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Keywords:

  • sildenafil;
  • mice;
  • fracture healing;
  • bone formation;
  • CYR61;
  • biomechanics

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Sildenafil, a cyclic guanosine monophosphate (cGMP)-dependent phospodiesterase-5 inhibitor, has been shown to be a potent stimulator of angiogenesis through upregulation of pro-angiogenic factors and control of cGMP concentration. Herein, we determined whether sildenafil also influences angiogenic growth factor expression and bone formation during the process of fracture healing. Bone healing was studied in a murine closed femur fracture model using radiological, biomechanical, histomorphometric, and protein biochemical analysis at 2 and 5 weeks after fracture. Thirty mice received 5 mg/kg body weight sildenafil p.o. daily. Controls (n = 30) received equivalent amounts of vehicle. After 2 weeks of fracture healing sildenafil significantly increased osseous fracture bridging, as determined radiologically and histologically. This resulted in an increased biomechanical stiffness compared to controls. A smaller callus area with a slightly reduced amount of cartilaginous tissue indicated an accelerated healing process. After 5 weeks the differences were found blunted, demonstrating successful healing in both groups. Western blot analysis showed a significantly higher expression of the pro-angiogenic and osteogenic cysteine-rich protein (CYR) 61, confirming the increase of bone formation. We show for the first time that sildenafil treatment accelerates fracture healing by enhancing bone formation, most probably by a CYR61-associated pathway. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:867–873

Fracture healing is a complex, sequentially orchestrated process, including inflammation, mesenchymal cell condensation, chondrogenesis, angiogenesis, and osteogenesis. Within this process, the vascularization of the tissue is a prerequisite for successful bone healing.1, 2 Reduced vascularity at the fracture site has been identified as one of the most significant parameters, accounting for delayed fracture healing and atrophic nonunion formation.3

Phosphodiesterase-5 (PDE5) catalyzes the breakdown of cyclic guanosine monophosphate (cGMP), one of the primary factors causing smooth muscle relaxation. Sildenafil is a selective inhibitor of PDE5. By means of its potent action of enhancing cGMP accumulation and ensuing vasodilation in corpus cavernosum, sildenafil has become the most widely used drug for erectile dysfunction treatment in men.4 During the last few years, several studies have shown that sildenafil exerts also angiogenic actions through upregulation of distinct pro-angiogenic growth factors.5, 6 Accordingly, the action of the drug has been analyzed in a variety of ischemic disease models.7, 8

Previous studies have shown that the angiogenic and osteogenic factors vascular endothelial growth factor (VEGF) and cysteine-rich 61 (CYR61) are involved in the process of bone formation and fracture healing.9 There is complete lack of information, however, whether sildenafil is capable of influencing these growth factors and thus the process of fracture healing. Therefore, we herein aimed at determining the effect of sildenafil treatment in fracture healing. We hypothesized that sildenafil accelerates fracture healing through stimulation of growth factor expression and bone formation.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Animals and Specimens

CD-1 mice were bred at the Institute for Clinical and Experimental Surgery, University of Saarland. They were kept at a 12-h light and dark cycle and were fed a standard diet with water ad libitum. All animal procedures were performed according to the National Institute of Health guidelines for the use of experimental animals and were approved by the German legislation on the protection of animals. For the present study a total of sixty 12- to 14-week-old animals were used. Thirty mice were treated daily with 5 mg/kg body weight (BW) sildenafil (Viagra®, Pfizer, Germany; dissolved in saline) p.o. using a gavage. After 2 weeks 10 of these mice were killed for radiological, biomechanical and histomorphometrical analysis, and another five mice were killed for biochemical analysis. In the remaining 15 animals daily sildenafil treatment was continued for a total of 5 weeks. Ten of these mice were then killed for radiological, biomechanical, and histomorphometrical analysis, and the remaining five mice were killed for protein biochemical analysis. Thirty vehicle-treated mice, receiving equivalent amounts of saline, served as controls, and were used for the different time point and parameter analysis in identical numbers as the sildenafil-treated animals.

The chosen dosage of sildenafil (5 mg/kg BW) is fivefold higher compared to the standard dose used in humans (0.7–1.5 mg/kg). This considers the higher rate of metabolism of sildenafil in rodents with an elimination half live of 1 h in mice compared to 4 h in man.

Surgical Procedure

Mice were anesthetized by an intraperitoneal injection of xylazine (25 mg/kg BW) and ketamine (75 mg/kg BW). Under sterile conditions a 4 mm medial parapatellar incision was performed at the right knee to dislocate the patella laterally. After drilling a hole (0.5 mm in diameter) into the intracondylar notch, an injection needle with a diameter of 0.4 mm was drilled into the intramedullary canal. Subsequently, a tungsten guide wire (0.2 mm in diameter) was inserted through the needle into the intramedullary canal. After removal of the needle, the femur was fractured by a 3-point bending device and an intramedullary titanium screw (18 mm length, 0.5 mm in diameter) was implanted over the guide wire to stabilize the fracture.10 The screw consisted of a cone-shaped distal head (diameter 0.8 mm) and a proximal thread (M 0.5 mm, length 4 mm) (AO Foundation, Research Implants Systems (RIS), Davos, Switzerland). After fracture fixation, the wound was closed using 6–0 synthetic sutures. Fracture and implant position were confirmed by radiography (MX-20, Faxitron X-ray Corporation, Wheeling, IL).

Radiological Analysis

At the end of the 2 and 5 weeks observation period the animals were re-anesthetized and ventro-dorsal X-rays (MX-20, Faxitron X-ray Corporation) of the healing femora were performed. Fracture healing was analyzed according to the classification of Goldberg with stage 0 indicating radiological nonunion, stage 1 indicating possible union and stage 2 indicating radiological union.11

Biomechanical Analysis

For biomechanical analysis, the right and the left femora were resected at 2 and 5 weeks and freed from soft tissue. After removing the implants, callus stiffness was measured with a nondestructive bending test using a 3-point bending device with a 20 N load cell (Mini-Zwick Z 2.5, Zwick GmbH, Ulm, Germany). Due to the different states of healing, the loads which had to be applied varied markedly between the individual animals. Loading was stopped individually in every case when the actual load-displacement curve deviated more than 1% from linearity.12 Control that the load was not destructive was performed macroscopically and microscopically (histology). To account for differences in prefracture bone stiffness of the individual animals, the unfractured left femora were also analyzed, serving as an internal control. Therefore, all values of the fractured femora are given in percent of the corresponding unfractured femora. To guarantee standardized measuring conditions, femora were mounted always with the ventral aspect upwards. The fracture zone was placed directly under the middle anvil with a working gauge length of 6 mm. Applying a gradually increasing bending force with 1 mm/min, the bending stiffness (N/mm) was calculated from the linear elastic part of the load-displacement diagram.13, 14

Histomorphometric Analysis

For histology, bones were fixed in IHC zinc fixative (BD Pharmingen, San Diego, CA) for 24 h, decalcified in 13 % EDTA solution for 2 weeks and then embedded in paraffin. Longitudinal sections of 5 µm thickness were stained according to the trichrome method. At a magnification of 1.25× (Olympus BX60 Microscope, Olympus, Tokyo, Japan; Zeiss Axio Cam and Axio Vision 3.1, Carl Zeiss, Oberkochen, Germany; ImageJ Analysis System, NIH, Bethesda, MD) structural indices were calculated according to the suggestions provided by Gerstenfeld et al.15 These included total callus area (bone, cartilaginous, and fibrous callus area)/femoral bone diameter (cortical width plus marrow diameter) at the fracture gap [CAr/BDm (mm)], callus diameter/femoral bone diameter [CDm/BDm], bone (total osseous tissue) callus area/total callus area [TOTAr/CAr (%)], cartilaginous callus area/total callus area [CgAr/CAr (%)] and fibrous tissue callus area/total callus area [FTAr/CAr (%)].

Additionally, we used a score system to evaluate the quality of fracture bridging.16 Both cortices were analyzed for bone bridging (2 points), cartilage bridging (1 point), or bridging with fibrous tissue (0 point). This score system results in a maximum of 4 points for each specimen, indicating complete bone bridging.

Western Blot Analysis

The callus tissue was frozen and stored at −80°C until required. For whole protein extracts and Western blot analysis of VEGF, CYR61, proliferating cell nuclear antigen (PCNA), cleaved caspase-3, receptor activator of NF-kappaB ligand (RANKL), osteoprotegerin (OPG), and endothelial nitric oxide synthase (eNOS), the callus tissue was homogenized in lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 0.1 mM EDTA, 0.5% Triton-X 100, 0.02% NaN3, 0.2 mM PMSF, and protease inhibitor cocktail (1:100, v/v; Sigma–Aldrich, Taufkirchen, Germany)), incubated for 30 min on ice and centrifuged for 30 min at 16,000g. Protein concentrations were determined using the Lowry assay. The whole protein extracts (10 µg protein per lane) were separated discontinuously on sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylendifluoride membranes. After blockade of nonspecific binding sites, membranes were incubated for 4 h with the following antibodies: rabbit anti-mouse VEGF (A20, 1:100, Santa Cruz Biotechnology, Heidelberg, Germany), goat anti-mouse CYR61 (1:100, Santa Cruz Biotechnology), mouse anti-mouse PCNA (1:500, Dako Cytomation, Hamburg, Germany), rabbit anti-mouse cleaved caspase-3 (1:400, Cell Signaling, Frankfurt, Germany), mouse anti-mouse RANKL (Abcam, Cambridge, United Kingdom), rabbit anti-mouse eNOS (1:300, BD Transduction, Heidelberg, Germany), and rabbit anti-mouse OPG (Santa Cruz Biotechnology). This was followed by corresponding horseradish peroxidase-conjugated secondary antibodies (1.5 h, 1:5,000, GE Healthcare Amersham, Freiburg, Germany). Protein expression was visualized using luminol-enhanced chemiluminescence (ECL, GE Healthcare Amersham). Signals were densitometrically assessed (Quantity one, Geldoc, BioRad, München, Germany) and normalized to β-actin signals (1:20,000, anti-β-actin, Sigma–Aldrich) to correct for unequal loading.

Statistical Analysis

All data are given as means ± SEM. After proving the assumption for normal distribution (Kolmogorov–Smirnov test) and equal variance (F-test), comparison between the two experimental groups was performed by Student's t-test. For nonparametrical data Mann–Whitney U-test was used. Statistics were performed using the GraphPad Prism 4.0 software package (GraphPad, San Diego, CA). A p-value <0.05 was considered to indicate significant differences.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Radiological Analysis of Fracture Healing

Radiological analyses 2 weeks after fracture indicated an improved healing in sildenafil-treated animals. In these animals osseous bridging of the fracture gap was achieved earlier compared to nontreated controls (p < 0.05). After 5 weeks, the fractures in both groups were completely healed radiologically (Table 1).

Table 1. Radiological Analysis 2 and 5 Weeks after Fracture Healing According to the Goldberg-Score of Sildenafil-Treated Animals and Vehicle-Treated Controls
Goldberg-ScoreSildenafil (5 mg/kg)Control
  1. Mean ± standard error of the mean (SEM); *p < 0.05 versus controls.

2 weeks2.0 ± 0.0*1.2 ± 0.6
5 weeks2.0 ± 0.02.0 ± 0.0

Biomechanical Analysis of Fracture Healing

Biomechanical analysis at 2 weeks after fracture healing showed a significantly higher bending stiffness in sildenafil-treated animals (p < 0.05) compared to controls (Fig. 1A). After 5 weeks, no significant differences could be detected between the two groups (Fig. 1B).

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Figure 1. Biomechanical analysis of the bending stiffness at 2 weeks (A) and 5 weeks (B) of fracture healing in control (white columns) and sildenafil-treated animals (black columns). Means ± SEM; *p < 0.05 versus control.

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Histological Analysis of Fracture Healing

All samples demonstrated a typical pattern of secondary fracture healing with callus formation involving both intramembranous and endochondral ossification. Nonetheless, at 2 weeks after fracture healing, the sildenafil-treated animals showed a significantly smaller total callus area (p < 0.05) when compared to controls (Fig. 2A). After 5 weeks callus size was found decreased also in controls, indicating appropriate remodeling at this time point also in the control group (Fig. 2B).

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Figure 2. Histomorphometric analysis of the total callus area (CAr) in relation to the diameter of the femur (BDm) (A,B) and the healing score (C,D) after 2 weeks (A,C) and 5 weeks (B,D) of fracture healing in control (white columns) and sildenafil-treated animals (black columns). Both cortices were analyzed for bone bridging (2 points), cartilage bridging (1 point), or bridging with fibrous tissue (0 point). Means ± SEM; *p < 0.05 versus control.

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In parallel, we observed a greater bridging score in sildenafil-treated animals at 2 weeks after fracture healing when compared to controls (p < 0.05; Fig. 2C). This difference was found blunted after 5 weeks of healing (Fig. 2D).

According to the radiological and biomechanical results, at 2 weeks total osseous tissue callus area/total callus area was found slightly increased in sildenafil-treated animals compared to controls (Fig. 3A). After 5 weeks, all animals of the two groups showed a comparable amount of bone tissue, that is, ∼90% (Fig. 3B). Accordingly, at 2 weeks cartilaginous callus area/total callus area was found reduced after sildenafil treatment (Fig. 3C), while at 5 weeks the remaining amount of cartilage within the callus did not differ between sildenafil-treated animals and controls (Fig. 3D).

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Figure 3. Histomorphometric analysis of the tissue distribution within the callus area. Total osseous tissue callus area/total callus area (TOTAr/CAr %) (A,B) and cartilaginous callus area/total callus area (CgAr/CAr %) (C,D) after 2 weeks (A,C) and 5 weeks (B,D) of fracture healing in control (white columns) and sildenafil-treated animals (black columns). Means ± SEM.

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Western Blot Analysis of Fracture Healing

After 2 weeks of fracture healing Western blot analysis of the callus tissue revealed that sildenafil did not significantly affect the expression of VEGF, PCNA, OPG, and RANKL (Figs. 4 and 5). In contrast, the angiogenic CYR61 was found significantly increased compared to controls (p < 0.05; Fig. 4), while eNOS, which is also capable of promoting angiogenesis, was found significantly lowered to ∼50% (p < 0.05; Fig. 5).

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Figure 4. Western blot analysis of VEGF (A,B) and CYR61 (A,C) expression in callus of control (white columns) and sildenafil-treated mice (black columns) after 2 weeks of fracture healing. Means ± SEM; *p < 0.05 versus control.

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thumbnail image

Figure 5. Western blot analysis of PCNA (A,B), RANKL (A,C), OPG (A,D), and eNOS (A,E) expression in callus of control (white columns) and sildenafil-treated mice (black columns) after 2 weeks of fracture healing. Means ± SEM; *p < 0.05 versus control.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In the present study we tested the hypothesis that sildenafil can accelerate fracture healing in mice. The data of our experiments confirmed our hypothesis. We herein demonstrate for the first time that sildenafil treatment enhances bone healing by accelerating osseous fracture bridging, resulting in an increased biomechanical stiffness. The reduced callus area with a decreased amount of cartilaginous tissue compared to controls further supports that sildenafil can accelerate the fracture healing process.

In the present study fracture healing was studied after 2 and 5 weeks. With the hypothesis that sildenafil accelerates healing, the early time point of 2 weeks was chosen because of the high activity of cellular and molecular events and the changes occurring within the callus tissue. The late 5-week time point was chosen to exclude side effects of sildenafil which may compromise definitive healing.

The accelerated bone healing with faster bone bridging in sildenafil-treated animals may be caused by CYR61, because it is well known that CYR61 is upregulated in fracture callus,9 stimulates endothelial cell migration and promotes osteoblast proliferation, differentiation and cell adhesion.17 Schütze et al.18 postulated that the fast and transient response of hCYR61 to 1,25-(OH)2D3, growth factors and cytokines suggests an important role of hCYR61 for osteoblast function and differentiation. In the present study sildenafil significantly increased the expression of the cysteine-rich protein 61 (CYR61) and enhanced bone healing by improving the quality of callus tissue, as demonstrated by a higher callus stiffness.

The increased CYR61 expression may influence fracture healing by acting in chondrocytes or osteocytes. In a rat fracture model, Hadjiargyrou et al.19 demonstrated that the highest peak of CYR61 expression correlates with chondrogenesis, indicating that CYR61 may play an important role during endochondral ossification. However, Lienau et al.9 comparing the course of healing after rigid and semi-rigid fixation in an ovine tibial fracture model, demonstrated that a prolonged persistence of cartilage in callus observed after less stable fixation is associated with a reduced CYR61 expression. They concluded that the reduced expression of CYR61 in cartilage led to a less effective or suboptimal chondrocyte differentiation, causing the longer persistence of cartilage in callus and, thus, a delay in healing.9 Accordingly, the increased CYR61 expression after sildenafil, observed in the present study, may have caused an accelerated cartilage resorption, promoting the process of fracture healing.

Besides its action in chondrocytes, CYR61 may also influence fracture healing by acting on osteocytes. Hadjiargyrou et al.19 could detect CYR61 in immature osteocytes within newly formed bone and suggested that some growth factors which regulate fracture repair may modulate CYR61 expression. This view is further supported by the fact that knockdown of CYR61 expression significantly diminishes Wnt3A-induced osteogenic differentiation.17 In addition, CYR61 has been shown to inhibit the formation of bone-resorbing osteoclasts20 and to stimulate the proliferation and differentiation of bone-forming osteoblasts.18, 21 Thus, the accelerated fracture healing after sildenafil, observed in the present study, may be due to an increase of proliferation and differentiation of bone-forming osteoblasts mediated by the elevated CYR61 expression.

Because there are no studies investigating a link between sildenafil and CYR61, little is known on a potential mechanism how sildenafil may upregulate CYR61. However, sildenafil is known to increase the cyclic AMP-responsive element binding protein (CREB)22 as well as CREB phosphorylation.23 In addition, CREB is capable of activating the CYR61 promotor24 as well as the angiogenic factor CYR61.25 Accordingly, sildenafil may have upregulated CYR61 via CREB.

Sildenafil did not significantly affect the balance between RANKL/RANK and OPG. RANKL is a potent stimulator of bone resorption by binding RANK in the cell membrane of osteoclasts. On the other hand, OPG is a soluble decoy receptor for RANKL that interferes with RANKL/RANK binding.26, 27 In the present study sildenafil induced an only slight increase of OPG expression, and did also not affect the expression of RANKL (p > 0.05). Thus, the accelerated bone healing after sildenafil is most probably not mediated by an OPG-associated inhibition of osteoclastogenesis.

Previous studies have shown that VEGF induces an upregulation of CYR61 in osteoblasts, promoting fracture healing by proangiogenic actions in endothelial cells.28 In the present study, however, we could not find an upregulation of VEGF after sildenafil treatment. Vidavalur et al.6 examined the effect of sildenafil on the angiogenic response in human coronary arteriolar endothelial cells and found that sildenafil induces the expression of VEGF. Moreover, Salloum et al.29 have demonstrated that sildenafil can increase eNOS and iNOS expression, indicating that the action of nitric oxide may represent an important molecular mechanism of sildenafil therapy. It has been reported that the ischemia-induced angiogenic activity is directly proportional to the amount of eNOS expression and activity.30 By contrast, Senthilkumar et al.31 found in eNOS- and iNOS-deficient mice that sildenafil enhances ischemia-induced angiogenesis independent of the nitric oxide synthases. Furthermore, in a study analyzing the effect of sildenafil on ischemia-induced neovascularization in hypercholesterolemic apolipoprotein E-deficient mice, Dussault et al.32 reported that sildenafil does not affect the expression of angiogenic factors such VEGF, HIF-1 alpha, and NO. They found that sildenafil significantly reduces oxidative stress and proposed that the antioxidative properties of sildenafil restore the angiogenic actions in hypercholesterolemic conditions. In the present study we determined for the first time VEGF and eNOS expression in callus tissue after sildenafil treatment. The fact that neither VEGF nor eNOS were found increased in the callus of healing bones after sildenafil treatment indicates that both molecules are most probably not responsible for the observed acceleration of fracture healing.

Taken together, the present study shows for the first time that sildenafil treatment accelerates fracture healing most probably by a CYR61-associated pathway.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Janine Becker for excellent technical assistance. The other authors have no affiliation or financial arrangement with an organization or company that has financial interests in the subject matter discussed in the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES