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Retendo (mucopolygen complex) effects on achille tendon healing
∗Corresponding author: Sezgin Bahadır Tekin. sezgintekin1988@hotmail.com
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Received: ,
Accepted: ,
This article was originally published by Reed Elsevier India Pvt. Ltd. and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Abstract
The injuries of achilles tendon which is among the most important tendons in the body include many problems. Limited tendon recovery is provided through the surgical procedures available. Re-rupture occurs during and after recovery and the long time it takes to return to work, impossibility of early movement of the affected ankle and not being able to provide a physiological recovery in tendon constitute a problem. Thus, our objective in this study was to demonstrate the recovery effects of Retendon (mucopolygen complex) on achilles tendon.
After taking the consent of the ethics board we applied for animal experiments, the experiment animals in our study were separated into two groups as those which were given Retendo® (Mucopolygen Complex - Mucopolysaccharides, Hydrolyzed Collagen Type I, Vitamin C) and the control group. These two groups were separated into four groups. In a total of eight groups, 40 female Wistar-Albino rats with an average weight of 200–350 gr. were included in the study.
The study had biomechanical and histological results. While there was no significant difference in vascularization, inflamation and fibroblastic level among the groups in histological terms, collagen formation was detected to be more regular in the experiment group than the control group. A significant efficiency couldn't be acquired in biomechanical terms.
Although Retendo® (mucopolygen complex) is commonly used in muscle and tendon diseases, its effect mechanism is not definitely known. But in the light of the findings we acquired in our study, we detected that Retendo® (mucopolygen complex) applied after achilles tendon ruptures positively effected collagen sequencing during recovery in histological terms but didn't have a significant effect on the mean load achilles tendon can tolerate before rupturing.
Keywords
Achille tendon
Retendo
Tendon healing
Rat
1 Introduction
Achilles tendon which is the strongest ligament of ankle joint posterior is among the most important biomechanical structures of human walking. Achilles tendon ruptures are more common in individuals between the ages of 25 and 40, especially in middle aged or older individuals who make sports.1,2 Quite a lot of studies were made on achilles tendon recovery until today. The treatment includes both conservative and surgical procedures. Untreated achilles tendon ruptures do not have good results.3 But limited tendon recovery is provided through the surgical procedures available. Re-rupture occurs during and after recovery and delays the patient's return to work. Also the mobilization support requirement during recovery causes a significant limitation in the continuation of the daily life activities of especially active and productive patients and most important of all, it also causes workforce loss. When all these factors combine, it is clearly understood that the starting of a stronger recovery, early tendon movement and physiological recovery caused the investigation of repair materials and alternative treatments other than available surgical procedures. The objective of our study was to investigate whether the preparation with the trade name Retendo® (ASSOS Pharmaceuticals, Bioiberica S.A., Spain) which includes mucopolysaccharide complexes, hydrolyzed type1 collagen and vitamin C and is commonly prescribed by sports doctors and orthopedists as it fastens tendon and ligament recovery is really efficient as this subject wasn't studied before.
2 Material-Method
This experimental study was planned as a biomechanical study. Since there is no study on this subject in literature, rat dose was determined by calculating the kg ratio according to the dose accepted for an average weight of 70 kg.
We applied for animal experiments, the experiment animals in our study were separated into two groups as those which were given Retendo® (Mucopolygen Complex - Mucopolysaccharides, Hydrolyzed Collagen Type I, Vitamin C) and the control group. These two groups were separated into four groups. In a total of eight groups, 40 female Wistar-Albino rats with an average weight of 200–350 gr. were included in the study. These rats were accepted as mature in terms of musculoskeletal structure. Rats were kept in eight separate cages during the experiment. Room temperature was kept constantly at 24°. Rats were fed with standard rodent feed. The cages were cleaned every day. They were also checked from time to time. The operators and staff used gloves, boxing shirts, masks and shoe covers during the applications.
2.1 Group 1 (0–7.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and at the same time, 2 × 1.5 mg oral Retendo® (mucopolygen complex) was started to be given to the rats with gastric lavage. The rats were sacrificed on the 7th day and right achilles tendon was excised.
2.2 Group 2 (0–7.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and no drug was started. The rats were sacrificed on the 7th day and right achilles tendon was excised.
2.3 Group 3 (0–14.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and at the same time, 2 × 1.5 mg oral Retendo® (mucopolygen complex) was started to be given to the rats with gastric lavage. The rats were sacrificed on the 14th day and right achilles tendon was excised.
2.4 Group 4 (0–14.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and no drug was started. The rats were sacrificed on the 14th day and right achilles tendon was excised.
2.5 Group 5 (0–21.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and at the same time, 2 × 1.5 mg oral Retendo® (mucopolygen complex) was started to be given to the rats with gastric lavage. The rats were sacrificed on the 21st day and right achilles tendon was excised.
2.6 Group 6 (0–21.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and no drug was started. The rats were sacrificed on the 21st day and right achilles tendon was excised.
2.7 Group 7 (0–28.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and also the rats were started to be given 2 × 1.5 mg Retendo® (mucopolygen complex) orally. The rats were sacrificed on the 28th day and right achilles tendon was excised.
2.8 Group 8 (0–28.days)
On the first day, right achilles tendons of the rats were cut from 0.5 cm proximal of calcaneus adhesion location. The cut achilles tendon was sutured the same day and no drug was started. The rats were sacrificed on the 28th day and right achilles tendon was excised.
2.9 Surgical technique
As surgical anesthetic, 50 mg/kg ketamine HCl and 5 mg/kg Xylazine Hcl were intraperitoneally applied together in the same injector. All subjects were operated by the same surgeon.
The hair of the rats on right cruris posterior was shaved. Aseptic surgical condition was provided and the lower right extremity was stained with betadine solution. 2 cm longitudinal standard incision was opened on the posterior and the subcutaneous tissues were cut.
Paratenon was opened after reaching achilles tendon and the tendon was exposed. Achilles tendon and plantaris tendons were separated from surrounding soft tissues. A full-layer transverse cut was formed from 0.5 cm proximal of calcaneus adhesion location on achilles tendon in all groups. Cut tendon ends were repaired from end to end with 4/0 PDS suture through Modified Kessler method. At the end of the surgery, a single dose of intraperitoneal 40 mg/kg cefazolin sodium was applied. Immobilization method was applied on the rats in postoperative period. The rats were followed up in metal cages at constant room temperature (23 °C) and under fluorescent light in postoperative period and were given water and standard rat feed. After surgery, it was ensured that all subjects could move freely in the cage.
2.10 Histological evaluation
Study groups (n = 20) and control group (n = 20) samples were kept in formalin solution and were delivered to KSU Faculty of Medicine Pathology Department. Vascularization, inflammation, fibroblastic level, collagen fiber arrangement and collagenization were evaluated in light microscope examination.
Histopathological changes were scored as 0,+,++ meaning no change, mild change and significant change respectively. Histopathological examination was performed by a single pathologist who wasn't informed on the groups before.
2.11 Biomechanical evaluation
Biomechanical tests were performed in the biomechanics laboratory in Kahramanmaraş Sütçü İmam University Textile department. ZWICK ROELL Z 5.0 universal test device was used for the test. 2 mm thick rubber anti-slip pads covered with sand paper were attached on the clamps with cyanoacrylate glue (303-A Bruno®) to prevent sliding and tendon damage. Tendons were fixed with clamps on both ends and compressed at 6 bar pressure. The clamps were arranged for the tensile test with a distance of 0.5 cm between them and so that they will split 0.1 mm per second. To provide equality of tendon lengths and parallelism of collagen fibrils 1 N standard stretching was performed before the test. Then the test was continued until the tendon ruptured. TestXpert 2 software was used for data analysis and stretching (change in length) against stress (applied force) and a maximum load was registered in Newton for each tendon (Figs. 1–3).
At the end of the first, second, third and fourth week, the rats were sacrificed under high dose ketamine anesthesia and achilles tendons were extracted with calcaneus in distal.
Since no biomechanical difference was formed between fresh frozen tendons and unstored tendons, all achilles tendons were kept at −23.0 °C at Kahramanmaraş Sütçü İmam University Faculty Microbiology Laboratory until they were subjected to biomechanical tests.4
Before starting the biomechanical tests, all achilles tendons were stored at room temperature to thaw. To prevent drying, achilles tendons were dampened with lactate ringer solution intermittently.4
Achilles tendons in the study and control groups were thawed and brought to the laboratory and they were put on the clamps of the test machine from both ends. Stretching at 0.1 mm/s was applied on the tendons. Rupturing forces of the tendons were registered in Newton
2.12 Statistical evaluation
Biomechanical characteristics of a total of 40 samples were evaluated and statistically analyzed. Mann-Whitney U test was used for comparison of the study groups since they didn't meet the parametric test conditions. “SPSS® 15.0 for Windows” (SPSS Inc. Chicago, IL, USA) statistics program was used for data analysis. p < 0.05 value was regarded as statistically significant (Table 1)
| Strength (Newton) | p | ||
| Group 1 (drug +)N = 20 | Group 2 (control)N = 20 | ||
| 1st week | 7.55 ± 1.71 | 18.24 ± 5.51 | 0.014 |
| 2nd week | 9.97 ± 4.03 | 8.25 ± 1.81 | 0.80 |
| 3rd week | 25.69 ± 9.02 | 20.62 ± 5.62 | 0.46 |
| 4th week | 28.78 ± 10.08 | 20.47 ± 15.84 | 0.32 |
3 Findings
3.1 Results of the histological evaluation
Study group (n = 20) and control group (n = 20) samples were grouped based on weeks and kept in formalin solution. They were evaluated for vascularization, inflammation, fibroblastic level and collagen fiber arrangement by a single pathologist using light microscopy in KSU Faculty of Medicine Pathology Department. While there was no significant difference in vascularization, inflammation and fibroblastic level among the groups, collagen formation was detected to be more regular in the experiment group than the control group.
3.2 Results of the biomechanical evaluation
Through biomechanical tests, the maximum forces reached at the rupturing moment of achilles tendons were acquired in Newton. In the study, mean and standard deviation values were calculated for the maximum forces reached in both experiment and control groups of eight different groups formed. Based on group weeks, achilles tendons of the experiment and control groups were statistically compared for the maximum forces they reached.
(7.55 ± 1.71) Newton was the mean of the maximum forces reached in achilles tendons of Group 1 rats. The achilles tendons of these rats were cut on the 0th day and these rats were sacrificed on the 7th day after oral Retendo® was given (mucopolygen complex). (18.24 ± 5.51) Newton was the mean of the maximum forces reached in achilles tendons of Group 2 rats. The achilles tendons of these rats were cut on the 0th day and these rats were sacrificed on the 7th day without giving any drugs. A statistically significant difference was detected among these two groups which were biomechanically compared (p < 0.05).
(9.97 ± 4.03) Newton was the mean of the maximum forces reached in achilles tendons of the rats in Group 3. The achilles tendons of these rats were cut on the 0th day and the rats were sacrificed on the 14th day after oral Retendo® was given (mucopolygen complex). (8.25 ± 1.81) Newton was the mean of the maximum forces reached in achilles tendons of Group 4 rats. The achilles tendons of these rats were cut on the 0th day and these rats were sacrificed on the 14th day without giving any drugs. A statistically significant difference wasn't detected among these two groups which were biomechanically compared (p > 0.05).
(25.69 ± 9.02) Newton was the mean of the maximum forces reached in achilles tendons of Group 5 rats. The achilles tendons of these rats were cut on the 0th day and these rats were sacrificed on the 21st day after oral Retendo® was given (mucopolygen complex). (20.62 ± 5.62) Newton was the mean of the maximum forces reached in achilles tendons of Group 6 rats. The achilles tendons of these rats were cut on the 0th day and these rats were sacrificed on the 21st day without giving any drugs. A statistically significant difference wasn't detected among these two groups which were biomechanically compared (p > 0.05).
(28.78 ± 10.08) Newton was the mean of the maximum forces reached in achilles tendons of Group 7 rats. The achilles tendons of these rats were cut on the 0th day and these rats were sacrificed on the 28th day after oral Retendo® was given (mucopolygen complex). (20.47 ± 15.84) Newton was the mean of the maximum forces reached in achilles tendons of Group 8 rats. The achilles tendons of these rats were cut on the 0th day and these rats were sacrificed on the 28th day without giving any drugs. A statistically significant difference wasn't detected among these two groups which were biomechanically compared (p > 0.05).
Drug given groups and the control groups were statistically compared among each other for the maximum forces they reached.
4 Discussion
Caused by sports or traumatic causes, tendon injuries are common conditions in daily life. Rehabilitation programs are started as soon as possible taking care not to damage the tendon stability acquired through repair. Early and slow loadings both positively effect the recovery phase and remove the adhesions which may limit joint movement range.5,6
Achilles tendon injuries treated through conservative or surgical technique may recur with re-ruptures at the late periods of recovery. Based on the studies on tendon repairs, re-ruptures in those with achilles tendons occur mostly in 4–12th weeks in humans.7 Many tendon studies show that controlled early movement following tendon repair fastened stretching force formation but also increased re-rupture incidence.8 There is a common recovery mechanism including tendon in all wound recoveries. First, fibrin formation occurs in wound area and blood cells, fibronectin and thrombocytes are confined inside this clot and they are demolished. Recovery phase starts with the effect of chemotactic factors released from demolished cells and the local growth factors reaching the environment.9 Apoptosis is a cellular death mechanism referred for the abduction of physiologically and pathologically unwanted, damaged or potentially neoplastic cells. Genetic control forms the basis of this mechanism. Many factors (such as heat, radiation, trauma, chemotherapeutic drugs and hypoxia) which may cause cell necrosis also cause apoptosis. In late phases of apoptosis, inflammatory cytokines released from these cells (such as insulin-like growth factor β) prevent inflammatory reaction.10,11 It is known that apoptosis occurs in tendon fibroblast cultures, animal models in which recurrent stretching were applied and also in chronic tendinopathy biopsy materials.12,13
In studies made with rat patellar tendon allografts, it was shown that allograft was the weakest in the fourth week (nearly 20% of its normal strength) (54). In another biomechanics study made with rat achilles tendons, the tendon strength was shown as 50% in the third week and 25% in the fourth week. It was stated that this change in the third week was caused by early fibrodysplasia and the weakening in the fourth week was caused by larger fibers replacing fibrodysplasia and the effort to build a longitudinal order among the fibers.14 Starting from the study performed by Steiner, Mae and Lui on apoptosis and other studies showing the presence of late re-ruptures, biomechanical results of oral Retendo® (mucopolygen complex) application were checked not only in the 4th week in which tendon is the weakest but also in the 1st, 2nd and 3rd weeks in our study.
Our study is an experimental study evaluating achilles tendon recovery in biomechanical and histological terms. In our study group, we tried to test whether Retendo® (mucoplygen complex) commonly used in muscle and tendon diseases had a positive contribution in tendon recovery or not.
In previous years, many studies were made on tendon recovery. In a study, it was shown that parenteral high dose vitamin c supplement increased early angiogenesis and type 1 collagen synthesis during achilles tendon recovery in rats. But strength wasn't measured in their studies, only histological evaluation was performed.15
In a study performed on flexor digitorum profundus tendon in chicken model, it was shown that vitamin c application with local injections on tendon repair area decreased fibrotic tissue and adhesions. Strength wasn't measured in these studies, only histological evaluation was performed.16
In a study on rat achilles model, it was shown that local tenocyte/hyaluronic acid application in repaired achilles tendon after repair positively effected tendon recovery.17
In another study made on rat achilles tendon model, the effect of oral glucosamine chondroitin sulphate application on achilles tendon recovery was examined and it was observed that this preparation positively effected tendon recovery in histological terms but there was no statistically significant difference in biomechanical measurements.18
Also the effect of resveratrol used as food supplement on achilles tendon recovery in diabetic rats was investigated and it was shown that the tendon fibrils provided more regular recovery of tendon fibrils in histological terms but didn't have a significant advantage in biomechanical test results.19 In another study made on rat achilles tendon model, it was shown that the subcutaneous application of low molecular weight heparin (nadroparin calcium) positively affected repaired achilles tendon cut recovery through biomechanical and histological inspections.20
In a study made on dogs, BMP-2 was applied around the tendon and a statistically significant recovery was performed in biomechanical measurements compared to the control group.21 Martinek et al. also made a similar study.22 A significant recovery compared to the control group was observed in BMP-2 gene transfer applied around the tendon. The effect of BMP-7 among growth factors on bone-tendon recovery was examined by Mihelic et al.23 and recovery increasing effect was shown. As we can understand from these studies, growth factors have significant positive effects on recovery. But despite all these positive effects, growth factors are expensive and difficult to acquire. Their manufacturing requires advanced laboratory conditions and technology. Because of these reasons, the use of growth factors is limited today. Starting from here, biomechanical evaluations were made from time to time by applying easy to use and cost-efficient (23 USA Dollars) Retendo® (mucopolygen complex) to increase biomechanical strength and decrease complications after achilles tendon repairs. But satisfying results couldn't be achieved.
In a study made in Hacettepe University, it was emphasized that paratenon and synovial tissues played a critical role both for blood formation and nutrition and as mesenchymal stem cell source in tendon recovery. While synovial structures provide the presence of certain growth factors in the environment in certain periods with their blood supply and stem cell potential, it was also detected that they are helpful for an ideal recovery by providing the viscosity of the tendon in the environment.24 With these positive effects, we think that paratenon and synovias shouldn't be damaged much in rat achilles tendon cutting models. Although Retendo® (mucopolygen complex) is commonly used in muscle and tendon diseases, its effect mechanism is not definitely known. But in the light of the findings we acquired in our study, we detected that Retendo® (mucopolygen complex) applied after achilles tendon ruptures positively effected collagen sequencing during recovery in histological terms but didn't have a significant effect on the mean load achilles tendon can tolerate before rupturing. As our study shows, Retendo® use after achilles tendon repair has a histologically limited advantage on tendon recovery and thus we think that it has no biomechanically significant effect.
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