Multiscale characterization of the musculoskeletal system

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Original Research
09 October 2023
Phosphorylated pullulan promotes calcification during bone regeneration in the bone defects of rat tibiae
Yasuhito Morimoto
12 more and 
Norio Amizuka
Elemental mapping of calcium (Ca) and phosphorus (P) in regenerated bone associated with PPL. (A–C) illustrate the composition (COMPO) images in the bone defect regions in the βTCP + PPL-grafted specimens at weeks 1 (A), 2 (B), and 4 (C). (D–F, J–L) show the EPMA images of Ca (D–F) and P (J–L) at weeks 1 (D,J), 2 (E,K), and 4 (F,L). Graphs (G–I, M–O) display the intensities of X-ray fluorescence from Ca (G–I) and P (M–O) at weeks 1 (G,M), 2 (H,N), and 4 (I,O) by EPMA analysis. The newly-formed PPL-associated bone exhibits a dense distribution of Ca and P at week 4 (F,I,L,O). Bar, (A–F, J–L): 50 μm.

The current study aimed to evaluate bone tissue regeneration using a combination of β-tricalcium phosphate (βTCP) 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), βTCP (βTCP group), or βTCP + PPL (βTCP + 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, βTCP-grafted specimens exhibited fewer but thicker trabeculae, whereas the βTCP + PPL group displayed many fine trabeculae at 4 weeks. In the βTCP + PPL group, new bone was associated with the βTCP granules and PPL. Similarly, PHOSPHO1-positive osteoblasts were localized on the βTCP granules as well as the PPL. On the other hand, TRAP-reactive osteoclasts predominantly localized on newly-formed bone and βTCP granules rather than on the PPL. No significant differences were observed in the expression of Alp, Integrin αv, 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 βTCP and PPL might promote bone regeneration and could be integrated into promising osteoconductive materials.

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