Effect of a Simple Collagen Type I Sponge for Achilles Tendon Repair in a Rat Model Sebastian A. Mu¨ller,*y MD, Lutz Du¨rselen,z PhD, Patricia Heisterbach,y MD, Chris Evans,§ PhD, and Martin Majewski,y MD Investigation performed at the University of Basel, Basel, Switzerland and the University of Ulm, Ulm, Germany Background: Several sophisticated approaches to tendon engineering have been investigated as ways to improve tendon healing with the early formation of repair tissue with possibly a high amount of type I collagen. Besides the new formation of collagen type I, there is evidence for the natural integration of surrounding collagen type I from healthy tendon parts into the healing defect. However, the simple application of a type I collagen sponge to the healing site to increase the amount of local collagen type I has not been investigated. Hypothesis: Healing of the rat Achilles tendon can be accelerated by an additional supply of collagen type I, resulting in increased tear resistance. Study Design: Controlled laboratory study. Methods: The right Achilles tendons of 42 rats were transected. In half of the animals, a type I collagen sponge was placed into the gap. Animals were allowed to move freely in their cages to simulate early functional therapy. After 1, 2, and 4 weeks, tendon length, width, maximal load to failure, and stiffness were measured and the healing site studied histologically according to the Bonar score. Inflammation was evaluated by the appearance of macrophages and neutrophilic and eosinophilic granulocytes. Results: Defects receiving collagen sponges showed improved healing, with significantly stronger (29.5 vs 5.0 N, respectively, at 1 week; P = .00003), shorter (11.6 vs 14.5 mm, respectively, at 4 weeks; P = .005), thicker (10.0 vs 1.8 mm2, respectively, at 1 week; P = .00002), and less stiff (19.5 vs 30.5 N/mm, respectively, at 4 weeks; P = .02) tendons than control tendons. Overall, the biomechanical properties of the collagen-treated tendons appeared to be significantly closer to those of native, uninjured tendons compared with tendons in the control group. Histologically, no inflammatory reaction due to the collagen sponge was found. Conclusion: Tendon healing was accelerated by the type I collagen sponge. Moreover, the mechanical properties of collagentreated tendons appeared to be significantly closer to those of normal, uninjured tendons compared with control tendons without collagen treatment. Clinical Relevance: As a simple type I collagen sponge seems to increase the amount of local collagen type I, the careful use of such sponges might be an option for tendon augmentation during Achilles tendon surgery. Keywords: Achilles tendon; tendon healing; tendon engineering; collagen type I; rat

Tendons biomechanically link muscle and bone, transferring muscle contraction to motion. The specialized

structural properties of tendons contribute to high tensile strength and tear resistance. These properties rely on the high collagen content of tendons, which accounts for 70% of the dry weight of this tissue. Collagen type I accounts for 95% of the collagen in tendons and is arranged in a parallel orientation of closely packed fibers.6 In cases of injuries, tendon healing runs through several stages of hemorrhaging, inflammation, tissue formation, and remodeling. Tissue formation first produces scar tissue with an increased cross-sectional area, mainly comprising collagen type III. In the remodeling stage, collagen type III is replaced by the mechanically more resistant collagen type I. This stage of healing, from biomechanically inferior scar tissue (tendon callus) to repaired tendon tissue, lasts for months and is associated with a high risk of reruptures.24 Moreover, healed tendon tissue rarely achieves the mechanical properties of those

*Address correspondence to Sebastian A. Mu¨ller, MD, Department of Orthopedic Surgery, University of Basel, Spitalstrasse 21, 4031 Basel, Switzerland (email: [email protected]). y Department of Orthopedic Surgery, University of Basel, Basel, Switzerland. z Institute of Orthopedic Research and Biomechanics, University of Ulm, Ulm, Germany. § Rehabilitation Medicine Research Center, Mayo Clinic, Rochester, Minnesota, USA. One or more of the authors has declared the following potential conflict of interest or source of funding: This work was funded by the Academy of Swiss Insurance Medicine. The American Journal of Sports Medicine, Vol. 44, No. 8 DOI: 10.1177/0363546516641942 Ó 2016 The Author(s)

1998

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of preinjured tendons, and the final tensile strength can be reduced by up to 30%.8,31 In addition to the described stages of tendon healing (hemorrhaging, inflammation, tissue formation, and remodeling), Ingraham et al9 have published a parallel ongoing process of ‘‘collagen recycling.’’ Collagen is liberated from the adjacent tendon end bridges the tendon’s defect zone.9 To improve healing, several approaches in tendon engineering have been described. Among these, scaffolds are placed into the defect zone with the idea to provide mechanical support and guide endogenous cells to promote matrix production and organization. Several highly engineered scaffolds with complex 3-dimensional structures, with or without stem cells, have been described.k However, these approaches focus on the support of an accelerated or improved natural course of tendon healing to remodeled tendons mainly containing type I collagen. Yet, what happens if type I collagen is additionally applied to the rupture site, supporting the ‘‘collagen recycling’’ process? To the best of our knowledge, the effect of such a type I collagen application has not yet been investigated in tendon engineering. Thus, the aim of this study was to biomechanically and histologically analyze the effect of a simple off-the-shelf collagen type I sponge for Achilles tendon repair in comparison with the natural course of healing. The hypothesis of this study was that tendon healing could be accelerated by an additional supply of collagen type I, resulting in increased tear resistance of healed tendons.

METHODS Animal Model A total of 42 adult male Sprague Dawley rats weighing 400 to 425 g (Harlan Netherlands) were used. The study was approved by the responsible animal committee. Animals were randomly assigned to 1 of 2 groups: collagen sponge and nonsponge. The nonsponge group served as the control. For each group, 5 animals were used for the biomechanical examination and 2 for the histological examination. Testing was performed at 1, 2, and 4 weeks after surgical transection of the right Achilles tendon. Under general anesthesia (isoflurane), the leg was shaved. Rats were placed on a heated surgery table, disinfected, and then covered with surgical drapes, leaving the right limb exposed. Skin, followed by the paratenon, was incised longitudinally. The tendon was completely transected perpendicular to the collagen fibers 5 mm proximal to the calcaneal insertion. To prevent internal splinting, the plantar tendon was transected as well. The collagen sponge (3 mm in length 3 2 mm in width 3 1 mm in thickness; collagen type I; Takeda) was placed into the gap (Figure 1). The paratenon and skin were closed with Prolene 4-0 (Ethicon) using single stitches. Within the nonsponge group, the tendon transection site was left empty. The paratenon and skin were sutured in the same manner as in the sponge group thereafter.

k

References 10, 12, 20, 22, 23, 28, 29, 33, 35.

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1999

Figure 1. Collagen sponge (3 mm in length 3 2 mm in width 3 1 mm in thickness) placed into the right Achilles tendon transection site of a rat. Postoperatively, the rats recovered on a heated pad. No cast immobilization was applied to the operated legs, and the animals were allowed to move within their cages.15,19 The skin sutures were removed at 1 week after surgery. After 1, 2, and 4 weeks, animals of both groups were euthanized with CO2 after isoflurane anesthesia. Whole muscle-tendon-bone units were explanted for testing. To do this, the skin was incised longitudinally along the whole thigh below the level of the knee down to the calcaneus. The Achilles tendon was harvested with the calcaneus and the distal two-thirds of the triceps surae muscle. The muscle-tendon-bone units were directly wrapped in gauze soaked with Ringer’s solution and frozen at 280°C until biomechanical and histological testing. In 5 randomly chosen animals, the uninjured left Achilles tendon was also harvested and biomechanically tested, serving as the ‘‘native tendon’’ control.

Biomechanical Testing The specimens were thawed in Ringer’s solution (25°C) for 4 hours. Before testing, tendon thickness at the healing site and tendon length were measured with a precision caliper (Digital Caliper; Tesa). To test the load to failure, the specimens were fastened with the muscle in a cryoclamp and the calcaneus in a copper clamp.34 The clamping devices were attached to a standard materials testing machine (Z010; Zwick). No preconditioning or cyclical stretching was applied to the tendons before testing. Room temperature was kept constant at 25°C, and the specimens were moistened with Ringer’s solution to prevent dehydration. Just before testing, the muscle was frozen in the cryoclamp with liquid nitrogen. The machine was started as soon as the muscle (but not the tendon) was frozen, which was confirmed manually with a metal needle.19 The displacement rate was constantly set at 1000 mm/min. Forcedisplacement curves were recorded digitally for subsequent data analysis. Load to failure (in N) and stiffness (in N/mm) were measured. Stiffness was assessed from the linear part of the force-elongation curve.

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Histological Examination

§

16 §

Statistical Analysis A software-based Student t test was used. Additionally, a Wilcoxon rank test was used, as normal distribution cannot be assumed because of the small sample size. The level of significance was set to P  .05 and only indicated if statistically significant for both tests. Means 6 SDs and P values are indicated.

RESULTS Tendon Length The sponge group showed a dynamic pattern in tendon length over time, with the longest tendons after 1 week (1 week: 12.4 6 2.3 mm; 2 weeks: 10.7 6 0.4 mm; 4 weeks: 11.6 6 0.5 mm). However, the nonsponge group showed significantly longer tendons after 2 and 4 weeks (1 week: 12.9 6 1.1 mm [P = .3]; 2 weeks: 13.3 6 1.0 mm [P = .0004]; 4 weeks: 14.5 6 1.8 mm [P = .005]). Interestingly, the length of collagen sponge tendons was in the range of that of native tendons (11.1 6 0.5 mm), whereas control tendons were significantly longer than native tendons at all time points (Figure 2).

Tendon Cross-sectional Area Starting with a cross-sectional area of 10.0 6 2.1 mm2 after 1 week, the sponge group showed an increase up to 13.3 6 3.2 mm2 after 2 weeks and a decrease to 12.6 6 1.9 mm2 after 4 weeks. In contrast, the cross-sectional area was significantly

14

* *

12

Length, mm

Thawed tendons were fixed in 4% buffered formalin (pH 7.4) for 24 hours, dehydrated, and embedded in paraffin wax. Longitudinal sections (5 mm) at the midsubstance of the tendon were stained with hematoxylin and eosin. Five sections of each animal at each time point were examined by 2 blinded investigators. If there was a mismatch in histological judgment of 1 specimen between the 2, the specimen was examined together. Specimens were assessed according to the Bonar criteria validated by Maffulli et al,18 including the judgment of tenocytes, the ground substance, collagen fibers, and the vascularity of each specimen. Tenocytes were evaluated for spindle shape, increased roundness, increased size, and amount of cytoplasm visible. The ground substance was judged for the stainability of mucins, the demarcation of bundles, and inconspicuous collagen staining. Collagen was judged for the organization and demarcation of bundles. Therefore, the separation of individual fibers, the loss of demarcation, and the loss of tendon architecture were evaluated. Vascularity was assessed by blood vessels between bundles and clusters of capillaries within the tendon tissue. Specific inflammation was evaluated by the appearance of macrophages and neutrophilic and eosinophilic granulocytes as a sign of acute and specific inflammation within the hematoxylin and eosin–stained specimens.

§

Nave tendon 10 8

Collagen sponge

6

Nonsponge

4 2 0

1 week

2 weeks

4 weeks

Figure 2. Mean Achilles tendon length in the collagen sponge and nonsponge groups at 1, 2, and 4 weeks after tenotomy. Error bars show the highest and lowest values measured. The black line indicates the mean native tendon length. Statistically significant difference *between groups and §with respect to the native tendon (P  .05). After 2 and 4 weeks, the tendons in the nonsponge group were significantly longer than those of the collagen sponge group. The length of collagen sponge tendons was comparable with that of native tendons, whereas control tendons were significantly longer than native tendons at 1, 2, and 4 weeks. smaller in the nonsponge group (1 week: 1.8 6 0.9 mm2 [P = .00002]; 2 weeks: 8.1 6 2.8 mm2 [P = .01]; 4 weeks: 9.7 6 2.1 mm2 [P = .03]). The cross-sectional area was larger than that of native tendons (3.4 6 0.2 mm2) for both the sponge and nonsponge groups at all time points except for control tendons at 1 week (Figure 3).

Load to Failure With 29.5 6 6.7 N after 1 week, the load to failure steadily increased to 53.8 6 13.0 N and 67.0 6 7.5 N in the sponge group after 2 and 4 weeks, respectively. Compared with the nonsponge group (1 week: 5.0 6 2.0 N [P = .00003]; 2 weeks: 41.2 6 1.5 N [P = .03]; 4 weeks: 75.1 6 10.3 N [P = .1]), the load to failure was significantly higher after 1 and 2 weeks. However, statistical significance was not attained by calculation with the Wilcoxon rank test for the 2-week value. After 2 weeks, the collagen sponge tendons had a load to failure comparable with native tendons (56.2 6 8.7 N). Control tendons were significantly weaker at 1 and 2 weeks than native tendons as well as collagen tendons at 1 week. However, both groups were significantly stronger than native tendons at 4 weeks (Figure 4).

Stiffness The sponge group showed early increased stiffness (1 week: 10.3 6 3.0 N/mm; 2 weeks: 18.3 6 4.9 N/mm; 4 weeks: 19.5 6 4.3 N/mm). In contrast, the nonsponge group further increased in stiffness after 2 weeks, with significantly lower stiffness after 1 week and significantly higher stiffness after 4 weeks (1 week: 2.8 6 0.7 N/mm [P = .0003]; 2 weeks: 15.4 6 2.6 N/mm [P = .1]; 4 weeks: 30.5 6 9.3 N/mm [P = .02]). At

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*§

16

*§

§

70

12 10 Collagen sponge

8

Nonsponge 6 4

§

80

§

2001

§

90

*§

Load to failure, N

Cross-seconal area, mm2

14

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Nave tendon

60 Nave tendon 50 40

§

Collagen sponge

§

Nonsponge

30 20

2

*§

10

0 1 week

2 weeks

0

4 weeks

1 week

2 and 4 weeks, stiffness of collagen sponge tendons was in the range of that of native tendons (18.5 6 2.4 N/mm) but significantly lower at 1 week. Stiffness of control tendons was significantly lower than for native tendons at 1 and 2 weeks but significantly higher at 4 weeks (Figure 5).

Histology After 1 week, a loose connective tissue matrix containing some inflammatory cells, including neutrophils, macrophages, and lymphocytes, was seen in all tendons. At 2 weeks, all tendons matured, with some remnants of the collagen sponge still seen in the sponge group. At 4 weeks, both groups showed a higher degree of organization and a more homogeneous pattern of collagen fibers. Collagen arrangement and maturation of fibroblasts to fibrocytes showed no major differences between the 2 groups. Tenocytes showed a decreased size without being spindle shaped and less stainable cytoplasm in both groups over time. No proinflammatory macrophages and neutrophilic and eosinophilic granulocytes as a sign of specific inflammation due to the collagen sponge were seen within the sponge group at any time point (Figure 6).

DISCUSSION The challenge in tendon engineering is the improvement of tendon healing toward mechanical properties as close as possible to those of normal tendon tissue.8,31 Several research groups have promoted complex collagen scaffolds to fill

2 weeks

4 weeks

Figure 4. Mean Achilles tendon load to failure in the collagen sponge and nonsponge groups at 1, 2, and 4 weeks after tenotomy. Error bars show the highest and lowest values measured. The black line indicates the mean native tendon load to failure. Statistically significant difference *between groups and §with respect to the native tendon (P  .05). The load to failure was significantly increased at 1 week in tendons receiving the collagen sponge. After 2 weeks, collagen sponge tendons were in the range of native tendons, whereas control tendons were significantly weaker than native tendons. At 4 weeks, both groups had higher loads to failure than native tendons.

*§

45 40 35

Sffness , N/mm

Figure 3. Mean Achilles tendon cross-sectional area in the collagen sponge and nonsponge groups at 1, 2, and 4 weeks after tenotomy. Error bars show the highest and lowest values measured. The black line indicates the mean native tendon cross-sectional area. Statistically significant difference *between groups and §with respect to the native tendon (P  .05). At all time points, tendons receiving the collagen sponge had a significantly greater cross-sectional area than controls (nonsponge group). The cross-sectional area was larger than that of native tendons for both the sponge and nonsponge groups at all time points except for the nonsponge group at 1 week.

30

Collagen sponge Nonsponge

25 §

20 15

Nave tendon §

10 5

*§

0 1 week

2 weeks

4 weeks

Figure 5. Mean Achilles tendon stiffness in the collagen sponge and nonsponge groups at 1, 2, and 4 weeks after tenotomy. Error bars show the highest and lowest values measured. The black line indicates the mean native tendon stiffness. Statistically significant difference *between groups and § with respect to the native tendon (P  .05). Tendons receiving collagen had higher initial stiffness than controls. Thereafter, the control tendons constantly increased their stiffness with time, such that their stiffness exceeded that of the collagen sponge group by 4 weeks. Collagen sponge tendons were in the range of native tendons at 2 and 4 weeks.

tendon defects in vitro and in vivo, with promising results in regeneration and biomechanical performance of the tendon. Scaffolds, including complex collagen structures, have

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Figure 6. Histology at 2 weeks after tendon transection. The tendons of both groups are healed together, showing dense, cellrich collagenous tissue with criss-cross collagen fibers running throughout. Compared with the control group (left), the sponge group (right) shows many vacuoles originating from the sponge texture. Black and striped scale bar in the hematoxylin and eosin images represents 200 mm. TABLE 1 Literature on Scaffolds Used in Tendon Engineeringa Study (Year)

Journal

Type of Scaffold

Type of Study

Awad et al2 (2000) Awad et al1 (2003) Hao et al7 (2010) Juncosa-Melvin et al11 (2005) Lee16 (2008) Kim et al13 (2008) Nicholson et al25 (2007)

J Biomed Mater Res J Orthop Res J Orthop Res Tissue Eng J Foot Ankle Surg Acta Biomater J Shoulder Elbow Surg

In vivo In vivo In vivo In vivo In vivo In vitro In vivo

Nillesen et al26 (2007) Provencher et al27 (2007) Sarrafian et al30 (2010)

Biomaterials Tech Orthop J Foot Ankle Surg

Collagen seeded with MSCs Collagen seeded with MSCs rASCs encapsulated in collagen/PLGA-beta-TCP scaffold Collagen seeded with MSCs Acellular human dermal tissue matrix 3-dimensional hyaluronic acid and collagen Cross-linked acellular porcine dermal vs porcine small intestine submucosa FGF2/VEGF/heparin/collagen Acellular dermal tissue matrix Cross-linked acellular porcine dermal vs platelet-rich plasma fibrin matrix

In vivo In vivo In vivo

a

FGF2, fibroblast growth factor 2; MSC, mesenchymal stem cell; PLGA-beta-TCP, poly(lactide-co-glycolide)/b-tricalcium phosphate; rASC, rabbit adipose-derived stem cells; VEGF, vascular endothelial growth factor.

also been used in combination with stem cells1,2,7,11,13,16,2527,30 (Table 1). It is well known that collagen type I is the main component of healthy tendons. However, during natural tendon healing, inferior scar tissue mainly consisting of collagen type III is formed first. Later, during the remodeling phase, type III collagen is replaced by the mechanically more resistant type I collagen. All attempts to accelerate tendon healing have aimed to hasten the natural healing process by using either a guiding matrix (scaffold) or growth stimulus (growth factors, genes, stem cells). It is unclear whether the natural healing course can be accelerated by providing collagen type I and whether this type I collagen can directly be integrated, according to the collagen recycling theory,9 or needs to be remodeled as well. To the best of our knowledge, a simple off-the-shelf type I collagen sponge without any tendon-like parallel orientation of fibers just consisting of the main tendon component has not yet been investigated in tendon engineering.

For proper tendon function, tendon length and tendon width are important mechanical parameters. During healing, a weak tendon callus, mainly formed by collagen type III, bridges the tendon defect, resulting in an increased cross-sectional area. This weak callus tends to elongate during healing, which usually results in impaired function of the muscle-tendon unit.4,5,17,21,32 Therefore, tendon cross-sectional area, length, and mechanical properties (load to failure and stiffness) are important measures of the healing process.8,21,31 In this study, the tendons in the nonsponge group were found to be significant longer than those of the sponge group at 2 and 4 weeks, with an elongation of 2.9 mm over 4 weeks. In contrast, the length of the tendons receiving the collagen implant decreased with time. This observation might reflect the early strength of the tendons receiving the collagen implant, which resists stretching. However, the collagen-treated tendons remained within the length of native tendons throughout the observation

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period. The fact that the control tendons elongated over time is consistent with findings by Konerding et al,14 who showed that nonsutured Achilles tendons appeared elongated and more slender compared with Achilles tendons protected by suture material during healing. The present study further showed a dynamic pattern with a typical initial increase in tendon width. The crosssectional area of tendons in the nonsponge group increased progressively during the course of the 4-week experiment, whereas the cross-sectional area of the tendons receiving sponges remained high during the entire period of the study. In agreement with these cross-sectional area data, tendons receiving the collagen inserts were much stronger after 1 and 2 weeks than control tendons, showing significantly higher loads to failure at 1 week. However, after 4 weeks, the control tendons had caught up, and there was no significant difference between the groups. Similar observations were described by Krapf et al,15 who compared nonoperated to operated Achilles tendons in a rat model during healing. Searching for mechanical properties comparable with those of uninjured tendons, the collagen-treated tendons reached the desired high tear resistance already at 2 weeks. At this time point, the control tendons were still significantly weaker than both the native tendons and the collagen-treated tendons. It took 4 weeks in our study until the control group was not weaker than native tendons or the collagen group. Thus, the hypothesis that a collagen supply results in early higher tear resistance of healing tendons can be affirmed. Besides the constant length over time and the early high tear resistance of collagen type I–treated tendons, each in the range of native tendons, tendon stiffness of the collagen group appeared to be in the range of that of normal tendon tissue at 2 and 4 weeks as well, which again is remarkable and desirable for tendon engineering. Moreover, at 4 weeks, the control tendons showed significantly higher tendon stiffness, resulting in less elastic but stiffer tendons, putting them at a higher risk to snap. Because of the decreased stiffness of the control tendons at 1 and 2 weeks, these are less efficient in force transfer. The histological analysis did not show evidence of an inflammatory response to the collagen sponge. This is in agreement with the report of Buchaim et al,3 who clearly demonstrated the high compatibility of collagen scaffolds in human tissue. From prior investigations in rats, it is known that the main changes in tendon healing happen 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. Additional experiments will be needed to find out whether tendon cross-sectional area tends to normalize toward native tendons and whether control tendons become even stiffer throughout a longer follow-up, meaning a higher risk for reruptures. Moreover, clinical studies on collagen-augmented tendon repair in patients will be needed in the future to evaluate whether collagen also supports the healing potential of human tendons. In conclusion, the additional supply of collagen type I in healing rat Achilles tendons resulted in a higher crosssectional area (1-4 weeks) and load to failure (1 week)

Collagen Sponge for Tendon Repair

2003

but shorter length (2-4 weeks) and smaller final stiffness (4 weeks). Overall, tendon healing was accelerated by a type I collagen sponge. The mechanical properties of collagen-treated tendons appeared to be significantly closer to those of normal, uninjured tendons than control tendons without collagen treatment. Histologically, no adverse affects due to the collagen sponge were found, supporting the collagen sponge as a simple but expectant approach in tendon engineering.

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