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Abstract
Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations—1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.
Highlights
Bone tissue engineering remains a highly active area of research largely because of complications associated with challenging healing scenarios [1]
This study aimed to determine the effects of HAp nanoparticles in polymer-mineral composite nanofiber scaffolds for bone regeneration
In order to assess the effects of HAp nanoparticles, scaffolds were fabricated with 1% or 10% w/w HAp nanoparticles, and HAp-free scaffolds were used as a control treatment
Summary
Bone tissue engineering remains a highly active area of research largely because of complications associated with challenging healing scenarios [1]. In order to direct progenitor cell differentiation, biomimetic designs have aimed to create conditions and signals that would be found in actively remodeling bone tissue. Soluble signals such as growth factors or cytokines have been shown to enhance osteoblastogenesis [6,7,8,9] by activating cell surface receptors. A colorimetric alkaline phosphatase (ALP) assay, total protein assay, quantitative polymerase chain reaction (qPCR), and immunofluorescent staining were used to evaluate the cell responses to nanoarchitecture containing different concentrations of HAp. In order to quantify intracellular ALP production after one, two, and three weeks in osteogenic conditions, the cells were lysed in CellLytic® solution (Sigma). The same lysate was used to determine the total intracellular protein content using a commercially available
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