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Combined Delivery of Two Different Bioactive Factors Incorporated in Hydroxyapatite Microcarrier for Bone Regeneration.

Tissue engineering and regenerative medicine (2020-08-18)
Tae-Woo Kim, Woo-Beom Ahn, Joong-Min Kim, Joong-Hyun Kim, Tae-Hyun Kim, Roman A Perez, Hyon-Seok Jang
ABSTRACT

The delivery of growth factors using a carrier system presents a promising and innovative tool in tissue engineering and dentistry today. Two of the foremost bioactive factors, bone morphogenetic protein-2 and vascular endothelial growth factor (VEGF), are widely applied using a ceramic scaffold. The aim of this study was to determine the use of hydroxyapatite microcarrier (MC) for dual delivery of osteogenic and angiogenic factors to accelerate hard tissue regeneration during the regenerative process. Two MCs of different sizes were fabricated by emulsification of gelatin and alpha-tricalcium phosphate (α-TCP). The experimental group was divided based on the combination of MC size and growth factors. For investigating the in vitro properties, rat mesenchymal stem cells (rMSCs) were harvested from bone marrow of the femur and tibia. For in vivo experiments, MC with/without growth factors was applied into the standardized, 5-mm diameter defects, which were made bilaterally on the parietal bone of the rat. The animals were allowed to heal for 8 weeks, and samples were harvested and analyzed by micro-computed tomography and histology. Improved proliferation of rat mesenchymal stem cells was observed with VEGF loaded MC. For osteogenic differentiation, dual growth factors delivered by MC showed higher osteogenic gene expression, alkaline phosphatse production and calcium deposition. The in vivo results revealed statistically significant increase in new bone formation when dual growth factors were delivered by MC. Dual growth factors administered on a calcium phosphate matrix showed significantly enhanced osteogenic potential. We propose this system has potential clinical utility in providing solutions for craniofacial bone defects, with the added benefit of early availability.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Calcium phosphate dibasic, 98.0-105.0%
Sigma-Aldrich
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate, ≥95%
Sigma-Aldrich
Phosphatase substrate, powder
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β-Glycerophosphate disodium salt hydrate, BioUltra, suitable for cell culture, suitable for plant cell culture, ≥99% (titration)
Sigma-Aldrich
Dexamethasone, powder, BioReagent, suitable for cell culture, ≥97%
Sigma-Aldrich
4-Nitrophenol solution, 10 mM
Sigma-Aldrich
Calcium carbonate, BioXtra, ≥99.0%
Sigma-Aldrich
Alizarin Red S, certified by the Biological Stain Commission