Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine http://pih.sagepub.com/
Silk and collagen scaffolds for tendon reconstruction Soon-Yong Kwon, Jin-Wha Chung, Hee-Jung Park, Yuan-Yuan Jiang, Jung-Keug Park and Young-Kwon Seo Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine 2014 228: 388 originally published online 4 April 2014 DOI: 10.1177/0954411914528890 The online version of this article can be found at: http://pih.sagepub.com/content/228/4/388
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Original Article
Silk and collagen scaffolds for tendon reconstruction
Proc IMechE Part H: J Engineering in Medicine 2014, Vol. 228(4) 388–396 Ó IMechE 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0954411914528890 pih.sagepub.com
Soon-Yong Kwon1, Jin-Wha Chung1, Hee-Jung Park2, Yuan-Yuan Jiang2, Jung-Keug Park2 and Young-Kwon Seo2
Abstract In this study, silk thread (Bombyx mori) was braided to a tube-like shape and sericin was removed from the silk tube. Thereafter, collagen/chondroitin-6-sulfate solution was poured into the silk tube, and the lyophilization process was performed. To assess the inflammatory response in vivo, raw silk and sericin-free silk tubes were implanted in the subcutaneous layer of mice. After 10 days of in vivo implantation, mild inflammatory responses were observed around the sericin-free silk tubes, and severe inflammation with the presence of neutrophils and macrophages was observed around the raw silk tubes. At 24 weeks post implantation, the regenerated tendon had a thick, cylindrical, grayish fibrous structure and a shiny white appearance, similar to that of the native tendon in the rabbit model of tendon defect. The average tensile strength of the native tendons was 220 6 20 N, whereas the average tensile strength of the regenerated tendons was 167 6 30 N and the diameter of the regenerated tendon (3 6 0.2 mm) was similar to that of the native tendons (4 6 0.3 mm). Histologically, the regenerated tendon resembled the native tendon, and all the regenerated tissues showed organized bundles of crimped fibers. Masson trichrome staining was performed for detecting collagen synthesis, and it showed that the artificial tendon was replaced by new collagen fibers and extracellular matrix. However, the regenerated tendon showed fibrosis to a certain degree. In conclusion, the artificial tendon, comprising a braided silk tube and lyophilized collagen sponge, was optimal for tendon reconstruction. Thus, this study showed an improved regeneration of neo-tendon tissues, which have the structure and tensile strength of the native tendon, with the use of the combination of collagen and silk scaffold.
Keywords Silk tube, collagen, composite scaffold, artificial tendon
Date received: 14 March 2013; accepted: 21 February 2014
Introduction Due to the increase in the popularity of sports, such as football, handball, and ice hockey, as well as due to the increase in the number of traffic accidents, the number of patients presenting with ligament and tendon injuries has increased in the recent years. The tendon healing process can last for several months, and the repaired tendon can achieve about 60% of the function.1 Each year in the United States, more than 300,000 rotator cuff reconstructions are performed after rotator cuff tears.2,3 In order to improve tendon repair, various surgical techniques using sutures and soft tissue anchors4 and some alternative therapies including biological grafts5,6 and nonbiodegradable artificial scaffolds are being employed.7,8 Although an autograft is ideal for the reconstruction of a tendon defect, very few tendons, such as palmaris longus and plantaris, can be harvested
for repairing tendon defects without causing a secondary functional disability. However, these extrasynovial tendons are known to cause more adhesions and scarring than intrasynovial tendons, and an additional surgery may also be necessary for releasing the scarred tendons.9 Donor site morbidity and availability can also be problematic. Therefore, if the immune reaction can be overcome, tendon allografts10 or xenografts can be the treatment option in the future.
1
Department of Orthopaedic Surgery, St. Mary’s Hospital, Seoul, Korea Department of Medical Biotechnology, Dongguk University, Seoul, Korea
2
Corresponding author: Young-Kwon Seo, Department of Medical Biotechnology, Dongguk University, 30, Pildong-ro l-gil Jung-gu, Seoul, 100 715, Korea. Email: [email protected]
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Recently, a new tissue engineering strategy has been developed for tendon and ligament reconstruction. The ideal scaffold should be biodegradable, porous, and biocompatible; should possess adequate mechanical strength; and should be able to promote the formation of ligament and tendon tissues. Tissue regeneration with the use of tissue engineering techniques not only restores the function and action of the defective tissue but also repairs the original tissue before it reaches the defect point. The synthetic nonbiodegradable scaffold currently available plays a role in mechanical function for a short period of time, but it eventually ruptures. Biodegradable ligaments and tendons do not possess both satisfactory biocompatibility and favorable physical properties. But the results of recent researches using silk materials were satisfactory in terms of biocompatibility and physical properties.11 Because of its high strength, toughness, and elasticity, silk has been used in various applications for clinical repair and in vitro tissue engineering, such as in bone repair,12,13 corneal and blood vessel tissue scaffolds,14,15 nerve regeneration,16 and ligament and tendon tissue engineering. But sufficient attachment or growth of cells is not achieved with the use of silk material alone. Therefore, Chen et al.17 tried to use an adhesion sequence or protein, and they found that arginine–glycine–aspartate (RGD)-binding silk matrices supported improved bone marrow stromal cell attachment and showed a higher cell density when compared with silk matrices in vitro. Studies were conducted for developing a structurally complex scaffold by using a synergistic combination of silk threads with porous silk sponges,18,19 and the results demonstrated that silk scaffolds had great potential in clinical applications. In the previous work of authors, collagen sponges were incorporated into a knitted silk to form a composite scaffold for ligament reconstruction. The knitted silk provided sufficient mechanical strength, while collagen sponges encouraged ingrowth of cells and enhanced neo-ligament regeneration by promoting extracellular matrix (ECM) secretion and angiogenesis in dog and rabbit models.20,21 Additionally, Chen et al. and Shen et al. investigated the use of a composite silk scaffold, which was composed of knitted silk and ECM, for ligament and tendon regeneration.22,23 The aim of this study was to generate a microporous scaffold for tendon tissues and to test the hypothesis that the combined collagen–silk scaffold may be suitable for tendon reconstruction. In the previous study, it was shown that knitted silk scaffolds covered by collagen sponge possess good internal communicating spaces and favor deposition of collagenous connective tissue matrix, which are crucial for ligament reconstruction in a dog model.20 The major matrix component of the tendon is collagen, and therefore, collagen sponge was used instead of silk sponge. Although the previously designed composite silk scaffold could improve ligament repair and reconstruction, because of the
roughness of orthopedic surgery compared to any another surgery, significant loss of collagen matrix occurred during surgery. Hence, there is a need to develop a composite scaffold for maintaining the stability of the structure during surgery. The fabrication of this composite silk scaffold was possible due to introduction of microporous silk sponges into its tube hole using the freeze-drying method. With the use of this technique, the issues associated with collagen and ECM loss could be overcome. In this study, silk thread was braided into a tube-like shape, and then collagen/chondroitin-6-sulfate (CS) solution was poured into the silk tube and the lyophilization process was performed. The tube-like shape of the braided silk fiber provided mechanical strength and protected the internal space of the collagen sponge. Thereafter, tendon repair was evaluated in a rabbit model.
Materials and methods Preparation of silk tubes and their implantation in mice Raw silk fibers (Bombyx mori) were purchased from Won Corporation (Seoul, South Korea), and silk tubes were made using a braiding machine. Scaffolds were prepared by removing the sericin (a glue-like protein that coats the native silk fiber) using an aqueous solution containing 0.02 M Na2CO3 and 0.3% IvoryÒ detergent.24 Four-week-old, male, BALB/c mice (n = 4) were used for the in vivo biocompatibility testing of raw silk and degummed silk. All the operations were performed under general anesthesia using ZoletilÒ injections (0.4 mL/kg; Virbac Laboratories, Carros, France) mixed with RompunÒ (10 mg/kg; Bayer Korea Ltd, Seoul, Korea). Hematoxylin and eosin (H&E) staining of the subcutaneous layer was evaluated at 10 days after implantation.
Preparation of the artificial tendon A type I atelocollagen powder (Bioland Corp., Cheongwon, Chungbuk, Korea) was dissolved in 0.001 N HCl at a concentration of 20 mg/mL, and CS (Sigma Chemical Company, St Louis, MO, USA) at a concentration of 2 mg/mL was added to the collagen solution. The silk tubes were then placed in molds and collagen/CS solution was injected into each mold. Then, they were lyophilized by freeze-drying (Samwon Freezing Engineering Co., Busan, South Korea) at 280 °C for 48 h to obtain artificial tendons. The composite silk scaffolds were incubated in 20 mL of 40% (v/v) ethanol containing 50 mM 2-(N-morpholino)ethanesulfonic acid (MES; Fluka Chemie AG, Buchs, Switzerland) for 30 min at room temperature and then immersed in 20 mL of 40% (v/v) ethanol containing 50 mM MES (pH 5.5), 24 mM 1-ethyl-3-(3-
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Figure 1. An Achilles tendon defect was created by excision at 2 cm above its (a, b) insertion site and an artificial tendon was implanted in the left rear paw (c, d, blue is the suture).
dimethylaminopropyl)carbodiimide (Fluka Chemie AG), and 5 mM N-hydroxysuccinimide (Fluka Chemie AG) for 12 h at room temperature. When the reaction was completed, the artificial tendons were washed twice in 0.1 M Na2HPO4 (pH 9.0) for 12 h, in 1 M NaCl for 6 h, in 2 M NaCl for 2 days, and finally rinsed with distilled water.25 Thereafter, the artificial tendons were lyophilized by freeze-drying and sterilized with g-irradiation at 15 kGy. The dimensions of the scaffold used in this study were ‘‘50 mm length and 3 mm diameter’’ (Figure 1).
Scanning electron microscopy For scanning electron microscopy (SEM; JSM-840A; JEOL Inc., Tokyo, Japan), scaffolds were fixed in a mixture of 4% glutaraldehyde and 2% formaldehyde in phosphate buffer (0.2 M mixture of monosodium phosphate and dipotassium phosphate, pH 7.4) for 2 h at room temperature. After thoroughly rinsing with 0.175 M phosphate buffer, specimens were immersed in 2% osmium tetroxide buffered with 0.2 M phosphate for 2 h, dehydrated with hexamethyldisilazane (HMDS; BioRad, Somerset, UK), sputter-coated with goldpalladium (E-5400; Bio-Rad), and observed by SEM at 15 kV.
Institutional Animal Care and Use Committee of Catholic University Hospital. The rabbits were randomly divided into the two groups, one is the control group and the other group was for Achilles tendon defect repair by silk scaffold. Macroscopic, histologic, and cell culture findings in the two study groups were compared with respect to appropriate tendon healing. The rabbits were anesthetized with an intramuscular injection of 1 mg/kg of Zoletil (Virbac SA, Carros cedex, France) mixed with Rompun (Bayer Korea Ltd) at a 1:1 ratio. Following anesthesia, left rear paws were anesthetized by administering 2% lidocaine with 1:100,000 epinephrine. The Achilles tendon was meticulously dissected from the adjacent tissues and transected approximately 1.5 cm above the calcaneal attachment (Figure 2(a)). The Achilles tendons (1.5 cm in length) in the left rear paws were excised. An artificial tendon was grafted and sutured with a 4-0 nonabsorbable monofilament using the pulvertaft repair technique (Figure 2(c)). At 24 weeks after implantation, the rabbits were sacrificed, and the specimens harvested from the left rear paws were examined by H&E and Masson’s trichrome (MT) staining. The degree of the inflammatory response and the level of collagen synthesis were compared.
Mechanical strength test Animal experiments In total, 20 adult New Zealand white rabbits (3.0 kg) were acclimated in a housing facility (1 rabbit/cage, 50% relative humidity, 12 h light/dark cycle) for 1 week after obtaining the study approval from the
To compare the mechanical properties after implantation, tensile strength test was performed at 6 months after transplantation. The regenerated tendons and normal Achilles tendons were tested using a material testing machine (H5KT; Salfords Redhill, Hounsfield,
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Figure 2. SEM of (a) raw silk and (b) sericin-free silk tubes. After 10 days of implantation into the subcutaneous layer of mice, (c) raw silk tubes containing sericin induced a more significant inflammatory reaction compared to (d) sericin-free silk tubes (original magnification: (a, b) 10003 and (c, d) 2003).
UK). For the test, the gage length (starting length) was set to 10 mm and the diameter of the used specimen was 5 mm. The crosshead speed was set to 60 mm/min (10% strain rate), and the chart speed was set to 60 mm/min. The ultimate tensile strength (N) was directly measured from the force–displacement graph.
Statistical analysis Data concerning the cell growth in vitro were evaluated using the Student’s t-test. The results are presented as means 6 standard deviations (SDs). Statistical significance was accepted for p values \ 0.05.
Results and discussion Various types of biomaterials for ligament and tendon repair have been developed and are being utilized. Biological responses to the proteins have raised questions regarding the biocompatibility of these materials. After implantation, host–material interactions that occur at the site of implantation can cause adverse inflammatory reactions. All the biomaterials derived from a nonautologous source will cause some degree of foreign body response after implantation in vivo. In this study, the collagen/CS substrate played an important role in increasing the cell attachment and proliferation. Collagen is known as a biocompatible and cytotropic natural material containing RGD sequences, and therefore, it has been utilized in
various clinical treatments, such as in artificial skin and injectable materials. However, the physical properties of collagen are very poor, and hence, it cannot bear the mechanical load of the materials. The artificial tendon designed in this study had the mechanical properties of a silk fiber, and lyophilized collagen/CS substrate increased the adhesion and proliferation of cells. Silk fibers from the B. mori silkworm have been used as surgical sutures for centuries. The main constituents of silk in its natural form are fibroin and sericin.26 Silk fibroin, the fibrous component, is one of the most studied structural proteins, and sericin is a group of glue proteins produced in the middle silk gland of the silkworm. The surface morphologies of raw silk tubes were observed with SEM. After sericin removal, the surface of the degummed silk fibers (Figure 3(b)) had an average diameter of about 500 mm and appeared smoother than the raw silk fibers (Figure 3(a)). To assess the inflammatory response in vivo, raw silk and sericin-free silk tubes were implanted in the subcutaneous layer of mice. After 10 days of in vivo implantation, mild inflammatory responses were observed around the sericin-free silk tubes (Figure 3(d)), but severe inflammation was noted around the raw silk tubes based on the presence of neutrophils and macrophages (Figure 3(c)). These ricin residues were visualized under SEM. Due to the presence of ricin residues, the silk fibers may not provide the desired effect in clinical
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Figure 3. (a) Appearance of the artificial tendon, (b) SEM of the cross section, and (c) end area of the artificial tendon (middle area: collagen sponge, original magnification: (b) 303 and (c) 403).
applications. Sericins have been described as the main cause of undesirable immunological responses.27 Liu et al. reported that the removal of sericin from the silk fibers causes a change in surface properties, and the sericin-free fibers showed a much higher water-binding and aqueous swelling ability. The loose and porous three-dimensional (3D) structure of silk fibers after degumming significantly expanded the surface area.28 Also, many investigators reported that sericin-free silk fibers are biocompatible in both the in vivo and in vitro cell culture models.29–31 The major concern with the use of silk for biomedical applications is the immune responses generated by sericin, and hence, the process of degumming is very important. A twisted, braided, and knitted silk scaffold has already been investigated in ligament or tendon reconstruction. However, braided or twisted scaffolds often encounter problems of poor cell attachment and migration, infiltration, and matrix production.17 In order to overcome these problems, several researchers have attempted to use a cell sheet technology or a composite technique for tissue engineering. Ouyang et al. demonstrated the use of cell sheet engineering technique for developing a tissue-engineered ligament. Mesenchymal stem cells (MSCs) were cultured to form a cell sheet, and it was assembled onto the knitted scaffold by a wrapping technique.32 However, these cells take a long time to form cell sheets. Other investigators developed a combined scaffold with microporous silk sponge or collagen sponges that were formed in the openings of a knitted or twisted scaffold to produce a composite matrix for direct cell seeding.18,19,22,23 Fan et al. and Liu et al. immersed the knitted silk scaffold in the silk solution and freeze-dried to allow formation of the microporous silk sponge in the opening of knitted silk matrix.18,19 Also, Chen et al. and Shen et al. developed a combined scaffold by incorporation of silk fibers, a knitted structure, and a microporous collagen matrix, which was very similar in structure when compared with the composite silk matrix developed by Goh.22,23 But such a combined 3D structure could not protect the collagen sponge because exposed collagen structures have weak properties for touch or pressure during the surgery.
Hence, this study tried to overcome these issues. The silk thread provided a braided tube-like structure, collagen/CS solution was injected into the silk tube, and it was then lyophilized. The braided artificial tendon had a tube-like structure, which was similar to that of the natural tendon. SEM imaging of the artificial tendon revealed that both the surface and internal spaces of the braided silk tubes were filled with collagen microsponge. The silk fibers, which were arranged in bundles, curled up in the scaffold (Figure 1(a)) and increased the mechanical strength. The collagen sponge caused an increased cell migration toward the inner layer of the artificial tendon (Figure 1(b)). Because the basic elements of tendons are collagen bundles (mostly type I collagen), tenocytes, and ECM, which are rich in proteoglycans (PGs), collagen appears to be the ideal foundation for artificial tendons.33 After implantation, three rabbits died due to wound infections, and other rabbits did not show any infection or ulceration. Also, no evidence of rupture progression was observed in any of the study groups at 24 weeks after implantation, although all the groups showed signs of mild synovitis. The specimens harvested at 24 weeks after implantation had a thick, cylindrical, grayish fibrous structure and a shiny white appearance (Figure 4(b)), similar to that of the natural tendon (Figure 4(a)). Also, the silk fiber could be discerned in the regenerated tendons until this time. Previously, other researchers reported that a silk sponge incorporated into a knitted silk mesh was implanted in the anterior cruciate ligament (ACL) of pigs, and the regenerated ligament resembled the native ACL. However, the silk fiber could not be discerned at 24 weeks postoperative.18 Silk is classified as a nondegradable material, but several studies demonstrated that the degradation time of silk is different depending on the type of animals and implantation site, and the degradation is usually mediated by an in vivo foreign body response. In general, silk fibers lose the majority of their tensile strength within 5 months in vivo, and their recognition in the implantation site is not possible within 2 years.34,35 Some investigators reported about the use of absorbable scaffolds such as polylactic acid (PLA), polycaprolactone (p-CL), and chitin scaffolds for tendon
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Figure 4. After transplantation of rabbit Achilles tendon tensile strength was measured. (a) Gross appearance of the native Achilles tendon and (b) grafted artificial tendon 24 weeks after implantation in the Achilles tendon defect of rabbits. The appearance of the regenerated tendon in the artificial tendon graft group was very similar to that of the native Achilles tendon. (e) Mechanical testing of the regenerated tendon (c, d: after tensile strength test).
reconstruction. Both PLA and the chitin/p-CL composite scaffolds showed a good initial tensile strength and an increased ingrowth of fibrous tissue.36 But PLA scaffolds showed a significant loss of tensile strength after 17 weeks. After 26 weeks, their tensile strength was only 27 6 9 N. Also, p-CL scaffolds showed a significant loss of tensile strength, and their tensile strength was only 109 6 8 N after 4 weeks. Normally, the mean tensile strength of rabbit tendons is 222 6 12.7 N.37 Figure 5 shows the data on mechanical strength of the artificial tendon at 24 weeks after implantation. The biomechanical tests indicated differences or similarities in the tensile properties between the healing tendons and the native tendons. Tensile strength is the maximum load that the tendon is able to withstand before rupturing, and it is measured in Newtons. The average tensile strength of the native tendons was 220 6 20 N, whereas the average tensile strength of the regenerated tendons was 167 6 30 N (n = 5) (Table 1). The mean tensile strength of the regenerated tendon was lower than that of the native tendon (p \ 0.05). Also, the diameter of the regenerated tendon (3 6 0.2 mm) was similar to that of the native tendon (4 6 0.3 mm), but the diameter of the regenerated tendon was decreased to some extent. These data showed that the tensile strength of the regenerated tendons recovered to almost 75% of that of the original tendon.
Table 1. Biomechanical properties of the regenerated tendon and the native tendon (n = 5). Biomechanical measurement
Regenerated tendon
Native tendon
Tensile strength (N) Stiffness (N/mm) Tensile stress (MPa)
167 6 30 15.8 6 3.2 22.1 6 3.0
220 6 20 11.4 6 2.0 15.9 6 2.0
Tensile stress indicates the maximum load per unit cross-sectional area of the tendon. The tensile stress at maximum load was 22.1 6 3.2 MPa for the regenerated tendon and 15.9 6 2 MPa for the native tendon. Also, the mean tensile stiffness of the regenerated tendon in the scaffold graft group (n = 5) and the native tendon group (n = 5) was 15.8 6 3.2 and 11.4 6 2 N/mm, respectively. The mean tensile stiffness of the regenerated tendon was higher than that of the native tendon (p \ 0.05). The tensile stiffness and stress of the regenerated tendon were higher than those of the native tendon, and these results indicated that the regenerated tendon may contain some amount of fibrosis tissue and silk scaffolds. Minimal inflammatory responses indicated by the presence of monocytes were observed around the silk fibers at 24 weeks post implantation and no foreign
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Figure 5. Comparison of mechanical properties between (a) the native tendon and the (b) regenerated tendon using an artificial tendon graft after 24 weeks. The tensile strength values are similar to native tendon and regenerated tendon (c).
body reaction was observed (Figure 6(d)). The histological appearance of the regenerated tendon was similar to that of the native tendon, and the completely regenerated tendon showed organized bundles of crimped fibers. MT staining was performed to detect collagen synthesis, and it was observed that the artificial tendon was replaced by new collagen fibers (blue color) and ECM. However, some of the regenerated tendons showed fibrosis. Collagen fibers are oriented in one direction. According to Kannus,38 collagen fibers in tendons are oriented not only longitudinally but also transversely and horizontally to form a 3D ultrastructure, and these findings indicate that the mechanical strength of artificial tendons depends on the orientation of collagen fibers.39 But in this study, the collagen sponge was produced by the lyophilization method only, without a collagen bundle and orientation of collagen fibers. The implanted collagen matrix was degraded by fibroblastic cells and macrophages, and the new collagen bundles and the direction of ECM were regenerated by tenocytes. A significant finding of this study was that silk tubes combined with collagen scaffolds were promising as artificial tendons.
Conclusion Maintenance of the mechanical properties of the scaffold until the newly regenerated tendons become mechanically competent is an important requirement for designing the scaffold that is used for regeneration of load-bearing tissues such as ligaments and tendons. For reconstruction, it is important to develop an implant that will resist the large amount of in vivo forces during the early part of the surgery when there is increasing cell migration.19 To design an effective artificial tendon, it is important to understand the mechanical properties and biocompatibility of the scaffolds. In this study, the in vitro cell cultures and biomechanical manifestations after grafting were examined in healing rabbit Achilles tendons during tissue remodeling. The artificial tendon used in this study was composed of braided silk tube and lyophilized collagen sponge, and it was found to be optimal for tendon reconstruction. This study demonstrated that the combination of collagen and silk scaffold improved the neo-tendon tissue regeneration, and this regenerated tendon had a similar structure and tensile strength to that of the native tendon. The collagen sponge was able to preserve the internal space in the
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Figure 6. (a, d) H&E and (b, c, e, f) MT staining of the implants at 24 weeks after surgery. The native tendon was populated with tenocytes and it showed the direction of orientation of collagen fibers (a). Thicker and dense collagen (c, blue color) was observed in the native tendon compared to that in the regenerated tendon using an artificial tendon graft (f) (original magnification: (a, d) 2003; (b, e) 403; (c, f) 2003).
silk tube scaffold under physical loading to allow the ingrowth of the neo-tendon tissue, and the silk tube scaffold provided mechanical support to resist the physical load. We believe that development of an artificial tendon is a realistic and worthwhile landmark that will advance tissue engineering. These artificial tendons are useful for tendon reconstruction, as seen in this study, and they may help to resolve the issues related to the mechanical properties, biocompatibility, and tendon regeneration in the future. But further investigations on the in vivo healing effect of these artificial tendons in a dog model are needed to evaluate the clinical relevance of these scaffolds. Declaration of conflicting interests The authors declare that there is no conflict of interest. Funding This work was supported by a grant from the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (0405-BO01-0204-0006). References 1. Pennisi E. Tending tender tendons. Science 2002; 295: 1011. 2. Audenaert E, Van Nuffel J, Schepens A, et al. Reconstruction of massive rotator cuff lesions with a synthetic interposition graft: a prospective study of 41 patients. Knee Surg Sports Traumatol Arthrosc 2006; 14(4): 360–364.
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25. Pieper JS, Oosterhof A, Dijkstra PJ, et al. Development of tailor-made collagen-glycosaminoglycan matrices: EDC/NHS crosslinking, and ultrastructural aspects. Biomaterials 2000; 21: 581–593. 26. Rajkhowa R, Hu X, Tsuzuki T, et al. Structure and biodegradation mechanism of milled Bombyx mori silk particles. Biomacromolecules 2012; 13(8): 2503–2512. 27. Dewair M, Baur X and Ziegler K. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk proteins. J Allergy Clin Immunol 1985; 76(4): 537–542. 28. Liu H, Ge Z, Wang Y, et al. Modification of sericin-free silk fibers for ligament tissue engineering application. J Biomed Mater Res B Appl Biomater 2007; 82(1): 129–138. 29. Meinel L, Hofmann S, Karageorgiou V, et al. The inflammatory responses to silk films in vitro and in vivo. Biomaterials 2005; 26(2): 147–155. 30. Panilaitis B, Altman GH, Chen J, et al. Macrophage responses to silk. Biomaterials 2003; 24(18): 3079–3085. 31. Wang X, Qiu Y, Carr AJ, et al. Improved human tenocyte proliferation and differentiation in vitro by optimized silk degumming. Biomed Mater 2011; 6(3): 035010. 32. Ouyang HW, Toh SL, Goh J, et al. Assembly of bone marrow stromal cell sheets with knitted poly (L-lactide) scaffold for engineering ligament analogs. J Biomed Mater Res B Appl Biomater 2005; 75(2): 264–271. 33. Franchi M, Trire` A, Quaranta E M, et al. Collagen structure of tendon relates to function. Scientific World Journal 2007; 30: 404–420. 34. Altman GH, Horan RL, Lu HH, et al. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002; 23: 4131–4141. 35. Postlethwait RW. Long-term comparative study of nonabsorbable sutures. Ann Surg 1970; 171: 892–898. 36. Sato M, Maeda M, Kurosawa H, et al. Reconstruction of rabbit Achilles tendon with three bioabsorbable materials: histological and biomechanical studies. J Orthop Sci 2000; 5(3): 256–267. 37. Reddy GK, Stehno-Bittel L and Enwemeka CS. Matrix remodeling in healing rabbit Achilles tendon. Wound Repair Regen 1999; 7(6): 518–527. 38. Kannus P. Structure of the tendon connective tissue. Scand J Med Sci Sports 2000; 10: 312–320. 39. Iannace S, Sabatini G, Ambrosio L, et al. Mechanical behaviour of composite artificial tendons and ligaments. Biomaterials 1995; 16: 675–680.
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Silk and collagen scaffolds for tendon reconstruction Soon-Yong Kwon, Jin-Wha Chung, Hee-Jung Park, Yuan-Yuan Jiang, Jung-Keug Park and Young-Kwon Seo Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine 2014 228: 388 originally published online 4 April 2014 DOI: 10.1177/0954411914528890 The online version of this article can be found at: http://pih.sagepub.com/content/228/4/388
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Original Article
Silk and collagen scaffolds for tendon reconstruction
Proc IMechE Part H: J Engineering in Medicine 2014, Vol. 228(4) 388–396 Ó IMechE 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0954411914528890 pih.sagepub.com
Soon-Yong Kwon1, Jin-Wha Chung1, Hee-Jung Park2, Yuan-Yuan Jiang2, Jung-Keug Park2 and Young-Kwon Seo2
Abstract In this study, silk thread (Bombyx mori) was braided to a tube-like shape and sericin was removed from the silk tube. Thereafter, collagen/chondroitin-6-sulfate solution was poured into the silk tube, and the lyophilization process was performed. To assess the inflammatory response in vivo, raw silk and sericin-free silk tubes were implanted in the subcutaneous layer of mice. After 10 days of in vivo implantation, mild inflammatory responses were observed around the sericin-free silk tubes, and severe inflammation with the presence of neutrophils and macrophages was observed around the raw silk tubes. At 24 weeks post implantation, the regenerated tendon had a thick, cylindrical, grayish fibrous structure and a shiny white appearance, similar to that of the native tendon in the rabbit model of tendon defect. The average tensile strength of the native tendons was 220 6 20 N, whereas the average tensile strength of the regenerated tendons was 167 6 30 N and the diameter of the regenerated tendon (3 6 0.2 mm) was similar to that of the native tendons (4 6 0.3 mm). Histologically, the regenerated tendon resembled the native tendon, and all the regenerated tissues showed organized bundles of crimped fibers. Masson trichrome staining was performed for detecting collagen synthesis, and it showed that the artificial tendon was replaced by new collagen fibers and extracellular matrix. However, the regenerated tendon showed fibrosis to a certain degree. In conclusion, the artificial tendon, comprising a braided silk tube and lyophilized collagen sponge, was optimal for tendon reconstruction. Thus, this study showed an improved regeneration of neo-tendon tissues, which have the structure and tensile strength of the native tendon, with the use of the combination of collagen and silk scaffold.
Keywords Silk tube, collagen, composite scaffold, artificial tendon
Date received: 14 March 2013; accepted: 21 February 2014
Introduction Due to the increase in the popularity of sports, such as football, handball, and ice hockey, as well as due to the increase in the number of traffic accidents, the number of patients presenting with ligament and tendon injuries has increased in the recent years. The tendon healing process can last for several months, and the repaired tendon can achieve about 60% of the function.1 Each year in the United States, more than 300,000 rotator cuff reconstructions are performed after rotator cuff tears.2,3 In order to improve tendon repair, various surgical techniques using sutures and soft tissue anchors4 and some alternative therapies including biological grafts5,6 and nonbiodegradable artificial scaffolds are being employed.7,8 Although an autograft is ideal for the reconstruction of a tendon defect, very few tendons, such as palmaris longus and plantaris, can be harvested
for repairing tendon defects without causing a secondary functional disability. However, these extrasynovial tendons are known to cause more adhesions and scarring than intrasynovial tendons, and an additional surgery may also be necessary for releasing the scarred tendons.9 Donor site morbidity and availability can also be problematic. Therefore, if the immune reaction can be overcome, tendon allografts10 or xenografts can be the treatment option in the future.
1
Department of Orthopaedic Surgery, St. Mary’s Hospital, Seoul, Korea Department of Medical Biotechnology, Dongguk University, Seoul, Korea
2
Corresponding author: Young-Kwon Seo, Department of Medical Biotechnology, Dongguk University, 30, Pildong-ro l-gil Jung-gu, Seoul, 100 715, Korea. Email: [email protected]
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Recently, a new tissue engineering strategy has been developed for tendon and ligament reconstruction. The ideal scaffold should be biodegradable, porous, and biocompatible; should possess adequate mechanical strength; and should be able to promote the formation of ligament and tendon tissues. Tissue regeneration with the use of tissue engineering techniques not only restores the function and action of the defective tissue but also repairs the original tissue before it reaches the defect point. The synthetic nonbiodegradable scaffold currently available plays a role in mechanical function for a short period of time, but it eventually ruptures. Biodegradable ligaments and tendons do not possess both satisfactory biocompatibility and favorable physical properties. But the results of recent researches using silk materials were satisfactory in terms of biocompatibility and physical properties.11 Because of its high strength, toughness, and elasticity, silk has been used in various applications for clinical repair and in vitro tissue engineering, such as in bone repair,12,13 corneal and blood vessel tissue scaffolds,14,15 nerve regeneration,16 and ligament and tendon tissue engineering. But sufficient attachment or growth of cells is not achieved with the use of silk material alone. Therefore, Chen et al.17 tried to use an adhesion sequence or protein, and they found that arginine–glycine–aspartate (RGD)-binding silk matrices supported improved bone marrow stromal cell attachment and showed a higher cell density when compared with silk matrices in vitro. Studies were conducted for developing a structurally complex scaffold by using a synergistic combination of silk threads with porous silk sponges,18,19 and the results demonstrated that silk scaffolds had great potential in clinical applications. In the previous work of authors, collagen sponges were incorporated into a knitted silk to form a composite scaffold for ligament reconstruction. The knitted silk provided sufficient mechanical strength, while collagen sponges encouraged ingrowth of cells and enhanced neo-ligament regeneration by promoting extracellular matrix (ECM) secretion and angiogenesis in dog and rabbit models.20,21 Additionally, Chen et al. and Shen et al. investigated the use of a composite silk scaffold, which was composed of knitted silk and ECM, for ligament and tendon regeneration.22,23 The aim of this study was to generate a microporous scaffold for tendon tissues and to test the hypothesis that the combined collagen–silk scaffold may be suitable for tendon reconstruction. In the previous study, it was shown that knitted silk scaffolds covered by collagen sponge possess good internal communicating spaces and favor deposition of collagenous connective tissue matrix, which are crucial for ligament reconstruction in a dog model.20 The major matrix component of the tendon is collagen, and therefore, collagen sponge was used instead of silk sponge. Although the previously designed composite silk scaffold could improve ligament repair and reconstruction, because of the
roughness of orthopedic surgery compared to any another surgery, significant loss of collagen matrix occurred during surgery. Hence, there is a need to develop a composite scaffold for maintaining the stability of the structure during surgery. The fabrication of this composite silk scaffold was possible due to introduction of microporous silk sponges into its tube hole using the freeze-drying method. With the use of this technique, the issues associated with collagen and ECM loss could be overcome. In this study, silk thread was braided into a tube-like shape, and then collagen/chondroitin-6-sulfate (CS) solution was poured into the silk tube and the lyophilization process was performed. The tube-like shape of the braided silk fiber provided mechanical strength and protected the internal space of the collagen sponge. Thereafter, tendon repair was evaluated in a rabbit model.
Materials and methods Preparation of silk tubes and their implantation in mice Raw silk fibers (Bombyx mori) were purchased from Won Corporation (Seoul, South Korea), and silk tubes were made using a braiding machine. Scaffolds were prepared by removing the sericin (a glue-like protein that coats the native silk fiber) using an aqueous solution containing 0.02 M Na2CO3 and 0.3% IvoryÒ detergent.24 Four-week-old, male, BALB/c mice (n = 4) were used for the in vivo biocompatibility testing of raw silk and degummed silk. All the operations were performed under general anesthesia using ZoletilÒ injections (0.4 mL/kg; Virbac Laboratories, Carros, France) mixed with RompunÒ (10 mg/kg; Bayer Korea Ltd, Seoul, Korea). Hematoxylin and eosin (H&E) staining of the subcutaneous layer was evaluated at 10 days after implantation.
Preparation of the artificial tendon A type I atelocollagen powder (Bioland Corp., Cheongwon, Chungbuk, Korea) was dissolved in 0.001 N HCl at a concentration of 20 mg/mL, and CS (Sigma Chemical Company, St Louis, MO, USA) at a concentration of 2 mg/mL was added to the collagen solution. The silk tubes were then placed in molds and collagen/CS solution was injected into each mold. Then, they were lyophilized by freeze-drying (Samwon Freezing Engineering Co., Busan, South Korea) at 280 °C for 48 h to obtain artificial tendons. The composite silk scaffolds were incubated in 20 mL of 40% (v/v) ethanol containing 50 mM 2-(N-morpholino)ethanesulfonic acid (MES; Fluka Chemie AG, Buchs, Switzerland) for 30 min at room temperature and then immersed in 20 mL of 40% (v/v) ethanol containing 50 mM MES (pH 5.5), 24 mM 1-ethyl-3-(3-
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Figure 1. An Achilles tendon defect was created by excision at 2 cm above its (a, b) insertion site and an artificial tendon was implanted in the left rear paw (c, d, blue is the suture).
dimethylaminopropyl)carbodiimide (Fluka Chemie AG), and 5 mM N-hydroxysuccinimide (Fluka Chemie AG) for 12 h at room temperature. When the reaction was completed, the artificial tendons were washed twice in 0.1 M Na2HPO4 (pH 9.0) for 12 h, in 1 M NaCl for 6 h, in 2 M NaCl for 2 days, and finally rinsed with distilled water.25 Thereafter, the artificial tendons were lyophilized by freeze-drying and sterilized with g-irradiation at 15 kGy. The dimensions of the scaffold used in this study were ‘‘50 mm length and 3 mm diameter’’ (Figure 1).
Scanning electron microscopy For scanning electron microscopy (SEM; JSM-840A; JEOL Inc., Tokyo, Japan), scaffolds were fixed in a mixture of 4% glutaraldehyde and 2% formaldehyde in phosphate buffer (0.2 M mixture of monosodium phosphate and dipotassium phosphate, pH 7.4) for 2 h at room temperature. After thoroughly rinsing with 0.175 M phosphate buffer, specimens were immersed in 2% osmium tetroxide buffered with 0.2 M phosphate for 2 h, dehydrated with hexamethyldisilazane (HMDS; BioRad, Somerset, UK), sputter-coated with goldpalladium (E-5400; Bio-Rad), and observed by SEM at 15 kV.
Institutional Animal Care and Use Committee of Catholic University Hospital. The rabbits were randomly divided into the two groups, one is the control group and the other group was for Achilles tendon defect repair by silk scaffold. Macroscopic, histologic, and cell culture findings in the two study groups were compared with respect to appropriate tendon healing. The rabbits were anesthetized with an intramuscular injection of 1 mg/kg of Zoletil (Virbac SA, Carros cedex, France) mixed with Rompun (Bayer Korea Ltd) at a 1:1 ratio. Following anesthesia, left rear paws were anesthetized by administering 2% lidocaine with 1:100,000 epinephrine. The Achilles tendon was meticulously dissected from the adjacent tissues and transected approximately 1.5 cm above the calcaneal attachment (Figure 2(a)). The Achilles tendons (1.5 cm in length) in the left rear paws were excised. An artificial tendon was grafted and sutured with a 4-0 nonabsorbable monofilament using the pulvertaft repair technique (Figure 2(c)). At 24 weeks after implantation, the rabbits were sacrificed, and the specimens harvested from the left rear paws were examined by H&E and Masson’s trichrome (MT) staining. The degree of the inflammatory response and the level of collagen synthesis were compared.
Mechanical strength test Animal experiments In total, 20 adult New Zealand white rabbits (3.0 kg) were acclimated in a housing facility (1 rabbit/cage, 50% relative humidity, 12 h light/dark cycle) for 1 week after obtaining the study approval from the
To compare the mechanical properties after implantation, tensile strength test was performed at 6 months after transplantation. The regenerated tendons and normal Achilles tendons were tested using a material testing machine (H5KT; Salfords Redhill, Hounsfield,
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Figure 2. SEM of (a) raw silk and (b) sericin-free silk tubes. After 10 days of implantation into the subcutaneous layer of mice, (c) raw silk tubes containing sericin induced a more significant inflammatory reaction compared to (d) sericin-free silk tubes (original magnification: (a, b) 10003 and (c, d) 2003).
UK). For the test, the gage length (starting length) was set to 10 mm and the diameter of the used specimen was 5 mm. The crosshead speed was set to 60 mm/min (10% strain rate), and the chart speed was set to 60 mm/min. The ultimate tensile strength (N) was directly measured from the force–displacement graph.
Statistical analysis Data concerning the cell growth in vitro were evaluated using the Student’s t-test. The results are presented as means 6 standard deviations (SDs). Statistical significance was accepted for p values \ 0.05.
Results and discussion Various types of biomaterials for ligament and tendon repair have been developed and are being utilized. Biological responses to the proteins have raised questions regarding the biocompatibility of these materials. After implantation, host–material interactions that occur at the site of implantation can cause adverse inflammatory reactions. All the biomaterials derived from a nonautologous source will cause some degree of foreign body response after implantation in vivo. In this study, the collagen/CS substrate played an important role in increasing the cell attachment and proliferation. Collagen is known as a biocompatible and cytotropic natural material containing RGD sequences, and therefore, it has been utilized in
various clinical treatments, such as in artificial skin and injectable materials. However, the physical properties of collagen are very poor, and hence, it cannot bear the mechanical load of the materials. The artificial tendon designed in this study had the mechanical properties of a silk fiber, and lyophilized collagen/CS substrate increased the adhesion and proliferation of cells. Silk fibers from the B. mori silkworm have been used as surgical sutures for centuries. The main constituents of silk in its natural form are fibroin and sericin.26 Silk fibroin, the fibrous component, is one of the most studied structural proteins, and sericin is a group of glue proteins produced in the middle silk gland of the silkworm. The surface morphologies of raw silk tubes were observed with SEM. After sericin removal, the surface of the degummed silk fibers (Figure 3(b)) had an average diameter of about 500 mm and appeared smoother than the raw silk fibers (Figure 3(a)). To assess the inflammatory response in vivo, raw silk and sericin-free silk tubes were implanted in the subcutaneous layer of mice. After 10 days of in vivo implantation, mild inflammatory responses were observed around the sericin-free silk tubes (Figure 3(d)), but severe inflammation was noted around the raw silk tubes based on the presence of neutrophils and macrophages (Figure 3(c)). These ricin residues were visualized under SEM. Due to the presence of ricin residues, the silk fibers may not provide the desired effect in clinical
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Figure 3. (a) Appearance of the artificial tendon, (b) SEM of the cross section, and (c) end area of the artificial tendon (middle area: collagen sponge, original magnification: (b) 303 and (c) 403).
applications. Sericins have been described as the main cause of undesirable immunological responses.27 Liu et al. reported that the removal of sericin from the silk fibers causes a change in surface properties, and the sericin-free fibers showed a much higher water-binding and aqueous swelling ability. The loose and porous three-dimensional (3D) structure of silk fibers after degumming significantly expanded the surface area.28 Also, many investigators reported that sericin-free silk fibers are biocompatible in both the in vivo and in vitro cell culture models.29–31 The major concern with the use of silk for biomedical applications is the immune responses generated by sericin, and hence, the process of degumming is very important. A twisted, braided, and knitted silk scaffold has already been investigated in ligament or tendon reconstruction. However, braided or twisted scaffolds often encounter problems of poor cell attachment and migration, infiltration, and matrix production.17 In order to overcome these problems, several researchers have attempted to use a cell sheet technology or a composite technique for tissue engineering. Ouyang et al. demonstrated the use of cell sheet engineering technique for developing a tissue-engineered ligament. Mesenchymal stem cells (MSCs) were cultured to form a cell sheet, and it was assembled onto the knitted scaffold by a wrapping technique.32 However, these cells take a long time to form cell sheets. Other investigators developed a combined scaffold with microporous silk sponge or collagen sponges that were formed in the openings of a knitted or twisted scaffold to produce a composite matrix for direct cell seeding.18,19,22,23 Fan et al. and Liu et al. immersed the knitted silk scaffold in the silk solution and freeze-dried to allow formation of the microporous silk sponge in the opening of knitted silk matrix.18,19 Also, Chen et al. and Shen et al. developed a combined scaffold by incorporation of silk fibers, a knitted structure, and a microporous collagen matrix, which was very similar in structure when compared with the composite silk matrix developed by Goh.22,23 But such a combined 3D structure could not protect the collagen sponge because exposed collagen structures have weak properties for touch or pressure during the surgery.
Hence, this study tried to overcome these issues. The silk thread provided a braided tube-like structure, collagen/CS solution was injected into the silk tube, and it was then lyophilized. The braided artificial tendon had a tube-like structure, which was similar to that of the natural tendon. SEM imaging of the artificial tendon revealed that both the surface and internal spaces of the braided silk tubes were filled with collagen microsponge. The silk fibers, which were arranged in bundles, curled up in the scaffold (Figure 1(a)) and increased the mechanical strength. The collagen sponge caused an increased cell migration toward the inner layer of the artificial tendon (Figure 1(b)). Because the basic elements of tendons are collagen bundles (mostly type I collagen), tenocytes, and ECM, which are rich in proteoglycans (PGs), collagen appears to be the ideal foundation for artificial tendons.33 After implantation, three rabbits died due to wound infections, and other rabbits did not show any infection or ulceration. Also, no evidence of rupture progression was observed in any of the study groups at 24 weeks after implantation, although all the groups showed signs of mild synovitis. The specimens harvested at 24 weeks after implantation had a thick, cylindrical, grayish fibrous structure and a shiny white appearance (Figure 4(b)), similar to that of the natural tendon (Figure 4(a)). Also, the silk fiber could be discerned in the regenerated tendons until this time. Previously, other researchers reported that a silk sponge incorporated into a knitted silk mesh was implanted in the anterior cruciate ligament (ACL) of pigs, and the regenerated ligament resembled the native ACL. However, the silk fiber could not be discerned at 24 weeks postoperative.18 Silk is classified as a nondegradable material, but several studies demonstrated that the degradation time of silk is different depending on the type of animals and implantation site, and the degradation is usually mediated by an in vivo foreign body response. In general, silk fibers lose the majority of their tensile strength within 5 months in vivo, and their recognition in the implantation site is not possible within 2 years.34,35 Some investigators reported about the use of absorbable scaffolds such as polylactic acid (PLA), polycaprolactone (p-CL), and chitin scaffolds for tendon
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Figure 4. After transplantation of rabbit Achilles tendon tensile strength was measured. (a) Gross appearance of the native Achilles tendon and (b) grafted artificial tendon 24 weeks after implantation in the Achilles tendon defect of rabbits. The appearance of the regenerated tendon in the artificial tendon graft group was very similar to that of the native Achilles tendon. (e) Mechanical testing of the regenerated tendon (c, d: after tensile strength test).
reconstruction. Both PLA and the chitin/p-CL composite scaffolds showed a good initial tensile strength and an increased ingrowth of fibrous tissue.36 But PLA scaffolds showed a significant loss of tensile strength after 17 weeks. After 26 weeks, their tensile strength was only 27 6 9 N. Also, p-CL scaffolds showed a significant loss of tensile strength, and their tensile strength was only 109 6 8 N after 4 weeks. Normally, the mean tensile strength of rabbit tendons is 222 6 12.7 N.37 Figure 5 shows the data on mechanical strength of the artificial tendon at 24 weeks after implantation. The biomechanical tests indicated differences or similarities in the tensile properties between the healing tendons and the native tendons. Tensile strength is the maximum load that the tendon is able to withstand before rupturing, and it is measured in Newtons. The average tensile strength of the native tendons was 220 6 20 N, whereas the average tensile strength of the regenerated tendons was 167 6 30 N (n = 5) (Table 1). The mean tensile strength of the regenerated tendon was lower than that of the native tendon (p \ 0.05). Also, the diameter of the regenerated tendon (3 6 0.2 mm) was similar to that of the native tendon (4 6 0.3 mm), but the diameter of the regenerated tendon was decreased to some extent. These data showed that the tensile strength of the regenerated tendons recovered to almost 75% of that of the original tendon.
Table 1. Biomechanical properties of the regenerated tendon and the native tendon (n = 5). Biomechanical measurement
Regenerated tendon
Native tendon
Tensile strength (N) Stiffness (N/mm) Tensile stress (MPa)
167 6 30 15.8 6 3.2 22.1 6 3.0
220 6 20 11.4 6 2.0 15.9 6 2.0
Tensile stress indicates the maximum load per unit cross-sectional area of the tendon. The tensile stress at maximum load was 22.1 6 3.2 MPa for the regenerated tendon and 15.9 6 2 MPa for the native tendon. Also, the mean tensile stiffness of the regenerated tendon in the scaffold graft group (n = 5) and the native tendon group (n = 5) was 15.8 6 3.2 and 11.4 6 2 N/mm, respectively. The mean tensile stiffness of the regenerated tendon was higher than that of the native tendon (p \ 0.05). The tensile stiffness and stress of the regenerated tendon were higher than those of the native tendon, and these results indicated that the regenerated tendon may contain some amount of fibrosis tissue and silk scaffolds. Minimal inflammatory responses indicated by the presence of monocytes were observed around the silk fibers at 24 weeks post implantation and no foreign
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Figure 5. Comparison of mechanical properties between (a) the native tendon and the (b) regenerated tendon using an artificial tendon graft after 24 weeks. The tensile strength values are similar to native tendon and regenerated tendon (c).
body reaction was observed (Figure 6(d)). The histological appearance of the regenerated tendon was similar to that of the native tendon, and the completely regenerated tendon showed organized bundles of crimped fibers. MT staining was performed to detect collagen synthesis, and it was observed that the artificial tendon was replaced by new collagen fibers (blue color) and ECM. However, some of the regenerated tendons showed fibrosis. Collagen fibers are oriented in one direction. According to Kannus,38 collagen fibers in tendons are oriented not only longitudinally but also transversely and horizontally to form a 3D ultrastructure, and these findings indicate that the mechanical strength of artificial tendons depends on the orientation of collagen fibers.39 But in this study, the collagen sponge was produced by the lyophilization method only, without a collagen bundle and orientation of collagen fibers. The implanted collagen matrix was degraded by fibroblastic cells and macrophages, and the new collagen bundles and the direction of ECM were regenerated by tenocytes. A significant finding of this study was that silk tubes combined with collagen scaffolds were promising as artificial tendons.
Conclusion Maintenance of the mechanical properties of the scaffold until the newly regenerated tendons become mechanically competent is an important requirement for designing the scaffold that is used for regeneration of load-bearing tissues such as ligaments and tendons. For reconstruction, it is important to develop an implant that will resist the large amount of in vivo forces during the early part of the surgery when there is increasing cell migration.19 To design an effective artificial tendon, it is important to understand the mechanical properties and biocompatibility of the scaffolds. In this study, the in vitro cell cultures and biomechanical manifestations after grafting were examined in healing rabbit Achilles tendons during tissue remodeling. The artificial tendon used in this study was composed of braided silk tube and lyophilized collagen sponge, and it was found to be optimal for tendon reconstruction. This study demonstrated that the combination of collagen and silk scaffold improved the neo-tendon tissue regeneration, and this regenerated tendon had a similar structure and tensile strength to that of the native tendon. The collagen sponge was able to preserve the internal space in the
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Figure 6. (a, d) H&E and (b, c, e, f) MT staining of the implants at 24 weeks after surgery. The native tendon was populated with tenocytes and it showed the direction of orientation of collagen fibers (a). Thicker and dense collagen (c, blue color) was observed in the native tendon compared to that in the regenerated tendon using an artificial tendon graft (f) (original magnification: (a, d) 2003; (b, e) 403; (c, f) 2003).
silk tube scaffold under physical loading to allow the ingrowth of the neo-tendon tissue, and the silk tube scaffold provided mechanical support to resist the physical load. We believe that development of an artificial tendon is a realistic and worthwhile landmark that will advance tissue engineering. These artificial tendons are useful for tendon reconstruction, as seen in this study, and they may help to resolve the issues related to the mechanical properties, biocompatibility, and tendon regeneration in the future. But further investigations on the in vivo healing effect of these artificial tendons in a dog model are needed to evaluate the clinical relevance of these scaffolds. Declaration of conflicting interests The authors declare that there is no conflict of interest. Funding This work was supported by a grant from the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (0405-BO01-0204-0006). References 1. Pennisi E. Tending tender tendons. Science 2002; 295: 1011. 2. Audenaert E, Van Nuffel J, Schepens A, et al. Reconstruction of massive rotator cuff lesions with a synthetic interposition graft: a prospective study of 41 patients. Knee Surg Sports Traumatol Arthrosc 2006; 14(4): 360–364.
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