Keywords
rivaroxaban - nadroparin calcium - Achilles tendon rupture - Achilles tendon healing
Introduction
Despite important developments in biological and biomechanical treatment modalities,
tendon healing remains a major orthopaedic challenge.[1] Achilles tendon rupture is one of the most common ruptures with an increasing incidence
and which often results in fibrotic scar tissue formation that has poor tissue quality
and inferior mechanical properties.[2]
[3] It has been reported that Achilles tendon injuries either fail to heal or demonstrate
a delay in healing due to complications, such as prolonged postoperative recovery
time, deep vein thrombosis, skin necrosis as well as infection around the surgical
site and re-rupture.[4]
[5]
[6] Several studies on Achilles tendon healing have investigated the efficiency of both
conservative and operative care, with each management strategy offering unique risks
and benefits.[7]
[8]
[9] The effects of stem cells,[10] and growth factors[11] platelet-rich plasma[12]
[13] on tendon healing, have been thoroughly studied over the past decades. However,
there remains insufficient evidence regarding which strategy is the most effective.
Venous thromboembolism is a severe condition that can occur as a result of musculoskeletal
system trauma and is a common source of postoperative morbidity and mortality, lest
appropriate steps are taken.[14] As with other orthopaedic injuries, Achilles tendon injury necessitates thromboembolism
prophylaxis after repair, or conservative treatment with long leg-cast immobilization.[14]
We believe that it is crucial to understand the effect of thromboembolism prophylaxis
on tendon healing, but there are a limited number of studies regarding the impact
of enoxaparin and rivaroxaban, which are widely used in thromboembolism prophylaxis
on tendon healing.[15]
[16]
[17] Among these, one study investigated the effect of rivaroxaban on tendon healing
with an unadjusted dosage of 3 mg/kg, whereas another focused on the effects of two
varying doses of nadroparin calcium on tendon healing: one dose was very close to
0.4 mL/daily with the other being 0.8 mL/daily. However, there are no studies investigating
the effect of rivaroxaban's (with an adjusted dose) on tendon healing as a novel oral
agent. This study aimed to investigate the effects of antithrombotic prophylactic
doses (adjusted doses) of nadroparin calcium and rivaroxaban on Achilles tendon healing
in a histopathological manner.
Materials and Methods
The present study was conducted at the Experimental Animal Breeding and Research Center
of Baskent University. The study was conducted between August 2013 and October 2013,
following the Declaration of Helsinki principles. This study's ethical approval was
obtained from the Ethics Committee for Animal Experiments of Baskent University (approval
number: DA13/36).
Experimental Design
Twenty-four young adult male Wistar Albino type rats of the same age, weighing 350
± 50 g, were randomly divided into three groups. All animals were housed in an environment
with 12-hour light and 12-hour dark cycles, 55% humidity, and 21 ± 2°C; they were
fed with standard feed. All rats underwent a full-thickness surgical incision of the
Achilles tendon, followed by primary repair (SA). After the procedure of the Achilles
tendon, group 1 was determined as the control group and received no medication. Group
2 received 2.03 mg/kg rivaroxaban equal to 0.6 mg rivaroxaban/daily (Xarelto, Bayer
HealthCare, Berlin, Germany) via gastric lavage once daily, for 28 days. The adjusted
dose of rivaroxaban for rats was calculated according to the study of Nair and Jacob.[18]
[19]
Group 3 was given subcutaneous 114 IU AXa nadroparin calcium (Fraxiparine, Glaxo SmithKline,
Canada) as low-molecular-weight heparin (LMWH), once daily for 28 days. The adjusted
dose of nadroparin calcium for the rats was calculated according to the study of Nair
and Jacob.[18]
[19] The rats were given free access to food and water as well as free movement within
their cages and the guidelines for the care and use of laboratory animals in biomedical
research were closely followed.[20]
Surgical Procedure
Before the surgery, 6 mg/kg xylazine and 70 mg/kg ketamine-hydrochloride were administered
for anesthesia by a researcher without clinical involvement. The right lower extremities
of the rats were wiped with povidone-iodine (Adeka İlaç ve Kimyasal Ürünler San. Ve
Tic. A.Ş) and shaved. An approximately-1.25 cm longitudinal incision was performed
under sterile conditions on the right Achilles tendon. The tendon was exposed and
cut transversely from approximately 0.5 cm proximal to the side of the right Achilles
tendon insertion site via the scalpel blade (no.10). The incised right Achilles tendon
was then repaired with the Kessler method using polydioxanone 4–0 (PDS II - Ethicon
Inc., Sommerville, United States), and the skin was sutured using 3–0 polyglactin
910 (Coated Vicryl - Ethicon Inc., Sommerville, United States) ([Fig. 1]).
Fig. 1 Calcaneus and gastrocnemius tips of Achilles tendon after partial tenotomy (A); primary repair of calcaneus and gastrocnemius tips of Achilles tendon after partial
tenotomy (B).
One week after the surgery, infection protection was applied twice a day by intraperitoneal
administration of 30 mg/kg cefazolin sodium (Iespor), and postoperative pain management
was performed by subcutaneous administration of 0.02 mg/kg fentanyl. Neither postoperative
immobilization nor postoperative exercise training was carried, and the wounds were
sterilely dressed for 5 days. The decision against the immobilization was based on
studies Murrell et al and Eliason et al. They reported that immobilization effects
negatively affected healing after Achilles tendon repair in a rat model.[21]
[22]
On the postoperative 29th day, euthanasia was performed through high-dose anesthesia
with 100 mg/kg thiopental sodium.
Histological Analysis
After euthanization, all Achilles tendons were resected proximal and distal ends of
the adhesion site via the scalpel blade (no.10) and sent for histopathological examination.
The Achilles tendon samples were fixed in 10% formaldehyde. The specimens were embedded
in paraffin, and sections of 5 mm thickness were prepared on slides. After deparaffinization,
the sections were stained with hematoxylin and eosin. The degrees of inflammation,
neovascularization, fibroblastic activity, and collagen fiber sequencing were examined
and scored for histopathological evaluation under a light microscope at 400 magnification.
The healing condition of the tendons was measured histopathologically by using Curtis
and Delee's staging method.[23]
The degree of inflammation and fibroblastic activity was rated as follows: none (0),
mild (1), moderate (2), and pronounced (3). Neovascularization was rated based on
the number of capillaries per high-powered field in a 0.45-micron diameter magnification
area. Less than five was rated as mild (1), 5 to 10 rated as moderate (2), and greater
than ten rated as pronounced (3). Collagen fiber sequencing was rated as follows:
scattered (1), slightly regular (2), and regular (3).
Healing tissue samples (5 mm diameter full-thickness biopsies from the Achilles tendon)
were also obtained for histologic evaluation. The number of inflammatory cells (excluding
histiocytes), capillary vessels, and fibroblasts were counted in three randomly selected
large 10 × magnification areas and were averaged. In each magnification area, counts
were performed within a total area of 0.0625 mm2, divided into 100 square areas. All histopathological analyses were performed by
a pathologist who was blinded to the study groups.
Statistical Analysis
The Statistical Package for Social Science (SPSS) version 21.0 for Windows software
(SPSS, Inc., Chicago, Illinois, United States) was used for all statistical analyses.
Data are shown as the mean ± standard deviation, median, minimum, and maximum values,
and interquartile range. Before statistical analysis, the Shapiro–Wilk test was used
to assess the distribution of the data. Levene's test analyzed homogeneity of variances
and the Kruskal-Wallis H test with a post hoc Dunn test was used to determine whether
there was a significant difference in the degree of inflammation, neovascularization,
fibroblastic activity, and collagen fiber sequencing between groups. The number of
inflammatory cells, capillary vessels, and fibroblasts, which met the parametric tests'
assumptions, were compared between three independent groups by one-way analysis of
variance and Tukey's honestly significant difference) test was used for multiple comparisons.
The significance level was set at p-value < 0.05.
Results
There were no perioperative complications during the surgical procedure. Following
rivaroxaban and nadroparin calcium administration, no early or late complications
were observed during the study, including systemic or local side effects. The degree
of inflammation, neovascularization, fibroblastic activity, and collagen fiber sequencing
is shown in [Table 1]. There was a statistically significant difference in the degree of inflammation
and collagen fiber sequencing among the groups. The degree of inflammation was higher
in group 1 compared than in groups 2 and 3 (p = 0.02 and p = 0.02, respectively). Otherwise, the score of collagen fiber sequencing was lower
in group 1 compared than in groups 2 and 3 (p = 0.04 and p = 0.001, respectively) ([Table 1]).
Table 1
Histological results of the study groups
|
Group 1 (sham-control)
|
Group 2
|
Group 3
|
|
No medication
|
Group 2 received 2.03 mg/kg rivaroxaban
|
Group 3 was given subcutaneous 114 IU AXa nadroparin calcium
|
|
The degree of inflammation[a]
|
1
|
1
|
1
|
|
2
|
1
|
1
|
|
3
|
1
|
1
|
|
2
|
1
|
1
|
|
1
|
1
|
1
|
|
1
|
1
|
1
|
|
2
|
1
|
1
|
|
1
|
1
|
1
|
|
Interpretation of the results
|
Higher compared with group 2, group 3 ([b]
p= 0.02 and p = 0.02)
|
Lower compared with group 1 ([b]
p = 0.02)
|
Lower compared with group 1 ([b]
p = 0.02)
|
|
Neovascularization[c]
|
2
|
1
|
1
|
|
2
|
2
|
1
|
|
2
|
1
|
2
|
|
2
|
2
|
2
|
|
2
|
2
|
2
|
|
2
|
1
|
2
|
|
2
|
1
|
1
|
|
1
|
2
|
2
|
|
Interpretation of the results[c]—There was no significant difference in the capillary vessels among groups.
|
|
Fibroblastic activity[a]
|
1
|
2
|
1
|
|
2
|
1
|
1
|
|
2
|
1
|
2
|
|
2
|
2
|
2
|
|
2
|
2
|
1
|
|
2
|
1
|
1
|
|
1
|
1
|
1
|
|
1
|
1
|
1
|
|
Interpretation of the results
|
Higher in group 1 compared with groups 2 and ([b]
p = 0.01 and p = 0.003, respectively)
|
Lower in group 2 compared with group 1 ([b]
p = 0.01)
|
Lower in group 3 compared with group 1 ([b]
p = 0.003)
|
|
Collagen fiber sequencing[d]
|
1
|
2
|
2
|
|
1
|
2
|
2
|
|
1
|
2
|
2
|
|
1
|
2
|
2
|
|
1
|
2
|
3
|
|
1
|
2
|
3
|
|
2
|
2
|
3
|
|
2
|
2
|
3
|
|
Interpretation of the results
|
Lower in group 1 compared with group 2, group 3 ([b]
p = 0,04 and p = 0.001, respectively)
|
Higher in group 2 compared with group 1 ([b]
p = 0.04)
|
Higher in group 3 compared with group 1 ([b]
p = 0.001)
|
Note: The healing condition of the tendons was measured histopathologically, by using
Curtis and Delee's staging method.[21]
Group 1: No medication; Group 2: rivaroxaban; Group 3: nadroparin calcium.
Data are expressed as median, interquartile range.
a Rated as follows: none (0), mild (1), moderate (2), and pronounced (3).
b Kruskal-Wallis H test.
c Number of capillaries, which were less than five was rated as mild (1), 5 to 10 rated
as moderate (2), and greater than 10 as pronounced (3).
d Collagen fiber sequencing were rated as follows: Scattered (1), slightly regular
(2), and regular (3).
Histological examination of the group 1 sample showed the presence of inflammatory
cells, an increase in the number of fibroblasts, and sequencing of collagen fibers
scattered ([Fig. 2A]). The presence of inflammatory cells, remarkable increases in the number of fibroblasts,
the presence of mature collagen fibers, and regular sequencing of collagen fibers
were shown in groups 2 and 3 ([Figs. 2B] and [C] ).
Fig. 2 Tissue samples with hematoxylin and eosin staining × 400 under light microscopy at
× 10 magnification. Group 1 received no medication (A); Group 2 received rivaroxaban (B); Group 3 received nadroparin calcium (C).
The findings obtained in accordance with the histological analysis are summarized
in [Table 2]). There was no significant difference in the capillary vessels among the groups
(p = 0.11). Conversely, there were statistically significant differences between the
groups regarding the number of inflammatory cells and fibroblasts (p = 0.01 and p = 0.002, respectively). In group 2, the number of inflammatory cells was lower than
groups 1 and 3 (p = 0.01 and p = 0.01, respectively). Elsewhere, the number of fibroblasts was higher in group 1
compared than in groups 2 and 3 (p = 0.01 and p = 0.003, respectively) ([Table 2]).
Table 2
Histological analysis of the number of inflammatory cells, vascular tissues, and fibroblasts
between groups
|
Group 1
|
Group 2
|
Group 3
|
One-way ANOVA
|
|
Mean ± SD
|
Mean ± SD
|
Mean ± SD
|
F
|
p-Value[a]
|
Tukey's HSD
|
|
(95% CI)
|
(95% CI)
|
(95% CI)
|
Group
|
p-Value[b]
|
|
Inflammatory cells
|
9.50 ± 9.06
|
2.37 ± 1.18
|
5.50 ± 2.67
|
3.35
|
0.01
|
1–2
|
0.01
|
|
(2.38–12.83)
|
(1.62–3.25)
|
(3.75–7.12)
|
1–3
|
0.45
|
|
|
|
2–3
|
0.01
|
|
Capillary vessels
|
7.88 ± 2.84
|
9.35 ± 3.24
|
6.38 ± 1.93
|
2.35
|
0.11
|
1–2
|
0.54
|
|
(6.25–9.83)
|
(7.51–11.48)
|
(5.06–7.60)
|
1–3
|
0.52
|
|
|
|
2–3
|
0.10
|
|
Fibroblasts
|
116.32 ± 24.20
|
84.61 ± 21.95
|
78.47 ± 12.47
|
8.09
|
0.002
|
1–2
|
0.01
|
|
(101.58–133.17)
|
(70.17–97.38)
|
(71.10–86.95)
|
1–3
|
0.003
|
|
|
|
2–3
|
0.81
|
Note: Group 1: No medication; Group 2: rivaroxaban; Group 3: nadroparin calcium.
Data are expressed as mean standard deviation (SD) (95% confidence interval [CI]).
a One-way analysis of variance (one-way ANOVA); significance level set at < 0.05.
b Tukey's honestly significant difference (HSD) test; significance level set at < 0.05.
Discussion
This study analyzed the histopathological effect of antithrombotic prophylactic-adjusted
doses of enoxaparin and rivaroxaban on Achilles tendon healing. The literature includes
a few clinical and experimental studies about antithrombotic agents' application and
its histological effect on tendon healing.[15]
[16]
[17]
Our findings revealed that antithrombotic agent administration with adjusted doses
decreased the degree of inflammation and increased regular collagen fiber sequencing
in rats after tendon repair. Furthermore, the density of inflammatory cells was lower
in rats who received rivaroxaban for 28 days than in rats who received nadroparin
calcium for 28 days. However, the density of fibroblasts was higher in rats who received
no medication at all, compared with the other groups.
There has been recent evidence that inflammation modulation at the early stages of
tendon reparation may lead to improved healing.[24]
However, in our experiment, we performed histopathological examination in the late
phase of tendon healing. Therefore, the evidence of inflammation and fibroblasts should
be interpreted as unregulated inflammation.
As discussed, and revealed in the past literature, controlled inflammation is largely
beneficial to tissue recovery, whereas excessive inflammation, or persistence, can
be detrimental.[25]
[26]
Although inflammatory cytokines draw fibroblasts to the repair site, excessive inflammation
may lead to poor clinical outcomes.[25]
[26]
Another question should be asked by readers why an experimental period of 28 days
has been chosen. Because, from prior investigations in rats, it is known that the
main changes in tendon healing occur within the first 2 weeks; therefore, this study
was designed with an observation period of 4 weeks, possibly limiting the significance
of the further progression of biomechanical properties.[27]
Another point of view regarding the time interval has been reported by Akamatsu et
al. They postulated that a period of 28 days is suggested as a period needed for collagen
recovery of the injured tendon.[28]
Tendon healing comprises three main phases. In the past, growth factors and cytokines
that have positive effects and that are involved in various stages have been identified
in these three main phases.[20] The positive effects of growth factors have also been demonstrated in animal experiments
and aside from these,[29]
[30] experimental studies have highlighted that cytokines from autologous conditioned
serum and cartilage-derived morphogenetic proteins contribute significantly to tendon
healing.[31]
[32]
In a study on the above-mentioned growth factors and cytokines, a cancer treatment
drug named bevacizumab, developed against vascular endothelial growth factor (VEGF),
has been reported to improve tendon healing in an experimental study as opposed to
the general philosophy.[33] However, if VEGF is a chemical that contributes to tendon healing, the antibody
developed against it would be expected to have an adverse effect.
In another study, Müller et al investigated the effect of a group of growth factors
(a validated combination of basic fibroblast growth factor, bone morphogenetic protein-12,
and transforming growth factor-β1) in the presence and absence of the paratenon layer.
They stressed that growth factors would only improve tendon healing in the presence
of the paratenon layer.[11]
Antithrombotic nadroparin and rivaroxaban are among the main medications we use of
antithrombotic prophylaxis of lower extremity trauma or orthopaedic surgery involving
the lower extremity.[34]
[35] Therefore, these agents' positive or negative contributions to Achilles tendon healing
are significant. In a previous study, Virchenko et al[36] established an experimental Achille's tendon rupture model in rats and focused on
the effect of thrombin on tendon healing.
Thrombin has remarkably similar molecular features as growth factors. The results
of this study revealed that thrombin had a positive effect on tendon healing. They
hypothesized that LMWH, which inhibits the activity and generation of thrombin, might
also inhibit tendon healing. They conducted an experimental study on rats that focused
on the dose-dependent effects of LMWH on tendon healing and concluded that LMWH slows
tendon repair if given continuously.[17] However, if injected twice daily, LMWH had no effect on tendon healing, presumably
because the antifactor Xa activity between injections returns to normal, allowing
sufficient thrombin stimulation for repair.
Following Virchenko et al's study, two other studies focused on the effect of antithrombotics,
one of them solely focused on various doses of nadroparin's effect, and the other
one focused on nadroparin's and rivaroxaban's effect.[15]
[16] Esen et al conducted an experimental study on rats and compared the effects of two
different daily doses of LMWH injections (nadroparin calcium) on tendon healing and
evaluated the results histologically as well as biomechanically.[16] All rats underwent full-thickness surgical incisions of the Achilles tendon, followed
by primary repair. After the operation, two groups received daily subcutaneous LMWH
injections (nadroparin calcium) for 4 weeks at high or low doses (group 1, 6 mg/kg,
170 IU AXa; group 2, 3 mg/kg, 85 IU AXa), and group 3 remained untreated as the control
group. The results showed that group 1, receiving 170 IU AXa daily, had substantially
higher overall failure and elongation values than groups 2 and 3. Histologically,
both groups 1 and 2 showed superior results when compared with the control group.[16] These findings also support our theory regarding the positive impact of nadroparin
calcium. In addition to this study, we found that rivaroxaban—human dosage 0.33 mg/mg/kg
divided by 0.162 Km and found animal equivalent dose with a dosage of 2.03 mg/kg also positively impacted
the tendon healing process. Nair and Jacob.[18]
[19] wrote a review article that provides basic information about the translation of
doses between species and estimation of starting dose for experimental studies, using
allometric scaling of US Food and Drug Administration (FDA). The animal equivalent
dose can also be calculated based on body surface area by either dividing or multiplying
the human dose (mg/kg) by the Km. In our study, we chose to apply 114 IU nadroparin because we adjusted the dose according
to the Nair and Jacob[18] study and FDA scale.[19] On the other hand, we suggest that the dosage of 170 IU/daily is more than the routine
practice.[18]
Eren et al conducted an experimental study on rats and investigated the effects of
nadroparin and rivaroxaban on tendon healing.[15] All rats underwent full-thickness surgical incision of the Achilles tendon followed
by primary repair. After the operation, their group 1 received daily subcutaneous
nadroparin calcium for 3 weeks at high doses (group 1, 170 IU AXa). Their group 2
received 3 mg/kg rivaroxaban daily for 21 days through gastric lavage, while group
3 remained untreated, as the control group. This study revealed that nadroparin and
rivaroxaban had positive histologic effects on tendon healing in a rat model, although
the same outcomes were not obtained in biomechanical assessments. In our study, the
effects of the same antithrombotic on tendon healing were investigated; however, we
administered the antithrombotic for 1 week longer than Eren et al's study.[15] One day in a rat's life was approximately equivalent to 34.8 human days; therefore,
an additional 1-week more follow-up (244 human days [8 months]) would reflect more
long-term follow-up data in which it will be more advantageous for research that seeks
the effect of some chemicals given systematically on the long-acting tendon healing
process. Their study roughly contained similar features as our work, but the following
details make a critical difference: first, in our experiment, adjuvants nadroparin
and rivaroxaban were administered to the subjects for 28 days rather than 21 days.
The difference in administration time makes an important distinction, since as we
pointed out, 1 week in the rat subjects' life corresponds to 8 months in human life,
which is equivalent to the 8 months postoperative follow-up time. This important difference
might make a significant difference in monitoring tendon healing. Another significant
distinction is that rivaroxaban and nadroparin were diluted in various proportions:
adjusted doses were calculated according to the allometric scale that provides basic
information about the translation of doses between species and an estimation of the
starting dose for experimental animal studies, which also corresponds to dosage suitable
for their daily use. A regular dosage of nadroparin calcium (114 IU) and rivaroxaban
(2.03 mg/kg) was applied in our research and led to a histologically better outcome
than the control group.
The strength of our study is the use of adjusted doses of rivaroxaban and nadroparin
calcium; however, its limitations should also be highlighted. First, Achilles tendon
ruptures in humans usually occur in the final event during the degeneration process.
In our study, we performed iatrogenic cuts on healthy rat tendons and repaired them;
therefore, the findings should be interpreted with caution. Second, our study had
a relatively small sample size; here again, the findings should be interpreted with
caution. Finally, the short-term or long-term biomechanical results of these agents
on tendon healing were not investigated. One of the weak points of this study is the
lack of biomechanical examination.
On the other hand, in this animal experiment, the artificially formed Achilles tendon
rupture was stitched with 4–0 (PDS II), suture material and the Kessler suture method
were used, so it should not be forgotten that there are two additional factors that
may affect the results of the experiment in testing the biomechanical strength of
the tendon healing. These are the suture holding capacity of suture material and suture
technique.
Therefore, according to us, it might be possible that biomechanical testing in research
aimed at testing the effect of some adjuvants on tendon healing would has been biased
by suture material and technique.
Conclusion
Both rivaroxaban and nadroparin calcium in their daily adjusted dosage have a beneficial
effect on rats' Achilles tendon healing, and thus, its use as an antithrombotic agent
will also contribute positively to the tendon healing process after Achilles tendon
repair. Further studies with larger sample sizes are needed to analyze the effects
of rivaroxaban and nadroparin calcium on the healing process after Achilles tendon
repair.
