Background
As a common injury, the incidence of Achilles tendon rupture has increased during the past few decades. Achilles tendon rupture which often locate on the left side occurs more frequently in men when they are 30 or 40 years old [
1]. Most Achilles tendon ruptures happen during sports [
2], and the incidence is increasing as a result of an increase of sport activities [
3].
Controversial discussion on treating acute Achilles tendon ruptures between nonoperative or operative repairs still remains [
4,
5]. Nonoperative treatment has been reported to be related to long periods of cast immobilization and high rates of rupture resulting in stiff ankles and weak calf muscles [
5,
6]. Surgical repair could minimize the gap between the ends of the tendon, giving rise to intensive tendon healing and decreased scar formation [
4,
5]. With these controversies, researchers suggest a tendency for operative treatment [
7,
8].
Many materials including human amniotic membrane (HAM), small intestinal submucosa (SIS), polyhydroxyethyl methacrylate membranes, and polypropylene mesh have been used as tendon graft materials to improve healing of tendon defects [
9]. As the innermost layer of the placenta, HAM has anti-inflammatory, anti-angiogenic, and anti-bacterial effects [
10] and has been known to support cellular migration, adhesion, and proliferation for healing in tissue defect sites [
11], which lead HAM to be used in a wide variety of clinical scenarios including tendon repair and peritendinous adhesion of flexor tendons impedance [
12,
13]. Nowadays, SIS has become a popular biological dressing in the reconstruction field. Researches have demonstrated that SIS can successfully remodel into functional tendon [
14] and SIS is also an ideal scaffold for tendon repair [
15]. Thus, according to the previous experiment, HAM and SIS could be applied to the Achilles tendon repair, but which one has more advantages is not fully understood.
In the current study, we aimed to test either HAM or SIS is preferable for the tendon repair. Thus, we investigated the healing effect of HAM and SIS on the ruptured Achilles tendon in rabbit models through adhesion evaluation, biomechanical, histological, and histomorphometrical analysis.
Methods
Animals
A total of 48 New Zealand white rabbits weighing 2.0–2.5 kg were purchased from Song Lian experimental animal farm (Shanghai, China). These animals were fed with normal fodder and tap water in standard hutches. The experiment was conducted under the protocol approved by our university. All studies were performed in agreement with the Principles of Laboratory Animal Care and guidelines established by the Institutional Animal Care and Use Committee of the university.
Preparation of biomaterials
Fresh HAM (purchased from Jiangxi Ruiji Biological Engineering Technology Co., Ltd.) was obtained from placentas in healthy donor mothers without infectious disease. Peeling the AM away from the chorion, the membrane was washed with sterile phosphate-buffered saline (PBS) to remove the blood clots and then permeated into 0.05 % trypsin + 0.02 % ethylenediaminetetraacetate (EDTA) at 37 °C for 2 h. After washing with sterile PBS containing streptomycin (0.1 mg/ml) and amphotericin B (0.25 μg/ml) for three times, the sterilized nitrocellulose filter paper with adherent HAM was stored in 50 % Dulbecco’s modified Eagle medium (DMEM) and 50 % glycerol at −80 °C.
The porcine small intestine was obtained and harvested from healthy pigs at the age of 3–6 months within 4 h of sacrifice. SIS was obtained using a longitudinal wiping motion with a scalpel handle and moistened gauze after removing the tunica serosa and muscularis. Submucous membrane was put into a solution containing methanol and chloroform (1:1, V/V) for 12 h and washed with ddH2O. After that, the membrane was incubated in 0.05 % trypsin and 0.05 % EDTA at 37 °C for 12 h and further treated with 0.5 % SDS in 0.9 % sodium chloride by shaking for 4 h. After removal of the detergent, the membrane was permeated through 0.1 % peroxyacetic acid and 20 % ethanol for 30 min and rinsed with saline solution.
Surgical procedure
Anesthesia was induced with an intravenous injection of 3 % pentobarbital sodium (1 ml/kg). After the right hind leg was shaved under sterile conditions, a 5-cm linear skin incision was created and a full-thickness transverse tenotomy was made at the mid-tendon point. Rabbits were divided into two groups in average (n = 24 in each group). Group P/A: a 2 cm × 2 cm section of the HAM soaked into 0.1 % rose bengal (RB) for 1 min. A single 5–0 Vicryl (Ethicon, USA) suture was placed to bring the tendon stumps into contact, and the HAM was permeated with 0.1 % RB wrapped around the tendon and bonded to the tendon surface. The group P/S was similar to the group P/A except that HAM was replaced with SIS. After a surgical wound is closed by 4–0 silk suture, the experimental leg was immobilized via plaster of paris bandage to make sure the hip joint is at 90° flexion and the ankle is at 180° straight. After a month, the plaster of paris bandage was removed, and animals were allowed to be active. At 7, 14, 28, and 56 days postoperation, six rabbits were respectively sacrificed. The experimental leg was opened again. After the repaired region is photographed, the Achilles tendon was harvested with part of the gastrocnemius muscle and calcaneus insertion. Then, the tendon samples were tested for mechanical properties. Finally, the sample was divided into two parts, one was immobilized by 4 % paraformaldehyde and another was frozen and stored at −80 °C.
Biomechanical analysis
The Achilles tendon came from gastrocnemius in the calcaneal stop and wrapped around a single layer of gauze at both ends to prevent glide of the Achilles tendon in the process of tension detection. After the width and thickness of the tendon were measured using a digital caliper, the tension of each repaired tendon was also tested by an Instron 4301 machine. The apparatus has a maximum load of 500 N and a minimum resolution of 0.01 N. The force-strain curve was collected for each sample by a connected computer with a speed of 10 mm/min. The apparatus quitted automatically when reached the maximum tension of each sample, and the vertex of the curve was regarded as the ultimate stress (US). The Young’s modulus (E), represented the slope coefficient of the stress-strain curve, is calculated by the equation: E = (FL)/(S△L), where F is the ultimate stress, L is the original in sample length, △L is the change length of the sample, and S is the cross-sectional area of the sample which is acquired by multiplied width with thickness.
Histology, histomorphometry, and immunofluorescent analysis
Samples were firstly immobilized by 4 % paraformaldehyde for 48 h at room temperature, then dehydrated in ethanol, and embedded in paraffin. Sagittal sections about 4 μm were cut for hematoxylin and eosin (HE) and immunohistochemistry at days 7, 14, 28, and 56 after operation and immunofluorescent staining analysis at postoperative days 3, 7, and 9. For HE, samples were stained with HE. For immunohistochemistry, tissue was fixed in paraformaldehyde and an enzymatic antigen retrieval step was performed. Samples were then blocked using 5 % bovine serum albumin (BSA) for 1 h at room temperature and incubated with collagen I antibody (1:200, Cambridge, MA, US) at 4 °C overnight. For immunofluorescent staining, vascularity was quantified by a morphometric stereological analysis on paraffin sections stained with anti-CD31 monoclonal antibody (1:50). A secondary antibody conjugated with fluorescein isothiocyanate (FITC) was applied at room temperature for 45 min.
Western blot analysis
The quantity of collagen I in each samples of repaired region was analyzed by western blot. Sample proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for 70 min after being electrophoresed through a 6 % sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel. Then, the membranes were incubated with collagen I antibody (1: 200) at 4 °C overnight after being blocked by 5 % milk for 1 h at room temperature. GAPDH was regarded as an internal reference.
Enzyme-linked immunosorbent assay (ELISA) analysis
The protein levels of IL-1β, IL-6, and TNF-α in the repair site were measured by ELISA, following the manufacturer’s instructions (USCN, Wuhan, China).
Adhesion evaluation
The adhesion grading system was as follows: grade 0, no adhesion; grade 1, filmy, transparent, and avascular; grade 2, opaque, translucent, and avascular; grade 3, opaque and capillaries present; grade 4, opaque and larger vessels present. The evaluation of scope adhesion formation was independently observed by two surgeons blinded to the details of the study when animals were sacrificed.
Statistical analysis
Statistical analysis of significant difference between groups was determined using independent sample t test that was used with SPSS 16.0 with significance set at a level of p < 0.05.
Discussion
In the present study, we investigated the healing effect of HAM and SIS on the ruptured Achilles tendon in rabbit models. We found that the biomechanical property of the tendons was significant higher in the P/S group at postoperative day 7 and day 14 than the P/A group. In addition, vascularity was significantly higher in the P/S group than that in the P/A group at days 3, 7, and 9. The results of the present study indicate that SIS applied to the laceracted tendons as a biologic scaffold has better treatment effect at Achilles tendon rupture than HAM in a rabbit model.
As a naturally occurring extracellular matrix, the porcine SIS plays an important role as a scaffold next to which the recipient site is acceptable to rebuild functionally and structurally suitable tissue [
16]. The SIS device is composed of collagen (about 90 %), glycosaminoglycans, fibronectin, and growth factors including transforming growth factor β (TGF-β), vascular endothelium growth factor, and fibroblast growth factor 2. SIS has been used in various different organ system repairs [
17]. Previous research has revealed promising findings in orthopedic soft tissue applications using SIS for Achilles tendon defect repair in dogs [
18] and for repair of a musculotendinous tissue in a canine Achilles tendon repair model [
15] . In this study, the SIS device was implanted as a resorbable scaffold and acted as a regenerative response, leading to collagen I-rich, connective tissue histologically similar with induced using HAM repair.
Biomechanical testing demonstrated that the US and
E values were significant higher in the P/S group at postoperative day 7 and at day 14 than those in the P/A group. Previous investigations applying HAM in tendon healing in a rabbit Achilles tendon model found that the biomechanical modulus of the HAM treatment group was higher at 4 weeks than that of the control group [
12], showing the treatment effect of HAM. In addition, Zalavras et al. [
14] revealed the progressive remodeling of the SIS-regenerated tendon in a rat model. In this research, we found no difference between the P/A and P/S groups in adhesion formation. Previous study has found that HAM could induce down-regulation of TGF-β signaling, which contributes to wound healing, such as the stimulation of collagen production and recruitment of fibroblasts [
19]. On the other hand, TGF-β has been found prospective to decrease the formation of peritendinous adhesions on the flexor tendons after surgery [
20]. It may hint that the effect of HAM and SIS on TGF-β may lead to less adhesion formation. Another important result is the higher strength with SIS after operation day 7 and day 14 biomechanical studies than the P/A group. These findings indicate that application of SIS is superior to HAM in tendon repairs.
The healthy Achilles tendon tissue is short of blood supply, and its nutrition mainly comes from the synovial membrane dispersion and blood vessels dispersion. When the Achilles tendon ruptures, the blood supply is interrupted and the blood vessel is damaged. New vascular network and granulation tissue formed by neovascularization provide the necessary nutrients for tendon repair. Generally, neovascularization occurs in the early stage of Achilles tendon healing, and the monitoring of new blood vessels can reflect the speed of Achilles tendon healing and indirectly reflect the impact of biological materials on the healing of Achilles tendon. In our present study, neovascularization in the P/S group was significantly higher compared with the P/A group. Previous investigator has revealed that transplantation of SIS is an effective bioscaffold as a body wall repair device [
21]. The SIS-regenerated tendons in our rabbit model showed fibroblastic ingrowth, collagen I deposition, reduction of inflammatory cytokines (IL-1β, IL-6 and TNF-α) levels, and neovascularization. It has been verified that fibroblastic cells appearing at the healing position are stemmed from undifferentiated mesenchymal cells which were derived from the recipient site [
22]. Thus, findings of the current study are consistent with the hypothesis of other studies that porcine xenograft acts as an agent with regenerative potential which plays an important role in host tissue ingrowth [
16,
23]. However, our study is observational, and more researches with set criteria quantified by blinded observers are still needed to be clarified.
There are still some limitations in the current study. First, the number of animals at each timepoint is small and quantitative evaluation of reparative cells and collagen proliferation using objective analyses are lacking. Second, the model in this study is not exactly clinically relevant to Achilles tendon ruptures in humans, and the Achilles tendon does not rupture in a clean fashion. Third, no verification in clinical human practice is one limitation of this study, and thus the long-term effects of SIS on tendon-healing are needed to be investigated in the future clinical practice. In addition, the addition of more bulk to the repair can, in humans, result in more difficult closure of the wound and give rise to a greater rate of infection.
Acknowledgements
This work was supported by Science and Technology Commission of Shanghai Municipality (134119b1300) and the National Natural Science Fund for Young Scholars of China (No. 81401585).