Effect of tendon hydrogel on healing of tendon injury

Introduction

Tendon repair is an important step in hand surgery and other types of plastic surgery. For a long time period post-surgery, tendons cannot be subjected to strain and their biomechanical properties are aberrant; micro tears, which may ultimately lead to full-thickness injury of tendon, can be characterized by microscopy (1,2). Common tendon injuries include the Achilles tendon, rotator cuff tendon and elbow lateral condyle, and due to their similar features and poor prognosis, numerous studies have assessed strategies to promote tendon healing, mainly including platelet-rich plasma, whole-blood injections, growth hormones and stem cells (3). Tendon healing after injury is usually affected by inflammation, collagen production and reshaping of. In the early stages after tendon damage, mononuclear cells and macrophages are activated, which release biomolecular factors promoting tendon cell proliferation and increase in collagen synthesis; these processes occurring as early as possible may provide sufficient mechanical strength to enable patients to perform basic activities. Studies have shown that early activity is of great significance for tendon performance and associated prognosis; early healing after tendon injury is therefore key (4–6). Tendon-derived extracellular matrix hydrogels containing connective tissue-specific proteins, including collagen I, can deliver tendon cells to the damage location and provide an environment supporting cell proliferation (7,8). Previous studies on rats showed that subcutaneous hydrogel injection was well tolerated with no nodules or allergic reaction and gradual degradation after 4 weeks (7,8). Based on these preliminary studies, the present study explored the ability of a tendon-derived hydrogel to promote tendon healing in rats after injury. The present study paved the road for the implementation of hydrogels for tendon healing in clinical practice.

Materials and methods

Tissue decellularization

The musculi flexor digitorum profundus, musculus flexor digitorum superficialis and flexor pollicis longus tendon were obtained from 36 rats (male, average weight, 350 g, 6–8 weeks) cadavers frozen directly after sacrifice. This study was approved by the ethics committee of the Ninth People's Hospital of Wuxi City, Wuxi, China. According to a previously described method (8), the epitenon, vaginae synoviales and musculature were extracted. The superficial flexor tendon was cut near the bifurcation at 2 cm from the end. The musculus flexor digitorum superficialis and flexor pollicis longus tendon were cut off near the bone tendon junction at 1 cm from the end. The tendons obtained were subjected to acellular treatment and subsequently immersed in 0.1% EDTA solution for 4 h (8). The tendons were then incubated in 0.1% lauryl sodium sulfate and 0.1% EDTA solution at room temperature for 24 h with stirring, followed by washing with phosphate buffer. The tendons were then frozen at −80°C. The acellular tendon tissue was then freeze-dried, ground into powder and kept at 4°C until use.

Preparation of tendon matrix hydrogel

The tendon powder was incubated with 1 mg/ml pepsin (Sigma-Aldrich, Merck KGaG, Darmstadt, Germany), 0.02M hydrochloric acid and aquae sterilisata (pH 2.2; powder concentration, 20 mg/ml) to digest the extracellular matrix. Following incubation at room temperature for 24 h with constant stirring, the sample was placed on ice and sodium carbonate hydrate was added to increase the pH to 8.0 to inactivate pepsin. Finally, 10 volumes of phosphate-buffered saline (PBS, adjusting pH 7.4) were added to obtain an isoosmotic solution.

Operative procedure and tendon treatment

A total of 36 Wistar rats (male, average weight, 350 g, 6–8 weeks) were obtained from the animal center of Ninth People's Hospital, and were fed at room temperature and 40–50% humidity, and with 12-h light/dark, and ad libitum access to food and water. The rats were fixed in the supine position after being anaesthetized by 2% isoflurane (Sigma-Aldrich; Merck KGaG). This study was approved by the Ethics Committee of the Ninth People's Hospital of Wuxi City (Wuxi, China). Following anti-bacterial treatment with buprenorphine and enrofloxacin (Sigma-Aldrich; Merck KGaG), a 1-cm curve cut was made around the Achilles tendon to ensure the completeness of the tendon. The tendon was carefully separated to avoid damaging the nervus cutaneus surae medialis and major vessels. Cyanoacrylate was used to bond two #15 surgical knives together to keep the incision parallel. Under a microscope, a damaged area of 0.5 mm in width and 5 mm in length was generated, reaching from the calcaneus to the central tendon of the bilateral lower extremities of each rat (Figs. 1 and 2). The site of injury on the left side was injected with 0.05 ml tendon hydrogel, while the right-hand side was injected with 0.05 ml phosphate-buffered saline as the control. The wound was then sutured with #4 absorb string (Ethicon, Inc., Somerville, NJ, USA). According to the findings of a preliminary experiment (8), animals were anaesthetized using 1 ml 2% isoflurane (20 mg/rat; Sigma-Aldrich; Merck KGaG) and then were sacrificed by decapitation at 2, 4 and 8 weeks after the surgery to obtain the tendons. A total of 10 tendon specimens were used for biomechanical performance tests at each time-point and 2 specimens were used for histopathological analysis.

Figure 1. Illustration of the location and mode of surgery. C, calcaneus; G, gastrocnemius; A, Achilles tendon; H, hydrogel.

Figure 2. Operation procedure. (A) The tendon was cut on both sides in a parallel fashion. (B) The damaged area except for the central part was put into shape under the microscope and (C) filled with hydrogel. Magnification, ×200.

Biomechanical performance test

The mini Bionix tset system (MTS Systems Corp., Eden Prairie, MN, USA) was used as a test load cell. From the rat feet obtained, the muscle was removed to expose the tendon junction. The foot was fixed, and abrasive paper and cyanoacrylate were used to fix the muscle-tendon junction (Fig. 3). Orthogonal images were captured with a digital camera (8.2 megapixels; Canon EOS 30D; Canon, Tokyo, Japan) and a digital caliper was used to determine the size of the tendon. Image J software version 2.0 (National Institutes of Health, Bethesda, MD, USA) was used to calculate the cross-sectional area and ultimate tensile stress of each tendon. At a tensile speed of 0.5 mm/sec, the load was increased until the destruction of the tendon and the ultimate failure load was recorded. The ultimate tensile stress was determined by limiting load and cross-sectional area (8). Tendon toughness was evaluated via a force/displacement curve.

Figure 3. Biomechanical test. For measurement of the ultimate failure load, tensile strength and toughness, a load was applied at the distance shown by the vernier caliper (5 mm). The arrowhead shows the direction of the applied load.

Histopathological analysis

The obtained tendon tissue was fixed for 10 min in 10% formalin (Sigma-Aldrich; Merck KGaG) at room temperature and embedded in ceresin wax and then cut into slices (6 µm). The morphological characteristics of the tendons were evaluated by hematoxylin and eosin staining. Adjacent sections were stained with Sirius red solution (Sigma-Aldrich; Merck KGaG) to determine the content of type-I and type-III collagen protein. Images were captured under a fluorescence microscope (Model: D-35578; Leica Microsystems, Wetzlar, Germany).

Statistical analysis

All of the data were analyzed by using the SPSS software 18.0 (SPSS Inc., Chicago, IL, USA). Comparisons among groups were performed by the paired Student's t-test. Values are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Results

Biomechanical test

For every biomechanical test, one rat tendon was rolled out from the forceps holder. The connection of the tendon muscle was broken, so that it was excluded. The results showed that 2 weeks after the operation, almost all tendon rupture positions were located in the tendon itself. By contrast at 4 and 8 weeks post-surgery, ruptures of the muscle-tendon junction were common (Table I). At 2 weeks after the operation, no obvious difference in ultimate failure load, ultimate tensile stress and toughness was observed between the two groups (P=0.151, 0.424 and 0.760, respectively). At 4 weeds after the operation, the ultimate failure load in the experimental group was increased by 28% of that of the control group (74.8±11.6 vs. 58.4±14.2 N; P=0.016; Fig. 4), while the ultimate tensile stress and toughness were not significantly different between the two groups (P=0.634 and 0.082, respectively). At 8 weeks post-surgery, the ultimate failure load, ultimate tensile stress and toughness did not show any significantly differences between the two groups (P=0.156, 0.385 and 0.748, respectively).

Figure 4. Ultimate failure load for each time point. *P<0.05 vs. Control group.

Table I. Results of biomechanical tests.

Table I.

Results of biomechanical tests.

Group	Treatment	Mechanical tests/tissue analyses (n)	Ultimate failure load (N)	Ultimate tensile stress (N/mm2)	Toughness (N/mm)	Tendon damage location
1	2 w, hydrogel	4/1	45.2±8.6	1.6±0.6	11.4±3.7	1TBI/8T/0B
	2 w, no hydrogel	9/2	39.8±6.3	1.4±0.2	10.9±2.7	2TBI/7T/0B
2	4 w, hydrogel	9/2	74.8±11.6a	3.0±0.7	17.9±3.5	5TBI/4T/0B
	4 w, no hydrogel	9/2	58.4±14.2a	2.8±1.0	14.7±3.9	7TBI/2T/0B
3	8 w, hydrogel	9/2	83.5±13.7	2.9±0.7	17.7±2.7	5TBI/4T/0B
	8 w, no hydrogel	9/2	74.0±13.2	2.6±0.7	17.3±4.5	6TBI/3T/0B

a P<0.05 represents the values compared to the 2 w, hydrogel or 2 w, no hydrogel. TBI, the junction of tendon and bone; T, tendon; B, bone; w, weeks.

Histopathological analysis

At 2 and 4 weeks after the operation, the cross-section of the tendons of the two groups was almost identical. In the hydrogel-treated group, the collagen aggregation in the injury position was visible (Figs. 5 and 6). At 2 weeks post-surgery, Sirius red staining showed that the type I collagen content in the experimental group was obviously higher than that in the control group (Fig. 5A). At week 4, the difference in collagen content in hydrogel group was more significant compared to control group, which suggests that hydrogel applied immediately after tendon injury can enhance the type-I collagen fibers (P<0.05, Fig. 5B). Meanwhile, type III collagen protein (green and yellow) in the control group was obvious higher than that in the experimental group (P<0.05, Fig. 5). Furthermore, type I collagen protein (red) in the experimental group was higher than that in the control (P<0.05, Fig. 5). At 8 weeks, no obvious difference was found in the collagen content between the two groups (Fig. 6).

Figure 5. H&E and Sirius red staining at 2 and 4 weeks after the operation. (A) H&E staining of (a) the control and (b) the experimental group as well as Sirius red staining of (c) the control and (d) the experimental group at 2 weeks after operation. Type I collagen protein (red staining) in the experimental group was shown to be higher than that in the control group. (B) H&E staining of (a) the control and (b) the experimental group as well as Sirius red staining of (c) the control and (d) the experimental group at 4 weeks after operation. Type III collagen protein (green and yellow) in the control group was obvious higher than that in the experimental group. Furthermore, type I collagen protein (red) in the experimental group was higher than that in the control group. T, tendon; *, injury position; H&E, hematoxylin and eosin. Magnification, ×200.

Figure 6. At 8 weeks after the operation, the content of type I collagen protein in (A) control group and (B) hydrogel group was almost the same. The damaged area has become invisible. The samples were identical, while an incision/scar is still visible in the experimental group. However, only one animal was assessed in here. Magnification, ×200.

Discussion

The results of the present study confirmed the former hypothesis that a tendon-derived extracellular matrix hydrogel can improve tissue regeneration and promote tendon healing. The use of tissue-derived hydrogel has been previously reported. Ventricle-derived extracellular matrix hydrogel has been applied in a model of cardiac damage, which improved the repair of myocardial cells and local metabolism, and skeletal muscle-derived extracellular matrix hydrogel has been applied in a model hindlimb ischemia which enhanced the distribution of skeletal muscle cells along the neovascularization (8,9). However, collagen hydrogel synthesized in vitro did not obviously improve the healing of tendon injury (10). In the present study, a hydrogel not only containing the matrix component of tendons, but also its proteins and biological factors, was prepared and applied. To the best of our knowledge, the present study was the first to prepare tendon matrix hydrogels and apply them to an animal model of tendon injury.

The biocompatibility of the extracellular matrix hydrogel has been shown to be good with degradation occurring after 4 weeks. The results of the present study showed that extracellular matrix hydrogel provides a suitable environment for cell metabolism and healing. For instance, strengthening of the phagocytic function of macrophages is beneficial for tissue remodeling (9). In the first stage of tendon healing, macrophages destroy and take up necrotic tissue, and release extracellular matrix, oligosaccharide derivatives and other factors. Macrophages can be used as a vasoactive and chemotactic factor to promote the aggregation of stem and progenitor cells. Furthermore, collagen synthesis is increased in the early stages of injury to repair damaged tissue (11). Certain studies have indicated that weakening of the tendon strength during the healing process is due to the natural collagen protein I being replaced by the thinner type III collagenous fibers (12,13). Sirius red staining at 4 weeks post-surgery showed that in the hydrogel group, the ratio of type I vs. type III collagen was obviously higher than that in the control group, which explains for the higher ultimate failure load in the hydrogel group. The type I collagen content was therefore consistent with the mechanical strength of the tendon. 4 weeks later, the hydrogel-treated tendon was thicker and more solid in hydrogel group (one animal) and the intersecting surface was greater compared to the control group. The toughness of the tendon arises from cross-linked collagenous fibers in the final stage of healing (14). Therefore, in the present study, not all of the differences were significant at different time-point (such as 4 and 8 weeks). The effect of the hydrogel was mainly shown in the early stages of injury and the hydrogel, as it was degraded after 4 weeks. Therefore, its effect of promoting tissue repair gradually decreased with time. The operation wound in the two groups was still visible at 8 weeks and the scars were covered by type I collagen, however, only one animal was assessed. The results indicated that the process of tissue repair and remodeling was almost finished at 8 weeks after tendon injury.

In clinical practice, the main application advantages of hydrogel may be that it accelerates the healing process of tendon injury in the early stage, enabling patients to perform early functional exercise. Numerous studies have shown that application of an appropriate mechanical force can promote fibroblast proliferation and the synthesis and arrangement of the original collagen protein (15,16). The hydrogel can be gathered at the physiological temperature, so that the targeted application to part of the tendon injury can be used by minimally invasive injection to improve the prognosis. Furthermore, substances including growth factors, platelet-rich plasma and stem cells can be mixed with hydrogel for injection; the efficacy of such preparations requires evaluation by further studies.

Of note, the present study had certain limitations. Sirius red staining used for the quantitative assessment of collagen content only provided one possible explanation for the differences in tendon strength demonstrated by the biomechanical test. However, the findings only provided limited information, as only one rat per group was used for histopathological analysis at each time-point. In further studies, analysis of changes in structure, collagen synthesis and content of associated biological factors will be helpful to confirm the effect of hydrogel on tendon healing. In the present study, a rat model of tendon injury was used, while there may be differences among various injury sites and species. Furthermore, an animal experiment using a larger sample is required for further comparison and confirmation of the results.

In conclusion, the present study showed that a tendon-derived extracellular matrix hydrogel was able to significantly improve the ultimate failure load of injured tendons from rats at 4 weeks after surgery. It was therefore suggested that the hydrogel may promote tendon healing. The present study paved the road for the development of a novel preparation for the clinical treatment of tendon injuries.

References