Temporal Effect of In Vivo Tendon Fatigue Loading on the Apoptotic Response Explained in the Context of Number of Fatigue Loading Cycles and Initial Damage Parameters Nelly Andarawis-Puri,1 Anaya Philip,1 Damien Laudier,1 Mitchell B. Schaffler,1,2 Evan L. Flatow1 1 Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, New York, 2Department of Biomedical Engineering, City College of New York, New York, New York
Received 16 October 2013; accepted 4 April 0214 Published online 16 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22639
ABSTRACT: Accumulation of damage is a leading factor in the development of tendinopathy. Apoptosis has been implicated in tendinopathy, but the biological mechanisms responsible for initiation and progression of these injuries are poorly understood. We assessed the relationship between initial induced damage and apoptotic activity 3 and 7 days after fatigue loading. We hypothesized that greater apoptotic activity (i) will be associated with greater induced damage and higher number of fatigue loading cycles, and (ii) will be higher at 7 than at 3 days after loading. Left patellar tendons were fatigue loaded for either 100 or 7,200 cycles. Diagnostic tests were applied before and after fatigue loading to determine the effect of fatigue loading on hysteresis, elongation, and loading and unloading stiffness (damage parameters). Cleaved Caspase-3 staining was used to identify and calculate the percent apoptosis in the patellar tendon. While no difference in apoptotic activity occurred between the 100 and 7,200 cycle groups, greater apoptotic activity was associated with greater induced damage. Apoptotic activity was higher at 7 than 3 days after loading. We expect that the decreasing number of healthy cells that can repair the induced damage in the tendon predispose it to further injury. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1097–1103, 2014. Keywords: damage accumulation; tendinopathy; apoptosis; cluster analysis; tendon fatigue
Tendinopathies are common and debilitating musculoskeletal injuries. Degenerative changes in ruptured tendons and macroscopically “healthy” tendons support an etiology wherein accumulation of sub-rupture fatigue damage ultimately leads to tendon rupture.1,2 The mechanisms responsible for initiation and progression of tendinopathies remain largely unknown, but the pace of accumulation of further degeneration outpaces the rate of repair, creating an environment that is conducive for further damage accumulation.3 Understanding the biological mechanisms associated with early onset of tendinopathy and further accumulation of damage is integral to development of effective therapeutic interventions. We previously developed a rat model of in vivo subrupture fatigue damage accumulation to investigate the early onset of tendinopathy.4 This model allows for precise control of the load and number of cycles directly applied to the tendon and is therefore conducive for investigating the progressive relationship between loading and damage. Since biological variability among animals can result in different levels of induced damage across tendons in response to the same loading, we previously identified damage parameters, such as hysteresis, stiffness of the loading and unloading load-displacement curves, and elongation, as effective initial indices of damage that can be used to analyze the effect of fatigue loading in the context of not only load levels and number of cycles, but also the initial damage induced.5 We found that inducing Conflict of interest: None. Grant sponsor: AR052743 (ELF) from NIAMS/NIH. Correspondence to: Nelly Andarawis-Puri (T: 212-241-1625; F: 212-876-3168; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
moderate level fatigue damage resulted in a 20% stiffness loss that was not recovered out to at least 6 weeks.6 Interestingly, we found that damaged tendons mount an anabolic molecular response that progressively shuts down with increasing damage severity.7 We expect that the diminished ability of the tendon to repair the induced damage may be attributable to an altered cell response to excessive damage and a decrease in the number of cells that can respond. While apoptosis has been implicated in tendinopathy, the biological mechanisms responsible for the initiation and progression of injury remain poorly understood.8–10 Therefore, we assessed the 3- and 7-day apoptotic activity in the context of initial induced damage and the number of loading cycles. We hypothesized that greater apoptotic activity (i) will be associated with greater induced damage and higher number of fatigue loading cycles at both time points, and (ii) will be higher at 7 than at 3 days after loading.
METHODS Following IACUC approval, left patellar tendons (PT) of anesthetized (isoflurane, 2–3% by volume, 0.4 L/min) adult female retired breeder Sprague-Dawley rats (n ¼ 28) (Charles River Laboratories, Ltd., Wilmington, MA) were surgically exposed and setup per our previously described protocols for fatigue loading.4,5 Briefly, under aseptic conditions, the tibia was fixed with a clamp, securing the limb at 30˚ knee flexion. The patella was clamped and connected in series to a 50-lb load cell and actuator of a servo-hydraulic loading system, allowing loading of the PT without damaging the tendon from clamping. Fatigue loading was applied by cycling between 1 and 40N for either 100 (n ¼ 13) or 7,200 (n ¼ 15) cycles at 1 Hz. Additional rats (n ¼ 6) were used as controls. Diagnostic tests (1–15N) were applied before (diag1: 420 cycles) and after (diag2: 120 cycles) fatigue loading. JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2014
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Hysteresis, stiffness of the loading and unloading loaddisplacement curves, and actuator position were calculated for the last 10 cycles of the pre-fatigue diag1 and the postfatigue diag2. The change in these parameters between diag1 and diag2 reflects the effect of fatigue loading and is referred to as the damage parameters.5 Previous studies showed no differences in initial parameters between diag2 and a third diagnostic that was applied 45 min after loading, suggesting that most of changes between diag1 and diag2 are nonrecoverable and can serve as indicators of the induced damage.5 Rats were euthanized 3 (n ¼ 6 and 8 for the 100 cycle and 7,200 cycle groups, respectively) or 7 (n ¼ 7 per cycle group) days after loading. The quadriceps-patella-PT-tibia complex was harvested and fixed in zinc buffered formalin under 2N tension. Blocks were decalcified and embedded in paraffin, and 5 mm sagittal sections were cut. Antigen retrieval in deparaffinized sections was achieved using DeCal solution (BioGenex Inc., Biogenex, Freemont, CA). Endogenous peroxidase activity was quenched using 3% H2O2. Non-specific binding was blocked with Dako Protein block. Immunohistochemical staining for cleaved Caspase-3 (Cell Signaling Technologies; diluted 1:1,000) was used to identify apoptotic cells. Incubation in rabbit serum without primary antibody was used as the negative staining control. Sections were counterstained with methylene blue to highlight negative cells. Multiple sister sections were visually compared to confirm the expected similarity. One of the sections was then used for all subsequent blinded quantitative analysis. Under 400X magnification, normal and apoptotic cells were counted at the insertion (tibial end), origin (patellar end), and midsubstance. A region at the insertion and the origin was defined for analysis by drawing an object that consisted of one side that outlined the border between the tendon and fibrocartilage (line 1), two sides at each end of line 1 that were perpendicular to line 1 with both ending at the bursal end of the tendon (lines 2 and 3), and a final line that traced the surface of the tendon and connected lines 2 and 3. The tendon length was measured, and the midpoint
was defined. Images throughout the full thickness of the tendon were captured at the midpoint to define the midsubstance region. For each region, the combined number of apoptotic cells and total cells was used to calculate the percent apoptotic and the apoptotic, alive, and total cell densities (cells/mm2). The repeatability of two trained graders was confirmed through three trials on a subset of five images. Control tendon analyses were averaged from both graders to minimize inter-observer variability. Relationship Between Number of Initial Loading Cycles and 3- or 7-Day Apoptotic Activity ANOVAs with post-hoc Bonferroni were used to compare apoptotic activity between 7,200-cycle, 100-cycle, and control tendons. The relationship between the number of loading cycles and apoptotic activity over time was evaluated with ANOVAs with post-hoc Bonferroni that compared the 3-day, 7-day, and control tendons for each loading group. To provide further context for the apoptotic activity, ANOVAs were also conducted to compare the number of alive, apoptotic, total cells/mm2 between loading groups and control tendons (Table 1). However, for simplicity, all further analyses were conducted with respect to percent apoptotic activity. Relationship Between Initial Damage Induced and 3- or 7-Day Apoptotic Activity The relationship between the induced damage and apoptotic activity, irrespective of the number of cycles, was evaluated. Linear relationships between each initial damage parameter and apoptotic activity at 3- or 7-days were evaluated with Pearson correlations. Non-linear relationships were identified using k-means cluster analysis, constraining the number of clusters to 2, since 2 outcomes were expected (low or high amount of damage).7 If the analysis yielded a cluster with
Received 16 October 2013; accepted 4 April 0214 Published online 16 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22639
ABSTRACT: Accumulation of damage is a leading factor in the development of tendinopathy. Apoptosis has been implicated in tendinopathy, but the biological mechanisms responsible for initiation and progression of these injuries are poorly understood. We assessed the relationship between initial induced damage and apoptotic activity 3 and 7 days after fatigue loading. We hypothesized that greater apoptotic activity (i) will be associated with greater induced damage and higher number of fatigue loading cycles, and (ii) will be higher at 7 than at 3 days after loading. Left patellar tendons were fatigue loaded for either 100 or 7,200 cycles. Diagnostic tests were applied before and after fatigue loading to determine the effect of fatigue loading on hysteresis, elongation, and loading and unloading stiffness (damage parameters). Cleaved Caspase-3 staining was used to identify and calculate the percent apoptosis in the patellar tendon. While no difference in apoptotic activity occurred between the 100 and 7,200 cycle groups, greater apoptotic activity was associated with greater induced damage. Apoptotic activity was higher at 7 than 3 days after loading. We expect that the decreasing number of healthy cells that can repair the induced damage in the tendon predispose it to further injury. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1097–1103, 2014. Keywords: damage accumulation; tendinopathy; apoptosis; cluster analysis; tendon fatigue
Tendinopathies are common and debilitating musculoskeletal injuries. Degenerative changes in ruptured tendons and macroscopically “healthy” tendons support an etiology wherein accumulation of sub-rupture fatigue damage ultimately leads to tendon rupture.1,2 The mechanisms responsible for initiation and progression of tendinopathies remain largely unknown, but the pace of accumulation of further degeneration outpaces the rate of repair, creating an environment that is conducive for further damage accumulation.3 Understanding the biological mechanisms associated with early onset of tendinopathy and further accumulation of damage is integral to development of effective therapeutic interventions. We previously developed a rat model of in vivo subrupture fatigue damage accumulation to investigate the early onset of tendinopathy.4 This model allows for precise control of the load and number of cycles directly applied to the tendon and is therefore conducive for investigating the progressive relationship between loading and damage. Since biological variability among animals can result in different levels of induced damage across tendons in response to the same loading, we previously identified damage parameters, such as hysteresis, stiffness of the loading and unloading load-displacement curves, and elongation, as effective initial indices of damage that can be used to analyze the effect of fatigue loading in the context of not only load levels and number of cycles, but also the initial damage induced.5 We found that inducing Conflict of interest: None. Grant sponsor: AR052743 (ELF) from NIAMS/NIH. Correspondence to: Nelly Andarawis-Puri (T: 212-241-1625; F: 212-876-3168; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
moderate level fatigue damage resulted in a 20% stiffness loss that was not recovered out to at least 6 weeks.6 Interestingly, we found that damaged tendons mount an anabolic molecular response that progressively shuts down with increasing damage severity.7 We expect that the diminished ability of the tendon to repair the induced damage may be attributable to an altered cell response to excessive damage and a decrease in the number of cells that can respond. While apoptosis has been implicated in tendinopathy, the biological mechanisms responsible for the initiation and progression of injury remain poorly understood.8–10 Therefore, we assessed the 3- and 7-day apoptotic activity in the context of initial induced damage and the number of loading cycles. We hypothesized that greater apoptotic activity (i) will be associated with greater induced damage and higher number of fatigue loading cycles at both time points, and (ii) will be higher at 7 than at 3 days after loading.
METHODS Following IACUC approval, left patellar tendons (PT) of anesthetized (isoflurane, 2–3% by volume, 0.4 L/min) adult female retired breeder Sprague-Dawley rats (n ¼ 28) (Charles River Laboratories, Ltd., Wilmington, MA) were surgically exposed and setup per our previously described protocols for fatigue loading.4,5 Briefly, under aseptic conditions, the tibia was fixed with a clamp, securing the limb at 30˚ knee flexion. The patella was clamped and connected in series to a 50-lb load cell and actuator of a servo-hydraulic loading system, allowing loading of the PT without damaging the tendon from clamping. Fatigue loading was applied by cycling between 1 and 40N for either 100 (n ¼ 13) or 7,200 (n ¼ 15) cycles at 1 Hz. Additional rats (n ¼ 6) were used as controls. Diagnostic tests (1–15N) were applied before (diag1: 420 cycles) and after (diag2: 120 cycles) fatigue loading. JOURNAL OF ORTHOPAEDIC RESEARCH SEPTEMBER 2014
1097
1098
ANDARAWIS-PURI ET AL.
Hysteresis, stiffness of the loading and unloading loaddisplacement curves, and actuator position were calculated for the last 10 cycles of the pre-fatigue diag1 and the postfatigue diag2. The change in these parameters between diag1 and diag2 reflects the effect of fatigue loading and is referred to as the damage parameters.5 Previous studies showed no differences in initial parameters between diag2 and a third diagnostic that was applied 45 min after loading, suggesting that most of changes between diag1 and diag2 are nonrecoverable and can serve as indicators of the induced damage.5 Rats were euthanized 3 (n ¼ 6 and 8 for the 100 cycle and 7,200 cycle groups, respectively) or 7 (n ¼ 7 per cycle group) days after loading. The quadriceps-patella-PT-tibia complex was harvested and fixed in zinc buffered formalin under 2N tension. Blocks were decalcified and embedded in paraffin, and 5 mm sagittal sections were cut. Antigen retrieval in deparaffinized sections was achieved using DeCal solution (BioGenex Inc., Biogenex, Freemont, CA). Endogenous peroxidase activity was quenched using 3% H2O2. Non-specific binding was blocked with Dako Protein block. Immunohistochemical staining for cleaved Caspase-3 (Cell Signaling Technologies; diluted 1:1,000) was used to identify apoptotic cells. Incubation in rabbit serum without primary antibody was used as the negative staining control. Sections were counterstained with methylene blue to highlight negative cells. Multiple sister sections were visually compared to confirm the expected similarity. One of the sections was then used for all subsequent blinded quantitative analysis. Under 400X magnification, normal and apoptotic cells were counted at the insertion (tibial end), origin (patellar end), and midsubstance. A region at the insertion and the origin was defined for analysis by drawing an object that consisted of one side that outlined the border between the tendon and fibrocartilage (line 1), two sides at each end of line 1 that were perpendicular to line 1 with both ending at the bursal end of the tendon (lines 2 and 3), and a final line that traced the surface of the tendon and connected lines 2 and 3. The tendon length was measured, and the midpoint
was defined. Images throughout the full thickness of the tendon were captured at the midpoint to define the midsubstance region. For each region, the combined number of apoptotic cells and total cells was used to calculate the percent apoptotic and the apoptotic, alive, and total cell densities (cells/mm2). The repeatability of two trained graders was confirmed through three trials on a subset of five images. Control tendon analyses were averaged from both graders to minimize inter-observer variability. Relationship Between Number of Initial Loading Cycles and 3- or 7-Day Apoptotic Activity ANOVAs with post-hoc Bonferroni were used to compare apoptotic activity between 7,200-cycle, 100-cycle, and control tendons. The relationship between the number of loading cycles and apoptotic activity over time was evaluated with ANOVAs with post-hoc Bonferroni that compared the 3-day, 7-day, and control tendons for each loading group. To provide further context for the apoptotic activity, ANOVAs were also conducted to compare the number of alive, apoptotic, total cells/mm2 between loading groups and control tendons (Table 1). However, for simplicity, all further analyses were conducted with respect to percent apoptotic activity. Relationship Between Initial Damage Induced and 3- or 7-Day Apoptotic Activity The relationship between the induced damage and apoptotic activity, irrespective of the number of cycles, was evaluated. Linear relationships between each initial damage parameter and apoptotic activity at 3- or 7-days were evaluated with Pearson correlations. Non-linear relationships were identified using k-means cluster analysis, constraining the number of clusters to 2, since 2 outcomes were expected (low or high amount of damage).7 If the analysis yielded a cluster with
