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The influence of compression on the healing of experimental tibial fractures
Injury, Int. J. Care Injured 42 (2011) 1152–1156
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Injury journal homepage: www.elsevier.com/locate/injury
The influence of compression on the healing of experimental tibial fractures Ulf Sigurdsen a,b,*, Olav Reikeras c, Stein Erik Utvag d,a a
Department of Orthopaedic Surgery, University of Oslo, Oslo, Norway Institute of Surgical Research, Oslo University Hospital Rikshospitalet, Oslo, Norway c Department of Orthopaedic Surgery, Oslo University Hospital Rikshospitalet, Oslo, Norway d Department of Orthopaedic Surgery, Akershus University Hospital, Nordbyhagen, Norway b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 18 August 2010
Purpose: Experimental studies of the effects of various mechanical conditions and stimuli on bone healing have disclosed an improvement potential in bone fracture mineralization and biomechanical properties. We therefore evaluated the effect of a clinically practicable application of a mechanical compressive interfragmentary stimulus on the healing of experimental tibial diaphyseal fractures. Methods: Sixty Male rats received a standardized tibial shaft osteotomy stabilized with a unilateral external fixator with a zero interfragmentary distance, and then randomly assigned to the compression (N = 20), control (N = 20) or distraction (N = 20) group. From days 4 to day 14, the external fixator was either tightened (compression group) or loosened (distraction group) once daily to gradually induce a total axial displacement of the external fixator pin clamps of 1.25 mm. Evaluation at 30 and 60 days postosteotomy included radiography, dual-energy X-ray absorptiometry (DXA), quantitative CT and mechanical testing. Results: All fractures healed radiographically with sparse callus. At 60 days, the compression and control groups exhibited significantly less amount of mineralized callus in terms of DXA measured callus area and bone mineral content (BMC) compared to the distraction group. These groups also demonstrated a smaller volume of low-mineralized bone tissue (callus) and a larger volume of highly mineralized bone tissue (cortical bone) measured by QCT than in the distraction group. Both mechanical strength and stiffness was significantly higher in the compression and control groups than in the distraction group at 60 days. Discussion: Compression did not enhance fracture healing in terms of mineralization, bending strength, or stiffness at the time of union, compared with the control condition. The compression and control groups exhibited improved healing in terms of mechanical strength and stiffness and a more mature callus mineralization compared with the distraction group. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Experimental rat model Tibial diaphyseal fracture healing External fixation Axial compression and axial distraction
Introduction Bone healing is a complicated physiologic process. The final result of fracture healing depends not only upon the fracture type, gap condition, host factors, blood supply and neural and hormonal regulation, but also upon the mechanobiological factors in the fracture environment.4,8,14,17–19,23,24 Research and publications on these factors are extensive, and mechanical fracture stimulation has previously been shown to significantly enhance bone healing. Early axial dynamization increased stiffness and tended to increase torsional strength in 2-mm gap transverse canine mid-tibial fractures stabilized with a unilateral external fixator.6 A short-term interfragmentary cyclic micromovement applied at a relatively high * Corresponding author at: Institute of Surgical Research, Oslo University Hospital Rikshospitalet, 0027 Oslo, Norway. Tel.: +47 23 07 35 17; fax: +47 23 07 25 30. E-mail address: [email protected] (U. Sigurdsen). 0020–1383/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2010.08.018
strain rate induced greater amount of periosteal callus and a higher maximum torque load.11 A difference between early and late application of micromotion within the fracture gap has also been demonstrated, with early cyclic micromotion increasing bone mineral density (BMD) and stiffness, and late micromotion diminishing (BMD).6,12,20 A combination of early compression and distraction may induce increased bone formation and mechanical bending stiffness.3 Interestingly, there is no consensus as to whether compression or distraction is the more effective stimulus.22 Most studies on fracture compression have employed various mounting and/or monitoring procedures, and torque, stiffness and energy absorption to failure have been found to be lower for simple, constant compression than for the more advanced cyclic compression.13,30 To our knowledge, no clinically practicable method of mechanical fracture stimulation that enhances bone healing has yet been identified. Fracture treatment with external fixation provides advantages as a method of stable mechanical fixation with minimal surgical
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insult to the patient, which is why it is the preferred treatment in certain fractures.2,5,15 Experimental animal external fixators have become more sophisticated,21 providing the ability to manipulate mechanical parameters and interfragmentary motion more precisely (e.g., axial compression, distraction and dynamization).3 The aims of this study were: (1) to evaluate the effect of a clinically applicable, mechanical compressive stimulus on fracture healing and (2) to compare healing under compression and static conditions with that effected using distraction, which induces slower consolidation of the callus segment. Materials and methods Animals and surgical procedures Sixty male Wistar rats (Møllega˚rds Avlslaboratorium, Eiby, Denmark), weighing approximately 350 g (312–392 g), were used in this study. The experiment conformed to the Norwegian Council of Animal Research Code for the Care and Use of Animals for Experimental Purposes and standard laboratory animal anaesthesia, analgesia and post-operative follow-up.28 The left tibia was exposed through an anterior incision from the tibial tuberosity and in the distal direction. The muscles on the medial and lateral aspects of the tibia were carefully elevated from the tibia and the anterior two-thirds of the tibia was cut at the level of the anterior ridge using a fine-toothed circular saw blade mounted on an electric drill. Then, the remaining one-third was manually broken, leaving the fibula intact. The aluminium/steel external fixator used in the study (Fig. 1) has been described previously by Mark et al. and by our research group.21,27 Four steel pins (diameter = 1.0 mm) were inserted: two proximal and two distal to the fracture. The core drill holes in the tibia were 0.8 mm in diameter. Fixator offset, which is the free length of the pins between the rat’s anterolateral tibial surface and the inner side of the fixator bar, was 6 mm. The position of the external fixator enabled free movement of the ankle and knee joints. The perioperative alignment and accurate fracture reduction with a zero interfragment distance were verified visually and manually. The rats were then randomly assigned to the compression (N = 20), control (N = 20) or distraction (N = 20) condition. The compressive and distractive stimuli were applied by manipulating of the external fixator from days 4 to day 14. Once daily during this early 10-day period, the tubular fixator steel screw connecting the pin clamps was either tightened (compression group) or loosened (distraction group) using a standardized tool, resulting in a daily screw rotation of 908 corresponding to a pin clamp displacement of 1/8 mm. Total displacement was thus 10 1/8 mm = 1.25 mm. The control group received similar sham manipulations. After day 14, no manipulation of the fixator was performed. Half of the animals in each study group were euthanised by an intra-peritoneal injection of pentobarbital at 30 days, and the other half at 60 days. The tibias were dissected free and examined visually, and the external fixators were carefully removed. The [(Fig._1)TD$IG]
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bones were kept frozen at 80 8C between dissection and radiological, dual-energy X-ray absorptiometry (DXA) and mechanical evaluations. Bone evaluation and statistics Radiographs were performed on a standard clinical digital system (Axiom Aristos; Siemens AG, Mu¨nchen, Germany). The Xray tube settings were 46 kV, 1.0 mAs and focus-to-film (sourceto-image-receptor) distance was 115 cm. DXA measurement was performed using a densitometer system for research animals (Piximus; Lunar Corp, Madison, WI, USA). The X-ray tube voltage, current, focal spot size and spot-to-imagereceptor distance were 80 kV, 400 mA, 0.25 mm 0.25 mm and 32 cm, respectively. The densitometric values29 for bone mineral content (BMC), BMD and callus area (CA) were calculated by the software from a 3.8-mm region of interest (ROI) at the fracture site, covering the entire fracture callus observed on the radiographs. Micro-computed tomography (mCT) scanning of the tibias and a LuciteTM phantom doped with hydroxyapatite (HA) with known mineral densities of 50, 250 and 750 mg cm 3 was performed on a Micro CAT II system (Imtek, now Siemens) with 300 steps and 2008 of rotation, and the X-ray camera detector size was 2048 2048 with a bin factor of 2. Exposure time was 500 ms and the voxels were cubic with a side length of 47.8 mm. The images were reconstructed and analysed with a commercially available reconstruction and visualization software package (Amira v4.1, Mercury Computer Systems Inc., Me´rignac Cedex, France). The analysed volume of interest was a 2-mm segment encompassing the fracture region including a smaller portion of the cortical bone ends in the fracture gap. Average Hounsfield values for areas within the different phantom densities on different cross-sections were analysed with linear regression and the regression line function was used to linearly convert calibrated CT attenuation in to HA density.26 Quantitative CT (QCT) measurements included soft callus (<171 mg cm 3), hard callus (<540 mg cm 3), cortical bone (<1200 mg cm 3) and volumetric bone mineral density (vBMD). Intact tibias (N = 5) were also scanned, and QCT measurements from a 1.25-mm ROI at the level of the osteotomy yielded 2.1 0.3 mm3 soft callus, 1.9 0.1 mm3 hard callus, 5.5 0.2 mm3 cortical bone and 936 6 mg cm 3 vBMD (mean SE). The tibias were placed between gauze pads soaked with normal saline before a bending test was performed using universal testing machine with a servo-hydraulic mechanical linear drive actuator with 100-mm of total vertical displacement and a maximum axial tension load of 250 N (MTS 858 Mini Bionix; MTS Systems Corp, Eden Prairie, MN, USA). The set-up included a three-point cantilever bending test10 designed to test the bone at the fracture site with the vertical travel speed set to 160 mm min 1. The obtained log file was then converted to a load-deformation curve7 and values for basic mechanical bone properties including strength, stiffness and work-to-fracture were obtained using a mathematical software package (Origin v 7.5; OriginLab Corp.,
Fig. 1. Schematic of the external fixator used in this study.
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Northampton, MA, USA).7,25 A similar bending test performed on ex vivo tibias with externally fixed osteotomies (N = 5) exhibited a stiffness of 3.2 0.6 N mm 1 (mean values standard error of mean (SEM)). In comparison, the stiffness of intact tibias (N = 5) was 3.6 0.3 N mm 1. A cadaveric test (Supplementary Table 1) showed a close to linear increase in measured interfragmentary compressive force in rat tibias (N = 5) with the external fixator mounted and manipulated as in the compression group reaching a maximum compressive axial load of around 27 N after a 9008 fixator-screw rotation. The bone-implant systems were observed for 24 h to measure system plasticity. The compressive force was measured repeatedly. After 24 h, the overall reduction in compressive force was less than 10% of the initial compressive force with minimal pin deformity. Neither the control nor the distraction situation was characterised ex vivo. Statistical data are expressed as mean and SEM values. To detect a clinically interesting difference in fracture strength of 10 N (SD 7.0) with a power of 80% and a level of significance of 0.05, the number of animals needed in each group for statistical analysis was preexperimentally calculated to be nine. Allowing for of expected surgical complications, ten animals were assigned to each of the six subgroups. Significant differences between the groups were tested by a one-way analysis of variance (ANOVA) with the Fischer’s least significant difference (LSD) pairwise multiple comparison test when applicable; the level of statistical significance was set at p < 0.05. Results Radiographs revealed healing of all fractures with sparse production of external callus (Fig. 2). mCT scans of all tibias in the three treatment groups of compression, control and distraction revealed bone lengths of 42.5 (0.2), 42.0 (0.2) and 43.8 (0.2), respectively (mean (SE), mm). There was no significant difference between compression and control (p = 0.133). Compression and control bones were significantly shorter than distracted bones (p < 0.001).
[(Fig._2)TD$IG]
Table 1 DXA measured Callus Area (CA, mm2), Bone Mineral Content (BMC, 10 3 g) and Bone Mineral Density (BMD, 10 3 g/cm2) in a 3.8 mm region of interest (ROI) at the fracture site in rat tibial diaphyseal fractures at 30 and 60 days post-osteotomy in the compression, control, and distraction groups.
CA 30 Days 60 Days BMC 30 Days 60 Days BMD 30 Days 60 Days
Compression
Control
Distraction
ANOVAa
22.4 1.0 19.8 0.9
21.3 0.6 19.3 0.6
23.7 0.9 22.4 0.9
0.181 0.025b
49.4 3.9 40.3 1.8
45.9 2.8 37.3 2.3
41.5 3.7 47.9 3.1
0.290 0.019c
22.2 1.6 20.5 1.1
21.7 1.3 19.3 0.9
17.5 1.3 21.6 1.3
0.049d 0.413
Mean SEM. a An LSD post-hoc comparison was used when applicable. b Compression versus distraction, p = 0.030; control versus distraction, p = 0.013. c Compression versus distraction, p = 0.041; control versus distraction, p = 0.007. d Compression versus distraction, p = 0.025; control versus distraction, p = 0.046.
DXA-measured (Table 1) BMD was significantly higher in both the compression and the control groups than in the distraction group at 30 days, while at 60 days it did not differ significantly between the groups. Both CA and BMC were significantly lower in both the compression and the control groups compared with the distraction at 60 days, not at 30 days. QCT of a 2.0-mm ROI at the fracture site (Table 2) at 30 days revealed the pronounced formation of soft and hard callus in all treatment groups with no significant differences between them. The volume of cortical bone in the distraction group was significantly than in the other two groups. At 60 days, the compression and the control groups had significantly less soft and hard callus and more cortical bone compared with the distraction group. vBMD was significantly higher in the compression and control groups than in the distraction group at both 30 and 60 days. Mechanical testing (Table 3) revealed no significant difference in strength and stiffness between the treatment groups at 30 days. However, both the compression and control groups had significantly more bending strength and stiffness at 60 days than in the distraction group. At 30 days, the fracture energy was lower in the compression group than in the control group. There were no other
Table 2 QCT measurement of a 2.0 mm ROI at the fracture site in rat tibial diaphyseal fractures at 30 and 60 days post-osteotomy in the compression, control, and distraction groups.
30 Days Soft callus (mm3) Hard callus (mm3) Cortical bone (mm3) vBMD (mg cm 3) 60 Days Soft callus (mm3) Hard callus (mm3) Cortical bone (mm3) vBMD (mg cm 3)
Fig. 2. X-ray of a rat tibia at 60 days after osteotomy and stabilization with an external fixator.
ANOVAa
Compression
Control
Distraction
10.0 1.8 20.1 2.3 3.8 0.8 764 37
9.0 2.0 19.5 2.3 5.2 0.9 836 46
8.7 1.1 15.7 1.6 1.6 0.4 693 24
0.844 0.292 0.006b 0.034c
3.2 0.4 8.6 1.0 9.1 0.7 946 27
3.2 0.5 6.4 0.8 10.3 0.5 965 30
6.4 1.1 16.6 1.7 5.0 0.6 783 37
0.004d <0.001e <0.001e 0.001f
Scan data have been converted in to mineral densities using a standard Lucite1 phantom and then segmented into soft callus (>171 mg cm 3), hard callus (>540 mg cm 3) and cortical bone (>1200 mg cm 3). Volumetric bone mineral density (vBMD) is also calculated. Mean SEM. a An LSD post-hoc comparison was used when applicable. b Compression versus distraction, p = 0.038; control versus distraction, p = 0.002. c Control versus distraction, p = 0.010. d Compression versus distraction, p = 0.003; control versus distraction, p = 0.004. e Both compression versus distraction and control versus distraction, p < 0.001. f Compression versus distraction, p = 0.001; control versus distraction, p < 0.001.
U. Sigurdsen et al. / Injury, Int. J. Care Injured 42 (2011) 1152–1156 Table 3 Bending strength (N), energy (N mm) and stiffness (N/mm) in rat tibial diaphyseal fractures at 30 and 60 days post-osteotomy in the compression, control, and distraction groups.
Strength 30 Days 60 Days Rigidity 30 Days 60 Days Energy 30 Days 60 Days
ANOVAa
Compression
Control
Distraction
5.0 1.3 18.0 2.5
7.6 1.0 19.2 2.8
7.2 1.5 11.4 1.4
0.340 0.047b
2.0 0.6 4.5 0.5
2.3 0.5 5,9 0.9
2.6 0.7 2.4 0.3
0.772 <0.001c
7.1 1.9 39.7 7.1
16.0 2.8 32.3 3.0
10.7 2.0 29.5 5.2
0.033d 0.423
Mean SEM. a An LSD post-hoc comparison was used when applicable. b Compression versus distraction, p = 0.046; control versus distraction, p = 0.013. c Compression versus distraction, p = 0.016; control versus distraction, p < 0.01. d Compression versus control, p = 0.010.
significant differences in mechanical characteristics between the compression and control groups at either 30 or 60 days. Discussion The results of earlier studies indicate that leg fractures in rats regain their mechanical properties similarly to intact bone at about 60 days post-injury.9,27 In the present study, we focused on the early and late phases of healing (30 days vs. 60 days). We found that the mechanical compressive interfragmentary force did not induce any positive stimulatory effect on bone healing in terms of fracture mineralization or mechanical bending strength and stiffness compared with static fixation (the control condition). On the other hand, the compressive stimuli did not delay fracture healing, as observed in the distraction bones exhibiting inferior strength and stiffness and a more immature callus mineralization in the late phase. Both the tight apposition of fragments and an intact fibula may explain the lack of shortening of bones exposed to compressive force and the densitometrically and biomechanically comparable results between those bones and the static bones (control). The mean group lengths indicate that the exact initial surgical reduction of the fracture fragments with a zero interfragmentary distance limited further compressive force-driven interfragmentary movement. In the static (control) group, interfragmentary contact between the bone ends and interfragmentary compressive stresses during function would have been possible, albeit perhaps at a lower magnitude than in the compression group. In addition, the fibula was left intact to increase torsional stability and prevent detrimental shear forces and rotational malunion.1,16 Increasing the compressive interfragmentary force in the boneimplant system, as in the compression group, would be expected to increase the system stiffness. While the stiffness of external fixators has a documented effect on healing,31 the role of the increased stiffness on fracture healing in our experiments is unclear since we did not include a control group without compression. Interestingly, at 30 days, the important biomechanical and densitometric properties were remarkably similar between the groups. Mechanical stimulation via the interfragmentary compressive force did not significantly alter the strength, stiffness or densitometric properties of the healing bone at this time point; only a significantly lower energy absorption before fracture in the compression group was recorded. Even though both BMD and vBMD were significantly higher in the compression and control groups, strength, stiffness and QCT-measured callus formation did not differ significantly at 30 days post-osteotomy. This confirms
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that the 30-day time point represents the early phase of fracture healing with a relatively large, immature and weak callus being present in all groups. Both the densitometric and mechanical properties suggest an ongoing remodelling activity in all groups in the late phase, and we observed two distinct densitometric patterns. First, in the compression and control groups, from days 30 to 60, there was a characteristic decrease in ‘immaturity’ in terms of the DXA parameters BMC and CA and the QCT parameters soft and hard callus with a simultaneous increase in ‘maturity’ in terms of strength, stiffness and other QCT parameters (cortical bone and vBMD) with values close to those of intact tibias. This indicates that the healing of the bones in these two groups was nearcompletion. Moreover, it is also evident that the two-dimensional DXA parameter BMD was a poor marker of this callus maturation in the compression and control groups. Second, in the distraction group, the pronounced callus that formed in the early phase was not reduced at 60 days. Even though the amount of cortical bone increased significantly between 30 and 60 days, there was still significantly less of it compared to that observed in the compression and control groups. The early mechanical distraction did induce a positive stimulatory effect in terms of a larger amount of soft and hard callus at 60 days, but the reduced strength, stiffness, cortical bone and vBMD indicate a postponement of callus maturation in this group. The presence of significantly longer tibias in the distraction group indicates that the early distraction creates a fracture gap. In addition, the longer distraction group tibias suggest that the intact fibula does not significantly limit the distraction. The bones of this group is significantly weaker with a more immature callus at 60 days. It has been shown previously that both the size of the fracture gap and the fracture stabilization stiffness influence bone healing.4,19 These factors may have played a role in the bone healing observed in the present study. The more than threefold increase in cortical bone in the distraction group between 30 and 60 days was indicative of a fracture gap that was not yet fully mineralized. As mentioned above, a 1.25-mm gap or transverse segment in the tibial diaphysis corresponds to approximately 5.5-mm3 of cortical bone, which is more than the mean difference between the distraction group and both of the other groups at both 30 and 60 days. Distracting the fragments caused cortical bone to be withdrawn from the ROI. The difference in cortical bone volume between the groups at 60 days is approximately equal to the amount of cortical bone withdrawn from the ROI adding 5.5-mm3 of cortical bone volume to the average cortical bone measured in the distraction group result in the distraction group no longer exhibiting an inferior in terms of cortical bone volume at 60 days. However, the clinically most interesting feature of the tibias is the significantly reduced strength, the distraction treatment thus representing a model of inferior bone healing compared with both the control and compression groups. In addition, there was a significant increase in strength and no significant reduction in rigidity in this group between days 30 and 60. Thus, no clear signs of failed healing were detected. The results in the distraction group therefore probably reflect the characteristic delayed but robust healing process associated with distraction osteogenesis.17,18 However, this process has the disadvantage of an extended healing time and the need to avoid a critical gap size. In short, given a longer healing time, the distracted bones would probably continue to remodel and, consequently, improve their mechanical properties. In this study, a slower maturation of the distracted callus segment was expected, and this provided a second reference for comparison. There are limitations to this study. The interfragmentary movement or compressive strain were not measured continously.
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However, there were no signs of pin or fixator loosening, which suggests that there was only minor interfragmentary motion.3,4 Moreover, the callus segment was not examined histologically. Such an examination would have necessitated a larger number of animals or the ability to perform non-destructive biomechanical evaluation of the bones. However, previous investigations of the distraction and compression regimens have not revealed significant differences in data from histological light-microscopy evaluations.3 The difference in the ROIs for DXA (length of 3.8 mm) and QCT (length of 2 mm) measurements prevents a direct comparison. Given the mean bone lengths in the compression and static groups, similar portions of callus and cortical boneends were probably measured in the two groups. It is difficult to separate the effects of the gap and the distractive force in the distraction group without including another group with an initial distraction gap of 1.25 mm. Similarly, yet another study group (of compression or distraction) with for example, a fibula osteotomy, could help to isolate and identify the effect of the intact fibula. The lengthening of the tibias in the distraction group may limit the application of the protocol. However, our aim was to investigate the process of healing in a fracture exposed to an axial compressive force and compare this to that in a fracture undergoing either static fixation or exposure to a distraction force. Future studies could combine defined periods with simultaneous distraction and compression of the fracture gap in order to prevent limb lengthening or shortening.3 In theory, a clinically applicable combination of early compression and distraction might stimulate callus formation without creating a fracture gap, avoiding the induction of delayed healing or nonunion, and potentially enhance the healing and recovery of mechanical properties. In conclusion, we found that (1) compression in externally fixed tibias did not enhance fracture healing in terms of mineralization of the fracture gap and mechanical properties at midterm and at the time of union compared with statically fixed bones, and (2) both compression and static fixation induced superior mechanical properties at 60 days and a more mature callus mineralization compared with distraction. Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgements Financial support for this study was provided by the Faculty Division Akershus University Hospital, University of Oslo and the Institute of Surgical Research, Oslo University Hospital Rikshospitalet, Oslo. The authors have no professional nor financial affiliations that may be perceived to have biased this presentation. We wish to thank engineer Per Ludvigsen for his assistance in the mechanical testing, researcher Lise Sofie Nissen-Meyer for technical guidance in the DXA measurements, chief engineer Hong Qu for assistance in the CT imaging, radiographer Camilla Stolp, Knut Rekdahl for producing the external fixators and pins and the Center for Comparative Medicine, Oslo University Hospital Rikshospitalet for housing our rodents. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.injury.2010.08.018.
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Injury journal homepage: www.elsevier.com/locate/injury
The influence of compression on the healing of experimental tibial fractures Ulf Sigurdsen a,b,*, Olav Reikeras c, Stein Erik Utvag d,a a
Department of Orthopaedic Surgery, University of Oslo, Oslo, Norway Institute of Surgical Research, Oslo University Hospital Rikshospitalet, Oslo, Norway c Department of Orthopaedic Surgery, Oslo University Hospital Rikshospitalet, Oslo, Norway d Department of Orthopaedic Surgery, Akershus University Hospital, Nordbyhagen, Norway b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 18 August 2010
Purpose: Experimental studies of the effects of various mechanical conditions and stimuli on bone healing have disclosed an improvement potential in bone fracture mineralization and biomechanical properties. We therefore evaluated the effect of a clinically practicable application of a mechanical compressive interfragmentary stimulus on the healing of experimental tibial diaphyseal fractures. Methods: Sixty Male rats received a standardized tibial shaft osteotomy stabilized with a unilateral external fixator with a zero interfragmentary distance, and then randomly assigned to the compression (N = 20), control (N = 20) or distraction (N = 20) group. From days 4 to day 14, the external fixator was either tightened (compression group) or loosened (distraction group) once daily to gradually induce a total axial displacement of the external fixator pin clamps of 1.25 mm. Evaluation at 30 and 60 days postosteotomy included radiography, dual-energy X-ray absorptiometry (DXA), quantitative CT and mechanical testing. Results: All fractures healed radiographically with sparse callus. At 60 days, the compression and control groups exhibited significantly less amount of mineralized callus in terms of DXA measured callus area and bone mineral content (BMC) compared to the distraction group. These groups also demonstrated a smaller volume of low-mineralized bone tissue (callus) and a larger volume of highly mineralized bone tissue (cortical bone) measured by QCT than in the distraction group. Both mechanical strength and stiffness was significantly higher in the compression and control groups than in the distraction group at 60 days. Discussion: Compression did not enhance fracture healing in terms of mineralization, bending strength, or stiffness at the time of union, compared with the control condition. The compression and control groups exhibited improved healing in terms of mechanical strength and stiffness and a more mature callus mineralization compared with the distraction group. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Experimental rat model Tibial diaphyseal fracture healing External fixation Axial compression and axial distraction
Introduction Bone healing is a complicated physiologic process. The final result of fracture healing depends not only upon the fracture type, gap condition, host factors, blood supply and neural and hormonal regulation, but also upon the mechanobiological factors in the fracture environment.4,8,14,17–19,23,24 Research and publications on these factors are extensive, and mechanical fracture stimulation has previously been shown to significantly enhance bone healing. Early axial dynamization increased stiffness and tended to increase torsional strength in 2-mm gap transverse canine mid-tibial fractures stabilized with a unilateral external fixator.6 A short-term interfragmentary cyclic micromovement applied at a relatively high * Corresponding author at: Institute of Surgical Research, Oslo University Hospital Rikshospitalet, 0027 Oslo, Norway. Tel.: +47 23 07 35 17; fax: +47 23 07 25 30. E-mail address: [email protected] (U. Sigurdsen). 0020–1383/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2010.08.018
strain rate induced greater amount of periosteal callus and a higher maximum torque load.11 A difference between early and late application of micromotion within the fracture gap has also been demonstrated, with early cyclic micromotion increasing bone mineral density (BMD) and stiffness, and late micromotion diminishing (BMD).6,12,20 A combination of early compression and distraction may induce increased bone formation and mechanical bending stiffness.3 Interestingly, there is no consensus as to whether compression or distraction is the more effective stimulus.22 Most studies on fracture compression have employed various mounting and/or monitoring procedures, and torque, stiffness and energy absorption to failure have been found to be lower for simple, constant compression than for the more advanced cyclic compression.13,30 To our knowledge, no clinically practicable method of mechanical fracture stimulation that enhances bone healing has yet been identified. Fracture treatment with external fixation provides advantages as a method of stable mechanical fixation with minimal surgical
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insult to the patient, which is why it is the preferred treatment in certain fractures.2,5,15 Experimental animal external fixators have become more sophisticated,21 providing the ability to manipulate mechanical parameters and interfragmentary motion more precisely (e.g., axial compression, distraction and dynamization).3 The aims of this study were: (1) to evaluate the effect of a clinically applicable, mechanical compressive stimulus on fracture healing and (2) to compare healing under compression and static conditions with that effected using distraction, which induces slower consolidation of the callus segment. Materials and methods Animals and surgical procedures Sixty male Wistar rats (Møllega˚rds Avlslaboratorium, Eiby, Denmark), weighing approximately 350 g (312–392 g), were used in this study. The experiment conformed to the Norwegian Council of Animal Research Code for the Care and Use of Animals for Experimental Purposes and standard laboratory animal anaesthesia, analgesia and post-operative follow-up.28 The left tibia was exposed through an anterior incision from the tibial tuberosity and in the distal direction. The muscles on the medial and lateral aspects of the tibia were carefully elevated from the tibia and the anterior two-thirds of the tibia was cut at the level of the anterior ridge using a fine-toothed circular saw blade mounted on an electric drill. Then, the remaining one-third was manually broken, leaving the fibula intact. The aluminium/steel external fixator used in the study (Fig. 1) has been described previously by Mark et al. and by our research group.21,27 Four steel pins (diameter = 1.0 mm) were inserted: two proximal and two distal to the fracture. The core drill holes in the tibia were 0.8 mm in diameter. Fixator offset, which is the free length of the pins between the rat’s anterolateral tibial surface and the inner side of the fixator bar, was 6 mm. The position of the external fixator enabled free movement of the ankle and knee joints. The perioperative alignment and accurate fracture reduction with a zero interfragment distance were verified visually and manually. The rats were then randomly assigned to the compression (N = 20), control (N = 20) or distraction (N = 20) condition. The compressive and distractive stimuli were applied by manipulating of the external fixator from days 4 to day 14. Once daily during this early 10-day period, the tubular fixator steel screw connecting the pin clamps was either tightened (compression group) or loosened (distraction group) using a standardized tool, resulting in a daily screw rotation of 908 corresponding to a pin clamp displacement of 1/8 mm. Total displacement was thus 10 1/8 mm = 1.25 mm. The control group received similar sham manipulations. After day 14, no manipulation of the fixator was performed. Half of the animals in each study group were euthanised by an intra-peritoneal injection of pentobarbital at 30 days, and the other half at 60 days. The tibias were dissected free and examined visually, and the external fixators were carefully removed. The [(Fig._1)TD$IG]
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bones were kept frozen at 80 8C between dissection and radiological, dual-energy X-ray absorptiometry (DXA) and mechanical evaluations. Bone evaluation and statistics Radiographs were performed on a standard clinical digital system (Axiom Aristos; Siemens AG, Mu¨nchen, Germany). The Xray tube settings were 46 kV, 1.0 mAs and focus-to-film (sourceto-image-receptor) distance was 115 cm. DXA measurement was performed using a densitometer system for research animals (Piximus; Lunar Corp, Madison, WI, USA). The X-ray tube voltage, current, focal spot size and spot-to-imagereceptor distance were 80 kV, 400 mA, 0.25 mm 0.25 mm and 32 cm, respectively. The densitometric values29 for bone mineral content (BMC), BMD and callus area (CA) were calculated by the software from a 3.8-mm region of interest (ROI) at the fracture site, covering the entire fracture callus observed on the radiographs. Micro-computed tomography (mCT) scanning of the tibias and a LuciteTM phantom doped with hydroxyapatite (HA) with known mineral densities of 50, 250 and 750 mg cm 3 was performed on a Micro CAT II system (Imtek, now Siemens) with 300 steps and 2008 of rotation, and the X-ray camera detector size was 2048 2048 with a bin factor of 2. Exposure time was 500 ms and the voxels were cubic with a side length of 47.8 mm. The images were reconstructed and analysed with a commercially available reconstruction and visualization software package (Amira v4.1, Mercury Computer Systems Inc., Me´rignac Cedex, France). The analysed volume of interest was a 2-mm segment encompassing the fracture region including a smaller portion of the cortical bone ends in the fracture gap. Average Hounsfield values for areas within the different phantom densities on different cross-sections were analysed with linear regression and the regression line function was used to linearly convert calibrated CT attenuation in to HA density.26 Quantitative CT (QCT) measurements included soft callus (<171 mg cm 3), hard callus (<540 mg cm 3), cortical bone (<1200 mg cm 3) and volumetric bone mineral density (vBMD). Intact tibias (N = 5) were also scanned, and QCT measurements from a 1.25-mm ROI at the level of the osteotomy yielded 2.1 0.3 mm3 soft callus, 1.9 0.1 mm3 hard callus, 5.5 0.2 mm3 cortical bone and 936 6 mg cm 3 vBMD (mean SE). The tibias were placed between gauze pads soaked with normal saline before a bending test was performed using universal testing machine with a servo-hydraulic mechanical linear drive actuator with 100-mm of total vertical displacement and a maximum axial tension load of 250 N (MTS 858 Mini Bionix; MTS Systems Corp, Eden Prairie, MN, USA). The set-up included a three-point cantilever bending test10 designed to test the bone at the fracture site with the vertical travel speed set to 160 mm min 1. The obtained log file was then converted to a load-deformation curve7 and values for basic mechanical bone properties including strength, stiffness and work-to-fracture were obtained using a mathematical software package (Origin v 7.5; OriginLab Corp.,
Fig. 1. Schematic of the external fixator used in this study.
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Northampton, MA, USA).7,25 A similar bending test performed on ex vivo tibias with externally fixed osteotomies (N = 5) exhibited a stiffness of 3.2 0.6 N mm 1 (mean values standard error of mean (SEM)). In comparison, the stiffness of intact tibias (N = 5) was 3.6 0.3 N mm 1. A cadaveric test (Supplementary Table 1) showed a close to linear increase in measured interfragmentary compressive force in rat tibias (N = 5) with the external fixator mounted and manipulated as in the compression group reaching a maximum compressive axial load of around 27 N after a 9008 fixator-screw rotation. The bone-implant systems were observed for 24 h to measure system plasticity. The compressive force was measured repeatedly. After 24 h, the overall reduction in compressive force was less than 10% of the initial compressive force with minimal pin deformity. Neither the control nor the distraction situation was characterised ex vivo. Statistical data are expressed as mean and SEM values. To detect a clinically interesting difference in fracture strength of 10 N (SD 7.0) with a power of 80% and a level of significance of 0.05, the number of animals needed in each group for statistical analysis was preexperimentally calculated to be nine. Allowing for of expected surgical complications, ten animals were assigned to each of the six subgroups. Significant differences between the groups were tested by a one-way analysis of variance (ANOVA) with the Fischer’s least significant difference (LSD) pairwise multiple comparison test when applicable; the level of statistical significance was set at p < 0.05. Results Radiographs revealed healing of all fractures with sparse production of external callus (Fig. 2). mCT scans of all tibias in the three treatment groups of compression, control and distraction revealed bone lengths of 42.5 (0.2), 42.0 (0.2) and 43.8 (0.2), respectively (mean (SE), mm). There was no significant difference between compression and control (p = 0.133). Compression and control bones were significantly shorter than distracted bones (p < 0.001).
[(Fig._2)TD$IG]
Table 1 DXA measured Callus Area (CA, mm2), Bone Mineral Content (BMC, 10 3 g) and Bone Mineral Density (BMD, 10 3 g/cm2) in a 3.8 mm region of interest (ROI) at the fracture site in rat tibial diaphyseal fractures at 30 and 60 days post-osteotomy in the compression, control, and distraction groups.
CA 30 Days 60 Days BMC 30 Days 60 Days BMD 30 Days 60 Days
Compression
Control
Distraction
ANOVAa
22.4 1.0 19.8 0.9
21.3 0.6 19.3 0.6
23.7 0.9 22.4 0.9
0.181 0.025b
49.4 3.9 40.3 1.8
45.9 2.8 37.3 2.3
41.5 3.7 47.9 3.1
0.290 0.019c
22.2 1.6 20.5 1.1
21.7 1.3 19.3 0.9
17.5 1.3 21.6 1.3
0.049d 0.413
Mean SEM. a An LSD post-hoc comparison was used when applicable. b Compression versus distraction, p = 0.030; control versus distraction, p = 0.013. c Compression versus distraction, p = 0.041; control versus distraction, p = 0.007. d Compression versus distraction, p = 0.025; control versus distraction, p = 0.046.
DXA-measured (Table 1) BMD was significantly higher in both the compression and the control groups than in the distraction group at 30 days, while at 60 days it did not differ significantly between the groups. Both CA and BMC were significantly lower in both the compression and the control groups compared with the distraction at 60 days, not at 30 days. QCT of a 2.0-mm ROI at the fracture site (Table 2) at 30 days revealed the pronounced formation of soft and hard callus in all treatment groups with no significant differences between them. The volume of cortical bone in the distraction group was significantly than in the other two groups. At 60 days, the compression and the control groups had significantly less soft and hard callus and more cortical bone compared with the distraction group. vBMD was significantly higher in the compression and control groups than in the distraction group at both 30 and 60 days. Mechanical testing (Table 3) revealed no significant difference in strength and stiffness between the treatment groups at 30 days. However, both the compression and control groups had significantly more bending strength and stiffness at 60 days than in the distraction group. At 30 days, the fracture energy was lower in the compression group than in the control group. There were no other
Table 2 QCT measurement of a 2.0 mm ROI at the fracture site in rat tibial diaphyseal fractures at 30 and 60 days post-osteotomy in the compression, control, and distraction groups.
30 Days Soft callus (mm3) Hard callus (mm3) Cortical bone (mm3) vBMD (mg cm 3) 60 Days Soft callus (mm3) Hard callus (mm3) Cortical bone (mm3) vBMD (mg cm 3)
Fig. 2. X-ray of a rat tibia at 60 days after osteotomy and stabilization with an external fixator.
ANOVAa
Compression
Control
Distraction
10.0 1.8 20.1 2.3 3.8 0.8 764 37
9.0 2.0 19.5 2.3 5.2 0.9 836 46
8.7 1.1 15.7 1.6 1.6 0.4 693 24
0.844 0.292 0.006b 0.034c
3.2 0.4 8.6 1.0 9.1 0.7 946 27
3.2 0.5 6.4 0.8 10.3 0.5 965 30
6.4 1.1 16.6 1.7 5.0 0.6 783 37
0.004d <0.001e <0.001e 0.001f
Scan data have been converted in to mineral densities using a standard Lucite1 phantom and then segmented into soft callus (>171 mg cm 3), hard callus (>540 mg cm 3) and cortical bone (>1200 mg cm 3). Volumetric bone mineral density (vBMD) is also calculated. Mean SEM. a An LSD post-hoc comparison was used when applicable. b Compression versus distraction, p = 0.038; control versus distraction, p = 0.002. c Control versus distraction, p = 0.010. d Compression versus distraction, p = 0.003; control versus distraction, p = 0.004. e Both compression versus distraction and control versus distraction, p < 0.001. f Compression versus distraction, p = 0.001; control versus distraction, p < 0.001.
U. Sigurdsen et al. / Injury, Int. J. Care Injured 42 (2011) 1152–1156 Table 3 Bending strength (N), energy (N mm) and stiffness (N/mm) in rat tibial diaphyseal fractures at 30 and 60 days post-osteotomy in the compression, control, and distraction groups.
Strength 30 Days 60 Days Rigidity 30 Days 60 Days Energy 30 Days 60 Days
ANOVAa
Compression
Control
Distraction
5.0 1.3 18.0 2.5
7.6 1.0 19.2 2.8
7.2 1.5 11.4 1.4
0.340 0.047b
2.0 0.6 4.5 0.5
2.3 0.5 5,9 0.9
2.6 0.7 2.4 0.3
0.772 <0.001c
7.1 1.9 39.7 7.1
16.0 2.8 32.3 3.0
10.7 2.0 29.5 5.2
0.033d 0.423
Mean SEM. a An LSD post-hoc comparison was used when applicable. b Compression versus distraction, p = 0.046; control versus distraction, p = 0.013. c Compression versus distraction, p = 0.016; control versus distraction, p < 0.01. d Compression versus control, p = 0.010.
significant differences in mechanical characteristics between the compression and control groups at either 30 or 60 days. Discussion The results of earlier studies indicate that leg fractures in rats regain their mechanical properties similarly to intact bone at about 60 days post-injury.9,27 In the present study, we focused on the early and late phases of healing (30 days vs. 60 days). We found that the mechanical compressive interfragmentary force did not induce any positive stimulatory effect on bone healing in terms of fracture mineralization or mechanical bending strength and stiffness compared with static fixation (the control condition). On the other hand, the compressive stimuli did not delay fracture healing, as observed in the distraction bones exhibiting inferior strength and stiffness and a more immature callus mineralization in the late phase. Both the tight apposition of fragments and an intact fibula may explain the lack of shortening of bones exposed to compressive force and the densitometrically and biomechanically comparable results between those bones and the static bones (control). The mean group lengths indicate that the exact initial surgical reduction of the fracture fragments with a zero interfragmentary distance limited further compressive force-driven interfragmentary movement. In the static (control) group, interfragmentary contact between the bone ends and interfragmentary compressive stresses during function would have been possible, albeit perhaps at a lower magnitude than in the compression group. In addition, the fibula was left intact to increase torsional stability and prevent detrimental shear forces and rotational malunion.1,16 Increasing the compressive interfragmentary force in the boneimplant system, as in the compression group, would be expected to increase the system stiffness. While the stiffness of external fixators has a documented effect on healing,31 the role of the increased stiffness on fracture healing in our experiments is unclear since we did not include a control group without compression. Interestingly, at 30 days, the important biomechanical and densitometric properties were remarkably similar between the groups. Mechanical stimulation via the interfragmentary compressive force did not significantly alter the strength, stiffness or densitometric properties of the healing bone at this time point; only a significantly lower energy absorption before fracture in the compression group was recorded. Even though both BMD and vBMD were significantly higher in the compression and control groups, strength, stiffness and QCT-measured callus formation did not differ significantly at 30 days post-osteotomy. This confirms
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that the 30-day time point represents the early phase of fracture healing with a relatively large, immature and weak callus being present in all groups. Both the densitometric and mechanical properties suggest an ongoing remodelling activity in all groups in the late phase, and we observed two distinct densitometric patterns. First, in the compression and control groups, from days 30 to 60, there was a characteristic decrease in ‘immaturity’ in terms of the DXA parameters BMC and CA and the QCT parameters soft and hard callus with a simultaneous increase in ‘maturity’ in terms of strength, stiffness and other QCT parameters (cortical bone and vBMD) with values close to those of intact tibias. This indicates that the healing of the bones in these two groups was nearcompletion. Moreover, it is also evident that the two-dimensional DXA parameter BMD was a poor marker of this callus maturation in the compression and control groups. Second, in the distraction group, the pronounced callus that formed in the early phase was not reduced at 60 days. Even though the amount of cortical bone increased significantly between 30 and 60 days, there was still significantly less of it compared to that observed in the compression and control groups. The early mechanical distraction did induce a positive stimulatory effect in terms of a larger amount of soft and hard callus at 60 days, but the reduced strength, stiffness, cortical bone and vBMD indicate a postponement of callus maturation in this group. The presence of significantly longer tibias in the distraction group indicates that the early distraction creates a fracture gap. In addition, the longer distraction group tibias suggest that the intact fibula does not significantly limit the distraction. The bones of this group is significantly weaker with a more immature callus at 60 days. It has been shown previously that both the size of the fracture gap and the fracture stabilization stiffness influence bone healing.4,19 These factors may have played a role in the bone healing observed in the present study. The more than threefold increase in cortical bone in the distraction group between 30 and 60 days was indicative of a fracture gap that was not yet fully mineralized. As mentioned above, a 1.25-mm gap or transverse segment in the tibial diaphysis corresponds to approximately 5.5-mm3 of cortical bone, which is more than the mean difference between the distraction group and both of the other groups at both 30 and 60 days. Distracting the fragments caused cortical bone to be withdrawn from the ROI. The difference in cortical bone volume between the groups at 60 days is approximately equal to the amount of cortical bone withdrawn from the ROI adding 5.5-mm3 of cortical bone volume to the average cortical bone measured in the distraction group result in the distraction group no longer exhibiting an inferior in terms of cortical bone volume at 60 days. However, the clinically most interesting feature of the tibias is the significantly reduced strength, the distraction treatment thus representing a model of inferior bone healing compared with both the control and compression groups. In addition, there was a significant increase in strength and no significant reduction in rigidity in this group between days 30 and 60. Thus, no clear signs of failed healing were detected. The results in the distraction group therefore probably reflect the characteristic delayed but robust healing process associated with distraction osteogenesis.17,18 However, this process has the disadvantage of an extended healing time and the need to avoid a critical gap size. In short, given a longer healing time, the distracted bones would probably continue to remodel and, consequently, improve their mechanical properties. In this study, a slower maturation of the distracted callus segment was expected, and this provided a second reference for comparison. There are limitations to this study. The interfragmentary movement or compressive strain were not measured continously.
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However, there were no signs of pin or fixator loosening, which suggests that there was only minor interfragmentary motion.3,4 Moreover, the callus segment was not examined histologically. Such an examination would have necessitated a larger number of animals or the ability to perform non-destructive biomechanical evaluation of the bones. However, previous investigations of the distraction and compression regimens have not revealed significant differences in data from histological light-microscopy evaluations.3 The difference in the ROIs for DXA (length of 3.8 mm) and QCT (length of 2 mm) measurements prevents a direct comparison. Given the mean bone lengths in the compression and static groups, similar portions of callus and cortical boneends were probably measured in the two groups. It is difficult to separate the effects of the gap and the distractive force in the distraction group without including another group with an initial distraction gap of 1.25 mm. Similarly, yet another study group (of compression or distraction) with for example, a fibula osteotomy, could help to isolate and identify the effect of the intact fibula. The lengthening of the tibias in the distraction group may limit the application of the protocol. However, our aim was to investigate the process of healing in a fracture exposed to an axial compressive force and compare this to that in a fracture undergoing either static fixation or exposure to a distraction force. Future studies could combine defined periods with simultaneous distraction and compression of the fracture gap in order to prevent limb lengthening or shortening.3 In theory, a clinically applicable combination of early compression and distraction might stimulate callus formation without creating a fracture gap, avoiding the induction of delayed healing or nonunion, and potentially enhance the healing and recovery of mechanical properties. In conclusion, we found that (1) compression in externally fixed tibias did not enhance fracture healing in terms of mineralization of the fracture gap and mechanical properties at midterm and at the time of union compared with statically fixed bones, and (2) both compression and static fixation induced superior mechanical properties at 60 days and a more mature callus mineralization compared with distraction. Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgements Financial support for this study was provided by the Faculty Division Akershus University Hospital, University of Oslo and the Institute of Surgical Research, Oslo University Hospital Rikshospitalet, Oslo. The authors have no professional nor financial affiliations that may be perceived to have biased this presentation. We wish to thank engineer Per Ludvigsen for his assistance in the mechanical testing, researcher Lise Sofie Nissen-Meyer for technical guidance in the DXA measurements, chief engineer Hong Qu for assistance in the CT imaging, radiographer Camilla Stolp, Knut Rekdahl for producing the external fixators and pins and the Center for Comparative Medicine, Oslo University Hospital Rikshospitalet for housing our rodents. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.injury.2010.08.018.
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