Combining technologies to create bioactive hybrid scaffolds for bone tissue engineering

Abstract

Combining technologies to engineer scaffolds that can offer physical and chemical cues to cells is an attractive approach in tissue engineering and regenerative medicine. In this study, we have fabricated polymer-ceramic hybrid scaffolds for bone regeneration by combining rapid prototyping (RP), electrospinning (ESP) and a biomimetic coating method in order to provide mechanical support and a physico-chemical environment mimicking both the organic and inorganic phases of bone extracellular matrix (ECM). Poly(ethylene oxide terephthalate)-poly(buthylene terephthalate) (PEOT/PBT) block copolymer was used to produce three dimensional scaffolds by combining 3D fiber (3DF) deposition, and ESP, and these constructs were then coated with a Ca-P layer in a simulated physiological solution. Scaffold morphology and composition were studied using scanning electron microscopy (SEM) coupled to energy dispersive X-ray analyzer (EDX) and Fourier Tranform Infrared Spectroscopy (FTIR). Bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on coated and uncoated 3DF and 3DF + ESP scaffolds for up to 21 d in basic and mineralization medium and cell attachment, proliferation, and expression of genes related to osteogenesis were assessed. Cells attached, proliferated and secreted ECM on all the scaffolds. There were no significant differences in metabolic activity among the different groups on days 7 and 21. Coated 3DF scaffolds showed a significantly higher DNA amount in basic medium at 21 d compared with the coated 3DF + ESP scaffolds, whereas in mineralization medium, the presence of coating in 3DF+ESP scaffolds led to a significant decrease in the amount of DNA. An effect of combining different scaffolding technologies and material types on expression of a number of osteogenic markers (cbfa1, BMP-2, OP, OC and ON) was observed, suggesting the potential use of this approach in bone tissue engineering.

Keywords: biomimetic coating; bone tissue engineering; calcium-phosphate; electrospinning; polymer; rapid prototyping.

Figure 1. A schematic illustration of the different technologies involved in fabricating the hybrid scaffolds used in this study. (A) 3-D fiber deposition (3DF) enables a controlled layer by layer deposition of extruded polymer, (B) Electrospinning to produce extra-cellular matrix like fibers and (C) Biomimetic calcium phosphate coating to enhance osteoconductivity of the scaffolds

Figure 2. Scaffold morphology using SEM (A) 3DF scaffold prepared by rapid prototyping. (B) 3DF + ESP scaffold prepared by combining rapid prototyping and electrospinning. The scaffold has been “opened” to enable visualization of the electrospun fiber meshes. Scale bar = 500 μm

Figure 3. Calcium-phosphate coated rapid prototyped scaffolds. Morphology and characterization. (A) SEM image of a 3DF scaffold coated with calcium-phosphate (scale bar = 200 μm). (B) EDX spectrum of the scaffold showing Ca and P peaks, (C and D) EDX elemental mapping of calcium and phosphorus respectively, (E) Electrospun fibers from a 3DF + ESP scaffold that have been coated with calcium-phosphate (scale bar = 100 μm). (F) High magnification SEM image showing the morphology of crystals formed during coating (scale bar = 10 μm), (G) FT-IR spectrum of calcium-phosphate coating.

Figure 4. Metabolic activity of cells seeded on different scaffolds in basic and mineralization medium on days 7 and 21 measured using Alamar Blue assay. Data are represented as mean ± standard deviation

Figure 5. Amount of DNA after 21 d on different scaffolds in basic and mineralization medium as measured using CyQuant assay. Data are represented as mean ± standard deviation. *Statistically significant differences (p < 0.05)

Figure 6. Cell morphology on scaffolds after 21 d. (A−D) represent 3DF (uncoated and coated) and 3DF + ESP (uncoated and coated scaffolds) in basic medium. Inset in C shows hMSCs on the electrospun layer (indicated by white arrow). (E) Higher magnification image of hMSCs attaching calcium-phosphate coatings. White arrow indicates coating. (F) Higher magnification image of hMSCs on electrospun fibers (white arrow). Scale bars A-D = 200 μm. Inset in C = 500 μm, E and F = 50 μm

Figure 7. qPCR analysis for osteogenic panel of genes after 7 d in culture. * represents p < 0.05. Data are represented as mean ± standard deviation

Figure 8. qPCR analysis for osteogenic panel of genes after 21 d in culture. * represents p < 0.05. Data are represented as mean ± standard deviation