The American Journal of Sports Medicine http://ajs.sagepub.com/
Effect of the Interposition of Calcium Phosphate Materials on Tendon-Bone Healing During Repair of Chronic Rotator Cuff Tear Song Zhao, Lingjie Peng, Guoming Xie, Dingfeng Li, Jinzhong Zhao and Congqin Ning Am J Sports Med 2014 42: 1920 originally published online May 22, 2014 DOI: 10.1177/0363546514532781 The online version of this article can be found at: http://ajs.sagepub.com/content/42/8/1920
Published by: http://www.sagepublications.com
On behalf of: American Orthopaedic Society for Sports Medicine
Additional services and information for The American Journal of Sports Medicine can be found at: Email Alerts: http://ajs.sagepub.com/cgi/alerts Subscriptions: http://ajs.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Aug 1, 2014 OnlineFirst Version of Record - May 22, 2014 What is This?
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Effect of the Interposition of Calcium Phosphate Materials on Tendon-Bone Healing During Repair of Chronic Rotator Cuff Tear Song Zhao,* MD, Lingjie Peng,y MS, Guoming Xie,* MD, Dingfeng Li,* MD, Jinzhong Zhao,*z MD, and Congqin Ning,yz PhD Investigation performed at Shanghai Sixth People’s Hospital, Shanghi, P.R. China, and Chinese Academy of Sciences, Shanghi, P.R. China Background: The current nature of tendon-bone healing after rotator cuff (RC) repair is still the formation of granulation tissue at the tendon-bone interface rather than the formation of fibrocartilage, which is the crucial structure in native tendon insertion and can be observed after knee ligament reconstruction. The interposition of calcium phosphate materials has been found to be able to enhance tendon-bone healing in knee ligament reconstruction. However, whether the interposition of these kinds of materials can enhance tendon-bone healing or even change the current nature of tendon-bone healing after RC repair still needs to be explored. Hypothesis: The interposition of calcium phosphate materials during RC repair would enhance tendon-bone healing or change its current nature of granulation tissue formation into a more favorable process. Study Design: Controlled laboratory study. Methods: A total of 144 male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon, followed by delayed repair after 3 weeks. The animals were allocated into 1 of 3 groups: (1) repair alone, (2) repair with Ca5(PO4)2SiO4 (CPS) bioceramic interposition, or (3) repair with hydroxyapatite (HA) bioceramic interposition at the tendon-bone interface. Animals were sacrificed at 2, 4, or 8 weeks postoperatively, and microcomputed tomography (micro-CT) was used to quantify the new bone formation at the repair site. New fibrocartilage formation and collagen organization at the tendon-bone interface was evaluated by histomorphometric analysis. Biomechanical testing of the supraspinatus tendon-bone complex was performed. Statistical analysis was performed using 1-way analysis of variance. Significance was set at P \ .05. Results: The micro-CT analysis demonstrated remarkable osteogenic activity and osteoconductivity to promote new bone formation and ingrowth of CPS and HA bioceramic, with CPS bioceramic showing better results than HA. Histological observations indicated that CPS bioceramic had excellent biocompatibility and biodegradability. At early time points after the RC repair, CPS bioceramic significantly increased the area of fibrocartilage at the tendon-bone interface compared with the control and HA groups. Moreover, CPS and HA bioceramics had significantly improved collagen organization. Biomechanical tests indicated that the CPS and HA groups have greater ultimate load to failure and stiffness than the control group at 4 and 8 weeks, and the CPS specimens exhibited the maximum ultimate load to failure, stiffness, and stress of the healing enthesis. Conclusion: Both CPS and HA bioceramics aid in cell attachment and proliferation and accelerate new bone formation, and CPS bioceramic has a more prominent effect on tendon-to-bone healing. Clinical Relevance: Local application of CPS and HA bioceramic at the tendon-bone interface shows promise in improving healing after rotator cuff tear repair. Keywords: rotator cuff tear; calcium phosphate–based bioceramic; rat model; tendon-bone healing
The rotator cuff (RC) is formed around the proximal humerus by the tendinous insertions of a group of muscles that dynamically stabilize the glenohumeral joint. Rotator
cuff tear (RCT) is among the most common injuries, leading to recurrent pain and dysfunction of the shoulder. Despite significant advances in surgical techniques that optimize the fixation of tendon to bone to achieve the highest possible initial strength, prior studies have reported a relatively high retear rate after RC repairs.9,26,28,29,39 Patient age, tear size, chronicity, and biologic healing response are described as important risk factors for
The American Journal of Sports Medicine, Vol. 42, No. 8 DOI: 10.1177/0363546514532781 Ó 2014 The Author(s)
1920 Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1921
retear.27 These risk factors indicate the insufficient healing capacity of these degenerative lesions, which is likely the main factor for the failure of the reconstruction performed in RC surgery. As a result, the study of improving the healing capacity of degenerative lesions has gained increasing interest. In humans, the RC often ruptures at the tendon-bone insertion site, which is transitioned by a well-defined zone of fibrocartilage. The normal tendon-bone insertion site of RC, which is similar to that of the knee ligament, is composed of 4 distinct transition zones: tendon, fibrocartilage, mineralized fibrocartilage, and bone.14 The fundamental function of the insertion site is to minimize stress concentration at the junction between soft tissue (tendon) and hard tissue (bone).1 Various methods have been reported to improve tendon-to-bone healing after RC repair. These methods included the reinforcement of tendon-to-bone fixation,11 introduction of growth factors (TGF-b1~3, BMP-2~7, bFGF, IGF-1, etc),33,42 application of bone marrow–derived mesenchymal stem cells,8 lowintensity pulsed ultrasound treatment,31 and so on. The results suggested that these methods improved the biomechanical strength of the tendon-bone construct to some extent. However, this improvement is typically caused by fibrovascular scar tissue rather than normal insertion site structure and composition, and thus the material properties of healing tissue are not improved.19 To change the formation of granulation tissue, the current nature of tendon-bone healing after RC repair, into fibrocartilage formation may greatly change the healing results. Therefore, the idea of redirecting the healing process away from scar formation and toward the regeneration of a native tendon-bone insertion site is an attractive strategy for improving the outcome of RC repairs. In previous studies regarding tendon-bone healing enhancement in ACL reconstruction models, calcium phosphate materials have been found to have positive effects. In a rabbit ACL reconstruction model, Tien et al38 filled the tendon-bone interface with calcium phosphate (CaP) cement, and Mutsuzaki et al24 hybridized CaP with the grafted tendon tissue. Histologic and mechanical examination found promotion of tendon-bone healing. In Huangfu and Zhao’s study in a canine model, it was found that adding a kind of mixture of tricalcium phosphate (TCP) and hydroxyapatite (HA) resulted in faster incorporation of the graft to the tunnel, with better biomechanical properties and a more mature histologic pattern.15 In addition, fibrocartilage and mineralized fibrocartilage formation have been found in all of these ACL reconstruction models. Tendon-to-bone healing depends on bone ingrowth into the granulation tissue at the healing tendon-bone
interface.40 To date, a limited number of studies have considered whether interposing the tendon-bone interface with CaP at the time of RC repair can improve tendonbone healing. Kovacevic et al18 augmented the treatment with CaP matrix and TGF-b3 in an acute rat RCT model and demonstrated meaningful improvement by the combination therapies. However, modeling of acute injury and repair in rats does not mimic clinical RCT in humans. In addition, the inferior resorbability of the CaP matrix may influence the process of tendon-to-bone healing. We suppose that an ideal tendon-bone enhancement material should be absorbed in time. Calcium phosphate–based biomaterials (CaPs) are attracting interest as substitutes for bone materials.4,20 The CaPs are similar in composition to the bone mineral (a calcium phosphate in the form of carbonate apatite) and exhibit bioactive (ability to directly bond to bone, thus forming a uniquely strong interface) and osteoconductive (ability to serve as a template or guide for the newly forming bone) properties. Interconnecting porosity (macroporosity and microporosity) similar to that of bone can be introduced by chemical or physical methods. The bioactive property promotes the formation of a carbonate HA layer, which attracts proteins to which cells bind or adhere, proliferate, and differentiate, leading to matrix production and biomineralization or the formation of new bone.20 Ca5(PO4)2SiO4 (CPS) bioceramic is one of the CaPs. Silicon is an essential trace element for bone development. Studies on silicon-based bioglasses and bioceramics have indicated that the high bioactivity of bioactive materials is attributed to the silicon component, which can form silanol groups (Si-OH) on the material surface through ion exchanges in physiological solutions.22 The silanol groups can act as bioactive sites to induce the formation of bonelike apatite by attracting calcium and phosphorus ions. Si-doped calcium phosphate materials have shown a great rate of in vivo dissolution and an improved ability to form bone.30 In previous studies, pure CPS bioceramic could be successfully synthesized by a sol-gel method.22 Compared with HA ceramic, CPS ceramic exhibited better bioactivity, cytocompatibility, and osteogenic activity in vitro.10,22 However, whether these favorable material properties can turn into favorable tendon-bone healing effects still needs to be explored. The purpose of this study was to evaluate whether the interposition of 2 kinds of calcium phosphate materials, the CPS ceramic and HA ceramic, during RC repair would improve the structural, histologic, and biomechanical outcomes in a rat model. Our hypothesis was that the interposition of calcium phosphate materials during RC repair would enhance tendon-bone healing or change its current
z Address correspondence to Jinzhong Zhao, MD, Department of Arthroscopic Surgery, Shanghai Jiao Tong University–Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai, 200233, P.R. China (e-mail: [email protected]); and Congqin Ning, PhD, State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, P.R. China (e-mail: [email protected]). *Department of Arthroscopic Surgery, Shanghai Jiao Tong University–Affiliated Sixth People’s Hospital, Shanghai, P.R. China. y State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, P.R. China. S.Z. and L.P. are co–first authors of this article. The authors declared that they have no conflicts of interest in the authorship and publication of this contribution.
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1922 Zhao et al
The American Journal of Sports Medicine
nature of granulation tissue formation into a more favorable process. Furthermore, we supposed that CPS ceramics, which have more favorable material characteristics than HA, should show greater promoting effects for tendon-bone healing.
MATERIALS AND METHODS Materials The CPS and HA bioceramic powders were synthesized as described in the literature.10,22 Powders ranging in size from 150 to 300 mm were used in the present study. Phase compositions of CPS and HA powders were identified by x-ray diffraction (D8 Advance, Bruker, Billerica, Massachusetts, USA). The powders were sterilized and stored at room temperature until surgery.
Establishment of RCT Model and RC Repair Chronic RCT models were first established. A total of 144 male Sprague-Dawley rats (obtained at 350-400 g) were used in this study. The surgical procedure was performed according to previously published studies.5,6,36 During the primary surgery, the left shoulder was operated on under general anesthesia to completely detach the supraspinatus tendon from its insertion site on the humerus. With the rat in the lateral decubitus position, a longitudinal incision was made on the anterolateral aspect of the shoulder, splitting the deltoid. Under adduction, retroversion, and slight internal rotation of the supraspinatus, insertion of the supraspinatus tendon on the greater tuberosity of the proximal humerus was visualized. The supraspinatus tendon was marked with a 5-0 Prolene suture (Ethicon, Blue Ash, Ohio, USA) approximately 3 mm medial from the insertion. Under tension of the suture, the tendon was detached sharply from the rotator interval anterior to the insertion of the infraspinatus posterior. Finally, the adhesions to the anterior and posterior tissue were released by longitudinal incisions oriented parallel to the fiber orientation of the supraspinatus tendon. The suture was knotted with 3 simple knots to facilitate the localization of the tendon stump at the time of scarification. The overlying deltoid muscle and skin were then closed, and the rats were allowed to engage in unrestricted cage activity. For 3 days postoperatively, the rats received weight-adjusted pain medication (metamizole oral and buprenorphine subcutaneous) every 12 hours. At 3 weeks postoperatively, the animals were randomized into 1 of 3 groups. In the control group, the tendon was repaired to its anatomic footprint (transosseous repair; n = 48). In the experimental groups, 96 rats underwent transosseous repair and were implanted with either CPS ceramic powder (n = 48; CPS group) or HA ceramic powder (n = 48; HA group) to augment the repair. For repair of a chronic degenerated tendon, the previous scar was reincised. Under humeroacromial extension, the marking suture was retrieved and the tendon was mobilized. The footprint was decorticated lightly with a No. 15 blade knife
to ensure complete debridement of the native enthesis. A small trough was made using an 18-gauge blunt needle and was located centrally in the footprint to allow for seating of the ceramic powder. All animals had the small trough present, with the experimental groups receiving 2 mg of either CPS or HA, while the trough remained empty in the control group. Bone tunnels were created at the anterior and posterior margins of the insertion site in a crossed fashion using a 22-gauge needle. A Mason-Allen stitch using a 3-0 Ethibond suture (Ethicon) was placed into the supraspinatus tendon. Suture ends from the tendon were then passed through the bone tunnels and firmly tied over the humeral metaphyseal cortex, anatomically repairing the supraspinatus tendon to its native footprint. The wound closure and postoperative treatment were performed according to the first operation.
Microcomputed Tomography Analysis Twenty-four animals (8 animals per group per time period) were allocated for microcomputed tomography (micro-CT) analysis at 2, 4, and 8 weeks after surgery. After necropsy, each shoulder girdle was carefully dissected to include only the supraspinatus tendon-bone complex and the proximal third of the humerus for fixation in 10% neutral buffered formalin. The bone density and new bone formation at the tendon insertion site on the greater tuberosity were assessed with micro-CT (eXplore Locus SP; GE Healthcare, London, Ontario, Canada). Each sample was placed in the holder surrounded by formalin and scanned using the conditions of 80 kV, 450 mA, and a 0.045-mm effective pixel size. The images were thresholded to distinguish bone voxels by use of a global threshold for each specimen. After the thresholded scan, 3-dimensional reconstructed images were obtained, with a customized 4 3 4-mm cylindrical region of interest (ROI) centered at the surface of the repaired supraspinatus tendon-bone footprint. The ROI contained the distal portion of the supraspinatus tendon and the bony footprint. The size of the ROI was determined based on postmortem observations, which indicated that the supraspinatus footprint measured approximately 3.5 3 3.5 mm. The bone mineral density (BMD), tissue mineral density (TMD), and bone volume fraction (bone volume/total volume; BV/TV) were calculated for a volume of interest at the greater tuberosity.
Histomorphometric Analysis After micro-CT analysis, the tissue specimens were decalcified with Immunocal (Decal, Congers, New York, USA) and embedded in paraffin. Sections 5-mm thick were cut in the coronal plane through the repaired supraspinatus tendon and the greater tuberosity. The tissue sections were stained with hematoxylin and eosin, safranin O/fast green, and picrosirius red. Using light microscopy (Leica DM4000B, Leica, Solms, Germany), tissue sections stained with safranin O/fast green were analyzed to determine the total area of new fibrocartilage formation at the insertion site of the RC repair. Digital images of the stained tissue sections were
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1923
taken using a Leica DFC420C camera (Leica). The ImageJ software program (National Institutes of Health, Bethesda, Maryland, USA) was used to determine the area of new fibrocartilage formation by manually outlining the area of metachromasia on the safranin O slides at a total magnification of 503. The total area of metachromasia was then recorded (in units of mm2) for each specimen.18 Tissue sections were stained with picrosirius red for semiquantitative analysis of the collagen deposition and maturation at the repair site. By quantifying the collagen bi-refringence under polarized light, differences in collagen maturity and organization in the healing tendon could be detected. Measurements were obtained by rotating the polarization plane until the maximum brightness was obtained. To facilitate comparisons between groups, tissues for all specimens were embedded and cut to a uniform thickness, and the light intensities were measured under exactly the same conditions of illumination and during the same sitting. After a photomicrograph was captured, the image was imported into ImageJ to be processed by 8-bit digitization, producing an image in which noncollagenous material was dark (zero) and collagenous material was depicted in gray scale with values from 1 to 255. Ten rectangular areas (50 3 50 mm) were randomly selected from the tendon region adjacent to the insertion site, and the grayscale values were measured and recorded.18
Biomechanical Testing Twenty-four animals were sacrificed at 2, 4, and 8 weeks after surgery and allocated for biomechanical testing (8 rats per group per time point). Each shoulder was removed from a –80°C freezer and thawed at room temperature on the day of biomechanical testing. The humerus with attached supraspinatus tendon was meticulously dissected from the surrounding tissues. The scar tissue was removed, and the supraspinatus muscle belly was removed from the tendon. The suture was left in situ. A digital caliper was used to measure the cross-sectional area of each supraspinatus tendon at the point of insertion into the humerus. The specimen was placed into a custom-designed uniaxial testing system. The tendon was secured in a screw grip using sandpaper and cyanoacrylate, while the humerus was secured into a vice grip that prevented fracture through the humeral physics. The supraspinatus tendon was secured to a 45-N load cell attached to a linear bearing, which allowed the alignment of the tendon in the direction of its pull. The humeral jig was secured to the linear stage, and the grip-to-grip distance was standardized across all specimens. Each specimen was preloaded to 0.1 N and then loaded to failure at a rate of 14 mm/s, corresponding to approximately 0.4% strain. The ultimate load to failure and the failure site were recorded. The displacement was measured using a 1-mm– resolution micrometer system attached to a linear stage. The linear region of the load-displacement curve was used to calculate the stiffness for each specimen. The ultimate stress at failure was calculated by dividing the ultimate load-to-failure by the cross-sectional area.2,12,18
Data Analysis Approval for this study was obtained from the related animal care committee of our institution. All authors were blinded to which study group or time interval the specimens belonged at the time of the histomorphometric analysis and biomechanical testing. Using data from previous reports,2,12,16,18 a priori power analysis with an error probability of .05 and power of .8 indicated that a minimum sample size of 8 rats per group per time point for biomechanical testing was necessary. All data are presented as the mean 6 standard deviation (SD). Statistical analysis was performed using 1-way analysis of variance with post hoc testing using the Holm-Sidak method. Significance was set at P \ .05.
RESULTS Macroscopic Observations No gross evidence of infection was observed at the surgical site in any of the specimens, and no adhesions or contractures limited the shoulder range of motion. The repaired tendon was in continuity with the bone in all animals at the time of sacrifice. No qualitative differences in the gross appearance of either the supraspinatus tendon or the healing enthesis were observed during necropsy. The shoulders in the HA group had remnants of the ceramic powder present at the tendon attachment site footprint at 2 and 4 weeks after surgery, while at later time points, no remnant was observed.
Micro-CT Analysis At each time point, the BMD of the experimental groups was significantly larger than that of the control group (2 weeks: 422.88 6 4.73 mg/mm3 [control], 432.00 6 3.96 mg/mm3 [HA], and 440.00 6 10.16 mg/mm3 [CPS]; 4 weeks: 434.00 6 4.99 mg/mm3 [control], 445.63 6 4.07 mg/mm3 [HA], and 452.13 6 5.64 mg/mm3 [CPS]; 8 weeks: 441.63 6 3.50 mg/mm3 [control], 454.38 6 5.55 mg/mm3 [HA], and 463.25 6 5.39 mg/mm3 [CPS]). At 4 and 8 weeks after surgery, the value of BMD of the CPS group was even greater than that of the HA group (Figure 1). Similarly, the values of TMD and BV/TV of the experimental groups were significantly larger than those of the control group at the 3 time points (2-week TMD: 521.25 6 5.45 mg/mm3 [control], 536.13 6 6.69 mg/mm3 [HA], and 546.13 6 7.75 mg/mm3 [CPS]; 4-week TMD: 534.25 6 5.90 mg/mm3 [control], 550.25 6 6.78 mg/mm3 [HA], and 559.13 6 4.94 mg/mm3 [CPS]; 8-week TMD: 543.38 6 7.39 mg/mm3 [control], 570.75 6 7.15 mg/mm3 [HA], and 580.25 6 4.98 mg/mm3 [CPS]; 2-week BV/TV: 73.0% 6 0.7% [control], 74.4% 6 0.4% [HA], and 75.2% 6 0.4% [CPS]; 4-week BV/TV: 73.8% 6 0.5% [control], 75.5% 6 0.7% [HA], and 76.4% 6 0.4% [CPS]; 8-week BV/TV: 74.7% 6 0.4% [control], 76.4% 6 0.5% [HA], 77.4% 6 0.4% [CPS]). Furthermore, the values of TMD and BV/TV of the CPS group were greater than those of the HA group at each time point (Figure 1). In addition, some remnants of
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1924 Zhao et al
The American Journal of Sports Medicine
Figure 1. (A) Representative microcomputed tomography images of the proximal humerus and (B) analysis of bone mineral density (BMD), tissue mineral density (TMD), and bone volume/total volume (BV/TV). Results are shown as mean 6 standard deviation. Effects of Ca5(PO4)2SiO4 on rotator cuff healing: *P \ .05 vs control; DP \ .05 vs HA; n = 8 for each group. CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite. HA bioceramic powder at the tendon-bone insertion site were found at 2, 4, and 8 weeks after surgery. (Because of the high sensitivity of micro-CT, it is different from macroscopic observation.) In contrast, no image of CPS remnant was observed at any time point. The CPS bioceramic exhibited a remarkable capacity to improve new bone formation at the tendon-bone interface. Furthermore, compared with HA, CPS exhibited superior degradability.
Histological Analysis
tissue was less cellular and more organized than was the case at 4 weeks. Fibrocartilage in small areas was observed. In the experimental groups, the larger areas of new bone and fibrocartilage observed at 4 weeks were remodeled and more organized than was the case at 2 weeks; the cells in the gap tissue were aligned with the tensile load on the tendon. The fibrocartilage in the tendon-to-bone insertion was remodeled into a fibrocartilage zone with a more natural appearance (Figure 2).
Metachromasia
Cellularity and Host Tissue Response. In the control group at 2 weeks after surgery, the inflammatory cells present consisted primarily of polymorphonuclear leukocytes; there was capillary proliferation, and the gap between the tendon and bone was filled in with fibrovascular granulation tissue. In the experimental groups, no obvious fibrovascular granulation tissue filled in the gap between the tendon and bone. Bone ingrowth into the interface between the tendon and bone was more pronounced than in the control group. Furthermore, chondrocytes were found between the tendon and bone in the HA group, and new fibrocartilage was observed in the CPS group. Specimens in the control group at 4 weeks after surgery contained fewer inflammatory cells, greater cellular organization, more collagen, and more bone ingrowth into the interface between tendon and bone than in the samples obtained at 2 weeks. There was no fibrocartilage formation in the tendon-bone insertion. Identical findings were made in the experimental groups, except that a larger area of new bone and fibrocartilage was found between the tendon and bone in both groups. In the control group at 8 weeks after surgery, the gap tissues were in the form of well-organized collagen fibers oriented in line with the tensile pull of the tendon; the
At 2 and 4 weeks after surgery, HA and CPS bioceramic significantly increased the area of glycosaminoglycan staining at the tendon-bone interface compared with the control group (2 weeks: 354,503 6 19,693 mm2 [control], 407,792 6 15,895 mm2 [HA], and 428,416 6 9621 mm2 [CPS]; 4 weeks: 402,729 6 14,704 mm2 [control], 444,700 6 16,966 mm2 [HA], and 467,040 6 16,710 mm2 [CPS]). The total area of metachromasia of the CPS group was significantly greater compared with that of the HA group. At 8 weeks after surgery, the experimental groups had a larger total area of metachromasia compared with the control group (409,909 6 14,257 mm2 [control], 472,470 6 13,300 mm2 [HA], and 488,879 6 11,111 mm2 [CPS]). There were no differences between the experimental groups (P = .057). CPS exhibited the capacity to improve cartilage regeneration at the tendon-bone interface, especially at 2 and 4 weeks after surgery (Figure 3).
Collagen Organization At all 3 time points, the experimental groups exhibited significantly improved collagen organization, based on birefringence under polarized light at the healing enthesis,
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1925
Figure 2. Representative hematoxylin and eosin–stained tissue sections (1003) of the tendon insertion site at 2, 4, and 8 weeks postoperatively. B, bone; CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite; I, interface; T, tendon. compared with the control group (2 weeks: 18.5 6 0.6 grayscale units [control], 20.5 6 0.9 grayscale units [HA], and 21.2 6 0.8 grayscale units [CPS]; 4 weeks: 29.8 6 0.9 grayscale units for controls, 31.5 6 0.7 grayscale units [HA], and 31.7 6 0.8 grayscale units [CPS]; 8 weeks: 42.1 6 1.3 grayscale units for controls, 43.8 6 1.0 grayscale units [HA], and 44.0 6 0.9 grayscale units [CPS]). There were no differences between the experimental groups at 2, 4, and 8 weeks, with P = .192, P = .885, and P = .973, respectively. These results indicate that CPS and HA are beneficial for collagen production (Figure 4).
Biomechanical Testing Cross-sectional Area of the Healing Enthesis. At each time point, no significant difference was observed in the cross-sectional area of the healing enthesis between the control group and the experimental groups (Figure 5A).
failure and stiffness of the supraspinatus tendon-bone construct in the experimental groups (ultimate load: 18.3 6 1.0 N [control], 20.7 6 1.6 N [HA], and 21.4 6 1.3 N [CPS]; stiffness: 8.0 6 0.8 N/mm [control], 9.4 6 0.8 N/mm [HA], and 9.7 6 0.6 N/mm [CPS]). There was no difference between the experimental CPS and HA groups (P = .597 and .852, respectively). At 8 weeks, there were significantly greater ultimate load to failure and stiffness of the supraspinatus tendon-bone construct in the CPS group compared with both the HA and control groups (ultimate load: 27.2 6 1.1 N [control], 28.8 6 1.0 N [HA], and 32.7 6 1.0 N [CPS], P \ .01 for both CPS vs control and CPS vs HA; stiffness: 13.2 6 0.7 N/mm [control], 14.0 6 0.6 N/mm [HA], and 14.9 6 0.4 N/mm [CPS]; P \ .01 for CPS vs control, P = .023 for CPS vs HA) (Figure 5, B and C). After 8 weeks, the mechanical strength of the supraspinatus tendon-bone construct in the CPS group obviously increased.
Ultimate Stress of the Healing Enthesis Ultimate Load to Failure and Stiffness From 2 to 8 weeks postoperatively, the ultimate load to failure and stiffness of the supraspinatus tendon-bone construct increased in all 3 groups. At 2 weeks after surgery, there was no significant difference between the groups (ultimate load: 8.4 6 0.6 N [control], 8.7 6 0.8 N [HA], and 9.1 6 0.9 N [CPS], with P = .888, P = .272, and P = .657, respectively; stiffness: 5.5 6 0.5 N/mm [control], 5.7 6 0.6 N/mm [HA], and 6.1 6 0.5 N/mm [CPS], with P = .892, P = .184, and P = .506, respectively). However, at 4 weeks, there were significantly greater values of the ultimate load to
The ultimate stress of the healing enthesis in the 3 groups increased over time. At 2 and 4 weeks, there was no significant difference between the groups (2 weeks: 1.00 6 0.03 MPa [control], 1.00 6 0.03 MPa [HA], and 1.01 6 0.03 MPa [CPS]; 4 weeks: 1.31 6 0.08 MPa [control], 1.34 6 0.07 MPa [HA], and 1.38 6 0.07 MPa [CPS]). At 8 weeks, there was significantly greater ultimate stress in the CPS group (1.65 6 0.09 MPa [control], 1.69 6 0.09 MPa [HA], and 1.92 6 0.09 MPa [CPS]) (Figure 5D). These results are consistent with the results of the ultimate load to failure and stiffness at 8 weeks.
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1926 Zhao et al
The American Journal of Sports Medicine
Figure 3. (A) Representative histology images of the cartilage at the insertion site (1003 magnification) and (B) the area of cartilage present at the insertion site as determined by metachromasia with safranin O–stained slides. Results are shown as mean 6 standard deviation (*P \ .05 vs control, DP \ .05 vs HA; n = 8 for each group). B, bone; CPS, Ca5(PO4)2 SiO4; HA, hydroxyapatite; I, interface; T, tendon.
DISCUSSION The pathogenesis of chronic RC degeneration is multifactorial and results from both mechanical and biological factors. Loss of cellularity, thinning and disorganization of tendon fibers, increased granulation tissue, and fibrocartilaginous changes may explain the relatively high rate of healing failure or recurrent RCTs after repair.3 Given the inadequate healing response, biomaterials offer the possibility to augment healing after RC repair and may be critical to improving clinical outcomes.23 While most of the prior studies used acute RCT models for evaluation, this does not reflect the degenerative, age-related RCTs commonly observed in humans, who tend to exhibit intrinsic degenerative changes in the torn RC tendon, tendon retraction, and osteoporosis of the greater tuberosity in the shoulders. In the present study, we established a chronic rat RCT model according to the Buchmann approach.5,6 His team demonstrated that chronic human RCT could be imitated in the rat model, in which tendon degeneration, inflammation, and muscle atrophy combined with a persisting defect at 3 weeks after detachment were comparable with the chronic tendon tears in humans.
Figure 4. (A) Representative picrosirius red–stained tissue sections of the healing enthesis (1003 magnification) and (B) analysis of the collagen bi-refringence. Results are shown as mean 6 standard deviation (*P \ .05 vs control; DP \ .05 vs HA; n = 8 for each group). B, bone; CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite; I, interface; T, tendon.
In this study, we tested the hypothesis that treatment with CPS bioceramic can promote new bone and fibrocartilage formation and strengthen the healing enthesis compared with repair alone in a chronic RCT model. Micro-CT analysis revealed that local application of CaP bioceramics significantly improved new bone formation at the tendon-bone interface. CPS bioceramic especially exhibited both remarkable osteogenic activity and osteoconductivity, which promoted new bone formation and ingrowth. In addition, CPS bioceramic exhibits excellent biodegradability. At early times after the repairs, the CPS material was degraded completely, thereby avoiding any interference with the healing process. We observed that the bioceramic materials exhibited a biocompatible host tissue response at the tendon repair site. No obvious inflammation was observed. Local application of CaP bioceramics are associated with improvements in the area of fibrocartilage and collagen organization at the healing enthesis compared with RC repair only at each time point, which underscores the importance of this material as a provisional matrix in the phase of tendon-bone healing. Moreover, there was a significantly
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1927
Figure 5. Biomechanical testing of the tendon at the insertion site: (A) cross-sectional areas, (B) ultimate load to failure, (C) stiffness, and (D) ultimate stress to failure values. Results are shown as mean 6 standard deviation (*P \ .05 vs control; DP \ .05 vs HA; n = 8 for each group). CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite. improved area of fibrocartilage with CPS bioceramic, indicating that CPS led to the formation of a more mature healing enthesis compared with either HA bioceramic or repair only. Biomechanical testing is critical to evaluate whether implantation of the bioceramics can improve the biomechanical properties of the repair constructs. In this study, local application of CaP bioceramic did result in greater ultimate load to failure and stiffness at 4 weeks. In addition, after 8 weeks, the mechanical strength of the supraspinatus tendon-bone construct augmented with CPS obviously increased. The biomechanical testing results confirmed the feasibility of the augmentation with CPS bioceramic for use in RC repair. Although the magnitudes of the biomechanical changes noted are relatively small, they are encouraging and would be clinically important. Because CaP has a chemical composition that is similar to bone, there is recent interest in its use as an osteoconductive material for bone growth. CaP is primarily for use as a bone void filler for the recontouring of non-weightbearing craniofacial skeletal defects.32 Leung et al21 reported the augmentation of screw fixation with injectable HA in the weightbearing region in an osteopenic goat. The material was highly osteoconductive and increased the screw pull-out force and energy required to failure when used in screw augmentation. In view of these favorable properties of calcium phosphate, it can be a good
candidate for the augmentation of healing. The osteoconductive nature of calcium phosphate might also suppress fibrous tissue formation and promote bone ingrowth into the interfacial gap at the tendon-bone insertion site.23 Injectable TCP,15 HA,38 and brushite CaP cement, which is composed of dicalcium phosphate dehydrate matrix with b-TCP granules,41 HA powder in collagen gel,17 magnesium-based bone adhesive,13 and hybridization of CaP onto the tendon graft,25 have been reported to augment grafted tendon-to-bone tunnel healing. In the present study, CPS bioceramic was demonstrated to be an ideal augmentative matrix for RCT repair in view of its cytocompatibility, osteogenic activity, osteoconductivity, and biodegradability. There are several limitations to this study. First, the RC reconstruction used in this model is different from that used in humans. However, the relevance of this animal model has been established in the literature.7,34-37 Second, the single load-to-failure test construct in this study does not replicate the clinical setting, which is more consistent with a cyclic load application.16 Third, this is a pilot study using small experimental animals, in which the observation period and evaluation tools are limited. That may be why some differences in the results are relatively small. However, the differences are statistically significant, and the results are encouraging. In our further study, we plan to choose large animal models to simulate human
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1928 Zhao et al
The American Journal of Sports Medicine
biomechanics more closely and observe in relatively longer time periods. Thus, the differences may be more significant. Finally, the tools for the bioceramics implantation require improvement. Custom tools are being researched that will ensure the material completely acts on the tendon-bone interface. Nevertheless, our study offered a novel approach to augment RCTs repairs with CPS bioceramic.
CONCLUSION Local application of CPS and HA bioceramic to the healing tendon-bone interface during RC repair in a rat chronic RCT model was found to strengthen the healing enthesis, increase new bone and fibrocartilage formation, and improve collagen organization compared with repair alone, with the CPS bioceramic showing better results than HA. Regeneration of RC using CPS bioceramic is a promising treatment for RCT. Further research will focus on loading the CPS bioceramic matrix with growth factors, stem cells, and so forth for RCT repair. Augmentation of the repair combined with concurrent drug delivery in this manner could enhance long-term healing after RC injury and repair and might eventually lead to improvement of the clinical surgical outcome by enhancing tissue regeneration.
REFERENCES 1. Angeline ME, Rodeo SA. Biologics in the management of rotator cuff surgery. Clin Sports Med. 2012;31(4):645-663. 2. Bedi A, Fox AJ, Kovacevic D, Deng XH, Warren RF, Rodeo SA. Doxycycline-mediated inhibition of matrix metalloproteinases improves healing after rotator cuff repair. Am J Sports Med. 2010;38(2):308-317. 3. Bedi A, Maak T, Walsh C, et al. Cytokines in rotator cuff degeneration and repair. J Shoulder Elbow Surg. 2012;21(2):218-227. 4. Bohner M. Calcium orthophosphates in medicine: from ceramics to calcium phosphate cements. Injury. 2000;31(suppl 4):37-47. 5. Buchmann S, Sandmann GH, Walz L, et al. Refixation of the supraspinatus tendon in a rat model—influence of continuous growth factor application on tendon structure. J Orthop Res. 2013;31(2):300-305. 6. Buchmann S, Walz L, Sandmann GH, et al. Rotator cuff changes in a full thickness tear rat model: verification of the optimal time interval until reconstruction for comparison to the healing process of chronic lesions in humans. Arch Orthop Trauma Surg. 2011;131(3):429-435. 7. Carpenter JE, Thomopoulos S, Flanagan CL, DeBano CM, Soslowsky LJ. Rotator cuff defect healing: a biomechanical and histologic analysis in an animal model. J Shoulder Elbow Surg. 1998;7(6):599-605. 8. Chong AK, Ang AD, Goh JC, et al. Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit achilles tendon model. J Bone Joint Surg Am. 2007;89(1):74-81. 9. Domb BG, Glousman RE, Brooks A, Hansen M, Lee TQ, ElAttrache NS. High-tension double-row footprint repair compared with reduced-tension single-row repair for massive rotator cuff tears. J Bone Joint Surg Am. 2008;90(suppl 4):35-39. 10. Duan W, Ning C, Tang T. Cytocompatibility and osteogenic activity of a novel calcium phosphate silicate bioceramic: silicocarnotite. J Biomed Mater Res A. 2013;101(7):1955-1961. 11. Gerber C, Schneeberger AG, Perren SM, Nyffeler RW. Experimental rotator cuff repair: a preliminary study. J Bone Joint Surg Am. 1999;81(9):1281-1290. 12. Gulotta LV, Kovacevic D, Montgomery S, Ehteshami JR, Packer JD, Rodeo SA. Stem cells genetically modified with the developmental gene MT1-MMP improve regeneration of the supraspinatus tendonto-bone insertion site. Am J Sports Med. 2010;38(7):1429-1437.
13. Gulotta LV, Kovacevic D, Ying L, Ehteshami JR, Montgomery S, Rodeo SA. Augmentation of tendon-to-bone healing with a magnesium-based bone adhesive. Am J Sports Med. 2008;36(7):1290-1297. 14. Gulotta LV, Rodeo SA. Growth factors for rotator cuff repair. Clin Sports Med. 2009;28(1):13-23. 15. Huangfu X, Zhao J. Tendon-bone healing enhancement using injectable tricalcium phosphate in a dog anterior cruciate ligament reconstruction model. Arthroscopy. 2007;23(5):455-462. 16. Ide J, Kikukawa K, Hirose J, Iyama K, Sakamoto H, Mizuta H. The effects of fibroblast growth factor-2 on rotator cuff reconstruction with acellular dermal matrix grafts. Arthroscopy. 2009;25(6):608-616. 17. Ishikawa H, Koshino T, Takeuchi R, Saito T. Effects of collagen gel mixed with hydroxyapatite powder on interface between newly formed bone and grafted achilles tendon in rabbit femoral bone tunnel. Biomaterials. 2001;22(12):1689-1694. 18. Kovacevic D, Fox AJ, Bedi A, et al. Calcium-phosphate matrix with or without TGF-beta3 improves tendon-bone healing after rotator cuff repair. Am J Sports Med. 2011;39(4):811-819. 19. Kovacevic D, Rodeo SA. Biological augmentation of rotator cuff tendon repair. Clin Orthop Relat Res. 2008;466(3):622–633. 20. LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev. 2008;108(11):4742-4753. 21. Leung KS, Siu WS, Li SF, et al. An in vitro optimized injectable calcium phosphate cement for augmenting screw fixation in osteopenic goats. J Biomed Mater Res B Appl Biomater. 2006;78(1):153-160. 22. Lu W, Duan W, Guo Y, Ning C. Mechanical properties and in vitro bioactivity of Ca5(PO4)2SiO4 bioceramic. J Biomater Appl. 2012;26(6):637-650. 23. Lui P, Zhang P, Chan K, Qin L. Biology and augmentation of tendonbone insertion repair. J Orthop Surg Res. 2010;5:59. 24. Mutsuzaki H, Sakane M, Ito A, et al. The interaction between osteoclast-like cells and osteoblasts mediated by nanophase calcium phosphate-hybridized tendons. Biomaterials. 2005;26(9):1027-1034. 25. Mutsuzaki H, Sakane M, Nakajima H, et al. Calcium-phosphatehybridized tendon directly promotes regeneration of tendon-bone insertion. J Biomed Mater Res A. 2004;70(2):319-327. 26. Nelson CO, Sileo MJ, Grossman MG, Serra-Hsu F. Single-row modified mason-allen versus double-row arthroscopic rotator cuff repair: a biomechanical and surface area comparison. Arthroscopy. 2008;24(8):941-948. 27. 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(1):30-39. 28. Ozbaydar M, Elhassan B, Esenyel C, et al. A comparison of singleversus double-row suture anchor techniques in a simulated repair of the rotator cuff: an experimental study in rabbits. J Bone Joint Surg Br. 2008;90(10):1386-1391. 29. Pietschmann MF, Froehlich V, Ficklscherer A, Wegener B, Jansson V, Muller PE. Biomechanical testing of a new knotless suture anchor compared with established anchors for rotator cuff repair. J Shoulder Elbow Surg. 2008;17(4):642-646. 30. Porter AE, Patel N, Skepper JN, Best SM, Bonfield W. Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics. Biomaterials. 2003;24(25):4609-4620. 31. Qin L, Lu H, Fok P, et al. Low-intensity pulsed ultrasound accelerates osteogenesis at bone-tendon healing junction. Ultrasound Med Biol. 2006;32(12):1905-1911. 32. Reddi SP, Stevens MR, Kline SN, Villanueva P. Hydroxyapatite cement in craniofacial trauma surgery: indications and early experience. J Craniomaxillofac Trauma. 1999;5(1):7-12. 33. Rodeo SA, Potter HG, Kawamura S, Turner AS, Kim HJ, Atkinson BL. Biologic augmentation of rotator cuff tendon-healing with use of a mixture of osteoinductive growth factors. J Bone Joint Surg Am. 2007;89(11):2485-2497. 34. Soslowsky LJ, Carpenter JE, DeBano CM, Banerji I, Moalli MR. Development and use of an animal model for investigations on rotator cuff disease. J Shoulder Elbow Surg. 1996;5(5):383-392. 35. Soslowsky LJ, Thomopoulos S, Tun S, et al. Neer Award 1999. Overuse activity injures the supraspinatus tendon in an animal model:
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
36.
37.
38.
39.
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1929
a histologic and biomechanical study. J Shoulder Elbow Surg. 2000;9(2):79-84. Thomopoulos S, Hattersley G, Rosen V, et al. The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study. J Orthop Res. 2002;20(3):454-463. Thomopoulos S, Soslowsky LJ, Flanagan CL, et al. The effect of fibrin clot on healing rat supraspinatus tendon defects. J Shoulder Elbow Surg. 2002;11(3):239-247. Tien YC, Chih TT, Lin JH, Ju CP, Lin SD. Augmentation of tendonbone healing by the use of calcium-phosphate cement. J Bone Joint Surg Br. 2004;86(7):1072-1076. Tocci SL, Tashjian RZ, Leventhal E, Spenciner DB, Green A, Fleming BC. Biomechanical comparison of single-row arthroscopic rotator
cuff repair technique versus transosseous repair technique. J Shoulder Elbow Surg. 2008;17(5):808-814. 40. Uhthoff HK, Sano H, Trudel G, Ishii H. Early reactions after reimplantation of the tendon of supraspinatus into bone: a study in rabbits. J Bone Joint Surg Br. 2000;82(7):1072-1076. 41. Wen CY, Qin L, Lee KM, Chan KM. The use of brushite calcium phosphate cement for enhancement of bone-tendon integration in an anterior cruciate ligament reconstruction rabbit model. J Biomed Mater Res B Appl Biomater. 2009;89(2):466-474. 42. Yamazaki S, Yasuda K, Tomita F, Tohyama H, Minami A. The effect of transforming growth factor-beta1 on intraosseous healing of flexor tendon autograft replacement of anterior cruciate ligament in dogs. Arthroscopy. 2005;21(9):1034-1041.
For reprints and permission queries, please visit SAGE’s Web site at http://www.sagepub.com/journalsPermissions.nav
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Effect of the Interposition of Calcium Phosphate Materials on Tendon-Bone Healing During Repair of Chronic Rotator Cuff Tear Song Zhao, Lingjie Peng, Guoming Xie, Dingfeng Li, Jinzhong Zhao and Congqin Ning Am J Sports Med 2014 42: 1920 originally published online May 22, 2014 DOI: 10.1177/0363546514532781 The online version of this article can be found at: http://ajs.sagepub.com/content/42/8/1920
Published by: http://www.sagepublications.com
On behalf of: American Orthopaedic Society for Sports Medicine
Additional services and information for The American Journal of Sports Medicine can be found at: Email Alerts: http://ajs.sagepub.com/cgi/alerts Subscriptions: http://ajs.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - Aug 1, 2014 OnlineFirst Version of Record - May 22, 2014 What is This?
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Effect of the Interposition of Calcium Phosphate Materials on Tendon-Bone Healing During Repair of Chronic Rotator Cuff Tear Song Zhao,* MD, Lingjie Peng,y MS, Guoming Xie,* MD, Dingfeng Li,* MD, Jinzhong Zhao,*z MD, and Congqin Ning,yz PhD Investigation performed at Shanghai Sixth People’s Hospital, Shanghi, P.R. China, and Chinese Academy of Sciences, Shanghi, P.R. China Background: The current nature of tendon-bone healing after rotator cuff (RC) repair is still the formation of granulation tissue at the tendon-bone interface rather than the formation of fibrocartilage, which is the crucial structure in native tendon insertion and can be observed after knee ligament reconstruction. The interposition of calcium phosphate materials has been found to be able to enhance tendon-bone healing in knee ligament reconstruction. However, whether the interposition of these kinds of materials can enhance tendon-bone healing or even change the current nature of tendon-bone healing after RC repair still needs to be explored. Hypothesis: The interposition of calcium phosphate materials during RC repair would enhance tendon-bone healing or change its current nature of granulation tissue formation into a more favorable process. Study Design: Controlled laboratory study. Methods: A total of 144 male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon, followed by delayed repair after 3 weeks. The animals were allocated into 1 of 3 groups: (1) repair alone, (2) repair with Ca5(PO4)2SiO4 (CPS) bioceramic interposition, or (3) repair with hydroxyapatite (HA) bioceramic interposition at the tendon-bone interface. Animals were sacrificed at 2, 4, or 8 weeks postoperatively, and microcomputed tomography (micro-CT) was used to quantify the new bone formation at the repair site. New fibrocartilage formation and collagen organization at the tendon-bone interface was evaluated by histomorphometric analysis. Biomechanical testing of the supraspinatus tendon-bone complex was performed. Statistical analysis was performed using 1-way analysis of variance. Significance was set at P \ .05. Results: The micro-CT analysis demonstrated remarkable osteogenic activity and osteoconductivity to promote new bone formation and ingrowth of CPS and HA bioceramic, with CPS bioceramic showing better results than HA. Histological observations indicated that CPS bioceramic had excellent biocompatibility and biodegradability. At early time points after the RC repair, CPS bioceramic significantly increased the area of fibrocartilage at the tendon-bone interface compared with the control and HA groups. Moreover, CPS and HA bioceramics had significantly improved collagen organization. Biomechanical tests indicated that the CPS and HA groups have greater ultimate load to failure and stiffness than the control group at 4 and 8 weeks, and the CPS specimens exhibited the maximum ultimate load to failure, stiffness, and stress of the healing enthesis. Conclusion: Both CPS and HA bioceramics aid in cell attachment and proliferation and accelerate new bone formation, and CPS bioceramic has a more prominent effect on tendon-to-bone healing. Clinical Relevance: Local application of CPS and HA bioceramic at the tendon-bone interface shows promise in improving healing after rotator cuff tear repair. Keywords: rotator cuff tear; calcium phosphate–based bioceramic; rat model; tendon-bone healing
The rotator cuff (RC) is formed around the proximal humerus by the tendinous insertions of a group of muscles that dynamically stabilize the glenohumeral joint. Rotator
cuff tear (RCT) is among the most common injuries, leading to recurrent pain and dysfunction of the shoulder. Despite significant advances in surgical techniques that optimize the fixation of tendon to bone to achieve the highest possible initial strength, prior studies have reported a relatively high retear rate after RC repairs.9,26,28,29,39 Patient age, tear size, chronicity, and biologic healing response are described as important risk factors for
The American Journal of Sports Medicine, Vol. 42, No. 8 DOI: 10.1177/0363546514532781 Ó 2014 The Author(s)
1920 Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1921
retear.27 These risk factors indicate the insufficient healing capacity of these degenerative lesions, which is likely the main factor for the failure of the reconstruction performed in RC surgery. As a result, the study of improving the healing capacity of degenerative lesions has gained increasing interest. In humans, the RC often ruptures at the tendon-bone insertion site, which is transitioned by a well-defined zone of fibrocartilage. The normal tendon-bone insertion site of RC, which is similar to that of the knee ligament, is composed of 4 distinct transition zones: tendon, fibrocartilage, mineralized fibrocartilage, and bone.14 The fundamental function of the insertion site is to minimize stress concentration at the junction between soft tissue (tendon) and hard tissue (bone).1 Various methods have been reported to improve tendon-to-bone healing after RC repair. These methods included the reinforcement of tendon-to-bone fixation,11 introduction of growth factors (TGF-b1~3, BMP-2~7, bFGF, IGF-1, etc),33,42 application of bone marrow–derived mesenchymal stem cells,8 lowintensity pulsed ultrasound treatment,31 and so on. The results suggested that these methods improved the biomechanical strength of the tendon-bone construct to some extent. However, this improvement is typically caused by fibrovascular scar tissue rather than normal insertion site structure and composition, and thus the material properties of healing tissue are not improved.19 To change the formation of granulation tissue, the current nature of tendon-bone healing after RC repair, into fibrocartilage formation may greatly change the healing results. Therefore, the idea of redirecting the healing process away from scar formation and toward the regeneration of a native tendon-bone insertion site is an attractive strategy for improving the outcome of RC repairs. In previous studies regarding tendon-bone healing enhancement in ACL reconstruction models, calcium phosphate materials have been found to have positive effects. In a rabbit ACL reconstruction model, Tien et al38 filled the tendon-bone interface with calcium phosphate (CaP) cement, and Mutsuzaki et al24 hybridized CaP with the grafted tendon tissue. Histologic and mechanical examination found promotion of tendon-bone healing. In Huangfu and Zhao’s study in a canine model, it was found that adding a kind of mixture of tricalcium phosphate (TCP) and hydroxyapatite (HA) resulted in faster incorporation of the graft to the tunnel, with better biomechanical properties and a more mature histologic pattern.15 In addition, fibrocartilage and mineralized fibrocartilage formation have been found in all of these ACL reconstruction models. Tendon-to-bone healing depends on bone ingrowth into the granulation tissue at the healing tendon-bone
interface.40 To date, a limited number of studies have considered whether interposing the tendon-bone interface with CaP at the time of RC repair can improve tendonbone healing. Kovacevic et al18 augmented the treatment with CaP matrix and TGF-b3 in an acute rat RCT model and demonstrated meaningful improvement by the combination therapies. However, modeling of acute injury and repair in rats does not mimic clinical RCT in humans. In addition, the inferior resorbability of the CaP matrix may influence the process of tendon-to-bone healing. We suppose that an ideal tendon-bone enhancement material should be absorbed in time. Calcium phosphate–based biomaterials (CaPs) are attracting interest as substitutes for bone materials.4,20 The CaPs are similar in composition to the bone mineral (a calcium phosphate in the form of carbonate apatite) and exhibit bioactive (ability to directly bond to bone, thus forming a uniquely strong interface) and osteoconductive (ability to serve as a template or guide for the newly forming bone) properties. Interconnecting porosity (macroporosity and microporosity) similar to that of bone can be introduced by chemical or physical methods. The bioactive property promotes the formation of a carbonate HA layer, which attracts proteins to which cells bind or adhere, proliferate, and differentiate, leading to matrix production and biomineralization or the formation of new bone.20 Ca5(PO4)2SiO4 (CPS) bioceramic is one of the CaPs. Silicon is an essential trace element for bone development. Studies on silicon-based bioglasses and bioceramics have indicated that the high bioactivity of bioactive materials is attributed to the silicon component, which can form silanol groups (Si-OH) on the material surface through ion exchanges in physiological solutions.22 The silanol groups can act as bioactive sites to induce the formation of bonelike apatite by attracting calcium and phosphorus ions. Si-doped calcium phosphate materials have shown a great rate of in vivo dissolution and an improved ability to form bone.30 In previous studies, pure CPS bioceramic could be successfully synthesized by a sol-gel method.22 Compared with HA ceramic, CPS ceramic exhibited better bioactivity, cytocompatibility, and osteogenic activity in vitro.10,22 However, whether these favorable material properties can turn into favorable tendon-bone healing effects still needs to be explored. The purpose of this study was to evaluate whether the interposition of 2 kinds of calcium phosphate materials, the CPS ceramic and HA ceramic, during RC repair would improve the structural, histologic, and biomechanical outcomes in a rat model. Our hypothesis was that the interposition of calcium phosphate materials during RC repair would enhance tendon-bone healing or change its current
z Address correspondence to Jinzhong Zhao, MD, Department of Arthroscopic Surgery, Shanghai Jiao Tong University–Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai, 200233, P.R. China (e-mail: [email protected]); and Congqin Ning, PhD, State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, P.R. China (e-mail: [email protected]). *Department of Arthroscopic Surgery, Shanghai Jiao Tong University–Affiliated Sixth People’s Hospital, Shanghai, P.R. China. y State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, P.R. China. S.Z. and L.P. are co–first authors of this article. The authors declared that they have no conflicts of interest in the authorship and publication of this contribution.
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1922 Zhao et al
The American Journal of Sports Medicine
nature of granulation tissue formation into a more favorable process. Furthermore, we supposed that CPS ceramics, which have more favorable material characteristics than HA, should show greater promoting effects for tendon-bone healing.
MATERIALS AND METHODS Materials The CPS and HA bioceramic powders were synthesized as described in the literature.10,22 Powders ranging in size from 150 to 300 mm were used in the present study. Phase compositions of CPS and HA powders were identified by x-ray diffraction (D8 Advance, Bruker, Billerica, Massachusetts, USA). The powders were sterilized and stored at room temperature until surgery.
Establishment of RCT Model and RC Repair Chronic RCT models were first established. A total of 144 male Sprague-Dawley rats (obtained at 350-400 g) were used in this study. The surgical procedure was performed according to previously published studies.5,6,36 During the primary surgery, the left shoulder was operated on under general anesthesia to completely detach the supraspinatus tendon from its insertion site on the humerus. With the rat in the lateral decubitus position, a longitudinal incision was made on the anterolateral aspect of the shoulder, splitting the deltoid. Under adduction, retroversion, and slight internal rotation of the supraspinatus, insertion of the supraspinatus tendon on the greater tuberosity of the proximal humerus was visualized. The supraspinatus tendon was marked with a 5-0 Prolene suture (Ethicon, Blue Ash, Ohio, USA) approximately 3 mm medial from the insertion. Under tension of the suture, the tendon was detached sharply from the rotator interval anterior to the insertion of the infraspinatus posterior. Finally, the adhesions to the anterior and posterior tissue were released by longitudinal incisions oriented parallel to the fiber orientation of the supraspinatus tendon. The suture was knotted with 3 simple knots to facilitate the localization of the tendon stump at the time of scarification. The overlying deltoid muscle and skin were then closed, and the rats were allowed to engage in unrestricted cage activity. For 3 days postoperatively, the rats received weight-adjusted pain medication (metamizole oral and buprenorphine subcutaneous) every 12 hours. At 3 weeks postoperatively, the animals were randomized into 1 of 3 groups. In the control group, the tendon was repaired to its anatomic footprint (transosseous repair; n = 48). In the experimental groups, 96 rats underwent transosseous repair and were implanted with either CPS ceramic powder (n = 48; CPS group) or HA ceramic powder (n = 48; HA group) to augment the repair. For repair of a chronic degenerated tendon, the previous scar was reincised. Under humeroacromial extension, the marking suture was retrieved and the tendon was mobilized. The footprint was decorticated lightly with a No. 15 blade knife
to ensure complete debridement of the native enthesis. A small trough was made using an 18-gauge blunt needle and was located centrally in the footprint to allow for seating of the ceramic powder. All animals had the small trough present, with the experimental groups receiving 2 mg of either CPS or HA, while the trough remained empty in the control group. Bone tunnels were created at the anterior and posterior margins of the insertion site in a crossed fashion using a 22-gauge needle. A Mason-Allen stitch using a 3-0 Ethibond suture (Ethicon) was placed into the supraspinatus tendon. Suture ends from the tendon were then passed through the bone tunnels and firmly tied over the humeral metaphyseal cortex, anatomically repairing the supraspinatus tendon to its native footprint. The wound closure and postoperative treatment were performed according to the first operation.
Microcomputed Tomography Analysis Twenty-four animals (8 animals per group per time period) were allocated for microcomputed tomography (micro-CT) analysis at 2, 4, and 8 weeks after surgery. After necropsy, each shoulder girdle was carefully dissected to include only the supraspinatus tendon-bone complex and the proximal third of the humerus for fixation in 10% neutral buffered formalin. The bone density and new bone formation at the tendon insertion site on the greater tuberosity were assessed with micro-CT (eXplore Locus SP; GE Healthcare, London, Ontario, Canada). Each sample was placed in the holder surrounded by formalin and scanned using the conditions of 80 kV, 450 mA, and a 0.045-mm effective pixel size. The images were thresholded to distinguish bone voxels by use of a global threshold for each specimen. After the thresholded scan, 3-dimensional reconstructed images were obtained, with a customized 4 3 4-mm cylindrical region of interest (ROI) centered at the surface of the repaired supraspinatus tendon-bone footprint. The ROI contained the distal portion of the supraspinatus tendon and the bony footprint. The size of the ROI was determined based on postmortem observations, which indicated that the supraspinatus footprint measured approximately 3.5 3 3.5 mm. The bone mineral density (BMD), tissue mineral density (TMD), and bone volume fraction (bone volume/total volume; BV/TV) were calculated for a volume of interest at the greater tuberosity.
Histomorphometric Analysis After micro-CT analysis, the tissue specimens were decalcified with Immunocal (Decal, Congers, New York, USA) and embedded in paraffin. Sections 5-mm thick were cut in the coronal plane through the repaired supraspinatus tendon and the greater tuberosity. The tissue sections were stained with hematoxylin and eosin, safranin O/fast green, and picrosirius red. Using light microscopy (Leica DM4000B, Leica, Solms, Germany), tissue sections stained with safranin O/fast green were analyzed to determine the total area of new fibrocartilage formation at the insertion site of the RC repair. Digital images of the stained tissue sections were
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1923
taken using a Leica DFC420C camera (Leica). The ImageJ software program (National Institutes of Health, Bethesda, Maryland, USA) was used to determine the area of new fibrocartilage formation by manually outlining the area of metachromasia on the safranin O slides at a total magnification of 503. The total area of metachromasia was then recorded (in units of mm2) for each specimen.18 Tissue sections were stained with picrosirius red for semiquantitative analysis of the collagen deposition and maturation at the repair site. By quantifying the collagen bi-refringence under polarized light, differences in collagen maturity and organization in the healing tendon could be detected. Measurements were obtained by rotating the polarization plane until the maximum brightness was obtained. To facilitate comparisons between groups, tissues for all specimens were embedded and cut to a uniform thickness, and the light intensities were measured under exactly the same conditions of illumination and during the same sitting. After a photomicrograph was captured, the image was imported into ImageJ to be processed by 8-bit digitization, producing an image in which noncollagenous material was dark (zero) and collagenous material was depicted in gray scale with values from 1 to 255. Ten rectangular areas (50 3 50 mm) were randomly selected from the tendon region adjacent to the insertion site, and the grayscale values were measured and recorded.18
Biomechanical Testing Twenty-four animals were sacrificed at 2, 4, and 8 weeks after surgery and allocated for biomechanical testing (8 rats per group per time point). Each shoulder was removed from a –80°C freezer and thawed at room temperature on the day of biomechanical testing. The humerus with attached supraspinatus tendon was meticulously dissected from the surrounding tissues. The scar tissue was removed, and the supraspinatus muscle belly was removed from the tendon. The suture was left in situ. A digital caliper was used to measure the cross-sectional area of each supraspinatus tendon at the point of insertion into the humerus. The specimen was placed into a custom-designed uniaxial testing system. The tendon was secured in a screw grip using sandpaper and cyanoacrylate, while the humerus was secured into a vice grip that prevented fracture through the humeral physics. The supraspinatus tendon was secured to a 45-N load cell attached to a linear bearing, which allowed the alignment of the tendon in the direction of its pull. The humeral jig was secured to the linear stage, and the grip-to-grip distance was standardized across all specimens. Each specimen was preloaded to 0.1 N and then loaded to failure at a rate of 14 mm/s, corresponding to approximately 0.4% strain. The ultimate load to failure and the failure site were recorded. The displacement was measured using a 1-mm– resolution micrometer system attached to a linear stage. The linear region of the load-displacement curve was used to calculate the stiffness for each specimen. The ultimate stress at failure was calculated by dividing the ultimate load-to-failure by the cross-sectional area.2,12,18
Data Analysis Approval for this study was obtained from the related animal care committee of our institution. All authors were blinded to which study group or time interval the specimens belonged at the time of the histomorphometric analysis and biomechanical testing. Using data from previous reports,2,12,16,18 a priori power analysis with an error probability of .05 and power of .8 indicated that a minimum sample size of 8 rats per group per time point for biomechanical testing was necessary. All data are presented as the mean 6 standard deviation (SD). Statistical analysis was performed using 1-way analysis of variance with post hoc testing using the Holm-Sidak method. Significance was set at P \ .05.
RESULTS Macroscopic Observations No gross evidence of infection was observed at the surgical site in any of the specimens, and no adhesions or contractures limited the shoulder range of motion. The repaired tendon was in continuity with the bone in all animals at the time of sacrifice. No qualitative differences in the gross appearance of either the supraspinatus tendon or the healing enthesis were observed during necropsy. The shoulders in the HA group had remnants of the ceramic powder present at the tendon attachment site footprint at 2 and 4 weeks after surgery, while at later time points, no remnant was observed.
Micro-CT Analysis At each time point, the BMD of the experimental groups was significantly larger than that of the control group (2 weeks: 422.88 6 4.73 mg/mm3 [control], 432.00 6 3.96 mg/mm3 [HA], and 440.00 6 10.16 mg/mm3 [CPS]; 4 weeks: 434.00 6 4.99 mg/mm3 [control], 445.63 6 4.07 mg/mm3 [HA], and 452.13 6 5.64 mg/mm3 [CPS]; 8 weeks: 441.63 6 3.50 mg/mm3 [control], 454.38 6 5.55 mg/mm3 [HA], and 463.25 6 5.39 mg/mm3 [CPS]). At 4 and 8 weeks after surgery, the value of BMD of the CPS group was even greater than that of the HA group (Figure 1). Similarly, the values of TMD and BV/TV of the experimental groups were significantly larger than those of the control group at the 3 time points (2-week TMD: 521.25 6 5.45 mg/mm3 [control], 536.13 6 6.69 mg/mm3 [HA], and 546.13 6 7.75 mg/mm3 [CPS]; 4-week TMD: 534.25 6 5.90 mg/mm3 [control], 550.25 6 6.78 mg/mm3 [HA], and 559.13 6 4.94 mg/mm3 [CPS]; 8-week TMD: 543.38 6 7.39 mg/mm3 [control], 570.75 6 7.15 mg/mm3 [HA], and 580.25 6 4.98 mg/mm3 [CPS]; 2-week BV/TV: 73.0% 6 0.7% [control], 74.4% 6 0.4% [HA], and 75.2% 6 0.4% [CPS]; 4-week BV/TV: 73.8% 6 0.5% [control], 75.5% 6 0.7% [HA], and 76.4% 6 0.4% [CPS]; 8-week BV/TV: 74.7% 6 0.4% [control], 76.4% 6 0.5% [HA], 77.4% 6 0.4% [CPS]). Furthermore, the values of TMD and BV/TV of the CPS group were greater than those of the HA group at each time point (Figure 1). In addition, some remnants of
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1924 Zhao et al
The American Journal of Sports Medicine
Figure 1. (A) Representative microcomputed tomography images of the proximal humerus and (B) analysis of bone mineral density (BMD), tissue mineral density (TMD), and bone volume/total volume (BV/TV). Results are shown as mean 6 standard deviation. Effects of Ca5(PO4)2SiO4 on rotator cuff healing: *P \ .05 vs control; DP \ .05 vs HA; n = 8 for each group. CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite. HA bioceramic powder at the tendon-bone insertion site were found at 2, 4, and 8 weeks after surgery. (Because of the high sensitivity of micro-CT, it is different from macroscopic observation.) In contrast, no image of CPS remnant was observed at any time point. The CPS bioceramic exhibited a remarkable capacity to improve new bone formation at the tendon-bone interface. Furthermore, compared with HA, CPS exhibited superior degradability.
Histological Analysis
tissue was less cellular and more organized than was the case at 4 weeks. Fibrocartilage in small areas was observed. In the experimental groups, the larger areas of new bone and fibrocartilage observed at 4 weeks were remodeled and more organized than was the case at 2 weeks; the cells in the gap tissue were aligned with the tensile load on the tendon. The fibrocartilage in the tendon-to-bone insertion was remodeled into a fibrocartilage zone with a more natural appearance (Figure 2).
Metachromasia
Cellularity and Host Tissue Response. In the control group at 2 weeks after surgery, the inflammatory cells present consisted primarily of polymorphonuclear leukocytes; there was capillary proliferation, and the gap between the tendon and bone was filled in with fibrovascular granulation tissue. In the experimental groups, no obvious fibrovascular granulation tissue filled in the gap between the tendon and bone. Bone ingrowth into the interface between the tendon and bone was more pronounced than in the control group. Furthermore, chondrocytes were found between the tendon and bone in the HA group, and new fibrocartilage was observed in the CPS group. Specimens in the control group at 4 weeks after surgery contained fewer inflammatory cells, greater cellular organization, more collagen, and more bone ingrowth into the interface between tendon and bone than in the samples obtained at 2 weeks. There was no fibrocartilage formation in the tendon-bone insertion. Identical findings were made in the experimental groups, except that a larger area of new bone and fibrocartilage was found between the tendon and bone in both groups. In the control group at 8 weeks after surgery, the gap tissues were in the form of well-organized collagen fibers oriented in line with the tensile pull of the tendon; the
At 2 and 4 weeks after surgery, HA and CPS bioceramic significantly increased the area of glycosaminoglycan staining at the tendon-bone interface compared with the control group (2 weeks: 354,503 6 19,693 mm2 [control], 407,792 6 15,895 mm2 [HA], and 428,416 6 9621 mm2 [CPS]; 4 weeks: 402,729 6 14,704 mm2 [control], 444,700 6 16,966 mm2 [HA], and 467,040 6 16,710 mm2 [CPS]). The total area of metachromasia of the CPS group was significantly greater compared with that of the HA group. At 8 weeks after surgery, the experimental groups had a larger total area of metachromasia compared with the control group (409,909 6 14,257 mm2 [control], 472,470 6 13,300 mm2 [HA], and 488,879 6 11,111 mm2 [CPS]). There were no differences between the experimental groups (P = .057). CPS exhibited the capacity to improve cartilage regeneration at the tendon-bone interface, especially at 2 and 4 weeks after surgery (Figure 3).
Collagen Organization At all 3 time points, the experimental groups exhibited significantly improved collagen organization, based on birefringence under polarized light at the healing enthesis,
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1925
Figure 2. Representative hematoxylin and eosin–stained tissue sections (1003) of the tendon insertion site at 2, 4, and 8 weeks postoperatively. B, bone; CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite; I, interface; T, tendon. compared with the control group (2 weeks: 18.5 6 0.6 grayscale units [control], 20.5 6 0.9 grayscale units [HA], and 21.2 6 0.8 grayscale units [CPS]; 4 weeks: 29.8 6 0.9 grayscale units for controls, 31.5 6 0.7 grayscale units [HA], and 31.7 6 0.8 grayscale units [CPS]; 8 weeks: 42.1 6 1.3 grayscale units for controls, 43.8 6 1.0 grayscale units [HA], and 44.0 6 0.9 grayscale units [CPS]). There were no differences between the experimental groups at 2, 4, and 8 weeks, with P = .192, P = .885, and P = .973, respectively. These results indicate that CPS and HA are beneficial for collagen production (Figure 4).
Biomechanical Testing Cross-sectional Area of the Healing Enthesis. At each time point, no significant difference was observed in the cross-sectional area of the healing enthesis between the control group and the experimental groups (Figure 5A).
failure and stiffness of the supraspinatus tendon-bone construct in the experimental groups (ultimate load: 18.3 6 1.0 N [control], 20.7 6 1.6 N [HA], and 21.4 6 1.3 N [CPS]; stiffness: 8.0 6 0.8 N/mm [control], 9.4 6 0.8 N/mm [HA], and 9.7 6 0.6 N/mm [CPS]). There was no difference between the experimental CPS and HA groups (P = .597 and .852, respectively). At 8 weeks, there were significantly greater ultimate load to failure and stiffness of the supraspinatus tendon-bone construct in the CPS group compared with both the HA and control groups (ultimate load: 27.2 6 1.1 N [control], 28.8 6 1.0 N [HA], and 32.7 6 1.0 N [CPS], P \ .01 for both CPS vs control and CPS vs HA; stiffness: 13.2 6 0.7 N/mm [control], 14.0 6 0.6 N/mm [HA], and 14.9 6 0.4 N/mm [CPS]; P \ .01 for CPS vs control, P = .023 for CPS vs HA) (Figure 5, B and C). After 8 weeks, the mechanical strength of the supraspinatus tendon-bone construct in the CPS group obviously increased.
Ultimate Stress of the Healing Enthesis Ultimate Load to Failure and Stiffness From 2 to 8 weeks postoperatively, the ultimate load to failure and stiffness of the supraspinatus tendon-bone construct increased in all 3 groups. At 2 weeks after surgery, there was no significant difference between the groups (ultimate load: 8.4 6 0.6 N [control], 8.7 6 0.8 N [HA], and 9.1 6 0.9 N [CPS], with P = .888, P = .272, and P = .657, respectively; stiffness: 5.5 6 0.5 N/mm [control], 5.7 6 0.6 N/mm [HA], and 6.1 6 0.5 N/mm [CPS], with P = .892, P = .184, and P = .506, respectively). However, at 4 weeks, there were significantly greater values of the ultimate load to
The ultimate stress of the healing enthesis in the 3 groups increased over time. At 2 and 4 weeks, there was no significant difference between the groups (2 weeks: 1.00 6 0.03 MPa [control], 1.00 6 0.03 MPa [HA], and 1.01 6 0.03 MPa [CPS]; 4 weeks: 1.31 6 0.08 MPa [control], 1.34 6 0.07 MPa [HA], and 1.38 6 0.07 MPa [CPS]). At 8 weeks, there was significantly greater ultimate stress in the CPS group (1.65 6 0.09 MPa [control], 1.69 6 0.09 MPa [HA], and 1.92 6 0.09 MPa [CPS]) (Figure 5D). These results are consistent with the results of the ultimate load to failure and stiffness at 8 weeks.
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1926 Zhao et al
The American Journal of Sports Medicine
Figure 3. (A) Representative histology images of the cartilage at the insertion site (1003 magnification) and (B) the area of cartilage present at the insertion site as determined by metachromasia with safranin O–stained slides. Results are shown as mean 6 standard deviation (*P \ .05 vs control, DP \ .05 vs HA; n = 8 for each group). B, bone; CPS, Ca5(PO4)2 SiO4; HA, hydroxyapatite; I, interface; T, tendon.
DISCUSSION The pathogenesis of chronic RC degeneration is multifactorial and results from both mechanical and biological factors. Loss of cellularity, thinning and disorganization of tendon fibers, increased granulation tissue, and fibrocartilaginous changes may explain the relatively high rate of healing failure or recurrent RCTs after repair.3 Given the inadequate healing response, biomaterials offer the possibility to augment healing after RC repair and may be critical to improving clinical outcomes.23 While most of the prior studies used acute RCT models for evaluation, this does not reflect the degenerative, age-related RCTs commonly observed in humans, who tend to exhibit intrinsic degenerative changes in the torn RC tendon, tendon retraction, and osteoporosis of the greater tuberosity in the shoulders. In the present study, we established a chronic rat RCT model according to the Buchmann approach.5,6 His team demonstrated that chronic human RCT could be imitated in the rat model, in which tendon degeneration, inflammation, and muscle atrophy combined with a persisting defect at 3 weeks after detachment were comparable with the chronic tendon tears in humans.
Figure 4. (A) Representative picrosirius red–stained tissue sections of the healing enthesis (1003 magnification) and (B) analysis of the collagen bi-refringence. Results are shown as mean 6 standard deviation (*P \ .05 vs control; DP \ .05 vs HA; n = 8 for each group). B, bone; CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite; I, interface; T, tendon.
In this study, we tested the hypothesis that treatment with CPS bioceramic can promote new bone and fibrocartilage formation and strengthen the healing enthesis compared with repair alone in a chronic RCT model. Micro-CT analysis revealed that local application of CaP bioceramics significantly improved new bone formation at the tendon-bone interface. CPS bioceramic especially exhibited both remarkable osteogenic activity and osteoconductivity, which promoted new bone formation and ingrowth. In addition, CPS bioceramic exhibits excellent biodegradability. At early times after the repairs, the CPS material was degraded completely, thereby avoiding any interference with the healing process. We observed that the bioceramic materials exhibited a biocompatible host tissue response at the tendon repair site. No obvious inflammation was observed. Local application of CaP bioceramics are associated with improvements in the area of fibrocartilage and collagen organization at the healing enthesis compared with RC repair only at each time point, which underscores the importance of this material as a provisional matrix in the phase of tendon-bone healing. Moreover, there was a significantly
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1927
Figure 5. Biomechanical testing of the tendon at the insertion site: (A) cross-sectional areas, (B) ultimate load to failure, (C) stiffness, and (D) ultimate stress to failure values. Results are shown as mean 6 standard deviation (*P \ .05 vs control; DP \ .05 vs HA; n = 8 for each group). CPS, Ca5(PO4)2SiO4; HA, hydroxyapatite. improved area of fibrocartilage with CPS bioceramic, indicating that CPS led to the formation of a more mature healing enthesis compared with either HA bioceramic or repair only. Biomechanical testing is critical to evaluate whether implantation of the bioceramics can improve the biomechanical properties of the repair constructs. In this study, local application of CaP bioceramic did result in greater ultimate load to failure and stiffness at 4 weeks. In addition, after 8 weeks, the mechanical strength of the supraspinatus tendon-bone construct augmented with CPS obviously increased. The biomechanical testing results confirmed the feasibility of the augmentation with CPS bioceramic for use in RC repair. Although the magnitudes of the biomechanical changes noted are relatively small, they are encouraging and would be clinically important. Because CaP has a chemical composition that is similar to bone, there is recent interest in its use as an osteoconductive material for bone growth. CaP is primarily for use as a bone void filler for the recontouring of non-weightbearing craniofacial skeletal defects.32 Leung et al21 reported the augmentation of screw fixation with injectable HA in the weightbearing region in an osteopenic goat. The material was highly osteoconductive and increased the screw pull-out force and energy required to failure when used in screw augmentation. In view of these favorable properties of calcium phosphate, it can be a good
candidate for the augmentation of healing. The osteoconductive nature of calcium phosphate might also suppress fibrous tissue formation and promote bone ingrowth into the interfacial gap at the tendon-bone insertion site.23 Injectable TCP,15 HA,38 and brushite CaP cement, which is composed of dicalcium phosphate dehydrate matrix with b-TCP granules,41 HA powder in collagen gel,17 magnesium-based bone adhesive,13 and hybridization of CaP onto the tendon graft,25 have been reported to augment grafted tendon-to-bone tunnel healing. In the present study, CPS bioceramic was demonstrated to be an ideal augmentative matrix for RCT repair in view of its cytocompatibility, osteogenic activity, osteoconductivity, and biodegradability. There are several limitations to this study. First, the RC reconstruction used in this model is different from that used in humans. However, the relevance of this animal model has been established in the literature.7,34-37 Second, the single load-to-failure test construct in this study does not replicate the clinical setting, which is more consistent with a cyclic load application.16 Third, this is a pilot study using small experimental animals, in which the observation period and evaluation tools are limited. That may be why some differences in the results are relatively small. However, the differences are statistically significant, and the results are encouraging. In our further study, we plan to choose large animal models to simulate human
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
1928 Zhao et al
The American Journal of Sports Medicine
biomechanics more closely and observe in relatively longer time periods. Thus, the differences may be more significant. Finally, the tools for the bioceramics implantation require improvement. Custom tools are being researched that will ensure the material completely acts on the tendon-bone interface. Nevertheless, our study offered a novel approach to augment RCTs repairs with CPS bioceramic.
CONCLUSION Local application of CPS and HA bioceramic to the healing tendon-bone interface during RC repair in a rat chronic RCT model was found to strengthen the healing enthesis, increase new bone and fibrocartilage formation, and improve collagen organization compared with repair alone, with the CPS bioceramic showing better results than HA. Regeneration of RC using CPS bioceramic is a promising treatment for RCT. Further research will focus on loading the CPS bioceramic matrix with growth factors, stem cells, and so forth for RCT repair. Augmentation of the repair combined with concurrent drug delivery in this manner could enhance long-term healing after RC injury and repair and might eventually lead to improvement of the clinical surgical outcome by enhancing tissue regeneration.
REFERENCES 1. Angeline ME, Rodeo SA. Biologics in the management of rotator cuff surgery. Clin Sports Med. 2012;31(4):645-663. 2. Bedi A, Fox AJ, Kovacevic D, Deng XH, Warren RF, Rodeo SA. Doxycycline-mediated inhibition of matrix metalloproteinases improves healing after rotator cuff repair. Am J Sports Med. 2010;38(2):308-317. 3. Bedi A, Maak T, Walsh C, et al. Cytokines in rotator cuff degeneration and repair. J Shoulder Elbow Surg. 2012;21(2):218-227. 4. Bohner M. Calcium orthophosphates in medicine: from ceramics to calcium phosphate cements. Injury. 2000;31(suppl 4):37-47. 5. Buchmann S, Sandmann GH, Walz L, et al. Refixation of the supraspinatus tendon in a rat model—influence of continuous growth factor application on tendon structure. J Orthop Res. 2013;31(2):300-305. 6. Buchmann S, Walz L, Sandmann GH, et al. Rotator cuff changes in a full thickness tear rat model: verification of the optimal time interval until reconstruction for comparison to the healing process of chronic lesions in humans. Arch Orthop Trauma Surg. 2011;131(3):429-435. 7. Carpenter JE, Thomopoulos S, Flanagan CL, DeBano CM, Soslowsky LJ. Rotator cuff defect healing: a biomechanical and histologic analysis in an animal model. J Shoulder Elbow Surg. 1998;7(6):599-605. 8. Chong AK, Ang AD, Goh JC, et al. Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit achilles tendon model. J Bone Joint Surg Am. 2007;89(1):74-81. 9. Domb BG, Glousman RE, Brooks A, Hansen M, Lee TQ, ElAttrache NS. High-tension double-row footprint repair compared with reduced-tension single-row repair for massive rotator cuff tears. J Bone Joint Surg Am. 2008;90(suppl 4):35-39. 10. Duan W, Ning C, Tang T. Cytocompatibility and osteogenic activity of a novel calcium phosphate silicate bioceramic: silicocarnotite. J Biomed Mater Res A. 2013;101(7):1955-1961. 11. Gerber C, Schneeberger AG, Perren SM, Nyffeler RW. Experimental rotator cuff repair: a preliminary study. J Bone Joint Surg Am. 1999;81(9):1281-1290. 12. Gulotta LV, Kovacevic D, Montgomery S, Ehteshami JR, Packer JD, Rodeo SA. Stem cells genetically modified with the developmental gene MT1-MMP improve regeneration of the supraspinatus tendonto-bone insertion site. Am J Sports Med. 2010;38(7):1429-1437.
13. Gulotta LV, Kovacevic D, Ying L, Ehteshami JR, Montgomery S, Rodeo SA. Augmentation of tendon-to-bone healing with a magnesium-based bone adhesive. Am J Sports Med. 2008;36(7):1290-1297. 14. Gulotta LV, Rodeo SA. Growth factors for rotator cuff repair. Clin Sports Med. 2009;28(1):13-23. 15. Huangfu X, Zhao J. Tendon-bone healing enhancement using injectable tricalcium phosphate in a dog anterior cruciate ligament reconstruction model. Arthroscopy. 2007;23(5):455-462. 16. Ide J, Kikukawa K, Hirose J, Iyama K, Sakamoto H, Mizuta H. The effects of fibroblast growth factor-2 on rotator cuff reconstruction with acellular dermal matrix grafts. Arthroscopy. 2009;25(6):608-616. 17. Ishikawa H, Koshino T, Takeuchi R, Saito T. Effects of collagen gel mixed with hydroxyapatite powder on interface between newly formed bone and grafted achilles tendon in rabbit femoral bone tunnel. Biomaterials. 2001;22(12):1689-1694. 18. Kovacevic D, Fox AJ, Bedi A, et al. Calcium-phosphate matrix with or without TGF-beta3 improves tendon-bone healing after rotator cuff repair. Am J Sports Med. 2011;39(4):811-819. 19. Kovacevic D, Rodeo SA. Biological augmentation of rotator cuff tendon repair. Clin Orthop Relat Res. 2008;466(3):622–633. 20. LeGeros RZ. Calcium phosphate-based osteoinductive materials. Chem Rev. 2008;108(11):4742-4753. 21. Leung KS, Siu WS, Li SF, et al. An in vitro optimized injectable calcium phosphate cement for augmenting screw fixation in osteopenic goats. J Biomed Mater Res B Appl Biomater. 2006;78(1):153-160. 22. Lu W, Duan W, Guo Y, Ning C. Mechanical properties and in vitro bioactivity of Ca5(PO4)2SiO4 bioceramic. J Biomater Appl. 2012;26(6):637-650. 23. Lui P, Zhang P, Chan K, Qin L. Biology and augmentation of tendonbone insertion repair. J Orthop Surg Res. 2010;5:59. 24. Mutsuzaki H, Sakane M, Ito A, et al. The interaction between osteoclast-like cells and osteoblasts mediated by nanophase calcium phosphate-hybridized tendons. Biomaterials. 2005;26(9):1027-1034. 25. Mutsuzaki H, Sakane M, Nakajima H, et al. Calcium-phosphatehybridized tendon directly promotes regeneration of tendon-bone insertion. J Biomed Mater Res A. 2004;70(2):319-327. 26. Nelson CO, Sileo MJ, Grossman MG, Serra-Hsu F. Single-row modified mason-allen versus double-row arthroscopic rotator cuff repair: a biomechanical and surface area comparison. Arthroscopy. 2008;24(8):941-948. 27. 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(1):30-39. 28. Ozbaydar M, Elhassan B, Esenyel C, et al. A comparison of singleversus double-row suture anchor techniques in a simulated repair of the rotator cuff: an experimental study in rabbits. J Bone Joint Surg Br. 2008;90(10):1386-1391. 29. Pietschmann MF, Froehlich V, Ficklscherer A, Wegener B, Jansson V, Muller PE. Biomechanical testing of a new knotless suture anchor compared with established anchors for rotator cuff repair. J Shoulder Elbow Surg. 2008;17(4):642-646. 30. Porter AE, Patel N, Skepper JN, Best SM, Bonfield W. Comparison of in vivo dissolution processes in hydroxyapatite and silicon-substituted hydroxyapatite bioceramics. Biomaterials. 2003;24(25):4609-4620. 31. Qin L, Lu H, Fok P, et al. Low-intensity pulsed ultrasound accelerates osteogenesis at bone-tendon healing junction. Ultrasound Med Biol. 2006;32(12):1905-1911. 32. Reddi SP, Stevens MR, Kline SN, Villanueva P. Hydroxyapatite cement in craniofacial trauma surgery: indications and early experience. J Craniomaxillofac Trauma. 1999;5(1):7-12. 33. Rodeo SA, Potter HG, Kawamura S, Turner AS, Kim HJ, Atkinson BL. Biologic augmentation of rotator cuff tendon-healing with use of a mixture of osteoinductive growth factors. J Bone Joint Surg Am. 2007;89(11):2485-2497. 34. Soslowsky LJ, Carpenter JE, DeBano CM, Banerji I, Moalli MR. Development and use of an animal model for investigations on rotator cuff disease. J Shoulder Elbow Surg. 1996;5(5):383-392. 35. Soslowsky LJ, Thomopoulos S, Tun S, et al. Neer Award 1999. Overuse activity injures the supraspinatus tendon in an animal model:
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
Vol. 42, No. 8, 2014
36.
37.
38.
39.
Effects of Calcium Phosphate Materials on Rotator Cuff Healing 1929
a histologic and biomechanical study. J Shoulder Elbow Surg. 2000;9(2):79-84. Thomopoulos S, Hattersley G, Rosen V, et al. The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study. J Orthop Res. 2002;20(3):454-463. Thomopoulos S, Soslowsky LJ, Flanagan CL, et al. The effect of fibrin clot on healing rat supraspinatus tendon defects. J Shoulder Elbow Surg. 2002;11(3):239-247. Tien YC, Chih TT, Lin JH, Ju CP, Lin SD. Augmentation of tendonbone healing by the use of calcium-phosphate cement. J Bone Joint Surg Br. 2004;86(7):1072-1076. Tocci SL, Tashjian RZ, Leventhal E, Spenciner DB, Green A, Fleming BC. Biomechanical comparison of single-row arthroscopic rotator
cuff repair technique versus transosseous repair technique. J Shoulder Elbow Surg. 2008;17(5):808-814. 40. Uhthoff HK, Sano H, Trudel G, Ishii H. Early reactions after reimplantation of the tendon of supraspinatus into bone: a study in rabbits. J Bone Joint Surg Br. 2000;82(7):1072-1076. 41. Wen CY, Qin L, Lee KM, Chan KM. The use of brushite calcium phosphate cement for enhancement of bone-tendon integration in an anterior cruciate ligament reconstruction rabbit model. J Biomed Mater Res B Appl Biomater. 2009;89(2):466-474. 42. Yamazaki S, Yasuda K, Tomita F, Tohyama H, Minami A. The effect of transforming growth factor-beta1 on intraosseous healing of flexor tendon autograft replacement of anterior cruciate ligament in dogs. Arthroscopy. 2005;21(9):1034-1041.
For reprints and permission queries, please visit SAGE’s Web site at http://www.sagepub.com/journalsPermissions.nav
Downloaded from ajs.sagepub.com at TEXAS A & M INTL UNIV on September 30, 2014
