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.
Highlights
Calcium phosphate (Ca-P) based synthetic bone substitutes are widely used in orthopaedic and maxillofacial surgeries [1,2]
In vivo Kinetics of bone morphogenetic protein-2 (BMP-2) Release from Scaffolds Based on the above results, we found that the addition of trehalose did not affect the release profile and bioactivity of Bone morphogenetic proteins (BMPs)-2 from the scaffolds when the BMP-2 was loaded by physical adsorption
Using the same amount of BMP-2 releasate to examine the bioactivity of BMP-2 loaded by this two loading methods, we found that the alkaline phosphatase (ALP) expression of lyophilized BMP-2 was lower than that of fresh BMP-2 which indicated the osteoinductive potential of BMP-2 might be partially subtracted by lyophilization
Summary
Calcium phosphate (Ca-P) based synthetic bone substitutes are widely used in orthopaedic and maxillofacial surgeries [1,2] These materials have proved to be biocompatible and osteoconductive, but lack the osteogenic potential to promote bone healing of critical-sized defects [3]. The implanted growth factors tend to be inactivated by heat, extreme pH, or proteases, resulting in unstable activity during the desired therapeutic period [15,16]. Increased concerns about these issues have led to the acceptance of a method for loading BMP-2 onto Ca-P scaffolds by coating it via lyophilization
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