Combination of Heel-strike like Mechanical Loading with Deproteinized Cancellous Bone Scaffold Implantation to Repair Segmental Bone Defects in Rabbits

Abstract

Under physiological conditions bone defects often occur at mechanical load bearing sites and bone substitutes used for regeneration should be similarly subjected to mechanical loading stress. In this study, we investigated whether a novel heel-strike like mechanical loading method can be used as a complementary therapy to promote bone regeneration following bone substitute grafting. To test this, three groups of rabbits with segmental bone defects in the tibia were implanted with bovine deproteinized cancellous bone scaffold (DCBS), with one group also receiving heel-strike like mechanical loading generated by a rap stress stimulator. From weeks 4-12 post-operation X-ray and micro-CT scanning showed that rabbits receiving combination therapy had significantly more callus at the bone defect. Moreover, bone defects in the combination group were completely replaced with new bone at week 12, while the DCBS implantation alone group healed only partially and rabbits receiving neither DCBS nor mechanical loading developed only small calluses throughout the observation period. Analysis of micro-CT scanning results demonstrated that new bone density in the combination group was significantly higher than the DCBS only group at weeks 4 and 12 (p<0.05). H&E staining results also indicated a significantly higher percentage of new bone in the bone defect area and a lower percentage of residual scaffold in the combination group compared to the DCBS only group (p<0.05). Thus, this heel-strike like mechanical loading method appears to accelerate bone regeneration following substitute implantation by restoring a local mechanical loading environment in segmental bone defects.

Keywords: bone regeneration; bone substitutes; deproteinized cancellous bone scaffold; heel-strike like mechanical loading; segmental bone defect.

Figure 1

Surgical procedure for creation of rabbit tibia segmental bone defects. A: A 5-mm tibia section was removed and the fracture ends were fixed tightly with steel plates and screws. B: A suitable DCBS was grafted into the bone defect area. C: The wounded leg was immobilized with tubular plaster to prevent the rabbits from walking.

Figure 2

Independently designed in vitro rap stress stimulator. A: Appearance and components. B: Application of in vitro rap stress stimulator to anesthetized rabbits to generate mechanical loading stimulation.

Figure 3

SEM analysis of DCBS. A: Gross appearance of DCBS. B: 3D structure showing uniform pore structures (6000×). C: Uniform particulate surface structure of DCBS (7000×).

Figure 4

X-ray examination and Lane-Sandhu scoring. X-ray images of A: Group A, B: Group B, and C: Group C at 2, 4, 8, and 12 weeks post-operation. D: Lane-Sandhu scoring indicating significantly higher scores in callus and bone regeneration in Group A compared to Groups B and C beginning 4 weeks post-operation. n=5, *p<0.05.

Figure 5

Gross observations 12 weeks after bone substitute grafting. A: The bone defect was completely repaired and the DCBS degraded in Group A, which received DCBS and mechanical loading stimulation group. B: In Group B, which only received DCBS grafting, the bone defect areas are partially repaired but some scaffold remains. C: The control group, Group C, received neither DCBS nor mechanical loading and shows only a small degree of callus formation. n=5.

Figure 6

Micro-CT scanning results and determination of regenerated bone volumes and densities. Micro-CT images of A: Group A and B: Group B bones at 4 and 12 weeks post-operation. At both time points Group A had significantly higher regenerated bone C: volumes and D: densities than Group B. n=5, *p<0.05.

Figure 7

Histological images and quantitative assessment of bone regeneration and DCBS degradation. Central histological slices taken from the fibula side of the defects show improved bone regeneration at 4 and 12 weeks post-operation in A: Group A compared to B: Group B (40×). Image analysis quantification showed C: significantly higher percentages of regenerated bones and D: significantly lower percentages of residual DCBS in the total bone defect area in Group A compared to Group B. Ic: interstitial cell, Nb: new bone, S: scaffold, F: fat tissue. n=5, *p<0.05.

Figure 8

Biomechanical strength of bone defect sites. At 12 w post-operation the maximum load strength of Group A was significantly higher than that of Group B and similar to that of normal tibia. n=5, *p<0.05.