Effects of low intensity pulsed ultrasound (LIPUS) and longitudinal cortical allograft perforation (LAP) on allograft healing: a biomechanical and histological investigation

Abstract

Massive cortical bone allografting suffers from a number of clinical complications including chronic infection, non-union, and fracture all due to a limited degree of graft remodeling within the host, even after extended periods of time in vivo. Despite being the preferred biologic alternative to autograft in cases where structural capacity is a clinical necessity, novel means that improve graft healing are significantly lacking. This project was established to investigate the effects of an exogenous, low-intensity pulsed ultrasound signal (LIPUS) either alone or in combination with increased graft cortical porosity (LAP) on allograft healing after four months in vivo. Results from this exploratory study will provide preliminary information on the mechanical and biologic response of limbs reconstructed with massive bone allograft using these therapies. The first part of this study (Chapter 2) was designed to evaluate improvements in the torsional capacity of 12 ovine hindlimbs reconstructed with 5 cm intercalary allografts. Treatment groups included reconstruction with allograft only (-)CTL and autograft only (+)CTL both with no adjuvant therapy, LIPUS, LAP, and LIPUS+LAP. Graft perforation was accomplished by creating 16,500 μm diameter conduits that extended 10 mm into the cortical endplates. Following euthanasia, the experimental and contralateral control limbs were harvested and tested to failure in torsion. Reconstructed limbs exposed daily to LIPUS resulted in a 30% increase in ultimate torque to failure (p=0.229) and stiffness (p=0.162) and promoted the development of 50% more callus with a radiodensity twice that quantified in the negative control limbs. Limbs reconstructed with perforated grafts demonstrated improved torsional capacity though to a lesser degree than LIPUS therapy. Combination therapy (LIPUS+LAP) was not statistically synergistic from a purely biomechanical standpoint but did improve reconstruction stiffness by 40% (p=0.063). (+)CTL limbs were completely healed at the four-months time point as demonstrated biomechanically. In the second part of the study (Chapter 3), decalcified and undecalcified histologic indices of allograft healing were quantified following exposure to LIPUS and LAP and compared to untreated controls. LIPUS and LAP therapies, alone and in combination, resulted in increases in periosteal callus formation and significantly greater bone formation rates (BFR) in this region of interest (p<0.01). LIPUS therapy appeared to accelerate endochondral bone formation in the callus producing a stronger, stiffer reconstruction after four months in vivo. Longitudinal perforations filled to varying degrees with appositional new bone that extended the length of the 10 mm conduit and demonstrated the efficacy of such a therapy at improving graft revitalization. The third and final portion of this study (Chapter 4) evaluated the effects of the longitudinal perforations on the structural integrity of the graft prior to transplantation. Uniaxial (n=13 pairs of limbs) and diametral compression tests (n=48 specimens) revealed insignificant changes in graft integrity as a result of LAP. Specifically, LAP modification reduced the compressive load to failure by approximately 6.7% (p=0.339) in uniaxial compression relative to non-perforated grafts. The same form of modification in diametral compression resulted in an 11.1% decrease in ultimate force to failure (p>0.05). Within the same diametral compression set-up, transversely perforated grafts (TAP) failed at 80% of the ultimate loads of their non-perforated controls and illustrated the deleterious effects of such a form of graft modification on integrity prior to transplantation. Overall, this exploratory study has demonstrated the potential of LIPUS and LAP as novel therapies to improve allograft healing. Biomechanics revealed no statistically significant improvements relative to the negative control at the =0.05 level, but 30%-40% increases in stiffness may be clinically meaningful. This was the first study to show that longitudinal perforations can improve graft revitalization without adversely affecting the integrity of the graft and the only study to date that has illustrated the potential of LIPUS as a stand-alone therapy to accelerate callus formation and graft incorporation without eliciting an adverse cellular response. Future studies are warranted to confirm the trends and significant findings of this pilot study.