Cytotherapy, 2014; 16: 857e867

Enhancement of tendon-bone healing with the use of bone morphogenetic protein-2 inserted into the suture anchor hole in a rabbit patellar tendon model

JAE GYOON KIM1,*, HAK JUN KIM2,*, SUNG EUN KIM3,*, JI HOON BAE1, YOU JIN KO1 & JUNG HO PARK1 1

Department of Orthopedic Surgery, Korea University College of Medicine, Ansan Hospital, Ansan, Gyeonggi-do, South Korea, 2Department of Orthopedic Surgery, Korea University College of Medicine, Guro Hospital, Seoul, South Korea, and 3Department of Orthopedic Surgery, Rare Disease Institute, Guro Hospital, Seoul, South Korea Abstract Background aims. Suture anchor fixation failure has been reported as a result of anchor loosening and migration during the tendon-bone repair. The aim of this study was to evaluate the effects of bone morphogenetic protein-2 (BMP-2) inserted into the suture anchor hole on bone formation and the tendon-bone healing. Methods. Both back legs of 24 New Zealand White rabbits (n ¼ 48) were used in this study. A metal suture anchor was then placed 5 mm below the cortex. In the control group, the space over the eyelet of the anchor (suture anchor hole) was not filled. In the experimental group, the suture anchor hole was filled with 0.1 mL of fibrin glue (group 2) or collagen gel (group 3) with 1 mg BMP-2. Histologic analysis, real-time-polymerase chain reaction, bone density and failure load measurement were performed, and differences were analyzed at 4 and 8 weeks. Results. Histologic analysis revealed more abundant new bone, mature bone and organized fibrocartilage at the tendon-bone interface at 4 and 8 weeks in groups in which BMP-2 was applied. At 8 weeks, the failure load of groups 1, 2 and 3 was significantly different among the three groups (P ¼ 0.01). After post hoc Tukey test, the failure load of group 2 was significantly higher than that of group 1 (P ¼ 0.01). Conclusions. BMP-2, administrated as described in this study, improved tendon-bone healing and bone formation, resulting in improved biomechanical strength of the tendonbone junction. Key Words: bone morphogenetic protein-2, collagen gel, fibrin glue, patellar tendon, suture anchor

Introduction The attachment of tendons to bone remains an integral aspect of orthopedic surgery (1). For the attachment of tendons or ligaments to bone, surgeons previously relied on the pullout suture technique, keyhole technique, staples and fixation with screws and washers (2). The development of suture anchors revolutionized soft-tissue fixation to bone and enabled the use of arthroscopic techniques in the attachment of tendon to bone. Suture anchors can be used in many procedures including rotator cuff repair, biceps tenodesis, flexor tendon repair and patellar tendon rupture (1,3,4). The ideal suture anchor should provide adequate primary strength to permit rehabilitation exercise during tendon-bone healing (5). Rotator cuff tears are more prevalent among elderly patients; therefore, the rotator cuff

tear is often associated with osteoporosis in the proximal humerus (6). Djurasovic et al. (7) reported that failure occurred in 10% of cases as a result of anchor loosening or migration. In some cadaver studies, authors reported that pull-out strength was strongly associated with bone mineral density (8,9). Therefore, if early bone healing around suture anchor occurred, it would strengthen pull-out strength of the suture anchor and prevent early failure of suture anchor fixation. To improve the biological tendon-bone healing, several trials have been performed with the application of bone morphogenetic protein-2 (BMP-2), demineralized bone matrix, platelet-rich plasma, low-intensity pulsed ultrasound, tricalcium phosphate, transforming growth factor-b1, and specific suture anchors made of collagen (10e16).

*These authors contributed equally to this work as co-first authors. Correspondence: Jung Ho Park, MD, Department of Orthopedic Surgery, Korea University College of Medicine, Ansan Hospital, 123, Jeokgeum-ro, Danwon-Gu, Ansan-si, Gyeongki-do, 425e707, Korea. E-mail: [email protected] (Received 2 August 2013; accepted 26 December 2013) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.12.012

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BMP-2 induces bone formation by regulating the recruitment and differentiation of osteoprogenitor cells (17,18). In addition, BMP-2 enhances the tendon-bone healing process and increases the biomechanical strength of tendon-bone junctions (11,19). However, BMP-2 has a short half-life and is structurally unstable and thus requires a high dose administration, which can cause various adverse effects such as extensive ectopic ossification, immune reactions, softtissue swelling, seroma formation and paradoxical osteolysis (20,21). Most of the previous studies aimed at improving tendon-bone healing through the use of suture anchors have ignored the suture anchor hole. To the best of our knowledge, only a few studies have investigated the suture anchor hole for fixation of the suture anchor (10,22). Giory et al. (22) suggested that injection of polymethyl methacrylate into the suture anchor hole before the placement of the anchor increases pull-out strength and the number of cycles to failure (22). Oshtory et al. (10) also suggested that tricalcium phosphate cement augmentation at the anchor hole increases the final load to failure. However, these studies were focused on increasing the pull-out strength of the suture anchor rather than improving tendon-bone healing. We assumed that BMP-2, with an appropriate carrier inserted into the suture anchor hole, would accelerate both the tendon-bone healing and bone formation at the suture anchor hole, which would also strengthen biomechanical strength of tendonbone junction and pull-out strength of the suture anchor. The aim of this study was to evaluate the effect of BMP-2 inserted into the suture anchor hole on tendonbone healing and bone formation. The hypothesis of this study was that the use of BMP-2 inserted into the suture anchor hole would accelerate tendon-bone healing and bone formation.

Methods Experimental design All experimental procedures with animals were performed under the guidelines for animal scientific procedures approved by institution’s ethics committee (KUIACUC-20110908-1). Twenty-four skeletally mature male New Zealand White rabbits (age, 13e15 weeks; mean body weight, 3.2  0.2 kg) were used in this study. National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH publication No. 82e23, revised 1985) were observed throughout the study. Both back legs were used in each rabbit

(n ¼ 48). Rabbits were randomly assigned to one of three groups through the use of permuted block randomization: group 1 was an untreated group without BMP-2 insertion (control group, n ¼ 16), group 2 was treated with a mixture of fibrin glue and BMP-2 (n ¼ 16) and group 3 (n ¼ 16) was treated with a mixture of collagen gel and BMP-2 before tendon reattachment. Each group was divided equally into 4- and 8-week subgroups (n ¼ 8 at each week). Surgical procedure Rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg) (Yuhan, Seoul, Korea) and xylazine (23.3 mg/kg) (Bayer, Leverkusen, Germany). Both limbs were then shaved and aseptically prepared for surgery. A longitudinal skin incision was made over the prepatellar area of the knee and exposed patella, the patellar tendon and the tibial tuberosity. The patellar tendon was then transected at its insertion on the tibial tuberosity. Next, the tibial tuberosity was denuded of all tendon, insertion fibrocartilage and periosteum with a high-speed burr without injury to the bone. One 4.5-mmediameter hole was then drilled into the center of the tibial tuberosity and flushed with sterile saline to ensure that it was clear of debris. Because the patellar tendon of rabbits is smaller than the rotator cuff tendon of humans, the original thick suture was removed from the eyelet of the 5-mm metal anchor (Wonnam Medical, Seoul, Korea), which was made of stainless steel, and reinserted a thinner and stronger Fiberwire suture 3-0 (Arthrex, Naples, FL, USA), allowing us to make a smaller knot. During insertion of the anchors, the anchor eyelet was placed 5 mm below the cortical surface, and four stitches were applied to the tendon insertion. Before the suture was tied, the space over the eyelet of the anchor (suture anchor hole) was filled with fibrin glue (Greenplast, Greencross Co, Yongin, Korea) [fibrinogen solution (100 mg/mL) containing thrombin (500 IU/mL)] and BMP-2 for group 2 and with collagen gel [collagen type I (3 mg/ mL), rat tail (Gibco, Life Technologies, Grand Island, NY, USA)] and BMP-2 for group 3. Among the carriers of BMP-2, collagen and fibrin have been effective in tendon-bone healing in animal studies. In addition, collagen can be made into a gel type, and the collagen gel and fibrin glue are easy to manipulate and can be formed to fit the small size of the suture anchor hole. Therefore, we chose these two carriers. The sutures were then tied, and the patellar tendon was reattached (Figure 1). Before insertion of BMP-2, 0.1 mL of fibrin glue or collagen gel was mixed with 1 mg of BMP-2 (total concentration,

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Figure 1. (A) The 5-mm metal anchor loaded with a Fiberwire suture 3e0. (B) The patellar tendon was transected at its insertion on the tibial tuberosity. The suture anchor was inserted after drilling one 4.5-mm-diameter hole at the center of the tibial tuberosity. (C) Illustration summarizes the experimental protocol. An anchor was placed 5 mm below the cortical surface, and four stitches were applied to insert the tendon. The space over the eyelet of the anchor (suture anchor hole) was then filled with fibrin glue or collagen gel with BMP-2.

10 mg/mL) (R & D Systems, Minneapolis, MN, USA). Intramuscular gentamicin 3 mg/kg (Kunwha, Seoul, Korea) was administered once a day for the first 3 days after surgery, and animals were monitored daily throughout the study. Sample preparation Animals were euthanized at 4 and 8 weeks in a CO2 chamber for 2 minutes. The previous surgical wounds were opened, and the proximal 7 cm of the tibia, patellar tendon, patellae and quadriceps tendon were isolated for evaluation. Specimens were then randomly allocated for each evaluation. For histologic evaluation, we allocated two knees at 4 and 8 weeks in each group. For real-time polymerase chain reaction (PCR), we allocated three knees at 4 and 8 weeks in each group. We also allocated three knees for microecomputed tomography (CT) evaluation; we then performed biomechanical testing with the use of the same knees at 4 and 8 weeks in each group. Histologic analysis Each specimen was fixed in 10% neutral-buffered formalin and was decalcified with the use of CalciClear Rapid (National Diagnostics, Yorkshire, UK) for 1 week before histology processing. The previously inserted anchor was removed from the opposite direction without damaging the suture anchor hole area. The specimen was then dehydrated in a graded ethanol series (Harleco, EMD Chemicals, NJ, USA), cleared with a xylene substitute (Thermo Electron Corporation, OH, USA) and embedded in paraffin for histological examination. Sagittal sections at the anchor insertion area were cut to a thickness of 4 mm and stained with hematoxylin and eosin and Masson trichrome stain. Stained slides were scanned, and individual sections for each sample were analyzed.

Real-time PCR Total RNA was isolated from the tissue samples obtained from the suture anchor hole with the use of TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Total RNA (1 mg) was then converted to complementary DNA with the use of a Maxime RT PreMix kit (iNtRON Biotechnology, Gyeonggi-do, Korea). The real-time PCR reactions were performed with the use of a SYBRGreen reaction kit (Roche Diagnostics, Almere, the Netherlands) according to the manufacturer’s instructions in a LightCycler 480 (Roche Diagnostics). The complementary DNA was used in a volume of 10 mL of PCR Mix (LightCycler DNA Master Fast Start kit; Roche Diagnostics) containing a final concentration of 1 pmol primers. The expression of collagen type I (COLI A2), alkaline phosphatase (ALP) and osteopontin genes were then examined, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as a housekeeping gene. The primer sequences used in the real-time PCR are listed in Table I. Each PCR was processed in triplicate. For real-time PCR, the values of the relative target gene expression were normalized to the geometric means of the relative GAPDH housekeeping gene expression. PCR efficiency was calculated by use Table I. Primers used for real-time PCR.

Gene

Primer sequence

5 -CTGAGAACGGGAAGCTGGT-30 50 -TTGATGTTGGCGGGATCT-30 ALP 50 -AATGAGGCTCTGACCAATGC-30 50 -TCTCCCAGGAAGAGGATGAG-30 Collagen 50 -AGGGAGAGCCTGGTGACAA-30 type I 50 -GAAGACCTTGCAATCCGTTG-30 Osteopontin 50 -ACCGCAGAATGCTATGTCCT-30 50 -GTGGTCATCGTCCTCATCCT-30

GAPDH

0

Product size (in base pairs) 73 77 72 121

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Figure 2. Bone density measurement and biomechanical testing. (A) Region of interest was set at the suture anchor hole. (B) Bone density was measured with the use of a micro-CT system tool. (C) The proximal tibiaepatellar tendonepatellaequadriceps tendon complexes were mounted in the custom jig, and tensile testing was performed with the use of a Shimadzu Universal Testing Machine.

of LightCycler software (Roche Applied Science, Penzberg, Germany). Relative quantification was performed relative to the control group. Quantitative micro-CT evaluation At 4 and 8 weeks after implantation, the bone density of the suture anchor hole was obtained with the use of a micro-CT system (Albira II imaging system, Carestream Health, MA, USA) operated at a voltage of 40 kV. A current of 250 mA was used with a nominal resolution of 9 mm/pixel. We determined the region of interest at the suture anchor hole and measured bone density with the use of a micro-CT system tool (Figure 2). Biomechanical testing Frozen specimens were thawed in normal saline at room temperature before testing. In each case, the proximal tibiaepatellar tendonepatellaequadriceps tendon complexes were mounted in a custom jig, and tensile testing was performed with the use of a Shimadzu Universal Testing Machine (Shimadzu Co, Kyoto, Japan) with a displacement rate of 10 mm/ minute after preloading with 10 N for 1 minute (Figure 2). To compare the biomechanical properties of the reattached tendon with those of the normal bone-tendon junction of the patellar tendon, three normal proximal tibiaepatellar tendonepatellae quadriceps tendon complexes were subjected to the same biomechanical testing. The failure load and mode of failure were then analyzed. Statistical analysis One-way analysis of variance was used to analyze differences in the failure load and total bone volume

among the three groups at 4 and 8 weeks, with statistically significant differences assessed by means of post hoc Tukey test to determine which two of the three groups differed significantly. An independent t test was used to evaluate differences within the groups at different time points at 4 and 8 weeks. All statistical analyses were performed with the use of SAS 9.2 (SAS Inc, Cary, NC, USA). Values are represented as mean  standard deviation, with a significance level of P < 0.05 for all analyses.

Results Histologic analysis For the interpretation of histologic findings, bone tissue that exhibited abundant osteoblastic rimming and a lack of lamellation was defined as “new bone” (23). At 4 weeks in group 1, new bone was more visible, with scant mature bone in the defect area (Figure 3A). The tendon-bone interface was predominantly fibrous, with small interpositional regions of fibrocartilage, although none of the fibrocartilage was mineralized (Figure 3B). In groups 2 and 3, new bone was more abundant compared with group 1, and mature lamellar bone was also visible (Figure 3C,E). The tendon-bone interface was predominantly fibrocartilaginous, with small amounts of mineralized fibrocartilage (Figure 3D,F). In addition, some new bone was also visible at 8 weeks in group 1, whereas mature lamellar bone was more visible than at 4 weeks (Figure 4A) and mineralized fibrocartilage was more visible at the bonetendon interface (Figure 4B). In groups 2 and 3, mature lamellar bone was thicker and more abundant compared with that in group 1 (Figure 4C,E), and large, distinct areas of organized fibrocartilage were observed with chondrocytes in their lacunae oriented in the direction of the collagen fibers (Figure 4D,F).

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Figure 3. Histologic findings of group 1 (A, B), group 2 (C, D) and group 3 (E, F) under a light microscope at 4 weeks (hematoxylin and eosin staining). (A) New bone was more visible than scant mature bone in the defect area (magnification 20). (B) The tendon-bone interface was predominantly fibrous, with small interpositional regions of fibrous cartilage, although none of the fibrous cartilage was mineralized (magnification 80). (C, E) New bone formation was more abundant compared with that in group 1, and mature trabecular bone with lamellation was also visible in the defect area, in contrast to group 1 (magnification 20). (D, F) The tendon-bone interface was predominantly fibrocartilaginous, with small amounts of mineralized fibrocartilage (magnification 80). *Location of suture anchor eyelets.

Masson trichrome staining indicated the presence of collagen-fibril formation in the tendon-bone junction and suture anchor hole during healing. At 4 weeks in group 1, loose collagen fibrils at the tendon-bone junction and suture anchor hole area were visible (Figure 5A). In groups 2 and 3, more concentrated and orderly collagen fibrils were visible in the tendon-bone junction, and more abundant collagen fibrils were visible at the suture anchor hole area compared with those in group 1 (Figure 5C,E). With respect to group 1, more concentrated and orderly collagen fibrils were visible at the tendon-bone junction and suture anchor hole area at 8 weeks compared with that at 4 weeks (Figure 5B). At 8 weeks, more concentrated collagen fibrillar structures were visible in the tendon-bone

junction and suture anchor hole area for groups 2 and 3 compared with group 1 (Figure 5D,F). Real-time PCR The levels of expression of ALP according to realtime PCR for the three groups are shown in Figure 6; the data are described in terms of the relative quantity (RQ) compared with the expression of group 1 at 4 and 8 weeks. The reference gene GAPDH remained stably expressed throughout the experiment. The mean expression of ALP in groups 2 and 3 was relatively weaker at 4 weeks (group 2, RQ ¼ 0.63; group 3, RQ ¼ 0.1) and 8 weeks (group 2, RQ ¼ 0.01; group 3, RQ ¼ 0.04) compared with that

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Figure 4. Histologic findings of group 1 (A, B), group 2 (C, D) and group 3 (E, F) under a light microscope at 8 weeks (hematoxylin and eosin staining). (A) Some new bone formation was visible, although mature bone with lamellation was more visible compared with that at 4 weeks in the defect area (magnification 20). (B) A more fibrocartilaginous area with mineralization was visible compared with that at 4 weeks at the bone-tendon interface (magnification 80). (C, E) Mature lamellar bone was thicker and more abundant in the defect area compared with that in group 1 (magnification 20). (D, F) Larger and more distinct areas of organized fibrocartilage were observed with chondrocytes in their lacunae oriented in the direction of the collagen fibers (magnification 80). *Location of suture anchor eyelets.

of group 1. The mean expression of collagen type I in group 2 was also weaker at 4 and 8 weeks (4 weeks, RQ ¼ 0.58; 8 weeks, RQ ¼ 0.78), although the expression in group 3 was stronger at 4 and 8 weeks (4 weeks, RQ ¼ 2.93; 8 weeks, RQ ¼ 1.69) compared with that of group 1. Compared with that of group 1, the expression of osteopontin in groups 2 and 3 was stronger at 4 weeks (group 2, RQ ¼ 2.45; group 3, RQ ¼ 1.75) and even further increased at 8 weeks (group 2, RQ ¼ 7.93; group 3, RQ ¼ 3.80). Quantitative micro-CT evaluation Bone density in the suture anchor hole area of all three groups is shown in Figure 7. There was no significant

difference among the three groups at 4 (P ¼ 0.56) and 8 weeks (P ¼ 0.39). The mean bone density in groups 1, 2 and 3 was 1.34  0.68 g/cm3, 0.88  0.73 g/cm3 and 1.75  0.52 g/cm3, respectively, at 4 weeks, and the mean bone density at 8 weeks in groups 1, 2 and 3 was 1.67  0.62 g/cm3, 3.20  0.57 g/cm3 and 2.50  0.59 g/cm3, respectively. With respect to group 2, there was a significant difference in the bone density between at 4 weeks (0.88  0.73 g/cm3) and 8 weeks (3.20  0.57 g/cm3) (P ¼ 0.01); however, there was no significant difference in the bone density between 4 and 8 weeks in group 1 (1.34  0.68 g/cm3 at 4 weeks; 1.67  0.62 g/cm3 at 8 weeks, P ¼ 0.43) and group 3 (1.75  0.52 g/cm3 at 4 weeks; 2.50  0.59 g/cm3 at 8 weeks, P ¼ 0.57).

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Figure 5. Histologic findings of group 1 (A, B), group 2 (C, D) and group 3 (E, F) under a light microscope at 4 and 8 weeks (Masson trichrome, magnification 100). (A) Loose collagen fibrils were visible in the tendon-bone junction and suture anchor hole area at 4 weeks. (B) At 8 weeks, more concentrated collagen fibrils were visible compared with that at 4 weeks at the tendon-bone junction and suture anchor hole area. (C, E) At 4 weeks, more concentrated and orderly collagen fibrils were visible at the tendon-bone junction compared with that of group 1. (D, F) At 8 weeks, more concentrated collagen fibrils were visible at the tendon-bone junction and suture anchor hole area compared with that of group 1.

Biomechanical testing Sixteen samples from three groups failed at the tendon-bone interface, although only two samples in group 1 failed at the tendon-bone avulsion-like interface at 4 weeks. There were no cases in which the

anchor was pulled out. The failure load of the normal rabbit patellar tendon (474.5  68.5 N) was higher than that for all three groups (P ¼ 0.001). At 4 weeks, there was no significant difference in the failure load among the three groups (P ¼ 0.36). The mean failure

Figure 6. In vivo gene expression of bone formation markers ALP, collagen type I and osteopontin. Ratios of target genes relative to the housekeeping gene GAPDH are expressed as a percentage of the control group at 4 and 8 weeks, which was set to 1. Values represent mean  standard deviation.

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Figure 7. Comparison of bone density among three groups at 4 and 8 weeks measured by micro-CT. Values represent mean  standard deviation. *P < 0.05.

load of groups 1, 2 and 3 was 94.5  13.6 N, 100.6  12.2 N and 73.0  18.7 N, respectively at 4 weeks. However, at 8 weeks, one-way analysis of variance revealed significant differences in the failure load among the three groups (P ¼ 0.01). In addition, the mean failure load of group 2 (194.6  15.7 N) was significantly higher than that of group 1 (105.6  19.8 N) after post hoc Tukey test (P ¼ 0.01). The mean failure load of group 3 (162.0  17.4 N) was not significantly higher than that of group 1 (105.6  19.8 N) (P ¼ 0.07). There was no significant difference in the mean failure load between groups 2 and 3 (P ¼ 0.32). Likewise, there was no significant difference for group 1 between 4 and 8 weeks (P ¼ 0.56). Conversely, the differences in mean failure load between 4 and 8 weeks for group 2 (P ¼ 0.002) and group 3 (P ¼ 0.002) were significant (Figure 8). Discussion The principal findings of this study were that BMP-2, used as described in this study with respect

Figure 8. Comparison of the failure load among three groups at 4 and 8 weeks after surgery and between 4 and 8 weeks in each group. Values represent mean  standard deviation. *P < 0.05.

to concentration and carriers (collagen gel or fibrin glue), improved the tendon-bone healing and bone formation of the suture anchor hole, which also increased biomechanical strength of the tendon-bone junction. Numerous studies have looked for methods to improve tendon-bone healing after suture anchor fixation, some of which have aimed to improve the design of the suture anchor or suture materials (24,25), whereas others have focused primarily on improving the biological process of tendon-bone healing (10,12e19). Insertion of a suture anchor of appropriate depth results in a vacant anchor hole remaining above the eyelet of the suture anchor, which we called suture anchor hole. However, only a few studies have explored the role of the suture anchor hole, which plays an important role in the fixation strength of the anchor-bone (10,22). In many studies, BMP-2 has been shown to induce bone formation and enhance tendon-bone healing (11,12,17e19,26). Therefore, we assumed that if the suture anchor hole was filled more quickly with bone after the application of BMP-2, it would enhance the pull-out strength of the suture anchor and improve tendon-bone healing. To the best of our knowledge, this is the first study to investigate the effects of BMP-2 in the suture anchor hole on biological tendon-bone healing and pull-out strength. Our histologic results showed that new bone was more visible and that the tendonbone interface was more mature after application of BMP-2. The tendon-bone healing consists of four normal histologic phases of the tendon-bone transition: tendon fiber, unmineralized fibrocartilage, mineralized fibrocartilage and bone (27). BMPs induce bone formation (17) and increase the tendonbone healing by increasing tenocyte activity and collagen type I expression and production, which is a primary component of tendon and bone (26,28). Pauly et al. (26) suggested a beneficial effect of BMP-2 on the regenerated tendon-bone unit that may be caused by improved collagen type I production of tenocyte-like cells and surrounding osteoblasts. Taken together, our results confirmed the effects of BMP-2 on tendon-bone healing. The bone formation marker ALP is an isoenzyme produced by osteoblasts during their proliferation in the early stages of bone healing. In the later stages of bone healing, osteopontin is released during the maturation of the extracellular matrix (29,30). Collagen type I is a component of the extracellular matrix in bone and tendon. BMP-2 expression reaches maximal levels within 24 h of injury, which suggests that BMP-2 plays a role in initiating the repair cascade (31). In the present study, the gene expression of collagen type I and ALP in groups 2 and 3 was less than that of group 1 at 4 and 8 weeks.

Tendon-bone healing with the use of bone morphogenetic protein-2 A possible explanation for this result is that the collagen type I and ALP genes in the BMP-2 groups were expressed earlier than were those of the control group, resulting in lower gene expression at 4 and 8 weeks. However, gene expression of collagen type I in group 3 was greater than that of group 1 at 4 and 8 weeks. The upregulation of collagen type I at 4 and 8 weeks in group 3 appeared to be a consequence of the degradation products of collagen type I. Anchor loosening or migration has been reported after tendon repair with the use of a suture anchor (7). The pull-out strength of the suture anchor was strongly associated with bone mineral density (8,9). Therefore, we assumed that if bone healing around the suture anchor and the tendon-bone healing progressed more rapidly by application of BMP-2, early pull-out of suture anchor would be prevented. In this study, there was no failure case with pull-out of the suture anchor. However, the difference in the failure load between the experimental and control groups in this study was not significant at 4 weeks. However, at 8 weeks, there was a significant difference in failure load among the three groups. This result may have been due to the 4-week period being too short to complete the tendon-bone healing, with more time needed for the tendon-bone junction to mature (32). The restoration of the interface between soft tissue and bone occurs over several weeks; however, the creation of a strong tendon-bone interface usually requires approximately 12 weeks (33). Therefore, the difference of failure load at 8 weeks should be higher than at 4 weeks. Interestingly, in the present study, the mean failure load of the experimental and control groups was 30e40% of the mean failure load of the normal patellar tendon. This result could also be explained by the time needed for tendon-bone healing and maturation. Consistent with this possibility, another study also showed that the failure load of the experimental group was approximately 40% of the normal patellar tendon at 4 weeks (34). There is currently no consensus regarding the appropriate concentration of BMP-2 for optimal healing. However, it is well known that the effectiveness of BMP-2, as indicated by the quantity of new bone induced, depends on BMP-2 concentration, the carrier used for delivery and the species of the experimental animal (17,35). In the present study, 1 mg (10 mg/mL) of BMP-2 with collagen gel or fibrin glue was used, which is considered to be a low dose. In the literature, doses of BMP-2 between 10 ng and 34 mg have been used, depending on the specific carrier and animal model (13,23,36,37). Relative to the current study, investigators have used similar or smaller amounts of BMP-2 in mouse calvaria and rabbit Achilles tendonebone models (23,37).

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The primary purpose of BMP-2 carrier is to retain the factors at the targeted site for a prolonged time, providing initial support for cells to attach and form regenerated tissue (38). BMP-2 has a short half-life and is structurally unstable. Therefore, it is necessary to administer a high dose, which can cause various adverse effects such as extensive ectopic ossification, immune reactions, soft-tissue swelling, seroma formation and paradoxical osteolysis (20,39). Therefore, carriers are needed that can release BMP-2 locally over an extended period and at sufficient concentrations. Such carriers include collagen sponges, collagen gels, fibrin glue, gelatin, hydroxyapatite, poly-L-lactic acid and hyaluronic acid (17,40). An optimal carrier should provide an osteoconductive scaffold for newly formed bone, which provides longterm local release of the growth factor and is gradually degraded (17,41), The efficacy of collagen sponges as carriers have been well established both in experimental and clinical settings (42). However, in the present study, collagen gel was used because it is easy to handle and avoids the burst release of BMP-2 caused by the compression of collagen sponges by the anchor suture when attaching tendon to bone. Importantly, several previous studies have demonstrated the effectiveness of collagen gel as a BMP-2 carrier (40,43). There was no significant difference between fibrin glue and collagen gel as BMP-2 carriers with respect to their effects on bone formation and the tendon-bone healing in our study. However, there was a tendency toward superior results with fibrin glue, although the difference was not significant. This result may have been caused by small but biologically significant differences in ambient temperature resulting in a longer time lapse for collagen phase transformation to occur in situ from a gel to a solid material (40). The collagen gel that we used was less solid than the fibrin glue at laboratory room temperature during experiments. In addition, delays in phase transformation could have caused the BMP-2 and collagen gel to leak out from the defect, preventing controlled and long-term release of BMP-2. There were some limitations in this study. First, the amount of BMP-2 administered was smaller than the amounts reported in previous studies. An optimal amount of BMP-2 has not been established and varies according to carriers and experimental animal model. Thus, it will be important in future studies to identify the minimal amount of BMP-2 that is effective in the tendon-bone healing. Nevertheless, previous studies have reported good results with similar or smaller amounts of BMP-2 than what was used in this study (23,37). A second limitation of this study was that the experimental duration was relatively short compared with the normal tendon-bone

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healing period, which has been reported to be approximately 12 weeks (32,33). However, we were interested in the possibility of early tendon-bone healing with the use of BMP-2, which was the reason that we chose 4 and 8 weeks for the experimental duration. Third, we did not evaluate the effect of fibrin glue or collagen gel alone on the bone-tendonehealing process. Thus, to evaluate the effects of BMP-2 alone, it would be necessary to use a carrier control group such as the fibrin glueeonly or collagen geleonly group. However, several previous studies have compared the difference between experimental and carrier control groups and reported that the effect of adding BMP-2 in a carrier is superior to that of the carrier-only group (11,23). Kim et al. (23) compared a fibrin glue group and a fibrin glue with BMP-2 group in a rabbit Achilles tendonebone model and reported that the effect of fibrin glue with BMP-2 is more significant than that of fibrin glue alone (23). On the basis of these findings, we did not use a carrier control group in this study. Finally, we were unable to analyze the concentration of the carrier (fibrin glue and collagen gel), which may affect the tendon-bone healing process. In our study, the concentration of collagen gel was 3 mg/mL, whereas that of fibrin gel was 100 mg/ mL (fibrinogen). In conclusion, BMP-2 administrated as described in this study with respect to concentration and delivery method (collagen gel or fibrin glue) improved tendon-bone healing and bone formation in the suture anchor hole, which improved biomechanical strength. Together, these results may help to improve tendon-bone healing with the use of suture anchors. Acknowledgments

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We thank So Yeon Song (Department of Dentistry, Korea University Guro Hospital) for technical assistance. 17.

Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

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