Effects of apatite particle size in two apatite/collagen composites on the osteogenic differentiation profile of osteoblastic cells

Int J Mol Med. 2013 Dec;32(6):1255-61. doi: 10.3892/ijmm.2013.1516. Epub 2013 Oct 2.


The development of new osteoconductive bone substitute materials is expected in medicine. In this study, we attempted to produce new hydroxylapatite (HAP)/collagen (Col) composites using two HAP particles of different sizes and porcine type I collagen. The two HAP particles were either nano-sized (40 nm in average diameter; n-HAP) or had macro-pore sizes of 0.5‑1.0 mm in length with fully interconnected pores (m-HAP). The aim of this study was to investigate the effects of apatite particle size in two HAP/Col composites on the osteogenic differentiation profile in osteoblast-like cells (SaOS-2). We created a collagen control sponge (Col) and two HAP/Col composite sponges (n-HAP/Col and m-HAP/Col) using freeze-drying and dehydrothermal cross-linking techniques, and then punched out samples of 6 mm in diameter and 1 mm in height. The SaOS-2 cells were cultured on three test materials for 1, 2, 3 and 4 weeks. Total RNA was extracted from the cultured cells and the expression of osteogenic differentiation-related genes was evaluated by reverse transcription PCR (RT-PCR) using primer sets of alkaline phosphatase (ALP), type 1 collagen (COL1), bone sialoprotein (BSP) and osteocalcin precursor [bone gamma-carboxyglutamate (gla) protein (BGLAP)] genes, as well as the β-actin gene. The cells were also cultured on Col, n-HAP/Col and m-HAP/Col specimens for 1 and 4 weeks, and were then observed under a scanning electron microscope (SEM). The experimental results were as follows: RT-PCR indicated that osteogenic differentiation, particularly the gene expression of BSP, was most accelerated when the cells were cultured on n-HAP/Col specimens, followed by m-HAP/Col, whilst the weakest accelaeration was observed when the cells were cultured on Col specimens. As shown by the SEM images, the SaOS-2 cells were fibroblastic when cultured on Col specimens for up to 4 weeks; they were fibroblastic when cultured on n-HAP/Col specimens for 1 week, but appeared as spheroids, while actively phagocytizing n-HAP particles at 4 weeks; however, they appeared as deformed fibroblasts when cultured on m-HAP/Col specimens, detached from the particles. Despite limited experimental results, our study suggests that n-HAP/Col may be employed as a new osteoconductive bone substitute material.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / genetics
  • Alkaline Phosphatase / metabolism
  • Animals
  • Apatites / chemistry
  • Apatites / pharmacology*
  • Biomarkers / metabolism
  • Cell Differentiation / drug effects*
  • Cell Differentiation / genetics
  • Cell Line
  • Collagen / pharmacology*
  • Gene Expression Regulation / drug effects
  • Humans
  • Nanoparticles / chemistry
  • Osteoblasts / cytology*
  • Osteoblasts / drug effects
  • Osteoblasts / enzymology
  • Osteoblasts / ultrastructure
  • Osteogenesis / drug effects*
  • Osteogenesis / genetics
  • Particle Size*
  • Porosity
  • Sus scrofa
  • Tissue Scaffolds / chemistry*


  • Apatites
  • Biomarkers
  • Collagen
  • Alkaline Phosphatase