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A Comparative Study on the Biomechanical and Histological Properties of Bone-to-Bone, Bone-to-Tendon, and Tendon-to-Tendon Healing: An Achilles Tendon−Calcaneus Model in Goats
Kwok-Sui Leung, Wai Sing Chong, Dick Ho Kiu Chow, Peng Zhang, Wing-Hoi Cheung, Margaret Wan Nar Wong and Ling Qin Am J Sports Med published online March 30, 2015 DOI: 10.1177/0363546515576904 The online version of this article can be found at: http://ajs.sagepub.com/content/early/2015/03/30/0363546515576904
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A Comparative Study on the Biomechanical and Histological Properties of Bone-to-Bone, Bone-toTendon, and Tendon-to-Tendon Healing An Achilles Tendon–Calcaneus Model in Goats Kwok-Sui Leung,* MD, FRCS, Wai Sing Chong,* MPhil, Dick Ho Kiu Chow,* PhD, Peng Zhang,*y PhD, Wing-Hoi Cheung,* PhD, Margaret Wan Nar Wong,* MD, FRCS, and Ling Qin,*yz§ PhD Investigation performed at Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Background: Surgical repair around the bone-tendon insertion (BTI) may involve bone-to-bone (BB), bone-to-tendon (BT), or tendon-to-tendon (TT) reattachment with varying healing outcome. Hypothesis: The repair of Achilles tendon–calcaneus (ATC) by reattachment of homogeneous tissue (BB or TT) would heal faster, with respect to tensile properties at the healing complex, than those of reattachment of heterogeneous tissues (BT) over time. Study Design: Controlled laboratory study. Methods: Forty-seven adolescent male Chinese goats were divided into BB, BT, and TT groups. Osteotomy of the calcaneus, reattachment of Achilles tendon to the calcaneus after removal of the insertion, and tenotomy of the Achilles tendon were performed to simulate BB, BT, and TT repair, respectively. The ATC healing complexes were harvested at 6, 12, or 24 weeks postoperatively. Mechanical and morphological properties of the healing ATC complexes were assessed by tensile testing and qualitative histology, respectively. The contralateral intact ATC complex was used as the control. Results: Failure load of BT was 33.4% lower than that of TT (P = .0243) at week 12. Ultimate strength of BT was 50.2% and 45.3% lower than that of TT at weeks 12 (P = .0002) and 24 (P = .0001), respectively. Tissue morphological characteristics of the BB and TT groups showed faster remodeling. The BT group showed limited regeneration of fibrocartilage zone and excessive formation of fibrous tissue at the healing interface. Conclusion: BTI repair between homogeneous tissues (BB and TT healing) showed better healing quality with respect to mechanical and histological assessments than did healing between heterogeneous tissues (BT healing). Clinical Relevance: Anatomic reconstruction of ATC complex injury may be a primary concern when selecting the proper surgical approach. However, it is recommended to select fracture fixation (BB) or tendon repair (TT) instead of bone-tendon reattachment (BT) if possible to ensure better outcome at the healing interface. Keywords: bone tendon insertion healing; Achilles tendon–calcaneus complex; goat model; mechanical test; histological morphology
absorber during mechanical loading.3,21,22,43 Surgical reattachment of tendon-to-bone is often observed in the repair of rotator cuff, flexor tendons in the hand, tibial insertion of the medial collateral ligament, patellar tendon to patella, and the Achilles tendon to calcaneus.43 Treatment protocols for BTI repair are diversified according to different injury sites, which may lead to different outcome and healing quality.
The bone-tendon insertion (BTI) is a complex structure that consists of bone, mineralized and nonmineralized fibrocartilage, and tendon. It serves as an interface for force transmission from bone to tendon and acts as a stress
The American Journal of Sports Medicine, Vol. XX, No. X DOI: 10.1177/0363546515576904 Ó 2015 The Author(s)
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The rate of healing among different tissues may vary. In an experiment using partial patellectomy model in rabbits, reattachment of homogeneous tissues (bone to bone [BB]) showed better healing quality in terms of mechanical properties than that of heterogeneous tissues (bone to tendon [BT]).18 Moreover, the tissue structure of the BT healing interface was overbridged by fibrous scar tissue, and limited fibrocartilage was observed.6,8,9,12,18,31,32,40,47 The surgical outcome in small-animal experiments, such as rats and rabbits, may not be replicable in large animals owing to differences in joint anatomy (structure) and function (mechanics) apart from variation in body size.4,29,41,50 Muscle contraction around the involved joint might also affect the healing at BTI.45 Mechanical loading may stimulate expression of genes10 and secretion of cytokines, such as bone morphogenetic protein11,33 and transforming growth factor beta,16 which may affect healing.5,7,15,21 Furthermore, the healing ability of small animals, such as rats and rabbits, is often faster than that of large animals or humans.2,34 Therefore, a largeanimal model of BTI healing that mimics the mechanical loading and healing ability of the patient is desirable to compare the quality and progression of healing within homogeneous tissue and heterogeneous tissues. The advantage of using the Achilles tendon–calcaneus (ATC) complex model in the goat is that the muscle-tendon forces, change of fascicle length, and muscle activation of goat distal hindlimb in response to locomotor grade are similar to those of the human.24 Therefore, the healing of the ATC in goats would be comparable with that of the human. Based on the complexity of tissues involved in the BTI region, the healing quality of the different BTI repairs—including BB, BT, and tendon to tendon (TT)—in the ATC complex of Chinese mountain goats was investigated with respect to restoration of their mechanical and histological properties. We hypothesized that the repair of ATC by reattachment of homogeneous tissue (BB or TT) would heal faster, with respect to tensile properties at the healing complex, than those of reattachment of heterogeneous tissues (BT) over time. The primary dependent variable of the current study was the tensile strength of the surgical reattachment site of ATC.
METHODS Surgical Repair of the ATC Complex Forty-seven adolescent male Chinese goats (body weight, 28.1 6 3.1 kg) were obtained and housed in the Laboratory of Animals Service Center of the Chinese University of
Hong Kong after animal experimental ethics was approved (97/054/ERG-CU97/6.68 and 14-085-MIS). The goats were divided into 3 groups (Appendix Table A1, available online at http://ajsm.sagepub.com/supplemental), and different surgical reattachment models (BB, BT, and TT) were performed according to the established protocols.30,46 Briefly, the goat was anesthetized by inhalation anesthesia with nitrous oxide and oxygen in a ratio of 1:2 by volume (Fluothane, Zeneca). Skin around the ATC complex was shaved and sterilized with 0.05% Hibitane (Zeneca). The ATC complex was approached through posterior midline skin incision aseptically. The overlaying tendon of flexor digitorum superficialis was incised to expose the ATC junction for the following 3 surgical interventions: BB repair, BT repair, and TT repair. The intact ATC without any surgical interventions was used as the control. BB Repair. Osteotomy with 0.9% saline irrigation was performed vertically at 1 cm distal to the ATC in junction. With the aid of bone forceps, 2 Kirschner wires (diameter, 0.9 mm) were drilled in parallel through the posteriorinferior poles of medial and lateral tuberosity at 30° with respect to the horizontal plane of calcaneus. A figure-of-8 tension band was applied to prevent dislocation of bone fragments before the connective tissue and skin were sutured for completion of the surgery (Figure 1A). BT Repair. The junction of ATC was explored surgically using a sharp scalpel. The calcaneus surface was partially decorticated to expose the osseous tissues. A No. 0 polydioxanone monofilament absorbable suture (PDS II, Ethicon Ltd) was applied in a Bunnell suture configuration to the Achilles tendon. A bone tunnel (diameter, 1.2 mm) was drilled from the lateral tuberosity to medial tuberosity horizontally. Both suture ends were passed through the drill hole. The Achilles tendon was pulled toward the calcaneus, and the complex was secured by knotting of the 2 suture ends. A figure-of-8 tension band wire (stainless steel surgical wire) was placed to protect the repair junction before the surgery was completed with skin suture (Figure 1B). TT Repair. The Achilles tendon was exposed and cut transversely at 2 cm proximal to its insertion to calcaneus using a sharp scalpel. Bunnell suture configuration with No. 0 polydioxanone monofilament absorbable suture (PDS II, Ethicon Ltd) was applied to join both ends of the tendon for fixation (Figure 1C). The advantage of using the Bunnell suture is minimal handling and care to avoid vascular interference. Even though other multistrand suture techniques are stronger and gap resistant, these suturing techniques may cause greater tissue trauma and be detrimental to the glide of the healing tendon.23 After the surgical reattachment, overlaying ligamentous capsule and skin were then closed with 3-0 sterile
§ Address correspondence to Ling Qin, PhD, Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Shatin, NT, 001, Hong Kong (email: [email protected]). *Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China. y Translational Medicine Research and Development Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China. z Lui Che Woo Institute of Innovative Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China. One or more of the authors has declared the following potential conflict of interest or source of funding: This project was supported by the Research Grants Council of the Hong Kong Special Administrative Regions (CUHK 4275/97M).
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Schering-Plough) was injected intramuscularly for analgesia. Cast (Scotchcast Plus, P-46325, 3M Health Care) was applied for immobilization of the operated limb for 6 weeks. The cast immobilization prevented dorsiflexion to reduce tension on the healing ATC. The goats were allowed full weightbearing and joint motion after cast removal. The goats were allowed for unrestricted cage activity after surgery in the animal house.
Sampling for Mechanical Testing and Histology
Figure 1. A schematic drawing of the establishment of the Achilles tendon–calcaneus (ATC) complex healing by reattachment of bone to bone (BB), bone to tendon (BT), and tendon to tendon (TT). (A) BB repair. A1: Osteotomy distal to the ATC complex. A2: Two Kirschner wires were drilled in parallel through the posterior-inferior poles of medial and lateral tuberosity. A3: A figure-of-8 tension band was applied to prevent dislocation of bone fragments. (B) BT repair. B1: The junction of ATC was explored surgically. B2: Bunnell suture configuration to Achilles tendon. A bone tunnel was drilled from lateral tuberosity to medial tuberosity horizontally. Both suture ends were passed through the drill hole. B3: A figure-of-8 tension band was placed to protect the repair junction. (C) TT repair. C1: Achilles tendon was exposed and cut transversely. C2: Bunnell suture configuration with suture. C3: The sutures were pulled, and the 2 ends of the Achilles tendon were secured against each other. Red line: figure-of-8 protection band. Blue line: suture. Black dotted line: incision site. Black bands: Kirschner wire. Solid lines: exposed wires. Dotted lines: nonexposed wires.
catgut (Chromic, Ethicon Ltd) and 3-0 braided silk suture (Mersilk, Ethicon Ltd). Antibiotic spray (Nebactin, Byk Gulden) was applied for disinfection. Temgesic (0.005 mg/kg;
The goats were euthanized at 6 (n = 17), 12 (n = 15), or 24 (n = 15) weeks postoperatively by overdose injection of sodium pentobarbital via the jugular vein. After the ATC complex was isolated, Kirschner wires and figure-of-8 tension band wires were removed from the ATC complex. For mechanical testing, the ATC complex was wrapped in gauze with saline, sealed in a freezer bag, and stored at 220°C freezer before mechanical testing. After thawing the samples at room temperature, the calcaneus of the ATC complex was placed vertically inside a mold and embedded with PMMA composed of Ureol 5202 B and Ureol 5202 A.17 After completion of polymerization, 2 holes (diameter, 5 mm) were drilled in parallel through the embedded calcaneus. The ATC complex was fixed on a custom-made testing jig consisting of an upper soft tissue clamp and lower stand. The upper clamp has serrated grooves, which grasp the Achilles tendon junction firmly. The lower stand has 2 horizontally arranged screws for fixing the polymer embedded calcaneus. Embedding the ends of the sample in MMA resin minimized slippage of the samples by providing better fixation between the lower clamp and sample. The ATC complex was fixed for tensile testing in a physiological position (ie, with a resting angle of 90° between Achilles tendon and calcaneus). The calcaneus was then screw fixed on the lower stand, and the Achilles muscle-tendon unit was carefully fixed on the upper clamp without creating premature failure at the contact point with the clamp (Appendix Figure A1, available online). For better fixation between specimens and metal clamps, the central one-third of the Achilles was isolated by transversely cutting over the lateral and medial one-third of the tendon for TT group. After the sample and jig were fitted onto a mechanical testing machine (H25KM, Hounsfield test equipment, Redhill), a tensile test was performed with a load cell of 2 kN at a testing speed of 50 mm/min. Failure load was recorded and the ultimate strength calculated by normalizing the failure load by the cross-section area (CSA) of the healing interface. Failure mode of each ATC was also recorded. The contralateral intact ATC complex was used as the control. Achilles tendon–calcaneus complexes used for histological study were fixed in 4% neutral buffered formalin, decalcified in 9% formic acid, and embedded in paraffin. Serial sagittal sections (7 mm) were cut using microtome (Leica, RM6165, Reichert-Jung GmbH) and stained with hematoxylin and eosin. A research scientist and an orthopaedic clinical scientist qualitatively evaluated the histological sections under transmitted light microscope (Leica Q500MC, Leica Cambridge Ltd).
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Figure 2. Midsagittal section of the Achilles tendon–calcaneus samples after operation compared among the bone-to-bone (BB), bone-to-tendon (BT), tendon-to-tendon (TT) groups at 3 healing time points. The white arrows indicate the healing interface for different groups.
Measurement of CSA of Healing Interface
RESULTS
The CSAs of the healing interface for different treatment groups were measured to calculate the ultimate strength from the failure load of each sample. A conventional caliper was used to measure the CSA of the healing interface of the tissue in the BB, BT, and TT groups. Other available methods for measuring CSA, such as ultrasound, magnetic resonance imaging, peripheral quantitative computed tomography, and micro–computed tomography, are advanced methods but are only suitable for a specific type of tissue (bone or tendon), while the healing BTI in the BT repair involves both hard and soft tissue. The CSA of the healing interface was calculated by multiplying the length and width of the remaining intact tissue. The CSA at the healing interface for each sample was measured 8 times and the mean used for statistical analysis. A single examiner performed all CSA measurements to minimize potential interindividual error.48 The coefficient of variation for repeated measures was calculated to be \5%.
Gross Observation of Specimens The healing interface of the BB group was identified at weeks 6 and 12 but not observable at week 24 postoperation (Figure 2). The bone marrow content in the BB group decreased with healing over time. For the BT group, the tendon surface was not shiny and was covered with white fibrous tissue surrounding the healing interface at week 6 postoperation. No parallel collagen bundles were observed at 12 weeks. At week 24, parallel alignment of collagen bundles were observed (Figure 2). For the TT group, transparent fibrous tissue with darker-red appearance was found at week 6 postoperation. At week 12, the parallel tendon fiber structure and fibrous scar tissue around tenotomy site became shiny looking. At week 24, fibrous tissue was found at the healing interface, but tenotomy margin was not identified (Figure 2).
Mechanical Testing Statistical Analysis All measurements were expressed as mean 6 SD, while the box plots showed median and quartiles. All analyses were performed using SPSS version 16.0 for Windows (SPSS Inc). Two-way analysis of variance with Bonferroni post hoc tests were used to determine the effect of different reattachment methods and time on the mechanical properties of ATC healing. Statistical significance was set at P \ .05.
The TT group showed a significantly higher failure load than that of the BT group at week 12 (50.1%, P = .0243) (Figure 3). However, no significant differences were detected among the 3 groups at weeks 6 and 24. There were also no significant differences in failure load between weeks 12 and 24 for the BB and TT groups. There was a significant time effect on failure load (P \ .0001) but no significant interaction between time effect and reattachment
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Figure 3. Maximum load of Achilles tendon–calcaneus complex before surgery (control) and at different time points after operation. Bone-to-tendon (BT) group showed lower maximum load at week 12 than the other 2 groups, while no significant difference was detected at week 24. *P \ .05. BB, bone to bone; TT, tendon to tendon.
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Figure 4. Ultimate tensile strength of Achilles tendon– calcaneus complex before surgery (control) and at different time points after operation. Bone-to-tendon (BT) group showed lower ultimate tensile strength at weeks 12 and 24 than the other 2 groups, while no significant difference was detected at week 6. **P \ .01. ***P \ .001. BB, bone to bone; TT, tendon to tendon.
method. With respect to ultimate strength, there were no significant differences among the 3 groups at week 6 (Figure 4). The ultimate strength of the TT group was higher than that of the BB and BT groups at week 12 (155.4% and 100.6%, respectively; P \ .0002) and week 24 (48.4% and 83.0%, respectively; P \ .0034). For ultimate strength, there was a significant time effect (P \ .0001) and significant interaction between time effect and reattachment methods (P = .0056). The CSAs of the healing interfaces for all groups were significantly different for all time points pre- and postoperation. For energy to failure, no significant difference was detected among the 3 reattachment methods at weeks 6, 12, and 24 (Figure 5). There was a significant time effect on energy to failure (P \ .0001) but no significant interaction between time effect and reattachment methods.
Failure Mode At week 6, all samples of the BB and TT groups failed at the healing interface. For the BT group, 75% (3 of 4) of the specimens failed at the healing interface, while 25% (1 of 4) failed at tendon midsubstance. At week 12, 80% (12 of 15) of the samples of the 3 groups failed at the healing interface. At week 24, 100% (5 of 5) of the samples of the BB group and 40% (2 of 5) of the sample in the TT group failed at the ends where the specimens were clamped instead of the healing interface. However, all samples (5 of 5) of the BT group failed at the tendon zone of the healing interface, while only 60% (3 of 5) of the samples of the TT group failed at the tendon tissue.
Histological Observation For the BB group, trabeculae near the osteotomy site were woven bone at week 6 (Figure 6). At week 12, active bone formation was found, and chondrocyte-like cells and
Figure 5. Energy to failure of Achilles tendon–calcaneus complex before surgery (control) and at different time points after operation. No significant difference was detected among the 3 reattachment methods at weeks 6, 12, and 24. BB, bone to bone; BT, bone to tendon; TT, tendon to tendon.
osteoblasts lining were present at the healing interface. At week 24, lamella trabecular bone with a decrease in osteocyte density was found at the osteotomy site (Figure 6). For the BT group, fibrous tissue was formed at the healing interface at week 6; at week 12, formation of new bone and fibrocartilage zone at the healing interface was minimal (Figure 7); at week 24, the newly formed tissues resembled normal ATC, including the 4 histologically different zones and the tidemark (Figure 7). The newly formed tendon tissue in the TT group showed increased cellularity with cells that were round at week 6 (Figure
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Figure 6. Representative images of bone-to-bone repair at weeks 6, 12, and 24 after operation. (A) At week 6, cartilage tissue was found at the osteotomy site. Woven bone was also observed surrounding the osteotomy site. (B) At week 12, woven bone was mainly found at the osteotomy site. (C) At week 24, the woven bone was remodeled to laminar bone, which provides better mechanical properties. Hematoxylin and eosin, magnification: 163. Ca, cartilage; LB, laminar bone; WB, woven bone. 8). At weeks 12 and 24, decrease of cellularity, presence of spindle-like cells, and organized alignment of the collagens were observed at the healing interface (Figure 8).
DISCUSSION This experiment used an ATC complex model in goats to demonstrate the effect of different surgical approaches on the healing quality of injury around the BTI with respect to mechanical and histological evaluations. Surgical repair for facilitating healing within homogeneous tissues (BB or TT) showed better healing quality—with respect to maximum loading at week 12, ultimate strength at week 24, and qualitative histological assessments—than the healing taking place between the heterogeneous tissues (BT). These results were comparable with previous studies such that extensive scar tissue, instead of fibrocartilage, overbridged the healing interface and remodeled with healing over time in the TB reattachment.16,18,29 The inferior mechanical properties and poor tissue integration of the BT group at the early time points, compared with the homogeneous tissues groups, may suggest that reattachment of heterogeneous tissue requires a long period of rehabilitation. Reattachment of homogeneous tissue showed higher ultimate strength than reattachment of BT, while no significant difference in failure load was detected among the 3 groups at week 24. Other treatments that enhanced tendon healing also reported significant differences at early time points but not at later time points. Farnebo et al12 used a reconstruction of ATC with decellularized BTI grafts and direct reattachment of Achilles tendon to calcaneus in rabbits. They reported a significant difference detected at the early time points (weeks 2 and 4) with respect to failure load and histological assessments but no significant differences detected at a later time point (week 12). Studies on the biological enhancement for Achilles tendon healing showed improvement on the failure load at early time points, while no significant difference was detected at later time points.1 In contrast, the significantly higher ultimate strength at week 24 observed in the BB and TT groups in our study might reflect the difference in the quality of the regenerated tissue. The BB and TT
Figure 7. Representative images of bone-to-tendon repair at different time points postoperation. At week 6, the cellular component of the interface was primarily fibroblastic and highly vascular. At week 12, the healing interface was not occupied by fibrous scar tissue. There was a limited amount of newly formed bone and minimal integration of the tissues. Moreover, fibrocartilage zone was not found at the healing interface between the bone and the tendon zone. At week 24, more fibrocartilage (FC) was observed between the newly formed bone and the tendon. The intact control showed a typical bone-tendon insertion at the Achilles tendon– calcaneus complex. Hematoxylin and eosin. B, bone; T, tendon. groups showed remodeled tissue with characteristics of laminar bone and normal tendon, respectively, while the
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Figure 8. Representative images of tendon to tendon before surgery (control) and its repair at different time points postoperation. (A) Normal intact Achilles tendon before tenotomy. (B) Healing tendon shows increased cellularity and vascularity at week 6 postoperation when compared with the normal intact tendon. (C) The cellularity of the healing interface at the tenotomy site was high at week 12. (D) Collagen fibers of the tendon remodeled into parallel arrays and the cellularity of the healing tendon decreased compared with week 12. Hematoxylin and eosin, magnification: 1003.
BT group showed minimal formation of new bone and fibrocartilage at the healing interface. The formation of the new bone and fibrocartilage at the healing interface is associated with the strength of the healing interface of BT reattachment.20,35,43 Therefore, the limited formation of native BTI tissue, such as bone and fibrocartilage, and the excess formation of scar tissue at the healing interface might have contributed to the significantly lower ultimate strength of the BT group. Inferior mechanical properties in the BT group at week 12—as demonstrated by lower failure load and ultimate strength in the BT group compared with BB group and TT group—may be related to the lack of transitional tissue and inferior extracellular matrix between the tendon and bone tissues. One of the possible explanations that healing for the reattachment of BT might take longer than BB and TT may be due to the lack of formation of the fibrocartilage zone and highly complex extracellular matrix found in the healing interface of the BTI. The fibrocartilage transition zone functions as a stress absorber by reducing the stiffness gradient between the bone and the tendon.3,43 Another layer of stress absorption is created by the chondrocytes located in the fibrocartilage.43 Collagen fibers create a figure-of-8 course around the chondrocytes. When the BTI is under tension, chondrocytes, which are located between the collagen fibers, get compressed.43 At week 12, a limited fibrocartilage zone was regenerated in the BT group. The properties of regenerated extracellular matrix, such as the orientation collagen fibers and the ratio of collagen subtypes, would determine the mechanical properties of the healing interface.12,41,42,44,51 Histological assessments showed that the extracellular matrix of the
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healing interface of the BB and TT groups was more organized as compared with that of the BT group. Regarding the collagen content of the newly formed matrix, collagen type III is often present at the healing interface between bone and tendon, while collagen type I is present at the healing interface of TT or BB.12,14 However, at a later time point, the BTI would have been remodeled, and the collagen type III would have been replaced with collagen type I.12,22 The ATC model is appropriate for investigating the healing of the extra-articular BTI. One of the advantages of extra-articular models, as compared with intra-articular models, is that when specific variables such as biological interventions or mechanical stimulations are being studied, the implanted tendon tissue does not remodel extensively.43 Therefore, the relationship between healing interface and its mechanical properties can be studied with less confounding factors with the ATC model. The current goat model showed comparable healing rate observed clinically and therefore could be regarded as an appropriate animal model to study the BTI healing. According to Nunley,27 2% to 3% of patients experience poor tendon regeneration during follow-up between 4 and 8 weeks, and 2% rerupture. Similarly, the ultimate strength of TT repair at weeks 12 and 24, which was higher than that of BB and BT repairs, implied a high healing capacity of the Achilles tendon in goat. The cell source and the mechanism for the formation of fibrocartilage remain for further investigation. In the current ATC healing model in goat, the fibrocartilage zone was removed surgically so that the cell source for the formation of fibrocartilage would originate from other progenitor cell sources. Therefore, some possible cell sources might include mesenchymal stem cells (MSCs), tendon-derived stem cells, and circulating stem cells.22,26,50 The introduction of bone MSCs to the bone tunnel have shown to improve the insertion healing of tendon to bone in a rabbit model through formation of fibrocartilagenous attachment at early time points.29 The mechanism for the healing of the BTI is not well understood, especially the formation of the fibrocartilage. The paracrine interaction between MSCs and different cell types in the healing interface affects the differentiation of the MSCs toward tendon, bone, or cartilage.25,36,38,39 Signals, including mechanical loading, would also affect the formation of fibrocartilage in BTI.16,28,37 Anatomic reconstruction of ATC complex injury may be the primary concern in decision making for selecting proper surgical approach. However, whenever possible, it is recommended to select BB or TT repair instead of BT repair; alternately, if BT reconstruction is indicated for surgical repair, then external healing enhancement for BT repair is recommended to ensure better outcome at the healing interface13,19,22 One of the study limitations was that it was not able to delineate the potential variation in length of BB, TT, and BT reattachment, which could have an effect on healing owing to potential differences in tension at the healing interface. To allow healing to occur after surgical repair, each surgical reattachment was ensured to have stable fixation, and the gap between the 2 ends of tissues was minimized.
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In conclusion, repair of BTI injury by reattachment of homogeneous tissue (BB and TT) displayed better recovery than that of the heterogeneous tissue (BT), mechanically and histologically, in a goat ATC injury model. Reconstruction of the ATC by initiating fracture repair or tendon repair might provide better and predictable healing than repair by reattachment of BT. The findings generated from the current ATC complex model may also be a relevant reference for repair of other types of BTI healing. However, further investigation is necessary to understand the similarity and difference in mechanism and signaling pathway involved in repair of homogeneous and heterogeneous tissues.
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17. Leung K, Qin L, Cheung WH. A Practical Manual for Musculoskeletal Research. Singapore: World Scientific; 2008. 18. Leung K-S, Qin L, Fu LK, Chan CW. A comparative study of bone to bone repair and bone to tendon healing in patella-patellar tendon complex in rabbits. Clin Biomech (Bristol, Avon). 2002;17(8):594-602. 19. Liu C, Wan P, Tan LL, Wang K, Yang K. Preclinical investigation of an innovative magnesium-based bone graft substitute for potential orthopaedic applications. J Orthop Transl. 2014;2(3):139-148. 20. Lu H, Hu J, Qin L, Chan KM, Li G, Li K. Area, length and mineralization content of new bone at bone-tendon junction predict its repair quality. J Orthop Res. 2011;29(5):672-677. 21. Lu HH, Thomopoulos S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu Rev Biomed Eng. 2013;15:201-226. 22. Lui P, Zhang P, Chan K, Qin L. Biology and augmentation of tendonbone insertion repair. J Orthop Surg Res. 2010;5:59. 23. McCoy BW, Haddad SL. The strength of achilles tendon repair: a comparison of three suture techniques in human cadaver tendons. Foot Ankle Int. 2010;31(8):701-705. 24. McGuigan MP, Yoo E, Lee DV, Biewener AA. Dynamics of goat distal hind limb muscle-tendon function in response to locomotor grade. J Exp Biol. 2009;212(pt 13):2092-2104. 25. Meretoja VV, Dahlin RL, Kasper FK, Mikos AG. Enhanced chondrogenesis in co-cultures with articular chondrocytes and mesenchymal stem cells. Biomaterials. 2012;33(27):6362-6369. 26. Ni M, Lui PPY, Rui YF, et al. Tendon-derived stem cells (TDSCs) promote tendon repair in a rat patellar tendon window defect model. J Orthop Res. 2012;30(4):613-619. 27. Nunley JA. The Achilles Tendon: Treatment and Rehabilitation. New York, NY: Springer; 2009. 28. O’Conor CJ, Case N, Guilak F. Mechanical regulation of chondrogenesis. Stem Cell Res Ther. 2013;4(4):61. 29. Plate JF, Brown PJ, Walters J, et al. Advanced age diminishes tendon-to-bone healing in a rat model of rotator cuff repair. Am J Sports Med. 2014;42(4):859-868. 30. Qin L, Leung KS, Chan CW, Fu LK, Rosier R. Enlargement of remaining patella after partial patellectomy in rabbits. Med Sci Sports Exerc. 1999;31(4):502-506. 31. Qin L, Wang L, Wong MWN, et al. Osteogenesis induced by extracorporeal shockwave in treatment of delayed osteotendinous junction healing. J Orthop Res. 2010;28(1):70-76. 32. Rodeo SA, Arnoczky SP, Torzilli PA, Hidaka C, Warren RF. Tendonhealing in a bone tunnel: a biomechanical and histological study in the dog. J Bone Joint Surg Am. 1993;75(12):1795-1803. 33. Rui YF, Lui PP, Ni M, Chan LS, Lee YW, Chan KM. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells. J Orthop Res. 2011;29(3):390-396. 34. Sah RL, Ratcliffe A. Translational models for musculoskeletal tissue engineering and regenerative medicine. Tissue Eng Part B Rev. 2010;16(1):1-3. 35. Schneider H. Zur Struktur der Sehnenansatzzonen. Z Anat Entwickl Gesch. 1956;119(5):431-456. 36. Schneider PR, Buhrmann C, Mobasheri A, Matis U, Shakibaei M. Three-dimensional high-density co-culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells. J Orthop Res. 2011;29(9):1351-1360. 37. Schwartz AG, Lipner JH, Pasteris JD, Genin GM, Thomopoulos S. Muscle loading is necessary for the formation of a functional tendon enthesis. Bone. 2013;55(1):44-51. 38. Sharma RI, Snedeker JG. Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone. Biomaterials. 2010;31(30):7695-7704. 39. Sharma RI, Snedeker JG. Paracrine interactions between mesenchymal stem cells affect substrate driven differentiation toward tendon and bone phenotypes. PLoS One. 2012;7(2):e31504. 40. Shi HF, Cheung WH, Qin L, Leung AH, Leung KS. Low-magnitude high-frequency vibration treatment augments fracture healing in ovariectomy-induced osteoporotic bone. Bone. 2010;46(5):12991305.
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41. 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):454463. 42. Thomopoulos S, Marquez JP, Weinberger B, Birman V, Genin GM. Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations. J Biomech. 2006;39(10):18421851. 43. Walsh WR. Repair and Regeneration of Ligaments, Tendons, and Joint Capsule. Totowa, NJ: Humana Press; 2006. 44. Wang I-NE, Mitroo S, Chen FH, Lu HH, Doty SB. Age-dependent changes in matrix composition and organization at the ligament-tobone insertion. J Orthop Res. 2006;24(8):1745-1755. 45. Wang W, Chen HH, Yang XH, Xu G, Chan KM, Qin L. Postoperative programmed muscle tension augmented osteotendinous junction repair. Int J Sports Med. 2007;28(8):691-696.
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46. Wong MW, Qin L, Lee KM, et al. Healing of bone-tendon junction in a bone trough: a goat partial patellectomy model. Clin Orthop Relat Res. 2003;413:291-302. 47. Wong MWN, Qin L, Lee KM, Leung K-S. Articular cartilage increases transition zone regeneration in bone-tendon junction healing. Clin Orthop Relat Res. 2009;467(4):1092-1100. 48. Woo SL, Gomez MA, Seguchi Y, Endo CM, Akeson WH. Measurement of mechanical properties of ligament substance from a boneligament-bone preparation. J Orthop Res. 1983;1(1):22-29. 49. Wren TA, Yerby SA, Beaupre GS, Carter DR. Influence of bone mineral density, age, and strain rate on the failure mode of human Achilles tendons. Clin Biomech (Bristol, Avon). 2001;16(6):529-534. 50. Xu L, Li G. Circulating mesenchymal stem cells and their clinical implications. J Orthop Transl. 2014;2(1):1-7. 51. Zhang J, Pan T, Wang JHC. Cryotherapy suppresses tendon inflammation in an animal model. J Orthop Transl. 2014;2(2):75-81.
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A Comparative Study on the Biomechanical and Histological Properties of Bone-to-Bone, Bone-to-Tendon, and Tendon-to-Tendon Healing: An Achilles Tendon−Calcaneus Model in Goats
Kwok-Sui Leung, Wai Sing Chong, Dick Ho Kiu Chow, Peng Zhang, Wing-Hoi Cheung, Margaret Wan Nar Wong and Ling Qin Am J Sports Med published online March 30, 2015 DOI: 10.1177/0363546515576904 The online version of this article can be found at: http://ajs.sagepub.com/content/early/2015/03/30/0363546515576904
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A Comparative Study on the Biomechanical and Histological Properties of Bone-to-Bone, Bone-toTendon, and Tendon-to-Tendon Healing An Achilles Tendon–Calcaneus Model in Goats Kwok-Sui Leung,* MD, FRCS, Wai Sing Chong,* MPhil, Dick Ho Kiu Chow,* PhD, Peng Zhang,*y PhD, Wing-Hoi Cheung,* PhD, Margaret Wan Nar Wong,* MD, FRCS, and Ling Qin,*yz§ PhD Investigation performed at Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Background: Surgical repair around the bone-tendon insertion (BTI) may involve bone-to-bone (BB), bone-to-tendon (BT), or tendon-to-tendon (TT) reattachment with varying healing outcome. Hypothesis: The repair of Achilles tendon–calcaneus (ATC) by reattachment of homogeneous tissue (BB or TT) would heal faster, with respect to tensile properties at the healing complex, than those of reattachment of heterogeneous tissues (BT) over time. Study Design: Controlled laboratory study. Methods: Forty-seven adolescent male Chinese goats were divided into BB, BT, and TT groups. Osteotomy of the calcaneus, reattachment of Achilles tendon to the calcaneus after removal of the insertion, and tenotomy of the Achilles tendon were performed to simulate BB, BT, and TT repair, respectively. The ATC healing complexes were harvested at 6, 12, or 24 weeks postoperatively. Mechanical and morphological properties of the healing ATC complexes were assessed by tensile testing and qualitative histology, respectively. The contralateral intact ATC complex was used as the control. Results: Failure load of BT was 33.4% lower than that of TT (P = .0243) at week 12. Ultimate strength of BT was 50.2% and 45.3% lower than that of TT at weeks 12 (P = .0002) and 24 (P = .0001), respectively. Tissue morphological characteristics of the BB and TT groups showed faster remodeling. The BT group showed limited regeneration of fibrocartilage zone and excessive formation of fibrous tissue at the healing interface. Conclusion: BTI repair between homogeneous tissues (BB and TT healing) showed better healing quality with respect to mechanical and histological assessments than did healing between heterogeneous tissues (BT healing). Clinical Relevance: Anatomic reconstruction of ATC complex injury may be a primary concern when selecting the proper surgical approach. However, it is recommended to select fracture fixation (BB) or tendon repair (TT) instead of bone-tendon reattachment (BT) if possible to ensure better outcome at the healing interface. Keywords: bone tendon insertion healing; Achilles tendon–calcaneus complex; goat model; mechanical test; histological morphology
absorber during mechanical loading.3,21,22,43 Surgical reattachment of tendon-to-bone is often observed in the repair of rotator cuff, flexor tendons in the hand, tibial insertion of the medial collateral ligament, patellar tendon to patella, and the Achilles tendon to calcaneus.43 Treatment protocols for BTI repair are diversified according to different injury sites, which may lead to different outcome and healing quality.
The bone-tendon insertion (BTI) is a complex structure that consists of bone, mineralized and nonmineralized fibrocartilage, and tendon. It serves as an interface for force transmission from bone to tendon and acts as a stress
The American Journal of Sports Medicine, Vol. XX, No. X DOI: 10.1177/0363546515576904 Ó 2015 The Author(s)
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The rate of healing among different tissues may vary. In an experiment using partial patellectomy model in rabbits, reattachment of homogeneous tissues (bone to bone [BB]) showed better healing quality in terms of mechanical properties than that of heterogeneous tissues (bone to tendon [BT]).18 Moreover, the tissue structure of the BT healing interface was overbridged by fibrous scar tissue, and limited fibrocartilage was observed.6,8,9,12,18,31,32,40,47 The surgical outcome in small-animal experiments, such as rats and rabbits, may not be replicable in large animals owing to differences in joint anatomy (structure) and function (mechanics) apart from variation in body size.4,29,41,50 Muscle contraction around the involved joint might also affect the healing at BTI.45 Mechanical loading may stimulate expression of genes10 and secretion of cytokines, such as bone morphogenetic protein11,33 and transforming growth factor beta,16 which may affect healing.5,7,15,21 Furthermore, the healing ability of small animals, such as rats and rabbits, is often faster than that of large animals or humans.2,34 Therefore, a largeanimal model of BTI healing that mimics the mechanical loading and healing ability of the patient is desirable to compare the quality and progression of healing within homogeneous tissue and heterogeneous tissues. The advantage of using the Achilles tendon–calcaneus (ATC) complex model in the goat is that the muscle-tendon forces, change of fascicle length, and muscle activation of goat distal hindlimb in response to locomotor grade are similar to those of the human.24 Therefore, the healing of the ATC in goats would be comparable with that of the human. Based on the complexity of tissues involved in the BTI region, the healing quality of the different BTI repairs—including BB, BT, and tendon to tendon (TT)—in the ATC complex of Chinese mountain goats was investigated with respect to restoration of their mechanical and histological properties. We hypothesized that the repair of ATC by reattachment of homogeneous tissue (BB or TT) would heal faster, with respect to tensile properties at the healing complex, than those of reattachment of heterogeneous tissues (BT) over time. The primary dependent variable of the current study was the tensile strength of the surgical reattachment site of ATC.
METHODS Surgical Repair of the ATC Complex Forty-seven adolescent male Chinese goats (body weight, 28.1 6 3.1 kg) were obtained and housed in the Laboratory of Animals Service Center of the Chinese University of
Hong Kong after animal experimental ethics was approved (97/054/ERG-CU97/6.68 and 14-085-MIS). The goats were divided into 3 groups (Appendix Table A1, available online at http://ajsm.sagepub.com/supplemental), and different surgical reattachment models (BB, BT, and TT) were performed according to the established protocols.30,46 Briefly, the goat was anesthetized by inhalation anesthesia with nitrous oxide and oxygen in a ratio of 1:2 by volume (Fluothane, Zeneca). Skin around the ATC complex was shaved and sterilized with 0.05% Hibitane (Zeneca). The ATC complex was approached through posterior midline skin incision aseptically. The overlaying tendon of flexor digitorum superficialis was incised to expose the ATC junction for the following 3 surgical interventions: BB repair, BT repair, and TT repair. The intact ATC without any surgical interventions was used as the control. BB Repair. Osteotomy with 0.9% saline irrigation was performed vertically at 1 cm distal to the ATC in junction. With the aid of bone forceps, 2 Kirschner wires (diameter, 0.9 mm) were drilled in parallel through the posteriorinferior poles of medial and lateral tuberosity at 30° with respect to the horizontal plane of calcaneus. A figure-of-8 tension band was applied to prevent dislocation of bone fragments before the connective tissue and skin were sutured for completion of the surgery (Figure 1A). BT Repair. The junction of ATC was explored surgically using a sharp scalpel. The calcaneus surface was partially decorticated to expose the osseous tissues. A No. 0 polydioxanone monofilament absorbable suture (PDS II, Ethicon Ltd) was applied in a Bunnell suture configuration to the Achilles tendon. A bone tunnel (diameter, 1.2 mm) was drilled from the lateral tuberosity to medial tuberosity horizontally. Both suture ends were passed through the drill hole. The Achilles tendon was pulled toward the calcaneus, and the complex was secured by knotting of the 2 suture ends. A figure-of-8 tension band wire (stainless steel surgical wire) was placed to protect the repair junction before the surgery was completed with skin suture (Figure 1B). TT Repair. The Achilles tendon was exposed and cut transversely at 2 cm proximal to its insertion to calcaneus using a sharp scalpel. Bunnell suture configuration with No. 0 polydioxanone monofilament absorbable suture (PDS II, Ethicon Ltd) was applied to join both ends of the tendon for fixation (Figure 1C). The advantage of using the Bunnell suture is minimal handling and care to avoid vascular interference. Even though other multistrand suture techniques are stronger and gap resistant, these suturing techniques may cause greater tissue trauma and be detrimental to the glide of the healing tendon.23 After the surgical reattachment, overlaying ligamentous capsule and skin were then closed with 3-0 sterile
§ Address correspondence to Ling Qin, PhD, Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Shatin, NT, 001, Hong Kong (email: [email protected]). *Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China. y Translational Medicine Research and Development Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China. z Lui Che Woo Institute of Innovative Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China. One or more of the authors has declared the following potential conflict of interest or source of funding: This project was supported by the Research Grants Council of the Hong Kong Special Administrative Regions (CUHK 4275/97M).
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Schering-Plough) was injected intramuscularly for analgesia. Cast (Scotchcast Plus, P-46325, 3M Health Care) was applied for immobilization of the operated limb for 6 weeks. The cast immobilization prevented dorsiflexion to reduce tension on the healing ATC. The goats were allowed full weightbearing and joint motion after cast removal. The goats were allowed for unrestricted cage activity after surgery in the animal house.
Sampling for Mechanical Testing and Histology
Figure 1. A schematic drawing of the establishment of the Achilles tendon–calcaneus (ATC) complex healing by reattachment of bone to bone (BB), bone to tendon (BT), and tendon to tendon (TT). (A) BB repair. A1: Osteotomy distal to the ATC complex. A2: Two Kirschner wires were drilled in parallel through the posterior-inferior poles of medial and lateral tuberosity. A3: A figure-of-8 tension band was applied to prevent dislocation of bone fragments. (B) BT repair. B1: The junction of ATC was explored surgically. B2: Bunnell suture configuration to Achilles tendon. A bone tunnel was drilled from lateral tuberosity to medial tuberosity horizontally. Both suture ends were passed through the drill hole. B3: A figure-of-8 tension band was placed to protect the repair junction. (C) TT repair. C1: Achilles tendon was exposed and cut transversely. C2: Bunnell suture configuration with suture. C3: The sutures were pulled, and the 2 ends of the Achilles tendon were secured against each other. Red line: figure-of-8 protection band. Blue line: suture. Black dotted line: incision site. Black bands: Kirschner wire. Solid lines: exposed wires. Dotted lines: nonexposed wires.
catgut (Chromic, Ethicon Ltd) and 3-0 braided silk suture (Mersilk, Ethicon Ltd). Antibiotic spray (Nebactin, Byk Gulden) was applied for disinfection. Temgesic (0.005 mg/kg;
The goats were euthanized at 6 (n = 17), 12 (n = 15), or 24 (n = 15) weeks postoperatively by overdose injection of sodium pentobarbital via the jugular vein. After the ATC complex was isolated, Kirschner wires and figure-of-8 tension band wires were removed from the ATC complex. For mechanical testing, the ATC complex was wrapped in gauze with saline, sealed in a freezer bag, and stored at 220°C freezer before mechanical testing. After thawing the samples at room temperature, the calcaneus of the ATC complex was placed vertically inside a mold and embedded with PMMA composed of Ureol 5202 B and Ureol 5202 A.17 After completion of polymerization, 2 holes (diameter, 5 mm) were drilled in parallel through the embedded calcaneus. The ATC complex was fixed on a custom-made testing jig consisting of an upper soft tissue clamp and lower stand. The upper clamp has serrated grooves, which grasp the Achilles tendon junction firmly. The lower stand has 2 horizontally arranged screws for fixing the polymer embedded calcaneus. Embedding the ends of the sample in MMA resin minimized slippage of the samples by providing better fixation between the lower clamp and sample. The ATC complex was fixed for tensile testing in a physiological position (ie, with a resting angle of 90° between Achilles tendon and calcaneus). The calcaneus was then screw fixed on the lower stand, and the Achilles muscle-tendon unit was carefully fixed on the upper clamp without creating premature failure at the contact point with the clamp (Appendix Figure A1, available online). For better fixation between specimens and metal clamps, the central one-third of the Achilles was isolated by transversely cutting over the lateral and medial one-third of the tendon for TT group. After the sample and jig were fitted onto a mechanical testing machine (H25KM, Hounsfield test equipment, Redhill), a tensile test was performed with a load cell of 2 kN at a testing speed of 50 mm/min. Failure load was recorded and the ultimate strength calculated by normalizing the failure load by the cross-section area (CSA) of the healing interface. Failure mode of each ATC was also recorded. The contralateral intact ATC complex was used as the control. Achilles tendon–calcaneus complexes used for histological study were fixed in 4% neutral buffered formalin, decalcified in 9% formic acid, and embedded in paraffin. Serial sagittal sections (7 mm) were cut using microtome (Leica, RM6165, Reichert-Jung GmbH) and stained with hematoxylin and eosin. A research scientist and an orthopaedic clinical scientist qualitatively evaluated the histological sections under transmitted light microscope (Leica Q500MC, Leica Cambridge Ltd).
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Figure 2. Midsagittal section of the Achilles tendon–calcaneus samples after operation compared among the bone-to-bone (BB), bone-to-tendon (BT), tendon-to-tendon (TT) groups at 3 healing time points. The white arrows indicate the healing interface for different groups.
Measurement of CSA of Healing Interface
RESULTS
The CSAs of the healing interface for different treatment groups were measured to calculate the ultimate strength from the failure load of each sample. A conventional caliper was used to measure the CSA of the healing interface of the tissue in the BB, BT, and TT groups. Other available methods for measuring CSA, such as ultrasound, magnetic resonance imaging, peripheral quantitative computed tomography, and micro–computed tomography, are advanced methods but are only suitable for a specific type of tissue (bone or tendon), while the healing BTI in the BT repair involves both hard and soft tissue. The CSA of the healing interface was calculated by multiplying the length and width of the remaining intact tissue. The CSA at the healing interface for each sample was measured 8 times and the mean used for statistical analysis. A single examiner performed all CSA measurements to minimize potential interindividual error.48 The coefficient of variation for repeated measures was calculated to be \5%.
Gross Observation of Specimens The healing interface of the BB group was identified at weeks 6 and 12 but not observable at week 24 postoperation (Figure 2). The bone marrow content in the BB group decreased with healing over time. For the BT group, the tendon surface was not shiny and was covered with white fibrous tissue surrounding the healing interface at week 6 postoperation. No parallel collagen bundles were observed at 12 weeks. At week 24, parallel alignment of collagen bundles were observed (Figure 2). For the TT group, transparent fibrous tissue with darker-red appearance was found at week 6 postoperation. At week 12, the parallel tendon fiber structure and fibrous scar tissue around tenotomy site became shiny looking. At week 24, fibrous tissue was found at the healing interface, but tenotomy margin was not identified (Figure 2).
Mechanical Testing Statistical Analysis All measurements were expressed as mean 6 SD, while the box plots showed median and quartiles. All analyses were performed using SPSS version 16.0 for Windows (SPSS Inc). Two-way analysis of variance with Bonferroni post hoc tests were used to determine the effect of different reattachment methods and time on the mechanical properties of ATC healing. Statistical significance was set at P \ .05.
The TT group showed a significantly higher failure load than that of the BT group at week 12 (50.1%, P = .0243) (Figure 3). However, no significant differences were detected among the 3 groups at weeks 6 and 24. There were also no significant differences in failure load between weeks 12 and 24 for the BB and TT groups. There was a significant time effect on failure load (P \ .0001) but no significant interaction between time effect and reattachment
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Figure 3. Maximum load of Achilles tendon–calcaneus complex before surgery (control) and at different time points after operation. Bone-to-tendon (BT) group showed lower maximum load at week 12 than the other 2 groups, while no significant difference was detected at week 24. *P \ .05. BB, bone to bone; TT, tendon to tendon.
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Figure 4. Ultimate tensile strength of Achilles tendon– calcaneus complex before surgery (control) and at different time points after operation. Bone-to-tendon (BT) group showed lower ultimate tensile strength at weeks 12 and 24 than the other 2 groups, while no significant difference was detected at week 6. **P \ .01. ***P \ .001. BB, bone to bone; TT, tendon to tendon.
method. With respect to ultimate strength, there were no significant differences among the 3 groups at week 6 (Figure 4). The ultimate strength of the TT group was higher than that of the BB and BT groups at week 12 (155.4% and 100.6%, respectively; P \ .0002) and week 24 (48.4% and 83.0%, respectively; P \ .0034). For ultimate strength, there was a significant time effect (P \ .0001) and significant interaction between time effect and reattachment methods (P = .0056). The CSAs of the healing interfaces for all groups were significantly different for all time points pre- and postoperation. For energy to failure, no significant difference was detected among the 3 reattachment methods at weeks 6, 12, and 24 (Figure 5). There was a significant time effect on energy to failure (P \ .0001) but no significant interaction between time effect and reattachment methods.
Failure Mode At week 6, all samples of the BB and TT groups failed at the healing interface. For the BT group, 75% (3 of 4) of the specimens failed at the healing interface, while 25% (1 of 4) failed at tendon midsubstance. At week 12, 80% (12 of 15) of the samples of the 3 groups failed at the healing interface. At week 24, 100% (5 of 5) of the samples of the BB group and 40% (2 of 5) of the sample in the TT group failed at the ends where the specimens were clamped instead of the healing interface. However, all samples (5 of 5) of the BT group failed at the tendon zone of the healing interface, while only 60% (3 of 5) of the samples of the TT group failed at the tendon tissue.
Histological Observation For the BB group, trabeculae near the osteotomy site were woven bone at week 6 (Figure 6). At week 12, active bone formation was found, and chondrocyte-like cells and
Figure 5. Energy to failure of Achilles tendon–calcaneus complex before surgery (control) and at different time points after operation. No significant difference was detected among the 3 reattachment methods at weeks 6, 12, and 24. BB, bone to bone; BT, bone to tendon; TT, tendon to tendon.
osteoblasts lining were present at the healing interface. At week 24, lamella trabecular bone with a decrease in osteocyte density was found at the osteotomy site (Figure 6). For the BT group, fibrous tissue was formed at the healing interface at week 6; at week 12, formation of new bone and fibrocartilage zone at the healing interface was minimal (Figure 7); at week 24, the newly formed tissues resembled normal ATC, including the 4 histologically different zones and the tidemark (Figure 7). The newly formed tendon tissue in the TT group showed increased cellularity with cells that were round at week 6 (Figure
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Figure 6. Representative images of bone-to-bone repair at weeks 6, 12, and 24 after operation. (A) At week 6, cartilage tissue was found at the osteotomy site. Woven bone was also observed surrounding the osteotomy site. (B) At week 12, woven bone was mainly found at the osteotomy site. (C) At week 24, the woven bone was remodeled to laminar bone, which provides better mechanical properties. Hematoxylin and eosin, magnification: 163. Ca, cartilage; LB, laminar bone; WB, woven bone. 8). At weeks 12 and 24, decrease of cellularity, presence of spindle-like cells, and organized alignment of the collagens were observed at the healing interface (Figure 8).
DISCUSSION This experiment used an ATC complex model in goats to demonstrate the effect of different surgical approaches on the healing quality of injury around the BTI with respect to mechanical and histological evaluations. Surgical repair for facilitating healing within homogeneous tissues (BB or TT) showed better healing quality—with respect to maximum loading at week 12, ultimate strength at week 24, and qualitative histological assessments—than the healing taking place between the heterogeneous tissues (BT). These results were comparable with previous studies such that extensive scar tissue, instead of fibrocartilage, overbridged the healing interface and remodeled with healing over time in the TB reattachment.16,18,29 The inferior mechanical properties and poor tissue integration of the BT group at the early time points, compared with the homogeneous tissues groups, may suggest that reattachment of heterogeneous tissue requires a long period of rehabilitation. Reattachment of homogeneous tissue showed higher ultimate strength than reattachment of BT, while no significant difference in failure load was detected among the 3 groups at week 24. Other treatments that enhanced tendon healing also reported significant differences at early time points but not at later time points. Farnebo et al12 used a reconstruction of ATC with decellularized BTI grafts and direct reattachment of Achilles tendon to calcaneus in rabbits. They reported a significant difference detected at the early time points (weeks 2 and 4) with respect to failure load and histological assessments but no significant differences detected at a later time point (week 12). Studies on the biological enhancement for Achilles tendon healing showed improvement on the failure load at early time points, while no significant difference was detected at later time points.1 In contrast, the significantly higher ultimate strength at week 24 observed in the BB and TT groups in our study might reflect the difference in the quality of the regenerated tissue. The BB and TT
Figure 7. Representative images of bone-to-tendon repair at different time points postoperation. At week 6, the cellular component of the interface was primarily fibroblastic and highly vascular. At week 12, the healing interface was not occupied by fibrous scar tissue. There was a limited amount of newly formed bone and minimal integration of the tissues. Moreover, fibrocartilage zone was not found at the healing interface between the bone and the tendon zone. At week 24, more fibrocartilage (FC) was observed between the newly formed bone and the tendon. The intact control showed a typical bone-tendon insertion at the Achilles tendon– calcaneus complex. Hematoxylin and eosin. B, bone; T, tendon. groups showed remodeled tissue with characteristics of laminar bone and normal tendon, respectively, while the
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Figure 8. Representative images of tendon to tendon before surgery (control) and its repair at different time points postoperation. (A) Normal intact Achilles tendon before tenotomy. (B) Healing tendon shows increased cellularity and vascularity at week 6 postoperation when compared with the normal intact tendon. (C) The cellularity of the healing interface at the tenotomy site was high at week 12. (D) Collagen fibers of the tendon remodeled into parallel arrays and the cellularity of the healing tendon decreased compared with week 12. Hematoxylin and eosin, magnification: 1003.
BT group showed minimal formation of new bone and fibrocartilage at the healing interface. The formation of the new bone and fibrocartilage at the healing interface is associated with the strength of the healing interface of BT reattachment.20,35,43 Therefore, the limited formation of native BTI tissue, such as bone and fibrocartilage, and the excess formation of scar tissue at the healing interface might have contributed to the significantly lower ultimate strength of the BT group. Inferior mechanical properties in the BT group at week 12—as demonstrated by lower failure load and ultimate strength in the BT group compared with BB group and TT group—may be related to the lack of transitional tissue and inferior extracellular matrix between the tendon and bone tissues. One of the possible explanations that healing for the reattachment of BT might take longer than BB and TT may be due to the lack of formation of the fibrocartilage zone and highly complex extracellular matrix found in the healing interface of the BTI. The fibrocartilage transition zone functions as a stress absorber by reducing the stiffness gradient between the bone and the tendon.3,43 Another layer of stress absorption is created by the chondrocytes located in the fibrocartilage.43 Collagen fibers create a figure-of-8 course around the chondrocytes. When the BTI is under tension, chondrocytes, which are located between the collagen fibers, get compressed.43 At week 12, a limited fibrocartilage zone was regenerated in the BT group. The properties of regenerated extracellular matrix, such as the orientation collagen fibers and the ratio of collagen subtypes, would determine the mechanical properties of the healing interface.12,41,42,44,51 Histological assessments showed that the extracellular matrix of the
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healing interface of the BB and TT groups was more organized as compared with that of the BT group. Regarding the collagen content of the newly formed matrix, collagen type III is often present at the healing interface between bone and tendon, while collagen type I is present at the healing interface of TT or BB.12,14 However, at a later time point, the BTI would have been remodeled, and the collagen type III would have been replaced with collagen type I.12,22 The ATC model is appropriate for investigating the healing of the extra-articular BTI. One of the advantages of extra-articular models, as compared with intra-articular models, is that when specific variables such as biological interventions or mechanical stimulations are being studied, the implanted tendon tissue does not remodel extensively.43 Therefore, the relationship between healing interface and its mechanical properties can be studied with less confounding factors with the ATC model. The current goat model showed comparable healing rate observed clinically and therefore could be regarded as an appropriate animal model to study the BTI healing. According to Nunley,27 2% to 3% of patients experience poor tendon regeneration during follow-up between 4 and 8 weeks, and 2% rerupture. Similarly, the ultimate strength of TT repair at weeks 12 and 24, which was higher than that of BB and BT repairs, implied a high healing capacity of the Achilles tendon in goat. The cell source and the mechanism for the formation of fibrocartilage remain for further investigation. In the current ATC healing model in goat, the fibrocartilage zone was removed surgically so that the cell source for the formation of fibrocartilage would originate from other progenitor cell sources. Therefore, some possible cell sources might include mesenchymal stem cells (MSCs), tendon-derived stem cells, and circulating stem cells.22,26,50 The introduction of bone MSCs to the bone tunnel have shown to improve the insertion healing of tendon to bone in a rabbit model through formation of fibrocartilagenous attachment at early time points.29 The mechanism for the healing of the BTI is not well understood, especially the formation of the fibrocartilage. The paracrine interaction between MSCs and different cell types in the healing interface affects the differentiation of the MSCs toward tendon, bone, or cartilage.25,36,38,39 Signals, including mechanical loading, would also affect the formation of fibrocartilage in BTI.16,28,37 Anatomic reconstruction of ATC complex injury may be the primary concern in decision making for selecting proper surgical approach. However, whenever possible, it is recommended to select BB or TT repair instead of BT repair; alternately, if BT reconstruction is indicated for surgical repair, then external healing enhancement for BT repair is recommended to ensure better outcome at the healing interface13,19,22 One of the study limitations was that it was not able to delineate the potential variation in length of BB, TT, and BT reattachment, which could have an effect on healing owing to potential differences in tension at the healing interface. To allow healing to occur after surgical repair, each surgical reattachment was ensured to have stable fixation, and the gap between the 2 ends of tissues was minimized.
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In conclusion, repair of BTI injury by reattachment of homogeneous tissue (BB and TT) displayed better recovery than that of the heterogeneous tissue (BT), mechanically and histologically, in a goat ATC injury model. Reconstruction of the ATC by initiating fracture repair or tendon repair might provide better and predictable healing than repair by reattachment of BT. The findings generated from the current ATC complex model may also be a relevant reference for repair of other types of BTI healing. However, further investigation is necessary to understand the similarity and difference in mechanism and signaling pathway involved in repair of homogeneous and heterogeneous tissues.
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