The development of a novel model of direct fracture healing in the rat

Cadaver and preliminary animal studies

A total of 11 adult ex-breeder male Sprague-Dawley rats (Harlan Laboratories, Blackthorn, United Kingdom) were killed, and the dimensions of the 22 cadaveric tibiae were measured in order to plan the size of the tibial plate for osteotomy fixation. Initial unpublished cadaver trials and preliminary animal experiments using various implant configurations and appropriate refinements in the custom-made jig identified the optimal implant dimensions with which to achieve stable compression plate fixation of a rat transverse diaphyseal tibial osteotomy. Table I details the implants used and the problems encountered at each stage of refinement while developing this model. These final implant dimensions enabled four screws with bicortical hold to be firmly placed in diaphyseal bone and allowed pre-bending¹³ of the plate for symmetric compression across the diaphyseal osteotomy site. This plating technique was tested in 15 cadavers.

Animals

A total of 22 skeletally mature ex-breeder male Sprague-Dawley rats (Harlan Laboratories) were used for the in vivo testing; they were aged between 11 and 13 months and had a mean weight of 512 g (sd 24.3). Animals were fed standard rat chow and provided with water ad libitum. Animals were housed in individual cages at constant temperature (21±1°C) and humidity (45% to 55%) using a 12-hour light/dark cycle.

A total of eight animals were used at the earlier stages of the model development during preliminary animal testing before recognition of the final arrangement for tibial plating (Table I).

A total of ten animals had compression plate fixation of a transverse tibial osteotomy using the final arrangement that was developed as is described here. Of this group, five animals were used for mechanical testing and the remaining five for histology.

In the remaining four animals, a transverse tibial osteotomy was stabilised with a uniaxial external fixator that used the same number and size of screw holes within the rat tibia to that employed in the plating model. Two animals underwent mechanical testing and two were assessed on histology. Animals were killed by induction of terminal anaesthesia at six weeks following surgery.

Surgical procedures

Compression plating

Isofluorane (1% to 3%) inhalational anaesthesia was used. Pre-operative antibiotic prophylaxis (1 ml/kg Synulox), fluids (10 ml/kg 0.9% saline) and analgesia (0.05 mg/kg buprenorphine) administered subcutaneously. The right hind leg was infiltrated with 0.4 ml/kg of 1% Xylocaine before skin incision. The anteromedial approach to the tibia was used, exploiting the interval between tibialis anterior and tibialis posterior.¹⁴ A custom-made four-hole jig was applied to the flat medial surface of the tibia (Fig. 1). The spacing between the second and third holes on the jig was 0.1 mm greater (to account for the subsequent osteotomy) than the corresponding holes on the plate, in order to ensure compression at the osteotomy site during plate application. Two holes on the lateral surface of the plate were used to passage a suture to help secure the jig to bone while drilling. Under constant cool saline irrigation, four holes were drilled, with the aid of the jig, using a shortened 1.0 mm drill bit powered by a hand-held Dremel Multitool (Dremel UK, Denham, United Kingdom). The periosteum was stripped circumferentially from the tibia at the level of the osteotomy^8,9 for the length of one diameter of the bone width at the osteotomy site before creation of a transverse tibial osteotomy with a fine circular saw of 0.1 mm thickness (RS Components, Corby, United Kingdom). The osteotomy was aligned manually. Hypodermic needles were placed through the pre-bent (convex)¹³ four-hole stainless steel plate (University of Edinburgh Physics Workshop, Edinburgh, United Kingdom), measuring 12 mm × 3 mm × 0.4 mm, into the bone holes to roughly position the plate on the medial surface of the tibia. Four 1.2 mm × 4 mm stainless steel screws (PTS Ltd, East Grinstead, United Kingdom) were used. The screws immediately adjacent to the fracture were inserted first. Then the two more distant holes were filled. Minor adjustments were made with screw tightening to permit plate application on to bone. The two screws adjacent to the fracture were securely tightened earlier than the distant two screws to obtain symmetric compression across the fracture. Then the tibia was manually tested to assess for movement at the fracture site. There was no mobility observed at surgery in any of the animals. 3-0 vicryl (ETHICON; Johnson & Johnson Medical Ltd, Livingston, United Kingdom) was used for wound closure. Post-operatively buprenorphine oral analgesia (0.3 mg/kg B.D) was administered in jelly cubes for 24 hours.

Figs. 1a - 1b

Photographs showing the custom-made four-hole jig, showing a) the superior surface, and b) the lateral surface, with two holes used for threading of fine wire to help secure the jig onto bone. There is also a roughened inferior surface (not seen) to prevent the jig slipping on bone during drilling.

Radiological analysis

Plain anteroposterior (AP) radiographs of the healing fractures were obtained at fortnightly intervals. A portable X-ray unit (Acu-Ray JR; Stern Manufacturing, Toronto, Canada) with an output of 60 kV, 2 mAs and exposure time of 0.1 ms was used. Images were captured on digital X-ray plates (Fuji CR Cassette; Fuji Photo Film Co Ltd, Tokyo, Japan). After death the tibiae were dissected free of soft tissues and the metalwork removed before capture of a standard contact lateral radiograph. Lateral radiographs were obtained to exclude the effect of fibula overlay. The visibility of the osteotomy was observed and scored as totally visible, partly visible, or absent, as previously reported^15,16 by the first author (TS) and an independent assessor (RT) at two separate time points in a blinded manner. Interobserver and intra-observer agreement was assessed using the linear weighted Kappa (κ) test.

Mechanical testing

The vertical distance from the lateral tibial plateau to the osteotomy site was measured on the injured tibia. A mark corresponding to the location of the tibial osteotomy was made with a pencil on the left contralateral tibia. Extricated tibiae from the uninjured left leg in all animals and from the injured limbs in the seven animals allocated to mechanical testing were placed in individual tubes and frozen at -20°C before mechanical testing within two weeks.¹⁷ Stress test to failure with a four-point bend was performed using a specially designed rig (University of Edinburgh Physics Workshop) in conjunction with a materials testing machine (Zwick/Roell, Ulm, Germany). The upper loading rig was mounted on a pivot in order to ensure symmetrical loading of the irregularly shaped rat tibiae at all four loading points. The upper loading span was spaced 10 mm apart and the lower static span was spaced 20 mm apart. Contact points were rounded to minimise notching of the bone after load application. Specimens were warmed in a water bath set at 37°C for a minimum of 30 minutes before mechanical testing. Immediately, following removal from the water bath tibiae were mounted on the rig resting on the lateral surface. Either, the fracture (operated side) or the pencil mark (un-operated side) was placed at the mid-point between the two lower loading points. The four-point bend was applied while the bone temperature remained at 37°C and well-hydrated. The tibial midshaft cross section following failure most closely resembled a triangle.

The triangular cross section area of the whole bone and intramedullary canal were derived from physical measurements made with sliding callipers and from scaled digital images. This was then used to obtain the cross sectional moment of inertia required to calculate the stress values at failure according to the Euler-Bernoulli Beam Theory, where the peak stress as a result of bending can be approximated by the bending moment multiplied by the distance from the neutral axis to the outermost fibres divided by the second moment of area.¹⁸

Histological analysis

The remaining extricated tibiae were immersed in 10% formalin in phosphate buffered saline at pH 7.2 to 7.4 for 48 hours, and then decalcified in 10% EDTA at pH 7 and 37°C for four weeks with weekly changes of ethylenediaminetetraacetic acid (EDTA). Three sets of three longitudinal 5 µm coronal sections spaced 100 µm apart were obtained from each specimen and labelled; anterior, middle and posterior. Sections were stained with Masson’s trichrome to assess the general morphology at the fracture site. With the aid of a grid, the number of osteoclast on either side of the osteotomy at a distance of 200 µm perpendicular to the line of the osteotomy was manually counted in all sections at × 20 magnification.

Cadaver and preliminary animal studies

The measurements of the 22 cadaveric tibiae (11 animals) revealed a mean sagittal width of 3.1 mm (sd 0.2), a mean coronal width of 2.9 mm (sd 0.12) and a mean length of 43.0 mm (sd 1.2). These values corresponded with previously observed tibial dimensions in the adult rat.^19,20 After placing four drill holes in bone with the custom-made jig (Fig. 1) and subsequent transverse tibial osteotomy with a 0.1 mm circular saw, it was observed that a pre-bent 12 mm × 3 mm × 0.4 mm plate secured with four stainless steel screws of 1.2 mm × 4 mm was best suited to achieve stable compression plate fixation (Fig. 2). Of the 15 cadaveric tibial osteotomies that were plated in this manner, none showed movement at the osteotomy site when these constructs were tested with manual bending.

Fig. 2

Intra-operative photograph of tibial plating, showing anatomical alignment and compression at the osteotomy site.

In the initial arrangement (Table I), the osteotomies were created at the proximal diaphysis of the tibia in order to allow the placement of four screws in areas where the tibia remains wide. Unfortunately, as the proximal screws were in metaphyseal bone, they loosened. This was noted on radiographs at five weeks post-operatively. External callus was seen on both histology and radiology. In order to overcome this, the plate was placed more distally on the tibia to allow for all four screws to be in diaphyseal bone. Smaller diameter 1.4 mm screws were used. Furthermore, the integrity of the fibula, despite contributing to stability,^21,22 may have impeded compression at the osteotomy. A narrower osteotomy was therefore created, which could be compressed during plate fixation but remaining in compression despite the effect of the intact fibula in preserving tibial length. These changes were implemented in the first change but between one and two weeks post-fixation the tibia had fractured through the most distal screw holes. The animals were noted to develop impaired mobility acutely and the fracture was evident on radiographs. Subsequently smaller diameter screws of 1.2 mm were used. The plate width was correspondingly reduced to 3 mm. Serial radiographs revealed no external callus but on histology at six weeks a wide gap was seen on the far cortex away from plate application. After this, plates were pre-bent to permit symmetrical compression across the fracture site.¹³ The thicker plates were difficult to pre-bend and occasionally either displaced the fracture or cracked the bone during screw tightening when attempting to conform the plate to the tibial surface in cadaver bone. These problems were not seen in the 15 cadaveric tibiae that were plated with the 0.4 mm plate. This implant was then used for the plate fixation in ten animals, for which the results are presented below.

Compression plating and external fixation: radiological assessment

Test for reliability of the scoring of osteotomy visibility between repeated measurements by the same assessor and between observations by the two separate assessors revealed good intra- and interobserver agreement. There was good intra-observer agreement for both the first author (TS) (κ = 0.77 (95% confidence interval (CI) 0.48 to 1)) and the independent assessor (RT) (κ = 0.73 (95% CI 0.42 to 1)). Similarly, interobserver agreement for fracture line visibility between the two observers was good with a κ value of 0.78 (95% CI 0.50 to 1).

In the compression plating group, serial radiographs at two, four and six weeks post-operatively revealed no visible external callus formation (Fig. 3). At six weeks, in the extricated tibia following plate removal, there was no external callus. On contact radiographs following plate removal there were no animals with totally visible osteotomy lines in the compression plating group (Table II).

Figs. 3a - 3b

Plain radiographs at six weeks after fracture in a) the plate fixation group and b) the external fixation group.

In contrast, in the external fixation group serial radiographs after surgery showed fracture healing with external callus formation in all cases. There were no animals with absent osteotomy lines (Table II) on contact radiographs in the external fixation group.

Compression plating and external fixation: mechanical testing

At six weeks the mean load at failure for the direct bone repair (plate fixation) group was 31.0 N (sd 8.5) and the stress at failure was 24.7 N/mm² (sd 6.2). The corresponding values for the intact contralateral tibiae were 169.7 N (sd 26.1) and 179.3 N/mm² (sd 41.5), respectively. At six weeks in the external fixation group the mean load and stress at failure for the healing tibial osteotomies was 25.9 N (sd 10.6) and 21.8 N/mm² (sd 3.0), respectively, and 166.4 N (sd 50.8) and 160.1 N/mm² (sd 50.8) for the intact contralateral tibiae, respectively.

Compression plating and external fixation: histology

In all animals in the direct fracture repair group, the Masson’s trichrome staining from all three levels confirmed the radiological findings of lack of external callus formation. ‘Contact healing’ predominated at the medial tibial cortical surface of plate application (5 of 5) and ‘gap healing’ was seen in the lateral cortex opposite to the plate (5 of 5) (Fig. 4). Osteonal units with leading osteoclasts (‘cutting-cones’) were seen to traverse the fracture site in all cases indicating direct fracture healing (Fig. 4). When viewed at × 20 magnification, there was a mean of three osteoclasts (2 to 4) seen in each specimen at a distance of 200 µm on either side of the osteotomy.

Figs. 4a - 4d

Histological images of Masson’s trichrome stained sections taken from specimens treated with plate fixation, showing a) the anterior section, b) the middle section and c) the posterior section (yellow box shows area magnified in Figure 4d) (all original magnification ×4). The osteotomy fragments are aligned with no external callus. On the upper aspect note ‘contact healing’ on the cortical surface of plate application and ‘gap healing’ on the lower cortex opposite plate application, best seen on middle section (b). Figure 4d – histological image at original magnification ×20, with the arrow showing the ‘cutting cone’ traversing the fracture site.

In contrast, in all of the animals in the external fixation group, fracture healing occurred with bridging external callus and new woven bone formation (Fig. 5).

Fig. 5

Histological images of Masson’s trichrome ×4 magnification in a specimen of external fixation. Note rim of external callus and new woven bone formation.