Fresh Frozen Bone Graft: An immunohistochemical Analysis on Angiogenic Proteins

Objectives: The present study aims to evaluate the neoangiogenic response of host tissue towards fresh frozen bone (FFB), by investigating differences in typical ECM molecules expression at different experimental times. Methods: FFB was used as grafting material in patients requiring bone regeneration for following implant supported prosthesis. Bone specimens were withdrawn at 4 and 6 months after grafting. Light Microscope analysis was performed after EM-EO staining. Immunohistochemycal analysis (DAB staining method) was performed to evaluate the expression of MMP2, MMP9, and VEGF. Wilcoxson-Mann-Witney Test was used to statistically evaluate differences in molecules expression. Results: Light microscope analysis showed a better integration only after 6 months from graft placement. Initially istological integration is not reached, bone grafts particles are clearly visible and differs from autologous bone, it appears as marble stones, because they have few blood vessels. MMP2, MMP9 and VEGF level of expression was high in FFB only from 6 months after the insertion of the grafting material. Conclusions: Even if FFB appeared clinically integrated with the host tissue, indicating it can be successfully used as graft in pre-prosthetic regenerative techniques, the expression of molecules specifically involved in neoangiogenesis was high only after 6 months. Neo-angiogenic phenomena, as the invasion of the biomaterials by new blood vessels, is indispensable for its following colonization by mesenchymal stem cells, which will lead the process of bone regeneration. A greater activity could be noticed in FFB graft just 6 months after grafting, so we can assume an implant positioning following classic protocol time of 6 months.