Improvement of bone regeneration capability of ceramic scaffolds by accelerated release of their calcium ions

Abstract

To regenerate the bone tissue, the fabrication of scaffolds for better tissue regeneration has attracted a great deal of attention. In fact, growth factors are already used in clinical practice and are being investigated for enhancing the capacity for bone tissue regeneration. However, despite their strong osteoinductive activity, these growth factors have several limitations: safety issues, high treatment costs, and the potential for ectopic bone formation. The aim of this study was therefore to develop ceramic scaffolds that could promote the capacity for bone regeneration without growth factors. Three-dimensional ceramic scaffolds were successfully fabricated from hydroxyapatite (HA) and tricalcium phosphate (TCP) using projection-based microstereolithography, which is an additive manufacturing technology. The effects of calcium ions released from ceramic scaffolds on osteogenic differentiation and bone regeneration were evaluated in vitro and in vivo. The osteogenesis-related gene expression and area of new bone formation in the HA/TCP scaffolds was higher than those in the HA scaffolds. Moreover, regenerated bone tissue in HA/TCP scaffolds were more matured than that in HA scaffolds. Through this study, we were able to enhance the bone regeneration capacity of scaffolds not by growth factors but by calcium ions released from the scaffolds. Ceramic scaffolds developed in this study might be useful for enhancing the capacity for regeneration in complex bone defects.

FIG. 1.

Macroscopic and microscopic images showing the ceramic scaffolds used in this study: (a) commercialized HABI-HAP-PD, (b, c) fabricated ceramic scaffolds, (a, b) for in vivo experiments, and (c) for in vitro experiments.

FIG. 2.

Calcium ions release over the course of 14 days.

FIG. 3.

Human turbinate mesenchymal stromal cell (hTMSC) proliferation on the ceramic scaffolds over the course of 14 days. *p<0.05.

FIG. 4.

Osteogenic differentiation of hTMSCs on ceramic scaffolds (Alizarin Red-S staining test). *p<0.05. Color images available online at www.liebertpub.com/tea

FIG. 5.

Quantitative real-time reverse transcription–polymerase chain reaction gene expression analysis of osteogenic markers. *p<0.05

FIG. 6.

Micro-computed tomography (CT) images showing new bone formation at the 16th week after implantation: (a) blank, (b) BABI-HAP-PD, (c) hydroxyapatite (HA), and (d) HA/tricalcium phosphate (TCP).

FIG. 7.

New bone formation area at the 16th week after implantation. *p<0.05.

FIG. 8.

The images (40×) of histological analysis using hematoxylin and eosin (H&E) (a, c, e, g) and Masson's trichrome (b, d, f, h) at the 16th week after implantation: (a, b) blank, (c, d) BABI-HAP-PD, (e, f ) HA, and (g, h) HA/TCP. Color images available online at www.liebertpub.com/tea

FIG. 9.

Magnified images (100× and 200×) of histological assay using H&E (a–d) and Masson's trichrome (e–h) at the 16th week after implantation: (a, b, e, f ) HA, and (c, d, g, h) HA/TCP. Color images available online at www.liebertpub.com/tea