Reconstruction of a Critical Sized Segmental Bone Defect in the Ovine Tibia by Tissue Engineering Methods

Currently, well-established clinical therapeutic approaches for segmental bone defect reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive, osteoinductive, and osteogenic properties, but face significant disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration leading to graft devitalisation and subsequent resorption resulting in decreased mechanical stability. As a result, recent research has focused on the development of alternative therapeutic concepts and the field of tissue engineering in particular has emerged as an important approach to bone regeneration. The objective of the current study was to develop an alternative therapeutic approach to bone grafting by evaluating the regenerative potential of medical grade polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds in combination with and without recombinant human bone morphogenetic protein 7 (rhBMP-7) in a clinically relevant large animal model and to compare the outcomes with the gold standard treatment, the application of autologous bone graft (ABG) from the iliac crest. Methods A 3 cm mid-diaphyseal tibial defect was created in the right hind limb of 32 merino sheep (average weight 42kg, age 7-8 years) and stabilized using a broad 4.5 mm dynamic compression plate (DCP, Synthes) with 4 screws proximally and 3 screws distally. Defects were left untreated (n=8), reconstructed with autologous cancellous bone graft from the left iliac crest (n=8) or a cylindrical mPCL-TCP scaffold fabricated via fused deposition modeling (height: 3cm, diameter: 2cm, porosity 70%; Osteopore, Singapore) with (n=8) or without (n=8) rhBMP-7 (3.5mg, OP-1 implant, Stryker). Animals were held for 12 weeks and allowed unrestricted weight-bearing. Conventional X-rays were taken after surgery and after 6 and 12 weeks and native CT scans were performed after sacrifice. 3D reconstructions were generated from the CT data and qualitative analysis was performed to assess mineralization in the defect and bridging. Mechanical testing was performed using an Instron 8874 biaxial testing machine. A torsion test was conducted at an angular velocity of 0.5 deg/s until the fracture point was reached. The contralateral tibia was used as a paired reference. The torsional moment (TM) and torsional stiffness (TS) were normalized against the intact contralateral tibiae. Statistical analysis was carried out using a one-tailed Mann-Whitney-Utest (SPSS) and p-values <0.05 were considered significant. Fig. 1: Micro CT images of a cylindrical mPCL-TCP scaffold fabricated via fused deposition modelling with a porosity of 70%. Dimensions: height 3cm, diameter 2cm. Results Conventional X-ray and CT analysis after 12 weeks confirmed the critical nature of the defect. None of the empty control defects showed signs of bridging and were filled with soft tissue only. Therefore no biomechanical testing could be carried out on these specimens. Only minor bone formation was observed in the scaffold group, however full defect bridging had occurred in all defects reconstructed with ABG or mPCL-TCP with rhBMP-7 (Fig.2). Scaffolds showed good osseointegration without any signs of resorption. Biomechanical testing revealed a significant higher torsional moment and stiffness for the ABG and rhBMP-7 groups when compared to the mPCL-TCP group. No significant differences were found between the ABG and rhBMP-7 group. However, the rhBMP-7 treatment tended to result in higher values for both torsional moment and stiffness (Fig. 3). Fig. 2: Representative 3D CT reconstructions of critical segmental bone defects, which were left untreated (A), reconstructed with ABG (B), a mPCL-TCP scaffold (C) or a mPCL-TCP scaffold combined with rhBMP-7 (D). Fig. 3: Median values of torsional moment (A) and stiffness (B) relative to the contralateral tibia (n=8). Error bars represent the 1 and 3 quartile. Discussion Our group has developed a standardized and reproducible ovine, tibial, critical-sized defect model as reflected by the non-union rate of 100% in the empty control group. The application of autografts from the iliac crest caused defect bridging in all cases. When compared to intact controls, the mechanical properties of the newly formed bone however were of inferior quality after 3 months. mPCL-TCP scaffolds didn’t evoke foreign body responses. The mPCL-TCP scaffolds alone did not result in any substantial mineralization of the defect area. However, when combined with rhBMP-7, bone formation within the defect equaled or even exceeded amounts observed with autografts. The study results suggest that mPCL-TCP scaffolds combined with a biologically active stimulus such as rhBMP-7 can serve as an equivalent alternative to autologous bone grafting in the early phase of defect regeneration. These findings however must be confirmed by long-term studies currently underway. ٭