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Department of Orthopedics, Balgrist University Hospital, Zurich, SwitzerlandDepartment of Orthopedic and Trauma Surgery, Cantonal Hospital Lucerne, Lucerne, Switzerland
Suture cut-through is a common cause of rotator cuff repair failure.
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Coating the suture with collagen cross-linker enhances strength of suture-tendon interface.
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Short-term in vitro culturing reduces tenocyte viability near suture.
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No difference in cell viability between treatment groups at 3 mm + from suture.
Abstract
Background
The suture-tendon interface often constitutes the point of failure in tendon suture repair. In the present study, we investigated the mechanical benefit of coating the suture with a cross-linking agent to strengthen the nearby tissue after suture placement in human tendons and we assessed the biological implications regarding tendon cell survival in-vitro.
Methods
Freshly harvested human biceps long head tendons were randomly allocated to control (n = 17) or intervention (n = 19) group. According to the assigned group, either an untreated or a genipin-coated suture was inserted into the tendon. 24 h after suturing, mechanical testing composed of cyclic and ramp-to-failure loading was performed. Additionally, 11 freshly harvested tendons were used for short-term in vitro cell viability assessment in response to genipin-loaded suture placement. These specimens were analyzed in a paired-sample setting as stained histological sections using combined fluorescent/light microscopy.
Findings
Tendons stitched with a genipin-coated suture sustained higher forces to failure. Cyclic and ultimate displacement of the tendon-suture construct remained unaltered by the local tissue crosslinking. Tissue crosslinking resulted in significant cytotoxicity in the direct vicinity of the suture (<3 mm). At larger distances from the suture, however, no difference in cell viability between the test and the control group was discernable.
Interpretation
The repair strength of a tendon-suture construct can be augmented by loading the suture with genipin. At this mechanically relevant dosage, crosslinking-induced cell death is confined to a radius of <3 mm from the suture in the short-term in-vitro setting. These promising results warrant further examination in-vivo.
Rotator cuff tears are common in the older population and can result in pain and disability, requiring surgical repair in which the torn tendon is reattached (
Retear rates after arthroscopic single-row, double-row, and suture bridge rotator cuff repair at a minimum of 1 year of imaging follow-up: a systematic review.
Arthrosc. J. Arthrosc. Relat. Surg.2015; 31: 2274-2281
Tissue engineering to enhance rotator cuff healing and reduce failure in arthroscopic shoulder surgery is an active field of research. Collagen-based scaffolds have shown improved histologic appearance but no increase in biomechanical strength in animal studies (
With tendon microstructure being a central contributor to its mechanical behavior, exogenous collagen cross-linking has shown potential in augmenting tendon resistance to various mechanical challenges. Genipin, a naturally occurring compound derived from Gardenia jasminoides, has been identified as inducing the lowest levels of cytotoxicity among substances that augment tendon strength. Genipin treatment successfully increased tendon strength following degenerative (
). Despite exhibiting a favorable outcome regarding cytotoxicity, genipin nevertheless induces concentration- and time-dependent cell death, indicating that mechanical augmentation of the tendon is inevitably associated with a certain degree of biological damage (
). Therefore, the demand for optimized local delivery strategies emerged. In an attempt to minimize exposure, genipin-coated sutures were developed with promising results for suture retention in healthy as well as in chemically degenerated animal tendons (
). In contrast to other approaches, the investigated method of collagen crosslinking induced by genipin-loaded surgical sutures adds minimal technical complexity to the surgical procedure. The current study aimed to assess (1) the potential mechanical benefit and (2) the biological implications regarding cytotoxicity of this approach in human shoulder tendons.
2. Methods
The study was approved by the responsible ethics committee (KEK-Zurich, project number: PB_2016–02665).
2.1 Tendon harvesting
We collected the intraarticular part of the long head of the biceps (LHB) tendon in patients who underwent total shoulder arthroplasty or procedures with a biceps tenodesis in our shoulder department (Department of Orthopedics, Balgrist University Hospital, Zurich, Switzerland). Both interventions require the sacrifice of the LHB tendon. For the biomechanical experiment, tendons were wrapped in gauze soaked with phosphate-buffered saline and frozen at −20 °C until testing. For the cytotoxicity experiment, immediately after harvesting tendons were incubated in Dulbecco's Modified Eagle's Medium (DMEM) and testing was commenced within the following hours.
2.2 Donor demographics and tendon sample descriptive statistics
Thirty-six tendon explants stemming from the proximal long head of the biceps tendon of 25 patients (median age 65 years [range, 51–82], 13 female, 12 male) were successfully assigned to testing (n = 17) or control group (n = 19). The tendon diameter was not different between the two groups (p = 0.785). Patient demographics and tendon baseline parameters are presented in Table 1.
Table 1Donor demographic data of samples used for mechanical testing.
Control group
Intervention group
n
17
19
age
66 (54–78, sd: 7.8)
64.5 (51–82, sd: 8.1)
sex (f/m)
8/9
13/6
Tendon diameter [mm]
3.6 (1.6–8.6, sd: 3.2)
4 (1.9–8.3, sd: 3.0)
Data are presented in median (range, standard deviation) or n. Note that in some instances tissue from one donor yielded two test samples and the reported patient frequencies do not coincide with the patient demographics reported in the text.
Of the 11 samples allocated to cell viability testing, two sample pairs were excluded from analysis due to low cell viability irrespective of the treatment group, and hence 9 sample pairs remained for the analysis. Median donor age was 52 (34–74) years and two donors were of female sex.
2.3 Suture coating
Coating formulation for sutures was prepared as described earlier (
). Dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) and acetone (50:50 v/v) were mixed as a cosolvent solution. As a second component genipin (500 mg/mL) (Challenge Bioproducts Co, Ltd., Taiwan, Republic of China) was added. For the preparation of the coating solution acid end-capped poly(lactic-co-glycolic acid) (Akina, Inc., West Lafayette, IN, USA) with a lactic acid: glycolic acid ratio of 50:50 and polyethylene glycol (Mn 400) was dissolved in the genipin-cosolvent solution at 1% and 35%, respectively. Sutures from the control group were coated with the analogous coating solution without genipin. A polyethylene suture (ORTHOCORD® USP #2; DePuy Synthes, New Brunswick, NJ, USA) with a needle attached was soaked in the coating solution for 10 min and subsequently vacuum-dried for 4 h to allow complete evaporation of the solvent.
2.4 Mechanical testing of suture pullout
Based on the estimated effect size of a previously performed study (
) we calculated the required sample size per group to be 17 samples to detect a significant difference with a probability of 80%. To account for the potential for procedural complications we allocated 20 tendon explants to each group. Four samples were excluded from analysis due to the tendon slipping at the clamping side (2 samples), data loss (1 sample), and sample damage during mounting (1 sample).
To assure comparable treatment groups the tendons were stratified by donor age and sex before randomization into either group. According to the allocated group, either a control-coated or a genipin-coated suture was inserted 1 cm from the tendon edge through its midsection. All suture placements were performed by the same researcher. The two suture ends were tied with a figure-of-eight knot and glued with cyanoacrylate adhesive. After suturing, each specimen was wrapped with a PBS-soaked cloth and stored at room temperature for 24 h after which mechanical testing was performed.
For an optimal fixation of the tendons, a PBS-soaked piece of cloth was glued to one end of the tendon using a cyanoacrylate adhesive and fixed in clamps. The suture loop was then connected to a 1-kN load cell (GTM Gassmann Theiss Messtechnik GmbH, Bickenbach, Germany) of a materials testing machine (Zwick 010; Zwick GmbH, Ulm, Germany). The mechanical testing protocol was comprised of a repeated cyclic test and a ramp-to-failure test. Specimens were preloaded at 5 N and then 20 cycles at a linear travel profile with axial tension force from 5 to 10 N were performed. After completion of this repeated cyclic testing, all samples underwent a ramp-to-failure test with a force increment of 1 N per second until the suture was completely pulled out (
). Force (N) and displacement (mm) were recorded using the corresponding software (Test Expert® 10; Zwick-Roell, Ulm, Germany). Maximum pull-out force was defined as the maximum force achieved during ramp-to-failure testing. Work to failure constitutes the energy absorbed by the construct from the end of cyclic testing until construct failure. Ultimate displacement was defined as the respective displacement at maximum force. Cyclic displacement was computed by averaging displacements at the lower force limits overall test cycles.
2.5 In-vitro cytotoxicity
Eleven tendon samples were used in a paired-sample design to assess local cytotoxicity of the treatment of the study. The tendon explants were cut to a length of 4 cm from the bony attachment side and cleaned from any soft tissue other than the tendon. The sample was cut again in half across its long axis and a genipin- and a control-coated suture was inserted each in one of the halves in an identical manner as the samples used for mechanical testing. The samples were then incubated in DMEM for 24 h. The medium was changed at 30 min intervals during the first 3 h followed by ∼7-h intervals for the remaining 21 h. After that, the samples were submerged in DMEM containing 1 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) for 3 h for live cell staining (blue/black precipitate) (
Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests.
). Tissues were washed with PBS, suspended in Tissue-Tek® O.C.T Compound (VWR), and frozen in liquid nitrogen. Histology samples were prepared following standard procedures (
). In short, cryosections were obtained by using a cryostat (Leica CM3050S) by cutting the samples along the tendon fibers, across the inserted suture into 10 μm slices, and placing them on glass slides. Samples were stored at −80 °C until further use.
2.6 Microscopy
To visualize cells (both alive and dead) sections were treated with a 5 μM solution of DAPI blue fluorescent nucleic acid stain (Thermo Fischer Scientific Inc., USA). Cells that were alive at the time of MTT staining are visible in absorption mode (in dark color), whereas cells (live and dead) are visible in the fluorescence channel (excitation wavelength: 405 nm). Three slices of each treatment sample were analyzed. The entire section was imaged using tile scanning and stitched together (10× magnification, Confocal A1 HD25, Nikon, Japan). Cells were first located in the fluorescence channel. An initial binary mask was created with a static threshold. Densely packed cell clusters were resolved into separate regions by erosion-dilation and watershed transformation. Cell viability was then determined based on the MTT signal strength in the absorption mode image at the corresponding locations that were identified in the fluorescent channel image using the DAPI staining (Fig. 1) (
Fig. 1A: Cross-sectional slides of the tendon samples were examined in absorption and fluorescence mode (Scale bar: 1 mm). B: Cells (Live or dead) were detected in the fluorescent channel with DAPI staining (marked by green circles) (Scale bar: 5 μm). C: Live cells were identified based on light absorption (marked by pink circles) due to MTT staining (Scale bar: 5 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Due to non-normality of the mechanical data. Group comparisons of the mechanical performance were analyzed using nonparametric Mann-Whitney U testing. The short-term cytotoxicity of the treatment under study was analyzed by inspecting the proportion of live cells as a function of distance from the suture (discretized into 9 bins) concerning the assigned testing group in a mixed-effects linear model (Diagonal covariance structure; Restricted maximum likelihood estimation). The analysis was performed in SPSS (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.) and R (
). Where not specified otherwise, data are reported in median and range. P-values smaller than 0.05 were considered statistically significant.
3. Results
3.1 Mechanical testing
In human biceps tendons treated with a genipin-coated suture maximum pullout force was greater with corresponding increases in absorbed energy. Cyclic and ultimate displacement of the tendon-suture construct was not significantly altered by genipin-coated suture treatment (Table 2).
Table 2Mechanical properties of the tendon-suture construct of both treatment groups.
Mechanical Characteristics
Control group (N = 17)
Intervention group (N = 19)
p-value*
Ultimate force [N]
15.7 (6.6, 77.9)
24.3 (12.1, 95.9)
0.032
Ultimate displacement [mm]
6.4 (1.5, 13.6)
8.9 (2.9, 7.7)
0.103
Ultimate stress [N/mm]
5.2 (2.5, 9.8)
6.1 (3.1, 19.3)
0.079
Ultimate energy [mJ]
102.7 (17.0, 688.5)
191.3 (83.3, 1054.9)
0.013
Cyclic displacement [mm]
2.2 (1.2, 7.6)
1.7 (1.1, 5.9)
0.570
Data are presented as median (range). *p-values are based on a Mann-Whitney U test.
In both treatment arms, cell viability was relatively low. Visual inspection of the cell viability as a function of the distance from the suture indicates the presence of a toxicity gradient in the genipin-treated samples, causing the cell viability to increase with distance from the suture, but not in the control samples (Fig. 2). Statistical modeling revealed a significant between-groups effect (p = 0.004) and a significant effect of the distance from the suture (p = 0.017) on cell viability.
Fig. 2Measured cell viabilities in the tendon samples as a function of suture distance for both groups (genipin-coated and untreated (control). While we found no gradient in cell viability as a function of the distance from the suture for control samples, genipin suture loading resulted in low cell viability in the proximity of the suture with an apparent absence of cytotoxicity further away. Line: Mean; Colored area around the mean: SD.
Cyclic loading of anchor-based rotator cuff repairs: confirmation of the tension overload phenomenon and comparison of suture anchor fixation with transosseous fixation.
). Degeneration of the collagen fibers in the tendon tissue may also lead to reduced suture retention strength in a repair. In an ex vivo sheep model of rotator cuff repair genipin markedly improved resistance to pullout at the tendon-suture interface for simple stitch patterns (
). With the recently introduced genipin-coated suture a simple stitch as it is used in most arthroscopically performed rotator cuff repairs was able to increase suture pullout strength in normal and chemically degenerated bovine tendon tissue by +60% (
). So far, no study has investigated the effect of genipin and more specifically genipin-coated sutures in human tendons. Since there exist interspecies differences in collagen properties, particularly collagen content and preexisting endogenous crosslinks (
). The current study corroborates these findings in a clinically more relevant model of human biceps tendon explants. We observed considerable improvement in repair ultimate strength of 55%. As reported previously (
), exogenous collagen cross-linking at mechanically relevant dosage inevitably comes with a cost of cytotoxicity. Loading the suture with the cross-linking agent as opposed to more diffusive modes of application aims to localize this chemical tissue alteration to the immediate vicinity of the suture and thereby limiting the induced biological damage. Indeed, at <3 mm distance from the suture, we observed low cell viability but above that, we found no notable additional degree of cell death compared to samples treated with control sutures. It, therefore, appears that this mode of application at the chosen dosage achieves a balance of mechanical augmentation at a limited biological effect. Toward clinical application of the treatment, the optimal dosage will have to be determined in more detailed dose-effect investigations.
) with an analysis of different suture types in living sheep after rotator cuff repair found a circumferential space around the sutures, forming an inner and outer capsule, separating the sutures from the surrounding tissue with a shift layer. In the same study suture pullout strength increased over time. This supports the fact that the initial stability of the suture-tendon interface can lead to better and faster tendon healing after repair despite a higher rate of cell death.
Compared to the recently presented study which showed a large increase of the suture pullout force by genipin-coated sutures (
) where the mechanical testing protocol reflected only one involuntary movement of the arm with an overload of the suture-tendon interface in early rehabilitation (
), we adapted the testing protocol for this study and added data of cyclic loading. This data showed no difference between the groups and indicates the primary benefit of genipin treatment to increase the suture-cut through resistance in the context of a single precipitating event leading to a potential re-tear rather than long-term tissue fatigue.
This study had several limitations. First, no assessment of the state of degeneration of the tendon explants was performed. However, a morphological and histochemical analysis of 55 patients (mean age 53 years [SD 10.5]) who underwent shoulder arthroscopy revealed advanced degeneration with disorganization of the longitudinal alignment of collagen fibers in all specimens (
Is the inflammation process absolutely absent in tendinopathy of the long head of the biceps tendon? Histopathologic study of the long head of the biceps tendon after arthroscopic treatment.
) tendinopathic changes to be present in most of our tendon samples. All collected tendons were inspected macroscopically and then randomly allocated into control and testing group. If there were two tendon parts collected from the same patients, they were allocated to different groups. However, the only objective criteria to assign the tendon to one group or another was the measurement of the diameter. In our analysis, there was no statistically significant difference found between the measured diameters between the two groups. Furthermore, we analyzed a single loop stitch, which is not the strongest repair option during arthroscopically managed biceps tendon refixation or rotator cuff repair. However, this type of repair stitch allows better inter-group comparability and comparability with earlier performed studies from our group where other tendon types were analyzed (
This study extends previous findings of increased tendon-suture pull-out strength with a genipin-loaded surgical suture to human biceps tendon tissue. The marked mechanical augmentation comes at the cost of cytotoxicity evident in a radius of 3 mm from the suture within the short term. Taken together, these results warrant investigation of the approach in the in vivo setting.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
We thank Barbara Niederöst and Rino Gantenbein (Institute for Biomechanics, ETH Zurich) for their help and contribution to the cell viability assessment.
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Cyclic loading of anchor-based rotator cuff repairs: confirmation of the tension overload phenomenon and comparison of suture anchor fixation with transosseous fixation.
Retear rates after arthroscopic single-row, double-row, and suture bridge rotator cuff repair at a minimum of 1 year of imaging follow-up: a systematic review.
Arthrosc. J. Arthrosc. Relat. Surg.2015; 31: 2274-2281
Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests.
Is the inflammation process absolutely absent in tendinopathy of the long head of the biceps tendon? Histopathologic study of the long head of the biceps tendon after arthroscopic treatment.
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