Sclerostin Monoclonal Antibody Enhanced Bone Fracture Healing in an Open Osteotomy Model in Rats Pui Kit Suen,1 Yi-Xin He,1 Dick Ho Kiu Chow,1 Le Huang,1 Chaoyang Li,2 Hua Zhu Ke,2 Michael S. Ominsky,2 Ling Qin1,3 1 Department of Orthopaedics and Traumatology, Lui Che Woo Institute of Innovation Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China, 2Metabolic Disorders Research, Amgen Inc., Thousand Oaks, California, 3Shenzhen Institute of Advanced Technology, Chinese Academy of Science, Shenzhen, China
Received 31 October 2013; accepted 4 March 2014 Published online 30 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22636
ABSTRACT: Sclerostin is a negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment promoted bone healing in various animal models. To further evaluate the healing efficiency of Scl-Ab in osteotomy healing, we investigated the time course effects of systemic administration of Scl-Ab on fracture repair in rat femoral osteotomy model. A total of 120 six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were treated with vehicle or Scl-Ab treatment for 3, 6, or 9 weeks. Fracture healing was evaluated by radiography, micro-CT, micro-CT based angiography, 4-point bending mechanical test and histological assessment. Scl-Ab treatment resulted in significantly higher total mineralized callus volume fraction, BMD and enhanced neovascularization. Histologically, Scl-Ab treatment resulted in a significant reduction in fracture callus cartilage at week 6 and increase in bone volume at week 9, associated with a greater proportion of newly formed bone area at week 6 and 9 by fluorescence microscopy. Mechanical testing showed significantly higher ultimate load in Scl-Ab treatment group at week 6 and 9. This study has demonstrated that Scl-Ab treatment enhanced bone healing in a rat femoral osteotomy model, as reflected in increased bone formation, bone mass and bone strength. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:997–1005, 2014. Keywords: sclerostin; sclerostin monoclonal antibody; osteotomy; fracture healing
Sclerostin is a protein secreted by osteocytes that inhibit bone formation.1 Sclerostin acts as an antagonist of the Wnt/beta-catenin signaling pathway in osteoblasts, and thus negatively regulates the differentiation and function of osteoblasts.1 Sclerostin deficiency leads to a high bone mass phenotype in humans2 and mice3; while over-expression of the sclerostin gene (SOST) in transgenic mouse model leads to osteopenia.4 These results indicated an inverse relationship between sclerostin and bone mass. Furthermore, systemic administration of sclerostin monoclonal antibody (Scl-Ab) inhibits sclerostin function,5 resulting in an increase in bone formation, bone mass, and strength in intact bone in multiple animal models, including rats, non-human primates, and also in unloading models associated with hindlimb-immobilization6 and spaceflight.7 Additionally, Scl-Ab has been shown to increase BMD in healthy men and postmenopausal women.8 Besides promoting bone formation in healthy animals and humans, Scl-Ab administration was also shown to improve bone healing in rodent models of drill hole defects,9,10 critical-size gap defects,11 closed fracture,6 bone defect healing in rats with type 2 diabetes mellitus12 and in cynomolgus monkeys after fibular osteotomy.6 Orthopedic surgeons often treat trauma patients surgically resulting in an “open fracture”, a more challenging clinical condition as compared with a closed fracture that has a better prognosis.13,14 The objective of this study is to investigate the time course effects of systemic administration Conflicts of interest: None. Grant sponsor: Amgen, Inc.; Grant sponsor: UCB Pharma; Grant number: TA095217. Correspondence to: Ling Qin (T: þ852-26323071; F: þ852-26377889; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
of Scl-Ab on fracture repair in an open fracture model using rat femoral osteotomy model, which this healing model resulted in delayed healing compared with closed fracture healing. The progression and quality of fracture healing were assessed by serial weekly radiography, bone mineral density (BMD) and bone volume fraction by micro-CT, angiogenesis by microCT-based angiography, callus composition and dynamics of bone formation by histomorphometric analysis, and fractured bone strength by mechanical testing.
MATERIALS AND METHODS Animals and Femoral Osteotomy Model A total of 120 six-month-old male Sprague–Dawley rats were obtained from the Laboratory Animal Services Center of the Chinese University of Hong Kong. The Animal Experimentation Ethics Committee of the University approved the experimental procedures (AEEC No. 09/042/MIS). Osteotomy was performed under general anesthesia at mid-shaft of the right femur using a circular saw with a diameter of 1.6 cm and a thickness of 0.1 mm (Fine Science Tools, Foster City, CA) and stabilized by intramedullary insertion of a sterilized 1.2 mm diameter Kirschner wire (Stryker China, Hong Kong, China) as described previously.15–17 Rats were randomly assigned to the sclerostin antibody treatment group (Scl-Ab VI, subcutaneous injection, 25 mg/kg, two times per week, as described previously18) or vehicle (saline) treatment group, and both treatments started from Day 1 post-operation. The progress of fracture healing for each animal was monitored weekly by digital radiography (MX-20; Faxitron Bioptics, Tucson, AZ). At week 3, 6, and 9 post-operation, 20 rats from each treatment group were terminated. For each treatment group, six rat femora were prepared and subjected to microCT based angiography prior to decalcification for histological analysis. The femora of the remaining 14 rats were subjected to micro-CT scanning (14 rats), mechanical testing (eight rats), and dynamic histomorphometric analysis (six rats) (Supplementary Fig. S1). JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Radiography and Micro-CT Assessment of the Fracture Callus Digital radiographic images acquired at 3, 6, and 9 weeks post-operation were analyzed by ImageJ (version 1.42q; NIH) to quantify the total area of the external fracture calluses. Femora were scanned by a desktop micro-CT system (mCT40, Scanco Medical, Bru¨ttisellen, Switzerland). The scan range was 13.2 mm (660 slides) centered on the fracture line and the 3D reconstruction of mineralized tissue was performed as described previously.15,19 Lower density bone tissue, representing less mature callus, and higher density bone tissue, representing original cortices and highly mineralized callus, were determined using two thresholds (low ¼ 200, high ¼ 350, Hounsfield units) according to our established protocol15,20 (Supplementary Fig. S2). The analysis determined total bone volume (BVt, mm3, threshold ¼ 200), high-density bone volume (BVh, mm3, threshold ¼ 350), lowdensity bone volume (BVl, mm3, equal to BVt BVh) the callus volume fractions for each of these parameters (BVh/ TV, BVl/TV, and BVt/TV) and the bone mineral density (BMD). A bone strength index (BSI) of the fracture callus was calculated from 1 mm proximal to 1 mm distal of the fracture line to obtain the average cross-sectional area (CSA) of the callus multiplied by the corresponding BMD (BSI ¼ CSA BMD).21 BVt, BVh, BVl, BVt/TV, BVh/TV, and BVl/TV of this sub-region were also determined. Micro-CT Based Angiography Perfusion with Microfil (Microfil MV-117; Flowtech, Carver, MA) was performed as described previously.20 Samples were decalcified in 5% formic acid for 2 weeks and scanned by a desktop micro-CT system. The scan range was 6 mm (300 slides), centered on the osteotomy line. The 3D reconstruction of the blood vessels was performed as previously described using a threshold of 100 Hounsfield units.20 The blood vessel volume (vessel volume, mm3) and connectivity of the blood vessels (connectivity, 1/mm3) were analyzed. Mechanical Test A destructive 4-point bending test was performed as described previously22 (H25KS; Hounsfield Test Equipment Ltd., Redhill, Surrey, UK). The femora were placed with anterior surface facing up with the upper and lower span lengths set to 10 and 26 mm, respectively. Load was applied at a rate of 5 mm/min until failure. Ultimate load (N), energy (J), and stiffness (N/mm) were calculated from the load– deformation curve using the test system’s software (QMAT Professional Material testing software, Hounsfield Test Equipment Ltd.). Histology and Histomorphometric Analysis For decalcified histology, phosphate buffered formalin fixed femora were decalcified by 5% formic acid for 2 weeks and embedded in paraffin. Five-micrometer thick sections were stained by Hematoxylin and Eosin (H&E), Safranin O, and toluidine blue for evaluation under light microscope (Zeiss Aixoplan with Spot RT digital camera; Zeiss, Oberkochen, Germany). Callus composition of the external callus was determined by ImageJ for evaluation of bone tissue area fraction and cartilage tissue area fraction. Sequential fluorescent labeling was used to study the dynamics of bone formation during fracture healing according to our established protocol,19 with calcein green and xylenol orange (10 and 90 mg/kg, respectively, Sigma– JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
Aldrich, St. Louis, MO) subcutaneously injected 2 and 1 week, respectively before euthanasia at each time point. Femora were embedded undecalcified in MMA, sectioned, ground, and polished to 100 mm and observed using a fluorescence microscope (Leica Q500MC; Leica Microsystems Cambridge Ltd, Cambridge, Cambridgeshire, United Kingdom). The temporal change in new bone formation was assessed for the total callus and periosteal callus within a 10 mm region centered on the fracture line; calculated as the ratio of the area labeled with xylenol orange to the area labeled with calcein green using ImageJ19 (Supplementary Fig. S3). Statistical Analysis All data were expressed as mean SD. Two-way ANOVA with Bonferroni posthoc test was used to compare the differences between the Scl-Ab treatment group and the vehicle group across time points. Pearson analysis was used to calculate the correlation coefficient between ultimate load and micro-CT based bone endpoints for each treatment group at week 6 and 9. Values of p < 0.05 were considered significant. All analyses were performed using GraphPad Prism 5 (California).
RESULTS Radiographic Analysis and Bone Mass of Fracture Calluses Radiographic images of week 3, 6, and 9 showed that the Scl-Ab treatment group had a larger fracture callus. Quantitative analysis showed that the Scl-Ab treatment group had a 23–30% larger callus (Fig. 1) compared with the vehicle group at all time points. Micro-CT analysis showed that BMD was 15–16% significantly higher in the Scl-Ab treatment group compared to vehicle groups at each time point. Similarly, the Scl-Ab treatment group had 16–23% significantly higher in BVt/TV at each time point and 20– 27% significantly higher in BVt at week 6 and 9 (p < 0.01), compared to their vehicle groups (Fig. 2). Furthermore, that the amount and proportion of mature (highly mineralized) bone volume were significantly greater with Scl-Ab treatment at week 6 and 9, as reflected in significant increases BVh/TV (26–33%) and BVh (38–42%) compared to their vehicle groups. Scl-Ab treatment was also associated with a significant increase the lower bone density-based BVl/TV (38%) and BVl (26%) at week 3, which may reflect an early progression in the early stage of fracture healing under the Scl-Ab treatment. Total tissue volume (TV) was not significantly affected by treatment at any time point (Table 1). Fractured Bone Strength Surrogates To study the correlation between the mechanical properties and the structural data as determined by micro-CT in the fracture site, the 2 mm sub-region centered on the fracture line was chosen for further analysis. At week 9, ultimate load was significantly correlated with CSA, BVh, BVt, BVl, and BSI in the Scl-Ab treatment group, with no significant effects observed for the week 9 vehicle group or either group at week 6 (Table 2).
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Callus Histology and Histomorphometric Analysis More bony tissue and less cartilage tissue was observed in fracture calluses across all time points in the Scl-Ab treatment groups compared with the vehicle groups by H&E, safranin O, and toluidine blue staining (Figs. 5A and S4). Quantitative analysis supported this observation, with a significant increase (32%) in bone area fraction at week 9, and a significant decrease in cartilage area fraction at week 6 in the Scl-Ab treatment groups compared with the vehicle groups (Fig. 5B). In addition, the Scl-Ab treatment group showed faster mineral deposition during the fracture healing process compared with the vehicle group as demonstrated by fluorescent microscopy (Fig. 6). At week 9, the Scl-Ab treatment group also showed an increase in the rate of new bone formation in both the total callus (þ41%) and the periosteal callus sub-region (þ42%).
DISCUSSIONS
Figure 1. Radiographic analysis of fracture healing progress at the osteotomy site of rat femora. Representative radiographs (A) and quantitative analysis of callus size (B) showed that larger fracture calluses were observed in Scl-Ab treatment group compared with the vehicle group at week 3, 6, and 9 postoperation. Mean SD, n ¼ 20/group/time point, p < 0.05 compared with Vehicle at the same time point.
Callus Angiogenesis Micro-CT based angiography demonstrated that calluses contained greater vessel volume with better connectivity in the Scl-Ab treatment group than the vehicle group at week 3 (Fig. 3). These differences were not observed at week 6 and 9. Callus Mechanical Properties by 4-Point Bending Mechanical Test Only week 6 and 9 groups were subjected to mechanical testing as the fractured femora at week 3 were too weak to test as confirmed in our pilot study. Scl-Ab treatment resulted in significantly higher ultimate load at week 6 (98%) and week 9 (53%). Stiffness and energy to failure also showed similar trends, with SclAb treatment group means that were higher at week 6 (stiffness, þ71%, p ¼ 0.08; energy to failure, þ124%, p < 0.05) and week 9 (stiffness, þ63%, p ¼ 0.08; energy to failure, þ51%, p ¼ 0.08) relative to controls (Fig. 4).
In this study, Scl-Ab improved open fracture healing in a rat femur osteotomy model by enhancing bone volume and mineralization, angiogenesis, and mechanical properties across the 9-week time course. Scl-Ab had previously been reported to improve closed fracture healing in rat femora, while the current data demonstrate similar improvements in a more challenging femoral osteotomy model of open fracture healing. In addition, Scl-Ab treatment improved neovascularization at week 3 post-fracture by micro-CT based angiography, a novel finding that has not previously been reported. Furthermore, Scl-Ab treatment specifically enhanced high mineralized bone volume fraction by micro-CT, which is similar to the treatment effect reported in cynomolgus monkeys by pQCT.18 Our results showed that Scl-Ab treatment increased fracture callus size (by digital radiography) and bone volume (by micro-CT and histology) as compared with vehicle treatment. The fracture callus and cortical bone work as a functional unit during fracture healing; therefore the pre-existing cortical bone was included in the micro-CT analysis of BMD and bone volume. Although the effects of Scl-Ab on the pre-existing cortical surfaces cannot be clearly separated from the effects on the periosteal callus, both should be considered important components of the healing response and contributors to bone strength. The histological assessment of bone volume and new bone formation in the periosteal callus alone does suggest a significant effect of Scl-Ab in this compartment alone. Further analysis of the micro-CT images using two different thresholds to delineate the higher and lower mineralized portions of fracture calluses revealed that, Scl-Ab treatment improved both the BVl/TV and BVh/ TV at week 3 and specifically enhanced the BVh/TV at week 6 and 9 post-operation. These indicated that Scl-Ab treatment may promote an early progression in osteogenesis during the early stage of fracture healing (week 3) and resulting in greater callus mineralization JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Figure 2. Scl-Ab treatment enhanced BMD and bone volume in the fracture callus. Representative micro-CT images (A), BMD (B), and total bone volume fraction (BVt/TV) (C) showed that Scl-Ab treatment resulted in more mineralized tissue in the fracture callus compared to the vehicle group. Mean SD, n ¼ 14/group/time point, p < 0.01 compared with Vehicle at the same time point. The preexisting cortex was also included in this analysis.
at the later stages (week 6 and 9). Although consistent callus bridging was not observed in either treatment group, a strong correlation was found between ultimate load and the bone volume parameters (BSI, CSA, BVh, BVt, and BVl) in the week 9 Scl-Ab treatment group. This result may be related to the greater maturity of
the fracture callus in the Scl-Ab treatment group at week 9, corresponding to greater functional strength. The increases in micro-CT based bone volume and mechanical properties in the Scl-Ab treated femora coincided with similar changes in the histologic composition of the fracture callus. The Scl-Ab treatment
Table 1. Comparison of Micro-CT Analysis of Fracture Calluses From Scl-Ab Treatment Group and Vehicle Group Across Time Points Week 3 Vehicle 3
BVh (mm ) BVl (mm3) TV (mm3) BVt/TV BVh/TV BVl/TV
108.91 12.84 58.21 20.51 350.57 45.56 0.48 0.07 0.32 0.06 0.16 0.04
Week 6 Scl-Ab
120.07 16.14 73.12 26.54 327.74 64.68 0.59 0.03 0.37 0.04 0.22 0.05
Vehicle 93.66 32.07 97.00 55.56 402.62 132.16 0.49 0.08 0.27 0.12 0.23 0.05
Week 9 Scl-Ab
133.42 29.16 96.03 44.99 385.59 74.32 0.60 0.04 0.36 0.10 0.24 0.07
Values are mean SD. p < 0.05; p < 0.01 compared with vehicle at the same time point. JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
Vehicle
Scl-Ab
135.12 12.26 61.35 13.93 351.93 61.70 0.56 0.05 0.39 0.05 0.17 0.02
187.05 16.60 61.74 11.78 384.48 47.71 0.65 0.04 , 0.49 0.04 0.16 0.02
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Table 2. Correlation Between Ultimate Load and Different Parameters in Micro-CT Analysis Correlation Coefficient (Pearson) Ultimate Load Treatment Week Week Week Week
6 6 9 9
Vehicle Scl-Ab Vehicle Scl-Ab
CSA
BVh
BVt
BVl
BVh/TV
BVt/TV
BVl/TV
BMD
BSI
0.267 0.07572 0.7178 0.8158
0.1296 0.3026 0.2207 0.8091
0.3897 0.3606 0.7502 0.818
0.318 0.3285 0.7615 0.7386
0.05519 0.2442 0.3272 0.2006
0.02022 0.6151 0.2472 0.3934
0.1973 0.5717 0.5530 0.5618
0.02019 0.4624 0.05540 0.3176
0.4223 0.3003 0.7527 0.8870
CSA, cross-sectional area; BVt, total bone volume; BVh, volume of high-density bone; BVl, volume of low-density bone; BVt/TV, total bone volume fraction; BVh/TV, volume fraction of high-density bone; BVl/TV, volume fraction of low-density bone; BMD, bone mineral density; BSI, bone strength index. p < 0.05; p < 0.01 significance of the correlation.
group contained more bony tissue and less cartilage tissue, which is similar to the previous reports in sclerostin knockout (SOST-KO) mice and after Scl-Ab treatment in cynomolgus monkeys undergoing fibular osteotomy.3,18 Based on our histomorphometric assessment, bone healing appears to be dominated by endochondral ossification in the rat osteotomy model. Therefore the increase in bone area fraction and decrease in cartilage area fraction in the Scl-Ab treat-
ment group would be consistent with an enhancement of endochondral ossification. Under this paradigm, the early increase in vascularization would accelerate endochondral ossification through a faster transition from cartilage through its hypertrophic stage to bone. Intramembranous bone formation at the periosteum distal to the osteotomy site may also contribute to the observed increases in bone volume, and could also be positively affected by increased vascularization.
Figure 3. Scl-Ab treatment enhanced neovascularization at callus at week 3 post-operation. Representative micro-CT images (A), vessel volume (B), and connectivity (C) showed that Scl-Ab treated samples contained more vessel volume and better connectivity compared to the vehicle group at week 3 by micro-CT based angiography. Mean SD, n ¼ 6/group/time point, p < 0.01 compared with Vehicle at the same time point. JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Figure 4. Mechanical test of the fractured femora. Four-point bending test showed that Scl-Ab treatment significantly enhanced the ultimate load (A) (p < 0.01), stiffness (B) (p ¼ 0.08), and energy to failure (C) (p < 0.05 at week 6 and p ¼ 0.08 at week 9) in fractured femora compared with the vehicle group at the same time point. Mean SD, n ¼ 8/group/time point, p < 0.05; p < 0.01 compared with vehicle at the same time point.
During fracture repair, early bone deposition is woven in nature, resulting in areas of fluorescent labeling rather than clear linear labeling on undecalcified MMA sections. Traditional mineral apposition rate (MAR) and bone formation rate (BFR) analysis based on linear labeling may therefore not be suitable for measurement. Therefore, areas labeled by fluorochromes were measured as a representative index for new bone growth as previously reported.19 The current study showed that Scl-Ab treatment resulted in an increase in the rate of bone formation at week 9, especially in the periosteal callus. Temporally, new bone formation continued to increase from week 6 to 9 in the Scl-Ab group, whereas it began to decline in the vehicle controls. These data coincided with the results of radiography, micro-CT analysis and histological callus composition. The continuous increase of bony callus volume fraction and BMD across time points indicates a faster mineralization of the fracture callus, and Scl-Ab treatment significantly induced bone formation and increased the mineral density in the fracture callus during fracture healing. The enhanced bone formation in the Scl-Ab treatment group was associated with improved mechanical properties via 4-point bending test. Although mechanical testing could not be performed on week 3 calluses due to the weakness of the fracture calluses, mechanical testing of week 6 and 9 calluses showed significant improvements in ultimate load with Scl-Ab treatment. These data indicate that Scl-Ab treatment significantly enhanced the healing rate of the fracture callus. The improvement in fracture healing depends on angiogenesis and it is observed that Scl-Ab treatment resulted in more and better connected vascular structure, as reflected by higher vessel volume and better connectivity, at the early time point (week 3) by microCT based angiography. Angiogenesis is an important process for fracture healing, and blood supply and its stability are the key factors that promote fracture healing process.23,24 Angiogenesis and osteogenesis is JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
a tightly coupled process. The vascular endothelial growth factor (VEGF) expression is positively regulated by the hypoxia-inducible factor-1 alpha (HIF-1alpha) and Osterix (Osx) in osteoblast cell cultures,25–27 and Wnt/beta-catenin signaling is involved in regulating VEGF-A expression for angiogenesis during retinal blood vessel formation28 and in colon cancer.29 Meanwhile, VEGF over-expression enhanced beta-catenin stabilization and resulted in a high bone mass phenotpye in a transgenic mice model.30 Besides regulating VEGF expression, both HIF-1alpha and Osx also inhibit the Wnt signaling pathway through inducing sclerostin and Dickkopf-1 expression, respectively31–33 (Supplementary Fig. S5). Neovascularization during fracture healing allows nutrient influx and delivers osteoprogenitors cells to the fracture site for osteogenesis to take place.34,35 In the current study, we observed that Scl-Ab treatment induced an early increase in neovascularization, which may contribute to enhanced fracture healing. The mechanism of an early increase in neovascularization by Scl-Ab required further investigation. In the healing efficiency, Scl-Ab treatment showed to improve 60% in peak load by 3-point bending test at week 7 post-operation in rat closed femoral fracture,18 whereas Scl-Ab treatment showed 50% increase in ultimate load by 4-point bending test at week 6 postoperation in this study. This might suggest a better improvement by Scl-Ab treatment in promoting open fracture healing. However, the mechanical test methodology, the age of rats and the time points are not matched in these two studies and therefore the results obtained cannot be directly compared with each other. In the future, a head-to-head comparison of Scl-Ab in open and closed fracture models is required to address the issue. The limitations of this study are: (1) external callus bridging was not observed by radiography or histology in the osteotomy model at week 9 and (2) compared with the ultimate load of the non-fractured femora
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Figure 5. Histological assessments of fracture calluses. Sagittal section of fracture calluses at the osteotomy site of femoral shaft in rats (A) and fracture callus composition (B). Scl-Ab treatment group showed more bony tissue and less cartilage area compared with the vehicle group starting at week 3. Magnification, 16. Scale bar ¼ 1 mm. Mean SD, n ¼ 6/group/time point, p < 0.05; p < 0.01 compare with Vehicle at the same time point.
from aged-matched rats (150 N, unpublished data), the ultimate load of femora after week 9 of Scl-Ab treatment only recovered around 40% of the original strength. Compared with closed fracture, open fracture is a more traumatic injury and therefore is more
challenging and may take longer to repair. Open fracture often results in wound infections, severe soft tissue injury and periosteal disruption, which contribute to slow healing and often in non-union; therefore, more care is needed for open fracture JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Figure 6. Dynamics of bone formation in Vehicle and Scl-Ab treatment groups at week 6 and 9. Representative fluorescent images (A) showing the new bone formed 2 weeks and 1 week prior to termination, as labeled by calcein green (green) and xylenol orange (red), respectively. The ratio of new bone formation in the total callus and periosteal callus was calculated as the ratio of area labeled in red versus green (B). Scl-Ab treatment groups showed an increased in rate of new bone formation in the total callus and periosteal callus at week 9. Magnification, 16. Scale bar ¼ 1 mm. Mean SD, n ¼ 6/group/time point, p < 0.05 compared with Vehicle at the same time point.
healing management.16,17,36–38 Among these factors, the periosteum is the most important for optimal bone healing, as it is one of the sources of osteoprogenitors cells and its damage or removal results in delayed bone healing.39,40 The damaged periosteum in the osteotomy surgery in this study likely resulted in delayed in the bridging of the fracture callus. The fracture gaps in rat closed femoral fractures are usually bridged at around week 6 after fracture,16 but in our femoral osteotomy model, the fracture callus in the radiographic and histological images were still not bridged at week 9. Therefore, a longer time point would be required to examine the effects of Scl-Ab treatment across the full time-course of functional recovery, as reflected in callus union and bone strength near intact levels. The continuing increases in new bone formation and strong correlation between the bone mass parameters to ultimate load at the fracture site in the week 9 Scl-Ab treatment group suggests a trend toward further improvements with Scl-Ab treatment at later stages of healing. Taken together, the systemic administration of Scl-Ab was able to enhance the fracture healing in rat femoral osteotomy model, as demonstrated by increased bone formation, bone mass and bone JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
strength. Our results support the therapeutic potential of sclerostin antibody to enhance open fracture healing.
ACKNOWLEDGMENTS We thank Zhang Ge for assistance in initial proposal preparation, Liu Zhong, Man Chi Wai, Zheng Li-Zhen, and Tang Tao for their help in animal surgery, Huang Panya, Lee Wai Chi, and Xu Zhe for preparing the paraffin samples and MMA samples. Ominsky M.S., Ke H.Z. and Li C.Y. are employed by Amgen, Inc.
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ing in ovariectomy-induced osteoporotic bone. Bone 46:1299– 1305. Fang TD, Salim A, Xia W, et al. 2005. Angiogenesis is required for successful bone induction during distraction osteogenesis. J Bone Miner Res 20:1114–1124. Jacobsen KA, Al-Aql ZS, Wan C, et al. 2008. Bone formation during distraction osteogenesis is dependent on both VEGFR1 and VEGFR2 signaling. J Bone Miner Res 23:596–609. Wang Y, Wan C, Deng L, et al. 2007. The hypoxia-inducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development. J Clin Invest 117:1616–1626. Chen D, Tian W, Li Y, et al. 2012. Osteoblast-specific transcription factor Osterix (Osx) and HIF-1alpha cooperatively regulate gene expression of vascular endothelial growth factor (VEGF). Biochem Biophys Res Commun 424: 176–181. Tang WJ, Yang F, Li Y, et al. 2012. Transcriptional regulation of vascular endothelial growth factor (VEGF) by osteoblast-specific transcription factor osterix (Osx) in osteoblasts. J Biol Chem 287:1671–1678. Zerlin M, Julius MA, Kitajewski J. 2008. Wnt/Frizzled signaling in angiogenesis. Angiogenesis 11:63–69. Easwaran V, Lee SH, Inge L, et al. 2003. beta-Catenin regulates vascular endothelial growth factor expression in colon cancer. Cancer Res 63:3145–3153. Maes C, Goossens S, Bartunkova S, et al. 2010. Increased skeletal VEGF enhances beta-catenin activity and results in excessively ossified bones. EMBO J 29:424–441. Chen D, Li Y, Zhou Z, et al. 2013. HIF-1alpha inhibits Wnt signaling pathway by activating Sost expression in osteoblasts. PLoS ONE 8:e65940. Zhang C, Cho K, Huang Y, et al. 2008. Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix. Proc Natl Acad Sci USA 105:6936–6941. Chen D, Li Y, Zhou Z, et al. 2012. Synergistic inhibition of Wnt pathway by HIF-1alpha and osteoblast-specific transcription factor osterix (Osx) in osteoblasts. PLoS ONE 7: e52948. Hankenson KD, Dishowitz M, Gray C, et al. 2011. Angiogenesis in bone regeneration. Injury 42:556–561. Marsell R, Einhorn TA. 2011. The biology of fracture healing. Injury 42:551–555. Okike K, Bhattacharyya T. 2006. Trends in the management of open fractures. A critical analysis. J Bone Joint Surg Am 88:2739–2748. Mathavan N, Bosemark P, Isaksson H, et al. 2013. Investigating the synergistic efficacy of BMP-7 and zoledronate on bone allografts using an open rat osteotomy model. Bone 56:440–448. Bosemark P, Isaksson H, McDonald MM, et al. 2013. Augmentation of autologous bone graft by a combination of bone morphogenic protein and bisphosphonate increased both callus volume and strength. Acta Orthop 84:106–111. Colnot C. 2009. Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration. J Bone Miner Res 24:274–282. Utvag SE, Grundnes O, Reikeraos O. 1996. Effects of periosteal stripping on healing of segmental fractures in rats. J Orthop Trauma 10:279–284.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site.
JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
Received 31 October 2013; accepted 4 March 2014 Published online 30 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22636
ABSTRACT: Sclerostin is a negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment promoted bone healing in various animal models. To further evaluate the healing efficiency of Scl-Ab in osteotomy healing, we investigated the time course effects of systemic administration of Scl-Ab on fracture repair in rat femoral osteotomy model. A total of 120 six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were treated with vehicle or Scl-Ab treatment for 3, 6, or 9 weeks. Fracture healing was evaluated by radiography, micro-CT, micro-CT based angiography, 4-point bending mechanical test and histological assessment. Scl-Ab treatment resulted in significantly higher total mineralized callus volume fraction, BMD and enhanced neovascularization. Histologically, Scl-Ab treatment resulted in a significant reduction in fracture callus cartilage at week 6 and increase in bone volume at week 9, associated with a greater proportion of newly formed bone area at week 6 and 9 by fluorescence microscopy. Mechanical testing showed significantly higher ultimate load in Scl-Ab treatment group at week 6 and 9. This study has demonstrated that Scl-Ab treatment enhanced bone healing in a rat femoral osteotomy model, as reflected in increased bone formation, bone mass and bone strength. ß 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:997–1005, 2014. Keywords: sclerostin; sclerostin monoclonal antibody; osteotomy; fracture healing
Sclerostin is a protein secreted by osteocytes that inhibit bone formation.1 Sclerostin acts as an antagonist of the Wnt/beta-catenin signaling pathway in osteoblasts, and thus negatively regulates the differentiation and function of osteoblasts.1 Sclerostin deficiency leads to a high bone mass phenotype in humans2 and mice3; while over-expression of the sclerostin gene (SOST) in transgenic mouse model leads to osteopenia.4 These results indicated an inverse relationship between sclerostin and bone mass. Furthermore, systemic administration of sclerostin monoclonal antibody (Scl-Ab) inhibits sclerostin function,5 resulting in an increase in bone formation, bone mass, and strength in intact bone in multiple animal models, including rats, non-human primates, and also in unloading models associated with hindlimb-immobilization6 and spaceflight.7 Additionally, Scl-Ab has been shown to increase BMD in healthy men and postmenopausal women.8 Besides promoting bone formation in healthy animals and humans, Scl-Ab administration was also shown to improve bone healing in rodent models of drill hole defects,9,10 critical-size gap defects,11 closed fracture,6 bone defect healing in rats with type 2 diabetes mellitus12 and in cynomolgus monkeys after fibular osteotomy.6 Orthopedic surgeons often treat trauma patients surgically resulting in an “open fracture”, a more challenging clinical condition as compared with a closed fracture that has a better prognosis.13,14 The objective of this study is to investigate the time course effects of systemic administration Conflicts of interest: None. Grant sponsor: Amgen, Inc.; Grant sponsor: UCB Pharma; Grant number: TA095217. Correspondence to: Ling Qin (T: þ852-26323071; F: þ852-26377889; E-mail: [email protected]) # 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
of Scl-Ab on fracture repair in an open fracture model using rat femoral osteotomy model, which this healing model resulted in delayed healing compared with closed fracture healing. The progression and quality of fracture healing were assessed by serial weekly radiography, bone mineral density (BMD) and bone volume fraction by micro-CT, angiogenesis by microCT-based angiography, callus composition and dynamics of bone formation by histomorphometric analysis, and fractured bone strength by mechanical testing.
MATERIALS AND METHODS Animals and Femoral Osteotomy Model A total of 120 six-month-old male Sprague–Dawley rats were obtained from the Laboratory Animal Services Center of the Chinese University of Hong Kong. The Animal Experimentation Ethics Committee of the University approved the experimental procedures (AEEC No. 09/042/MIS). Osteotomy was performed under general anesthesia at mid-shaft of the right femur using a circular saw with a diameter of 1.6 cm and a thickness of 0.1 mm (Fine Science Tools, Foster City, CA) and stabilized by intramedullary insertion of a sterilized 1.2 mm diameter Kirschner wire (Stryker China, Hong Kong, China) as described previously.15–17 Rats were randomly assigned to the sclerostin antibody treatment group (Scl-Ab VI, subcutaneous injection, 25 mg/kg, two times per week, as described previously18) or vehicle (saline) treatment group, and both treatments started from Day 1 post-operation. The progress of fracture healing for each animal was monitored weekly by digital radiography (MX-20; Faxitron Bioptics, Tucson, AZ). At week 3, 6, and 9 post-operation, 20 rats from each treatment group were terminated. For each treatment group, six rat femora were prepared and subjected to microCT based angiography prior to decalcification for histological analysis. The femora of the remaining 14 rats were subjected to micro-CT scanning (14 rats), mechanical testing (eight rats), and dynamic histomorphometric analysis (six rats) (Supplementary Fig. S1). JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Radiography and Micro-CT Assessment of the Fracture Callus Digital radiographic images acquired at 3, 6, and 9 weeks post-operation were analyzed by ImageJ (version 1.42q; NIH) to quantify the total area of the external fracture calluses. Femora were scanned by a desktop micro-CT system (mCT40, Scanco Medical, Bru¨ttisellen, Switzerland). The scan range was 13.2 mm (660 slides) centered on the fracture line and the 3D reconstruction of mineralized tissue was performed as described previously.15,19 Lower density bone tissue, representing less mature callus, and higher density bone tissue, representing original cortices and highly mineralized callus, were determined using two thresholds (low ¼ 200, high ¼ 350, Hounsfield units) according to our established protocol15,20 (Supplementary Fig. S2). The analysis determined total bone volume (BVt, mm3, threshold ¼ 200), high-density bone volume (BVh, mm3, threshold ¼ 350), lowdensity bone volume (BVl, mm3, equal to BVt BVh) the callus volume fractions for each of these parameters (BVh/ TV, BVl/TV, and BVt/TV) and the bone mineral density (BMD). A bone strength index (BSI) of the fracture callus was calculated from 1 mm proximal to 1 mm distal of the fracture line to obtain the average cross-sectional area (CSA) of the callus multiplied by the corresponding BMD (BSI ¼ CSA BMD).21 BVt, BVh, BVl, BVt/TV, BVh/TV, and BVl/TV of this sub-region were also determined. Micro-CT Based Angiography Perfusion with Microfil (Microfil MV-117; Flowtech, Carver, MA) was performed as described previously.20 Samples were decalcified in 5% formic acid for 2 weeks and scanned by a desktop micro-CT system. The scan range was 6 mm (300 slides), centered on the osteotomy line. The 3D reconstruction of the blood vessels was performed as previously described using a threshold of 100 Hounsfield units.20 The blood vessel volume (vessel volume, mm3) and connectivity of the blood vessels (connectivity, 1/mm3) were analyzed. Mechanical Test A destructive 4-point bending test was performed as described previously22 (H25KS; Hounsfield Test Equipment Ltd., Redhill, Surrey, UK). The femora were placed with anterior surface facing up with the upper and lower span lengths set to 10 and 26 mm, respectively. Load was applied at a rate of 5 mm/min until failure. Ultimate load (N), energy (J), and stiffness (N/mm) were calculated from the load– deformation curve using the test system’s software (QMAT Professional Material testing software, Hounsfield Test Equipment Ltd.). Histology and Histomorphometric Analysis For decalcified histology, phosphate buffered formalin fixed femora were decalcified by 5% formic acid for 2 weeks and embedded in paraffin. Five-micrometer thick sections were stained by Hematoxylin and Eosin (H&E), Safranin O, and toluidine blue for evaluation under light microscope (Zeiss Aixoplan with Spot RT digital camera; Zeiss, Oberkochen, Germany). Callus composition of the external callus was determined by ImageJ for evaluation of bone tissue area fraction and cartilage tissue area fraction. Sequential fluorescent labeling was used to study the dynamics of bone formation during fracture healing according to our established protocol,19 with calcein green and xylenol orange (10 and 90 mg/kg, respectively, Sigma– JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
Aldrich, St. Louis, MO) subcutaneously injected 2 and 1 week, respectively before euthanasia at each time point. Femora were embedded undecalcified in MMA, sectioned, ground, and polished to 100 mm and observed using a fluorescence microscope (Leica Q500MC; Leica Microsystems Cambridge Ltd, Cambridge, Cambridgeshire, United Kingdom). The temporal change in new bone formation was assessed for the total callus and periosteal callus within a 10 mm region centered on the fracture line; calculated as the ratio of the area labeled with xylenol orange to the area labeled with calcein green using ImageJ19 (Supplementary Fig. S3). Statistical Analysis All data were expressed as mean SD. Two-way ANOVA with Bonferroni posthoc test was used to compare the differences between the Scl-Ab treatment group and the vehicle group across time points. Pearson analysis was used to calculate the correlation coefficient between ultimate load and micro-CT based bone endpoints for each treatment group at week 6 and 9. Values of p < 0.05 were considered significant. All analyses were performed using GraphPad Prism 5 (California).
RESULTS Radiographic Analysis and Bone Mass of Fracture Calluses Radiographic images of week 3, 6, and 9 showed that the Scl-Ab treatment group had a larger fracture callus. Quantitative analysis showed that the Scl-Ab treatment group had a 23–30% larger callus (Fig. 1) compared with the vehicle group at all time points. Micro-CT analysis showed that BMD was 15–16% significantly higher in the Scl-Ab treatment group compared to vehicle groups at each time point. Similarly, the Scl-Ab treatment group had 16–23% significantly higher in BVt/TV at each time point and 20– 27% significantly higher in BVt at week 6 and 9 (p < 0.01), compared to their vehicle groups (Fig. 2). Furthermore, that the amount and proportion of mature (highly mineralized) bone volume were significantly greater with Scl-Ab treatment at week 6 and 9, as reflected in significant increases BVh/TV (26–33%) and BVh (38–42%) compared to their vehicle groups. Scl-Ab treatment was also associated with a significant increase the lower bone density-based BVl/TV (38%) and BVl (26%) at week 3, which may reflect an early progression in the early stage of fracture healing under the Scl-Ab treatment. Total tissue volume (TV) was not significantly affected by treatment at any time point (Table 1). Fractured Bone Strength Surrogates To study the correlation between the mechanical properties and the structural data as determined by micro-CT in the fracture site, the 2 mm sub-region centered on the fracture line was chosen for further analysis. At week 9, ultimate load was significantly correlated with CSA, BVh, BVt, BVl, and BSI in the Scl-Ab treatment group, with no significant effects observed for the week 9 vehicle group or either group at week 6 (Table 2).
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Callus Histology and Histomorphometric Analysis More bony tissue and less cartilage tissue was observed in fracture calluses across all time points in the Scl-Ab treatment groups compared with the vehicle groups by H&E, safranin O, and toluidine blue staining (Figs. 5A and S4). Quantitative analysis supported this observation, with a significant increase (32%) in bone area fraction at week 9, and a significant decrease in cartilage area fraction at week 6 in the Scl-Ab treatment groups compared with the vehicle groups (Fig. 5B). In addition, the Scl-Ab treatment group showed faster mineral deposition during the fracture healing process compared with the vehicle group as demonstrated by fluorescent microscopy (Fig. 6). At week 9, the Scl-Ab treatment group also showed an increase in the rate of new bone formation in both the total callus (þ41%) and the periosteal callus sub-region (þ42%).
DISCUSSIONS
Figure 1. Radiographic analysis of fracture healing progress at the osteotomy site of rat femora. Representative radiographs (A) and quantitative analysis of callus size (B) showed that larger fracture calluses were observed in Scl-Ab treatment group compared with the vehicle group at week 3, 6, and 9 postoperation. Mean SD, n ¼ 20/group/time point, p < 0.05 compared with Vehicle at the same time point.
Callus Angiogenesis Micro-CT based angiography demonstrated that calluses contained greater vessel volume with better connectivity in the Scl-Ab treatment group than the vehicle group at week 3 (Fig. 3). These differences were not observed at week 6 and 9. Callus Mechanical Properties by 4-Point Bending Mechanical Test Only week 6 and 9 groups were subjected to mechanical testing as the fractured femora at week 3 were too weak to test as confirmed in our pilot study. Scl-Ab treatment resulted in significantly higher ultimate load at week 6 (98%) and week 9 (53%). Stiffness and energy to failure also showed similar trends, with SclAb treatment group means that were higher at week 6 (stiffness, þ71%, p ¼ 0.08; energy to failure, þ124%, p < 0.05) and week 9 (stiffness, þ63%, p ¼ 0.08; energy to failure, þ51%, p ¼ 0.08) relative to controls (Fig. 4).
In this study, Scl-Ab improved open fracture healing in a rat femur osteotomy model by enhancing bone volume and mineralization, angiogenesis, and mechanical properties across the 9-week time course. Scl-Ab had previously been reported to improve closed fracture healing in rat femora, while the current data demonstrate similar improvements in a more challenging femoral osteotomy model of open fracture healing. In addition, Scl-Ab treatment improved neovascularization at week 3 post-fracture by micro-CT based angiography, a novel finding that has not previously been reported. Furthermore, Scl-Ab treatment specifically enhanced high mineralized bone volume fraction by micro-CT, which is similar to the treatment effect reported in cynomolgus monkeys by pQCT.18 Our results showed that Scl-Ab treatment increased fracture callus size (by digital radiography) and bone volume (by micro-CT and histology) as compared with vehicle treatment. The fracture callus and cortical bone work as a functional unit during fracture healing; therefore the pre-existing cortical bone was included in the micro-CT analysis of BMD and bone volume. Although the effects of Scl-Ab on the pre-existing cortical surfaces cannot be clearly separated from the effects on the periosteal callus, both should be considered important components of the healing response and contributors to bone strength. The histological assessment of bone volume and new bone formation in the periosteal callus alone does suggest a significant effect of Scl-Ab in this compartment alone. Further analysis of the micro-CT images using two different thresholds to delineate the higher and lower mineralized portions of fracture calluses revealed that, Scl-Ab treatment improved both the BVl/TV and BVh/ TV at week 3 and specifically enhanced the BVh/TV at week 6 and 9 post-operation. These indicated that Scl-Ab treatment may promote an early progression in osteogenesis during the early stage of fracture healing (week 3) and resulting in greater callus mineralization JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Figure 2. Scl-Ab treatment enhanced BMD and bone volume in the fracture callus. Representative micro-CT images (A), BMD (B), and total bone volume fraction (BVt/TV) (C) showed that Scl-Ab treatment resulted in more mineralized tissue in the fracture callus compared to the vehicle group. Mean SD, n ¼ 14/group/time point, p < 0.01 compared with Vehicle at the same time point. The preexisting cortex was also included in this analysis.
at the later stages (week 6 and 9). Although consistent callus bridging was not observed in either treatment group, a strong correlation was found between ultimate load and the bone volume parameters (BSI, CSA, BVh, BVt, and BVl) in the week 9 Scl-Ab treatment group. This result may be related to the greater maturity of
the fracture callus in the Scl-Ab treatment group at week 9, corresponding to greater functional strength. The increases in micro-CT based bone volume and mechanical properties in the Scl-Ab treated femora coincided with similar changes in the histologic composition of the fracture callus. The Scl-Ab treatment
Table 1. Comparison of Micro-CT Analysis of Fracture Calluses From Scl-Ab Treatment Group and Vehicle Group Across Time Points Week 3 Vehicle 3
BVh (mm ) BVl (mm3) TV (mm3) BVt/TV BVh/TV BVl/TV
108.91 12.84 58.21 20.51 350.57 45.56 0.48 0.07 0.32 0.06 0.16 0.04
Week 6 Scl-Ab
120.07 16.14 73.12 26.54 327.74 64.68 0.59 0.03 0.37 0.04 0.22 0.05
Vehicle 93.66 32.07 97.00 55.56 402.62 132.16 0.49 0.08 0.27 0.12 0.23 0.05
Week 9 Scl-Ab
133.42 29.16 96.03 44.99 385.59 74.32 0.60 0.04 0.36 0.10 0.24 0.07
Values are mean SD. p < 0.05; p < 0.01 compared with vehicle at the same time point. JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
Vehicle
Scl-Ab
135.12 12.26 61.35 13.93 351.93 61.70 0.56 0.05 0.39 0.05 0.17 0.02
187.05 16.60 61.74 11.78 384.48 47.71 0.65 0.04 , 0.49 0.04 0.16 0.02
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Table 2. Correlation Between Ultimate Load and Different Parameters in Micro-CT Analysis Correlation Coefficient (Pearson) Ultimate Load Treatment Week Week Week Week
6 6 9 9
Vehicle Scl-Ab Vehicle Scl-Ab
CSA
BVh
BVt
BVl
BVh/TV
BVt/TV
BVl/TV
BMD
BSI
0.267 0.07572 0.7178 0.8158
0.1296 0.3026 0.2207 0.8091
0.3897 0.3606 0.7502 0.818
0.318 0.3285 0.7615 0.7386
0.05519 0.2442 0.3272 0.2006
0.02022 0.6151 0.2472 0.3934
0.1973 0.5717 0.5530 0.5618
0.02019 0.4624 0.05540 0.3176
0.4223 0.3003 0.7527 0.8870
CSA, cross-sectional area; BVt, total bone volume; BVh, volume of high-density bone; BVl, volume of low-density bone; BVt/TV, total bone volume fraction; BVh/TV, volume fraction of high-density bone; BVl/TV, volume fraction of low-density bone; BMD, bone mineral density; BSI, bone strength index. p < 0.05; p < 0.01 significance of the correlation.
group contained more bony tissue and less cartilage tissue, which is similar to the previous reports in sclerostin knockout (SOST-KO) mice and after Scl-Ab treatment in cynomolgus monkeys undergoing fibular osteotomy.3,18 Based on our histomorphometric assessment, bone healing appears to be dominated by endochondral ossification in the rat osteotomy model. Therefore the increase in bone area fraction and decrease in cartilage area fraction in the Scl-Ab treat-
ment group would be consistent with an enhancement of endochondral ossification. Under this paradigm, the early increase in vascularization would accelerate endochondral ossification through a faster transition from cartilage through its hypertrophic stage to bone. Intramembranous bone formation at the periosteum distal to the osteotomy site may also contribute to the observed increases in bone volume, and could also be positively affected by increased vascularization.
Figure 3. Scl-Ab treatment enhanced neovascularization at callus at week 3 post-operation. Representative micro-CT images (A), vessel volume (B), and connectivity (C) showed that Scl-Ab treated samples contained more vessel volume and better connectivity compared to the vehicle group at week 3 by micro-CT based angiography. Mean SD, n ¼ 6/group/time point, p < 0.01 compared with Vehicle at the same time point. JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Figure 4. Mechanical test of the fractured femora. Four-point bending test showed that Scl-Ab treatment significantly enhanced the ultimate load (A) (p < 0.01), stiffness (B) (p ¼ 0.08), and energy to failure (C) (p < 0.05 at week 6 and p ¼ 0.08 at week 9) in fractured femora compared with the vehicle group at the same time point. Mean SD, n ¼ 8/group/time point, p < 0.05; p < 0.01 compared with vehicle at the same time point.
During fracture repair, early bone deposition is woven in nature, resulting in areas of fluorescent labeling rather than clear linear labeling on undecalcified MMA sections. Traditional mineral apposition rate (MAR) and bone formation rate (BFR) analysis based on linear labeling may therefore not be suitable for measurement. Therefore, areas labeled by fluorochromes were measured as a representative index for new bone growth as previously reported.19 The current study showed that Scl-Ab treatment resulted in an increase in the rate of bone formation at week 9, especially in the periosteal callus. Temporally, new bone formation continued to increase from week 6 to 9 in the Scl-Ab group, whereas it began to decline in the vehicle controls. These data coincided with the results of radiography, micro-CT analysis and histological callus composition. The continuous increase of bony callus volume fraction and BMD across time points indicates a faster mineralization of the fracture callus, and Scl-Ab treatment significantly induced bone formation and increased the mineral density in the fracture callus during fracture healing. The enhanced bone formation in the Scl-Ab treatment group was associated with improved mechanical properties via 4-point bending test. Although mechanical testing could not be performed on week 3 calluses due to the weakness of the fracture calluses, mechanical testing of week 6 and 9 calluses showed significant improvements in ultimate load with Scl-Ab treatment. These data indicate that Scl-Ab treatment significantly enhanced the healing rate of the fracture callus. The improvement in fracture healing depends on angiogenesis and it is observed that Scl-Ab treatment resulted in more and better connected vascular structure, as reflected by higher vessel volume and better connectivity, at the early time point (week 3) by microCT based angiography. Angiogenesis is an important process for fracture healing, and blood supply and its stability are the key factors that promote fracture healing process.23,24 Angiogenesis and osteogenesis is JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
a tightly coupled process. The vascular endothelial growth factor (VEGF) expression is positively regulated by the hypoxia-inducible factor-1 alpha (HIF-1alpha) and Osterix (Osx) in osteoblast cell cultures,25–27 and Wnt/beta-catenin signaling is involved in regulating VEGF-A expression for angiogenesis during retinal blood vessel formation28 and in colon cancer.29 Meanwhile, VEGF over-expression enhanced beta-catenin stabilization and resulted in a high bone mass phenotpye in a transgenic mice model.30 Besides regulating VEGF expression, both HIF-1alpha and Osx also inhibit the Wnt signaling pathway through inducing sclerostin and Dickkopf-1 expression, respectively31–33 (Supplementary Fig. S5). Neovascularization during fracture healing allows nutrient influx and delivers osteoprogenitors cells to the fracture site for osteogenesis to take place.34,35 In the current study, we observed that Scl-Ab treatment induced an early increase in neovascularization, which may contribute to enhanced fracture healing. The mechanism of an early increase in neovascularization by Scl-Ab required further investigation. In the healing efficiency, Scl-Ab treatment showed to improve 60% in peak load by 3-point bending test at week 7 post-operation in rat closed femoral fracture,18 whereas Scl-Ab treatment showed 50% increase in ultimate load by 4-point bending test at week 6 postoperation in this study. This might suggest a better improvement by Scl-Ab treatment in promoting open fracture healing. However, the mechanical test methodology, the age of rats and the time points are not matched in these two studies and therefore the results obtained cannot be directly compared with each other. In the future, a head-to-head comparison of Scl-Ab in open and closed fracture models is required to address the issue. The limitations of this study are: (1) external callus bridging was not observed by radiography or histology in the osteotomy model at week 9 and (2) compared with the ultimate load of the non-fractured femora
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Figure 5. Histological assessments of fracture calluses. Sagittal section of fracture calluses at the osteotomy site of femoral shaft in rats (A) and fracture callus composition (B). Scl-Ab treatment group showed more bony tissue and less cartilage area compared with the vehicle group starting at week 3. Magnification, 16. Scale bar ¼ 1 mm. Mean SD, n ¼ 6/group/time point, p < 0.05; p < 0.01 compare with Vehicle at the same time point.
from aged-matched rats (150 N, unpublished data), the ultimate load of femora after week 9 of Scl-Ab treatment only recovered around 40% of the original strength. Compared with closed fracture, open fracture is a more traumatic injury and therefore is more
challenging and may take longer to repair. Open fracture often results in wound infections, severe soft tissue injury and periosteal disruption, which contribute to slow healing and often in non-union; therefore, more care is needed for open fracture JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
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Figure 6. Dynamics of bone formation in Vehicle and Scl-Ab treatment groups at week 6 and 9. Representative fluorescent images (A) showing the new bone formed 2 weeks and 1 week prior to termination, as labeled by calcein green (green) and xylenol orange (red), respectively. The ratio of new bone formation in the total callus and periosteal callus was calculated as the ratio of area labeled in red versus green (B). Scl-Ab treatment groups showed an increased in rate of new bone formation in the total callus and periosteal callus at week 9. Magnification, 16. Scale bar ¼ 1 mm. Mean SD, n ¼ 6/group/time point, p < 0.05 compared with Vehicle at the same time point.
healing management.16,17,36–38 Among these factors, the periosteum is the most important for optimal bone healing, as it is one of the sources of osteoprogenitors cells and its damage or removal results in delayed bone healing.39,40 The damaged periosteum in the osteotomy surgery in this study likely resulted in delayed in the bridging of the fracture callus. The fracture gaps in rat closed femoral fractures are usually bridged at around week 6 after fracture,16 but in our femoral osteotomy model, the fracture callus in the radiographic and histological images were still not bridged at week 9. Therefore, a longer time point would be required to examine the effects of Scl-Ab treatment across the full time-course of functional recovery, as reflected in callus union and bone strength near intact levels. The continuing increases in new bone formation and strong correlation between the bone mass parameters to ultimate load at the fracture site in the week 9 Scl-Ab treatment group suggests a trend toward further improvements with Scl-Ab treatment at later stages of healing. Taken together, the systemic administration of Scl-Ab was able to enhance the fracture healing in rat femoral osteotomy model, as demonstrated by increased bone formation, bone mass and bone JOURNAL OF ORTHOPAEDIC RESEARCH AUGUST 2014
strength. Our results support the therapeutic potential of sclerostin antibody to enhance open fracture healing.
ACKNOWLEDGMENTS We thank Zhang Ge for assistance in initial proposal preparation, Liu Zhong, Man Chi Wai, Zheng Li-Zhen, and Tang Tao for their help in animal surgery, Huang Panya, Lee Wai Chi, and Xu Zhe for preparing the paraffin samples and MMA samples. Ominsky M.S., Ke H.Z. and Li C.Y. are employed by Amgen, Inc.
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