, a decrease of viability of 40% at 1 minute, and a decrease in viability of 50% at 3 minutes. After 1 week in culture, chondrocyte viability after 30 seconds of thermal exposure at 55°C was 68% and after 1 minute of thermal exposure at 55°C was 52%.27 By use of another assay to determine chondrocyte viability in that study, investigators determined results similar to the results for chondrocyte viability identified in the study reported here. In addition, at the times and temperature combinations tested in that study,27 there was no recovery of viability during the 7-day culture period. The increased viability was most likely secondary to shorter treatment times (3 minutes, compared with the 5 minutes for our study). Therefore, we believe that these results further validate the use of the green fluorescent–ethidium homodimer staining system for determination of chondrocyte viability in osteochondral sections.
An additional confounding factor among stud-4,26,27,29 is the thickness of cartilage sections used. Sections were a thickness of 200 to 800 μm in 1 study29 and 2 mm in another study,27 whereas the sections were a thickness of 1.5 mm in the study reported here. Thicker sections will result in uneven heating and the potential for areas that do not reach the desired temperature when they are not heated for a sufficient amount of time. It is unclear from the results reported in 1 study27 whether those investigators were able to determine the location of the viable cells; furthermore, it is not possible to use flow cytometry with thicker sections, as was the case in another study.29 In addition, investigators in 2 studies26,27 reported the metabolic rate as a percentage of the control sample, and as they acknowledged, this can be accounted for by increased metabolic activity of a few chondrocytes.
Metabolic activity of chondrocytes appears to be more sensitive to thermal insult than is viability of chondrocytes. Protein and proteoglycan synthesis have been evaluated by use of 3H-serine and 35S-sulfate pulse labeling, respectively. Temperatures as low as 45°C significantly reduced proteoglycan synthesis at treatment times > 1 minute.27 Proteoglycan production was reduced by > 90% after treatment at 55°C for 1 and 3 minutes, respectively, when synthesis was evaluated immediately after treatment. Neither protein nor proteoglycan synthesis provided any evidence of recovery after 7 days of culture.
In a subsequent study,26 investigators evaluated the metabolic response of cartilage treated at 50°, 53°, 55°, and 60°C. Proteoglycan synthesis was suppressed by 40% for treatment at 55°C for 5 seconds and > 90% for treatment at 60°C for 5 seconds. After 7 days of culture, proteoglycan synthesis recovered to 90% of control values for the treatment temperature of 55°C and 60% for the treatment temperature of 60°C. Treatment times > 5 seconds reduced proteoglycan synthesis at 7 days from 50% to > 90%, depending on the time and temperature combination. Finally, treatment times of 30 seconds or longer did not result in a significant change in proteoglycan or protein production at any time point. As the authors of that study26 stated, additional experiments need to be conducted to determine which cells are producing the protein and proteoglycan that is measured in these analyses. In situ labeling of osteochondral sections would allow correlation of chondrocyte viability and metabolic activity measured through protein or proteoglycan production.23
One of the reasons that RFE quickly gained acceptance for use in chondroplasty was the ability to rapidly contour the articular surface with minimal removal of adjacent normal articular cartilage. The probes currently used clinically reach temperatures that range from 70° to > 100°C.30,31 Although these devices can rapidly and accurately contour the surface, concern has arisen as to the effect these devices have on chondrocyte viability. On the basis of the study reported here and other studies, there is a critical temperature and exposure time that will result in irreversible injury to chondrocytes. The temperature required to induce morphologic changes to normal and chondromalacic cartilage has been investigated.4 Microscopically, morphologic changes to 250-μm sections of articular cartilage separated from the subchondral bone were evident in chondromalacic cartilage treated at 57°C and normal articular cartilage treated at 61°C. It may be difficult to equate the temperatures that resulted in morphologic changes in the study reported here directly to clinical arthroscopy because of confounding factors, such as the cooling effect of the arthroscopy irrigation fluid32 and accuracy for determination of the temperature of the cartilage surface. However, when surgeons use RFE for thermal chondroplasty, they should remember that exposure of cartilage to temperatures higher than 55°C for 5 minutes may result in irreversible injury to chondrocytes.
Several suggestions should be kept in mind when comtemplating the use of RFE for thermal chondroplasty. First, clinicians should understand the basic principles of RFE-based technology (monopolar or bipolar RFE) and the capability of RFE for fine excision of tissues and potential for excessive heating when removal of large flaps or fronds is contemplated. Second, on the basis of these principles, large flaps and fibrillated materials should first be removed by use of a mechanical shaver or grasps, which would then by followed by use of RFE to fine-tune the treatment area. Third, the clinicians should make as few passes as possible during treatment. Fourth, clinicians should adhere to the manufacturer's recommendations with regard to whether the RFE probe should be in light contact (monopolar RFE) or noncontact (most bipolar RFEs). Finally, clinicians should keep the activated RFE devices moving and never let them remain in 1 location.
Matrix properties, chondrocyte morphology, and the metabolic activity of chondrocytes vary on the basis of their distance (depth) from the surface of the articular cartilage.10,11,23,33–35 The location within the matrix affects cellular function, metabolic rate, and response to injury.10 Analysis of results reported here revealed that the location within articular cartilage affects chondrocyte susceptibility to thermal energy. Analysis of the percentage of live cells revealed a clear decrease in viability between the superficial layers and the deepest layer. In addition, the ratio of the percentage of live cells in the deepest layer, compared with the percentage of live cells in the most superficial layer for treatment temperatures that ranged from 37° to 65°C decreased from 0.71 to 0.06. We hypothesize that the high metabolic rates of the chondrocytes in the deepest layers may result in increased susceptibility to thermal injury; however, further investigation is required. Investigators in another study27 also reported that viability and metabolic activity were more susceptible to thermal insult in chondrocytes from osteoarthritic cartilage than in normal cartilage.
In several studies,19,32,36–40 our laboratory group has determined the amount of chondrocyte death, including penetration of the subchondral bone, by use of both monopolar and bipolar RFE. Other investigators41 questioned the validity of those studies, specifically viability testing at time 0 in comparison with viability at various time points after thermal exposure. Results of the study reported here support the fact that initial chondrocyte viability may overestimate cell death for treatments at temperatures below a certain temperature-time threshold, which is also supported by results reported26,27 by other investigators who used different techniques.
We elected to use a 5-minute incubation time to ensure that the entire osteochondral section was heated to the prescribed temperature. This is greater than the amount of time recommended for thermal chondroplasty. A study design that could investigate specific temperatures delivered to the cartilage matrix for the clinically appropriate period and still ensure that this temperature is reached within the matrix would be ideal; however, we have not yet determined a way to perform such a study to investigate a wide range of temperatures and times. The study reported here indicates that relatively low temperatures cause a significant death loss of chondrocytes and supports the results reported27–29 by other investigators who have determined the effect of time and temperature on chondrocyte death by use of other measures of viability and metabolic activity.
Cartilage exposed to thermal energy exceeding 50°C for 5 minutes results in the greatest decrease in chondrocyte viability, and exposure to temperatures of 55°C or higher results in minimal recovery of viability during a 14-day culture period. In addition, to limit chondrocyte death, differences among cartilage layers with regard to the response to heating need to be considered when developing tools that heat or ablate the cartilage matrix.
ABBREVIATIONS
| RFE | Radiofrequency energy |
Baker CL. Current use of radiofrequency in arthroscopy and sports medicine (oral presentation). 27th Annu Meet Am Orthop Soc Sports Med, Keystone, Colo, June 2001
Beuthanasia-D Special, Schering-Plough Animal Health Corp, Kenilworth, NJ
HL-1 cell culture media, Bio-Whittaker Inc, Walkersville, Md
Gentamicin sulfate, Fischer Scientific, Pittsburgh, Pa
Calcein-AM, Molecular Probes, Eugene, Ore.
Ethidium homodimer, Molecular Probes, Eugene, Ore.
MRC-1024, Bio-Rad Laboratories, Hemel Hempstead, UK.
522DF32, Bio-Rad Laboratories, Hercules, Calif.
585EFLP, Bio-Rad Laboratories, Hercules, Calif
Adobe PhotoShop, version 5.0.2, Adobe, San Jose, Calif
Proc GLM, version 8.02, SAS Institute Inc, Cary, NC.
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