In atrophic non-union models, a minimally invasive technique is used to deliver stem cells into the fracture site via percutaneous injection. This technique is significantly affected by a backflow leakage and the net number of cells might be reduced. The Z-track method is a technique used in clinical practice for intramuscular injections to prevent backflow leakage.

We evaluated the potential of the Z-track injection technique for preventing cell loss in non-union models by determining the behaviour of observable marker fluids. Firstly, toluene blue stain was used as an injection material to allow visual detection of its distribution. Rat's cadaver legs were used and tibias were kept unbroken to ensure intact skin and overlying soft tissue. Technique includes pulling the skin over the shin of tibia towards the ankle and injection of the dye around the mid-shaft. The needle was then partially pulled back, the skin was returned to its normal position and a complete extraction of the needle was followed. Secondly, a mixture of contrast material and toluene blue was used to allow direct visual and radiological detection of the injected material into the fracture site. Ante-grade nailing of tibia via tibial tuberosity was carried out followed by a 3 point closed fracture. Injection was performed into the fracture gap similarly to the steps above. X-rays were taken to visualise the location and distribution of the injected material.

Observation revealed no blue stain could be detected over the skin, X -rays revealed that the radiopaque dye remained around the tibia with no escape of the material into the superficial layers or onto the skin surface. Therefore, the number of cells delivered and maintained at a target site could be increased by the Z-track method and therefore, the therapeutic benefit of stem cell injections could be optimised with this simple technique.

Appropriate in vivo models can be used to understand atrophic non-union pathophysiology. In these models, X-ray assessment is essential and a reliable good quality images are vital in order to detect any hidden callus formation or deficiency. However, the radiographic results are often variable and highly dependent on rotation and positioning from the detector/film. Therefore, standardised A-P and lateral x-ray views are essential for providing a full radiological picture and for reliably assessing the degree of fracture union.

We established and evaluated a method for standardised imaging of the lower limb and for reliably obtaining two perpendicular views (e.g. true A-P and true lateral views). The normal position of fibula in murine models is posterolateral to the tibia, therefore, a proper technique must show fibula in both views. In order to obtain the correct position, the knee joint and ankle joints were flexed to 90 degrees and the foot was placed in a perpendicular direction with the x-ray film. To achieve this, a leg holder was made and used to hold the foot and the knee while the body was in the supine position. Lateral views were obtained by putting the foot parallel to the x-ray film. Adult Wister rat cadavers were used and serial x-rays were taken.

A-P view in supine position showed the upper part of the fibula clearly, however, there was an unavoidable degree of external rotation in the whole lower limb, and the lower part of the fibula appeared behind the tibia. Therefore, a true A-P view whilst the body was in the supine position was difficult. To overcome this, a P-A view of the leg was performed with the body prone position, this allowed both upper and lower parts of the fibula to appear clearly in both views. This method provides two true perpendicular views (P-A and lateral) and helped to optimise radiological assessment.

There is a growing trend towards using pre-clinical models of atrophic non-union. This study investigated different fixation devices, by comparing the mechanical stability at the fracture site of tibia bone fixed by either intramedullary nail, compression plate or external fixator. 40 tibias from adult male Wistar rats' cadavers were osteotomised at the mid-shaft and a gap of 1 mm was created and maintained at the fracture site to simulate criteria of atrophic non-union model. These were divided into five groups (n=8 in each): the first group was fixed with 20G intramedullary nail, the second group with 18G nail, the third group with 4-hole plate, the fourth group with 6-hole plate, and the fifth group with external fixator. Tibia was harvested by leg disarticulation from the knee and ankle joints, the soft tissues were carefully removed from the leg, and tibias were kept hydrated throughout the experiment. Each group was then subdivided into two subgroups for mechanical testing: one for axial loading (n=4) and one for 4-point bending (n=4). Statistical analysis was carried out by ANOVA with a fisher post-hoc comparison between groups. A p-value less than 0.05 was considered statistically significant. Axial load to failure data and stiffness data revealed that intramedullary nails are significantly stronger and stiffer than other devices, however there was no statistically significant difference axially between the nail thicknesses. In bending, load to failure revealed that 18G nails are significantly stronger than 20G. We concluded that 18G nail is superior to the other fixation devices, therefore it has been used for in-vivo experiments to create a novel model of atrophic non-union with stable fixation.

This review is aimed at clinicians appraising preclinical trauma studies and researchers investigating compromised bone healing or novel treatments for fractures. It categorises the clinical scenarios of poor healing of fractures and attempts to match them with the appropriate animal models in the literature.

We performed an extensive literature search of animal models of long bone fracture repair/nonunion and grouped the resulting studies according to the clinical scenario they were attempting to reflect; we then scrutinised them for their reliability and accuracy in reproducing that clinical scenario.

Models for normal fracture repair (primary and secondary), delayed union, nonunion (atrophic and hypertrophic), segmental defects and fractures at risk of impaired healing were identified. Their accuracy in reflecting the clinical scenario ranged greatly and the reliability of reproducing the scenario ranged from 100% to 40%.

It is vital to know the limitations and success of each model when considering its application.

There is a growing trend towards using pre-clinical models of atrophic non-union. This study investigated different fixation devices, by comparing the mechanical stability at the fracture site of tibia bone fixed by either intramedullary nail, compression plate or external fixator. 40 tibias from adult male Wistar rats' cadavers were osteotomised at the mid-shaft and a gap of 1 mm was created and maintained at the fracture site to simulate criteria of atrophic non-union model. These were divided into five groups (n=8 in each): the first group was fixed with 20G intramedullary nail, the second group with 18G nail, the third group with 4-hole plate, the fourth group with 6-hole plate, and the fifth group with external fixator. Tibia was harvested by leg disarticulation from the knee and ankle joints, the soft tissues were carefully removed from the leg, and tibias were kept hydrated throughout the experiment. Each group was then subdivided into two subgroups for mechanical testing: one for axial loading (n=4) and one for 4-point bending (n=4).

Statistical analysis was carried out by ANOVA with a fisher post-hoc comparison between groups. A p-value less than 0.05 was considered statistically significant. Axial load to failure data and stiffness data revealed that intramedullary nails are significantly stronger and stiffer than other devices, however there was no statistically significant difference axially between the nail thicknesses. In bending, load to failure revealed that 18G nails are significantly stronger than 20G. We concluded that 18G nail is superior to the other fixation devices, therefore it has been used for in-vivo experiments to create a novel model of atrophic non-union with stable fixation.

Our aim was to develop a clinically relevant model of atrophic nonunion in the rat to test the hypothesis that the vessel density of atrophic nonunion reaches that of normal healing bone, but at a later time-point. Atrophic nonunion is usually attributed to impaired blood supply and is poorly understood. We determined the number of blood vessels at the site of an osteotomy using immunolocalisation techniques in both normally healing bones and in atrophic nonunion. At one week after operation there were significantly fewer blood vessels in the nonunion group than in the healing group. By eight weeks, the number in the atrophic nonunion group had reached the same level as that in the healing group. Our findings suggest that the number of blood vessels in atrophic nonunion reaches the same level as that in healing bone, but at a later time-point. Diminished vascularity within the first three weeks, but not at a later time-point, may prevent fractures from uniting

Objectives

To explore the therapeutic potential of combining bone marrow-derived mesenchymal stem cells (BM-MSCs) and hydroxyapatite (HA) granules to treat nonunion of the long bone.