Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo

Jun Zhao; Shaoyi Wang; Jianqiang Bao; Xiaojuan Sun; Xiaochen Zhang; Xiuli Zhang; Dongxia Ye; Jie Wei; Changsheng Liu; Xinquan Jiang; Gang Shen; Zhiyuan Zhang

doi:10.1371/journal.pone.0054645

Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo

PLoS One. 2013;8(1):e54645. doi: 10.1371/journal.pone.0054645. Epub 2013 Jan 24.

Authors

Jun Zhao¹, Shaoyi Wang, Jianqiang Bao, Xiaojuan Sun, Xiaochen Zhang, Xiuli Zhang, Dongxia Ye, Jie Wei, Changsheng Liu, Xinquan Jiang, Gang Shen, Zhiyuan Zhang

Affiliation

¹ Department of Orthodontics, College of Stomatology, Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.

Abstract

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ± 5.32%) and area (40.71% ± 7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ± 0.44%, calcein: 6.08% ± 1.37%) and mineral apposition rate (4.13 ± 0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17 ± 15.02 Mpa) and load of fracture (144.67 ± 16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bone Morphogenetic Protein 2 / metabolism*
Calcium / chemistry*
Cell Proliferation
Coculture Techniques
Durapatite / chemistry*
Freeze Drying
Kinetics
Mesenchymal Stem Cells / cytology
Osteogenesis*
Tissue Scaffolds*
Trehalose / pharmacology*

Substances

Bone Morphogenetic Protein 2
Durapatite
Trehalose
Calcium

Grants and funding

This work is supported by the National Natural Science Foundation of China (81200815, 81271182, 30973363, 81000420), Science and technology fund, Shanghai Jiao Tong University, School of Medicine 11XJ21024. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.