Knee Surg Sports Traumatol Arthrosc DOI 10.1007/s00167-014-2923-7
SHOULDER
Rotator cuff repair using a decellularized tendon slices graft: an in vivo study in a rabbit model Juan Pan • Guo-Ming Liu • Liang-Ju Ning • Yi Zhang • Jing-Cong Luo • Fu-Guo Huang • Ting-Wu Qin
Received: 7 March 2013 / Accepted: 20 February 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Although varieties of surgical repair techniques and materials have been used to repair rotator cuff defects, re-tearing frequently occurs. The purpose of this study is to evaluate the postoperative outcomes of rotator cuff repairs with a decellularized tendon slices (DTSs) graft in a rabbit model. Methods Large defects in the infraspinatus tendons were created bilaterally in 21 rabbits. The graft group underwent reconstruction of the defects with the DTSs grafts, while the defect group did not undergo any treatment. The specimens underwent histological observation, biomechanical testing, and magnetic resonance imaging (MRI) detection at 4, 8, and 12 weeks after surgery. In addition, 2
Juan Pan and Guo-Ming Liu have contributed equally to this work. J. Pan Department of Rehabilitation Medicine, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China G.-M. Liu F.-G. Huang Department of Orthopaedic Surgery, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China e-mail: [email protected] L.-J. Ning J.-C. Luo T.-W. Qin (&) Institute of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China e-mail: [email protected] Y. Zhang Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China
rabbits that were not operated on were used for MRI detection as a normal reference. Results Histological analysis revealed that the graft promoted host cell ingrowth and tissue integration, and a tendon-like structure developed at 12 weeks. The ultimate tensile load had a significant difference between specimens at 4 and 12 weeks in the graft group, but there was no significant difference between the graft group and the defect group. In the graft group, the stiffness at 12 weeks was significantly greater than that at 4 or 8 weeks, and it was also greater than the stiffness in the defect group at 12 weeks. MRI demonstrated that the signal strength of the regenerative tissue from the graft group at 12 weeks was similar to that of normal infraspinatus tendon. Conclusion The DTSs graft allowed for incorporation of host tendon and improved the biomechanical performance of the regenerative tendon. Therefore, the graft could be a promising bioscaffold to enhance the surgical repair of large rotator cuff defects and consequently improve the clinical outcome of rotator cuff tears. Keywords Rotator cuff tears Decellularized tendon slices Graft Repair In vivo Rabbit
Introduction Injury to the rotator cuff is one of the most common aetiologies of pain and disability of the upper extremity. The size of rotator cuff tears has a significant effect on the clinical result after a surgical repair [9]. Large or massive rotator cuff tears are often associated with all kinds of pathologic degenerations [6, 25], which makes the treatment more difficult. The main complication of large or massive rotator cuff repairs is the re-tear of the tendon, and
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the incidence of re-rupture rate is high [31]. Numerous studies have described different treatment methods for large or massive rotator cuff tears, including non-operative management [22], surgical debridement and decompression [26], direct repair [28], tendon transfers [13, 30], and the use of various tendon grafts [11]. However, the results to date have not been entirely satisfactory. Therefore, selection of an optimal treatment for individual patients is still a significant challenge. Tissue engineering has gradually evolved as one of the promising approaches for rotator cuff repair and regeneration [15, 39], and it aims to optimize the interaction among cells, extracellular matrix (ECM) scaffolds and growth factors to promote rotator cuff tendon regeneration [5]. There are many materials [1, 7, 37, 43] used to augment or bridge the defects of rotator cuff for the purpose of accelerating the healing of rotator cuff repairs. Although recent studies have made some progresses, the technology needed to achieve the goal of regenerating a mechanical, biological, and functional tendon is still in its infancy. An important area of tendon tissue engineering is to explore ideal scaffolds that can almost recapitulate the biological properties of native ECM as well as features which allow it as the scaffold to be used for cell ingrowth and tendon regeneration [23]. The use of ECM derived from decellularized tissue as a biological scaffold is increasingly frequent in regenerative medicine and tissue engineering strategies [8]. The scaffolds derived from ECM are believed to provide a chemically and structurally instructive environment for host cells, via their natural composition, 3-dimensional structure, and/or remodelling by-products, which may improve the biology during healing [10]. Recently, a novel ECM scaffold with multilayer tendon slices was reported that the seeded cells aligned between the collagen fibres of the tendon slices, and expressed a marker of tendon phenotype [29]. In a previous study, it has been demonstrated that native tendon slices 300 lm or more in thickness had similar biomechanical characteristics to the intact tendon bundle [34]. More recently, further study illustrated that decellularized tendon slices (DTSs) with a thickness of 300 lm were thin sheet scaffolds with the elemental mechanical strength, inherent ultrastructure, specific proteoglycans, and multiple growth factors of tendon ECM [27]. Based on these reports, it appears that the new tissue engineering strategy provided a possibility to use the native DTSs as the scaffold for tendon regeneration. However, no research has been carried out to consider the efficiency of using this graft to repair rotator cuff defects in vivo. The purpose of this study is to assess the feasibility of a DTSs graft as a patch to repair the large defect of the rotator cuff in a rabbit model. It is hypothesized that the DTSs graft would improve the biomechanical performance and accelerate tendon healing.
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Fig. 1 Appearance of DTSs (*40 mm long, 4 mm wide, and 0.3 mm thick)
Materials and methods DTSs were fabricated using previously published protocol [27]. In brief, preparation of the DTSs included the following procedures: repetitive freeze/thaw treatment for Achilles tendons from adult beagle dogs, frozen section with a thickness of 300 lm (Fig. 1), nuclease treatment, and rinsing in PBS. Then, the DTSs were freeze-dried and packaged in polythene wrappers and finally, sterilized with ethylene oxide gas. Study groups Twenty-three male New Zealand white rabbits with a body weight between 2.5 and 3 kg were used in this study. Among them 21 rabbits (in total of 42 sites) were used to create large U-shaped defects by surgically removing partial infraspinatus tendons on bilateral shoulders and randomly allocated into both the graft group and the defect group. The graft group (n = 21) underwent reconstruction of the infraspinatus tendon defect with a DTSs graft, whereas the defect group (n = 21), used as the control group, did not undergo any treatment in order to evaluate the spontaneous healing capacity. At 4, 8, and 12 weeks after surgery, two rabbits from each group were evaluated in vivo by magnetic resonance imaging (MRI) detection. At each time point, seven rabbits were killed and bilateral shoulders were harvested. The harvested specimens from five shoulders for each group were used for biomechanical testing and the other two specimens were subjected to histological examination. In addition, two rabbits that were not operated on were used for MRI detection as a normal reference.
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Fig. 2 a A U-shaped defect (arrowhead) of infraspinatus tendon in the rabbit model. b Reconstruction of infraspinatus tendon defect with a DTSs graft
Operative procedures All animal experiments were carried out at the Institute of Animal Experimentation of Sichuan University. Rabbits were anesthetized by an intraperitoneal injection of chloral hydrate (3 ml/kg). Under sterile surgical conditions, a 3 cm longitudinal anterolateral skin incision was made on alternate right and left side. Dissection proceeded to the deltoid muscle, which was split in line with its fibres, and the infraspinatus tendon was identified. Then, an 8-mm-long full-thickness infraspinatus tendon U-shaped defect from the insertion onto the greater tuberosity was created by performing a sharp dissection. The width of the U-shaped defect was approximately 50 % of the width of the infraspinatus tendon (Fig. 2a), correlating to a large tear by some definition [3]. The footprint of rotator cuff on the greater tuberosity was prepared to a rough surface with bleeding. Two bone tunnels (0.8 mm in diameter) were created from the footprint to the proximal humeral metaphysis. In the graft group, the DTSs graft was superposed by three layers of DTSs with a thickness of 0.9 mm that approximates the thickness of the native infraspinatus tendon and rehydrated in saline. With use of the defect as a template, the graft was cut to the size, and modified Kessler sutures were placed with 5-0 Ethibond suture between the graft and the tendon. The graft was then attached to the insertion on the greater tuberosity, and sutures were tied to the lateral aspect of the cortex through the two bone tunnels (Fig. 2b). In the defect group, no repair of the tear was performed. The deltoid muscle was re-approximated and the incision was closed. Each rabbit was returned to its cage and allowed to perform normal cage activities without immobilization. The rabbits were evaluated daily for signs of illness, the presence of surgical wound infection, or dehiscence. At the predetermined time points, rabbits were killed by intraperitoneal injection of excessive pentobarbital. All specimens were obtained by harvesting proximal humerus, the
entire infraspinatus tendon, and removing the surrounding tissues. Bilateral shoulders were evaluated macroscopically for signs of infection, adhesions, and rotator cuff integrity. Histological observation Specimens for histological analysis were immediately transferred to a 4 % buffered formaldehyde solution for approximately 2 days, and decalcified in 10 % EDTA for 4 weeks, after complete decalcification, the tissue was longitudinally split to obtain full-thickness sections from the regenerated tissues, and embedded in paraffin. Longitudinal sections of 5 lm in thickness were obtained at the repair sites, including tendon defect region and tendon– bone junction region and then stained with haematoxylin and eosin. Histological sections were viewed under a microscope to evaluate the cellular and tissue responses, vascularity, new matrix deposition, collagen fibre organization as well as the presence or absence of inflammatory cells. Biomechanical testing Specimens were kept hydrated with saline during preparation and frozen at -80 °C until mechanical testing could be performed. After being thawed at the room temperature, the proximal humerus was embedded in polymethyl methacrylate, and positioned at the lower clamp in a material testing system (Instron 8874, USA). Specifically, the accuracy of measurement was ±0.5 % for load and ±0.04 % for displacement. The free end of the tendon was fixed in a holding clamp lined-grit sandpaper to prevent it from slipping during tensile testing. Mechanical testing was performed at a rate of 10 mm/min, and the direction of distraction was aligned with the fibres of the infraspinatus tendon. Before tensile testing, a 5.0 N traction was applied ten times to each specimen for precondition. Mechanical testing was performed at the room
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temperature and specimens were moistened with saline during testing. The tensile testing stopped when the tensile load was lower than 50 % of its maximum value. A loaddeformation curve was obtained from the load and displacement data. The linear stiffness was defined by the slope of the linear part after the toe region of the curve. The ultimate tensile load at which the repair failed was determined from the curve. The modes of failure in the specimens during tensile testing were also recorded. MRI evaluation Two rabbits at each time point from the two groups, as well as two normal rabbits, were randomly selected to undergo MRI detection. These rabbits were imaged in a 3.0-T clinical MR unit (Philips Corp, Holland). Shoulders were placed in the coil in lateral position and transverse imaging of the shoulder was performed routinely through the long axis of the rotator cuff tendon. The scanning protocol included the location of infraspinatus tendon using SE/T2WI sequence and PDWI sequences on transverse view for obtaining accurate images. Pulse sequences were chosen for sensitivity to fluid to serve as an internal comparison for signal properties of the tendon. On all pulse sequences obtained, fluid in the glenohumeral joint had high signal intensity. The signal characteristics and thickness of the regenerative tissue as well as continuity of the tendon were evaluated by a radiologist blinded to the infraspinatus tendon. IRB approval All animal operations were approved by the Institution of Animal Experiments Ethics Committee of West China Hospital, Sichuan University (Approval Number: 2011033), and animal care in this current study was conducted in accordance with the rules and regulations of research facilities for laboratory animal science in our university. Statistical analysis All data were presented as mean ± SD. Statistical differences were measured using an analysis of variance and a Tukey post hoc test was conducted for multiple comparisons between individual groups. In all cases, the significance level of P \ 0.05 was used.
Results Gross examination Gross examination demonstrated that all rabbits had full range of motion. The animals were able to ambulate in their cages
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Fig. 3 Gross appearance of specimens both the graft and the control group at 12 weeks. IS Infraspinatus tendon; H humerus
without limping and also maintained the ability to climb and reach out for food with the operative extremity. Three rabbits had wound swelling or dehiscence but no signs of infection. At 4 weeks, the upper surface of the graft was covered with sparse connective tissue in the graft group; however, defects in all specimens of the defect group were visible and not completely bridged by sparse connective tissue. Specimens from the graft group showed a secure connection of the graft complex which was covered with thick scar tissue over the graft at 8 weeks and the graft could not be discerned at 12 weeks (Fig. 3). In the control group, the sparse connective tissue became denser at 8 weeks and the scar formation was most prominent at 12 weeks (Fig. 3). No gross failure of specimens was noted before testing for the two groups. Histological observation Histological evaluation of the DTSs graft in the tendon defect region was carried out at 4, 8, and 12 weeks after surgery. At 4 weeks, the DTSs graft remained intact and there were evidences of three slices of the DTSs graft with obvious inflammatory cells surrounding each slice of the graft in the graft group (Fig. 4a, b). At 8 weeks, a large number of tendon-like cells were distributed evenly in parallel rows between the decellularized tendon fibres within the outer layer of the graft. A few vascular structures were evident, but residual unpopulated graft is present centrally and inflammatory cells almost disappeared around the graft in the graft group (Fig. 4c, d). At 12 weeks, tendon-like cells diffused into the graft and new tissue infiltration in a typical wavy pattern was apparent, similar to native tendon structure. The graft seemed to be completely incorporated with host tissue, and it was difficult to identify the margin between the graft and tendon (Fig. 4e, f).
Knee Surg Sports Traumatol Arthrosc Fig. 4 Representative slides of the graft group in the tendon defect region at 4, 8, and 12 weeks. a, b 4 Weeks. Three slices of DTSs with obvious inflammatory cells (arrowhead) surrounding each slice of the DTSs graft. c, d 8 Weeks. Fibroblasts ingrowth (pentagram) within the outer layer of the graft. e, f 12 Weeks. Tendon-like cells infiltration and new tissue ingrowth into the graft. Haematoxylin eosin staining, 940 (a, c, e); Haematoxylin eosin staining, 9400 (b, d, f)
At 4 weeks, the tendon–bone junction region in the graft group was filled with granulation tissue (Fig. 5a, b), which contained a large number of new blood vessels and connective tissue. In the defect group, loose fibrous tissue was noted at the tendon defect area, but the defect was still clearly visible and there was no indication of inflammatory response (Fig. 5c, d). At 8 weeks, there were abundant angiogenesis and collagen fibres filled in the tendon–bone junction region. Near the bone region, newly formed fibrocartilage was observed (Fig. 6a, b). In the defect group, tight fibrous connective tissue filled the defect. This was characterized by an increase in collagen fibres, cellularity, and neoangiogenesis. However, collagen fibres aligned disorderly and no fibrocartilage was formed in the tendon-to-bone insertion (Fig. 6c, d).
At 12 weeks, in the graft group, the fibrocartilage-like structures were obvious in the tendon–bone junction region compared with that at 8 weeks (Fig. 7a, b). In the control specimens, it was observed that the fibrous tissue at the tendon defect area became denser, extending from the edge of the bone, but fibroblasts in disorder, and there was a loss of polarity. No fibrocartilage formation was seen in the tendon-to-bone region (Fig. 7c, d). Biomechanical testing The result of biomechanical testing is shown in Table 1. All specimens from two groups demonstrated an overall increase in ultimate tensile load over time. In the graft group, the difference between specimens obtained at 4 and
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Fig. 5 Representative slides in the tendon–bone junction region at 4 weeks. a, b Graft group. Granulation tissue within the graft–bone region was composed of new blood vessels (arrowhead) and fibrous
connective tissue (arrow). c, d Defect group. Loose connective tissue (arrow) was observed. Haematoxylin eosin staining, 940 (a, c); Haematoxylin eosin staining, 9200 (b, d)
12 weeks was significant. At all time points, the ultimate tensile load of the graft group was greater than that of the defect group, but the difference was not statistically significant. All specimens from the graft group demonstrated an overall increase in linear stiffness over time, and at 12 weeks, the linear stiffness was significantly greater than that at 4 or 8 weeks (P \ 0.05). At 12 weeks, the linear stiffness of the graft group was significantly higher than that of the defect group (P \ 0.05). The modes of failure are shown in Table 2. At 4 weeks, most specimens failed at the midsubstance of tendons, and most of the shoulders ruptured at the tendon–bone junction at 8 weeks for the two groups. At 12 weeks, 2/5 specimens in the graft group had failed due to humeral fracture and the remaining specimens from this time point failed at the tendon–bone interface. In the defect group, one humeral fracture occurred during mechanical testing while the others failed at the tendon–bone interface at 12 weeks.
of signal intensity and continuity could be observed by MRI. At 4 week, MRI showed the continuity and high signal intensity of the repaired tendon that indicated tissue oedema in the graft group, similar to the control group (Fig. 9a, b). At 8 weeks, there was decreased signal intensity in both groups compared with that at 4 week, but the repaired tendon exhibited more regular sign compared with that of the control group (Fig. 9c, d). MRI demonstrated that the signal intensity of infraspinatus tendon was similar to normal low signal intensity at 12 weeks. The image finding of the graft group showed that the repaired tendon had homogeneous, regular, and low signal intensity, with characteristics of normal infraspinatus tendon. The reparative structure of the tissue in the defect group was disordered, hypertrophic and might be indicative of a scar tissue (Fig. 9e, f).
Discussion MRI evaluation Magnetic resonance imaging allowed clear distinction among the normal tendon, the newly formed tissue, and the humerus. The native tendon had homogeneous low signal intensity (Fig. 8). When the tendon was injured, the change
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The most important finding of the present study was that the DTSs graft allowed for incorporation of host tendon tissue and consequently improved the biomechanical performance of the regenerative tendon when the graft was used to repair large rotator cuff tears in a rabbit model.
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Fig. 6 Representative slides in the tendon–bone region at 8 weeks. a, b Graft group. Fibroblasts ingrowth (arrow), neovascularization (arrowhead) within the outer layer of the graft. Newly formed fibrocartilage was observed. c, d Defect group. Disorganized fibrous
connective tissue and neoangiogenesis (arrowhead) filled the defect. Haematoxylin eosin staining, 9100 (a, c); Haematoxylin eosin staining, 9400 (b, d)
Multiple techniques have been proposed to treat large or massive rotator cuff tears; however, each procedure has considerable limitations [13, 22, 26, 28, 30, 33]. Biological scaffolds, including small intestine submucosa [32], acellular dermal matrix [41], and chitin fabric [12], were used for repairing rotator cuff defects, and the results showed that these scaffolds were remodelled to tendon-like architecture, with homogeneous distribution of fibroblasts and parallel alignment of collagen fibres; however, disastrous inflammatory reaction, poor biomechanical characteristics, and fast degradation need to be taken into consideration when using the material as an option for replacing tissue in irreparable rotator cuff tears [11]. The current study explored the feasibility of repairing the larger rotator cuff defect using DTSs graft in rabbits. The DTS scaffold was chosen due to its unique characterizations, including the native tendon ECM architecture, sufficient tensile strength, collagen matrices, and growth factors. It was not crosslinked chemically and was minimally modified during the preparation process that could create a microenvironment favourable to the infiltration of cells. Histologically, it was shown that the defect of rotator cuff was filled with orientated fibrous tissue in the specimens at 8 and 12 weeks in the graft group. The fibrous
tissue in a typical wavy pattern was apparent at 12 weeks, similar to native tendon fibres. This study indicated that the DTSs graft appeared to provide an appropriate microenvironment for migration and proliferation of host cells as well as to enhance the integration between host tissue and graft compared with other materials [2, 4, 19, 42, 44]. It’s worth noting that the continuity of large defects was restored in rabbits; however, specimens from the defect group were histologically inferior to the graft group and the new tissue was considered to be scar tissue. Sano et al. [36] used fascial autograft to repair rotator cuff defects in rabbits and a normal tendon structure was simulated by 8 weeks postoperatively. Similar to our result, histological incorporation of the graft into a structure resembling normal tendon was present by 12 weeks; however, their use of an autograft resulted in donor site morbidity. The attachment site between the tendon and bone was a weak link during the early healing process in repaired rotator cuff tears [17]. Several methods for augmenting the tendon–bone junction showed the promotion of healing potential. TGF-b3 was applied to reconstruct tendon–bone insertion and the results revealed that local delivery of this growth factor led to the formation of a mature enthesis with a strong supraspinatus tendon–bone repair construct [20].
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Fig. 7 Representative slides in the tendon–bone region at 12 weeks. a, b Graft group. Fibrocartilage (arrow) and neovascularization (arrowhead) were observed. c, d Defect group. Fibrous tissue and
neovascularization (arrowhead) were seen. Haematoxylin eosin staining, 9100 (a, c); Haematoxylin eosin staining, 9400 (b, d)
Table 1 Biomechanical tests of the ultimate load and stiffness of all specimens
Table 2 Modes of failure in the specimens during mechanical testing
Time (weeks)
Group (n = 5 for each group)
4
Graft group
78.2 ± 12.0a
27.6 ± 7.9a
Defect group
76.7 ± 20.8
25.4 ± 8.0
8
Graft group
96.3 ± 32.9
28.9 ± 8.4b
Defect group
96.2 ± 33.1
31.7 ± 4.8
12
Graft group
136.0 ± 24.5a
Defect group
Ultimate tensile load (N)
111.0 ± 38.7
Linear stiffness (N/mm)
44.6 ± 9.7a,b,c 29.2 ± 6.9
c
a Statistically significant difference between the 4-week and the 12-week group b
Statistically significant difference between the 8-week and the 12-week group
Time point and treatment
Mode of failure in all specimens Midsubstance
Tendon–bone junction
Humeral fracture
4 Weeks Graft group
4
1
0
Defect group
4
1
0
8 Weeks Graft group
1
4
0
Defect group
1
4
0
Graft group
0
3
2
Defect group
0
4
1
12 Weeks
c
Statistically significant difference between the graft group and the defect group
IGF-1 and VEGF also had the potential of promotion tendon–bone healing. DTSs retained more than 90 % of the content of TGF-b, IGF-1, and VEGF after the process of removing cells [18, 27]. Hence, our use of DTSs was considered an alternative choice for enhancing tendon– bone healing.
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In this study, the fibrous tissue which connected the graft to the bone was different from the normal tendon– bone structure; however, fibrocartilage, which is essential composition of tendon–bone interface, was observed at the graft–bone junction region at 8 weeks. Therefore, DTSs graft may offer an advantage in reconstructing secure tendon–bone insertion. It is speculated that DTSs graft retains the native extracellular architecture that could
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Fig. 8 MRI of the normal shoulder joint. The image showed the infraspinatus tendon (white arrow) and head of humerus (plum blossom star)
induce the integration between host tissue and the graft. This graft also contains a variety of growth factors showing they could accelerate the healing process at tendon–bone insertion [16, 35]. However, it should be noted that final healing of tendon–bone structure is a long-term process, and a long-term study to evaluate the regenerated tendon characteristics is therefore necessary [14, 21]. In the current study, biomechanical testing revealed that the ultimate tensile load was not statistically significant between the graft and defect groups at each time point. However, the difference in ultimate tensile load was significant between specimens at 4 and 12 weeks in the graft group. Moreover, the linear stiffness of the graft group gradually increased with the healing time, and especially it was significantly higher than that of the control at 12 weeks. These results indicated that DTSs graft improved the biomechanical performance of the regenerative tendon tissue when the graft was used to repair the rotator cuff defects. Actually, the stiffness is more relevant than the failure load when assessing soft tissue healing to bone [38, 40]. In this study, the linear stiffness of the graft group increased sharply compared with that of the defect group at 12 weeks, but small values for the linear stiffness during the early recovery period indicated the possibility of elongation of the construct. It was demonstrated from mechanical testing that most specimens from the 4-week group failed at the midsubstance and specimens from other groups mostly failed at the tendon–bone junction. The modes of failure were consistent with the histological results. At 4 weeks, numerous inflammatory cells infiltration and graft degradation led to the result that strength of the graft was lower than that of the tendon–bone junction. With the extension of survival time, a growing number of tendon-like cells
infiltrated in the DTSs graft and tendon-like structure was observed; however, the normal tendon–bone insertion was not completely developed, so the strength of the graft was greater than that of tendon–bone junction. Interestingly, 2/5 specimens in the graft group at 12 weeks failed due to humeral fracture. This finding suggested that the strength of the tendon–bone interface in these specimens might exceed the strength of the cortical bone of the humeral diaphysis. Therefore, the results of ultimate load to fail might not accurately reflect the strength of the bone–tendon and graft interface [24]. Magnetic resonance imaging detection demonstrated that signal intensity of infraspinatus tendon was gradually reduced from a high signal to a low one in two groups, and the signal strength was similar to normal low signal intensity at 12 weeks. In the graft group, as revealed by MRI detection, the greater thickness of the 4-, 8-week samples than in normal tendon may imply a process of neotendon formation. At 12 weeks, the repaired tendon had become slimmer, and was similar to normal infraspinatus tendon, suggesting that it had gone through the remodelling and maturation phase of tendon healing. Although there was abundant and irregular new tissue formation in the control group, the quality of reparative tissues was relatively poor, indicative of scar tissue as evidenced by the histological finding at 12 weeks. In this study, MRI detection demonstrated that there was hypertrophic scar formation in the defect group as opposed to the regenerative tissue being homogeneous and regular in the graft group. This study provided in vivo data to support the use of DTSs grafts for repairing of large rotator cuff defects in rabbits. It is limited by the difficulty of replicating human disease in animal models. Our experiment created acute rotator cuff defects and do not replicate chronic rotator cuff tears, in which the remaining tendon and muscle may be attenuated or atrophied and retracted. Another limitation of our study is that the comparison of the DTSs graft with other reconstruction techniques and normal infraspinatus tendon was not performed because our study focused on assessing the feasibility of reconstructing large rotator cuff defects with this novel DTSs graft. In addition, the sample size was relatively small, resulting in low power for some comparisons. Nonetheless, the use of DTSs for repairing rotator cuff obtained positive results in animal. Further study is needed to determine the role of this bioscaffold in the treatment for humans with rotator cuff tears.
Conclusion The DTSs graft was capable of improving biomechanical properties of the repaired tendon as measured in increased
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Fig. 9 MRI of the shoulder joint after surgery. a, c, e Graft group at 4, 8, and 12 weeks, respectively. b, d, f Control group at 4, 8, and 12 weeks, respectively. The images showed the infraspinatus tendon (white arrow) and head of humerus (plum blossom star)
stiffness values, providing an appropriate microenvironment for migration and proliferation of host cells, and enhancing the integration between host tissue and graft as revealed by histological and MRI results. Thereby, DTSs graft was useful as a scaffold in the reconstruction of large rotator cuff defects in rabbits. This study provides important and fundamental information for the development of rotator cuff regeneration by the use of the DTSs graft. Acknowledgments This work was supported by the grants from National High Technology Research and Development Program of China (2012AA020502) and National Natural Science Foundation of China (31370988). We thank Ms. Jiang P. Fan and Jamie M. Kimball for the linguistic assistance during the preparation of this manuscript. Conflict of interest
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We declare that we have no conflict of interest.
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24. MacGillivray JD, Fealy S, Terry MA, Koh JL, Nixon AJ, Warren R (2006) Biomechanical evaluation of a rotator cuff defect model augmented with a bioresorbable scaffold in goats. J Shoulder Elbow Surg 15:639–644 25. Matthews TJ, Hand GC, Rees JL, Athanasou NA, Carr AJ (2006) Pathology of the torn rotator cuff tendon. Reduction in potential for repair as tear size increases. J Bone Joint Surg Br 88:489–495 26. Neri BR, Chan KW, Kwon YW (2009) Management of massive and irreparable rotator cuff tears. J Shoulder Elbow Surg 18:808–818 27. Ning LJ, Zhang Y, Chen XH, Luo JC, Li XQ, Yang ZM, Qin TW (2012) Preparation and characterization of decellularized tendon slices for tendon tissue engineering. J Biomed Mater Res A 100:1448–1456 28. Oizumi N, Suenaga N, Fukuda K, Minami A (2007) Massive rotator cuff tears repaired on top of humeral head by McLaughlin’s procedure. J Shoulder Elbow Surg 16:321–326 29. Omae H, Zhao C, Sun YL, An KN, Amadio PC (2009) Multilayer tendon slices seeded with bone marrow stromal cells: a novel composite for tendon engineering. J Orthop Res 27:937–942 30. Omid R, Lee B (2013) Tendon transfers for irreparable rotator cuff tears. J Am Acad Orthop Surg 21:492–501 31. Papadopoulos P, Karataglis D, Boutsiadis A, Fotiadou A, Christoforidis J, Christodoulou A (2010) Functional outcome and structural integrity following mini-open repair of large and massive rotator cuff tears: a 3–5 year follow-up study. J Shoulder Elbow Surg 20:131–137 32. Perry SM, Gupta RR, Van Kleunen J, Ramsey ML, Soslowsky LJ, Glaser DL (2007) Use of small intestine submucosa in a rat model of acute and chronic rotator cuff tear. J Shoulder Elbow Surg 16:S179–S183 33. Phipatanakul WP, Petersen SA (2009) Porcine small intestine submucosa xenograft augmentation in repair of massive rotator cuff tears. Am J Orthop 38:572–575 34. Qin TW, Chen Q, Sun YL, Steinmann SP, Amadio PC, An KN, Zhao C (2012) Mechanical characteristics of native tendon slices for tissue engineering scaffold. J Biomed Mater Res B Appl Biomater 100:752–758 35. Rodeo SA, Potter HG, Kawamura S, Turner AS, Kim HJ, Atkinson BL (2007) Biologic augmentation of rotator cuff tendon-healing with use of a mixture of osteoinductive growth factors. J Bone Joint Surg Am 89:2485–2497 36. Sano H, Kumagai J, Sawai T (2002) Experimental fascial autografting for the supraspinatus tendon defect: remodeling process of the grafted fascia and the insertion into bone. J Shoulder Elbow Surg 11:166–173 37. Santoni BG, McGilvray KC, Lyons AS, Bansal M, Turner AS, Macgillivray JD, Coleman SH, Puttlitz CM (2010) Biomechanical analysis of an ovine rotator cuff repair via porous patch augmentation in a chronic rupture model. Am J Sports Med 38:679–686 38. Schlegel TF, Hawkins RJ, Lewis CW, Motta T, Turner AS (2006) The effects of augmentation with swine small intestine submucosa on tendon healing under tension: histologic and mechanical evaluations in sheep. Am J Sports Med 34:275–280 39. Shen W, Chen J, Yin Z, Chen X, Liu H, Heng BC, Chen W, Ouyang HW (2012) Allogenous tendon stem/progenitor cells in silk scaffold for functional shoulder repair. Cell Transplant 21:943–958 40. Sherman SL, Lin EC, Verma NN, Mather RC, Gregory JM, Dishkin J, Harwood DP, Wang VM, Shewman EF, Cole BJ, Romeo AA (2012) Biomechanical analysis of the pectoralis major tendon and comparison of techniques for tendo-osseous repair. Am J Sports Med 40:1887–1894 41. Xu H, Sandor M, Qi S, Lombardi J, Connor J, McQuillan DJ, Iannotti JP (2012) Implantation of a porcine acellular dermal
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defects with porcine small intestinal submucosa in an animal model. J Shoulder Elbow Surg 15:224–231 44. Zheng MH, Chen J, Kirilak Y, Willers C, Xu J, Wood D (2005) Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation. J Biomed Mater Res B Appl Biomater 73:61–67
SHOULDER
Rotator cuff repair using a decellularized tendon slices graft: an in vivo study in a rabbit model Juan Pan • Guo-Ming Liu • Liang-Ju Ning • Yi Zhang • Jing-Cong Luo • Fu-Guo Huang • Ting-Wu Qin
Received: 7 March 2013 / Accepted: 20 February 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Although varieties of surgical repair techniques and materials have been used to repair rotator cuff defects, re-tearing frequently occurs. The purpose of this study is to evaluate the postoperative outcomes of rotator cuff repairs with a decellularized tendon slices (DTSs) graft in a rabbit model. Methods Large defects in the infraspinatus tendons were created bilaterally in 21 rabbits. The graft group underwent reconstruction of the defects with the DTSs grafts, while the defect group did not undergo any treatment. The specimens underwent histological observation, biomechanical testing, and magnetic resonance imaging (MRI) detection at 4, 8, and 12 weeks after surgery. In addition, 2
Juan Pan and Guo-Ming Liu have contributed equally to this work. J. Pan Department of Rehabilitation Medicine, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China G.-M. Liu F.-G. Huang Department of Orthopaedic Surgery, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China e-mail: [email protected] L.-J. Ning J.-C. Luo T.-W. Qin (&) Institute of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China e-mail: [email protected] Y. Zhang Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China
rabbits that were not operated on were used for MRI detection as a normal reference. Results Histological analysis revealed that the graft promoted host cell ingrowth and tissue integration, and a tendon-like structure developed at 12 weeks. The ultimate tensile load had a significant difference between specimens at 4 and 12 weeks in the graft group, but there was no significant difference between the graft group and the defect group. In the graft group, the stiffness at 12 weeks was significantly greater than that at 4 or 8 weeks, and it was also greater than the stiffness in the defect group at 12 weeks. MRI demonstrated that the signal strength of the regenerative tissue from the graft group at 12 weeks was similar to that of normal infraspinatus tendon. Conclusion The DTSs graft allowed for incorporation of host tendon and improved the biomechanical performance of the regenerative tendon. Therefore, the graft could be a promising bioscaffold to enhance the surgical repair of large rotator cuff defects and consequently improve the clinical outcome of rotator cuff tears. Keywords Rotator cuff tears Decellularized tendon slices Graft Repair In vivo Rabbit
Introduction Injury to the rotator cuff is one of the most common aetiologies of pain and disability of the upper extremity. The size of rotator cuff tears has a significant effect on the clinical result after a surgical repair [9]. Large or massive rotator cuff tears are often associated with all kinds of pathologic degenerations [6, 25], which makes the treatment more difficult. The main complication of large or massive rotator cuff repairs is the re-tear of the tendon, and
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the incidence of re-rupture rate is high [31]. Numerous studies have described different treatment methods for large or massive rotator cuff tears, including non-operative management [22], surgical debridement and decompression [26], direct repair [28], tendon transfers [13, 30], and the use of various tendon grafts [11]. However, the results to date have not been entirely satisfactory. Therefore, selection of an optimal treatment for individual patients is still a significant challenge. Tissue engineering has gradually evolved as one of the promising approaches for rotator cuff repair and regeneration [15, 39], and it aims to optimize the interaction among cells, extracellular matrix (ECM) scaffolds and growth factors to promote rotator cuff tendon regeneration [5]. There are many materials [1, 7, 37, 43] used to augment or bridge the defects of rotator cuff for the purpose of accelerating the healing of rotator cuff repairs. Although recent studies have made some progresses, the technology needed to achieve the goal of regenerating a mechanical, biological, and functional tendon is still in its infancy. An important area of tendon tissue engineering is to explore ideal scaffolds that can almost recapitulate the biological properties of native ECM as well as features which allow it as the scaffold to be used for cell ingrowth and tendon regeneration [23]. The use of ECM derived from decellularized tissue as a biological scaffold is increasingly frequent in regenerative medicine and tissue engineering strategies [8]. The scaffolds derived from ECM are believed to provide a chemically and structurally instructive environment for host cells, via their natural composition, 3-dimensional structure, and/or remodelling by-products, which may improve the biology during healing [10]. Recently, a novel ECM scaffold with multilayer tendon slices was reported that the seeded cells aligned between the collagen fibres of the tendon slices, and expressed a marker of tendon phenotype [29]. In a previous study, it has been demonstrated that native tendon slices 300 lm or more in thickness had similar biomechanical characteristics to the intact tendon bundle [34]. More recently, further study illustrated that decellularized tendon slices (DTSs) with a thickness of 300 lm were thin sheet scaffolds with the elemental mechanical strength, inherent ultrastructure, specific proteoglycans, and multiple growth factors of tendon ECM [27]. Based on these reports, it appears that the new tissue engineering strategy provided a possibility to use the native DTSs as the scaffold for tendon regeneration. However, no research has been carried out to consider the efficiency of using this graft to repair rotator cuff defects in vivo. The purpose of this study is to assess the feasibility of a DTSs graft as a patch to repair the large defect of the rotator cuff in a rabbit model. It is hypothesized that the DTSs graft would improve the biomechanical performance and accelerate tendon healing.
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Fig. 1 Appearance of DTSs (*40 mm long, 4 mm wide, and 0.3 mm thick)
Materials and methods DTSs were fabricated using previously published protocol [27]. In brief, preparation of the DTSs included the following procedures: repetitive freeze/thaw treatment for Achilles tendons from adult beagle dogs, frozen section with a thickness of 300 lm (Fig. 1), nuclease treatment, and rinsing in PBS. Then, the DTSs were freeze-dried and packaged in polythene wrappers and finally, sterilized with ethylene oxide gas. Study groups Twenty-three male New Zealand white rabbits with a body weight between 2.5 and 3 kg were used in this study. Among them 21 rabbits (in total of 42 sites) were used to create large U-shaped defects by surgically removing partial infraspinatus tendons on bilateral shoulders and randomly allocated into both the graft group and the defect group. The graft group (n = 21) underwent reconstruction of the infraspinatus tendon defect with a DTSs graft, whereas the defect group (n = 21), used as the control group, did not undergo any treatment in order to evaluate the spontaneous healing capacity. At 4, 8, and 12 weeks after surgery, two rabbits from each group were evaluated in vivo by magnetic resonance imaging (MRI) detection. At each time point, seven rabbits were killed and bilateral shoulders were harvested. The harvested specimens from five shoulders for each group were used for biomechanical testing and the other two specimens were subjected to histological examination. In addition, two rabbits that were not operated on were used for MRI detection as a normal reference.
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Fig. 2 a A U-shaped defect (arrowhead) of infraspinatus tendon in the rabbit model. b Reconstruction of infraspinatus tendon defect with a DTSs graft
Operative procedures All animal experiments were carried out at the Institute of Animal Experimentation of Sichuan University. Rabbits were anesthetized by an intraperitoneal injection of chloral hydrate (3 ml/kg). Under sterile surgical conditions, a 3 cm longitudinal anterolateral skin incision was made on alternate right and left side. Dissection proceeded to the deltoid muscle, which was split in line with its fibres, and the infraspinatus tendon was identified. Then, an 8-mm-long full-thickness infraspinatus tendon U-shaped defect from the insertion onto the greater tuberosity was created by performing a sharp dissection. The width of the U-shaped defect was approximately 50 % of the width of the infraspinatus tendon (Fig. 2a), correlating to a large tear by some definition [3]. The footprint of rotator cuff on the greater tuberosity was prepared to a rough surface with bleeding. Two bone tunnels (0.8 mm in diameter) were created from the footprint to the proximal humeral metaphysis. In the graft group, the DTSs graft was superposed by three layers of DTSs with a thickness of 0.9 mm that approximates the thickness of the native infraspinatus tendon and rehydrated in saline. With use of the defect as a template, the graft was cut to the size, and modified Kessler sutures were placed with 5-0 Ethibond suture between the graft and the tendon. The graft was then attached to the insertion on the greater tuberosity, and sutures were tied to the lateral aspect of the cortex through the two bone tunnels (Fig. 2b). In the defect group, no repair of the tear was performed. The deltoid muscle was re-approximated and the incision was closed. Each rabbit was returned to its cage and allowed to perform normal cage activities without immobilization. The rabbits were evaluated daily for signs of illness, the presence of surgical wound infection, or dehiscence. At the predetermined time points, rabbits were killed by intraperitoneal injection of excessive pentobarbital. All specimens were obtained by harvesting proximal humerus, the
entire infraspinatus tendon, and removing the surrounding tissues. Bilateral shoulders were evaluated macroscopically for signs of infection, adhesions, and rotator cuff integrity. Histological observation Specimens for histological analysis were immediately transferred to a 4 % buffered formaldehyde solution for approximately 2 days, and decalcified in 10 % EDTA for 4 weeks, after complete decalcification, the tissue was longitudinally split to obtain full-thickness sections from the regenerated tissues, and embedded in paraffin. Longitudinal sections of 5 lm in thickness were obtained at the repair sites, including tendon defect region and tendon– bone junction region and then stained with haematoxylin and eosin. Histological sections were viewed under a microscope to evaluate the cellular and tissue responses, vascularity, new matrix deposition, collagen fibre organization as well as the presence or absence of inflammatory cells. Biomechanical testing Specimens were kept hydrated with saline during preparation and frozen at -80 °C until mechanical testing could be performed. After being thawed at the room temperature, the proximal humerus was embedded in polymethyl methacrylate, and positioned at the lower clamp in a material testing system (Instron 8874, USA). Specifically, the accuracy of measurement was ±0.5 % for load and ±0.04 % for displacement. The free end of the tendon was fixed in a holding clamp lined-grit sandpaper to prevent it from slipping during tensile testing. Mechanical testing was performed at a rate of 10 mm/min, and the direction of distraction was aligned with the fibres of the infraspinatus tendon. Before tensile testing, a 5.0 N traction was applied ten times to each specimen for precondition. Mechanical testing was performed at the room
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temperature and specimens were moistened with saline during testing. The tensile testing stopped when the tensile load was lower than 50 % of its maximum value. A loaddeformation curve was obtained from the load and displacement data. The linear stiffness was defined by the slope of the linear part after the toe region of the curve. The ultimate tensile load at which the repair failed was determined from the curve. The modes of failure in the specimens during tensile testing were also recorded. MRI evaluation Two rabbits at each time point from the two groups, as well as two normal rabbits, were randomly selected to undergo MRI detection. These rabbits were imaged in a 3.0-T clinical MR unit (Philips Corp, Holland). Shoulders were placed in the coil in lateral position and transverse imaging of the shoulder was performed routinely through the long axis of the rotator cuff tendon. The scanning protocol included the location of infraspinatus tendon using SE/T2WI sequence and PDWI sequences on transverse view for obtaining accurate images. Pulse sequences were chosen for sensitivity to fluid to serve as an internal comparison for signal properties of the tendon. On all pulse sequences obtained, fluid in the glenohumeral joint had high signal intensity. The signal characteristics and thickness of the regenerative tissue as well as continuity of the tendon were evaluated by a radiologist blinded to the infraspinatus tendon. IRB approval All animal operations were approved by the Institution of Animal Experiments Ethics Committee of West China Hospital, Sichuan University (Approval Number: 2011033), and animal care in this current study was conducted in accordance with the rules and regulations of research facilities for laboratory animal science in our university. Statistical analysis All data were presented as mean ± SD. Statistical differences were measured using an analysis of variance and a Tukey post hoc test was conducted for multiple comparisons between individual groups. In all cases, the significance level of P \ 0.05 was used.
Results Gross examination Gross examination demonstrated that all rabbits had full range of motion. The animals were able to ambulate in their cages
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Fig. 3 Gross appearance of specimens both the graft and the control group at 12 weeks. IS Infraspinatus tendon; H humerus
without limping and also maintained the ability to climb and reach out for food with the operative extremity. Three rabbits had wound swelling or dehiscence but no signs of infection. At 4 weeks, the upper surface of the graft was covered with sparse connective tissue in the graft group; however, defects in all specimens of the defect group were visible and not completely bridged by sparse connective tissue. Specimens from the graft group showed a secure connection of the graft complex which was covered with thick scar tissue over the graft at 8 weeks and the graft could not be discerned at 12 weeks (Fig. 3). In the control group, the sparse connective tissue became denser at 8 weeks and the scar formation was most prominent at 12 weeks (Fig. 3). No gross failure of specimens was noted before testing for the two groups. Histological observation Histological evaluation of the DTSs graft in the tendon defect region was carried out at 4, 8, and 12 weeks after surgery. At 4 weeks, the DTSs graft remained intact and there were evidences of three slices of the DTSs graft with obvious inflammatory cells surrounding each slice of the graft in the graft group (Fig. 4a, b). At 8 weeks, a large number of tendon-like cells were distributed evenly in parallel rows between the decellularized tendon fibres within the outer layer of the graft. A few vascular structures were evident, but residual unpopulated graft is present centrally and inflammatory cells almost disappeared around the graft in the graft group (Fig. 4c, d). At 12 weeks, tendon-like cells diffused into the graft and new tissue infiltration in a typical wavy pattern was apparent, similar to native tendon structure. The graft seemed to be completely incorporated with host tissue, and it was difficult to identify the margin between the graft and tendon (Fig. 4e, f).
Knee Surg Sports Traumatol Arthrosc Fig. 4 Representative slides of the graft group in the tendon defect region at 4, 8, and 12 weeks. a, b 4 Weeks. Three slices of DTSs with obvious inflammatory cells (arrowhead) surrounding each slice of the DTSs graft. c, d 8 Weeks. Fibroblasts ingrowth (pentagram) within the outer layer of the graft. e, f 12 Weeks. Tendon-like cells infiltration and new tissue ingrowth into the graft. Haematoxylin eosin staining, 940 (a, c, e); Haematoxylin eosin staining, 9400 (b, d, f)
At 4 weeks, the tendon–bone junction region in the graft group was filled with granulation tissue (Fig. 5a, b), which contained a large number of new blood vessels and connective tissue. In the defect group, loose fibrous tissue was noted at the tendon defect area, but the defect was still clearly visible and there was no indication of inflammatory response (Fig. 5c, d). At 8 weeks, there were abundant angiogenesis and collagen fibres filled in the tendon–bone junction region. Near the bone region, newly formed fibrocartilage was observed (Fig. 6a, b). In the defect group, tight fibrous connective tissue filled the defect. This was characterized by an increase in collagen fibres, cellularity, and neoangiogenesis. However, collagen fibres aligned disorderly and no fibrocartilage was formed in the tendon-to-bone insertion (Fig. 6c, d).
At 12 weeks, in the graft group, the fibrocartilage-like structures were obvious in the tendon–bone junction region compared with that at 8 weeks (Fig. 7a, b). In the control specimens, it was observed that the fibrous tissue at the tendon defect area became denser, extending from the edge of the bone, but fibroblasts in disorder, and there was a loss of polarity. No fibrocartilage formation was seen in the tendon-to-bone region (Fig. 7c, d). Biomechanical testing The result of biomechanical testing is shown in Table 1. All specimens from two groups demonstrated an overall increase in ultimate tensile load over time. In the graft group, the difference between specimens obtained at 4 and
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Fig. 5 Representative slides in the tendon–bone junction region at 4 weeks. a, b Graft group. Granulation tissue within the graft–bone region was composed of new blood vessels (arrowhead) and fibrous
connective tissue (arrow). c, d Defect group. Loose connective tissue (arrow) was observed. Haematoxylin eosin staining, 940 (a, c); Haematoxylin eosin staining, 9200 (b, d)
12 weeks was significant. At all time points, the ultimate tensile load of the graft group was greater than that of the defect group, but the difference was not statistically significant. All specimens from the graft group demonstrated an overall increase in linear stiffness over time, and at 12 weeks, the linear stiffness was significantly greater than that at 4 or 8 weeks (P \ 0.05). At 12 weeks, the linear stiffness of the graft group was significantly higher than that of the defect group (P \ 0.05). The modes of failure are shown in Table 2. At 4 weeks, most specimens failed at the midsubstance of tendons, and most of the shoulders ruptured at the tendon–bone junction at 8 weeks for the two groups. At 12 weeks, 2/5 specimens in the graft group had failed due to humeral fracture and the remaining specimens from this time point failed at the tendon–bone interface. In the defect group, one humeral fracture occurred during mechanical testing while the others failed at the tendon–bone interface at 12 weeks.
of signal intensity and continuity could be observed by MRI. At 4 week, MRI showed the continuity and high signal intensity of the repaired tendon that indicated tissue oedema in the graft group, similar to the control group (Fig. 9a, b). At 8 weeks, there was decreased signal intensity in both groups compared with that at 4 week, but the repaired tendon exhibited more regular sign compared with that of the control group (Fig. 9c, d). MRI demonstrated that the signal intensity of infraspinatus tendon was similar to normal low signal intensity at 12 weeks. The image finding of the graft group showed that the repaired tendon had homogeneous, regular, and low signal intensity, with characteristics of normal infraspinatus tendon. The reparative structure of the tissue in the defect group was disordered, hypertrophic and might be indicative of a scar tissue (Fig. 9e, f).
Discussion MRI evaluation Magnetic resonance imaging allowed clear distinction among the normal tendon, the newly formed tissue, and the humerus. The native tendon had homogeneous low signal intensity (Fig. 8). When the tendon was injured, the change
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The most important finding of the present study was that the DTSs graft allowed for incorporation of host tendon tissue and consequently improved the biomechanical performance of the regenerative tendon when the graft was used to repair large rotator cuff tears in a rabbit model.
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Fig. 6 Representative slides in the tendon–bone region at 8 weeks. a, b Graft group. Fibroblasts ingrowth (arrow), neovascularization (arrowhead) within the outer layer of the graft. Newly formed fibrocartilage was observed. c, d Defect group. Disorganized fibrous
connective tissue and neoangiogenesis (arrowhead) filled the defect. Haematoxylin eosin staining, 9100 (a, c); Haematoxylin eosin staining, 9400 (b, d)
Multiple techniques have been proposed to treat large or massive rotator cuff tears; however, each procedure has considerable limitations [13, 22, 26, 28, 30, 33]. Biological scaffolds, including small intestine submucosa [32], acellular dermal matrix [41], and chitin fabric [12], were used for repairing rotator cuff defects, and the results showed that these scaffolds were remodelled to tendon-like architecture, with homogeneous distribution of fibroblasts and parallel alignment of collagen fibres; however, disastrous inflammatory reaction, poor biomechanical characteristics, and fast degradation need to be taken into consideration when using the material as an option for replacing tissue in irreparable rotator cuff tears [11]. The current study explored the feasibility of repairing the larger rotator cuff defect using DTSs graft in rabbits. The DTS scaffold was chosen due to its unique characterizations, including the native tendon ECM architecture, sufficient tensile strength, collagen matrices, and growth factors. It was not crosslinked chemically and was minimally modified during the preparation process that could create a microenvironment favourable to the infiltration of cells. Histologically, it was shown that the defect of rotator cuff was filled with orientated fibrous tissue in the specimens at 8 and 12 weeks in the graft group. The fibrous
tissue in a typical wavy pattern was apparent at 12 weeks, similar to native tendon fibres. This study indicated that the DTSs graft appeared to provide an appropriate microenvironment for migration and proliferation of host cells as well as to enhance the integration between host tissue and graft compared with other materials [2, 4, 19, 42, 44]. It’s worth noting that the continuity of large defects was restored in rabbits; however, specimens from the defect group were histologically inferior to the graft group and the new tissue was considered to be scar tissue. Sano et al. [36] used fascial autograft to repair rotator cuff defects in rabbits and a normal tendon structure was simulated by 8 weeks postoperatively. Similar to our result, histological incorporation of the graft into a structure resembling normal tendon was present by 12 weeks; however, their use of an autograft resulted in donor site morbidity. The attachment site between the tendon and bone was a weak link during the early healing process in repaired rotator cuff tears [17]. Several methods for augmenting the tendon–bone junction showed the promotion of healing potential. TGF-b3 was applied to reconstruct tendon–bone insertion and the results revealed that local delivery of this growth factor led to the formation of a mature enthesis with a strong supraspinatus tendon–bone repair construct [20].
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Fig. 7 Representative slides in the tendon–bone region at 12 weeks. a, b Graft group. Fibrocartilage (arrow) and neovascularization (arrowhead) were observed. c, d Defect group. Fibrous tissue and
neovascularization (arrowhead) were seen. Haematoxylin eosin staining, 9100 (a, c); Haematoxylin eosin staining, 9400 (b, d)
Table 1 Biomechanical tests of the ultimate load and stiffness of all specimens
Table 2 Modes of failure in the specimens during mechanical testing
Time (weeks)
Group (n = 5 for each group)
4
Graft group
78.2 ± 12.0a
27.6 ± 7.9a
Defect group
76.7 ± 20.8
25.4 ± 8.0
8
Graft group
96.3 ± 32.9
28.9 ± 8.4b
Defect group
96.2 ± 33.1
31.7 ± 4.8
12
Graft group
136.0 ± 24.5a
Defect group
Ultimate tensile load (N)
111.0 ± 38.7
Linear stiffness (N/mm)
44.6 ± 9.7a,b,c 29.2 ± 6.9
c
a Statistically significant difference between the 4-week and the 12-week group b
Statistically significant difference between the 8-week and the 12-week group
Time point and treatment
Mode of failure in all specimens Midsubstance
Tendon–bone junction
Humeral fracture
4 Weeks Graft group
4
1
0
Defect group
4
1
0
8 Weeks Graft group
1
4
0
Defect group
1
4
0
Graft group
0
3
2
Defect group
0
4
1
12 Weeks
c
Statistically significant difference between the graft group and the defect group
IGF-1 and VEGF also had the potential of promotion tendon–bone healing. DTSs retained more than 90 % of the content of TGF-b, IGF-1, and VEGF after the process of removing cells [18, 27]. Hence, our use of DTSs was considered an alternative choice for enhancing tendon– bone healing.
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In this study, the fibrous tissue which connected the graft to the bone was different from the normal tendon– bone structure; however, fibrocartilage, which is essential composition of tendon–bone interface, was observed at the graft–bone junction region at 8 weeks. Therefore, DTSs graft may offer an advantage in reconstructing secure tendon–bone insertion. It is speculated that DTSs graft retains the native extracellular architecture that could
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Fig. 8 MRI of the normal shoulder joint. The image showed the infraspinatus tendon (white arrow) and head of humerus (plum blossom star)
induce the integration between host tissue and the graft. This graft also contains a variety of growth factors showing they could accelerate the healing process at tendon–bone insertion [16, 35]. However, it should be noted that final healing of tendon–bone structure is a long-term process, and a long-term study to evaluate the regenerated tendon characteristics is therefore necessary [14, 21]. In the current study, biomechanical testing revealed that the ultimate tensile load was not statistically significant between the graft and defect groups at each time point. However, the difference in ultimate tensile load was significant between specimens at 4 and 12 weeks in the graft group. Moreover, the linear stiffness of the graft group gradually increased with the healing time, and especially it was significantly higher than that of the control at 12 weeks. These results indicated that DTSs graft improved the biomechanical performance of the regenerative tendon tissue when the graft was used to repair the rotator cuff defects. Actually, the stiffness is more relevant than the failure load when assessing soft tissue healing to bone [38, 40]. In this study, the linear stiffness of the graft group increased sharply compared with that of the defect group at 12 weeks, but small values for the linear stiffness during the early recovery period indicated the possibility of elongation of the construct. It was demonstrated from mechanical testing that most specimens from the 4-week group failed at the midsubstance and specimens from other groups mostly failed at the tendon–bone junction. The modes of failure were consistent with the histological results. At 4 weeks, numerous inflammatory cells infiltration and graft degradation led to the result that strength of the graft was lower than that of the tendon–bone junction. With the extension of survival time, a growing number of tendon-like cells
infiltrated in the DTSs graft and tendon-like structure was observed; however, the normal tendon–bone insertion was not completely developed, so the strength of the graft was greater than that of tendon–bone junction. Interestingly, 2/5 specimens in the graft group at 12 weeks failed due to humeral fracture. This finding suggested that the strength of the tendon–bone interface in these specimens might exceed the strength of the cortical bone of the humeral diaphysis. Therefore, the results of ultimate load to fail might not accurately reflect the strength of the bone–tendon and graft interface [24]. Magnetic resonance imaging detection demonstrated that signal intensity of infraspinatus tendon was gradually reduced from a high signal to a low one in two groups, and the signal strength was similar to normal low signal intensity at 12 weeks. In the graft group, as revealed by MRI detection, the greater thickness of the 4-, 8-week samples than in normal tendon may imply a process of neotendon formation. At 12 weeks, the repaired tendon had become slimmer, and was similar to normal infraspinatus tendon, suggesting that it had gone through the remodelling and maturation phase of tendon healing. Although there was abundant and irregular new tissue formation in the control group, the quality of reparative tissues was relatively poor, indicative of scar tissue as evidenced by the histological finding at 12 weeks. In this study, MRI detection demonstrated that there was hypertrophic scar formation in the defect group as opposed to the regenerative tissue being homogeneous and regular in the graft group. This study provided in vivo data to support the use of DTSs grafts for repairing of large rotator cuff defects in rabbits. It is limited by the difficulty of replicating human disease in animal models. Our experiment created acute rotator cuff defects and do not replicate chronic rotator cuff tears, in which the remaining tendon and muscle may be attenuated or atrophied and retracted. Another limitation of our study is that the comparison of the DTSs graft with other reconstruction techniques and normal infraspinatus tendon was not performed because our study focused on assessing the feasibility of reconstructing large rotator cuff defects with this novel DTSs graft. In addition, the sample size was relatively small, resulting in low power for some comparisons. Nonetheless, the use of DTSs for repairing rotator cuff obtained positive results in animal. Further study is needed to determine the role of this bioscaffold in the treatment for humans with rotator cuff tears.
Conclusion The DTSs graft was capable of improving biomechanical properties of the repaired tendon as measured in increased
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Fig. 9 MRI of the shoulder joint after surgery. a, c, e Graft group at 4, 8, and 12 weeks, respectively. b, d, f Control group at 4, 8, and 12 weeks, respectively. The images showed the infraspinatus tendon (white arrow) and head of humerus (plum blossom star)
stiffness values, providing an appropriate microenvironment for migration and proliferation of host cells, and enhancing the integration between host tissue and graft as revealed by histological and MRI results. Thereby, DTSs graft was useful as a scaffold in the reconstruction of large rotator cuff defects in rabbits. This study provides important and fundamental information for the development of rotator cuff regeneration by the use of the DTSs graft. Acknowledgments This work was supported by the grants from National High Technology Research and Development Program of China (2012AA020502) and National Natural Science Foundation of China (31370988). We thank Ms. Jiang P. Fan and Jamie M. Kimball for the linguistic assistance during the preparation of this manuscript. Conflict of interest
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We declare that we have no conflict of interest.
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