glass fibers facilitate proliferation and osteogenesis through Runx2 transcription in murine osteoblastic cells

2 Cell-material interactions and compatibility are important aspects of bioactive materials for 3 bone tissue engineering. Phosphate glass fiber (PGF) is an attractive inorganic filler with fibrous 4 structure and tunable composition, which has been widely investigated as a bioactive filler for 5 bone repair applications. However, the interaction of osteoblasts with PGFs has not been widely 6 investigated to elucidate the osteogenic mechanism of PGFs. In this study, different concentrations 7 of short PGFs with interlaced oriented topography were co-cultured with MC3T3-E1 cells for 8 different periods, and the synergistic effects of fiber topography and ionic product of PGFs on 9 osteoblast responses including cell adhesion, spreading, proliferation and osteogenic 10 differentiation were investigated. It was found that osteoblasts were more prone to adhere on 11 PGFs through vinculin protein, leading to enhanced cell proliferation with polygonal cell shape 12 and spreading cellular actin filaments. In addition, osteoblasts incubated on PGF meshes showed 13 enhanced alkaline phosphatase (ALP) activity, extracellular matrix mineralization, and increased 14 expression of osteogenesis-related marker genes, which could be attributed to the 15 Wnt/β-catenin/Runx2 signaling pathway. This study elucidated the possible mechanism of PGF on 16 triggering specific osteoblast behavior, which would be highly beneficial for designing PGF-based 17 bone graft substitutes with excellent osteogenic functions. 18

1. Introduction Bone loss is one of the most important causes of skeletal disease such as congenital defects, 3 oral and maxillofacial pathologies and osteoporosis leading to bone fractures 1 . Many studies focus 4 on bone tissue engineering strategies for skeletal tissue regeneration. In bone tissue engineering, 5 engineered scaffolds can provide specific microenvironment and architecture for cell adhesion, 6 migration, proliferation and even bone formation by regulating osteogenic differentiation 2 . The 7 stiffness, elasticity, topography, morphology and specific degradation products of the scaffold can 8 regulate cell-cell, cell-ECM and cell-material interactions 3 . Scaffold architecture is reported to be 9 critically important for bone regeneration as it could be made to mimic the native 3D environment 10 for osteoblasts in order to promote cell adhesion and activation 4, 5 . In addition, bioactive 11 constituents of the scaffold are also important characteristics of bone tissue engineering materials 6 .

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On the one hand, materials can provide bioactivity to interact with surrounding cells and tissues 13 and avoid the host immune response 7 . On the other hand, biomaterial surfaces can present 14 osteo-inductive factors such as calcium ions and growth factors to the local microenvironment in 15 order to enhance bone formation 7, 8 .

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Phosphate-based glasses have gained increasing attention 9, 10 , because of their similar 17 composition to the inorganic component of native bone, biocompatibility, bioactivity, and 18 osteo-conductivity 11, 12 . It has been previously reported that biodegradable composites reinforced 19 with phosphate glass fibers (PGFs) could promote the proliferation of preosteoblasts 13 . PGFs have 20 also been shown to promote neuronal polarization and directional growth of axons in vitro and in 21 vivo, potentially repairing peripheral nerve injury in vivo [14][15][16] . In addition, fibrous chitosan-glued 22 phosphate glass fiber scaffolds were shown to be non-cytotoxic against bone marrow stromal 23 cells 17 . Furthermore, nanocomposite polymer scaffolds using sacrificial phosphate glass fibers 24 have shown support for the growth of human tenocytes cells 18 . It was also recently reported that

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PGFs can be deposited as coating on bulk metallic surfaces to enhance the proliferation and 26 expression of osteogenic-related genes in MC3T3-E1 cells 19 . These attractive characteristics and 27 higher bioactivity of PGFs suggest that they can be considered suitable building blocks for bone 28 scaffolds and can function as an excellent tissue engineering material. 3 (in mol%) --denoted as P50, and then melted in a Pt-Au crucible at 1100 ℃ for 90 min.

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Continuous fibers with a diameter in the range ~10-20 µm were then produced from the molten 5 glass via an in-house melt-draw spinning facility at ~1600 rpm, followed by annealing at 5 ℃ 6 below the glass transition temperature (Tg=479℃) for 90 min. The as-prepared PGF bundles were 7 chopped into short PGF meshes with the length of 2-3 mm before further utilization. Chopped PGFs were immersed in 75 vol% ethanol, subjected to ultrasonication treatment for 10 20 min, and then dried in vacuum desiccator at 50℃ overnight. The samples were adhered onto a 11 sample holder using conductive carbon tape and then sputter-coated with gold for 1.5 min.

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The protein expression was observed under fluorescence microscope (Nikon 80i, Japan).

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The morphology and elemental composition of the synthesized PGFs are showed in Figure 1.

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The SEM result showed that PGFs possessed smooth surface with a diameter of 12.48±0.19 μm 23 ( Figure 1A). Surface EDX analysis was also performed to measure the elemental composition of 24 the produced PGFs. The EDX pattern of the selected area in Figure 1B  release profiles of calcium ions from PGFs, which is closely associated with the formation of HA 28 phase, are plotted in Figure 1C. A burst ion release up to 2.69 μg/mg was detected during the first  Figure 2A).

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The cytoskeleton of MC3T3-E1 cells on PGF fibers was performed by labeling the 11 organization of F-actin stress fiber using immunofluorescence assay. The results showed that

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MC3T3-E1 cells on PGFs possessed wide spreading cellular actin filaments and polygonal cell 13 shape, especially in the 1 mg/ml of PGF group (indicated by the arrows). Whilst the orientated 14 cellular actin filaments and fibroblast-like osteoblasts were seen in the control group ( Figure 2B).

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These results indicated that MC3T3-E1 cells preferred to spread on the PGF fibers with polygonal 16 cell shape, probably due to the interlaced morphology of the PGF meshes and preferential 17 adhesion of osteoblasts on the PGFs.  7 Moreover, compared with the control group, the MC3T3-E1 cells cultured with PGFs exhibited 8 lower ALP activity at day 3 and day 7, but higher ALP activity at day 14 and day 21. In addition,

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ALP activity of cells on 1 mg/ml of PGFs was significantly higher than for cells on 0.5 mg/ml of 10 PGFs at 14 and 21 days ( Figure 4A). These results suggested that PGFs could promote osteoblast 11 osteogenesis and delay the differentiation process by a concentration-dependent manner.

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To confirm the effect of PGFs on osteogenic differentiation, alizarin red staining was 13 performed to detect the possible mineral deposits on cell surfaces during osteogenic induction 14 after 0, 3, 7, 14 and 21 days ( Figure 4B). The scanning images showed that more mineral 15 deposition was observed on the cells cultured with 0.5 mg/ml and 1 mg/ml of PGF and no mineral 16 formed in PGFs without cells ( Figure S1). These data indicate that the PGFs could promote the 17 osteogenic differentiation of MC3T3-E1 cells.    were also significantly upregulated in the PGF group at 21 days ( Figure 6A).

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MC3T3-E1 cells were cultured on PGFs in osteogenic induction medium for 21 days and the 8 impact of PGFs on the protein expression of Runx2 was then examined by immunofluorescence 9 assay. The results showed that the protein expression of Runx2 was upregulated in the cells 10 cultured with PGFs ( Figure 6B). Thus, we suggest that PGFs could promote osteogenic 11 differentiation by upregulating Runx2 expression through Wnt/β-catenin signal pathway. the control group, osteoblasts spread well on PGFs and the cytoskeleton displayed a better shape 7 that allowed the cells to be more prone to adhere on PGF surfaces tightly through higher expressed 8 vinculin ( Figure 3A). Furthermore, the mRNA expression of adhesion-related genes including 9 Vinculin and Fibronectin 1 was increased significantly in PGF groups ( Figure 3B). These