The study of the relationship which exists between bone and its mechanical environment can be conducted using cellular in vitro models, but this does not always accurately represent the cells in situ environment, and limits investigation to the cellular level. It is therefore important to have experimental models, in which loading can be carefully controlled, and the resultant bone response measured. Much of the early work into bone mechanoadaptation was carried out by surgical methods in sheep and turkeys (Rubin and Lanyon 1984; Lanyon and Rubin 1984). Clearly surgical interventions are more straightforward on larger animals. However the cost of housing large animals is far larger than for rodents. Increasingly rats and mice are becoming the animals of choice for skeletal research, largely due to their low cost to acquire and maintain, rapid growth and reproduction rates, and the extensive genetic knockouts available. One genetic knock-out which has been used in bone-muscle relationships is the myostatin deficient mouse, myostatin is a
negative regulator of muscle development, and the myostatin (GDF-8) deficient mouse has double the muscle mass of wild type mice. The skeletons of myostain- deficient mice show increased bone mineral density, and trabecular area (Elkasrawy and Hamrick 2010).
Despite the established relationship between muscle and bone (see section 1.8) previous work investigating mechanoadaptation of the skeleton has largely avoided the use of muscular contraction. The earliest investigations into bone adaptation often utilised drastic surgical methods such as, the removal of one in a set of paired bones, to increase loads upon the other (Lanyon et al. 1982). This was found to result in substantial bone growth. However surgical interference with the periosteum has been found to cause osteogenesis, and without the ability to perform a sham osteotomy it is not possible to conclude that bone growth arises exclusively from mechanical loads (Churches et al. 1980). The alternative technique of pin-loading has also been used extensively (Lanyon and Rubin 1984). In this model metal pins
53 are surgically inserted in to long bones. Under anaesthesia the pins could then be pulled together statically or dynamically by engaging the pins in a loading apparatus which pulled the pins together creating a compressive load. This technique also showed dramatic bone growth under dynamic loading conditions (Lanyon and Rubin 1984), with the advantage of being able to be run in conjunction with sham
experiments, in which pins are inserted but not loaded.
Whilst sham experiments provide a level of certainty in results, using non-surgical techniques is often preferential in terms of animal welfare, and can create a more easily reproducible loading condition. A number of such methods have been developed. The main loading models can be broadly divided into bending, axial compression and oscillation. Bending methods include, three-point bending (Fig.1.15a) (Sakai et al. 2011), four-point bending (Turner et al. 1994), and cantilever bending (Gross et al. 2002). Axial compression comprises of the distal and proximal ends of a bone constrained in cups, whilst a compressive force is transmitted between the two (Fig.1.15b) (Poulet et al. 2011; Torrance et al. 1994). Axial compression is often regarded as the gold-standard for studying
mechanoadaptation in rodents, as it mimics physiological loading through the joints (McBride and Silva 2012). Furthermore mechanical loads can be easily controlled and manipulated, and the contra-lateral limb can be used as a control. All of these models require anesthesia and do not allow the investigation of muscular forces upon bone. Placing rodents upon low-magnitude, high-frequency oscillating platforms has been found to provide robust osteogenic responses (Rubin et al. 2001) however the forces being applied in this model are not clear, and are substantially complicated by muscular interaction (Judex and Rubin 2010).
54 Figure 1.15 Diagrammatic representations of established bone loading
techniques, a) diagram of 3-point bending, in which a long bone of an anesthetised animal is secured longitudinally between three points. Loads are applied by the superior point, figure modified from Sakai et al (2011). b) a diagram of axial compression, in which the an anesthetised animal has their long bone secured between an upper and lower cup, and forces are applied axially, figure modified from Poulet et al (2011).
Mechanoadaptation occurs in response to decreased as well as increased loading. Two contrasting forms of unloading frequently investigated are, loss of muscular contraction, as seen in spinal cord injury, and removal of reaction forces, as seen in space flight and bed rest. Spinal cord injury is very simple to bring about in an experimental animal, by dennervation of muscles (Tuukkanen et al. 1991).
Alternatively paralysis can be induced by the injection of botulinum toxin A (botox) (Manske et al. 2010; Poliachik et al. 2010) (see section 1.8.1). Tail or hindlimb suspension is a widely used technique to study the effect of unloading upon the rodent skeleton (Morey-Holton et al. 2005). In this model a rodent has its tail suspended to the top of its cage, so its hindlimbs do not touch the floor of its cage, removing ground reaction forces. The forelimbs remain fully weight bearing and support locomotion. More recently a partial weight suspension model has been developed (Wagner et al. 2010). This model allows quadrupedal suspension of the mouse, and so preserves normal gait characteristics, achieving a model closer to spaceflight conditions than hindlimb suspension. So whilst methods exist for studying the removal of muscular forces there is still little work looking at the controlled application of muscular forces upon the skeletal system.
55 The most popular animals used for both loading and unloading experiments are rats and mice. Therefore experimental outcomes in rats and mice can be reviewed in the context of a vast body of previous work.
1.13 Anatomical Overview of the Rodent Hindlimb