Endothelial cell and osteoblast co-cultures on biphasic composite scaffolds for bone tissue engineering

Date
2010
Authors
Shah, Amita
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Abstract

Large segmental bone defects pose a difficult challenge in bone tissue engineering since both bone formation and vessel formation are required in order to successfully regenerate the tissue. The objectives of this research were to develop a biphasic composite scaffold for bone tissue engineering and to examine endothelial and osteoblast interactions in co-culture, culminating in an in vivo model which utilized both the biphasic composite scaffold and endothelial/osteoblast co-cultures.

The biphasic composite scaffold was designed for regeneration of segmental long bone defects. It consists of a hydroxyapatite (HA) ring with an oxygen gas plasma treated polylactic (PLA) scaffold core. The oxygen gas plasma treatment was found to improve endothelial cell attachment, viability, and proliferation on the PLA scaffolds. Microarray analysis showed that the mechanism behind the improvements induced by the treatment were through enhancements of cell signaling and the cell cycle. Studies of the scaffold system with cells confirmed that that it allows for cell attachment and proliferation, as well as migration between the hydroxyapatite and polylactic acid phases of the scaffold.

Endothelial cell and osteoblast co-cultures were investigated in different ratios to determine which endothelial cell to osteoblast co-culture ratios resulted in increased angiogenesis and mineralization. Angiogenesis was enhanced in co-culture ratios of 5:1 and 1:1 endothelial cells to osteoblasts, while mineralization was present only in co-culture ratios of 1:5 and 1:10. It was found that contact between the cells is important for mineralization to occur. The increase in alkaline phosphatase and vascular endothelial growth factor secretion in the co-cultures with contact between the cell types is not seen in cultures without contact between the cells. It was also found that there is increased osteoblast migration and decreased endothelial cell migration into and out of the central areas of the scaffold when osteoblasts and endothelial cells are co-cultured.

A preliminary in vivo study was done with PLA scaffolds in a rat calvarial defect. The scaffolds were seeded with adipose-derived stem cells differentiated into endothelial cells or osteoblasts. The scaffolds seeded with the endothelial cells had improved vascularization, but significantly decreased bone formation. The scaffolds seeded with the osteoblasts had bone formation and evidence of vascularization. From these results, a rat calvarial defect model was developed which utilized the biphasic composite scaffold with co-cultures in a 1:1 ratio or a mixed co-culture system where the HA phase was seeded with the 1:5 co-culture ratio and the PLA phase was seeded with the 5:1 ratio. At 12 weeks there was significant migration and damage of the HA phase of the scaffold so analysis of bone growth was compromised. The rat calvarial defect is not an appropriate model for evaluating the biphasic composite scaffold and in the future, a segmental defect model should be used.

The results of this research gives further insight into endothelial cell and osteoblast interactions and demonstrates that co-cultures can be designed such that they improve vascularization and mineralization for bone tissue engineering in vitro. The biphasic composite scaffold is a viable option for segmental bone defect regeneration and further studies incorporating these two technologies should be pursued.

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Keywords
Bone Regeneration, Co-Culture, Hydroxyapatite, Osteoblast, Polylactic Acid, Tissue Engineering
Citation
Department
Biomedical Engineering