VEGF Serum Concentrations in Patients with Long Bone Fractures: A Comparison between Impaired and Normal Fracture Healing Kambiz Sarahrudi,1 Anita Thomas,2 Tomas Braunsteiner,1 Harald Wolf,1 Vilmos Ve´csei,1 Seyedhossein Aharinejad2 1
Department of Traumatology, Medical University of Vienna, Waehringer Guertel 18-20, A- 1090 Vienna, Austria, 2Laboratory for Cardiovascular Research, Center of Anatomy and Cell Biology, Medical University of Vienna, Waehringerstr. 13, A-1090 Vienna, Austria Received 28 November 2008; accepted 18 March 2009 Published online 28 April 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20906
ABSTRACT: Vascular endothelial growth factor (VEGF) plays an important role in the bone repair process as a potent mediator of angiogenesis and it influences directly osteoblast differentiation. Inhibiting VEGF suppresses angiogenesis and callus mineralization in animals. However, no data exist so far on systemic expression of VEGF with regard to delayed or failed fracture healing in humans. One hundred fourteen patients with long bone fractures were included in the study. Serum samples were collected over a period of 6 months following a standardized time schedule. VEGF serum concentrations were measured. Patients were assigned to one of two groups according to their course of fracture healing. The first group contained 103 patients with physiological fracture healing. Eleven patients with delayed or nonunions formed the second group of the study. In addition, 33 healthy volunteers served as controls. An increase of VEGF serum concentration within the first 2 weeks after fracture in both groups with a following decrease within 6 months after trauma was observed. Serum VEGF concentrations in patients with impaired fracture healing were higher compared to the patients with physiological healing during the entire observation period. However, statistically significant differences were not observed at any time point between both groups. VEGF concentrations in both groups were significantly higher than those in controls. The present results show significantly elevated serum concentrations of VEGF in patients after fracture of long bones especially at the initial healing phase, indicating the importance of VEGF in the process of fracture healing in humans. ß 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1293–1297, 2009 Keywords: fracture; bone; VEGF; delayed/nonunion; growth factors
Fracture healing is a unique process. Fractured bone is regenerated by a complex mechanism, which involves several stages.1 Within the last 10 years the bone remodeling process has been intensively investigated. Numerous cytokines, angiogenic factors, proteases, and morphogens with significant roles in fracture healing have been described.2 However, despite intensive research, most of the regulation mechanisms involved in bone healing are still unknown.3 Most of the current knowledge on growth factors associated with fracture healing is based on animal experiments or in vitro studies.4 Vascular endothelial growth factor (VEGF) seems to play an important role in the bone repair process as a potent mediator of angiogenesis.5–7 In vitro studies suggest that VEGF couples angiogenesis to the formation of bone through an intimate interplay with bone morphogenetic proteins (BMPs) and direct activation of osteoblasts.8 VEGF deposition at the site of bone damage has been shown to enhance the formation of bone in burr hole defects9 of murine femoral fractures and in critical-sized defects of the radius in rabbits.10 Exogenous VEGF application for the treatment of tibial nonunions was as useful as autologous bone-grafting in a rabbit model.11 The primary action of VEGF is to mediate new blood vessel formation.10 Furthermore, osteoblast differentiation has been shown to be directly influenced by VEGF.10,12 Inhibiting VEGF not only suppresses angiogenesis, but also bone formation and callus mineralization in rabbits and mice.10 These
Correspondence to: S. Aharinejad (T: þ43-1-4277-61119; F: þ43-14277-61120; E-mail:
[email protected]) ß 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
results suggest that VEGF is involved in mineralization of the cartilaginous callus during fracture repair. Many investigators have suggested that not only local release of growth factors at the fracture site but also a systemic reaction are relevant for triggering local effects.13–16 An insufficient systemic supply of growth factors leads to a loss of bone substance and to reduced differentiation of osteoblasts.17,18 This hypothesis is supported by a study of Gazit et al.,15 who showed that systemic application of TGF-b increased bone density in osteopenic mice.15 Further studies on elective osteotomies in animal experiments showed simultaneous local and systemic increase of growth factors.19 Another recent study showed a systemic increase of TGF-b1 during the process of fracture healing in humans.20 To our knowledge, no data exist so far on systemic measurement of VEGF with regard to delayed or failed fracture healing in humans. The aim of this study was to investigate possible differences in circulating VEGF concentrations between patients with regular and failed fracture healing.
MATERIALS AND METHODS Patients This study was approved by the Ethics Committee of the Medical University of Vienna Nr.466/2005. A consecutive series of 114 patients with meta/diaphyseal fractures of long bone (humerus, femur, lower leg, and forearm) treated surgically at our institution between April 2006 and April 2008 were included in the study. Patients gave informed consent to be enrolled in the study and were 18 to 90 years old. Exclusion criteria were: open fractures type III according to the Gustilo classification, previous bone operations, preexisting bone diseases except for osteoporosis, renal/liver insufficiency, malignant tumors, long term steroid treatment, JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2009
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Table 1. Fracture Localization, Soft Tissue Damage, and Treatment Modalities of Patients with Normal Fracture Healing Fixation
Fracture Femur Lower leg Femur þ lower leg (n ¼ 4) Humerus Forearm
Plate
IM
Ex. Fix.
Screw
Soft-Tissue Damage 18
1 14 2
17 32 4
1 3 2
0 1 0
3 12 0
13 6
8 3
0 0
0 0
0 2
IM, intramedullary fixation; Ex. fix., external fixator; soft-tissue damage 18, type I fractures due to the Gustilo classification.
immunosuppression, and long term treatment with nonsteroidal anti-inflammatory drugs. Patients were assigned to two groups according to their course of fracture healing. The first group contained 103 patients (male n ¼ 50, female n ¼ 53, mean age: 54.2 20.4) with normal fracture healing (Table 1). Eleven patients with impaired fracture healing (delayed or nonunion) formed the second group of the study (Table 2). Two of the 11 patients developed a hypertrophic type of delayed union with breakage of the implant. Another two patients had implant breakage because of an atrophic type of delayed union. Seven patients developed an atrophic type of delayed union (n ¼ 2) or nonunion (n ¼ 5). Additional injuries contained finger fracture in one, metacarpal fractures in two, radius fractures in four, and patella and ankle fracture both in one patient. One patient suffered from multiple bone fractures. This and the other nine patients with accompanying injuries belonged to the group of patients with physiological fracture healing. Only one patient had multiple injuries. The diagnosis of bony consolidation or delayed union was based on exerciseinduced pain and conventional x-rays or computed tomography. Delayed union was defined as failed fracture healing without radiological signs of bony consolidation after 4 months postoperatively. Nonunion was defined as the absence of complete consolidation at 6 months after surgery. In addition, 33 healthy volunteers (16 males, 17 females, mean age: 35.6 years) served as controls. All patients were followed up for at least 6 months after the operation at our out-patient clinic. The follow up examination
was based on clinical and radiological examination at 1, 2, 4, 6, 8, 12, and 24 weeks after trauma. Blood Samples Peripheral venous blood was obtained from each patient in periodical intervals of 1, 2, 4, 6, 8, 12, and 24 weeks after surgery and stored at 80 8C until analysis. Moreover, VEGF serum concentration was measured in 11 patients immediately after trauma at the time of hospital admission. Each control individual donated one blood sample. Measurement of VEGF VEGF serum concentrations were measured by a commercially available antibody (Quantikine; RD Systems, Minneapolis, MN) in enzyme-linked immuno sorbent assay (ELISA). All analytical steps were performed according to the manufacturer’s recommended protocol. The VEGF assay detects specifically the biologic active form of the protein. All samples were measured in duplicates to avoid interassay variability. The comparison of the measurements utilizing different kits for the same time points of the study measurements indicates the low range of variability of the assays. Concentrations are presented as mean of duplicate measurements. Statistical Analysis Statistical analyses were performed using the SAS system for Windows, v 9.1, and the Enterprise Guide, v 4.1 (SAS Institute, Inc., Cary, NC). Data are presented as means standard deviation (SD). The statistical significance level was set at p < 0.05. Comparisons between groups of continuous variables were made using nonparametric ANOVA (Wilcoxon rank-sum test for two variables or Kruskal-Wallis test for more than two variables).
RESULTS VEGF Serum Concentrations in Patients with Physiological Fracture Healing Initial VEGF serum concentration immediately after injury measured in 11 patients was 681.0 483.6 pg/mL. VEGF serum concentration was 848.1 500.3 pg/mL in the first week and peaked to its maximum of 952.2 549.2 pg/mL at 2 weeks after trauma. The serum concentration decreased after 2 weeks to reach minimum concentrations at 4 (633.2 412.1 pg/mL) and 6 weeks (631.9 452.8 pg/mL) after trauma. A slight increase was observed after 8 weeks (694.9 428.2 pg/mL). This
Table 2. Demographics of Patients with Long Bone Fractures and Delayed/Nonunion Nr.
Sex
Age (Years)
Type (ASIF-Classification)
Location
Soft-Tissue Damage
Fixation
1 2 3 4 5 6 7 8 9 10 11
Female Male Male Male Male Male Male Female Male Female Male
52 24 63 41 42 66 35 64 55 82 63
42-A1 32-A2 42-A1 33-C3 13-C3 12-B1 33-A3 43-A1 11-A2 31-A3 42-A1
Tibia/fibula Femur Tibia/fibula Femur Humerus Humerus Femur Tibia/fibula Humerus Femur Tibia/fibula
0 0 0 0 0 0 0 0 0 0 0
Screw/plate Nail Nail Plate Plate Nail External fixator Plate Plate Nail External fixator
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Figure 3. VEGF serum concentrations (mean SD) in patients with normal fracture healing (unions), and initial (preoperative) values (n ¼ 11).
Figure 1. VEGF serum concentrations (mean SD) in controls and in patients with long-bone fractures and normal (unions) or delayed consolidation (nonunions). Number sign indicates significant changes of VEGF concentrations compared to the controls in nonunions (p < 0.0001 for day 7; p ¼ 0.0001 for day 14; p ¼ 0.0057 for day 28; p ¼ 0.0083 for day 42; p ¼ 0.0091 for day 56; p ¼ 0.011 for day 168). Asterisks indicate significant changes of VEGF concentrations compared to the controls in unions (p < 0.0001 for day 7; p ¼ 0.0001 for day 14; p ¼ 0.0024 for day 28; p ¼ 0.0051 for day 42; p ¼ 0.0048 for day 56; p ¼ 0.012 for day 168).
increase was followed by a continuous approach to the minimum concentration level after the 8th week until the end of the observation period at 6 months (597.7 389) after trauma (Fig. 1). Significant differences between the highest level at 2 weeks and the levels at week 4 (p ¼ 0.0001), 6 (p ¼ 0.0001), 12 (p ¼ 0.0003), and 24 (p ¼ 0.0005) were observed (Fig. 2). Figure 3 shows the VEGF time course in those 11 patients who had a preoperative analysis. VEGF Serum Concentrations in Patients with Impaired Fracture Healing Serum VEGF concentrations in patients with impaired fracture healing were higher compared to the patients
Figure 2. VEGF serum concentrations (mean SD) in patients with normal fracture healing (unions). Significant time-related differences in VEGF concentrations compared to the highest level (2 weeks) are demonstrated.
with physiological healing during the entire observation period. However, statistically significant differences were not observed at any time point between patients with normal compared to those with impaired fracture healing (Fig. 1). Serum VEGF concentrations in patients with impaired fracture healing were slightly higher during the first 2 weeks, with the maximum VEGF concentration at the second week. Thereafter the VEGF serum concentration continuously decreased and reached a minimum at the end of the observation period. Differences in VEGF serum concentrations between patients with atrophic and hypertrophic types of nonunion are demonstrated in Figure 4. Comparison to Controls During the whole study period the VEGF concentrations in both groups were significantly higher than those in controls (387.5 191. pg/mL) (Fig. 1).
DISCUSSION The fracture repair process relies on the formation of new blood vessels in the fracture site.21 Angiogenic cytokines play a crucial role in the formation and growth of new blood vessels. VEGF, a dimeric heparin-binding glycoprotein, plays an increasing central role in the development and modulation of angiogenesis. Different animal models have demonstrated the importance of VEGF in the process of fracture healing.10,22,23 VEGF has been shown to induce the formation of new blood vessels and to influence osteoblast differentiation.10,12
Figure 4. VEGF serum concentrations in patients with atrophic and hypertrophic types of nonunion. JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2009
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In a rabbit model, VEGF inhibition led to a reduction of angiogenesis on the one hand and caused a decrease of new bone formation and callus mineralization on the other.10 The use of VEGF inhibitors in a cortical defect model in rabbits led to a reduction of callus size and mineralization when compared to control animals.10 In another study VEGF treatment improved bone healing in a critical-size defect model in rabbits. The investigators found that there was no difference in the relevant biomechanical properties of the callus between the animals that received VEGF and those who were treated by autografting after 7 weeks.11 Tarkka et al.23 demonstrated that the transfer of VEGF gene through an adenovirus vector to the defect site in the rat femur improved the healing of the defect. Geiger et al.22 supported the findings of the latter study by implantation of a special matrix into critical-size defects of rabbit radius. This matrix contained a plasmid coding for VEGF. After 6 weeks VEGF-treated animals showed new bone formation, while control animals showed no defect filling. All these experiments demonstrate the pivotal role of VEGF in the processes of fracture healing. The present study demonstrates for the first time the temporal systemic expression pattern of VEGF in patients with long bone fractures. It further compares the differences in the systemic expression of VEGF between patients with normal fracture healing and patients with impaired fracture healing. Weiss et al.24 reported a collective of 20 patients with either distraction osteogenesis for limb correction or patients who underwent elective osteotomies for axis correction of the lower limb. Their results indicated, similar to ours, significantly elevated VEGF serum levels in rigid osteotomy and after callus distraction. They concluded that bone regeneration in osteotomy healing is accompanied by systemic increase of VEGF. Later, Weiss et al.25 reported the VEGF levels of a cohort of 30 patients with long bone fractures in another more recent study. Fifteen patients with physiological healing were matched to 15 patients with failed fracture healing. VEGF serum levels of these patients were further compared to a healthy control group.25 They found higher VEGF serum concentrations in patients with bone fracture compared to the control collective. Peak concentrations were observed during early fracture healing at the first and second weeks after trauma.25 Serum VEGF levels were higher in patients with failed fracture healing; nevertheless, no statistically significant differences were observed.25 Our results are nearly identical and confirm the previously reported data by Weis et al.24,25 In the present study, VEGF serum concentrations in patients with fractures of long bones were significantly higher than in controls. A continuous increase of VEGF serum concentration was observed immediately after fracture. This trend continued as highest VEGF concentrations were measured in the first 2 weeks after trauma. Serum concentration decreased after 2 weeks to reach minimum concentrations at 4 and 6 weeks after trauma. A slight JOURNAL OF ORTHOPAEDIC RESEARCH OCTOBER 2009
increase was observed after 8 weeks. This increase was followed by a continuous approach to the minimum concentration level after the eighth week until the end of the observation period at 6 months after trauma. These results are in accordance with other reports showing a high concentration of VEGF during the inflammatory phase of fracture healing in humans.26,27 Street et al.26 demonstrated highly elevated VEGF concentrations in fracture hematoma and peripheral blood of 32 patients immediately after trauma. Grad et al.27 also observed highly elevated VEGF concentrations in 55 polytraumatized patients within the first week after trauma, with the highest level in the second week. We believe that the initial increase of VEGF is part of the angiogenic response of the bone to the high demand for new blood vessels at the fracture site during inflammatory phase. The initial increase of VEGF and other angiogenic factors (FGF basic and PDGF-AB) was also observed by Weiss et al.25 in a similar study. The high VEGF levels at 2 weeks after trauma reflect the regulatory role of VEGF for the formation of new blood vessels. The initial peak, as also observed in our study, however, might also be attributable to the surgical trauma, as also shown by Street et al.10 The transformation of soft callus tissue into hard callus formation takes place between 4–6 weeks after injury. At this time VEGF levels decreased to reach a plateau level. In this ‘‘plateau’’ phase VEGF levels were significantly lower than at 2 weeks. The plateau phase was also observed by Weiss et al.24 in distraction osteogenesis and rigid osteotomies. The authors suggested that the plateau phase reflects the ongoing remodeling of callus tissue. Another question addressed in our study was whether VEGF serum concentrations in patients with delayed or nonunions differ from the patients with normal fracture healing. Patients with impaired fracture healing had higher serum levels of VEGF compared to patients with normal fracture healing. Although the differences were not statistically significant, there seems to be a general increase of the serum VEGF concentration in patients with impaired fracture healing. While we observed a significant decline of VEGF concentration after the second week in patients with normal fracture healing, the decline of the VEGF serum concentration in patients with impaired fracture healing after the second week was less pronounced. Higher VEGF levels in patients with impaired fracture healing might reflect the effort to enhance the vascularization of the fracture site. However, delayed or nonunions are multicausal and the detailed reason for their development cannot be explained by our results. The present study is limited by the absence of data on local VEGF concentration at different time points and by the low number of delayed/nonunions. The strengths of the present study are the prospective manner of data collection over the whole period of fracture healing and the large number of patients with normal fracture healing. Significantly elevated serum VEGF concentrations in patients with fracture of long bones, especially at
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the initial healing phase, indicate the important role of this molecule in the process of fracture healing. Moreover, these are the first comparative systemic measurements of VEGF in patients suffering from delayed union and normal fracture healing. An increase of the systemic VEGF levels in patients with delayed or nonunions could be demonstrated. Definitely, further studies with a higher number of patients with impaired fracture healing are needed to clarify the role of VEGF in fracture healing.
ACKNOWLEDGMENTS This study was supported by grants from the Lorenz Bo¨ hler Foundation and the Austrian Science Fund (FWF grant P19188-B09) to K. Sarahrudi.
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