This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.
This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.
Distraction osteogenesis (DO) is a surgical technique widely used clinically1,2,3,4 in humans for lower1,2 and upper3 limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision as well as in maxillofacial reconstruction4. DO leads to bone regeneration after placement of an external fixator in bone and osteotomy. The osteotomized extremities are moved away from each other by gradual distraction2 to reach the desired elongation. A consolidation period follows, during which there is no more elongation.
The DO procedure is divided into three distinct phases: latency, distraction, and consolidation. Generally, a 7-day latency period starts just after osteotomy4. This allows bone repair to begin the initial step of the healing process4. The latency period is followed by a distraction period where traction forces are applied to the regenerated callus and surrounding soft tissues1,2,4. When the desired elongation is reached, distraction stops and the consolidation period begins. During this period, the external fixator is maintained until the regenerated bone is functional enough to support its removal.
Various parameters of DO influence bone repair such as length and rate of lengthening, type of external fixator, frequency of distraction, length of the consolidation period, or type of mechanical stress applied to the distracted callus. As an example, the rate and frequency of lengthening can lead either to premature consolidation5 or disruption of the process by creating non-recoverable damage like necrotic tissue or cysts within the callus6,7.
Many DO protocols have been applied to different animal models8,9,10 to study bone repair processes and to maximize bone consolidation. In rats, most studies11,12,13,14,15 focused on how to shorten the DO protocol by speeding up callus consolidation. Some of these experimental studies used external fixators already commercially available for human clinical applications5,13,15,16. However, these types of external fixator are not suitable for DO on the rat femur, which exhibits different anatomical characteristics from the human femur. Moreover, only a few studies clearly demonstrate the efficiency of their protocols in obtaining a functional regenerated bone7,16. It is therefore difficult to compare results from various DO studies, due to their differing protocols and lack of information regarding the external fixator12,13,14,17.
Thus, the aim of this study was to describe, in a rat model, an efficient and reproducible protocol for DO on the femur that leads to a functional regenerated bone. To this end, we designed a homemade and easy-to-use external fixator especially for the rat femur, which we have described in detail in this protocol. In drafting the technical specifications for this device, we took into account all the fundamental constraints for a good distribution of mechanical stresses and avoiding the production of residual stress. The technical specification included an appropriate geometry for the device to allow pure traction force on bones and surrounding tissue, an appropriate weight for the gait of the animal, control of the length of bone elongation, and a good alignment of bone segments without production of shear stress at the intersection of pins and bone. Moreover, this device had to be usable without sedation of the animal during distraction, biocompatible, and sterilizable without damage. After 7 weeks of consolidation, this protocol for DO on the rat femur led to a functional regenerated bone, demonstrated by the animals' physiological weight-bearing without fracture of the regenerated callus after removal of the external fixator. The physiological gait of the animals was consistent with architectural parameters obtained from micro-CT analysis of regenerated callus and X-ray analysis.
All procedures described were approved by the University of Aix-Marseille institutional animal care and use committee and the French research ministry and performed in the conventional animal house of Marseille Medical Faculty (France).
1. Define Functional Specifications of the External Fixator Based on the Following Guidelines
2. Surgery
NOTE: An assistant is required for all surgical procedures. Four 12 week-old male Sprague Dawley rats were fed a standard laboratory diet ad libitum.
3. Distraction
The X-ray images taken from the end of surgical procedure to the end of consolidation showed no loosening of the half-threaded pins in the femur, indicating stable anchorage. The pins were parallel and well-preserved. The osteotomized extremities were well-aligned along the longitudinal axis of the bone during the DO process (Figure 2). At the end of the latency period, no calcified areas were visible (Figure 2B). At the end of the distraction period, a few calcified areas were visible close to the preexisting cortices (Figure 2C). After 28 days of consolidation (Day 45), the non-calcified region of the gap between the osteotomized extremities was smaller and we observed a periosteal callus not only near the gap but also at the level of the pins (Figure 2D). After 47 days of consolidation (Day 64), the regenerating callus was completely bridged (Figure 2E). After removal of the external fixator and 2 days of physiological weight-bearing, the animals had physiological gait and there was no evidence of fracture (Figure 2F).
3D micro-CT analysis of the serial longitudinal sections of the regenerating callus demonstrated that calcified bridging was always present (Figure 3A-D). A continuous outer cortical was observed at the periphery of the regenerating callus (Figure 3C-D). According to the longitudinal profiles, the regenerating callus was completely filled by a network of bony trabeculae. After 49 days of consolidation (Day 66), a less mineralized region remained at the center of the regenerated bone (Figure 3E-F). The architectural parameters of the micro-CT indicated that the average volume fraction (BV/TV) for the regenerating callus and the contralateral femur were respectively 55% ± 13 and 97.85% ± 1.7 (Table 1 and Table 2). The mean value of volumetric bone mineral density (vBMD) for the regenerating callus was 750 mg/cm3 ± 25. The mean value of the cross-sectional area was higher for the regenerating callus than for the contralateral femur (17.23 mm2 ± 9.3 vs 9.5 mm2 ± 1.2) (Table 1 and Table 2). The mean callus cortical thickness was thinner than the cortical thickness of contralateral femur (0.317 mm ± 0.04 vs 0.6 mm ± 0.05) (Tables 1 and 2).
Figure 1: Computer-aided design (CAD) of the external fixator and the drilling guide. (A) CAD of the external fixator with the pins. (B) CAD of the drilling guide. Scale bar = 5 mm. Please click here to view a larger version of this figure.
Figure 2: Representative X-rays of a rat femur at various time-points. X-ray radiographs of the distracted callus at 0, 7, 17, 45, 64 and 66 days. (A) X-rays taken after the surgery (Day 0). (B) X-ray radiograph taken after the 7-day latency period. No calcified callus is apparent near the osteotomized extremities (OE). (C) At the end of distraction, a small, poorly calcified zone is apparent near the osteotomized extremities (rectangular zone). (D) After 28 days of consolidation (Day 45), the gap between the osteotomized extremities (*) and a second cortical is apparent (yellow arrow). (E) Radiograph after 47 days of consolidation (Day 64) (F) X-ray radiograph taken after 49 days of consolidation (Day 66), with two days of physiological weight-bearing. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 3: 3D micro-CT reconstruction of the distracted callus of a rat femur. The mineralization of the bone is illustrated by color ranging from yellow to blue. (A,B) Representation of the bridging cortices of the distracted callus following the anteroposterior and longitudinal axis. (C,D) Proximal and distal cross-section revealing the initial cortical bone (black arrow) and the bridging cortices of the distracted callus (yellow arrow) (E,F) 3D longitudinal profiles of the distracted callus after 7 weeks of consolidation: the callus is filled by a network of trabecular bone. Scale bar = 1000 µm. Calibration bar = 0 to 2.54 g/cm3 (yellow = 1.73 g/cm3, pink = 0.84 g/cm3, and blue = 0.17 g/cm3). Please click here to view a larger version of this figure.
Table 1: Callus parameters of distracted femur after 49 days of consolidation. The region of interest (ROI) selected to calculate all the parameters was the zone between the osteotomized extremities. All the values obtained for each rat were calculated on each slice of the image stack. Results are expressed as mean ± standard deviation. BV/TV: volume fraction; CSA: cross-sectional area; vBMD: bone mineral density; Ct.Th: cortical thickness.
Table 2: Contralateral parameters of intact femur after 49 days of consolidation. All the values obtained for each rat were calculated on each slice of the image stack. Results are expressed as mean ± standard deviation. BV/TV: volume fraction; CSA: cross-sectional area; Ct.Th: cortical thickness.
This study describes a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator. Our DO protocol led to a functional regenerated bone. After 47 days of consolidation, removal of the homemade external fixator and 2 days of physiological weight-bearing by the animal did not induce any fracture of the regenerating callus. Thanks to the micro-CT reconstruction, the evidence of a complete bridging confirmed that the regenerated callus was functional. A previous study clearly demonstrated a positive correlation between the presence of calcified bridging and the load that a regenerated callus can sustain19. Moreover, the vBMD found for the regenerated callus was about 67% of the vBMD found for the diaphysis part of the femur from the controlateral limb20. We also found an average vBMD of the regenerated callus that was close to the value previously described21. This highlights the stable environment created by the homemade external fixator leading to efficient bone repair. In addition, the fixator was well tolerated by the animals. The positioning and the light weight of the external fixator did not disturb the animals’ gaits, so they were able to walk just after surgery.
Throughout the protocol, no breaking or loosening of pins occurred, evidence that the threading procedure ensured an effective and stable anchoring of the pins in the femur. It is crucial that pins are well-anchored for good stability of the DO micro-environment: loosening one pin has been shown to decrease by half the stiffness of the device18. Moreover, rats must be housed singly in cages with a specially designed flat cover to prevent the animal from hooking up the external fixator. The manual and easy-to-perform distraction of the regenerating callus limits pin-loosening but also avoids having to sedate animals every 12 h for several days and contributes to the reliability and efficiency of the homemade external fixator. Another important point to check is that the pins penetrate both cortices and do not protrude more than 1 mm. This step is sensitive, and if X-ray radiographs do not confirm appropriate positioning of the pins, this needs to be corrected manually using a Mayo-Hegar needle-holder while the animal is still anesthetized. The protocol has some limitations. It is a time-consuming and constraining protocol, which limits the number of animals it can be used on. Moreover, given the dexterity required and the meticulousness of the surgery, the procedure could take a couple of practice runs to master.
Throughout the DO procedure, the alignment of the osteotomized extremities was maintained. When implanting pins in the femur, it is very important to use a drilling guide so that the half-threaded pins are parallel, thereby ensuring good distribution of the mechanical stress without production of shear stress throughout the distraction and consolidation phases. It is also very important to clean the wound daily to prevent any blocking of the square nut that would make manual distraction difficult, which could affect the alignment of osteotomized extremities and the proper anchoring of pins. How to proceed to the osteotomy represents the last key point for the regeneration. The osteotomy must be performed with a piezotome to ensure a uniform and regular osseous section and to prevent thermic-induced necrosis and damage to surrounding soft tissues. Moreover, excessive heat is sometimes generated when drilling the pre-holes. An irrigating system coupled with the electric drill can be used to prevent thermic-induced necrosis.
To conclude, we describe an efficient and reproducible protocol, in a rat femoral model, for DO that leads to a functional regenerated bone. Designing a homemade external fixator based on the anatomical characteristics of the rat femur enabled a stable environment to be generated and allowed the formation of a calcified regenerated callus that was sufficiently consolidated to support physiological weight-bearing by the animals. Our next objective is to use this reproducible protocol to determine the shortest consolidation period compatible with a functional regenerated bone. The subsequent aim will be to improve the DO technique, especially seeking ways to shorten the consolidation period. This reproducible protocol could also be useful to identify mechanisms involved in bone repair. Finally, the characteristics of the home-made external fixator make it usable in clinical practice for human finger elongation.
The authors have nothing to disclose.
This work was supported and funded by the CNRS Mecabio challenge.
The authors thank the animal care technician for taking care of the animals throughout the procedure. The authors also acknowledge IVTV central Lyon, through Thierry Hoc. Thanks to Marjorie Sweetko for language revision.
We are greatful to Marylène Lallemand, Cécile Génovésio, and Patrick Laurent for their contribution to this experimental study.
Kétamine | Renaudin | 578 540-2 | Supply by animal house |
Médétomidine | Virbac | 6799091 | Supply by animal house |
Sevoflurane | Centravet | 567 477-2 | Supply by animal house |
Buprenorphine | Indivor France | 3400932731060 | Supply by animal house |
Enrofloxacine | ChannelPharmaceutical Facturing | FR/V/4955220 | Supply by animal house |
Piezotome | Satelec Acteon | F57510 | |
Heating pet pad | Therasage | AL8365936 | Supply by the animal house |
Dental X-ray | S.A.R.L Innovation médicales et dentaires | WYZ – BLUEX | |
Winiwix Software | Softys Dental | PFT | |
Micro-CT system | nanoScan SPECT/CT | GEIT-31105EN (05/14) | Subcontract by IVTV central Lyon |
Micro-CT analysis Software | phoenix datos X2 reconstruction | none | Free software |
Electric razor | Brawn | GT415 | Supply by animal house |
Senn’s retractors | Word Precision Instruments | 501718 | Blunt version |
Betadine Solution | Mundipharma Medical Company | D08AG02 | Supply by animal house |
Resorbable suture thread (5.0) | Ethicon | JV1023 | Supply by animal house |
Rugine | Word Precision Instruments | 503406 | |
Mayo-Hegar needle holder | Word Precision Instruments | V503382 | |
Metal drill | Beuterlock | V020944018003 | |
Micro Olsen-Hegar Needle-holder | Word Precision Instruments | 501989 | |
Mayo scissor | Word Precision Instruments | 501752 | |
Scalpel | Word Precision Instruments | 500236 | |
Sprague-Dawley | Janvier | none | 12 weaks and male |