Phosphorylated pullulan promotes calcification during bone regeneration in the bone defects of rat tibiae

The current study aimed to evaluate bone tissue regeneration using a combination of beta-tricalcium phosphate (beta TCP) and phosphorylated pullulan (PPL, a phosphate-rich polysaccharide polymer consisting of maltotriose units). Round defects of 2 mm diameter were created in the arterial center of rat tibiae, which were further treated with vehicle (control group), beta TCP (beta TCP group), or beta TCP + PPL (beta TCP + PPL group) grafts. The control specimens without bone grafts exhibited rapid bone formation after 1 week; however, the regenerated bone was not resorbed until 4 weeks. In contrast, beta TCP-grafted specimens exhibited fewer but thicker trabeculae, whereas the beta TCP + PPL group displayed many fine trabeculae at 4 weeks. In the beta TCP + PPL group, new bone was associated with the beta TCP granules and PPL. Similarly, PHOSPHO1-positive osteoblasts were localized on the beta TCP granules as well as the PPL. On the other hand, TRAP-reactive osteoclasts predominantly localized on newly-formed bone and beta TCP granules rather than on the PPL. No significant differences were observed in the expression of Alp, Integrin alpha v, Osteopontin, Osteocalcin, and Dmp-1 in PPL-treated MC3T3-E1 osteoblastic cells, suggesting that PPL did not facilitate osteoblastic differentiation. However, von Kossa staining identified abundant needle-like calcified structures extending inside the PPL. Furthermore, transmission electron microscopy (TEM) revealed many globular structures identical to calcified nodules. In addition, calcified collagen fibrils were observed in the superficial layer of the PPL. Thus, PPL may serve as a scaffold for osteoblastic bone formation and promotes calcification on its surface. In conclusion, we speculated that beta TCP and PPL might promote bone regeneration and could be integrated into promising osteoconductive materials.