Understanding cell-material interactions is pivotal for the success of tissue engineering. In this study, we explore how osteoblasts respond to 3D nanofibrous gelatin scaffolds (3D-NF-GS). We use high resolution confocal microscopy to visualize, analyze, and quantify cell-matrix interactions and tissue formation.
Primary osteoblasts were seeded onto 3D-NF-GS and cultured for 5 days and 14 days. They were immunolabeled with specific antibodies: Integrin b1, vinculin, tyrosine phosphorylated paxillin and collagen type I and visualized with a Leica Confocal SP5 microscope. Alizarin red staining was used for mineral detection.
Primary osteoblasts cultured on 3D-NF-GS exhibited uniform migration after 5 days with the cells getting increasingly confluent by the 14th day of culture. The cells started adhering to 3D-NF-GS via focal adhesions by 5 days depicted by vinculin and phosphorylated paxillin. Significantly low integrin b1 expression was present in the form of fibrillar adhesions overlapping actin stress fibers. The integrin b1 and phosphorylated paxillin adhesions increased in number at 14 days. However, vinculin adhesions did not increase but had a phenotypic shift from classical focal adhesions to fibrillar adhesions. ECM production depicted by collagen type I secretion was detected on the 3D-NF-GS at 14 days. The osteoblasts started making nodular aggregations by 14 days and abundant mineralization of 3D-NF-GS was demonstrated.
Conclusion: Osteoblasts adhered to the 3D-NF-GS showing an overall increase in focal adhesions from 5 to 14 days. They migrated and proliferated on 3D-NF-GS leading to secretion of the primary osteoid protein, type I collagen. Mineral deposition was demonstrated on 3D-NF-GS by 14 days indicating that 3D-NF-GS could serve as an optimum material for bone tissue engineering.
Keywords: Adhesion, Confocal Imaging, Osteoblasts/osteoclasts, Regeneration and Tissue engineering
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