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Calcium phosphate-hybridised tendon graft to reduce bone-tunnel enlargement after ACL reconstruction in goats
The Knee 19 (2012) 455–460
Contents lists available at ScienceDirect
The Knee
Calcium phosphate-hybridised tendon graft to reduce bone-tunnel enlargement after ACL reconstruction in goats Hirotaka Mutsuzaki a, Masataka Sakane b,⁎, Hiromi Nakajima c, Naoyuki Ochiai b a
Department of Orthopaedic Surgery, Ibaraki Prefectural University of Health Sciences, 4669–2 Ami Ami-machi, Inashiki-gun, Ibaraki 300–0394, Japan Department of Orthopaedic Surgery, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305–8575, Japan c Department of Agriculture, Ibaraki University, 3-21-1 Chuuou, Ami, Ibaraki 300–0393, Japan b
a r t i c l e
i n f o
Article history: Received 22 September 2010 Received in revised form 9 March 2011 Accepted 24 March 2011 Keywords: Anterior cruciate ligament Tendon-bone healing Calcium phosphate hybridisation Bone tunnel enlargement Osteoclast
s u m m a r y Bone-tunnel enlargement can have a negative impact on long-term clinical success. To solve the problem, we developed a novel technique to improve tendon–bone healing by hybridising calcium phosphate (CaP) with a tendon graft using an alternate soaking process. The objective of this study was to analyse bone-tunnel enlargement, mechanical properties and histological features, especially the number of osteoclasts at the tendon–bone interface using a CaP-hybridised tendon graft and an untreated tendon graft 6 months after anterior cruciate ligament (ACL) reconstruction in goats. The percentage of bone–tunnel enlargement for the CaP group was decreased compared with that for the control group for the femoral side (p b 0.05). The failure load was not statistically different between the CaP group and the control group, and was all midsubstance rupture for both groups. In the CaP group, cartilage layer was more observed at the tendon–bone interface of the joint aperture site than in the control group (p b 0.05). Many osteoclasts on the femoral side of the tendon– bone interface in the control were observed compared with that in the CaP group (p b 0.05). At the femoral side, the CaP-hybridised tendon graft reduced bone-tunnel enlargement associated with tendon–bone healing 6 months after ACL reconstruction in goats. Clinically, the CaP-hybridised tendon graft for ACL reconstruction can reduce bone-tunnel enlargement. © 2011 Elsevier B.V. All rights reserved.
It is well established that injury to the anterior cruciate ligament (ACL) results in knee instability and functional disability. As a result, these injuries frequently require ligament reconstruction using a tendon graft [1–3]. However, a tendon graft requires soft-tissue-to-bone healing within both bone tunnels. Grana et al [4]. evaluated a tendon autograft within bone tunnels in a rabbit ACL reconstruction model. An indirect tendon insertion with fibrous connective tissue was observed at the tendon–bone interface. Moreover, it has frequently been shown that bone-tunnel enlargement occurs following ACL reconstruction using soft-tissue grafts [5–9]. Increased knee laxity in correlation with radiographic femoral tunnel enlargement in ACL reconstructions using soft-tissue grafts has been demonstrated [10]. Moreover, enlargement of the articular end of the bone tunnels owing to bone resorption associated with osteoclasts is a common problem in ACL reconstruction [11,12]. We considered that firm tendon–bone healing near the joint and prevention of bone-tunnel enlargement can be important for good clinical outcome of ACL reconstruction. Therefore, we developed a novel technique to improve tendon–bone attachment by hybridising calcium phosphate (CaP) with tendons using an alternate soaking process [13].
⁎ Corresponding author. Tel.: + 81 29 853 3219; fax: + 81 29 853 3214. E-mail address: [email protected] (M. Sakane). 0968-0160/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.knee.2011.03.008
Using the CaP-hybridised tendon, we observed a scarless direct bonding area between the grafted tendon and the newly formed bone without inflammatory reaction 2–3 weeks after ACL reconstruction in rabbits [14,15], which we also observed in goats 6 weeks after the reconstruction [16], owing to its osteoconductive potential and affinity for living tissues. However, long-term results regarding bone-tunnel enlargement and the interface between the CaP-hybridised tendon and the bone for ACL reconstruction are unclear. We used a goat model of ACL reconstruction to solve these problems because long-term studies using goat knees have shown effective restoration of knee stability and minimal articular cartilage degeneration after ACL reconstruction [17,18]. We hypothesise that in the case of using CaP-hybridised tendon grafts, bone-tunnel enlargement associated with osteoclasts at the joint aperture site is minimised owing to improved tendon–bone healing compared with that using untreated tendon grafts 6 months after the operation. Moreover, the mechanical properties at the interface in the CaP group may be superior to those in the control group because of its firm anchoring formation. The objective of this study was to analyse bone-tunnel enlargement, mechanical properties and histological features, especially the number of osteoclasts at the tendon–bone interface using a CaP-hybridised tendon graft and an untreated tendon graft 6 months after ACL reconstruction in goats.
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1. Materials and Methods This study design is a controlled laboratory study. 1.1. CaP hybridisation method Twelve skeletally mature female Saanen breed goats (6 months of age) were used in this study. Conditions were the same as in our previous study [16]. The goats were maintained in accordance with the guidelines of the Ethical Committee of the Biomaterial Center of the National Institute for Materials Science and the National Institutes of Health for the care and use of laboratory animals (NIH Pub. No. 85–23 Rev. 1985). The CaP hybridisation method was the same as in previous studies [14–16]. The flexor digitorum longus (FDL) tendons and hamstring tendons were used as tendon grafts. Double-ruple-strand tendons of 45-mm length and 6.5-mm diameter were prepared. The tibial end of the grafts was secured with a non-absorbable suture, and a polyester tape suture tied over the EndoButton® (Smith & Nephew, USA) was passed through the looped femoral end of the grafts. Then, the central third of the grafts at the intra-articular portion were covered with the sleeve of a rubber glove tied on each side to prevent CaP hybridisation [16]. After these procedures, the grafts were soaked in 100 ml of a Ca solution (100 mM CaCl2 + 30 mM L-histidine, pH 7.4, 25 °C) for 30 s. The grafts were subsequently soaked in 100 ml of a NaHPO4 solution (116.4 mM NaH2PO4:128.7 mM Na2HPO4 12H2O = 15%:85%, pH 7.4, 25 °C) for 30 s. Before each soaking, the grafts were washed in a saline solution. This cycle was repeated 10 times [14–16]. In the control group, the tendon was soaked in a saline solution for 10 min. 1.2. Surgical procedure The surgical procedures were the same as in our previous study [16]. All the surgical procedures were performed under aseptic conditions with the animals under general anaesthesia. On the right knee, an anterior lateral skin incision was made. We exposed the knee joint by the lateral para-patella approach. ACL was then completely transected, and gross anterior subluxation of the tibia was confirmed by manual examination. The tibial and femoral tunnels were drilled to form holes of 6.5 mm diameter on the tibial and femoral insertions of the ACL. The length of the tunnel was at least 20 mm. The graft described above was passed through the femoral and tibial tunnels. The graft fixation was achieved via the EndoButton® at the femoral side and a 4.5-mmdiameter cortex screw (MEIRA Corporation, Nagoya, Japan) at the tibial side with 20 N as the initial tension (Fig. 1) [16]. After reconstruction, we sutured the capsule and skin wound. Postoperatively, all the goats were allowed free activity in their cages (cage area, 50 m2) without receiving any antibiotics. We did not perform daily wound dressing. Visual inspection revealed that normal gait patterns did not return until 3–4 weeks after surgery. The goats were sacrificed (1 ml of sodium pentobarbital per 4 kg goat mass administered intravenously) exactly 6 months after the surgery. The hind limbs were disarticulated at the hip joint. A total of six specimens each from the CaP and control groups were obtained. The six specimens in each group were sealed in double plastic bags and immediately stored at − 20 °C until use in biomechanical analysis. 1.3. Ex vivo computed tomography (CT) evaluation The six frozen specimens from each group were used for computed tomography (CT) evaluation. CT scans of the specimens were performed (Brilliance CT 64, Philips, Amsterdam, the Netherlands) to assess femoral and tibial bone tunnels. The CT scans (voltage: 120 kV, current: 230 mA) in the full-extension knee position were supervised and examined by a single radiologist. A standard protocol was used consistently throughout the study. Initial volume acquisition was with 0.9 mm cuts from 30 mm above the femoral tunnel to 30 mm below the
Fig. 1. ACL reconstruction. The graft (black arrow) was pulled taut through the drill hole with both ends fixed at the distal part of the femur (EndoButton) and proximal part of the tibia (screw).
tibial tunnel. Using a Virtual Place Lexus (AZE Ltd., Tokyo, Japan) workstation, three-dimensional images were reconstructed. Coronal, sagittal and axial images of the femoral and tibial bone tunnels were obtained. We measured the anterior–posterior diameter (APD), medial– lateral diameter (MLD) and tunnel cross-sectional area (CSA) of the femur and tibia at the main joint aperture site after sacrifice (6 months APD, MLD or CSA) (Fig. 2). The percentage of bone-tunnel enlargement in tunnel APD, MLD and CSA was calculated using the following formula: Percentage of bone−tunnel enlargement of APD; MLD; or CSAð%Þ = ð6 months APD; MLD or CSA– Original APD; MLD or CSAÞ × 100 = Original APD; MLD or CSA: Original APD and MLD are the diameters of the drill using in the operation. Original CSA is the calculated area of CSA based on the diameter. 1.4. Biomechanical analysis After CT evaluation, biomechanical analysis was performed. Before biomechanical testing, each specimen was thawed for 24 h at room temperature. The tibia and femur were cut 20 cm from the knee joint line, and surrounding tissue except the grafted tendon was removed. The cross-sectional area of the graft at the joint line was measured using an apparatus for measuring the cross-sectional area of the grafted tendon (Jintai Danmenseki Sokuteiki, Meira Corporation, Nagoya, Japan). For biomechanical testing, the experimental femur– ACL graft–tibia complexes were then fixed to custom-designed clamps that allowed tensile loading along the long axis of the graft on a material testing machine (AGS-N 1kN, Shimadzu Corporation, Kyoto, Japan). To ensure that we were determining the tensile properties of the graft and its interface within the tunnels, the additional fixation devices (EndoButton and fixation screw) were removed. After a 3 N preload was applied, load-to-failure testing at an elongation rate of 30 mm min–1 was performed. The load–elongation curves of the experimental femur–ACL graft–tibia complexes were
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Fig. 2. CT images. (a) Coronal image of femoral bone tunnel. Medial-lateral diameter (MLD: black arrow) was measured. (b) Sagittal image of femoral bone tunnel. Anterior-posterior diameter (APD: black arrow) was measured. (c) Axial image of femoral bone tunnel. Tunnel cross-sectional area (CSA) was measured. (d) Coronal image of tibial bone tunnel. Medial-lateral diameter (MLD: black arrow) was measured. (e) Sagittal image of tibial bone tunnel. Anterior-posterior diameter (APD: black arrow) was measured. (f) Axial image of tibial bone tunnel. Tunnel cross-sectional area (CSA) was measured.
recorded. Stiffness (N mm–1) was calculated from the slope of the linear region between 1 and 2.5 mm of elongation. Failure load was divided by the cross-sectional area of the graft at the joint line of each specimen as the value of stress [16].
1.5. Histological analysis After the biomechanical analysis, histological evaluations were performed. The tendon–bone interface was completely intact after the biomechanical analysis, as all the specimens failed in the tendon midsubstance on histological observation. Therefore, we were able to perform histological analysis at the tendon–bone interface in the femoral and tibial side in both groups. The specimens were fixed in 10% neutralbuffered formalin, decalcified and embedded in paraffin. The specimens were sliced 5-μm-thick sagittal to the bone tunnel. Haematoxylin and eosin (H–E) staining, safranin-O staining for identification of the cartilage layer in the interface, Masson's trichrome (MT) staining for identification of collagenous fibre tissue and cathepsin K immunostaining (cathepsin K mouse monoclonal antibody; Novocastra Laboratories Ltd., UK) for identification of osteoclasts were performed. The specimens were examined by light microscopy after staining. Histological comparisons of the tendon–bone interface anterior and posterior bone tunnel surface at the joint aperture site (cartilaginous insertion or fibrous insertion) in the femoral and tibial side were carried out between the CaP group and the control group. They were blinded by an orthopaedic surgeon. To determine the failure modes at 6 months, microscopy observations were performed after the biomechanical test. The failure modes (pull out or midsubstance rupture) of the experimental femur– ACL graft–tibia complexes were recorded.
Using a Mac Scope program (Mitani Co., Japan) on a Macintosh computer, the numbers of osteoclasts in the tendon–bone interface at the femoral and tibial sides at the joint aperture site (10 mm from the joint line) were measured. Next, the obtained numbers of osteoclasts were divided by the length of each femoral and tibial bone tunnel of each specimen to correct the values. We defined the corrected value of the number of osteoclasts as the number of osteoclasts per millimetre. 1.6. Statistical Analyses The bone-tunnel enlargement data (APD, MLD and CSA), biomechanical data (ultimate failure load, stiffness and stress), and osteoclast data of the two groups were compared using the Student's t-test at a p b 0.05 significance value. Concerning the bone-tunnel enlargement data and the osteoclast data in each group, statistical analysis of differences between the femoral side and tibial side were performed using the Student's t-test at a p b 0.05 significance value. To compare the histological difference between the CaP and control groups, Mann– Whitney's U test was used at a p b 0.05 significance value. 2. Results 2.1. CT evaluation The bone-tunnel enlargement rates obtained using CT (CaP group, n = 6; control group, n = 6) are shown in Table 1. The average percentage of bone-tunnel enlargement in the six femoral bone-tunnel APD for the untreated tendon graft (53.8± 16.1%) was significantly greater than that of the CaP-hybridised tendon graft (41.0 ± 6.6%, p = 0.0488). The average percentage of bone-tunnel enlargement of MLD in the femoral side for the untreated tendon graft (53.8± 18.4%) was also significantly greater than that of the CaP-hybridised tendon graft (30.8 ± 23.3%, p = 0.0431). The average percentage of bone-tunnel enlargement of CSA in the femoral side was greater in the control group (170.4 ± 54.8%) than in the CaP group (118.1 ± 20.7%, p = 0.0269). However, there was no
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Table 1 CT evaluation: percentage of bone tunnel enlargement. Femoral side
CaP group (n = 6) Control (n = 6)
Tibial side
APD(%)
MLD(%)
CSA(%)
APD(%)
MLD(%)
CSA(%)
41.0 ± 6.6 * # 53.8 ± 16.1
30.8 ± 23.3 * 53.8 ± 18.4
118.1 ± 20.7 * # 170.4 ± 54.8
23.6 ± 13.2 38.2 ± 23.9
23.6 ± 15.1 36.9 ± 28.1
66.3 ± 23.8 113.6 ± 73.4
APD: anterior-posterior diameter, MLD: medial-lateral diameter, CSA: cross-sectional area. Results are the mean ± SD. * p b 0.05 compared with control values. # p b 0.05 compared with tibial values.
Table 2 Biomechanical results.
CaP group (n = 6) Control (n = 6)
Ultimate failure load (N)
Stiffness (N/mm)
Stress (N/mm2)
Failure mode (tendon midsubstance: pulled out)
562.3 ± 144.1
50.5 ± 15.0
13.7 ± 2.3
6:0
575.4 ± 115.1
43.5 ± 13.7
15.2 ± 2.4
6:0
Histological observation
2.2. Biomechanical analysis All the specimens failed in the tendon midsubstance on histological observation. The results of biomechanical analysis (CaP, n = 6; control, n = 6) are shown in Table 2. No significant difference in the ultimate failure load between the CaP-hybridised tendon graft (562.3 ± 144.1 N) and the control graft (575.4± 115.1 N) was found (p = 0.4312). Moreover, no significant difference in stiffness between the CaP-hybridised tendon graft (50.5± 15.0 N mm–1) and the control graft (43.5 ± 13.7 N mm–1) was found (p = 0.2095). No significant difference in the stress between the CaP-hybridised tendon graft (13.7 ± 2.3 N mm–2) and the control graft (15.2 ± 2.4 N mm–2) was found (p = 0.1430).
Results are the mean ± SD.
Table 3 Histological observation (cartilage layer formation).
CaP group (n = 6) Control (n = 6)
greater than those of the tibial bone tunnel (p = 0.0199 and p = 0.0015, respectively). However, there was no significant difference between the femoral and tibial side in the MLD in the CaP group, and the APD, MLD and CSA in the control group (p = 0.2634, p = 0.0615, p = 0.1202 and p = 0.0799, respectively).
Femur (number / 6)
Tibia (number / 6)
Anterior
Posterior
Anterior
Posterior
1/6 0/6
2/6 0/6
4/6 2/6
2/6 0/6
significant difference between the CaP and control groups in the tibial bone tunnel (APD: 23.6± 13.2% vs. 38.2 ± 23.9%, p = 0.1095, MLD: 23.6± 15.1% vs. 36.9± 28.1%, p = 0.1647, CSA: 66.3± 23.8% vs. 113.6 ± 73.4%, p = 0.0823). In the CaP group, the average percentage of bone-tunnel enlargement of APD and CSA in the femoral bone tunnel was significantly
2.3. Histological analysis The results of histological analysis (CaP, n = 6; control, n = 6) are shown in Table 3. In the CaP group, on the femoral and tibial sides at the anterior and posterior surface of the bone tunnel of the joint aperture site, cartilage layers that were approximately 1–2 mm in length and 100–800 μm in thickness containing glycosaminoglycan stained by safranin-O were observed (Fig. 3). At the joint aperture site, cartilage layers between the tendon and the bone were observed at the anterior surface of the femoral bone tunnel in one of six specimens, at the posterior surface of the femoral bone tunnel in two of six specimens, at the anterior surface of the tibial bone tunnel in four of six specimens and at the posterior surface of the tibial bone tunnel in two of six specimens.
Fig. 3. Histological sections of CaP group. This area is at the posterior surface of bone tunnel of the joint aperture site in the tibia. (a) H-E staining (x 100), (b) safranin-O staining (x 100), and (c) MT staining (x 100). T = tendon graft, B = bone, C = cartilage tissue. Cartilage layer with red-stained glycosaminoglycan was observed between the grafted tendon and the bone.
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Fig. 4. Histological sections of the control group. This area is at the posterior surface of bone tunnel of the aperture site in the tibia. (a) H-E staining (x 100), (b) safranin-O staining (x 100), and (c) MT staining (x 100). T = tendon graft, B = bone, F = fibrous tissue. Fibrous tissue between the tendon and the bone was observed.
Table 4 Osteoclast analysis.
CaP group (n = 6) Control (n = 6)
Femur (number / mm)
Tibia (number / mm)
0.29 ± 0.12 * 1.68 ± 1.47
0.43 ± 0.72 0.74 ± 0.51
p = 0.0221). The number of osteoclasts per millimetre in the interface of the tibial bone tunnel in the CaP group (0.43 ± 0.72) was not significantly different from that in the control group (0.74 ± 0.51, p = 0.2088). There was no significant difference between the femoral and tibial side in each group (p = 0.3264 in the CaP group and p = 0.0525 in the control group, respectively). Osteoclasts were scattered in the interface in the CaP group (Fig. 5(a)). However, osteoclasts in the control group clustered in the interface (Fig. 5(b)).
Results are the mean ± SD. * p b 0.05 compared with control values. # p b 0.05 compared with tibial values.
3. Discussion In the control group, a fibrous connective tissue between the grafted tendon and the bone interface was observed by H–E and MT staining (Fig. 4). No cartilage layer at the interface was observed at the anterior or posterior surface of the femoral bone tunnel, or at the posterior surface of the tibial bone tunnel. At the anterior surface of the tibial bone tunnel in two of six specimens, a cartilage layer at the interface was observed. Cartilage layers formed at the interface near the joint in the CaP group were larger than those in the control group (p = 0.0416). Cartilage layer formation was observed on the tibial side more frequently than on the femoral side. The results of osteoclast analysis (CaP, n = 6; control, n = 6) are shown in Table 4. The number of osteoclasts per millimetre in the interface of the femoral bone tunnel in the CaP group (0.29 ± 0.12) was smaller than that in the control group (1.68 ± 1.47,
Bone-tunnel enlargement is observed particularly on the femoral side in conventional ACL reconstruction [10]. The width of the tendon–bone interface is greater at the intra-articular aperture than at the tunnel exit, particularly in the femoral tunnel – using a rabbit ACL reconstruction model [11,12]. Osteoclasts were frequently found at the femoral tunnel apertures. In our study, bone-tunnel enlargement and the number of osteoclasts on the femoral side in the case of using the CaP-hybridised tendon graft for ACL reconstruction at 6 months reduced compared with that in the case of using the
Fig. 5. Immunohistological sections (cathepsin K staining) of the interface between grafted tendon and bone (x 400). T = tendon graft, B = bone. (a) CaP-hybridized tendon graft. Only one osteoclast (arrow) was observed in the interface. (b) Untreated tendon graft. Osteoclasts (arrows) clustered in the interface.
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untreated graft. Reducing bone-tunnel enlargement using the CaPhybridised tendon graft can be achieved owing to inhibition of the appearance of osteoclasts. ACL insertion consists of four distinguishable tissue layers of transition, that is, ligaments, fibrocartilage, mineralised fibrocartilage and bone [19,20]. The cartilaginous layers function as stress or shock brakes by reducing the stiffness gradient between the ligament and the bone [21,22]. In the CaP group, a cartilage layer at the interface on the femoral and tibial sides at the anterior and posterior surface of the bone tunnel of the joint aperture site was observed. However, the cartilage layer was observed less frequently in the femoral side than in the tibial side. In the CaP group, the APD and CSA in the tibial bone tunnel were significantly smaller than those of the femoral bone tunnel. Therefore, the cartilage layer between the tendon and bone at the joint aperture site after ACL reconstruction may prevent bone tunnel enlargement to absorb the stress between the grafted tendon and bone. We considered that tensile stress and/or compressive stress at the interface can promote the formation of the cartilage layer after direct bonding with the bone in the CaP group [14–16]. On the other hand, shear stress between the grafted tendon and the bone associated with the bungee effect [23] can form a fibrous tissue at the interface in the control group, as similarly observed in previous studies [4,14]. The cartilage layers were observed at the anterior tibial surface of the bone tunnel in the control group. The compressive and/or tensile stress may act at this area during tendon–bone healing even in the control group. In our study, the mechanical test results were not significantly different between both groups. The failure mode in the CaP and control groups was always tendon midsubstance rupture, as shown by histological observation. We could not determine which interface was strong in the CaP group or the control group. The weakest link of the replacement graft begins to shift from the soft-tissue–bone interface to the midsubstance of the graft at 6 weeks [16]. For maturation of the graft, appropriate tensile stress may be required. However, the technique of ACL reconstruction in an animal model is less accurate than that in humans, for example, graft placement, initial tension and multiple bundle graft. Therefore, the graft maturation in an animal model may be slower than that in humans after tendon–bone healing. A limitation of this study is that it is unclear whether the appearance of osteoclasts is the cause or effect of bone-tunnel enlargement. Further study is needed to understand the mechanism of bone resorption. Moreover, an investigation into the relationship between bone-tunnel enlargement and knee instability is necessary. Examination of non-significant results of this study may be necessary to check for possible false negatives due to the small number of samples. If we use a larger number of specimens, a clearer significant difference may have appeared. In conclusion, the CaP-hybridised tendon graft reduced bonetunnel enlargement in the femoral side associated with tendon–bone healing with cartilage layer and decreasing number of osteoclasts at the tendon–bone interface 6 months after ACL reconstruction in goats. Clinically, the CaP-hybridised tendon graft for ACL reconstruction can reduce bone-tunnel enlargement. 4. Conflict of Interest Statement No author has received any financial benefit for this study.
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Contents lists available at ScienceDirect
The Knee
Calcium phosphate-hybridised tendon graft to reduce bone-tunnel enlargement after ACL reconstruction in goats Hirotaka Mutsuzaki a, Masataka Sakane b,⁎, Hiromi Nakajima c, Naoyuki Ochiai b a
Department of Orthopaedic Surgery, Ibaraki Prefectural University of Health Sciences, 4669–2 Ami Ami-machi, Inashiki-gun, Ibaraki 300–0394, Japan Department of Orthopaedic Surgery, Institute of Clinical Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305–8575, Japan c Department of Agriculture, Ibaraki University, 3-21-1 Chuuou, Ami, Ibaraki 300–0393, Japan b
a r t i c l e
i n f o
Article history: Received 22 September 2010 Received in revised form 9 March 2011 Accepted 24 March 2011 Keywords: Anterior cruciate ligament Tendon-bone healing Calcium phosphate hybridisation Bone tunnel enlargement Osteoclast
s u m m a r y Bone-tunnel enlargement can have a negative impact on long-term clinical success. To solve the problem, we developed a novel technique to improve tendon–bone healing by hybridising calcium phosphate (CaP) with a tendon graft using an alternate soaking process. The objective of this study was to analyse bone-tunnel enlargement, mechanical properties and histological features, especially the number of osteoclasts at the tendon–bone interface using a CaP-hybridised tendon graft and an untreated tendon graft 6 months after anterior cruciate ligament (ACL) reconstruction in goats. The percentage of bone–tunnel enlargement for the CaP group was decreased compared with that for the control group for the femoral side (p b 0.05). The failure load was not statistically different between the CaP group and the control group, and was all midsubstance rupture for both groups. In the CaP group, cartilage layer was more observed at the tendon–bone interface of the joint aperture site than in the control group (p b 0.05). Many osteoclasts on the femoral side of the tendon– bone interface in the control were observed compared with that in the CaP group (p b 0.05). At the femoral side, the CaP-hybridised tendon graft reduced bone-tunnel enlargement associated with tendon–bone healing 6 months after ACL reconstruction in goats. Clinically, the CaP-hybridised tendon graft for ACL reconstruction can reduce bone-tunnel enlargement. © 2011 Elsevier B.V. All rights reserved.
It is well established that injury to the anterior cruciate ligament (ACL) results in knee instability and functional disability. As a result, these injuries frequently require ligament reconstruction using a tendon graft [1–3]. However, a tendon graft requires soft-tissue-to-bone healing within both bone tunnels. Grana et al [4]. evaluated a tendon autograft within bone tunnels in a rabbit ACL reconstruction model. An indirect tendon insertion with fibrous connective tissue was observed at the tendon–bone interface. Moreover, it has frequently been shown that bone-tunnel enlargement occurs following ACL reconstruction using soft-tissue grafts [5–9]. Increased knee laxity in correlation with radiographic femoral tunnel enlargement in ACL reconstructions using soft-tissue grafts has been demonstrated [10]. Moreover, enlargement of the articular end of the bone tunnels owing to bone resorption associated with osteoclasts is a common problem in ACL reconstruction [11,12]. We considered that firm tendon–bone healing near the joint and prevention of bone-tunnel enlargement can be important for good clinical outcome of ACL reconstruction. Therefore, we developed a novel technique to improve tendon–bone attachment by hybridising calcium phosphate (CaP) with tendons using an alternate soaking process [13].
⁎ Corresponding author. Tel.: + 81 29 853 3219; fax: + 81 29 853 3214. E-mail address: [email protected] (M. Sakane). 0968-0160/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.knee.2011.03.008
Using the CaP-hybridised tendon, we observed a scarless direct bonding area between the grafted tendon and the newly formed bone without inflammatory reaction 2–3 weeks after ACL reconstruction in rabbits [14,15], which we also observed in goats 6 weeks after the reconstruction [16], owing to its osteoconductive potential and affinity for living tissues. However, long-term results regarding bone-tunnel enlargement and the interface between the CaP-hybridised tendon and the bone for ACL reconstruction are unclear. We used a goat model of ACL reconstruction to solve these problems because long-term studies using goat knees have shown effective restoration of knee stability and minimal articular cartilage degeneration after ACL reconstruction [17,18]. We hypothesise that in the case of using CaP-hybridised tendon grafts, bone-tunnel enlargement associated with osteoclasts at the joint aperture site is minimised owing to improved tendon–bone healing compared with that using untreated tendon grafts 6 months after the operation. Moreover, the mechanical properties at the interface in the CaP group may be superior to those in the control group because of its firm anchoring formation. The objective of this study was to analyse bone-tunnel enlargement, mechanical properties and histological features, especially the number of osteoclasts at the tendon–bone interface using a CaP-hybridised tendon graft and an untreated tendon graft 6 months after ACL reconstruction in goats.
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1. Materials and Methods This study design is a controlled laboratory study. 1.1. CaP hybridisation method Twelve skeletally mature female Saanen breed goats (6 months of age) were used in this study. Conditions were the same as in our previous study [16]. The goats were maintained in accordance with the guidelines of the Ethical Committee of the Biomaterial Center of the National Institute for Materials Science and the National Institutes of Health for the care and use of laboratory animals (NIH Pub. No. 85–23 Rev. 1985). The CaP hybridisation method was the same as in previous studies [14–16]. The flexor digitorum longus (FDL) tendons and hamstring tendons were used as tendon grafts. Double-ruple-strand tendons of 45-mm length and 6.5-mm diameter were prepared. The tibial end of the grafts was secured with a non-absorbable suture, and a polyester tape suture tied over the EndoButton® (Smith & Nephew, USA) was passed through the looped femoral end of the grafts. Then, the central third of the grafts at the intra-articular portion were covered with the sleeve of a rubber glove tied on each side to prevent CaP hybridisation [16]. After these procedures, the grafts were soaked in 100 ml of a Ca solution (100 mM CaCl2 + 30 mM L-histidine, pH 7.4, 25 °C) for 30 s. The grafts were subsequently soaked in 100 ml of a NaHPO4 solution (116.4 mM NaH2PO4:128.7 mM Na2HPO4 12H2O = 15%:85%, pH 7.4, 25 °C) for 30 s. Before each soaking, the grafts were washed in a saline solution. This cycle was repeated 10 times [14–16]. In the control group, the tendon was soaked in a saline solution for 10 min. 1.2. Surgical procedure The surgical procedures were the same as in our previous study [16]. All the surgical procedures were performed under aseptic conditions with the animals under general anaesthesia. On the right knee, an anterior lateral skin incision was made. We exposed the knee joint by the lateral para-patella approach. ACL was then completely transected, and gross anterior subluxation of the tibia was confirmed by manual examination. The tibial and femoral tunnels were drilled to form holes of 6.5 mm diameter on the tibial and femoral insertions of the ACL. The length of the tunnel was at least 20 mm. The graft described above was passed through the femoral and tibial tunnels. The graft fixation was achieved via the EndoButton® at the femoral side and a 4.5-mmdiameter cortex screw (MEIRA Corporation, Nagoya, Japan) at the tibial side with 20 N as the initial tension (Fig. 1) [16]. After reconstruction, we sutured the capsule and skin wound. Postoperatively, all the goats were allowed free activity in their cages (cage area, 50 m2) without receiving any antibiotics. We did not perform daily wound dressing. Visual inspection revealed that normal gait patterns did not return until 3–4 weeks after surgery. The goats were sacrificed (1 ml of sodium pentobarbital per 4 kg goat mass administered intravenously) exactly 6 months after the surgery. The hind limbs were disarticulated at the hip joint. A total of six specimens each from the CaP and control groups were obtained. The six specimens in each group were sealed in double plastic bags and immediately stored at − 20 °C until use in biomechanical analysis. 1.3. Ex vivo computed tomography (CT) evaluation The six frozen specimens from each group were used for computed tomography (CT) evaluation. CT scans of the specimens were performed (Brilliance CT 64, Philips, Amsterdam, the Netherlands) to assess femoral and tibial bone tunnels. The CT scans (voltage: 120 kV, current: 230 mA) in the full-extension knee position were supervised and examined by a single radiologist. A standard protocol was used consistently throughout the study. Initial volume acquisition was with 0.9 mm cuts from 30 mm above the femoral tunnel to 30 mm below the
Fig. 1. ACL reconstruction. The graft (black arrow) was pulled taut through the drill hole with both ends fixed at the distal part of the femur (EndoButton) and proximal part of the tibia (screw).
tibial tunnel. Using a Virtual Place Lexus (AZE Ltd., Tokyo, Japan) workstation, three-dimensional images were reconstructed. Coronal, sagittal and axial images of the femoral and tibial bone tunnels were obtained. We measured the anterior–posterior diameter (APD), medial– lateral diameter (MLD) and tunnel cross-sectional area (CSA) of the femur and tibia at the main joint aperture site after sacrifice (6 months APD, MLD or CSA) (Fig. 2). The percentage of bone-tunnel enlargement in tunnel APD, MLD and CSA was calculated using the following formula: Percentage of bone−tunnel enlargement of APD; MLD; or CSAð%Þ = ð6 months APD; MLD or CSA– Original APD; MLD or CSAÞ × 100 = Original APD; MLD or CSA: Original APD and MLD are the diameters of the drill using in the operation. Original CSA is the calculated area of CSA based on the diameter. 1.4. Biomechanical analysis After CT evaluation, biomechanical analysis was performed. Before biomechanical testing, each specimen was thawed for 24 h at room temperature. The tibia and femur were cut 20 cm from the knee joint line, and surrounding tissue except the grafted tendon was removed. The cross-sectional area of the graft at the joint line was measured using an apparatus for measuring the cross-sectional area of the grafted tendon (Jintai Danmenseki Sokuteiki, Meira Corporation, Nagoya, Japan). For biomechanical testing, the experimental femur– ACL graft–tibia complexes were then fixed to custom-designed clamps that allowed tensile loading along the long axis of the graft on a material testing machine (AGS-N 1kN, Shimadzu Corporation, Kyoto, Japan). To ensure that we were determining the tensile properties of the graft and its interface within the tunnels, the additional fixation devices (EndoButton and fixation screw) were removed. After a 3 N preload was applied, load-to-failure testing at an elongation rate of 30 mm min–1 was performed. The load–elongation curves of the experimental femur–ACL graft–tibia complexes were
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Fig. 2. CT images. (a) Coronal image of femoral bone tunnel. Medial-lateral diameter (MLD: black arrow) was measured. (b) Sagittal image of femoral bone tunnel. Anterior-posterior diameter (APD: black arrow) was measured. (c) Axial image of femoral bone tunnel. Tunnel cross-sectional area (CSA) was measured. (d) Coronal image of tibial bone tunnel. Medial-lateral diameter (MLD: black arrow) was measured. (e) Sagittal image of tibial bone tunnel. Anterior-posterior diameter (APD: black arrow) was measured. (f) Axial image of tibial bone tunnel. Tunnel cross-sectional area (CSA) was measured.
recorded. Stiffness (N mm–1) was calculated from the slope of the linear region between 1 and 2.5 mm of elongation. Failure load was divided by the cross-sectional area of the graft at the joint line of each specimen as the value of stress [16].
1.5. Histological analysis After the biomechanical analysis, histological evaluations were performed. The tendon–bone interface was completely intact after the biomechanical analysis, as all the specimens failed in the tendon midsubstance on histological observation. Therefore, we were able to perform histological analysis at the tendon–bone interface in the femoral and tibial side in both groups. The specimens were fixed in 10% neutralbuffered formalin, decalcified and embedded in paraffin. The specimens were sliced 5-μm-thick sagittal to the bone tunnel. Haematoxylin and eosin (H–E) staining, safranin-O staining for identification of the cartilage layer in the interface, Masson's trichrome (MT) staining for identification of collagenous fibre tissue and cathepsin K immunostaining (cathepsin K mouse monoclonal antibody; Novocastra Laboratories Ltd., UK) for identification of osteoclasts were performed. The specimens were examined by light microscopy after staining. Histological comparisons of the tendon–bone interface anterior and posterior bone tunnel surface at the joint aperture site (cartilaginous insertion or fibrous insertion) in the femoral and tibial side were carried out between the CaP group and the control group. They were blinded by an orthopaedic surgeon. To determine the failure modes at 6 months, microscopy observations were performed after the biomechanical test. The failure modes (pull out or midsubstance rupture) of the experimental femur– ACL graft–tibia complexes were recorded.
Using a Mac Scope program (Mitani Co., Japan) on a Macintosh computer, the numbers of osteoclasts in the tendon–bone interface at the femoral and tibial sides at the joint aperture site (10 mm from the joint line) were measured. Next, the obtained numbers of osteoclasts were divided by the length of each femoral and tibial bone tunnel of each specimen to correct the values. We defined the corrected value of the number of osteoclasts as the number of osteoclasts per millimetre. 1.6. Statistical Analyses The bone-tunnel enlargement data (APD, MLD and CSA), biomechanical data (ultimate failure load, stiffness and stress), and osteoclast data of the two groups were compared using the Student's t-test at a p b 0.05 significance value. Concerning the bone-tunnel enlargement data and the osteoclast data in each group, statistical analysis of differences between the femoral side and tibial side were performed using the Student's t-test at a p b 0.05 significance value. To compare the histological difference between the CaP and control groups, Mann– Whitney's U test was used at a p b 0.05 significance value. 2. Results 2.1. CT evaluation The bone-tunnel enlargement rates obtained using CT (CaP group, n = 6; control group, n = 6) are shown in Table 1. The average percentage of bone-tunnel enlargement in the six femoral bone-tunnel APD for the untreated tendon graft (53.8± 16.1%) was significantly greater than that of the CaP-hybridised tendon graft (41.0 ± 6.6%, p = 0.0488). The average percentage of bone-tunnel enlargement of MLD in the femoral side for the untreated tendon graft (53.8± 18.4%) was also significantly greater than that of the CaP-hybridised tendon graft (30.8 ± 23.3%, p = 0.0431). The average percentage of bone-tunnel enlargement of CSA in the femoral side was greater in the control group (170.4 ± 54.8%) than in the CaP group (118.1 ± 20.7%, p = 0.0269). However, there was no
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Table 1 CT evaluation: percentage of bone tunnel enlargement. Femoral side
CaP group (n = 6) Control (n = 6)
Tibial side
APD(%)
MLD(%)
CSA(%)
APD(%)
MLD(%)
CSA(%)
41.0 ± 6.6 * # 53.8 ± 16.1
30.8 ± 23.3 * 53.8 ± 18.4
118.1 ± 20.7 * # 170.4 ± 54.8
23.6 ± 13.2 38.2 ± 23.9
23.6 ± 15.1 36.9 ± 28.1
66.3 ± 23.8 113.6 ± 73.4
APD: anterior-posterior diameter, MLD: medial-lateral diameter, CSA: cross-sectional area. Results are the mean ± SD. * p b 0.05 compared with control values. # p b 0.05 compared with tibial values.
Table 2 Biomechanical results.
CaP group (n = 6) Control (n = 6)
Ultimate failure load (N)
Stiffness (N/mm)
Stress (N/mm2)
Failure mode (tendon midsubstance: pulled out)
562.3 ± 144.1
50.5 ± 15.0
13.7 ± 2.3
6:0
575.4 ± 115.1
43.5 ± 13.7
15.2 ± 2.4
6:0
Histological observation
2.2. Biomechanical analysis All the specimens failed in the tendon midsubstance on histological observation. The results of biomechanical analysis (CaP, n = 6; control, n = 6) are shown in Table 2. No significant difference in the ultimate failure load between the CaP-hybridised tendon graft (562.3 ± 144.1 N) and the control graft (575.4± 115.1 N) was found (p = 0.4312). Moreover, no significant difference in stiffness between the CaP-hybridised tendon graft (50.5± 15.0 N mm–1) and the control graft (43.5 ± 13.7 N mm–1) was found (p = 0.2095). No significant difference in the stress between the CaP-hybridised tendon graft (13.7 ± 2.3 N mm–2) and the control graft (15.2 ± 2.4 N mm–2) was found (p = 0.1430).
Results are the mean ± SD.
Table 3 Histological observation (cartilage layer formation).
CaP group (n = 6) Control (n = 6)
greater than those of the tibial bone tunnel (p = 0.0199 and p = 0.0015, respectively). However, there was no significant difference between the femoral and tibial side in the MLD in the CaP group, and the APD, MLD and CSA in the control group (p = 0.2634, p = 0.0615, p = 0.1202 and p = 0.0799, respectively).
Femur (number / 6)
Tibia (number / 6)
Anterior
Posterior
Anterior
Posterior
1/6 0/6
2/6 0/6
4/6 2/6
2/6 0/6
significant difference between the CaP and control groups in the tibial bone tunnel (APD: 23.6± 13.2% vs. 38.2 ± 23.9%, p = 0.1095, MLD: 23.6± 15.1% vs. 36.9± 28.1%, p = 0.1647, CSA: 66.3± 23.8% vs. 113.6 ± 73.4%, p = 0.0823). In the CaP group, the average percentage of bone-tunnel enlargement of APD and CSA in the femoral bone tunnel was significantly
2.3. Histological analysis The results of histological analysis (CaP, n = 6; control, n = 6) are shown in Table 3. In the CaP group, on the femoral and tibial sides at the anterior and posterior surface of the bone tunnel of the joint aperture site, cartilage layers that were approximately 1–2 mm in length and 100–800 μm in thickness containing glycosaminoglycan stained by safranin-O were observed (Fig. 3). At the joint aperture site, cartilage layers between the tendon and the bone were observed at the anterior surface of the femoral bone tunnel in one of six specimens, at the posterior surface of the femoral bone tunnel in two of six specimens, at the anterior surface of the tibial bone tunnel in four of six specimens and at the posterior surface of the tibial bone tunnel in two of six specimens.
Fig. 3. Histological sections of CaP group. This area is at the posterior surface of bone tunnel of the joint aperture site in the tibia. (a) H-E staining (x 100), (b) safranin-O staining (x 100), and (c) MT staining (x 100). T = tendon graft, B = bone, C = cartilage tissue. Cartilage layer with red-stained glycosaminoglycan was observed between the grafted tendon and the bone.
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Fig. 4. Histological sections of the control group. This area is at the posterior surface of bone tunnel of the aperture site in the tibia. (a) H-E staining (x 100), (b) safranin-O staining (x 100), and (c) MT staining (x 100). T = tendon graft, B = bone, F = fibrous tissue. Fibrous tissue between the tendon and the bone was observed.
Table 4 Osteoclast analysis.
CaP group (n = 6) Control (n = 6)
Femur (number / mm)
Tibia (number / mm)
0.29 ± 0.12 * 1.68 ± 1.47
0.43 ± 0.72 0.74 ± 0.51
p = 0.0221). The number of osteoclasts per millimetre in the interface of the tibial bone tunnel in the CaP group (0.43 ± 0.72) was not significantly different from that in the control group (0.74 ± 0.51, p = 0.2088). There was no significant difference between the femoral and tibial side in each group (p = 0.3264 in the CaP group and p = 0.0525 in the control group, respectively). Osteoclasts were scattered in the interface in the CaP group (Fig. 5(a)). However, osteoclasts in the control group clustered in the interface (Fig. 5(b)).
Results are the mean ± SD. * p b 0.05 compared with control values. # p b 0.05 compared with tibial values.
3. Discussion In the control group, a fibrous connective tissue between the grafted tendon and the bone interface was observed by H–E and MT staining (Fig. 4). No cartilage layer at the interface was observed at the anterior or posterior surface of the femoral bone tunnel, or at the posterior surface of the tibial bone tunnel. At the anterior surface of the tibial bone tunnel in two of six specimens, a cartilage layer at the interface was observed. Cartilage layers formed at the interface near the joint in the CaP group were larger than those in the control group (p = 0.0416). Cartilage layer formation was observed on the tibial side more frequently than on the femoral side. The results of osteoclast analysis (CaP, n = 6; control, n = 6) are shown in Table 4. The number of osteoclasts per millimetre in the interface of the femoral bone tunnel in the CaP group (0.29 ± 0.12) was smaller than that in the control group (1.68 ± 1.47,
Bone-tunnel enlargement is observed particularly on the femoral side in conventional ACL reconstruction [10]. The width of the tendon–bone interface is greater at the intra-articular aperture than at the tunnel exit, particularly in the femoral tunnel – using a rabbit ACL reconstruction model [11,12]. Osteoclasts were frequently found at the femoral tunnel apertures. In our study, bone-tunnel enlargement and the number of osteoclasts on the femoral side in the case of using the CaP-hybridised tendon graft for ACL reconstruction at 6 months reduced compared with that in the case of using the
Fig. 5. Immunohistological sections (cathepsin K staining) of the interface between grafted tendon and bone (x 400). T = tendon graft, B = bone. (a) CaP-hybridized tendon graft. Only one osteoclast (arrow) was observed in the interface. (b) Untreated tendon graft. Osteoclasts (arrows) clustered in the interface.
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untreated graft. Reducing bone-tunnel enlargement using the CaPhybridised tendon graft can be achieved owing to inhibition of the appearance of osteoclasts. ACL insertion consists of four distinguishable tissue layers of transition, that is, ligaments, fibrocartilage, mineralised fibrocartilage and bone [19,20]. The cartilaginous layers function as stress or shock brakes by reducing the stiffness gradient between the ligament and the bone [21,22]. In the CaP group, a cartilage layer at the interface on the femoral and tibial sides at the anterior and posterior surface of the bone tunnel of the joint aperture site was observed. However, the cartilage layer was observed less frequently in the femoral side than in the tibial side. In the CaP group, the APD and CSA in the tibial bone tunnel were significantly smaller than those of the femoral bone tunnel. Therefore, the cartilage layer between the tendon and bone at the joint aperture site after ACL reconstruction may prevent bone tunnel enlargement to absorb the stress between the grafted tendon and bone. We considered that tensile stress and/or compressive stress at the interface can promote the formation of the cartilage layer after direct bonding with the bone in the CaP group [14–16]. On the other hand, shear stress between the grafted tendon and the bone associated with the bungee effect [23] can form a fibrous tissue at the interface in the control group, as similarly observed in previous studies [4,14]. The cartilage layers were observed at the anterior tibial surface of the bone tunnel in the control group. The compressive and/or tensile stress may act at this area during tendon–bone healing even in the control group. In our study, the mechanical test results were not significantly different between both groups. The failure mode in the CaP and control groups was always tendon midsubstance rupture, as shown by histological observation. We could not determine which interface was strong in the CaP group or the control group. The weakest link of the replacement graft begins to shift from the soft-tissue–bone interface to the midsubstance of the graft at 6 weeks [16]. For maturation of the graft, appropriate tensile stress may be required. However, the technique of ACL reconstruction in an animal model is less accurate than that in humans, for example, graft placement, initial tension and multiple bundle graft. Therefore, the graft maturation in an animal model may be slower than that in humans after tendon–bone healing. A limitation of this study is that it is unclear whether the appearance of osteoclasts is the cause or effect of bone-tunnel enlargement. Further study is needed to understand the mechanism of bone resorption. Moreover, an investigation into the relationship between bone-tunnel enlargement and knee instability is necessary. Examination of non-significant results of this study may be necessary to check for possible false negatives due to the small number of samples. If we use a larger number of specimens, a clearer significant difference may have appeared. In conclusion, the CaP-hybridised tendon graft reduced bonetunnel enlargement in the femoral side associated with tendon–bone healing with cartilage layer and decreasing number of osteoclasts at the tendon–bone interface 6 months after ACL reconstruction in goats. Clinically, the CaP-hybridised tendon graft for ACL reconstruction can reduce bone-tunnel enlargement. 4. Conflict of Interest Statement No author has received any financial benefit for this study.
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