To examine the osteogenic capacity of stem/progenitor cells in bone defects, two local factors are critical: the length of the bony defect and the mechanical stability of the bone. Since bone is capable of self-regeneration to a certain length, numerous studies have explored the concept of critical size of defects (CSD) at different anatomical locations and in various animal species with the goal of preventing concomitant bone healing.1
For rats, a 6-mm defect in femur has been suggested as an appropriate CSD.5,6
In the current animal model, when only collagen gel was implanted, slight callus formation was observed at each bone-end of the defects in week 3, and no further propagation of healing was seen in the following weeks, a sign of atrophic nonunion. The defects were unchanged at week 8 in all 8 animals that were implanted with collagen gel alone. Therefore, the femoral CSD for this particular species of immunodeficient rats fixed with an external fixator is 6 mm. Rats that received BMP-2 implantation at the defect site had full healing of the defect, confirming the applicability of this model for investigating bone regeneration ().
Figure 2. (a) Radiograph of a rat that was implanted with BMP-2 in the femoral defect shows callus within the segmental defect at week 1. (b) Histological section of hematoxylin and eosin (H & E) staining shows endochondral ossification in the femoral defect (more ...)
After CSD is created, fixation to sustain the length of defect and stabilize bone-end is required. Various methods have been developed using common orthopedic techniques to fix bone defects in animals, including intramedullary Kirschner wire fixation, internal (plate) fixation,7,8
and external fixation.1
Intramedullary Kirschner wire fixation lacks a locking mechanism to prevent interfragmentary movement, which causes the reduction of the length of the defect.9,10
Internal plate fixation, if applied appropriately, can provide adequate mechanical stability to the bone defect.5
However, if the hardware is not specially manufactured for rat femurs and not applied with specialized tools, the fixation may not be able to achieve the desired stability. In addition, the skills and scientific principles of internal fixation require vigorous training. Importantly, intramedullary pins and plates are foreign materials within the bony defect, which interferes with the local physiological environment.11
Alterations of tissue environment are particular concerns for stem/progenitor cells, which are multipotent and differentiated according to environmental cues.12
The use of external fixation avoids the contact between fixation materials and local tissues within the defects, thus sparing the implanted stem/progenitor cells from potential exposure to nonphysiological stimulations. The application of an external fixator is also relatively easy and does not require specialized training.
To our knowledge, there are no commercial products available for external fixation of rat bony defects. This has led many investigators to develop their own systems of external fixation.7,10,13
For practical reasons, the external fixator must be lightweight, inexpensive, relatively easy to apply, and stable through the duration of the experiment. An assortment of external fixation frames and techniques has been developed for this purpose. While bone cement is convenient to apply and provides initial solid fixation to the pins, it has been found that there is often loosening in late stage of the fixation. The external fixator used in this study was designed for convenient assembly without the use of highly specialized tools or skills. The parts for this external fixator can be purchased from any hardware stores. The external fixator provided sufficient strength of bone fixation using a lightweight material (aluminum alloy) that the animals were able to tolerate without difficulty. The ability to bear weight early is important for bone regeneration in long bone defects as it provides mechanical stimulation.14
In this study, all animals had the ability to bear weight immediately postoperatively and walked without difficulty. The external fixator used in the current study is also adjustable, and the M4 fasteners can be tightened as needed to prevent loosening of the frame.
The most common complications of external fixation are pin loosening and pin tract infection.15
Pin tract infection was observed in nine rats and all healed after local daily application of antibiotic ointment. Two rats developed deep tissue infection, one of which necessitated euthanasia. Pin loosening was observed in nine rats. Most of the cases were corrected by adjustment of the external fixator and did not affect the overall stability of the device. Only two of the external fixators failed, both of these failures occurred at 7 weeks post operation. One rat died 4 weeks post operation of unknown causes; however, it did have deep infection found on necropsy. One rat developed a femur fracture at the proximal pin site at 3 weeks, which was likely iatrogenic during the insertion of pins.
It is evident that this self-designed external fixator provided a stable mechanical environment for bone regeneration in the defects. The fact that BMP-2 implantation had healing of the defect proves that there was adequate mechanical stability of the defect to allow for potential healing. Defects implanted with stem/progenitor cells showed various degrees of bone regeneration at the defect site (). On histologic analysis, regenerated woven bone and fibrocartilage were seen within the defects implanted with human stem/progenitor cells (). This indicates new bone was regenerated through the process of endochondral ossification. In the tissues surrounding the defect, there were no obvious signs of inflammatory reaction.
When an external fixator is applied to immunodeficient animals, such as the athymic rats used in this study, infection is a concern. Among the 35 athymic rats that had an external fixator applied to stabilize the critical-sized femoral defects, only two rats had deep infection. None of the rats with superficial pin tract infection developed uncontrollable systematic infection or required additional antibiotic treatment beyond topical antibiotic ointment. The rate of the failure of external fixation in this study was consistent with the 5% rate of failure for applying external fixators to the bone defects (without cell implantation) in immunocompetent laboratory rats.16
Being able to tighten the external fixator during the course of follow-up is one of the advantages of this type of external fixator and attributes to the low failure rate of external fixation, as compared to a high failure rate (8/8) of internal fixation in 8 weeks.7
In summary, an external fixator has been developed and successfully applied to critical-sized femoral defects in athymic rats. This model is particularly useful for studying bone regeneration with human stem/progenitor cells in vivo.