Nano-level Mechanical And Morphological Study Of Low-magnitude Vibration In Bone Healing

Introduction: With aging and the development of global economy, bone fractures,usually caused by traffic accidents, falling or osteoporosis, are becoming morecommon in daily life. Serious economic burdens or body pain are often presented inthe cases of unexpected bone nonunion or delayed union during the rehabilitationprocess. Medication is the most popular regime at present in bone healing, but withsevere side effects in the long duration of treatment. Thus, a non-pharmacologic andnon-invasive treatment for improving fracture healing should be discussed urgently.Some previous studies showed that mechanical vibration could accelerate fracturehealing, and prevent bone nonunion or delayed union during bone healing process. Inthis present study, low-magnitude and high-frequency vibration (35Hz,0.25g,15min/day) was loaded on the sheep subjected to low limb fracture. Various mechanicalloading regimens with continuous or intervals modes on fracture healing werecompared. Different from most studies on macro-level, nano-level morphological andmechanical properties of callus tissue were investigated in this protocol. The effectsand mechanism of these two kinds of mechanical stimulus in improving bone healingwere discussed.Methods: Nine female small tail han sheep,12-month-old, were randomly assignedinto three groups averagely: base control group (BCG), continuous vibration group(CVG),7days interval vibration group (7consecutive days of vibration,7consecutive days rest;7DVG). Transverse fractures of metatarsi of right hind limbwere established by surgery, with limit contact dynamic compression plate (LC-DCP)being implanted for fixation. The treatment of vertical mechanical stimulus withfrequency of35Hz and acceleration of0.25g was loaded on the right hind limb twoweek post-fracture. The tested animals were loaded by mechanical regime for15minper day, lasting for8weeks. All the metatarsi of right hind limb were harvested aftersacrificed by overdose of anaesthetics after8weeks treatment of vibration. After the removal of LC-DCP, samples in four positions of proximal or distal fracture line weretaken. Nano-morphological and mechanical properties were determined by atomicforce microscopy (AFM) analyses and nanoindentation testing.Results and conclusion:1. AFM test: Grain sizes of the four positions near the fracture line area werecompared. The BCG presented the maximum in the grain sizes, while the minimumvalue was shown in the7DVG (p<0.05). The grain sizes of CVG and7DVG weresignificantly smaller than that of BCG, respectively(p<0.05). Comparing with CVG,the grain size of7DVG was smaller obviously (p<0.05). In addition, the surfaceroughnesses of the four samples were also tested in the present protocol. However, nostatistical differences were observed among groups (p>0.05). The roughest surfacewas detected in the7DVG, while the BCG and CVG showed smoother surface in thefour positions relatively. An active bone remodeling is reflected by the smallermineral grain sizes and higher roughnesses of bone materials. Therefore,nano-morphological analysis results showed that the vibration could accelerate boneremodeling and promote fracture healing. Compared with daily loading, themechanical regime with intervals showed more active bone remodeling.2. Nanoindentation testing: In the proximal external callus, the maximum elasticmodulus was observed in the CVG, while the maximum hardness and E/H ratio wereshown in BCG and CVG, respectively. However, there was no statistical difference inthe comparison of these three tested or calculated parameters(p>0.05). In the proximalinternal calluses, the7DVG showed the maximum in elastic modulus and E/H ratio,while the maximum hardness was detected in BCG despite of the non-statisticaldifferences. In addition, the7DVG showed the maximum E/H ratio in both distalexternal calluses and distal cortical bone non-significantly (p>0.05). Compared withthe maximum of elastic modulus and hardness shown in7DVG of the external callus,the CVG and BCG presented the maximum of these two parameters in cortical boneseparately. However, no significant difference were found (p>0.05). With the higherE/H ratio, the bone showed a better toughness to mechanical loading. Although there was no significant difference among all the groups, the CVG or7DVG still showed ahigher elastic modulus, hardness and E/H ratio slightly. This phenomenon mayindicate a great potential of mechanical loading in bone healing. More analyses wouldbe performed in future.In summary, mechanical regime could promote bone healing procedure, whilethe stimulus with intervals showed a better activity than daily loading in thenano-morphological and mechanical properties close to the traumaposition.