Stochastic amplitude-modulated stretching of rabbit flexor digitorum profundus tendons reduces stiffness compared to cyclic loading but does not affect tenocyte metabolism
© Steiner et al.; licensee BioMed Central Ltd. 2012
Received: 13 February 2012
Accepted: 8 November 2012
Published: 14 November 2012
It has been demonstrated that frequency modulation of loading influences cellular response and metabolism in 3D tissues such as cartilage, bone and intervertebral disc. However, the mechano-sensitivity of cells in linear tissues such as tendons or ligaments might be more sensitive to changes in strain amplitude than frequency. Here, we hypothesized that tenocytes in situ are mechano-responsive to random amplitude modulation of strain.
We compared stochastic amplitude-modulated versus sinusoidal cyclic stretching. Rabbit tendon were kept in tissue-culture medium for twelve days and were loaded for 1h/day for six of the total twelve culture days. The tendons were randomly subjected to one of three different loading regimes: i) stochastic (2 – 7% random strain amplitudes), ii) cyclic_RMS (2–4.42% strain) and iii) cyclic_high (2 - 7% strain), all at 1 Hz and for 3,600 cycles, and one unloaded control.
At the end of the culture period, the stiffness of the “stochastic” group was significantly lower than that of the cyclic_RMS and cyclic_high groups (both, p < 0.0001). Gene expression of eleven anabolic, catabolic and inflammatory genes revealed no significant differences between the loading groups.
We conclude that, despite an equivalent metabolic response, stochastically stretched tendons suffer most likely from increased mechanical microdamage, relative to cyclically loaded ones, which is relevant for tendon regeneration therapies in clinical practice.
KeywordsTendon Tensile stiffness Stochastic amplitude-modulation Strain control Proteoglycan production Gene expression Cell activity
Tendinopathy is the term used to describe the pathological conditions resulting from tendon overuse [1, 2]. The morbidity of tendon injuries, especially in sports and in manual occupations, is relatively high in our society [3, 4]. Chronic tendon injuries are often associated with forceful or repetitive loading, which leads to the accumulation of micro-tears [2, 5]. The relationship between repetitive mechanical loading and tenocyte metabolism has been previously investigated in several in vitro studies to investigate the influence of frequency, amplitude and time on the biochemical and biological response . Recently, the biomechanical response of tenocytes was modeled under a variety of physiologically relevant frequency-modulated loading regimes [7–9]. Several studies demonstrate the regulation of MMP through the interaction of mechanical loading [6, 10, 11].
Thus, the mechano-biological response for linearly-oriented, viscoelastic tissues loaded with frequency modulation has been relatively well studied. However, from a patient’s perspective, stochastic loading may be a much more relevant scenario, since it mimics the random, physiological motions experienced in daily activities. Previous applied loading regimes found in the literature are based on a regular cyclic loading applied at different frequencies with different magnitudes [6, 10–13]. Smooth and regular amplitudes do not reflect the situation in vivo. This has been demonstrated in in vivo gate analysis in rabbit, a common model selected for tendon studies, which revealed that the frequency in “relaxed” hopping is approximately 1Hz but  variable. Another study used the rabbit flexor digitorum profundus model for flexor tendon tissue engineering, where the authors found bioreactor cyclic strain increases construct strength . Thus, this rabbit tendon has been successfully evaluated for a model system for the study of tendon mechano-biology multiple times in the literature [5, 14].
Tendon source and tissue harvest
Two hind paws of eight six-month old female rabbits (Oryctolagus cuniculus) were obtained from a local butcher within 24 h post mortem. First the hair of the hind paws was shaved and then the skin was aseptically cut and removed. After a general surface disinfection step with 1% betadine B solution (Mundipharma, Basel, Switzerland), the flexor digitorum profundus tendons (6 tendons per animal) were aseptically isolated by dissecting the muscles and immediately placed in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Invitrogen, Basel, Switzerland) with 10% penicillin/streptomycin (1 mg/mL, Sigma) for 30 min at 37°C. Then, the specimens were washed with phosphate buffered saline (PBS) and randomly assigned to the three specified loading regimes and an unloaded control group, which was maintained in static culture conditions. The tendons were then cultured in high-glucose DMEM containing 5 μg/mL amphotericin B (Sigma) and 100 μg/mL penicillin/streptomycin containing 10% Fetal Calf Serum (FCS) at 37°C, 5% CO2 and 100% humidity. Media changes were performed every two days.
Tendon stretching protocols
This resulted in a loading regime comprising 3600 stretching cycles between 2–4.4% strain. The third group (“cyclic_high”) provided a comparison to a loading regime comprising sinusoidal stretching between the same maximal strain peaks of 2-7% strain included in the stochastic loading regime.
A pre-load of 2.5 N was applied to define a consistent zero strain point. The recorded output parameters were time, displacement and force response of the specimen under strain control. The data were analyzed using a custom analysis script in Matlab (Mathworks inc., MA, USA). For each of the 3,600 loading cycles, the stiffness was calculated by a linear regression of the linear portion of the loading curve. To exclude background noise from the load cell, the data was filtered and cycles with forces < 3N were omitted. Tendons that ruptured during the experiments were re-clamped (thus, shortened) and the same loading protocol was applied.
A predefined mid-section of the tendon was used for biochemical analysis. Half of the tissue was used to assess gene expression and the other half served for the measurement of cell viability, matrix production and the DNA/GAG assay. A day 0 control was taken after the unloaded equilibration phase and processed similarly.
RNA Extraction and Real-Time RT-PCR
Primers used for RT-PCR
Reference gene 18S ribosomal RNA
AGT GCG GGT CAT AAG CTT GC
GGT GTG TAC AAA GGG CAG GG
GAG GTC GTG GTG AAA GGT GT
GTG TGG ATG GGG TAC CTG AC
Collagen type 1
TTC TTG GTG CTC CTG GCA TTC
GCA ATC CGT TGT GTC CCT TTA TG
Collagen type 2
GAC CCC ATG CAG TAC ATG CG
CCA GTA GTC ACC GCT CTT CC
ATA CCT GGA AAA CTA CTA CA ATC TG
TCT TCA GGG TT TCA GCA TCT
AGC CAA TGG AAA TGA AAA CTC TTC
CCA GTG GAT AGG CTG AGC AAA
TGC CCC TCC TCA ACA GTA AC
GAG CCC GCT GCA TTC TTC TT
Tissue inhibitor of metalloproteinase 1
AGC AGA GCC TGC ACC TGT GT
CCA CAA ACT TGG CCC TGA TG
Tissue inhibitor of metalloproteinase 3
TCT GCA ACT CCG ACA TCG TG
CGG ATG CAG GCG TAG TGT T
A disintegrin and metalloproteinase with thrombospondin motifs 4
GAC CTT CCG TGA AGA GCA GTG T
CCT GGC AGG TGA GTT TGC AT
Tumor necrosis factor alpha
CAG CCT CTT CTC TTT CCT GCT
CCG ATC ACC CTG AAG TGC
TCC AGA CGA GGG CAT CCA
CTG CCG GAA GCT CTT GTT G
CTG GTG GTG GCT ACC GCT TT
ATG GTC TCC AGG ATG CTC CG
CAA CCT TCC TGC TGT CTC TG
GGT CCA CTC TCA ATC ACT CT
DNA and GAG Quantification
The tendon samples were digested in 1 mL proteinase K solution for 16 h at 56°C and 300 rpm to assess both the DNA and the GAG content of the tendon. For DNA analysis, samples were stained with Hoechst dye and the fluorescent emission was measured at 457 nm with an excitation wavelength of 368 nm (Tecan Reader Infinite 200; Tecan, Männedorf, Switzerland). To measure the GAG content, the 1,9-dimethylmethylene blue (DMMB) assay, adjusted for low pH, was performed as described in Enobakhare  and Farndale  and absorbance was read at 600 nm (SpectraMax 190, Molecular Devices, Sunnyvale, California, United States, distributed by Bucher inc., Switzerland). Since the DNA content is constant per cell (~7 pg), this parameter was used to normalize both matrix production and cell activity.
Alamar blue© cell activity test
To assess tenocyte viability after the 12-day tissue culture period, an Alamar Blue© test (Invitrogen) was performed, where the tissue was allowed to react for 2 h at 37°C and the absorbance at 570nm was measured using an absorbance reader (Tecan).
Stiffness was analyzed using two-way ANOVA, with culture time and loading regime as the two independent factors. The gene expression, GAG/DNA and the Alamar Blue data were analyzed with non-parametric Kruskal-Wallis test using GraphPad Prism v. 6.0a, GraphPad Software, San Diego California USA, http://http//:www.graphpad.com. A p-value < 0.05 was considered significant.
Generally, proliferation and cell activity, i.e. DNA content and Alamar blue assay, both confirmed that the tenocytes were metabolically active and alive. The "cyclic_high" group showed a slight decrease of DNA content, whereas the tendons in the other groups showed similar cell activity, but no significant difference could be found. The different tendons showed a high variance, not only the specimens from different animals, but also tendons from the same rabbit.
Cell activity (Figure 5B), as measured from Alamar blue assay, was not significantly different between all groups.
Mechanical properties of randomly amplitude-modulated tendons
The primary goal of this study was to test the importance of amplitude modulation for the mechanical stimulation of linearly-oriented tissues. We found significant differences in tensile stiffness between the stochastically loaded and the cyclic, sinusoidal loaded tendons (with equivalent RMS amplitude) in the first two days of loading (Figure 4). Generally, the stiffness of tendons of the stochastically stretched group was reduced, compared to the cyclically loaded tendons (Figure 4). We cannot explain this difference in stiffness strictly by biological changes, such as cell viability or activity of tenocytes (Figures 5 and 6), since we did not see any significant changes in cell viability, activity or matrix production. Furthermore, it is unlikely that metabolic changes would immediately result in observable matrix degradation. Thus, the differences are probably purely mechanical, by microfracture of collagen fibers. This should be investigated in future experiments using histological analysis at the μm scale or by SEM.
Biological response of tenocytes
Relative to the day 0 control, all three groups of tenocytes responded with a minor down-regulation of ACAN and collagen type 1 (Figure 6). However, tenocytes of the stochastic loading regime tended to down-regulate ACAN, collagen type I, ADAMTS4 and MMP13 relative to the cyclic_RMS and cyclic_high group. An increase in collagen I with cyclic loading was also found by Wang et al. and Parkinson 2010 et al.  observed that there is a net proteoglycan content increase in injured tendons, due to an altered metabolism rather than due to changes in gene expression levels. However, there was no difference between the loading groups and the unloaded control in the present experiments, which is also true for the up-regulation of other genes. It should be mentioned that the measured gene expression is possibly a mixture of tenocytes and progenitor cells due to the relatively young age of the rabbits.
Culture time was certainly a limit of the study; it is possible that changes to the extracellular matrix (ECM) cannot be seen with a culture period of only twelve days. On the one hand, any changes in gene expression should be still detectable, since RNA changes can be found within hours upon mechanical loading . The timing of the culture start (here allowing an equilibration period of 3 days) will most likely have a detrimental influence on the mRNA transcript level, not so for col 1, but definitely for MMP3 and MMP13; these transcript levels have been shown to increase over time in an explant model of rat tail tendon fascicles . With respect to tissue homeostasis, we did not find any significant differences among the three loading regimes. On the other hand, it may also be that the sampling window for gene expression was delayed and thus, no changes in RNA could be detected after the stimuli. However, it has been reported that changes in mRNA persist after 24h incubation time [23, 25]. The time point of harvest after the loading regime still seems to be critical, there have been significant changes found if tissue is analyzed after 1h or longer time periods
An up-regulation of the pro-inflammatory genes that could lead to apoptosis was not evident at the RNA level in our study. Further investigation by histology or scanning electron microscopy would allow inferring definitive conclusions on the microstructure of the tendons. During the first 1–2 days of mechanical loading, 8 out of 40 tendons experienced partial ruptures and had to be re-clamped (3 in the stochastic; 2 in the cyclic_RMS and 3 in the cyclic_high group). Thus, the re-clamping of the tendons might have had an influence on the stiffness results, but may also indicate the accumulation of micro-damage. Improved clamping techniques may allow a more unbiased comparison of the clamped versus unclamped regions .
The cell density of the tendon is relatively low compared to other musculoskeletal tissues. This also includes the vascular cells and synovial cells of the tendon sheath that encloses each tendon . The tissue is sparsely vascularized and the main constituent is collagen type 1  Collagen is the main component of most organic matrices like bones, ligaments, tendons and the intervertebral disc . A remarkable 60-85% of a tendons dry weight is assigned to type I collagen. A small, mechanically important portion (2%) is elastin and 4-5% are different proteins. The extracellular substance is dominated by proteoglycans (PG) and, in combination with water, they are thought to have a spacing and lubricating role for tendon [27–29]. The mechano-biological response might be masked by the generally very rich culture media, which has an abundance of growth factors, high glucose content and vitamins. Results from the matrix production at the protein level should also be reflected by the gene expression data. For all 11 genes studied, there were no statistical and biologically significant changes amongst the loading groups. These results are consistent with studies in human achilles tendon, where no changes in the expression for genes of the major collagens and proteoglycans could be found . The same study also did not see any change for ADAMTS-4, MMP3, MMP13 and TIMP3 with the exception of the up-regulation of TIMP1. The authors hypothesize that the matrix turnover is favored for degeneration rather than matrix generation. However, another limitation of this study is that we did not look at tenocyte specific transcription factors such as scleraxis, which have been shown to respond to mechanical stimulation, especially with increased cyclic compression [30–32] nor did we look at tenomodulin and tenascin-C , two marker genes, which are important for maintaining tenocyte phenotype [34, 35]. For MMP1 and MMP3, it was found that cyclic mechanical loading inhibits their expression [6, 36]. It is generally accepted that training promotes both synthesis and degeneration and the process is highly dynamic . It is important to state that by analysis of only RNA expression-levels, conclusions on protein expression are limited. Translation efficiency, post-translational modification and -activation, protein turnover rates or inhibitory proteins that may have a large influence on how much protein is actually synthesized. MMP could be present in the tissue as pro-MMP, and thus in an inactive form, or they might be bound to TIMPs. An up-regulation of a MMP does therefore not necessarily mean matrix degeneration . Due to these potential effects it would be crucial to also include quantification on the protein level to support real-time PCR data if longer loading / culture times will be chosen in further experiments.
Stochastic modulation of amplitude in strain-controlled stretching of tendons resulted in a reduced tendon stiffness, compared to sinusoidal, cyclic loading regimes, with equivalent RMS amplitude, or sinusoidal, cyclic loading between the same peak strain magnitudes. The change in stiffness was not associated with changes in cell activity, cell density (DNA) or GAG content.
Ladina Ettinger assisted with laboratory assays. The project was supported by the Orthopaedic Department, Insel University Hospital, Bern, Switzerland.
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