| Background: Treatment of large bone defect in routine orthopaedic clinics requires bone-grafting and/or scaffold materials, especially desirable with composite material combined with therapeutic and bioactive agents for achieving better treatment outcome. The strategy of this study was to develop such a bioactive biodegradable composite bone scaffold incorporating a phytomolecule icaritin as an exogenous growth factor or bone morphogenetic protein-2 (BMP-2) as a known endogenous growth factor for bone regeneration. Based on polylactide-co-glycolide (PLGA) and Tricalcium Phosphate (TCP), we fabricated innovative BMP-2 or icaritin incorporated scaffold materials, namely PLGA/TCP (Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and highicaritin with three different dosages of icaritin (Treatment groups) by an advanced prototyping technology.;Aims: The overall aim of the study was to establish and evaluate a local delivery system with slow release of bioactive agents for acceleration of bone regeneration in a bone defect model in rabbits. In vivo efficacy study served as end-point of this comparative study between composite scaffold incorporating exogenous growth factor icaritin and endogenous growth factor BMP-2.;Materials & Methods: Composite scaffolds were fabricated at -28ºC by a low-temperature rapid-prototyping machine. PLGA and TCP were used as basic carrier materials, and icaritin or BMP-2 was incorporated as exogenous or endogenous bioactive growth factors, respectively. The incorporated scaffolds were named by PLGA/TCP (P/T, Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and high-icaritin (Treatment groups). In addition, we prepared BMP-2 and icaritin loading scaffolds, namely PLGA/TCP+BMP-2 as positive control group and PLGA/TCP+middle-icaritin as comparative group by entire immersion in the solution and dry in vacuum cabinet for 24 hours. In vitro osteogenic potentials of the designed bioactive composite scaffolds were tested in scaffold-co-cultured rabbit bone marrow stem cells (BMSCs) for measurement of cell seeding and proliferation efficiency, alkaline phosphatase (ALP) activity, calcium deposition, and quantitative mRNA expression of relative osteogenic genes. In vivo efficacy investigation was designed to evaluate osteogenesis and angiogenesis in a bilateral ulna bone segmental defect model implanted with composite scaffold in rabbits, with radiography and in vivo micro-CT for studying new bone regeneration and micro-CT-based angiography and histology for neovascularization, dynamic MRI for local blood perfusion function, as well as host tissue and scaffold material interactions.;Results: The in vitro characterization and osteogenic potential of the fabricated scaffolds were performed and confirmed, respectively. Micro-CT quantitation showed that the scaffolds had interconnected macropores with an average porosity of 75+/-3.27 % and pore size or diameter of 458+/-25.6 microm. Compared to P/T, P/T+icaritin and P/T/BMP-2 scaffolds, P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin (n=6, p<0.01) presented higher cell seeding efficiency, ALP activity and calcium nodules and up-regulated mRNA expressions of Collagen type I and Osteopontin of co-cultured BMSCs. P/T+BMP-2 showed the best osteogenic effects among all groups (n=6, p<0.01). In vivo measurement of x-ray and micro-CT in rabbit ulna bone defect model at week 2, 4 and 8 post-surgery showed more newly formed bone in the defects treated with P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin scaffold (n=6, p<0.01) compared with that of P/T, P/T+icaritin and P/T/BMP-2 groups. P/T+BMP-2 also showed the best bone formation among all groups (n=6, p<0.01). Histological results also demonstrated that there were more osteoid tissues and typical lamellar bone in surface and internal of the implants, as well as along the adjacent host bone in P/T/icaritin groups (n=5, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01). P/T+BMP-2 group showed the most newly formed bone (n=6, p<0.01). In addition, newly formed vessels in the defects were identified with micro-CT-based angiography and functionally supported by dynamic MRI for reflecting blood perfusion. The results showed more ingrowing new vessels in P/T/icaritin groups (n=6, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01), compared to P/T and P/T/BMP-2 groups. For comparing dose effects among three scaffolds incorporating different concentration of icaritin, we found that middle dose PLGA/TCP/icaritin composite scaffold showed the best osteogenic potential.;Conclusion: PLGA/TCP scaffolds incorporating exogenous phytomolecule icaritin demonstrated the desired osteogenic differentiation potential and bone regeneration capability as investigated in vitro and in vivo, where the middle dose of icaritin incorporating PLGA/TCP composite scaffold showed the best effects. These findings may form a good foundation for potential clinical validation of this innovative bioactive composite scaffold with either conventional endogenous BMP-2 for in vitro loading before application or more attractively and user-friendly incorporated with exogenous phytomolecule icaritin as a ready product for enhancing bone defect repair.;Keywords: Ulna bone segmental defect; bone healing; PLGA/TCP composite scaffolds; icaritin; BMP-2. |
