J Shoulder Elbow Surg (2014) 23, 445-455
www.elsevier.com/locate/ymse
BASIC SCIENCE
2013 Neer Award: Effect of the adipose-derived stem cell for the improvement of fatty degeneration and rotator cuff healing in rabbit model Joo Han Oh, MD, PhDa, Seok Won Chung, MDb,*, Sae Hoon Kim, MD, PhDc, Jin Young Chung, DVM, PhDd, Joon Yub Kim, MDe a
Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea b Department of Orthopaedic Surgery, Konkuk University School of Medicine, Konkuk University Medical Center, Seoul, South Korea c Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, South Korea d Department of Neurology, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, South Korea e Department of Orthopaedic Surgery, Myongji Hospital, Goyang, South Korea Background: This study was conducted to verify the effects of adipose-derived stem cells (ADSCs) on tendon healing and reversal of fatty infiltration in a chronic rotator cuff tear model by using the rabbit subscapularis (SSC). Methods: The SSC insertions in 32 rabbits were cut bilaterally. After 6 weeks, secondary procedures were performed bilaterally, dividing the rabbits into 4 groups of 8 rabbits each as follows: the ADSCþrepair group, salineþrepair group, ADSC-only group, and saline-only group. A fifth group of 8 rabbits served as normal controls (control group). Electromyographic, biomechanical, and histologic analyses were performed 6 weeks after the secondary procedures. Results: All SSC tendons in the ADSC-only and saline-only groups failed to heal and were excluded from the electromyographic and biomechanical tests. On electromyographic evaluation, the ADSCþrepair group exhibited a larger compound muscle action potential area than the salineþrepair group (11.86 2.97 ms $ mV vs 9.42 3.57 ms $ mV, P ¼ .029), and this response was almost at the level of the control group (13.17 6.6 3 ms $ mV, P ¼ .456). Biomechanically, the load-to-failure of the ADSCþrepair group (87.02 29.81 N) was higher than that of the salineþrepair group (59.85 37.77 N), although this difference did not reach statistical significance (P ¼ .085). Histologically, the mean proportions of fatty infiltration in the SSC muscles were 29% 15%, 43% 9%, 51% 14%, 63% 10%, and 18% 9% for the ADSCþrepair, salineþrepair, ADSC-only, saline-only, and control groups, respectively (P < .001). The
This study was approved by the Seoul National University Bundang Hospital (SNUBH) Institutional Animal Care and Use Committee (Approval: No. BA1005-062/030-01). This study was supported by the Basic Science Research Program through the National Research Foundation of Koreafunded by the Ministry of Education, Science and Technology (2010-000850).
*Reprint requests: Seok Won Chung, MD, Shoulder and Elbow Surgery Division, Department of Orthopedic Surgery, Konkuk University School of Medicine, Konkuk University Medical Center, 120-1 Neungdong-ro, Gwangjin-gu, Seoul 143-729, South Korea. E-mail address: [email protected] (S.W. Chung).
1058-2746/$ - see front matter Ó 2014 Journal of Shoulder and Elbow Surgery Board of Trustees. http://dx.doi.org/10.1016/j.jse.2013.07.054
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degree of fat staining increased from the ADSCþrepair group (unclear or weak) to the saline-only group (strongly present). Conclusion: Local administration of ADSCs might have the possibility to improve muscle function and tendon healing and decrease fatty infiltration after cuff repair. Level of evidence: Basic Science Study, Biomechanics and Histology, Animal Model. Ó 2014 Journal of Shoulder and Elbow Surgery Board of Trustees. Keywords: Adipose-derived stem cell; rotator cuff tear; fatty infiltration; tendon-to-bone healing; compound muscle action potential
Rotator cuff tears are a common condition that causes shoulder pain and dysfunction that has been reported to occur in more than 50% of people aged older than 60 years.30,39 Although rotator cuff repair techniques and instrumentation have rapidly improved, rotator cuff healing failure after surgical repair remains one of the most common and well-known complications. Indeed, current clinical data suggest that approximately 50% of surgically repaired rotator cuffs do not heal properly, independent of the surgical procedures used.6,7,35 Rotator cuff tendon and muscle quality is a key predisposing factor of healing failure and poor functional outcome.12,33 Unfortunately, rotator cuff tears usually occur in elderly individuals who exhibit inherent tissue aging19 and usually present with chronic tears with disorganized tendon scar tissue and fatty infiltration in the muscle.12,29 Fatty infiltration represents fat deposition in tissues, particularly between cells, and preoperative fatty infiltration of the rotator cuff muscles is an important predisposing factor to repair failure and poor functional outcomes.18,19 However, fatty infiltration in chronic rotator cuff tears has been regarded as an irreversible phenomenon even after successful cuff repair,17,34 and nonreversible fatty infiltration was reported to persist even at long-term follow-up.51 The development of fatty infiltration after rotator cuff tendon detachment is well established in a rabbit model. Bjorkenheim et al5 demonstrated that fatty infiltration of the rabbit supraspinatus was most prominent at 6 weeks after injury, and Gupta et al22 showed that fatty infiltration of the muscle increased significantly at 6 weeks after tenotomy of the rabbit subscapularis (SSC). However, a few studies that investigated the effect of reattachment of the rotator cuff tendon on fatty infiltration in a rabbit model failed to show a decrease in fatty infiltration after reattachment.43 Stem cell–based therapies for the repair and regeneration of tendon and muscle represent a paradigm shift and show great promise as alternative therapeutic solutions for a number of diseases.45 Several investigators have attempted to demonstrate the effect of stem cells on rotator cuff healing; however, these effects remain unproven and controversial. Yokoya et al48 showed that mesenchymal stem cells (MSCs) were capable of regenerating rotator cuff tendon-to-bone insertions and the tendon belly in a rabbit
model. However, Gulotta et al21 suggested that the addition of MSCs to the insertion site of the repaired rotator cuff tendon did not improve the structure, composition, or strength of the healing tendon attachment site in a rat model. Moreover, no study to date has investigated the effect of stem cells on fatty infiltration in rotator cuff muscles. The adipose-derived stem cell (ADSC) is a progenitor cell that can proliferate and differentiate into different types of mesenchymal cells, such as tenocytes or myocytes, as well as locally release growth factors and cytokines.26 This ability may be beneficial in enhancing tendon-to-bone healing and improving fatty infiltration. Therefore, the purpose of this study was to verify the effects of ADSCs on tendon-to-bone healing and improvement of fatty infiltration in a chronic rotator cuff tear model by using rabbit SSC muscle. We hypothesized that local administration of ADSCs in the SSC muscle enhances tendon-to-bone healing and improves fatty infiltration.
Materials and methods All experiments in this study were conducted in accordance with the Seoul National University Bundang Hospital guidelines for the care and use of laboratory animals.
Allocation of experimental animals This was a controlled laboratory study involving animals. Power analysis indicated that a sample size of 7 was required for biomechanical studies to detect a significant difference in ultimate failure load (mean difference, 90 N; standard deviation, 40 N; aerror, 0.05; b-error, 0.2; drop-out rate, 20%), and a sample size of 8 was necessary for histology studies to detect a 10% difference in fat-to-muscle proportion (a-error, 0.05; b-error, 0.2; drop-out rate, 20%), based on previous studies.28,44 We randomly allocated 40 mature New Zealand White male rabbits (average age, 24 weeks; weight, 3.5-4.0 kg) into 5 groups (8 rabbits per group) as follows: the ADSCþrepair group, salineþrepair group, ADSC-only group, saline-only group, and control group. The surgeries on each of the 40 animals were bilateral; thus, the number of SSC muscles in each group was 16, for a total of 80 SSC muscles included in the study. The study design is illustrated in Figure 1.
Stem cell in rabbit subscapularis model
Figure 1
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Overview of the study time course. ADSC, adipose-derived stem cell; EMG, electromyelography.
Isolation and culture of rabbit ADSCs ADSCs were obtained from adipose tissue in the inguinal fat pad of 2 New Zealand White male rabbits that were not included in the study. The laboratory protocol for the isolation and culture of rabbit ADSCs is described in Appendix 1.
Chronic rotator cuff tear model The chronic tear model in the ADSCþrepair, salineþrepair, ADSC-only, and saline-only groups was created in both rabbit shoulders by completely severing the SSC tendon at the insertion site, and the torn tendon was wrapped with a 10-mm-long silicone Penrose drain with an 8-mm outer diameter (Yushin Corp, Bucheon, South Korea) to inhibit adhesion to the surrounding soft tissue. The detailed surgical procedures are depicted in Appendix 2. Postoperatively, 2 rabbits (1 in the saline only group and 1 in the control group) caused self-inflicted wound dehiscence, which required irrigation and repeat closure without complications. These superficial dehiscence wounds caused no problems in the study, because they eventually healed without further incident.
SSC repair and ADSC injection After 6 weeks, under the same anesthesia and with the same approach, we removed the Penrose drain wrapped around the SSC tendon and reattached it to the lesser tuberosity by using 2 singleloaded 1.9-mm Mini Quick Anchor Plus (DePuy Mitek, Raynham, MA, USA) minisuture anchors loaded with 2-0 Ethibond braided polyester suture (Ethicon, Somerville, NJ, USA). This was performed in the ADSCþrepair and salineþrepair groups after creating a bleeding bed at the footprint of the lesser tuberosity. Both sutures were tied in a mattress fashion over the lateral aspect of the SSC tendon, approximately 3 mm from the end of the tendon. In the ADSCþrepair and ADSC-only groups, we injected the previously cultured ADSCs (1 107) in 500 mL Hank’s Balanced Salt Solution (Sigma-Aldrich, St. Louis, MO, USA) into the SSC muscle adjacent to the musculotendinous junction area.
In the salineþrepair group, we injected the same volume of saline into the same area (musculotendinous junction area of the SSC muscle) after repair. In the saline-only and control groups, we performed a sham surgery without repair or ADSC injection, and instead injected the same volume of saline into the musculotendinous junction area of the SSC muscle. The subcutaneous tissue and skin wounds were closed using the same method, and the rabbits were permitted usual cage activity without immobilization.
Electromyographic evaluation At 6 weeks after the secondary procedures (repair and ADSC injection for the ADSCþrepair group, repair only for the salineþrepair group, ADSC injection only for the ADSC-only group, and sham surgery for the saline-only and control groups), we measured the compound muscle action potential (CMAP) 5 times for each rabbit SSC muscle by using a portable electrostimulator (Compex Medical Systems/DJO Global, Ecublens, Switzerland) under anesthesia. The electromyographic (EMG) evaluation method is described in Appendix 3. From the CMAP data, we calculated the average of the areas under the negative CMAP phase as a parameter; this indicates the level of motor unit recruitment (Fig. 2).41
Biomechanical evaluation After EMG examination, the rabbits were fully anesthetized and euthanized with carbon dioxide, and the entire SSC muscle and tendon of the right shoulder along with the humeral head of each rabbit was harvested. The mode of tear and the load to failure at a rate of 1 mm/s with a preload of 5 N after 5 consecutive preconditioning cycles (from 5 to 50 N at a loading rate of 15 N/s) were evaluated by using a custom fixture-clamping system (Appendix 4) and an Instron 5565A materials testing machine (Instron, Norwood, MA, USA; Fig. 3). The tendon was loaded until it pulled apart from the bone or ruptured at its midsubstance (Fig. 4). The SSC tendon was fixed to this system along its anatomic direction to allow the tensile loading and tendon-to-bone
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Figure 2 (A) Measurements and (B) the results of compound muscle action potential in rabbit subscapularis. The upper and lower subscapularis nerve area was stimulated with a needle electrode (white arrow), and the compound muscle action potential was recorded from an active needle electrode (black arrow) and a reference surface electrode (curved arrow). The average areas (ms $ mV) under the negative phase of the compound muscle action potential formed by numbers 1, 2, and 3 (gray triangle) were calculated.
Figure 3 (A) The materials testing machine and (B) a custom fixture-clamping system are shown. (C) Illustration of the custom fixtureclamping system comprising a humeral head fixation unit and cryogenic tendon fixation unit. The humeral head fixation unit rigidly fixes the humeral head with screws and permits the subscapularis tendon attached to the humeral head to emerge through a hole that is bigger than the subscapularis tendon width but smaller than the humeral head, thus blocking the head’s escape (curved arrow). The tendon emerging from the hole is later fixed to the cryogenic tendon fixation unit, which secures the proximal part of the tendon by using a sandpaper-faced clamp (white arrow) and liquid nitrogen introduced through a funnel (black arrow) to the inside of the metal to prevent slippage.
interface to form a right angle. Data from the tensile load-tofailure testing were automatically collected with a data acquisition system on a personal computer.
Histologic evaluation We harvested the SSC muscle and sliced it transversely at 1 cm proximal to the musculotendinous junction (distal portion of the muscle). Tissue sections stained with hematoxylin and eosin (H&E) and Oil Red O, and the proportion of fat to muscle and the degree of fat-cell staining were evaluated at original magnification 200. The detailed laboratory procedure is summarized in Appendix 5. Each slide was independently examined twice for a total of 10 scanned sections per slide, and each was analyzed by 2 examiners (1 orthopedic surgeon and 1 musculoskeletal specialty pathologist) in a randomized and blinded fashion to eliminate observer bias. We used the average of 10 measured values of the proportion of fatty infiltration for quantitative
analysis. The slides were coded by using randomly generated numbers, and only after all of the slides had been assessed was the original identification of each slide revealed. Intraobserver and interobserver reliability was assessed, and these reliabilities were excellent, with interclass correlation coefficients of 0.93 (P < .001) and 0.84 (P < .001), respectively.
Statistical analysis Kruskal-Wallis testing, followed by post-hoc Mann-Whitney testing, was used to evaluate the EMG, biomechanical, and histologic differences in H&E stain between groups. Data are presented as the mean standard deviation. Interclass correlation coefficients were used to assess intraobserver and interobserver reliability for the histologic evaluation of the fat-to-muscle ratio. All statistical analyses were performed by using SPSS 12.0 software (SPSS Inc, Chicago, IL, USA), and a P value < .05 was set as the level of statistical significance.
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Figure 4 Image of (A) the insertional tear and (B) the midsubstance tear of the rabbit subscapularis tendon obtained by supramaximal tensile loading applied to the tendon by using an Instron materials testing machine.
Table I
The results of electromyographic and biomechanical evaluation
Variable
ADSCþrepair
Salineþrepair
Control
Area under CMAP, mean SD ms$mV Insertional tear, No. Midsubstance tear, No. Load to failure, mean SD, N
11.86 2.97) 4 3 87.02 29.81y
9.42 3.57 5 2 59.85 37.77
13.17 6.63 3 5 122.97 50.78
ADSCþrepair group, repair þ injection of adipose-derived stem cells at 6 weeks after initial subscapularis tear; CMAP, compound muscle action potential; Salineþrepair group, repair þ saline injection at 6 weeks after subscapularis tear; SD, standard deviation. ) The ADSCþrepair group showed a larger area than the salineþrepair group (P ¼ .029) and almost reached the level of the control group (P ¼ .456). y The loads to failure were higher in the ADSCþrepair group than in the salineþrepair group, even though it did not reach significant level (P ¼ .085).
Results One rabbit in the ADSCþrepair group and 1 in the salineþrepair group showed active infection with pus discharge at the final evaluation and were excluded from the study. All SSC tendons in the ADSC-only and salineonly groups failed to heal, with no connection between tendon and bone; therefore, EMG and biomechanical testing was completed only for the ADSCþrepair, salineþrepair, and control groups. Visual inspection showed no dehiscence or incomplete healing in the ADSCþrepair and salineþrepair groups.
EMG evaluation The EMG evaluation is reported in Table I. The mean areas under the negative CMAP phase were 11.86 2.97 ms $ mV, 9.42 3.57 ms $ mV, and 13.17 6.63 ms $ mV in the ADSCþrepair, salineþrepair, and control groups, respectively. The ADSCþrepair group showed a larger area than the salineþrepair group (P ¼ .029), nearly reaching the level of the control group (P ¼ .456).
midsubstance tears in the ADSCþrepair group, 5 insertional tears and 2 midsubstance tears in the salineþrepair group, and 3 insertional tears and 5 midsubstance tears in the control group (Fig. 4). Midsubstance tearing suggests strong tendon-to-bone healing, whereas insertional tearing suggests relatively weak tendon-to-bone healing.42 Midsubstance tearing (strong tendon-to-bone healing) was more prevalent in the ADSCþrepair group (42.9%) than in the salineþrepair group (28.6%), albeit not to the level of the control group (62.5%). The load-to-failure values were higher in the ADSCþrepair group (87.02 29.81 N) than in the salineþrepair group (59.85 37.77 N), although this difference did not reach statistical significance (P ¼ .085). The load to failure of normal SSC tendon (control group) was 122.97 50.78 N, higher than that of the other groups (P ¼ .019 compared with the ADSCþrepair group and P ¼ .004 compared with the salineþrepair group). One rabbit in the control group showed an extraordinary high load-tofailure value (195.34 N) in the biomechanical evaluation as well as high area value (32.62 ms $ mV) in the EMG evaluation, causing high variability of the standard deviation in the control group.
Biomechanical evaluation
Histologic evaluation
Results of the biomechanical evaluation are summarized in Table I. The modes of failure were 4 insertional tears and 3
Histologic evaluations are reported in Table II and shown in Figure 5. The mean proportions of fatty infiltration in the
450 Table II
J.H. Oh et al. The amount of stained portion in Oil Red O stain)
Group
Not present
Unclear or weak
Moderately present
Strongly present
ADSCþrepair Salineþrepair ADSC-only Saline-only Control
0 0 0 0 2
3 2 1 0 5
3 4 2 2 1
1 1 5 6 0
ADSC-only, adipose-derived stem cell injection alone at 6 weeks after subscapularis tear; ADSCþrepair, repair þ injection of adipose-derived stem cells at 6 weeks after initial subscapularis tear; Saline-only group, saline injection alone with no subsequent procedures after initial subscapularis tear; Salineþrepair group, repair þ saline injection at 6 weeks after subscapularis tear; control group, normal control. ) Weakly present means that less than 10% of the slide section was stained with Oil Red O, moderately present means 10% to 30% was stained, and strongly present means more than 30% was stained. More high-grade of staining in number (strongly present > moderately present > unclear or weak > not present) means increased Oil Red O staining and fatty infiltration.
SSC muscles were 29% 15%, 43% 9%, 51% 14%, 63% 10%, and 18% 9% in the ADSCþrepair, salineþrepair, ADSC-only, saline-only, and control groups, respectively, for grade 1 (P < .001), and 30% 15%, 44% 11%, 51% 14%, 64% 11%, and 16% 8%, respectively, for grade 2 (P < .001). The mean proportions of both grades were 30% 14%, 43% 10%, 51% 14%, 64% 10%, and 17% 8% in the ADSCþrepair, salineþrepair, ADSC-only, saline-only, and control groups, respectively (P < .001). The ADSCþrepair group showed a lower fat proportion than the salineþrepair group (grade 1, P ¼ .029; grade 2, P ¼ .036; and average of both grades, P ¼ .028), and that of the ADSC only group was lower than that of the saline-only group (grade 1, P ¼ .044; grade 2, P ¼ .035; and the average of both grades, P ¼ .038). In addition, when we assessed the stained portion qualitatively by using Oil Red O, the degree of fat staining increased from the ADSCþrepair group (unclear or weak) to the saline-only group (strongly present). Individually, the degree of fat staining was lower in the ADSCþrepair group compared with the salineþrepair group and in the ADSConly group compared with the saline-only group. The ADSCþrepair group showed more ‘‘unclear or weak’’ and less ‘‘moderately and strongly present’’ staining than the salineþrepair group; similarly, the ADSC only group showed more ‘‘unclear or weak’’ and less ‘‘moderately and strongly present’’ staining than the saline-only group.
Discussion Through local administration of ADSCs into repaired rabbit SSC muscle, we demonstrated the possibility of
improvement in tendon healing and fatty infiltration. To the best of our knowledge, this is the first study to investigate the positive effects of ADSCs on tendon healing according to pullout strength and histologic fatty infiltration by using a rabbit model of chronic SSC tear. Despite rapid improvements in surgical techniques and instrumentation, the healing failure rate after rotator cuff repair is considerably high,18 and the effects of repair on fatty infiltration, a known negative prognostic factor for rotator cuff healing and functional outcome, remain controversial.13,17 Thus, numerous biologic strategies, such as local injection of growth factors,50 platelet concentrate,1 and MSCs,23,27 have been recently used to enhance the quality of tendon and muscle and, therefore, to improve tendon healing and reduce or minimize fatty infiltration: Zhang et al50 demonstrated that the administration of exogenous vascular endothelial growth factor can significantly improve tensile strength early in the course of rat Achilles tendon healing, and Aspenberg et al1 showed that platelet concentrate injection after rat Achilles tendon repair improved the material characteristics and maturation of the tendon callus. In addition, Li et al27 showed that bone marrow–derived MSCs played a role in promoting tendon-to-bone tunnel healing, and Chen et al8 revealed that the injectable hydrogel made with periosteal progenitor cells and bone morphogenic protein-2 provides a powerful inductive ability between the rotator cuff tendon and the bone and enhances tendonto-bone healing through the neoformation of fibrocartilage. Furthermore, Kawiak et al23 showed that the active participation of satellite cells or MSCs is involved in damaged muscle regeneration and muscle atrophy improvement. Among these approaches, MSCs have received particular attention owing to their potential for self-renewal and totipotency. Although bone marrow–derived MSCs are well known in clinical practice, fatty tissue as a source of MSCs remains a subject of current research. ADSCs, however, have the merits of less-invasive harvesting (such as by minimally invasive liposuction) from adipose tissue, an abundant, easily accessible, and reproducible cell source, and higher yield of multipotent stem cells with good viability.26 Moreover, unlike hematopoietic cells, ADSCs do not elicit a robust lymphocyte reaction and instead release immunosuppressive factors that enable the allogeneic ADSCs to engraft successfully.15 In the present study, no signs of rejection were observed in the ADSCinjected SSC, despite the use of allogeneic ADSCs isolated from donor rabbits. This result may in part indicate the immunomodulatory properties of ADSCs. The positive effects of ADSCs on tendon healing regarding pullout strength and histological fatty infiltration may be due to direct differentiation of the ADSCs and their subsequent release of growth factors and cytokines, such as transforming growth factor-b or vascular endothelial growth factor.31,47 ADSCs are capable not only of selfdifferentiation but also of promoting differentiation of
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Figure 5 Fatty infiltration assessment by (A) hematoxylin and eosin and (B) Oil Red O staining using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and AxioCam-MRC5 (Carl Zeiss Microscopy GmbH, Jena, Germany) at original magnification 200. The mean proportion of fatty infiltration in the rabbit subscapularis muscle (average of both grades) on hematoxylin and eosin stain was 30% 14% in the ADSCþrepair group, 43% 10% in the ADSC-only group, 51% 14% in the salineþrepair group, 64% 10% in the saline-only group, and 17% 8% in the control group. The degree of fat staining by Oil Red O was increased from the ADSCþrepair group (unclear or weak) to the saline-only group (strongly present).
other endogenous stem cells along the required lineage pathway by stimulating their recruitment.16 The potential of ADSCs to differentiate and stimulate differentiation of other endogenous stem cells into bone, tendon, muscle, and ligament may be beneficial for enhancement of tendon-to-bone healing, the potential to regain the physiologic tendon-to-bone interface, and to reverse fatty infiltration. In addition, the growth factors and cytokines produced by ADSCs can regulate biologic processes, including cell proliferation, migration, differentiation, and extracellular matrix deposition, which may also play an important role in tendon healing and improvement of fatty infiltration.31 Moreover, ADSCs may provide antioxidant chemicals or free-radical scavengers to an ischemic site, thereby aiding in removal of toxic substances from the torn tendon and muscle and promoting recovery of the surviving cells.40 Although controversial, several animal studies have shown the beneficial effects of stem cells on tendon healing. Uysal et al46 showed that ADSCs enhanced Achilles tendon regeneration and healing in a rabbit model, and Awad et al2 suggested that autologous bone marrow MSCs significantly increased biomechanical and histologic function after 4 weeks in a rabbit patellar tendon model. Similarly, Young et al49 also showed that MSCs improved the biomechanics, structure, and function of the rabbit Achilles tendon after injury. In addition, Nourissat et al32 revealed that local injection of MSCs stimulated the
production of a neoenthesis at the bone-to-tendon junction that did not occur after surgery alone. These studies, however, only investigated the effect of stem cells or growth factors on tendon healing and did not show the effects on muscle regeneration or improvement of fatty infiltration. The current study investigated these effects and was further strengthened by the use of the chronic SSC tear model in rabbit and performance of a unique EMG evaluation for muscle function. The anatomy of the rabbit SSC muscle is an accurate and reliable model for the study of rotator cuff disease, given its unique anatomic, histologic, and mechanical properties.20 That is, the rabbit SSC tendon complex, constrained by a bony tunnel and coracobrachialis muscle, is quite similar to the human supraspinatus tendon complex that is similarly constrained by a subacromial bony tunnel and coracoacromial ligament.20 In the rabbit SSC model, the increases in fatty infiltration and interstitial connective tissue of the muscle after tenotomy are similar to the human environment after tenotomy.22 Another consideration is that the healing pathway and muscle changes fundamentally differ between acute and chronic injury. Chronic lesions may have decreased healing potential due to poor-quality tissues.12 Considering that rotator cuff tears usually develop over a long period of time as a result of intrinsic or extrinsic factors and that most rotator cuff surgeries are performed on chronic tears, the use of the chronic tear model is essential for this research;
452 nevertheless, most animal models evaluate acute injury and repair conditions. Finally, the CMAP represents the sum of a group of nearly simultaneous action potentials from several muscle fibers in the same area, evoked by stimulation of the supplying motor nerve, which indicates the level of recruitment of functioning muscle fibers.11 From the EMG evaluation of the repaired SSC muscle with and without ADSC injection, we were able to assess the functional and anatomic effects of ADSCs on repaired SSC muscle. Fatty infiltration of the rotator cuff muscle has been considered an irreversible phenomenon, even after successful rotator cuff repair.17,18 In the present study, however, the degree of fatty infiltration was lower in the rabbits administered ADSCs than in those who were not. The ADSCþrepair group showed less fatty infiltration than the salineþrepair group, and the ADSC-only group showed less than fatty infiltration the saline-only group. This implies the possibility of improvement in fatty infiltration and muscular atrophy by ADSC administration. Several trials have investigated the contribution of ADSCs to skeletal muscle regeneration. Kim et al24 found that ADSCs attached to polylactic glycolic acid spheres injected subcutaneously into the necks of nude mice were able to generate muscle tissue. Similarly, Di Rocco et al9 provided evidence that ADSCs possess an intrinsic myogenic potential and can spontaneously differentiate into skeletal muscle. In our previous study, we also reported that the injected ADSCs might assist in regeneration of the rotator cuff muscle by way of the insulin-like growth factor 1 signaling pathway.25 We think that the ability of ADSCs for muscle regeneration may have a relationship with the reduction of fatty infiltration in muscle tissue. Although the biomechanical pullout strength and histologic fatty infiltration were significantly better in rabbits that received local administration of ADSCs into the repaired SSC muscle, these values, particularly for pullout strength, which might be related to tendon-to-bone healing, did not reach the levels exhibited by the normal controls. Chronic rotator cuff tearing causes various changes, including decreased muscle volume, increased fat content of the muscle, altered muscle fiber composition, increased fibrotic infiltration of the muscle, and alterations in the muscle architecture, over a long period of time.4,38 Some of these chronic changes may not reverse fully after surgical repair and ADSC injection. In addition, we believe that the fibrovascular scar tissue formed between the tendon and the bone, which remains even after ADSC administration, may result in decreased tensile strength and render repaired tendons prone to failure. Moreover, it is possible that the tendon does not heal in any location where the muscle is ideally preloaded or stretched. Furthermore, type III collagen, which is formed during tendon healing, may be a factor that causes repaired tendon weakness: newly formed type III collagen is thinner and more extensible than type I collagen,
J.H. Oh et al. a mature form of collagen that is mainly responsible for the mechanical strength of the tendon.10 Nevertheless, the contraction, a functional measure of the SSC muscle of the ADSCþrepair group on EMG evaluation, was markedly improveddalmost to the level of the normal controls. We believe that the recovery of sarcomere length and histologic muscle fiber structure, as well as load-induced muscular hypertrophy, after reattachment of the torn SSC tendon may play a partial role in the marked improvement in muscle function.3 The present study has several limitations that require consideration. First, we repaired the torn SSC tendons only 6 weeks after acute surgical detachment, whereas human chronic rotator cuff tears typically develop slowly over a long period of time as a result of intrinsic and extrinsic factors, and in clinical practice, much longer delays have been reported before surgical repair of rotator cuff tendons.36 However, the time course of injury and healing for this animal model is most likely shorter than that of humans.14 Although the exact difference in the time course of injury and healing between rabbits and humans is unknown, we believe that 6 weeks is a reasonable interval for the chronic changes and healing of rabbit rotator cuff tendon to occur after repair; this duration was determined based on previous studies.37,42 Second, we studied a single time point (at 6 weeks after repair or ADSC injection), and thus had no information on the initial degrees of fatty infiltration and further changes with time. We did not prove whether fatty infiltration was reversed after ADSC injection, and further changes in muscle recovery and fatty infiltration are possible; therefore, future investigations comparing before and after repair or ADSC injection, or both, evaluated over longer periods may be necessary. Third, we only measured the biomechanical pullout strength, without histologic evaluation of tendon-to-bone healing. Histologic evaluation of collagen fiber orientation, fiber type, fibrocartilage, new enthesis formation, and microarchitecture of the muscle and tendon fibers may be beneficial. Fourth, we evaluated the level of fatty infiltration in a single section of muscle by histologic evaluation. A more quantitative method, such as evaluation of genes related to the adipocyte differentiation in the entire muscle or triglyceride quantification to assess the amount of fat, would be helpful. Fifth, muscle stimulation and contraction on EMG testing may not fully reflect the functional aspects of repaired and ADSC-injected rotator cuff muscle. A more robust test, such as ex vivo in-bath skeletal muscle functional testing, may be needed for better feedback on the functional strength of the rotator cuff muscle. Sixth, we did not track the injected ADSCs and did not identify the molecular pathways involved in muscle regeneration or improvement of fatty infiltration. Further studies, such as immunohistochemical assay of ADSC-associated
Stem cell in rabbit subscapularis model gene markers to detect accelerated myogenic differentiation and the expression of ADSC-related growth factors or cytokines, may be needed.
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Conclusion We demonstrated the positive effects of locally administered ADSCs on tendon-to-bone healing and fatty infiltration, as assessed by electrophysiologic, biomechanical, and histologic changes in a rabbit chronic SSC tear model. Considering the high failure rate and poor functional outcomes associated with severe fatty infiltration, the findings of the current study will have a farreaching effect on the treatment of rotator cuff tears. This approach might represent another strategy to increase the cuff-healing rate and improve fatty infiltration of rotator cuff muscle, both of which remain unsolved even after successful cuff repair.
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Disclaimer The authors, their immediate families, and any research foundations with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article.
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References 21. 1. Aspenberg P, Virchenko O. Platelet concentrate injection improves Achilles tendon repair in rats. Acta Orthop Scand 2004;75:93-9. http:// dx.doi.org/10.1080/00016470410001708190 2. Awad HA, Butler DL, Boivin GP, Smith FN, Malaviya P, Huibregtse B, et al. Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng 1999;5:267-77. 3. Baar K, Nader G, Bodine S. Resistance exercise, muscle loading/ unloading and the control of muscle mass. Essays Biochem 2006;42: 61-74. http://dx.doi.org/10.1042/bse0420061 4. Baker JH, Hall-Craggs EC. Changes in sarcomere length following tenotomy in the rat. Muscle Nerve 1980;3:413-6. 5. Bjorkenheim JM, Paavolainen P, Ahovuo J, Slatis P. Resistance of a defect of the supraspinatus tendon to intraarticular hydrodynamic pressure: an experimental study on rabbits. J Orthop Res 1990;8: 175-9. 6. Boileau P, Brassart N, Watkinson DJ, Carles M, Hatzidakis AM, Krishnan SG. Arthroscopic repair of full-thickness tears of the supraspinatus: does the tendon really heal? J Bone Joint Surg Am 2005;87:1229-40. http://dx.doi.org/10.2106/JBJS.D.02035 7. Burkhart SS, Lo IK. Arthroscopic rotator cuff repair. J Am Acad Orthop Surg 2006;14:333-46. 8. Chen CH, Chang CH, Wang KC, Su CI, Liu HT, Yu CM, et al. Enhancement of rotator cuff tendon-bone healing with injectable periosteum progenitor cells-BMP-2 hydrogel in vivo. Knee Surg
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Sports Traumatol Arthrosc 2011;19:1597-607. http://dx.doi.org/10. 1007/s00167-010-1373-0 Di Rocco G, Iachininoto MG, Tritarelli A, Straino S, Zacheo A, Germani A, et al. Myogenic potential of adipose-tissue-derived cells. J Cell Sci 2006;119:2945-52. http://dx.doi.org/10.1242/jcs.03029 Eriksen HA, Pajala A, Leppilahti J, Risteli J. Increased content of type III collagen at the rupture site of human Achilles tendon. J Orthop Res 2002;20:1352-7. http://dx.doi.org/10.1016/S0736-0266(02)00064-5 Gantz BJ, Gmuer AA, Holliday M, Fisch U. Electroneurographic evaluation of the facial nerve. Method and technical problems. Ann Otol Rhinol Laryngol 1984;93:394-8. Gazielly DF, Gleyze P, Montagnon C. Functional and anatomical results after rotator cuff repair. Clin Orthop Relat Res 1994;304:43-53. Gerber C, Fuchs B, Hodler J. The results of repair of massive tears of the rotator cuff. J Bone Joint Surg Am 2000;82:505-15. Gimbel JA, Van Kleunen JP, Mehta S, Perry SM, Williams GR, Soslowsky LJ. Supraspinatus tendon organizational and mechanical properties in a chronic rotator cuff tear animal model. J Biomech 2004;37:739-49. http://dx.doi.org/10.1016/j.jbiomech Gimble JM, Grayson W, Guilak F, Lopez MJ, Vunjak-Novakovic G. Adipose tissue as a stem cell source for musculoskeletal regeneration. Front Biosci (Schol Ed) 2011;3:69-81. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res 2007;100:1249-60. http://dx.doi.org/ 10.1161/01.RES.0000265074.83288.09 Gladstone JN, Bishop JY, Lo IK, Flatow EL. Fatty infiltration and atrophy of the rotator cuff do not improve after rotator cuff repair and correlate with poor functional outcome. Am J Sports Med 2007;35: 719-28. http://dx.doi.org/10.1177/0363546506297539 Goutallier D, Postel JM, Gleyze P, Leguilloux P, Van Driessche S. Influence of cuff muscle fatty degeneration on anatomic and functional outcomes after simple suture of full-thickness tears. J Shoulder Elbow Surg 2003;12:550-4. http://dx.doi.org/10.1016/S1058274603002118 Goutallier D, Postel JM, Van Driessche S, Voisin MC. [Histological lesions of supraspinatus tendons in full thickness tears of the rotator cuff]. Rev Chir Orthop Reparatrice Appar Mot 2005;91:109-13. http:// dx.doi.org/MDOI-RCO-04-2005-91-2-0035-1040-101019-200513185 Grumet RC, Hadley S, Diltz MV, Lee TQ, Gupta R. Development of a new model for rotator cuff pathology: the rabbit subscapularis muscle. Acta Orthop 2009;80:97-103. http://dx.doi.org/10.1080/ 17453670902807425 Gulotta LV, Kovacevic D, Ehteshami JR, Dagher E, Packer JD, Rodeo SA. Application of bone marrow-derived mesenchymal stem cells in a rotator cuff repair model. Am J Sports Med 2009;37:212633. http://dx.doi.org/10.1177/0363546509339582 Gupta R, Lee TQ. Contributions of the different rabbit models to our understanding of rotator cuff pathology. J Shoulder Elbow Surg 2007; 16:S149-57. http://dx.doi.org/10.1016/j.jse.2007.05.002 Kawiak J, Brzoska E, Grabowska I, Hoser G, Streminska W, Wasilewska D, et al. Contribution of stem cells to skeletal muscle regeneration. Folia Histochem Cytobiol 2006;44:75-9. Kim M, Choi YS, Yang SH, Hong HN, Cho SW, Cha SM, et al. Muscle regeneration by adipose tissue-derived adult stem cells attached to injectable PLGA spheres. Biochem Biophys Res Commun 2006;348:386-92. http://dx.doi.org/10.1016/j.bbrc.2006.07.063 Kim SH, Chung SW, Oh JH. Expression of insulin-like growth factor type 1 receptor and myosin heavy chain in rabbit’s rotator cuff muscle after injection of adipose-derived stem cell. Knee Surg Sports Traumatol Arthrosc 2013; http://dx.doi.org/10.1007/s00167-013-2560-6 [Epub ahead of print]. Kuhbier JW, Weyand B, Sorg H, Radtke C, Vogt PM, Reimers K. [Stem cells from fatty tissue: A new resource for regenerative medicine?]. Chirurg 2010;81:826-32. http://dx.doi.org/10.1007/ s00104-010-1962-y Li YG, Wei JN, Lu J, Wu XT, Teng GJ. Labeling and tracing of bone marrow mesenchymal stem cells for tendon-to-bone tunnel healing.
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Knee Surg Sports Traumatol Arthrosc 2011;19:2153-8. http://dx.doi. org/10.1007/s00167-011-1506-0 Machin D, Campbell MJ, Fayers P, Pinol A. Statistical tables for the design of clinical studies. 2nd edition. Oxford: Blackwell; 1998. Matsen FA 3rd, Arntz CT. Rotator cuff tendon failure. In: Rockwood CA Jr, Matsen FA 3rd, editors. The shoulder. 1st ed. Philadelphia, PA: WB Saunders Company; 1990. p. 647-77. Milgrom C, Schaffler M, Gilbert S, van Holsbeeck M. Rotator-cuff changes in asymptomatic adults. The effect of age, hand dominance and gender. J Bone Joint Surg Br 1995;77:296-8. Molloy T, Wang Y, Murrell G. The roles of growth factors in tendon and ligament healing. Sports Med 2003;33:381-94. http://dx.doi.org/ 10.2165/00007256-200333050-00004 Nourissat G, Diop A, Maurel N, Salvat C, Dumont S, Pigenet A, et al. Mesenchymal stem cell therapy regenerates the native bonetendon junction after surgical repair in a degenerative rat model. PLoS One 2010;5:e12248. http://dx.doi.org/10.1371/journal.pone. 0012248 Oh JH, Kim SH, Ji HM, Jo KH, Bin SW, Gong HS. Prognostic factors affecting anatomic outcome of rotator cuff repair and correlation with functional outcome. Arthroscopy 2009;25:30-9. http://dx.doi.org/10. 1016/j.arthro.2008.08.010 Oh JH, Kim SH, Kim JH, Shin YH, Yoon JP, Oh CH. The level of vitamin D in the serum correlates with fatty degeneration of the muscles of the rotator cuff. J Bone Joint Surg Br 2009;91:1587-93. http://dx.doi.org/10.1302/0301-620X.91B12.22481 Park JY, Lhee SH, Choi JH, Park HK, Yu JW, Seo JB. Comparison of the clinical outcomes of single- and double-row repairs in rotator cuff tears. Am J Sports Med 2008;36:1310-6. http://dx.doi.org/10.1177/ 0363546508315039 Romeo AA, Hang DW, Bach BR Jr, Shott S. Repair of full thickness rotator cuff tears. Gender, age, and other factors affecting outcome. Clin Orthop Relat Res 1999;367:243-55. Rubino LJ, Sprott DC, Stills HF Jr, Crosby LA. Fatty infiltration does not progress after rotator cuff repair in a rabbit model. Arthroscopy 2008;24:936-40. http://dx.doi.org/10.1016/j.arthro.2008.03.004 Safran O, Derwin KA, Powell K, Iannotti JP. Changes in rotator cuff muscle volume, fat content, and passive mechanics after chronic detachment in a canine model. J Bone Joint Surg Am 2005;87:266270. http://dx.doi.org/10.2106/JBJS.D.02421 Sher JS, Uribe JW, Posada A, Murphy BJ, Zlatkin MB. Abnormal findings on magnetic resonance images of asymptomatic shoulders. J Bone Joint Surg Am 1995;77:10-5. Spees JL, Olson SD, Whitney MJ, Prockop DJ. Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A 2006;103:1283-8. http://dx.doi.org/10.1073/pnas.0510511103 Stalberg E. Skeletal muscle: structure and function. Clinical neurophysiology of disorders of muscle and neuromuscular junction, including fatigue. Handbook of clinical neurophysiology. Vol. 2. Philadelphia, PA: Elsevier; 2003. Trudel G, Ramachandran N, Ryan SE, Rakhra K, Uhthoff HK. Supraspinatus tendon repair into a bony trough in the rabbit: mechanical restoration and correlative imaging. J Orthop Res 2010;28: 710-5. http://dx.doi.org/10.1002/jor.21045 Uhthoff HK, Matsumoto F, Trudel G, Himori K. Early reattachment does not reverse atrophy and fat accumulation of the supraspinatus–an experimental study in rabbits. J Orthop Res 2003;21:386-92. http://dx. doi.org/10.1016/S0736-0266(02)00208-5 Uhthoff HK, Seki M, Backman DS, Trudel G, Himori K, Sano H. Tensile strength of the supraspinatus after reimplantation into a bony trough: an experimental study in rabbits. J Shoulder Elbow Surg 2002; 11:504-9. http://dx.doi.org/S1058274602000721 Uysal AC, Mizuno H. Tendon regeneration and repair with adipose derived stem cells. Curr Stem Cell Res Ther 2010;5:161-7. Uysal CA, Tobita M, Hyakusoku H, Mizuno H. Adipose-derived stem cells enhance primary tendon repair: biomechanical and
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immunohistochemical evaluation. J Plast Reconstr Aesthet Surg 2012; 65:1712-9. http://dx.doi.org/10.1016/j.bjps.2012.06.011 Wang XJ, Dong Z, Zhong XH, Shi RZ, Huang SH, Lou Y, et al. Transforming growth factor-beta1 enhanced vascular endothelial growth factor synthesis in mesenchymal stem cells. Biochem Biophys Res Commun 2008;365:548-54. http://dx.doi.org/10.1016/j.bbrc.2007. 11.014 Yokoya S, Mochizuki Y, Natsu K, Omae H, Nagata Y, Ochi M. Rotator cuff regeneration using a bioabsorbable material with bone marrowderived mesenchymal stem cells in a rabbit model. Am J Sports Med 2012;40:1259-68. http://dx.doi.org/10.1177/0363546512442343 Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res 1998;16:406-13. Zhang F, Liu H, Stile F, Lei MP, Pang Y, Oswald TM, et al. Effect of vascular endothelial growth factor on rat Achilles tendon healing. Plast Reconstr Surg 2003;112:1613-9. http://dx.doi.org/10.1097/01.PRS. 0000086772.72535.A4 Zumstein MA, Jost B, Hempel J, Hodler J, Gerber C. The clinical and structural long-term results of open repair of massive tears of the rotator cuff. J Bone Joint Surg Am 2008;90:2423-31. http://dx.doi.org/ 10.2106/JBJS.G.00677
Appendix 1 Adipose tissue was digested in collagenase type I solution (Invitrogen, Carlsbad, CA, USA) with gentle agitation for 1 hour at 37 C. Upper adipocyte fractions were removed from the stromal fractions by centrifugation at 1200g for 10 minutes. The remaining stromal fractions were treated with 3 mL red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes at room temperature, filtered through a 100-mm nylon mesh, and centrifuged at 1200g for 10 minutes. The combined stromal fractions of the samples were resuspended and cultured in Dulbecco’s Modified Eagle’s Medium (Welgene, Daegu, Korea) containing 5% fetal bovine serum. Cells were allowed to adhere to the flask for 24 hours, after which fresh media was added. The cells were incubated at 37 C in 5% CO2. Culture media was changed every 2 to 3 days. Passage 3 ADSCs were detached with 0.25% trypsinethylene diamine tetraacetic acid (Invitrogen, Carlsbad, CA, USA), then rinsed with phosphate-buffered saline and incubated with the a fluorescent cell membrane marker Vybrant DiI (Molecular Probes, Eugene, OR, USA) for 20 minutes at 37 C in accordance with the manufacturer’s protocol. For transplantation, cells were suspended at a concentration of 1 107 labeled cells in 500 mL Hank’s Balanced Salt Solution (Sigma-Aldrich, St. Louis, MO, USA).
Appendix 2 Animals were maintained in conventional plastic cages and fed laboratory food and tap water. Anesthesia was induced with intramuscular injection of 15 mg/kg Zoletil 20 comprising 20 mg/mL Zoletil, 50 mg Tiletamine, and 50 mg Zolazepam (Virbac, Carros, France) and 5 mg/kg Rompun (23.32 mg/mL xylazine hydrochloride; Bayer Korea, Seoul,
Stem cell in rabbit subscapularis model Korea). The shoulders were shaved, and the animals were positioned supine with a pillow between the shoulder and the operating table. Under aseptic conditions, a deltopectoral approach was adopted with an approximately 3-cm oblique skin incision, exposing the SSC tendon. For the adipose-derived stem cell (ADSC)þrepair, salineþrepair, ADSC-only, and saline-only groups, the chronic tear model was created in both rabbit shoulders by completely severing the SSC tendon at the insertion site to allow free retraction from the lesser tuberosity and then wrapping the torn tendon with a 10-mm-long silicone Penrose drain with an 8 mm outer diameter (Yushin Corp, Bucheon, Korea) to inhibit adhesion to the surrounding soft tissue for 6 weeks until the secondary procedures could be performed. For the control group, a sham surgery was performed to create a similar biologic environment. The fascia and subcutaneous tissues were sutured by using interrupted 3-0 Vicryl sutures (Ethicon, Somerville, NJ, USA), and the skin was sutured with interrupted 3-0 nylon sutures. The limbs were not immobilized postoperatively, and the rabbits were allowed to exercise as desired in their respective cages. They had free access to water and food.
Appendix 3 To elicit the compound muscle action potential (CMAP) of the subscapularis (SSC) muscle, the posterior cord area of the brachial plexus, branching to the upper and lower SSC nerves, was exposed and then stimulated with a needle electrode at a supramaximal intensity (1 Hz frequency, 0.1 millisecond stimulation duration, 30 mA or greater current). CMAPs were recorded from an active electrode (a needle electrode placed in the SSC muscle belly) and from a reference electrode (a surface electrode at the SSC tendon) with a filtering frequency of 10 Hz to 10 kHz, sweep speed of 1 millisecond/division, and sensitivity of 1 mV/division, by using a portable Medelec Synergy 2-channel electromyography/nerve conduction velocity system (Oxford Instrument Medical Systems, Oxford, UK).
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Appendix 4 For tensile testing of the subscapularis (SSC) tendon, we designed and manufactured a custom clamping system consisting of 2 separate fixtures: (1) a humeral head fixation unit, which rigidly fixes the humeral head and permits the SSC tendon and muscle attached to the humeral head to emerge through a hole, and (2) a cryogenic tendon fixation unit, which secures the myotendinous junction of the tendon by using a sandpaper-faced clamp and liquid nitrogen introduced through a funnel to the inside of the metal to prevent slippage.
Appendix 5 The samples were mounted in optimal cutting temperature (OCT) compound on filter paper and frozen in liquid nitrogen. Serial transverse sections (5-mm thick) were cut on a Leica CM 1900 cryostat (Leica Microsystems Inc, Buffalo Grove, IL USA), mounted on glass slides (1 section/slide), and fixed with acetone for 5 minutes at –70 C. These samples were used to make assessments of the fat-to-muscle proportion using hematoxylin and eosin (H&E) staining and of the degree of fat cell staining by Oil Red O stain under original magnification 200 by using an Olympus BX51 microscope (Olympus, Tokyo, Japan). On H&E stain, the amount of fatty infiltration present on the slides was calculated by subtracting the area of stained muscle and connective tissue from the total area by using Image-Pro Plus digital analyzing software (Media Cybernetics Inc, Rockville, MD, USA), and the proportion of fatty infiltration was calculated as the amount of fat divided by the total area. Four degrees of Oil Red O stain were assessed: not present, weakly present (less than 10% of the slide section was stained), moderately present (10% to 30% was stained), and strongly present (more than 30% was stained). Images were captured and acquired by using an AxioCam MRC-5, with AxioVision 4.4 acquisition software (Carl Zeiss Microscopy GmbH, Jena, Germany).
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BASIC SCIENCE
2013 Neer Award: Effect of the adipose-derived stem cell for the improvement of fatty degeneration and rotator cuff healing in rabbit model Joo Han Oh, MD, PhDa, Seok Won Chung, MDb,*, Sae Hoon Kim, MD, PhDc, Jin Young Chung, DVM, PhDd, Joon Yub Kim, MDe a
Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea b Department of Orthopaedic Surgery, Konkuk University School of Medicine, Konkuk University Medical Center, Seoul, South Korea c Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, South Korea d Department of Neurology, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, South Korea e Department of Orthopaedic Surgery, Myongji Hospital, Goyang, South Korea Background: This study was conducted to verify the effects of adipose-derived stem cells (ADSCs) on tendon healing and reversal of fatty infiltration in a chronic rotator cuff tear model by using the rabbit subscapularis (SSC). Methods: The SSC insertions in 32 rabbits were cut bilaterally. After 6 weeks, secondary procedures were performed bilaterally, dividing the rabbits into 4 groups of 8 rabbits each as follows: the ADSCþrepair group, salineþrepair group, ADSC-only group, and saline-only group. A fifth group of 8 rabbits served as normal controls (control group). Electromyographic, biomechanical, and histologic analyses were performed 6 weeks after the secondary procedures. Results: All SSC tendons in the ADSC-only and saline-only groups failed to heal and were excluded from the electromyographic and biomechanical tests. On electromyographic evaluation, the ADSCþrepair group exhibited a larger compound muscle action potential area than the salineþrepair group (11.86 2.97 ms $ mV vs 9.42 3.57 ms $ mV, P ¼ .029), and this response was almost at the level of the control group (13.17 6.6 3 ms $ mV, P ¼ .456). Biomechanically, the load-to-failure of the ADSCþrepair group (87.02 29.81 N) was higher than that of the salineþrepair group (59.85 37.77 N), although this difference did not reach statistical significance (P ¼ .085). Histologically, the mean proportions of fatty infiltration in the SSC muscles were 29% 15%, 43% 9%, 51% 14%, 63% 10%, and 18% 9% for the ADSCþrepair, salineþrepair, ADSC-only, saline-only, and control groups, respectively (P < .001). The
This study was approved by the Seoul National University Bundang Hospital (SNUBH) Institutional Animal Care and Use Committee (Approval: No. BA1005-062/030-01). This study was supported by the Basic Science Research Program through the National Research Foundation of Koreafunded by the Ministry of Education, Science and Technology (2010-000850).
*Reprint requests: Seok Won Chung, MD, Shoulder and Elbow Surgery Division, Department of Orthopedic Surgery, Konkuk University School of Medicine, Konkuk University Medical Center, 120-1 Neungdong-ro, Gwangjin-gu, Seoul 143-729, South Korea. E-mail address: [email protected] (S.W. Chung).
1058-2746/$ - see front matter Ó 2014 Journal of Shoulder and Elbow Surgery Board of Trustees. http://dx.doi.org/10.1016/j.jse.2013.07.054
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degree of fat staining increased from the ADSCþrepair group (unclear or weak) to the saline-only group (strongly present). Conclusion: Local administration of ADSCs might have the possibility to improve muscle function and tendon healing and decrease fatty infiltration after cuff repair. Level of evidence: Basic Science Study, Biomechanics and Histology, Animal Model. Ó 2014 Journal of Shoulder and Elbow Surgery Board of Trustees. Keywords: Adipose-derived stem cell; rotator cuff tear; fatty infiltration; tendon-to-bone healing; compound muscle action potential
Rotator cuff tears are a common condition that causes shoulder pain and dysfunction that has been reported to occur in more than 50% of people aged older than 60 years.30,39 Although rotator cuff repair techniques and instrumentation have rapidly improved, rotator cuff healing failure after surgical repair remains one of the most common and well-known complications. Indeed, current clinical data suggest that approximately 50% of surgically repaired rotator cuffs do not heal properly, independent of the surgical procedures used.6,7,35 Rotator cuff tendon and muscle quality is a key predisposing factor of healing failure and poor functional outcome.12,33 Unfortunately, rotator cuff tears usually occur in elderly individuals who exhibit inherent tissue aging19 and usually present with chronic tears with disorganized tendon scar tissue and fatty infiltration in the muscle.12,29 Fatty infiltration represents fat deposition in tissues, particularly between cells, and preoperative fatty infiltration of the rotator cuff muscles is an important predisposing factor to repair failure and poor functional outcomes.18,19 However, fatty infiltration in chronic rotator cuff tears has been regarded as an irreversible phenomenon even after successful cuff repair,17,34 and nonreversible fatty infiltration was reported to persist even at long-term follow-up.51 The development of fatty infiltration after rotator cuff tendon detachment is well established in a rabbit model. Bjorkenheim et al5 demonstrated that fatty infiltration of the rabbit supraspinatus was most prominent at 6 weeks after injury, and Gupta et al22 showed that fatty infiltration of the muscle increased significantly at 6 weeks after tenotomy of the rabbit subscapularis (SSC). However, a few studies that investigated the effect of reattachment of the rotator cuff tendon on fatty infiltration in a rabbit model failed to show a decrease in fatty infiltration after reattachment.43 Stem cell–based therapies for the repair and regeneration of tendon and muscle represent a paradigm shift and show great promise as alternative therapeutic solutions for a number of diseases.45 Several investigators have attempted to demonstrate the effect of stem cells on rotator cuff healing; however, these effects remain unproven and controversial. Yokoya et al48 showed that mesenchymal stem cells (MSCs) were capable of regenerating rotator cuff tendon-to-bone insertions and the tendon belly in a rabbit
model. However, Gulotta et al21 suggested that the addition of MSCs to the insertion site of the repaired rotator cuff tendon did not improve the structure, composition, or strength of the healing tendon attachment site in a rat model. Moreover, no study to date has investigated the effect of stem cells on fatty infiltration in rotator cuff muscles. The adipose-derived stem cell (ADSC) is a progenitor cell that can proliferate and differentiate into different types of mesenchymal cells, such as tenocytes or myocytes, as well as locally release growth factors and cytokines.26 This ability may be beneficial in enhancing tendon-to-bone healing and improving fatty infiltration. Therefore, the purpose of this study was to verify the effects of ADSCs on tendon-to-bone healing and improvement of fatty infiltration in a chronic rotator cuff tear model by using rabbit SSC muscle. We hypothesized that local administration of ADSCs in the SSC muscle enhances tendon-to-bone healing and improves fatty infiltration.
Materials and methods All experiments in this study were conducted in accordance with the Seoul National University Bundang Hospital guidelines for the care and use of laboratory animals.
Allocation of experimental animals This was a controlled laboratory study involving animals. Power analysis indicated that a sample size of 7 was required for biomechanical studies to detect a significant difference in ultimate failure load (mean difference, 90 N; standard deviation, 40 N; aerror, 0.05; b-error, 0.2; drop-out rate, 20%), and a sample size of 8 was necessary for histology studies to detect a 10% difference in fat-to-muscle proportion (a-error, 0.05; b-error, 0.2; drop-out rate, 20%), based on previous studies.28,44 We randomly allocated 40 mature New Zealand White male rabbits (average age, 24 weeks; weight, 3.5-4.0 kg) into 5 groups (8 rabbits per group) as follows: the ADSCþrepair group, salineþrepair group, ADSC-only group, saline-only group, and control group. The surgeries on each of the 40 animals were bilateral; thus, the number of SSC muscles in each group was 16, for a total of 80 SSC muscles included in the study. The study design is illustrated in Figure 1.
Stem cell in rabbit subscapularis model
Figure 1
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Overview of the study time course. ADSC, adipose-derived stem cell; EMG, electromyelography.
Isolation and culture of rabbit ADSCs ADSCs were obtained from adipose tissue in the inguinal fat pad of 2 New Zealand White male rabbits that were not included in the study. The laboratory protocol for the isolation and culture of rabbit ADSCs is described in Appendix 1.
Chronic rotator cuff tear model The chronic tear model in the ADSCþrepair, salineþrepair, ADSC-only, and saline-only groups was created in both rabbit shoulders by completely severing the SSC tendon at the insertion site, and the torn tendon was wrapped with a 10-mm-long silicone Penrose drain with an 8-mm outer diameter (Yushin Corp, Bucheon, South Korea) to inhibit adhesion to the surrounding soft tissue. The detailed surgical procedures are depicted in Appendix 2. Postoperatively, 2 rabbits (1 in the saline only group and 1 in the control group) caused self-inflicted wound dehiscence, which required irrigation and repeat closure without complications. These superficial dehiscence wounds caused no problems in the study, because they eventually healed without further incident.
SSC repair and ADSC injection After 6 weeks, under the same anesthesia and with the same approach, we removed the Penrose drain wrapped around the SSC tendon and reattached it to the lesser tuberosity by using 2 singleloaded 1.9-mm Mini Quick Anchor Plus (DePuy Mitek, Raynham, MA, USA) minisuture anchors loaded with 2-0 Ethibond braided polyester suture (Ethicon, Somerville, NJ, USA). This was performed in the ADSCþrepair and salineþrepair groups after creating a bleeding bed at the footprint of the lesser tuberosity. Both sutures were tied in a mattress fashion over the lateral aspect of the SSC tendon, approximately 3 mm from the end of the tendon. In the ADSCþrepair and ADSC-only groups, we injected the previously cultured ADSCs (1 107) in 500 mL Hank’s Balanced Salt Solution (Sigma-Aldrich, St. Louis, MO, USA) into the SSC muscle adjacent to the musculotendinous junction area.
In the salineþrepair group, we injected the same volume of saline into the same area (musculotendinous junction area of the SSC muscle) after repair. In the saline-only and control groups, we performed a sham surgery without repair or ADSC injection, and instead injected the same volume of saline into the musculotendinous junction area of the SSC muscle. The subcutaneous tissue and skin wounds were closed using the same method, and the rabbits were permitted usual cage activity without immobilization.
Electromyographic evaluation At 6 weeks after the secondary procedures (repair and ADSC injection for the ADSCþrepair group, repair only for the salineþrepair group, ADSC injection only for the ADSC-only group, and sham surgery for the saline-only and control groups), we measured the compound muscle action potential (CMAP) 5 times for each rabbit SSC muscle by using a portable electrostimulator (Compex Medical Systems/DJO Global, Ecublens, Switzerland) under anesthesia. The electromyographic (EMG) evaluation method is described in Appendix 3. From the CMAP data, we calculated the average of the areas under the negative CMAP phase as a parameter; this indicates the level of motor unit recruitment (Fig. 2).41
Biomechanical evaluation After EMG examination, the rabbits were fully anesthetized and euthanized with carbon dioxide, and the entire SSC muscle and tendon of the right shoulder along with the humeral head of each rabbit was harvested. The mode of tear and the load to failure at a rate of 1 mm/s with a preload of 5 N after 5 consecutive preconditioning cycles (from 5 to 50 N at a loading rate of 15 N/s) were evaluated by using a custom fixture-clamping system (Appendix 4) and an Instron 5565A materials testing machine (Instron, Norwood, MA, USA; Fig. 3). The tendon was loaded until it pulled apart from the bone or ruptured at its midsubstance (Fig. 4). The SSC tendon was fixed to this system along its anatomic direction to allow the tensile loading and tendon-to-bone
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Figure 2 (A) Measurements and (B) the results of compound muscle action potential in rabbit subscapularis. The upper and lower subscapularis nerve area was stimulated with a needle electrode (white arrow), and the compound muscle action potential was recorded from an active needle electrode (black arrow) and a reference surface electrode (curved arrow). The average areas (ms $ mV) under the negative phase of the compound muscle action potential formed by numbers 1, 2, and 3 (gray triangle) were calculated.
Figure 3 (A) The materials testing machine and (B) a custom fixture-clamping system are shown. (C) Illustration of the custom fixtureclamping system comprising a humeral head fixation unit and cryogenic tendon fixation unit. The humeral head fixation unit rigidly fixes the humeral head with screws and permits the subscapularis tendon attached to the humeral head to emerge through a hole that is bigger than the subscapularis tendon width but smaller than the humeral head, thus blocking the head’s escape (curved arrow). The tendon emerging from the hole is later fixed to the cryogenic tendon fixation unit, which secures the proximal part of the tendon by using a sandpaper-faced clamp (white arrow) and liquid nitrogen introduced through a funnel (black arrow) to the inside of the metal to prevent slippage.
interface to form a right angle. Data from the tensile load-tofailure testing were automatically collected with a data acquisition system on a personal computer.
Histologic evaluation We harvested the SSC muscle and sliced it transversely at 1 cm proximal to the musculotendinous junction (distal portion of the muscle). Tissue sections stained with hematoxylin and eosin (H&E) and Oil Red O, and the proportion of fat to muscle and the degree of fat-cell staining were evaluated at original magnification 200. The detailed laboratory procedure is summarized in Appendix 5. Each slide was independently examined twice for a total of 10 scanned sections per slide, and each was analyzed by 2 examiners (1 orthopedic surgeon and 1 musculoskeletal specialty pathologist) in a randomized and blinded fashion to eliminate observer bias. We used the average of 10 measured values of the proportion of fatty infiltration for quantitative
analysis. The slides were coded by using randomly generated numbers, and only after all of the slides had been assessed was the original identification of each slide revealed. Intraobserver and interobserver reliability was assessed, and these reliabilities were excellent, with interclass correlation coefficients of 0.93 (P < .001) and 0.84 (P < .001), respectively.
Statistical analysis Kruskal-Wallis testing, followed by post-hoc Mann-Whitney testing, was used to evaluate the EMG, biomechanical, and histologic differences in H&E stain between groups. Data are presented as the mean standard deviation. Interclass correlation coefficients were used to assess intraobserver and interobserver reliability for the histologic evaluation of the fat-to-muscle ratio. All statistical analyses were performed by using SPSS 12.0 software (SPSS Inc, Chicago, IL, USA), and a P value < .05 was set as the level of statistical significance.
Stem cell in rabbit subscapularis model
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Figure 4 Image of (A) the insertional tear and (B) the midsubstance tear of the rabbit subscapularis tendon obtained by supramaximal tensile loading applied to the tendon by using an Instron materials testing machine.
Table I
The results of electromyographic and biomechanical evaluation
Variable
ADSCþrepair
Salineþrepair
Control
Area under CMAP, mean SD ms$mV Insertional tear, No. Midsubstance tear, No. Load to failure, mean SD, N
11.86 2.97) 4 3 87.02 29.81y
9.42 3.57 5 2 59.85 37.77
13.17 6.63 3 5 122.97 50.78
ADSCþrepair group, repair þ injection of adipose-derived stem cells at 6 weeks after initial subscapularis tear; CMAP, compound muscle action potential; Salineþrepair group, repair þ saline injection at 6 weeks after subscapularis tear; SD, standard deviation. ) The ADSCþrepair group showed a larger area than the salineþrepair group (P ¼ .029) and almost reached the level of the control group (P ¼ .456). y The loads to failure were higher in the ADSCþrepair group than in the salineþrepair group, even though it did not reach significant level (P ¼ .085).
Results One rabbit in the ADSCþrepair group and 1 in the salineþrepair group showed active infection with pus discharge at the final evaluation and were excluded from the study. All SSC tendons in the ADSC-only and salineonly groups failed to heal, with no connection between tendon and bone; therefore, EMG and biomechanical testing was completed only for the ADSCþrepair, salineþrepair, and control groups. Visual inspection showed no dehiscence or incomplete healing in the ADSCþrepair and salineþrepair groups.
EMG evaluation The EMG evaluation is reported in Table I. The mean areas under the negative CMAP phase were 11.86 2.97 ms $ mV, 9.42 3.57 ms $ mV, and 13.17 6.63 ms $ mV in the ADSCþrepair, salineþrepair, and control groups, respectively. The ADSCþrepair group showed a larger area than the salineþrepair group (P ¼ .029), nearly reaching the level of the control group (P ¼ .456).
midsubstance tears in the ADSCþrepair group, 5 insertional tears and 2 midsubstance tears in the salineþrepair group, and 3 insertional tears and 5 midsubstance tears in the control group (Fig. 4). Midsubstance tearing suggests strong tendon-to-bone healing, whereas insertional tearing suggests relatively weak tendon-to-bone healing.42 Midsubstance tearing (strong tendon-to-bone healing) was more prevalent in the ADSCþrepair group (42.9%) than in the salineþrepair group (28.6%), albeit not to the level of the control group (62.5%). The load-to-failure values were higher in the ADSCþrepair group (87.02 29.81 N) than in the salineþrepair group (59.85 37.77 N), although this difference did not reach statistical significance (P ¼ .085). The load to failure of normal SSC tendon (control group) was 122.97 50.78 N, higher than that of the other groups (P ¼ .019 compared with the ADSCþrepair group and P ¼ .004 compared with the salineþrepair group). One rabbit in the control group showed an extraordinary high load-tofailure value (195.34 N) in the biomechanical evaluation as well as high area value (32.62 ms $ mV) in the EMG evaluation, causing high variability of the standard deviation in the control group.
Biomechanical evaluation
Histologic evaluation
Results of the biomechanical evaluation are summarized in Table I. The modes of failure were 4 insertional tears and 3
Histologic evaluations are reported in Table II and shown in Figure 5. The mean proportions of fatty infiltration in the
450 Table II
J.H. Oh et al. The amount of stained portion in Oil Red O stain)
Group
Not present
Unclear or weak
Moderately present
Strongly present
ADSCþrepair Salineþrepair ADSC-only Saline-only Control
0 0 0 0 2
3 2 1 0 5
3 4 2 2 1
1 1 5 6 0
ADSC-only, adipose-derived stem cell injection alone at 6 weeks after subscapularis tear; ADSCþrepair, repair þ injection of adipose-derived stem cells at 6 weeks after initial subscapularis tear; Saline-only group, saline injection alone with no subsequent procedures after initial subscapularis tear; Salineþrepair group, repair þ saline injection at 6 weeks after subscapularis tear; control group, normal control. ) Weakly present means that less than 10% of the slide section was stained with Oil Red O, moderately present means 10% to 30% was stained, and strongly present means more than 30% was stained. More high-grade of staining in number (strongly present > moderately present > unclear or weak > not present) means increased Oil Red O staining and fatty infiltration.
SSC muscles were 29% 15%, 43% 9%, 51% 14%, 63% 10%, and 18% 9% in the ADSCþrepair, salineþrepair, ADSC-only, saline-only, and control groups, respectively, for grade 1 (P < .001), and 30% 15%, 44% 11%, 51% 14%, 64% 11%, and 16% 8%, respectively, for grade 2 (P < .001). The mean proportions of both grades were 30% 14%, 43% 10%, 51% 14%, 64% 10%, and 17% 8% in the ADSCþrepair, salineþrepair, ADSC-only, saline-only, and control groups, respectively (P < .001). The ADSCþrepair group showed a lower fat proportion than the salineþrepair group (grade 1, P ¼ .029; grade 2, P ¼ .036; and average of both grades, P ¼ .028), and that of the ADSC only group was lower than that of the saline-only group (grade 1, P ¼ .044; grade 2, P ¼ .035; and the average of both grades, P ¼ .038). In addition, when we assessed the stained portion qualitatively by using Oil Red O, the degree of fat staining increased from the ADSCþrepair group (unclear or weak) to the saline-only group (strongly present). Individually, the degree of fat staining was lower in the ADSCþrepair group compared with the salineþrepair group and in the ADSConly group compared with the saline-only group. The ADSCþrepair group showed more ‘‘unclear or weak’’ and less ‘‘moderately and strongly present’’ staining than the salineþrepair group; similarly, the ADSC only group showed more ‘‘unclear or weak’’ and less ‘‘moderately and strongly present’’ staining than the saline-only group.
Discussion Through local administration of ADSCs into repaired rabbit SSC muscle, we demonstrated the possibility of
improvement in tendon healing and fatty infiltration. To the best of our knowledge, this is the first study to investigate the positive effects of ADSCs on tendon healing according to pullout strength and histologic fatty infiltration by using a rabbit model of chronic SSC tear. Despite rapid improvements in surgical techniques and instrumentation, the healing failure rate after rotator cuff repair is considerably high,18 and the effects of repair on fatty infiltration, a known negative prognostic factor for rotator cuff healing and functional outcome, remain controversial.13,17 Thus, numerous biologic strategies, such as local injection of growth factors,50 platelet concentrate,1 and MSCs,23,27 have been recently used to enhance the quality of tendon and muscle and, therefore, to improve tendon healing and reduce or minimize fatty infiltration: Zhang et al50 demonstrated that the administration of exogenous vascular endothelial growth factor can significantly improve tensile strength early in the course of rat Achilles tendon healing, and Aspenberg et al1 showed that platelet concentrate injection after rat Achilles tendon repair improved the material characteristics and maturation of the tendon callus. In addition, Li et al27 showed that bone marrow–derived MSCs played a role in promoting tendon-to-bone tunnel healing, and Chen et al8 revealed that the injectable hydrogel made with periosteal progenitor cells and bone morphogenic protein-2 provides a powerful inductive ability between the rotator cuff tendon and the bone and enhances tendonto-bone healing through the neoformation of fibrocartilage. Furthermore, Kawiak et al23 showed that the active participation of satellite cells or MSCs is involved in damaged muscle regeneration and muscle atrophy improvement. Among these approaches, MSCs have received particular attention owing to their potential for self-renewal and totipotency. Although bone marrow–derived MSCs are well known in clinical practice, fatty tissue as a source of MSCs remains a subject of current research. ADSCs, however, have the merits of less-invasive harvesting (such as by minimally invasive liposuction) from adipose tissue, an abundant, easily accessible, and reproducible cell source, and higher yield of multipotent stem cells with good viability.26 Moreover, unlike hematopoietic cells, ADSCs do not elicit a robust lymphocyte reaction and instead release immunosuppressive factors that enable the allogeneic ADSCs to engraft successfully.15 In the present study, no signs of rejection were observed in the ADSCinjected SSC, despite the use of allogeneic ADSCs isolated from donor rabbits. This result may in part indicate the immunomodulatory properties of ADSCs. The positive effects of ADSCs on tendon healing regarding pullout strength and histological fatty infiltration may be due to direct differentiation of the ADSCs and their subsequent release of growth factors and cytokines, such as transforming growth factor-b or vascular endothelial growth factor.31,47 ADSCs are capable not only of selfdifferentiation but also of promoting differentiation of
Stem cell in rabbit subscapularis model
451
Figure 5 Fatty infiltration assessment by (A) hematoxylin and eosin and (B) Oil Red O staining using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and AxioCam-MRC5 (Carl Zeiss Microscopy GmbH, Jena, Germany) at original magnification 200. The mean proportion of fatty infiltration in the rabbit subscapularis muscle (average of both grades) on hematoxylin and eosin stain was 30% 14% in the ADSCþrepair group, 43% 10% in the ADSC-only group, 51% 14% in the salineþrepair group, 64% 10% in the saline-only group, and 17% 8% in the control group. The degree of fat staining by Oil Red O was increased from the ADSCþrepair group (unclear or weak) to the saline-only group (strongly present).
other endogenous stem cells along the required lineage pathway by stimulating their recruitment.16 The potential of ADSCs to differentiate and stimulate differentiation of other endogenous stem cells into bone, tendon, muscle, and ligament may be beneficial for enhancement of tendon-to-bone healing, the potential to regain the physiologic tendon-to-bone interface, and to reverse fatty infiltration. In addition, the growth factors and cytokines produced by ADSCs can regulate biologic processes, including cell proliferation, migration, differentiation, and extracellular matrix deposition, which may also play an important role in tendon healing and improvement of fatty infiltration.31 Moreover, ADSCs may provide antioxidant chemicals or free-radical scavengers to an ischemic site, thereby aiding in removal of toxic substances from the torn tendon and muscle and promoting recovery of the surviving cells.40 Although controversial, several animal studies have shown the beneficial effects of stem cells on tendon healing. Uysal et al46 showed that ADSCs enhanced Achilles tendon regeneration and healing in a rabbit model, and Awad et al2 suggested that autologous bone marrow MSCs significantly increased biomechanical and histologic function after 4 weeks in a rabbit patellar tendon model. Similarly, Young et al49 also showed that MSCs improved the biomechanics, structure, and function of the rabbit Achilles tendon after injury. In addition, Nourissat et al32 revealed that local injection of MSCs stimulated the
production of a neoenthesis at the bone-to-tendon junction that did not occur after surgery alone. These studies, however, only investigated the effect of stem cells or growth factors on tendon healing and did not show the effects on muscle regeneration or improvement of fatty infiltration. The current study investigated these effects and was further strengthened by the use of the chronic SSC tear model in rabbit and performance of a unique EMG evaluation for muscle function. The anatomy of the rabbit SSC muscle is an accurate and reliable model for the study of rotator cuff disease, given its unique anatomic, histologic, and mechanical properties.20 That is, the rabbit SSC tendon complex, constrained by a bony tunnel and coracobrachialis muscle, is quite similar to the human supraspinatus tendon complex that is similarly constrained by a subacromial bony tunnel and coracoacromial ligament.20 In the rabbit SSC model, the increases in fatty infiltration and interstitial connective tissue of the muscle after tenotomy are similar to the human environment after tenotomy.22 Another consideration is that the healing pathway and muscle changes fundamentally differ between acute and chronic injury. Chronic lesions may have decreased healing potential due to poor-quality tissues.12 Considering that rotator cuff tears usually develop over a long period of time as a result of intrinsic or extrinsic factors and that most rotator cuff surgeries are performed on chronic tears, the use of the chronic tear model is essential for this research;
452 nevertheless, most animal models evaluate acute injury and repair conditions. Finally, the CMAP represents the sum of a group of nearly simultaneous action potentials from several muscle fibers in the same area, evoked by stimulation of the supplying motor nerve, which indicates the level of recruitment of functioning muscle fibers.11 From the EMG evaluation of the repaired SSC muscle with and without ADSC injection, we were able to assess the functional and anatomic effects of ADSCs on repaired SSC muscle. Fatty infiltration of the rotator cuff muscle has been considered an irreversible phenomenon, even after successful rotator cuff repair.17,18 In the present study, however, the degree of fatty infiltration was lower in the rabbits administered ADSCs than in those who were not. The ADSCþrepair group showed less fatty infiltration than the salineþrepair group, and the ADSC-only group showed less than fatty infiltration the saline-only group. This implies the possibility of improvement in fatty infiltration and muscular atrophy by ADSC administration. Several trials have investigated the contribution of ADSCs to skeletal muscle regeneration. Kim et al24 found that ADSCs attached to polylactic glycolic acid spheres injected subcutaneously into the necks of nude mice were able to generate muscle tissue. Similarly, Di Rocco et al9 provided evidence that ADSCs possess an intrinsic myogenic potential and can spontaneously differentiate into skeletal muscle. In our previous study, we also reported that the injected ADSCs might assist in regeneration of the rotator cuff muscle by way of the insulin-like growth factor 1 signaling pathway.25 We think that the ability of ADSCs for muscle regeneration may have a relationship with the reduction of fatty infiltration in muscle tissue. Although the biomechanical pullout strength and histologic fatty infiltration were significantly better in rabbits that received local administration of ADSCs into the repaired SSC muscle, these values, particularly for pullout strength, which might be related to tendon-to-bone healing, did not reach the levels exhibited by the normal controls. Chronic rotator cuff tearing causes various changes, including decreased muscle volume, increased fat content of the muscle, altered muscle fiber composition, increased fibrotic infiltration of the muscle, and alterations in the muscle architecture, over a long period of time.4,38 Some of these chronic changes may not reverse fully after surgical repair and ADSC injection. In addition, we believe that the fibrovascular scar tissue formed between the tendon and the bone, which remains even after ADSC administration, may result in decreased tensile strength and render repaired tendons prone to failure. Moreover, it is possible that the tendon does not heal in any location where the muscle is ideally preloaded or stretched. Furthermore, type III collagen, which is formed during tendon healing, may be a factor that causes repaired tendon weakness: newly formed type III collagen is thinner and more extensible than type I collagen,
J.H. Oh et al. a mature form of collagen that is mainly responsible for the mechanical strength of the tendon.10 Nevertheless, the contraction, a functional measure of the SSC muscle of the ADSCþrepair group on EMG evaluation, was markedly improveddalmost to the level of the normal controls. We believe that the recovery of sarcomere length and histologic muscle fiber structure, as well as load-induced muscular hypertrophy, after reattachment of the torn SSC tendon may play a partial role in the marked improvement in muscle function.3 The present study has several limitations that require consideration. First, we repaired the torn SSC tendons only 6 weeks after acute surgical detachment, whereas human chronic rotator cuff tears typically develop slowly over a long period of time as a result of intrinsic and extrinsic factors, and in clinical practice, much longer delays have been reported before surgical repair of rotator cuff tendons.36 However, the time course of injury and healing for this animal model is most likely shorter than that of humans.14 Although the exact difference in the time course of injury and healing between rabbits and humans is unknown, we believe that 6 weeks is a reasonable interval for the chronic changes and healing of rabbit rotator cuff tendon to occur after repair; this duration was determined based on previous studies.37,42 Second, we studied a single time point (at 6 weeks after repair or ADSC injection), and thus had no information on the initial degrees of fatty infiltration and further changes with time. We did not prove whether fatty infiltration was reversed after ADSC injection, and further changes in muscle recovery and fatty infiltration are possible; therefore, future investigations comparing before and after repair or ADSC injection, or both, evaluated over longer periods may be necessary. Third, we only measured the biomechanical pullout strength, without histologic evaluation of tendon-to-bone healing. Histologic evaluation of collagen fiber orientation, fiber type, fibrocartilage, new enthesis formation, and microarchitecture of the muscle and tendon fibers may be beneficial. Fourth, we evaluated the level of fatty infiltration in a single section of muscle by histologic evaluation. A more quantitative method, such as evaluation of genes related to the adipocyte differentiation in the entire muscle or triglyceride quantification to assess the amount of fat, would be helpful. Fifth, muscle stimulation and contraction on EMG testing may not fully reflect the functional aspects of repaired and ADSC-injected rotator cuff muscle. A more robust test, such as ex vivo in-bath skeletal muscle functional testing, may be needed for better feedback on the functional strength of the rotator cuff muscle. Sixth, we did not track the injected ADSCs and did not identify the molecular pathways involved in muscle regeneration or improvement of fatty infiltration. Further studies, such as immunohistochemical assay of ADSC-associated
Stem cell in rabbit subscapularis model gene markers to detect accelerated myogenic differentiation and the expression of ADSC-related growth factors or cytokines, may be needed.
453
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10.
Conclusion We demonstrated the positive effects of locally administered ADSCs on tendon-to-bone healing and fatty infiltration, as assessed by electrophysiologic, biomechanical, and histologic changes in a rabbit chronic SSC tear model. Considering the high failure rate and poor functional outcomes associated with severe fatty infiltration, the findings of the current study will have a farreaching effect on the treatment of rotator cuff tears. This approach might represent another strategy to increase the cuff-healing rate and improve fatty infiltration of rotator cuff muscle, both of which remain unsolved even after successful cuff repair.
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Disclaimer The authors, their immediate families, and any research foundations with which they are affiliated have not received any financial payments or other benefits from any commercial entity related to the subject of this article.
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Appendix 1 Adipose tissue was digested in collagenase type I solution (Invitrogen, Carlsbad, CA, USA) with gentle agitation for 1 hour at 37 C. Upper adipocyte fractions were removed from the stromal fractions by centrifugation at 1200g for 10 minutes. The remaining stromal fractions were treated with 3 mL red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes at room temperature, filtered through a 100-mm nylon mesh, and centrifuged at 1200g for 10 minutes. The combined stromal fractions of the samples were resuspended and cultured in Dulbecco’s Modified Eagle’s Medium (Welgene, Daegu, Korea) containing 5% fetal bovine serum. Cells were allowed to adhere to the flask for 24 hours, after which fresh media was added. The cells were incubated at 37 C in 5% CO2. Culture media was changed every 2 to 3 days. Passage 3 ADSCs were detached with 0.25% trypsinethylene diamine tetraacetic acid (Invitrogen, Carlsbad, CA, USA), then rinsed with phosphate-buffered saline and incubated with the a fluorescent cell membrane marker Vybrant DiI (Molecular Probes, Eugene, OR, USA) for 20 minutes at 37 C in accordance with the manufacturer’s protocol. For transplantation, cells were suspended at a concentration of 1 107 labeled cells in 500 mL Hank’s Balanced Salt Solution (Sigma-Aldrich, St. Louis, MO, USA).
Appendix 2 Animals were maintained in conventional plastic cages and fed laboratory food and tap water. Anesthesia was induced with intramuscular injection of 15 mg/kg Zoletil 20 comprising 20 mg/mL Zoletil, 50 mg Tiletamine, and 50 mg Zolazepam (Virbac, Carros, France) and 5 mg/kg Rompun (23.32 mg/mL xylazine hydrochloride; Bayer Korea, Seoul,
Stem cell in rabbit subscapularis model Korea). The shoulders were shaved, and the animals were positioned supine with a pillow between the shoulder and the operating table. Under aseptic conditions, a deltopectoral approach was adopted with an approximately 3-cm oblique skin incision, exposing the SSC tendon. For the adipose-derived stem cell (ADSC)þrepair, salineþrepair, ADSC-only, and saline-only groups, the chronic tear model was created in both rabbit shoulders by completely severing the SSC tendon at the insertion site to allow free retraction from the lesser tuberosity and then wrapping the torn tendon with a 10-mm-long silicone Penrose drain with an 8 mm outer diameter (Yushin Corp, Bucheon, Korea) to inhibit adhesion to the surrounding soft tissue for 6 weeks until the secondary procedures could be performed. For the control group, a sham surgery was performed to create a similar biologic environment. The fascia and subcutaneous tissues were sutured by using interrupted 3-0 Vicryl sutures (Ethicon, Somerville, NJ, USA), and the skin was sutured with interrupted 3-0 nylon sutures. The limbs were not immobilized postoperatively, and the rabbits were allowed to exercise as desired in their respective cages. They had free access to water and food.
Appendix 3 To elicit the compound muscle action potential (CMAP) of the subscapularis (SSC) muscle, the posterior cord area of the brachial plexus, branching to the upper and lower SSC nerves, was exposed and then stimulated with a needle electrode at a supramaximal intensity (1 Hz frequency, 0.1 millisecond stimulation duration, 30 mA or greater current). CMAPs were recorded from an active electrode (a needle electrode placed in the SSC muscle belly) and from a reference electrode (a surface electrode at the SSC tendon) with a filtering frequency of 10 Hz to 10 kHz, sweep speed of 1 millisecond/division, and sensitivity of 1 mV/division, by using a portable Medelec Synergy 2-channel electromyography/nerve conduction velocity system (Oxford Instrument Medical Systems, Oxford, UK).
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Appendix 4 For tensile testing of the subscapularis (SSC) tendon, we designed and manufactured a custom clamping system consisting of 2 separate fixtures: (1) a humeral head fixation unit, which rigidly fixes the humeral head and permits the SSC tendon and muscle attached to the humeral head to emerge through a hole, and (2) a cryogenic tendon fixation unit, which secures the myotendinous junction of the tendon by using a sandpaper-faced clamp and liquid nitrogen introduced through a funnel to the inside of the metal to prevent slippage.
Appendix 5 The samples were mounted in optimal cutting temperature (OCT) compound on filter paper and frozen in liquid nitrogen. Serial transverse sections (5-mm thick) were cut on a Leica CM 1900 cryostat (Leica Microsystems Inc, Buffalo Grove, IL USA), mounted on glass slides (1 section/slide), and fixed with acetone for 5 minutes at –70 C. These samples were used to make assessments of the fat-to-muscle proportion using hematoxylin and eosin (H&E) staining and of the degree of fat cell staining by Oil Red O stain under original magnification 200 by using an Olympus BX51 microscope (Olympus, Tokyo, Japan). On H&E stain, the amount of fatty infiltration present on the slides was calculated by subtracting the area of stained muscle and connective tissue from the total area by using Image-Pro Plus digital analyzing software (Media Cybernetics Inc, Rockville, MD, USA), and the proportion of fatty infiltration was calculated as the amount of fat divided by the total area. Four degrees of Oil Red O stain were assessed: not present, weakly present (less than 10% of the slide section was stained), moderately present (10% to 30% was stained), and strongly present (more than 30% was stained). Images were captured and acquired by using an AxioCam MRC-5, with AxioVision 4.4 acquisition software (Carl Zeiss Microscopy GmbH, Jena, Germany).
