Three-dimensional dynamic culture of pre-osteoblasts seeded in HA-CS/Col/nHAP composite scaffolds and treated with α-ZAL

Abstract

Pre-osteoblast MC3T3-E1 cells were cultured in hyaluronic acid-modified chitosan/collagen/nano-hydroxyapatite (HA-CS/Col/nHAP) composite scaffolds and treated with phytoestrogen a-zearalanol (a-ZAL) to improve bone tissue formation for bone tissue engineering. Perfusion and dynamic strain were applied to three-dimensional (3D) cultured cells, which simulates mechanical microenvironment in bone tissue and solves mass transfer issues. The morphology of cell-scaffold constructs in vitro was then examined and markers of osteogenesis were assessed by immunohistochemistry staining and western blotting. The results showed that cells expanded their pseudopodia in an irregular manner and dispersed along the walls in 3D-dynamic culture. Osteogenic phenotype was increased or maintained by enhanced collagen I (COLI) levels, decreased osteopontin expression and having little effect on osteocalcin expression during the 12 days of in vitro culture. In response to a-ZAL, the cell-scaffold constructs showed inhibited cellular proliferation, enhanced the alkaline phosphatase (ALP) activity and increased ratio of osteoprotegerin to receptor activator of nuclear factor kappa B (NF-kB) ligand (RANKL). Application of perfusion and dynamic strain to cells-scaffold constructs treated with a-ZAL represents a promising approach in the studies of osteogenesis stimulation of bone tissue engineering.

Enhanced osteoblastogenesis in three-dimensional collagen gels

“…4 Collagen I provides a tissue-derived natural polymer; it is biocompatible and biodegradable and its mechanical and biological properties can be controlled and varied as required. 3,5 As a scaffold for bone regeneration, collagen can be used alone or in combination with various natural polymers and minerals that provide better representation of the extracellular environment, including hyaluronic acid-modified chitosan/collagen/hydroxyapatite composites 6 and collagen/hydroxyapatite scaffolds. 4 Although a number of studies have investigated osteoblasts cultured in 3D collagen gels, they mostly used microscopy for visual analysis of the osteoblast phenotype and alkaline phosphatase activity as a measure of osteoblast function.…”

Effects of constrained dynamic loading, CKIP‑1 gene knockout and combination stimulations on bone loss caused by mechanical unloading

“…Our previous study revealed that constrained dynamic loading could strengthen bone structure and reduce bone fractures. Apparent strain (1,200 µε, 2,500-2,800 µε) can improve osteoblast proliferation and differentiation, increase osteoblast activity and counter osteoporosis in ovariectomized mice in vitro and MC3T3-E1 cells in vivo (30)(31)(32). In the present study, we first explored the effect of constrained dynamic loading in combination with CKIP-1 knockout in a tail suspension mouse model.…”

Evaluation of in vitro and in vivo osteogenic differentiation of nano‐hydroxyapatite/chitosan/poly(lactide‐co‐glycolide) scaffolds with human umbilical cord mesenchymal stem cells

“…23 The higher exposure of HA nanoparticles at the scaffold surface may result in significantly higher mineralization and enhanced bone formation 24 The osteogenic differentiation effect of CS have be mentioned in some literatures. [25][26][27][28][29] It has been reported that the combination of HA and CS can enhance bone regeneration and conductivity. 28 Wu et al 7 found that CS applied over the porous PLGA scaffold facilitated the differentiation of osteoblasts on the scaffold, possibly being an accelerator for such differentiation.…”

“…The osteoconductivity of the scaffold is positively correlated to the similarity of the nHA and natural bone minerals in composition, size, and morphology . The higher exposure of HA nanoparticles at the scaffold surface may result in significantly higher mineralization and enhanced bone formation The osteogenic differentiation effect of CS have be mentioned in some literatures …”