Osteoformation potential of an allogenic partially demineralized bone matrix in critical-size defects in the rat calvarium.

Allogenic demineralized bone matrix has been developed as a reliable alternative to the autologous bone graft. In the present study, we assessed the osteoformation potential of a partially demineralized bone matrix (PDBM) in a paste form obtained without an added carrier. This formulation included the preparation of cancelous bone from femoral heads after decellularision, delipidation, demineralization in HCl and autoclaving at 121 °C. Structural and biochemical characteristics of PDBM were determined using FTIR (Fourier transform infrared spectroscopy), hydroxyproline, DNA content assays, and optical ellipsometry. The osteoformation potential was evaluated in 8-, 6-, and 4-mm-diameter rat-calvarial bone defects by in vivo micro-CT analysis, performed immediately after surgery on days 0, 15, 30, 45, and 60. Moreover, histological and histomorphometric analyses were done on day 60. PDBM was compared to cancelous bone powder (BP) before its partial demineralization. The expression levels of selected inflammation-, angiogenesis-, and bone-related genes were also investigated by RT-PCR, 3, 7, and 14 days after surgery. Compared to the control group, the PDBM group exhibited a significant increase (p < 0.05) in radiopacity in 8-mm- and 6-mm-diameter defects at all time points tested. On day 60, the amount of newly-formed bone was greater (16 and 1.6 folds; p < 0.001; respectively) compared to that in control defects. No bone formation was observed in defects filled with BP regardeless of the size. In 8-mm-diameter defect, PDBM was effective enough to induce the upregulation of genes pertinent to inflammation (i.e., TNFα, IL-6, and IL-8), angiogenesis (i.e., VEGF, VWF), and osteogenesis (ALP, RUNX2, BGLAP, SP7) by day 3 after surgery. This study showed that the tested PDBM deeply influences the early critical events involved in bone regeneration and exhibits efficient osteoformation capacity, making it an attractive graft option for treating defects in periodontal and maxillofacial areas.

Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions.

Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. STATEMENT OF SIGNIFICANCE: Upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.