Osteogenic Effects of Dipyridamole Versus rhBMP-2 Using 3D-Printed Bioceramic Scaffolds in a Growing Alveolar Cleft Model

Purpose: Alveolar clefts affect three out of four patients with cleft lip and palate (CL/P) resulting in maxillary arch instability, inability to support dentition, and facial asymmetry. Secondary bone grafting is the standard of care for alveolar clefts, but the procedure is associated with donor site morbidity and graft resorption. Although rhBMP-2 is under investigation for alveolar cleft repair, safety concerns remain regarding pathologic effects on the growing suture. This provides the impetus for investigating alternative osteogenic molecules. Dipyridamole (DIPY) is an adenosine receptor indirect agonist with known osteogenic potential. This study compared the regenerative capacity and side effects of DIPY to rhBMP-2 at alveolar cleft defects delivered via 3D-printed bio-ceramic (3DBC) scaffolds.

Methods: 23 skeletally immature New Zealand White rabbits underwent unilateral, 3.5mm x 3.5mm alveolar resection adjacent to the growing suture. Five defects without intervention served as negative controls. The remaining defects were reconstructed with 3DBC scaffolds coated with the following agents: 1000μm DIPY (n=6), 10,000μm DIPY (n=7), and 0.2 mg/mL rhBMP-2 (n=5). Animals were euthanized at t=8 weeks. Samples were scanned using microCT and new bone volume within scaffold was quantified using Amira 6.1 software (Visage Imaging GmbH, Berlin, Germany). Non-decalcified histology was performed and new bone within scaffold pores were evaluated for mechanical properties (i.e. reduced elastic modulus, hardness) and compared to internal controls of non-injured bone. Statistical analysis was performed using a generalized linear mixed model between groups and Wilcoxon rank sum test within samples.

Results: MicroCT revealed a lack of bridging bone healing in negative controls. There was bridging bone formation across all 3DBC treatment groups. As a function of bone growth within the scaffold interstices, 1,000μm-DIPY scaffolds regenerated 28.03±7.38% bone, 10,000μm-DIPY scaffolds regenerated 36.18±6.83% bone (p=0.104 1,000μm vs. 10,000μm DIPY), and rhBMP-2 coated scaffolds regenerated 37.17±16.69% bone (p=0.124 vs. 1,000μm-DIPY and p=0.938 vs. 10,000μm-DIPY). MicroCT also revealed rhBMP-2 to be associated with active bone resorption at the area of the suture and evidence of early suture fusion, which was again not seen in DIPY group or controls. On histology and electron microscopy, no changes in suture biology were evident in DIPY groups, while the rhBMP-2 group demonstrated early signs of suture fusion as well as osteoclastic activity at suture borders suggestive of osteolysis. Healing was intramembranous-like and endochondral-like, with highly cellular and vascularized structure across all groups. Reduced elastic moduli of new bone was significantly less than native bone, irrespective of DIPY or BMP augmentation (p<0.01).

Conclusion: Dipyridamole augments bone regeneration similarly to rhBMP-2 at both 1,000μm and 10,000μm concentrations. Dipyridamole generates new bone without osteolysis and early suture fusion associated with rhBMP-2. 3DBC scaffolds are effective at local osteogenic agent delivery.