Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo.
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.
Department of Orthodontics, College of Stomatology, Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Jun
J
Zhao
Shaoyi
S
Wang
Jianqiang
J
Bao
Xiaojuan
X
Sun
Xiaochen
X
Zhang
Xiuli
X
Zhang
Dongxia
D
Ye
Jie
J
Wei
Changsheng
C
Liu
Xinquan
X
Jiang
Gang
G
Shen
Zhiyuan
Z
Zhang
D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry*; D049109:Cell Proliferation; D018920:Coculture Techniques; D017886:Durapatite / Q000737:chemistry*; D005612:Freeze Drying; D007700:Kinetics; D059630:Mesenchymal Stem Cells / Q000166:cytology; D010012:Osteogenesis; D054457:Tissue Scaffolds; D014199:Trehalose / Q000494:pharmacology*
D055396:Bone Morphogenetic Protein 2; D017886:Durapatite; D014199:Trehalose; D002118:Calcium