Introduction: In the field of bone tissue engineering, the recent focus has been on composites combining polymeric matrices with inorganic components[1],[2]. Composite grafts are often seeded with growth factors and signaling molecules to augment osteogenic differentiation[3]. However, elevated and/or prolonged dosage of inductive molecules can have undesirable cytotoxic and inflammatory effects[4]-[6].
Nanofibrous scaffolds have potential for use in tissue engineering applications due to their structural similarity to natural extracellular matrix (ECM)[7].Studies have shown that cells respond acutely to physical stimuli in the nanoscale range[8],[9]. In addition, electrospun nanofibers have high surface area-to-volume ratios[10], allowing for maximal cellular attachment and proliferation.
The aim of this study was to design a synthetic, bioresorbable scaffold that promotes osteogenic differentiation in vivo, without the use of supplemental proteins and growth factors. It was hypothesized that in vivo efficacy may be enhanced through the incorporation of stem cells.
Materials and Methods: Poly(ϵ-caprolactone) (PCL)/poly(ethylene glycol) diacrylate (PEGDA) nanofibers were fabricated by electrospinning. Scaffolds were biomineralized in CaCl2 and Na2HPO4. Multipotent adult progenitor cell (MAPC®) attachment and viability were evaluated using calcein and ethidium bromide. Biomineralized scaffolds were seeded with MAPCs and cultured in basal medium or osteogenic inductive medium for 4 weeks to evaluate in vitro osteogenic differentiation. Early differentiation was assessed by alkaline phosphatase (ALP) expression through day 7. Osteonectin expression was measured at 3 and 4 weeks via an enzyme-linked immunosorbent assay (ELISA). To substantiate the in vitro data, MAPC-seeded scaffolds were implanted subcutaneously in Sprague-Dawley rats. Scaffold immunogenicity and osteoinductivity were evaluated.
Results: On both biomineralized (BM) and non-biomineralized (NBM) scaffolds, MAPC attachment occurred within 2 hours, and viability was 99% after 7 days. In vitro studies showed an increase in early bone marker ALP during the first 7 days of MAPC culture on BM scaffolds, with and without inductive medium. Osteocalcin expression levels, analyzed via ELISA, indicated enhanced osteogenic differentiation on the BM scaffolds. BM scaffolds increased MAPC expression of osteogenic markers without the use of inductive medium. Cells seeded on NBM scaffolds had lower expression of osteogenic markers compared to BM scaffolds.
Discussion: In vitro results confirmed that non-biomineralized scaffolds support cellular growth, while the process of biomineralization introduces osteoinductivity. The ability to induce osteogenic differentiation in vivo without growth factors mitigates many of the risks associated with delivery of growth factors and signaling proteins. In addition, the presented scaffold is low-cost and scalable. The nanofiber parameters are easily tuned, and versatility of the scaffold makes it suitable for use in a wide range of applications.
Conclusion: The biomineralized nanofibrous scaffold presents a customizable, low-cost approach to synthetic osteoinductive bone matrix fabrication. The scaffold has potential application for a variety of uses in bone tissue engineering, resulting from its ability to promote osteogenic differentiation without embedded growth factors and signaling molecules.
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