International Immunopharmacology 24 (2015) 7–13
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Systemic administration of sclerostin monoclonal antibody accelerates fracture healing in the femoral osteotomy model of young rats Gao Feng a, Zhang Chang-Qing b, Chai Yi-Min b, Li Xiao-Lin b,⁎ a b
Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Jiaotong University, China Department of Orthopedic Surgery, Shanghai Jiao Tong University affiliated Sixth People's Hospital, Shanghai Jiaotong University, China
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 26 October 2014 Accepted 10 November 2014 Available online 18 November 2014 Keywords: Sclerostin Monoclonal antibody Osteotomy Fracture healing
a b s t r a c t Genetic studies have demonstrated that sclerostin was a key negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment enhanced bone healing in experimental fracture healing. The purpose was to investigate the effects of systemic Scl-Ab administration on open fracture healing in young rats. Unilateral femoral fractures were generated in eight-week-old Sprague–Dawley rats. Rats were treated with vehicle or Scl-Ab for 6 weeks. Fracture healing was evaluated by western blotting, immunohistochemistry, histology, radiography, micro-CT, and biomechanical testing. In addition, the bone mass of intact femur was also evaluated by micro-CT. The results showed that, at 1 and 2 weeks after fracture, proliferating cell nuclear antigen (PCNA) score and bone morphogenetic protein-2 (BMP-2) expression in the Scl-Ab group were significantly increased compared with the control group. A decrease in cartilage in the Scl-Ab group was also observed after fracture, and this was accompanied by more rapider fracture healing. At 4 and 6 weeks, there were significant increases in bone mass and mechanical properties in the calluses from Scl-Ab group compared with control group. In addition, Scl-Ab treatment also showed significant anabolic effects in intact femur. In conclusion, systemic Scl-Ab administration has a significant enhancement in a rat femoral osteotomy model. These results support the therapeutic potential of Scl-Ab as a noninvasive strategy to enhance open fracture healing. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fracture repair is a complex process, which consists mainly of granulation tissue formation, callus formation, and bone remodeling [1]. Most fracture healing completely restores bone structure and function. However, approximately 10% of fracture patients eventually experience impaired healing, which includes delayed union, non-union, and other complications. This creates a severe public health problem, and results to vast social burden [2]. Cytokines such as bone morphogenetic proteins (BMPs) have been used to improve bone healing in open fractures and treat fractureassociated complications in patients [3]. However, the negative aspects of BMPs are obvious. BMPs must be surgically implanted at the site of fracture, and may result in local complications such as edema, infection, and heterotopic bone formation [4]. Thus far, no noninvasive therapy improving fracture repair and reducing the risk of fracture-associated complications has been approved in clinical trials.
⁎ Corresponding author at: Department of Orthopedic Surgery, Shanghai Jiao Tong University affiliated Sixth People's Hospital, Shanghai Jiaotong University, 600 Yishan Road, Xuhui District, Shanghai, China. Zip code: 200233. Tel./fax: +86 2164369181. E-mail address: [email protected] (L. Xiao-Lin).
http://dx.doi.org/10.1016/j.intimp.2014.11.010 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Recently, the role of canonical Wnt pathway in fracture healing has get attention [5,6]. The activation of this pathway may improve fracture healing [5–7]. Sclerostin, which binds to lipoprotein receptor-related protein 5/6 (LRP5/6), is a key inhibitor in the canonical Wnt signaling. As an antagonist of the Wnt pathway in osteoblasts, sclerostin negatively regulates osteoblast proliferation and differentiation [8–10]. The mice with sclerostin gene knockout showed increased bone formation, bone mass, and bone strength [11]. Sclerostin deficiency also results in a high bone mass phenotype in humans [12,13]. In contrast, the overexpression of the sclerostin gene results in osteopenia in transgenic mouse model [14]. These studies showed an inverse relationship between sclerostin and bone formation. Sclerostin antibody (Scl-Ab) can neutralize the inhibitory effects of sclerostin on Wnt pathway [15]. The treatment of Scl-Ab resulted in increased bone formation, bone mass, and bone anabolic activity in a rat model of postmenopausal osteoporosis [15]. Additionally, Scl-Ab treatment also increased bone formation and bone mineral density in postmenopausal women [16]. The primary purpose of this study was to investigate the effects of Scl-Ab treatment in an open fracture model in young rats. We hypothesized that Scl-Ab treatment would accelerate open fracture healing. In addition, the anabolic effects of Scl-Ab treatment in intact femora were also investigated.
8
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
2. Materials and methods 2.1. Animals A total of 48 eight-week-old male Sprague–Dawley rats were purchased from the Laboratory Animal Center of the Shanghai Jiaotong University. The experimental protocol was approved by the Animal Study Committee of the University (Ref. No. 59/012). Rats were acclimated to 12 hour light/12 hour dark cycle at 22 °C for 1 week. During the experimental period, rats were housed in cages (25 × 30 × 60 cm2) with ad libitum access to water and pelleted commercial rodent diet. 2.2. Femoral osteotomy model Femoral diaphyseal fractures were produced in a standard manner as previously described [17]. All osteotomies were performed under general anesthesia. The right femur was fixed intramedullarily using a Kirschner wire (diameter 1.5 mm, Stryker China, Hong Kong, China). Then, the right femoral diaphysis was transversely fractured with a semi-lunar osteotome at the midshaft of the femur. The fracture fragments of femur were contacted and stabilized after fracture. All wires were cut level with the condyles to avoid restriction of motion of the knee joint. After recovery from anesthesia, unrestricted activity was allowed. Post-operation, animals were randomly divided into the SclAb treatment group (subcutaneous injection, 25 mg/kg body weight, two times per week) or vehicle (saline) treatment group. This dosage (25 mg/kg body weight, two times per week) has been previously reported to be effective to enhance fracture healing in rats [18]. In both groups, treatments started from day 1 after fracture and lasted until sacrifice. The rats were sacrificed at 1 (n = 6), 2 (n = 6), 4 (n = 6), and 6 (n = 6) weeks after fracture. At 1 and 2 weeks, six right femora from each group, which have been X-rayed, were immediately bisected along sagittal plane. The two parts of the femur were respectively used for western blotting and histology (including immunohistochemistry) analysis. At 4 and 6 weeks, both the right and left femora were collected; after X-ray and micro-CT analysis, the right femora were used for biomechanical testing. The left femora were only used for micro-CT analysis at 4 and 6 weeks (Table 1). 2.3. Western blotting analysis At 1 and 2 weeks, the protein expression of proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) in the callus was determined by Western blotting analysis. For extraction of the protein fraction, frozen tissue samples, which have been harvested from the callus at the fracture site, were homogenized. After that, the proteins were extracted in lysis buffer. Proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (PAGE). The protein bands were transferred to a nitrocellulose membrane by standard protocols and probed using anti-PCNA (1:1000; Dako Cytomation, Hamburg, Germany) and anti-BMP-2 (1:200; Abcam, Cambridge, UK). Table 1 Number of animals per study group. 1 week
Control Scl-Ab
2 weeks
4 weeks
The secondary horse radish peroxidase-labeled anti-IgG antibody (1:5000; GE Healthcare, Freiburg, Germany) was applied and incubated at room temperature. Protein expression was visualized by luminol-enhanced chemiluminescence reaction (Hyperfilm ECL, AmershamBiosciences, Freiburg, Germany). The optical densities of bands were assessed densitometrically (Geldoc, Quantity one software, BioRad, Hercules, CA, USA). After a stripping procedure, the Western blots of β-actin were performed on the same membrane. 2.4. Histology At 1 and 2 weeks, one part of each right femur, which had been bisected along sagittal plane, was fixed with 4% paraformaldehyde overnight at room temperature, and decalcified in 14% EDTA for 6 weeks (liquid refreshed weekly). After sufficient decalcification was achieved (judged by the easy penetration of a needle), the sample was dehydrated through an ethanol series, embedded in paraffin, and 5 μm sections were cut longitudinally. The sections were stained with hematoxylin and eosin (HE) for light microscopic examination (Zeiss Aixoplan, Zeiss, Oberkochen, Baden-Württemberg, Germany). The bone tissue area fraction and cartilage tissue area fraction were calculated by software using an analysis image window, which measures 1.6 mm2, in the central part of external callus [7]. The average of ten tissue sections was used to determine callus area fraction. The area fraction of the cartilage and bone was evaluated in a blinded manner by an independent investigator. 2.5. Immunohistochemical staining Five micrometer sections were deparaffinized, rehydrated, and treated (95 °C, 10 min) in an antigen retrieval buffer of sodium citrate (pH 6.0). Sections were cooled (room temperature, 20 min) and washed in dH2O. Endogenous peroxidase activity was blocked by 3% H2O2 (37 °C, 10 min). Sections were blocked with 10% bovine serum (room temperature, 30 min). Then, sections were incubated with mouse monoclonal PCNA primary antibody (1:100; Southern Biotech, Birmingham, AB, USA) or mouse monoclonal BMP-2 primary antibody (1:300; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 °C. The next day, sections were incubated with the secondary antibody peroxidaselabeled goat anti-mouse IgGs (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), then visualized with diaminobenzidine for a predetermined time, and counterstained with hexatoxylin. For negative controls, the primary antibody was replaced with 0.01 mol/L phosphate buffered saline. In the external callus, the number of PCNA-positive cells was analyzed. Four regions (0.9 mm × 1.5 mm in size), distributed in 2 corner and 2 central areas of external callus were used to calculate PCNA-positive cells in each section [19]. The ratio of PCNA-positive to total cells was calculated and expressed as a percentage. The average ratio of ten sections was used as the PCNA score. The integrated optical density (IOD) of the BMP-2 positive staining was analyzed using an image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Bethesda, MD, USA). The IOD was also analyzed using four regions (0.9 mm × 1.5 mm in size), which were also distributed in 2 corner and 2 central areas of external callus, and the average IOD of 10 sections was used as the IOD score.
6 weeks
WB
Histo
WB
Histo
μCT
Bm
μCT
Bm
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
The number of animals in Scl-Ab group and control group. Analysis methods included Western blotting (WB), histology (Histo, including immunohistochemistry), micro-CT (μCT), and biomechanics (Bm). At 1 and 2 weeks, six right femora from each group were bisected along sagittal plane. The two parts of the femur were respectively used for Western blotting and histology (including immunohistochemistry) analyses. At 4 and 6 weeks, both the right and left femora were collected; after μCT analysis, the right femora were used for biomechanical testing. The left femora were only used for microCT analysis at 4 and 6 weeks.
2.6. X-ray At 1, 2, 4, and 6 weeks, the anteroposterior X-ray of the right femora was examined by digital radiography (55 kv, 3 mas; MX-20; Faxitron Bioptics, Tucson, AZ, USA) to assess the progress of fracture repair. 2.7. Micro-computed tomography (μCT) At 4 and 6 weeks, both the right and left femora were scanned using a Micro-CT system (μCT40, Scanco Medical, Brüttisellen, Switzerland).
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
9
For all measurements, the matrix size was 1024 × 1024, and the isotropic voxel resolution was 12 μm. For the right femur, the scan line was placed at the central of fracture callus. The maximum callus crosssectional area (C.Ar), total bone mineral content (Tt.BMC) and total bone mineral density (Tt.BMD) of the central 1 mm of the fracture callus were assessed [20]. The threshold ≥ 464 mg/cm3 was defined to separate the bone/callus from the marrow [21]. For the left femur, the midshaft of femur spanning 10% of the femur height (threshold 710 mg/cm3) was investigated [17]. The total bone mineral area (Tt.BMA), Tt.BMC, and Tt.BMD were also calculated, respectively. 2.8. Biomechanical testing At 4 and 6 weeks, after the measurement of micro-CT, a four-point bending machine (Avalon Technologies, Rochester, MN, USA) was used to evaluate the biomechanical property of the right femora at the fracture site. The femur was installed on fixture (anterior surface facing upward on two lower support points 20 mm apart, and the two upper bars 8 mm apart). Load was applied at a rate of 5 mm/min until fracture. The stiffness (N/mm, defined as the slope of the linear portion of the load–deformation curve), ultimate load (N; defined as the maximum force that the specimen sustained), and energy (N × mm; defined as the area under the load–deformation curve) were calculated according to the load–deformation curve. 2.9. Statistical analysis Data were expressed as mean ± standard deviation (SD). Statistical computation of data was performed using the statistical package SPSS 10.0 (SPSS, Chicago, IL, USA). Differences between two groups were tested by a student's t-test. For comparisons across three or more groups, two-way ANOVA followed by Bonferroni test was used. A Pvalue less than 0.05 was considered significant. 3. Results Throughout this study, no difference in the mean weight was observed between two groups. The rats in both groups resumed normal activity within three days after operation. All rats survived the study. There were no technical failure (poor fracture fixation) and infection that happened during or after surgery. 3.1. Systematic Scl-Ab administration increased PCNA and BMP-2 expression within the callus At 1 and 2 weeks, the expression of PCNA and BMP-2 was detected in the callus of the Scl-Ab group and control group. At 1 week, the PCNA and BMP-2 levels measured from the Scl-Ab group were significantly higher than that of the control group (all P b 0.01) (Fig. 1). At 2 weeks, both the PCNA and BMP-2 levels in callus from the Scl-Ab group were still significantly higher than that of the control group (all P b 0.01) (Fig. 1). At 1 and 2 weeks, for both groups, a lot of PCNA-positive cells were observed in the external callus. Compared with the control group, the Scl-Ab group had 61% and 98% higher PCNA score at 1 week and 2 weeks, respectively (all P b 0.01) (Fig. 2). The positive signals of BMP-2 were also observed in callus tissues of both groups (Fig. 3). At 1 and 2 weeks, the Scl-Ab group demonstrated a substantially increased IOD of BMP-2 positive signals in the external callus compared with the control group (Fig. 3). 3.2. Systematic Scl-Ab administration accelerated fracture healing At 1 and 2 weeks post-fracture, compared with the control group, a decrease in cartilage callus in Scl-Ab group was seen (Fig. 4). The callus in Scl-Ab group was more mature and contained less cartilage and more dense bone. Correspondingly, a more rapider fracture healing in the
Fig. 1. Western blotting analyses of proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) in rats treated with Scl-Ab and rats treated with vehicle at 1 and 2 weeks post-fracture. Data were given as mean ± SD, * b 0.01.
Scl-Ab group was also conformed by X-ray compared with the control group at 4 weeks. At this time point, the fracture line was still clearly visible in the control group (Fig. 5). In comparison, the fracture line had disappeared in the Scl-Ab group. The fracture appeared to have healed.
3.3. Systematic Scl-Ab administration increased bone mass and biomechanical properties in callus At 4 and 6 weeks after fracture, the C.Ar, Tt.BMC, and Tt.BMD of the callus were significantly different between two groups. At both 4 and 6 weeks, C.Ar, Tt.BMC, and Tt.BMD were significantly higher in Scl-Ab group than in control group (all P b 0.01) (Fig. 6). Biomechanical testing showed that the Scl-Ab group exhibited 19% (P b 0.01) and 29% (P b 0.01) higher stiffness at 4 weeks and 6 weeks, respectively, 56% (P b 0.01) and 97% (P b 0.01) higher ultimate load at 4 weeks and 6 weeks, respectively and 32% (P b 0.01) and 56% (P b 0.01) higher energy at 4 weeks and 6 weeks, respectively compared with the control group (Table 2).
3.4. Systematic Scl-Ab administration showed anabolic effects in intact femur Micro-CT analysis of the intact left femora showed that the bone mass of diaphysis was significantly increased by Scl-Ab treatment (Fig. 7). At 4 weeks, in the midshaft of diaphysis, the effects of the SclAb treatment could be observed as a significant increase in Tt.BMA of 10% (P b 0.01), Tt.BMC of 14% (P b 0.05), and Tt.BMD of 9% (P b 0.01). Moreover, at 6 weeks, Tt.BMA was increased by 12% (P b 0.01), Tt.BMC was increased by 65% (P b 0.01), and Tt.BMD was increased by 17% (P b 0.01), respectively.
10
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
Fig. 2. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in Scl-Ab group and control group at 1 and 2 weeks after fracture. At 1 and 2 weeks, PCNApositive staining was found predominantly in external callus surrounding the fracture site. Old cortical bone did not contain PCNA-positive cells. Scale bars represent 50 μm. (A) PCNA-positive cells in the control (a, c) and Scl-Ab (b, d) groups at 1 week (a, b) and 2 weeks (c, d). The Scl-Ab group had more PCNA-positive cells in the external callus compared with the control group. (B) The PCNA score in each group was calculated. Data were given as mean ± SD, n = 6/group/time point, * b 0.01.
4. Discussion At the early stage of fracture healing, abundant undifferentiated mesenchymal cells and osteoprogenitor cells aggregate, proliferate, differentiate, and express various cytokines to participate in the fracture healing [22]. Therefore, it is very important to closely regulate the early stage in order to accelerate fracture healing. Our results clearly showed that Scl-Ab improved the cell proliferation and expression of BMP-2 at the early stage post-fracture, and accelerated the process of fracture healing. It also enhanced callus volume, mineralization, and mechanical properties across the 6-week time course. The activation of Wnt pathway increases the β-catenin (a key positive regulator of Wnt pathway) level in cultured osteoblasts [23]. Robinson et al. also reported that β-catenin expression was increased in pre-osteoblasts and osteoblasts in the inner periosteum by injecting SB-415286 (a Wnt pathway activator) into the calvaria of growing rats [24]. Scl-Ab can neutralize the inhibitory effects of sclerostin on Wnt/b-catenin pathway in vitro. The expression of β-catenin was significantly enhanced in sclerostin knockout mice than in sclerostin wildtype mice [25]. Therefore, the Scl-Ab administration may stimulate the Wnt/β-catenin pathway to promote β-catenin level. In this study, at 1 and 2 weeks, the callus specimens from the Scl-Ab group contained more PCNA-positive cells than did the control group (Fig. 2). This showed that the systematic Scl-Ab administration significantly stimulated the proliferation of cells in the callus. Axin is also an intracellular inhibitor in the canonical Wnt/β-catenin pathway. It has been conformed that, compared with the Axin+/+ type counterpart, calvarial osteoblasts in Axin−/− mice had a higher level of β-catenin
Fig. 3. Immunohistochemical staining of bone morphogenetic protein-2 (BMP-2) in control (a, c) and Scl-Ab (b, d) groups at 1 (a, b) and 2 (c, d) weeks following fracture. Scale bars represent 50 μm. (A) BMP-2 positive signals in the control (a, c) and Scl-Ab (b, d) groups at 1 week (a, b) and 2 weeks (c, d). At 1 and 2 weeks, the enhanced BMP-2 expression was clearly observed in Scl-Ab group. (B) The IOD of BMP-2 positive signals in each group was calculated. Data were given as mean ± SD, n = 6/group/time point, * b 0.01, ** b 0.05.
expression level. Correspondingly, the proliferation of osteoblast was substantially increased [26]. LRP5 gene is a Wnt protein receptor. The knockout mice (LRP5−/−) displayed impaired β-catenin expression and reduced osteoblast proliferation in long bones [27]. The increases of regenerating bone in sclerostin knockout mice were due to an increase in the osteoblast numbers [25]. In our study, in Scl-Ab group, the higher cell proliferation can be observed. Based on early studies, we hypothesized that the more PCNA-positive cells in Scl-Ab group were likely to be mainly caused by a higher number of proliferating osteoblasts or osteoblast precursors. As the most potent osteoinductive agents, BMP-2 plays a critical role in bone repair by mobilizing osteoprogenitor cells, stimulating the differentiation processes of mesenchymal cells to an osteoblastic lineage, and enhancing bone formation [28]. Therefore, to induce the callus at the fracture site to increase BMP-2 expression will be very conducive to fracture healing [29]. In addition, as the most effective inducer of osteogenesis, endogenous BMP-2 is usually regarded as one of the indicators to evaluate the biological environment in bone formation [30]. The present study revealed that, at 1 and 2 weeks, the expression of endogenous BMP-2 signal in the Scl-Ab group was significantly increased than in the control group (Figs. 1, 3). The significantly higher bone mass, C.Ar and biomechanical properties also conformed increased fracture healing in the Scl-Ab group (Fig. 6) (Table 2). This enhanced bone formation was partly contributed to improved expression of endogenous BMP-2 at least. The mechanism of an early increase in endogenous BMP-2 by Scl-Ab required further investigation. In similar fracture models, previous studies reported fracture healing with both endochondral and direct bone formation [17]. In this study, at weeks 1 and 2, the calluses in Scl-Ab group contained less cartilage.
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
11
Fig. 4. Histological assessments of fracture callus. (A) These images showed a decrease in cartilage tissue and an enhanced bone formation in Scl-Ab group (b, d) compared with control group (a, c) at 1 week (a, b) and 2 weeks (c, d). (B) Scl-Ab group showed less cartilage area compared with the vehicle group at 1 and 2 weeks. Scale bars represent 50 μm. Data were given as mean ± SD, n = 6/group/time point, * b 0.01.
Fig. 6. Scl-Ab treatment improved the bone mass in the callus at 4 and 6 weeks after fracture. (A) Representative axial micro-CT images in control group (a, c) and Scl-Ab group (b, d) at 4 weeks (a, b) and 6 weeks (c, d) after fracture. Micro-CT images represent the group medians for peak load. (B) Micro-CT analysis showed that Scl-Ab treatment resulted in improved bone mass in the right femora compared with the control group. Data were given as mean ± SD, n = 6/group/time point, * b 0.01.
Although the expression of sclerostin has been detected in chondrocytes in human, it is unclear whether Scl-Ab administration had any effects on the formation of cartilage [10]. Interestingly, rat fractures treated with a Wnt activator healed without the formation of cartilage islets in the callus [17]. The activation of Wnt pathway causes the mesenchymal progenitor cells to differentiate into osteogenic cells instead of chondrocytes [31,32]. In this study, there was a less pronounced effect of reducing cartilage callus. This may be explained that the potency of Scl-Ab to activate the Wnt pathway is lower compared with those Table 2 Mechanical test of the fractured femora in rats at 4 and 6 weeks after fracture.
Fig. 5. Systemic administration of Scl-Ab increased bone healing in rat femoral fracture. The radiographs of the fractured femora in control group (a, c, e, g) and Scl-Ab group (b, d, f, h) at 1 week (a, b), 2 weeks (c, d), 4 weeks (e, f) and 6 weeks (g, h). Note the union in Scl-Ab group and the fracture line in the control group at 4 weeks.
Parameters
Time
n
Control
Ultimate load (N) Energy (N*mm) Stiffness (N/mm)
4 6 4 6 4 6
6 6 6 6 6 6
80.2 109.2 32.2 52.6 231.2 303.6
weeks weeks weeks weeks weeks weeks
± ± ± ± ± ±
5.3 8.2 3.2 4.3 11.3 12.1
n
Scl-Ab
6 6 6 6 6 6
125.2 215.3 42.5 82.3 274.8 392.5
± ± ± ± ± ±
P 8.2 10.3 3.9 5.2 11.7 14.6
b0.01 b0.01 b0.01 b0.01 b0.01 b0.01
Mechanical test of the fractured femora. The four-point bending tests showed enhanced mechanical properties in Scl-Ab group compared with control group at 4 and 6 weeks post-fracture. Ultimate load, energy, and stiffness were derived from load/displacement curves. Data were given as mean ± SD.
12
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
Fig. 7. Scl-Ab treatment improved the bone mass in the intact femora at 4 and 6 weeks after fracture. Micro-CT analysis showed that Scl-Ab treatment resulted in apparent anabolic effects in the intact femora compared with the control group. Data were given as mean ± SD, n = 6/group/time point, * b 0.01, ** b 0.05.
Wnt activators [33]. After the fracture has been stabilized sufficiently, the transition from cartilaginous callus to bone at the fracture would begin in no time [20]. Scl-Ab administration led to an earlier stabilization of the fracture [18]. Perhaps, the progress from cartilage to woven bone was also accelerated in the Scl-Ab group (Fig. 4). This was beneficial to produce more mature calluses at the fracture site. In this study, the systemic Scl-Ab administration increased Tt.BMA, Tt.BMC, and Tt.BMD in the non-operated femur of rats. In ovariectomized rats, Scl-Ab treatment showed these skeletal effects. In cynomolgus monkeys, Scl-Ab treatment significantly increased bone mass at the lumbar spine, femoral neck, and distal radius, at which osteoporotic fractures occur frequently [34]. This demonstrated a profound systemic effect of Scl-Ab on bone tissue. The anabolic effect of Scl-Ab treatment that increases bone mass in the non-operated bone should be advantageous to prevent a secondary osteoporotic fracture. Until now, no systemic therapy has been found that indeed improves fracture repair in patients. As an approved anabolic therapy in osteoporosis, intermittent PTH has been conformed to improve open fracture healing in rat model [19,35]. However, in clinical trials, this therapy failed to achieve its major objective of reducing time to radiographic healing [36]. In addition, although enhancing callus remodeling, intermittent PTH did not increase whole-femur strength in cynomolgus monkeys [37]. Whether the improvements in bone mass and strength in rat open fracture models treated with Scl-Ab will result in improved fracture healing in humans required clinical trials to verify. Taken together, the systemic administration of Scl-Ab increased bone formation, bone mass, and bone strength in fractured and intact bones in rat open fracture models. These results support the therapeutic potential of Scl-Ab as a noninvasive strategy to enhance open fracture healing.
References [1] Wigner NA, Luderer HF, Cox MK, Sooy K, Gerstenfeld LC, Demay MB. Acute phosphate restriction leads to impaired fracture healing and resistance to BMP-2. J Bone Miner Res 2010;25:724–33. [2] Einhorn TA. Enhancement of fracture-healing. J Bone Joint Surg Am 1995;77: 940–56. [3] Mont MA, Ragland PS, Biggins B, Friedlaender G, Patel T, Cook S, et al. Use of bone morphogenetic proteins for musculoskeletal applications. An overview. J Bone Joint Surg Am 2004;86-A(Suppl. 2):41–55. [4] Garrison KR, Donell S, Ryder J, Shemilt I, Mugford M, Harvey I, et al. Clinical effectiveness and cost-effectiveness of bone morphogenetic proteins in the non-healing of fractures and spinal fusion: a systematic review. Health Technol Assess 2007;11: 1–150 [iii-iv]. [5] Macsai CE, Foster BK, Xian CJ. Roles of Wnt signalling in bone growth, remodelling, skeletal disorders and fracture repair. J Cell Physiol 2008;215:578–87. [6] Silkstone D, Hong H, Alman BA. Beta-catenin in the race to fracture repair: in it to Wnt. Nat Clin Pract Rheumatol 2008;4:413–9. [7] Chen Y, Whetstone HC, Lin AC, Nadesan P, Wei Q, Poon R, et al. Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing. PLoS Med 2007;4:e249.
[8] Balemans W, Ebeling M, Patel N, Van Hul E, Olson P, Dioszegi M, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001;10:537–43. [9] Brunkow ME, Gardner JC, Van Ness J, Paeper BW, Kovacevich BR, Proll S, et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001;68:577–89. [10] Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22: 6267–76. [11] Li X, Ominsky MS, Niu QT, Sun N, Daugherty B, D'Agostin D, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res 2008;23:860–9. [12] Hamersma H, Gardner J, Beighton P. The natural history of sclerosteosis. Clin Genet 2003;63:192–7. [13] Vanhoenacker FM, Balemans W, Tan GJ, Dikkers FG, De Schepper AM, Mathysen DG, et al. Van Buchem disease: lifetime evolution of radioclinical features. Skeletal Radiol 2003;32:708–18. [14] Kramer I, Loots GG, Studer A, Keller H, Kneissel M. Parathyroid hormone (PTH)induced bone gain is blunted in SOST overexpressing and deficient mice. J Bone Miner Res 2010;25:178–89. [15] Li X, Ominsky MS, Warmington KS, Morony S, Gong J, Cao J, et al. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res 2009;24:578–88. [16] Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single-dose, placebo-controlled, randomized study of AMG 785, a sclerostin monoclonal antibody. J Bone Miner Res 2011;26: 19–26. [17] Sisask G, Marsell R, Sundgren-Andersson A, Larsson S, Nilsson O, Ljunggren O, et al. Rats treated with AZD2858, a GSK3 inhibitor, heal fractures rapidly without endochondral bone formation. Bone 2013;54:126–32. [18] Suen PK, He YX, Chow DH, Huang L, Li C, Ke HZ, et al. Sclerostin monoclonal antibody enhanced bone fracture healing in an open osteotomy model in rats. J Orthop Res 2014;32:997–1005. [19] Nakajima A, Shimoji N, Shiomi K, Shimizu S, Moriya H, Einhorn TA, et al. Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1–34). J Bone Miner Res 2002;17:2038–47. [20] Ominsky MS, Li C, Li X, Tan HL, Lee E, Barrero M, et al. Inhibition of sclerostin by monoclonal antibody enhances bone healing and improves bone density and strength of nonfractured bones. J Bone Miner Res 2011;26:1012–21. [21] Komatsubara S, Mori S, Mashiba T, Nonaka K, Seki A, Akiyama T, et al. Human parathyroid hormone (1–34) accelerates the fracture healing process of woven to lamellar bone replacement and new cortical shell formation in rat femora. Bone 2005;36:678–87. [22] Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects of bone healing. Injury 2005;36:1392–404. [23] Marsell R, Sisask G, Nilsson Y, Sundgren-Andersson AK, Andersson U, Larsson S, et al. GSK-3 inhibition by an orally active small molecule increases bone mass in rats. Bone 2012;50:619–27. [24] Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, Cullen DM, Zhao W, Li C, et al. Wnt/ beta-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem 2006;281:31720–8. [25] McGee-Lawrence ME, Ryan ZC, Carpio LR, Kakar S, Westendorf JJ, Kumar R. Sclerostin deficient mice rapidly heal bone defects by activating beta-catenin and increasing intramembranous ossification. Biochem Biophys Res Commun 2013; 441:886–90. [26] Yu HM, Jerchow B, Sheu TJ, Liu B, Costantini F, Puzas JE, et al. The role of Axin2 in calvarial morphogenesis and craniosynostosis. Development 2005;132:1995–2005. [27] Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass 2nd DA, et al. Cbfa1independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 2002; 157:303–14. [28] Eriksson C, Nygren H, Ohlson K. Implantation of hydrophilic and hydrophobic titanium discs in rat tibia: cellular reactions on the surfaces during the first 3 weeks in bone. Biomaterials 2004;25:4759–66.
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13 [29] Stucki U, Schmid J, Hammerle CF, Lang NP. Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans. Clin Oral Implants Res 2001;12:121–7. [30] Zheng LW, Ma L, Cheung LK. Changes in blood perfusion and bone healing induced by nicotine during distraction osteogenesis. Bone 2008;43:355–61. [31] Bennett CN, Longo KA, Wright WS, Suva LJ, Lane TF, Hankenson KD, et al. Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci U S A 2005;102: 3324–9. [32] Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrowderived mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci 2009;66:236–53. [33] Baron R, Rawadi G, Roman-Roman S. Wnt signaling: a key regulator of bone mass. Curr Top Dev Biol 2006;76:103–27.
13
[34] Ominsky MS, Vlasseros F, Jolette J, Smith SY, Stouch B, Doellgast G, et al. Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength. J Bone Miner Res 2010;25:948–59. [35] Tagil M, McDonald MM, Morse A, Peacock L, Mikulec K, Amanat N, et al. Intermittent PTH (1-–34) does not increase union rates in open rat femoral fractures and exhibits attenuated anabolic effects compared to closed fractures. Bone 2010;46:852–9. [36] Aspenberg P, Genant HK, Johansson T, Nino AJ, See K, Krohn K, et al. Teriparatide for acceleration of fracture repair in humans: a prospective, randomized, double-blind study of 102 postmenopausal women with distal radial fractures. J Bone Miner Res 2010;25:404–14. [37] Manabe T, Mori S, Mashiba T, Kaji Y, Iwata K, Komatsubara S, et al. Human parathyroid hormone (1–34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys. Bone 2007;40:1475–82.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Systemic administration of sclerostin monoclonal antibody accelerates fracture healing in the femoral osteotomy model of young rats Gao Feng a, Zhang Chang-Qing b, Chai Yi-Min b, Li Xiao-Lin b,⁎ a b
Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Jiaotong University, China Department of Orthopedic Surgery, Shanghai Jiao Tong University affiliated Sixth People's Hospital, Shanghai Jiaotong University, China
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 26 October 2014 Accepted 10 November 2014 Available online 18 November 2014 Keywords: Sclerostin Monoclonal antibody Osteotomy Fracture healing
a b s t r a c t Genetic studies have demonstrated that sclerostin was a key negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment enhanced bone healing in experimental fracture healing. The purpose was to investigate the effects of systemic Scl-Ab administration on open fracture healing in young rats. Unilateral femoral fractures were generated in eight-week-old Sprague–Dawley rats. Rats were treated with vehicle or Scl-Ab for 6 weeks. Fracture healing was evaluated by western blotting, immunohistochemistry, histology, radiography, micro-CT, and biomechanical testing. In addition, the bone mass of intact femur was also evaluated by micro-CT. The results showed that, at 1 and 2 weeks after fracture, proliferating cell nuclear antigen (PCNA) score and bone morphogenetic protein-2 (BMP-2) expression in the Scl-Ab group were significantly increased compared with the control group. A decrease in cartilage in the Scl-Ab group was also observed after fracture, and this was accompanied by more rapider fracture healing. At 4 and 6 weeks, there were significant increases in bone mass and mechanical properties in the calluses from Scl-Ab group compared with control group. In addition, Scl-Ab treatment also showed significant anabolic effects in intact femur. In conclusion, systemic Scl-Ab administration has a significant enhancement in a rat femoral osteotomy model. These results support the therapeutic potential of Scl-Ab as a noninvasive strategy to enhance open fracture healing. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fracture repair is a complex process, which consists mainly of granulation tissue formation, callus formation, and bone remodeling [1]. Most fracture healing completely restores bone structure and function. However, approximately 10% of fracture patients eventually experience impaired healing, which includes delayed union, non-union, and other complications. This creates a severe public health problem, and results to vast social burden [2]. Cytokines such as bone morphogenetic proteins (BMPs) have been used to improve bone healing in open fractures and treat fractureassociated complications in patients [3]. However, the negative aspects of BMPs are obvious. BMPs must be surgically implanted at the site of fracture, and may result in local complications such as edema, infection, and heterotopic bone formation [4]. Thus far, no noninvasive therapy improving fracture repair and reducing the risk of fracture-associated complications has been approved in clinical trials.
⁎ Corresponding author at: Department of Orthopedic Surgery, Shanghai Jiao Tong University affiliated Sixth People's Hospital, Shanghai Jiaotong University, 600 Yishan Road, Xuhui District, Shanghai, China. Zip code: 200233. Tel./fax: +86 2164369181. E-mail address: [email protected] (L. Xiao-Lin).
http://dx.doi.org/10.1016/j.intimp.2014.11.010 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Recently, the role of canonical Wnt pathway in fracture healing has get attention [5,6]. The activation of this pathway may improve fracture healing [5–7]. Sclerostin, which binds to lipoprotein receptor-related protein 5/6 (LRP5/6), is a key inhibitor in the canonical Wnt signaling. As an antagonist of the Wnt pathway in osteoblasts, sclerostin negatively regulates osteoblast proliferation and differentiation [8–10]. The mice with sclerostin gene knockout showed increased bone formation, bone mass, and bone strength [11]. Sclerostin deficiency also results in a high bone mass phenotype in humans [12,13]. In contrast, the overexpression of the sclerostin gene results in osteopenia in transgenic mouse model [14]. These studies showed an inverse relationship between sclerostin and bone formation. Sclerostin antibody (Scl-Ab) can neutralize the inhibitory effects of sclerostin on Wnt pathway [15]. The treatment of Scl-Ab resulted in increased bone formation, bone mass, and bone anabolic activity in a rat model of postmenopausal osteoporosis [15]. Additionally, Scl-Ab treatment also increased bone formation and bone mineral density in postmenopausal women [16]. The primary purpose of this study was to investigate the effects of Scl-Ab treatment in an open fracture model in young rats. We hypothesized that Scl-Ab treatment would accelerate open fracture healing. In addition, the anabolic effects of Scl-Ab treatment in intact femora were also investigated.
8
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
2. Materials and methods 2.1. Animals A total of 48 eight-week-old male Sprague–Dawley rats were purchased from the Laboratory Animal Center of the Shanghai Jiaotong University. The experimental protocol was approved by the Animal Study Committee of the University (Ref. No. 59/012). Rats were acclimated to 12 hour light/12 hour dark cycle at 22 °C for 1 week. During the experimental period, rats were housed in cages (25 × 30 × 60 cm2) with ad libitum access to water and pelleted commercial rodent diet. 2.2. Femoral osteotomy model Femoral diaphyseal fractures were produced in a standard manner as previously described [17]. All osteotomies were performed under general anesthesia. The right femur was fixed intramedullarily using a Kirschner wire (diameter 1.5 mm, Stryker China, Hong Kong, China). Then, the right femoral diaphysis was transversely fractured with a semi-lunar osteotome at the midshaft of the femur. The fracture fragments of femur were contacted and stabilized after fracture. All wires were cut level with the condyles to avoid restriction of motion of the knee joint. After recovery from anesthesia, unrestricted activity was allowed. Post-operation, animals were randomly divided into the SclAb treatment group (subcutaneous injection, 25 mg/kg body weight, two times per week) or vehicle (saline) treatment group. This dosage (25 mg/kg body weight, two times per week) has been previously reported to be effective to enhance fracture healing in rats [18]. In both groups, treatments started from day 1 after fracture and lasted until sacrifice. The rats were sacrificed at 1 (n = 6), 2 (n = 6), 4 (n = 6), and 6 (n = 6) weeks after fracture. At 1 and 2 weeks, six right femora from each group, which have been X-rayed, were immediately bisected along sagittal plane. The two parts of the femur were respectively used for western blotting and histology (including immunohistochemistry) analysis. At 4 and 6 weeks, both the right and left femora were collected; after X-ray and micro-CT analysis, the right femora were used for biomechanical testing. The left femora were only used for micro-CT analysis at 4 and 6 weeks (Table 1). 2.3. Western blotting analysis At 1 and 2 weeks, the protein expression of proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) in the callus was determined by Western blotting analysis. For extraction of the protein fraction, frozen tissue samples, which have been harvested from the callus at the fracture site, were homogenized. After that, the proteins were extracted in lysis buffer. Proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (PAGE). The protein bands were transferred to a nitrocellulose membrane by standard protocols and probed using anti-PCNA (1:1000; Dako Cytomation, Hamburg, Germany) and anti-BMP-2 (1:200; Abcam, Cambridge, UK). Table 1 Number of animals per study group. 1 week
Control Scl-Ab
2 weeks
4 weeks
The secondary horse radish peroxidase-labeled anti-IgG antibody (1:5000; GE Healthcare, Freiburg, Germany) was applied and incubated at room temperature. Protein expression was visualized by luminol-enhanced chemiluminescence reaction (Hyperfilm ECL, AmershamBiosciences, Freiburg, Germany). The optical densities of bands were assessed densitometrically (Geldoc, Quantity one software, BioRad, Hercules, CA, USA). After a stripping procedure, the Western blots of β-actin were performed on the same membrane. 2.4. Histology At 1 and 2 weeks, one part of each right femur, which had been bisected along sagittal plane, was fixed with 4% paraformaldehyde overnight at room temperature, and decalcified in 14% EDTA for 6 weeks (liquid refreshed weekly). After sufficient decalcification was achieved (judged by the easy penetration of a needle), the sample was dehydrated through an ethanol series, embedded in paraffin, and 5 μm sections were cut longitudinally. The sections were stained with hematoxylin and eosin (HE) for light microscopic examination (Zeiss Aixoplan, Zeiss, Oberkochen, Baden-Württemberg, Germany). The bone tissue area fraction and cartilage tissue area fraction were calculated by software using an analysis image window, which measures 1.6 mm2, in the central part of external callus [7]. The average of ten tissue sections was used to determine callus area fraction. The area fraction of the cartilage and bone was evaluated in a blinded manner by an independent investigator. 2.5. Immunohistochemical staining Five micrometer sections were deparaffinized, rehydrated, and treated (95 °C, 10 min) in an antigen retrieval buffer of sodium citrate (pH 6.0). Sections were cooled (room temperature, 20 min) and washed in dH2O. Endogenous peroxidase activity was blocked by 3% H2O2 (37 °C, 10 min). Sections were blocked with 10% bovine serum (room temperature, 30 min). Then, sections were incubated with mouse monoclonal PCNA primary antibody (1:100; Southern Biotech, Birmingham, AB, USA) or mouse monoclonal BMP-2 primary antibody (1:300; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 °C. The next day, sections were incubated with the secondary antibody peroxidaselabeled goat anti-mouse IgGs (1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), then visualized with diaminobenzidine for a predetermined time, and counterstained with hexatoxylin. For negative controls, the primary antibody was replaced with 0.01 mol/L phosphate buffered saline. In the external callus, the number of PCNA-positive cells was analyzed. Four regions (0.9 mm × 1.5 mm in size), distributed in 2 corner and 2 central areas of external callus were used to calculate PCNA-positive cells in each section [19]. The ratio of PCNA-positive to total cells was calculated and expressed as a percentage. The average ratio of ten sections was used as the PCNA score. The integrated optical density (IOD) of the BMP-2 positive staining was analyzed using an image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Bethesda, MD, USA). The IOD was also analyzed using four regions (0.9 mm × 1.5 mm in size), which were also distributed in 2 corner and 2 central areas of external callus, and the average IOD of 10 sections was used as the IOD score.
6 weeks
WB
Histo
WB
Histo
μCT
Bm
μCT
Bm
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
n=6 n=6
The number of animals in Scl-Ab group and control group. Analysis methods included Western blotting (WB), histology (Histo, including immunohistochemistry), micro-CT (μCT), and biomechanics (Bm). At 1 and 2 weeks, six right femora from each group were bisected along sagittal plane. The two parts of the femur were respectively used for Western blotting and histology (including immunohistochemistry) analyses. At 4 and 6 weeks, both the right and left femora were collected; after μCT analysis, the right femora were used for biomechanical testing. The left femora were only used for microCT analysis at 4 and 6 weeks.
2.6. X-ray At 1, 2, 4, and 6 weeks, the anteroposterior X-ray of the right femora was examined by digital radiography (55 kv, 3 mas; MX-20; Faxitron Bioptics, Tucson, AZ, USA) to assess the progress of fracture repair. 2.7. Micro-computed tomography (μCT) At 4 and 6 weeks, both the right and left femora were scanned using a Micro-CT system (μCT40, Scanco Medical, Brüttisellen, Switzerland).
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
9
For all measurements, the matrix size was 1024 × 1024, and the isotropic voxel resolution was 12 μm. For the right femur, the scan line was placed at the central of fracture callus. The maximum callus crosssectional area (C.Ar), total bone mineral content (Tt.BMC) and total bone mineral density (Tt.BMD) of the central 1 mm of the fracture callus were assessed [20]. The threshold ≥ 464 mg/cm3 was defined to separate the bone/callus from the marrow [21]. For the left femur, the midshaft of femur spanning 10% of the femur height (threshold 710 mg/cm3) was investigated [17]. The total bone mineral area (Tt.BMA), Tt.BMC, and Tt.BMD were also calculated, respectively. 2.8. Biomechanical testing At 4 and 6 weeks, after the measurement of micro-CT, a four-point bending machine (Avalon Technologies, Rochester, MN, USA) was used to evaluate the biomechanical property of the right femora at the fracture site. The femur was installed on fixture (anterior surface facing upward on two lower support points 20 mm apart, and the two upper bars 8 mm apart). Load was applied at a rate of 5 mm/min until fracture. The stiffness (N/mm, defined as the slope of the linear portion of the load–deformation curve), ultimate load (N; defined as the maximum force that the specimen sustained), and energy (N × mm; defined as the area under the load–deformation curve) were calculated according to the load–deformation curve. 2.9. Statistical analysis Data were expressed as mean ± standard deviation (SD). Statistical computation of data was performed using the statistical package SPSS 10.0 (SPSS, Chicago, IL, USA). Differences between two groups were tested by a student's t-test. For comparisons across three or more groups, two-way ANOVA followed by Bonferroni test was used. A Pvalue less than 0.05 was considered significant. 3. Results Throughout this study, no difference in the mean weight was observed between two groups. The rats in both groups resumed normal activity within three days after operation. All rats survived the study. There were no technical failure (poor fracture fixation) and infection that happened during or after surgery. 3.1. Systematic Scl-Ab administration increased PCNA and BMP-2 expression within the callus At 1 and 2 weeks, the expression of PCNA and BMP-2 was detected in the callus of the Scl-Ab group and control group. At 1 week, the PCNA and BMP-2 levels measured from the Scl-Ab group were significantly higher than that of the control group (all P b 0.01) (Fig. 1). At 2 weeks, both the PCNA and BMP-2 levels in callus from the Scl-Ab group were still significantly higher than that of the control group (all P b 0.01) (Fig. 1). At 1 and 2 weeks, for both groups, a lot of PCNA-positive cells were observed in the external callus. Compared with the control group, the Scl-Ab group had 61% and 98% higher PCNA score at 1 week and 2 weeks, respectively (all P b 0.01) (Fig. 2). The positive signals of BMP-2 were also observed in callus tissues of both groups (Fig. 3). At 1 and 2 weeks, the Scl-Ab group demonstrated a substantially increased IOD of BMP-2 positive signals in the external callus compared with the control group (Fig. 3). 3.2. Systematic Scl-Ab administration accelerated fracture healing At 1 and 2 weeks post-fracture, compared with the control group, a decrease in cartilage callus in Scl-Ab group was seen (Fig. 4). The callus in Scl-Ab group was more mature and contained less cartilage and more dense bone. Correspondingly, a more rapider fracture healing in the
Fig. 1. Western blotting analyses of proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) in rats treated with Scl-Ab and rats treated with vehicle at 1 and 2 weeks post-fracture. Data were given as mean ± SD, * b 0.01.
Scl-Ab group was also conformed by X-ray compared with the control group at 4 weeks. At this time point, the fracture line was still clearly visible in the control group (Fig. 5). In comparison, the fracture line had disappeared in the Scl-Ab group. The fracture appeared to have healed.
3.3. Systematic Scl-Ab administration increased bone mass and biomechanical properties in callus At 4 and 6 weeks after fracture, the C.Ar, Tt.BMC, and Tt.BMD of the callus were significantly different between two groups. At both 4 and 6 weeks, C.Ar, Tt.BMC, and Tt.BMD were significantly higher in Scl-Ab group than in control group (all P b 0.01) (Fig. 6). Biomechanical testing showed that the Scl-Ab group exhibited 19% (P b 0.01) and 29% (P b 0.01) higher stiffness at 4 weeks and 6 weeks, respectively, 56% (P b 0.01) and 97% (P b 0.01) higher ultimate load at 4 weeks and 6 weeks, respectively and 32% (P b 0.01) and 56% (P b 0.01) higher energy at 4 weeks and 6 weeks, respectively compared with the control group (Table 2).
3.4. Systematic Scl-Ab administration showed anabolic effects in intact femur Micro-CT analysis of the intact left femora showed that the bone mass of diaphysis was significantly increased by Scl-Ab treatment (Fig. 7). At 4 weeks, in the midshaft of diaphysis, the effects of the SclAb treatment could be observed as a significant increase in Tt.BMA of 10% (P b 0.01), Tt.BMC of 14% (P b 0.05), and Tt.BMD of 9% (P b 0.01). Moreover, at 6 weeks, Tt.BMA was increased by 12% (P b 0.01), Tt.BMC was increased by 65% (P b 0.01), and Tt.BMD was increased by 17% (P b 0.01), respectively.
10
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
Fig. 2. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in Scl-Ab group and control group at 1 and 2 weeks after fracture. At 1 and 2 weeks, PCNApositive staining was found predominantly in external callus surrounding the fracture site. Old cortical bone did not contain PCNA-positive cells. Scale bars represent 50 μm. (A) PCNA-positive cells in the control (a, c) and Scl-Ab (b, d) groups at 1 week (a, b) and 2 weeks (c, d). The Scl-Ab group had more PCNA-positive cells in the external callus compared with the control group. (B) The PCNA score in each group was calculated. Data were given as mean ± SD, n = 6/group/time point, * b 0.01.
4. Discussion At the early stage of fracture healing, abundant undifferentiated mesenchymal cells and osteoprogenitor cells aggregate, proliferate, differentiate, and express various cytokines to participate in the fracture healing [22]. Therefore, it is very important to closely regulate the early stage in order to accelerate fracture healing. Our results clearly showed that Scl-Ab improved the cell proliferation and expression of BMP-2 at the early stage post-fracture, and accelerated the process of fracture healing. It also enhanced callus volume, mineralization, and mechanical properties across the 6-week time course. The activation of Wnt pathway increases the β-catenin (a key positive regulator of Wnt pathway) level in cultured osteoblasts [23]. Robinson et al. also reported that β-catenin expression was increased in pre-osteoblasts and osteoblasts in the inner periosteum by injecting SB-415286 (a Wnt pathway activator) into the calvaria of growing rats [24]. Scl-Ab can neutralize the inhibitory effects of sclerostin on Wnt/b-catenin pathway in vitro. The expression of β-catenin was significantly enhanced in sclerostin knockout mice than in sclerostin wildtype mice [25]. Therefore, the Scl-Ab administration may stimulate the Wnt/β-catenin pathway to promote β-catenin level. In this study, at 1 and 2 weeks, the callus specimens from the Scl-Ab group contained more PCNA-positive cells than did the control group (Fig. 2). This showed that the systematic Scl-Ab administration significantly stimulated the proliferation of cells in the callus. Axin is also an intracellular inhibitor in the canonical Wnt/β-catenin pathway. It has been conformed that, compared with the Axin+/+ type counterpart, calvarial osteoblasts in Axin−/− mice had a higher level of β-catenin
Fig. 3. Immunohistochemical staining of bone morphogenetic protein-2 (BMP-2) in control (a, c) and Scl-Ab (b, d) groups at 1 (a, b) and 2 (c, d) weeks following fracture. Scale bars represent 50 μm. (A) BMP-2 positive signals in the control (a, c) and Scl-Ab (b, d) groups at 1 week (a, b) and 2 weeks (c, d). At 1 and 2 weeks, the enhanced BMP-2 expression was clearly observed in Scl-Ab group. (B) The IOD of BMP-2 positive signals in each group was calculated. Data were given as mean ± SD, n = 6/group/time point, * b 0.01, ** b 0.05.
expression level. Correspondingly, the proliferation of osteoblast was substantially increased [26]. LRP5 gene is a Wnt protein receptor. The knockout mice (LRP5−/−) displayed impaired β-catenin expression and reduced osteoblast proliferation in long bones [27]. The increases of regenerating bone in sclerostin knockout mice were due to an increase in the osteoblast numbers [25]. In our study, in Scl-Ab group, the higher cell proliferation can be observed. Based on early studies, we hypothesized that the more PCNA-positive cells in Scl-Ab group were likely to be mainly caused by a higher number of proliferating osteoblasts or osteoblast precursors. As the most potent osteoinductive agents, BMP-2 plays a critical role in bone repair by mobilizing osteoprogenitor cells, stimulating the differentiation processes of mesenchymal cells to an osteoblastic lineage, and enhancing bone formation [28]. Therefore, to induce the callus at the fracture site to increase BMP-2 expression will be very conducive to fracture healing [29]. In addition, as the most effective inducer of osteogenesis, endogenous BMP-2 is usually regarded as one of the indicators to evaluate the biological environment in bone formation [30]. The present study revealed that, at 1 and 2 weeks, the expression of endogenous BMP-2 signal in the Scl-Ab group was significantly increased than in the control group (Figs. 1, 3). The significantly higher bone mass, C.Ar and biomechanical properties also conformed increased fracture healing in the Scl-Ab group (Fig. 6) (Table 2). This enhanced bone formation was partly contributed to improved expression of endogenous BMP-2 at least. The mechanism of an early increase in endogenous BMP-2 by Scl-Ab required further investigation. In similar fracture models, previous studies reported fracture healing with both endochondral and direct bone formation [17]. In this study, at weeks 1 and 2, the calluses in Scl-Ab group contained less cartilage.
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
11
Fig. 4. Histological assessments of fracture callus. (A) These images showed a decrease in cartilage tissue and an enhanced bone formation in Scl-Ab group (b, d) compared with control group (a, c) at 1 week (a, b) and 2 weeks (c, d). (B) Scl-Ab group showed less cartilage area compared with the vehicle group at 1 and 2 weeks. Scale bars represent 50 μm. Data were given as mean ± SD, n = 6/group/time point, * b 0.01.
Fig. 6. Scl-Ab treatment improved the bone mass in the callus at 4 and 6 weeks after fracture. (A) Representative axial micro-CT images in control group (a, c) and Scl-Ab group (b, d) at 4 weeks (a, b) and 6 weeks (c, d) after fracture. Micro-CT images represent the group medians for peak load. (B) Micro-CT analysis showed that Scl-Ab treatment resulted in improved bone mass in the right femora compared with the control group. Data were given as mean ± SD, n = 6/group/time point, * b 0.01.
Although the expression of sclerostin has been detected in chondrocytes in human, it is unclear whether Scl-Ab administration had any effects on the formation of cartilage [10]. Interestingly, rat fractures treated with a Wnt activator healed without the formation of cartilage islets in the callus [17]. The activation of Wnt pathway causes the mesenchymal progenitor cells to differentiate into osteogenic cells instead of chondrocytes [31,32]. In this study, there was a less pronounced effect of reducing cartilage callus. This may be explained that the potency of Scl-Ab to activate the Wnt pathway is lower compared with those Table 2 Mechanical test of the fractured femora in rats at 4 and 6 weeks after fracture.
Fig. 5. Systemic administration of Scl-Ab increased bone healing in rat femoral fracture. The radiographs of the fractured femora in control group (a, c, e, g) and Scl-Ab group (b, d, f, h) at 1 week (a, b), 2 weeks (c, d), 4 weeks (e, f) and 6 weeks (g, h). Note the union in Scl-Ab group and the fracture line in the control group at 4 weeks.
Parameters
Time
n
Control
Ultimate load (N) Energy (N*mm) Stiffness (N/mm)
4 6 4 6 4 6
6 6 6 6 6 6
80.2 109.2 32.2 52.6 231.2 303.6
weeks weeks weeks weeks weeks weeks
± ± ± ± ± ±
5.3 8.2 3.2 4.3 11.3 12.1
n
Scl-Ab
6 6 6 6 6 6
125.2 215.3 42.5 82.3 274.8 392.5
± ± ± ± ± ±
P 8.2 10.3 3.9 5.2 11.7 14.6
b0.01 b0.01 b0.01 b0.01 b0.01 b0.01
Mechanical test of the fractured femora. The four-point bending tests showed enhanced mechanical properties in Scl-Ab group compared with control group at 4 and 6 weeks post-fracture. Ultimate load, energy, and stiffness were derived from load/displacement curves. Data were given as mean ± SD.
12
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13
Fig. 7. Scl-Ab treatment improved the bone mass in the intact femora at 4 and 6 weeks after fracture. Micro-CT analysis showed that Scl-Ab treatment resulted in apparent anabolic effects in the intact femora compared with the control group. Data were given as mean ± SD, n = 6/group/time point, * b 0.01, ** b 0.05.
Wnt activators [33]. After the fracture has been stabilized sufficiently, the transition from cartilaginous callus to bone at the fracture would begin in no time [20]. Scl-Ab administration led to an earlier stabilization of the fracture [18]. Perhaps, the progress from cartilage to woven bone was also accelerated in the Scl-Ab group (Fig. 4). This was beneficial to produce more mature calluses at the fracture site. In this study, the systemic Scl-Ab administration increased Tt.BMA, Tt.BMC, and Tt.BMD in the non-operated femur of rats. In ovariectomized rats, Scl-Ab treatment showed these skeletal effects. In cynomolgus monkeys, Scl-Ab treatment significantly increased bone mass at the lumbar spine, femoral neck, and distal radius, at which osteoporotic fractures occur frequently [34]. This demonstrated a profound systemic effect of Scl-Ab on bone tissue. The anabolic effect of Scl-Ab treatment that increases bone mass in the non-operated bone should be advantageous to prevent a secondary osteoporotic fracture. Until now, no systemic therapy has been found that indeed improves fracture repair in patients. As an approved anabolic therapy in osteoporosis, intermittent PTH has been conformed to improve open fracture healing in rat model [19,35]. However, in clinical trials, this therapy failed to achieve its major objective of reducing time to radiographic healing [36]. In addition, although enhancing callus remodeling, intermittent PTH did not increase whole-femur strength in cynomolgus monkeys [37]. Whether the improvements in bone mass and strength in rat open fracture models treated with Scl-Ab will result in improved fracture healing in humans required clinical trials to verify. Taken together, the systemic administration of Scl-Ab increased bone formation, bone mass, and bone strength in fractured and intact bones in rat open fracture models. These results support the therapeutic potential of Scl-Ab as a noninvasive strategy to enhance open fracture healing.
References [1] Wigner NA, Luderer HF, Cox MK, Sooy K, Gerstenfeld LC, Demay MB. Acute phosphate restriction leads to impaired fracture healing and resistance to BMP-2. J Bone Miner Res 2010;25:724–33. [2] Einhorn TA. Enhancement of fracture-healing. J Bone Joint Surg Am 1995;77: 940–56. [3] Mont MA, Ragland PS, Biggins B, Friedlaender G, Patel T, Cook S, et al. Use of bone morphogenetic proteins for musculoskeletal applications. An overview. J Bone Joint Surg Am 2004;86-A(Suppl. 2):41–55. [4] Garrison KR, Donell S, Ryder J, Shemilt I, Mugford M, Harvey I, et al. Clinical effectiveness and cost-effectiveness of bone morphogenetic proteins in the non-healing of fractures and spinal fusion: a systematic review. Health Technol Assess 2007;11: 1–150 [iii-iv]. [5] Macsai CE, Foster BK, Xian CJ. Roles of Wnt signalling in bone growth, remodelling, skeletal disorders and fracture repair. J Cell Physiol 2008;215:578–87. [6] Silkstone D, Hong H, Alman BA. Beta-catenin in the race to fracture repair: in it to Wnt. Nat Clin Pract Rheumatol 2008;4:413–9. [7] Chen Y, Whetstone HC, Lin AC, Nadesan P, Wei Q, Poon R, et al. Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing. PLoS Med 2007;4:e249.
[8] Balemans W, Ebeling M, Patel N, Van Hul E, Olson P, Dioszegi M, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001;10:537–43. [9] Brunkow ME, Gardner JC, Van Ness J, Paeper BW, Kovacevich BR, Proll S, et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001;68:577–89. [10] Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22: 6267–76. [11] Li X, Ominsky MS, Niu QT, Sun N, Daugherty B, D'Agostin D, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res 2008;23:860–9. [12] Hamersma H, Gardner J, Beighton P. The natural history of sclerosteosis. Clin Genet 2003;63:192–7. [13] Vanhoenacker FM, Balemans W, Tan GJ, Dikkers FG, De Schepper AM, Mathysen DG, et al. Van Buchem disease: lifetime evolution of radioclinical features. Skeletal Radiol 2003;32:708–18. [14] Kramer I, Loots GG, Studer A, Keller H, Kneissel M. Parathyroid hormone (PTH)induced bone gain is blunted in SOST overexpressing and deficient mice. J Bone Miner Res 2010;25:178–89. [15] Li X, Ominsky MS, Warmington KS, Morony S, Gong J, Cao J, et al. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res 2009;24:578–88. [16] Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single-dose, placebo-controlled, randomized study of AMG 785, a sclerostin monoclonal antibody. J Bone Miner Res 2011;26: 19–26. [17] Sisask G, Marsell R, Sundgren-Andersson A, Larsson S, Nilsson O, Ljunggren O, et al. Rats treated with AZD2858, a GSK3 inhibitor, heal fractures rapidly without endochondral bone formation. Bone 2013;54:126–32. [18] Suen PK, He YX, Chow DH, Huang L, Li C, Ke HZ, et al. Sclerostin monoclonal antibody enhanced bone fracture healing in an open osteotomy model in rats. J Orthop Res 2014;32:997–1005. [19] Nakajima A, Shimoji N, Shiomi K, Shimizu S, Moriya H, Einhorn TA, et al. Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1–34). J Bone Miner Res 2002;17:2038–47. [20] Ominsky MS, Li C, Li X, Tan HL, Lee E, Barrero M, et al. Inhibition of sclerostin by monoclonal antibody enhances bone healing and improves bone density and strength of nonfractured bones. J Bone Miner Res 2011;26:1012–21. [21] Komatsubara S, Mori S, Mashiba T, Nonaka K, Seki A, Akiyama T, et al. Human parathyroid hormone (1–34) accelerates the fracture healing process of woven to lamellar bone replacement and new cortical shell formation in rat femora. Bone 2005;36:678–87. [22] Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects of bone healing. Injury 2005;36:1392–404. [23] Marsell R, Sisask G, Nilsson Y, Sundgren-Andersson AK, Andersson U, Larsson S, et al. GSK-3 inhibition by an orally active small molecule increases bone mass in rats. Bone 2012;50:619–27. [24] Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, Cullen DM, Zhao W, Li C, et al. Wnt/ beta-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem 2006;281:31720–8. [25] McGee-Lawrence ME, Ryan ZC, Carpio LR, Kakar S, Westendorf JJ, Kumar R. Sclerostin deficient mice rapidly heal bone defects by activating beta-catenin and increasing intramembranous ossification. Biochem Biophys Res Commun 2013; 441:886–90. [26] Yu HM, Jerchow B, Sheu TJ, Liu B, Costantini F, Puzas JE, et al. The role of Axin2 in calvarial morphogenesis and craniosynostosis. Development 2005;132:1995–2005. [27] Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass 2nd DA, et al. Cbfa1independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol 2002; 157:303–14. [28] Eriksson C, Nygren H, Ohlson K. Implantation of hydrophilic and hydrophobic titanium discs in rat tibia: cellular reactions on the surfaces during the first 3 weeks in bone. Biomaterials 2004;25:4759–66.
G. Feng et al. / International Immunopharmacology 24 (2015) 7–13 [29] Stucki U, Schmid J, Hammerle CF, Lang NP. Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans. Clin Oral Implants Res 2001;12:121–7. [30] Zheng LW, Ma L, Cheung LK. Changes in blood perfusion and bone healing induced by nicotine during distraction osteogenesis. Bone 2008;43:355–61. [31] Bennett CN, Longo KA, Wright WS, Suva LJ, Lane TF, Hankenson KD, et al. Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci U S A 2005;102: 3324–9. [32] Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrowderived mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci 2009;66:236–53. [33] Baron R, Rawadi G, Roman-Roman S. Wnt signaling: a key regulator of bone mass. Curr Top Dev Biol 2006;76:103–27.
13
[34] Ominsky MS, Vlasseros F, Jolette J, Smith SY, Stouch B, Doellgast G, et al. Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength. J Bone Miner Res 2010;25:948–59. [35] Tagil M, McDonald MM, Morse A, Peacock L, Mikulec K, Amanat N, et al. Intermittent PTH (1-–34) does not increase union rates in open rat femoral fractures and exhibits attenuated anabolic effects compared to closed fractures. Bone 2010;46:852–9. [36] Aspenberg P, Genant HK, Johansson T, Nino AJ, See K, Krohn K, et al. Teriparatide for acceleration of fracture repair in humans: a prospective, randomized, double-blind study of 102 postmenopausal women with distal radial fractures. J Bone Miner Res 2010;25:404–14. [37] Manabe T, Mori S, Mashiba T, Kaji Y, Iwata K, Komatsubara S, et al. Human parathyroid hormone (1–34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys. Bone 2007;40:1475–82.
