Customized Ca-P/PHBV nanocomposite scaffolds for bone tissue engineering: design, fabrication, surface modification and sustained release of growth factor

Abstract

Integrating an advanced manufacturing technique, nanocomposite material and controlled delivery of growth factor to form multifunctional tissue engineering scaffolds was investigated in this study. Based on calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) nanocomposite microspheres, three-dimensional Ca-P/PHBV nanocomposite scaffolds with customized architecture, controlled porosity and totally interconnected porous structure were successfully fabricated using selective laser sintering (SLS), one of the rapid prototyping technologies. The cytocompatibility of sintered Ca-P/PHBV nanocomposite scaffolds, as well as PHBV polymer scaffolds, was studied. For surface modification of nanocomposite scaffolds, gelatin was firstly physically entrapped onto the scaffold surface and heparin was subsequently immobilized on entrapped gelatin. The surface-modification improved the wettability of scaffolds and provided specific binding site between conjugated heparin and the growth factor recombinant human bone morphogenetic protein-2 (rhBMP-2). The surface-modified Ca-P/PHBV nanocomposite scaffolds loaded with rhBMP-2 significantly enhanced the alkaline phosphatase activity and osteogenic differentiation markers in gene expression of C3H10T1/2 mesenchymal stem cells. Together with osteoconductive nanocomposite material and controlled growth factor delivery strategies, the use of SLS technique to form complex scaffolds will provide a promising route towards individualized bone tissue regeneration.

Figure 1.

Schematic illustration of the processing for Ca–P/PHBV nanocomposite scaffold fabrication. Left panel: (a) synthesis of Ca–P nanoparticles based on Ca and P sources and at pH 11, (b) Ca–P nanoparticles observed under SEM, (c) TEM image and SAD pattern of Ca–P nanoparticles. Middle panel: (d) fabrication of Ca–P/PHBV microspheres using S/O/W emulsion–solvent evaporation method, (e) Ca–P/PHBV nanocomposite microspheres, (f) high magnification view of the Ca–P nanocomposite microsphere surface. Right panel: (g) schematic illustration for the principle of SLS, (h) a Sinterstation illustration 2000 SLS system, (i) the miniature sintering platform for modified Sinterstation 2000 SLS system.

Figure 2.

(a) Complex models designed and shared by Hart (2009): (i) salamanders, (ii) elevated icosidodecahedron, (iii) snarl; (b) sintered Ca–P/PHBV nanocomposite porous structures based on the Hart models; (c) three-dimensional model of a human proximal femoral condyle reconstructed from CT images and then processed into porous scaffold using cubic cells; (d) sintered Ca–P/PHBV nanocomposite proximal femoral condyle scaffold. (The real size of the sintered proximal femoral condyle scaffold was 40% of the design model.) Scale bar, 1 cm.

Figure 3.

Sintered PHBV polymer scaffolds and Ca–P/PHBV nanocomposite scaffolds for cytocompatibility tests. (a) Schematic of the computer-aided design model in trimetric view; (b) sintered scaffolds: (i) PHBV polymer scaffold, and (ii) Ca–P/PHBV nanocomposite scaffold; (c) micro-CT image of Ca–P/PHBV nanocomposite scaffold; (d) SEM image of one layer of sintered Ca–P/PHBV nanocomposite scaffold; (e) morphology of SaOS-2 cells cultured for 7 days on Ca–P/PHBV nanocomposite scaffold; (f) SaOS-2 cell proliferation determined by MTT assay (*p < 0.05); (g) ALP activity of SaOS-2 cells (**p < 0.01); (f,g) bar with dots, PHBV; bar with lines, Ca–P/PHBV; bar with crosses, control.

Figure 4.

(a) Ca–P/PHBV scaffolds for surface modification: (i) design model and (ii) sintered Ca–P/PHBV nanocomposite scaffold; (b) SEM image of sintered Ca–P/PHBV nanocomposite scaffold (top view); (c) illustration of physical entrapment of gelatin and immobilization of heparin on the surface of sintered Ca–P/PHBV nanocomposite scaffold and the binding strategy of rhBMP-2; (d) contact angle images for PHBV film (left) and surface modified PHBV film (right); (e) images of Ca–P/PHBV nanocomposite scaffolds after toludine blue staining: (i) scaffold with immobilized heparin in the as-produced state, (ii) scaffold with immobilized heparin after 14-day immersion in PBS, and (iii) scaffold control sample without immobilized heparin.

Figure 5.

(a,b) Osteogenic differentiation of C3H10T1/2 cells in osteogenic medium with 1000 ng ml⁻¹ rhBMP-2 for 21 days, evidenced by (a) positive ALP staining and (b) calcium deposition stained by Alizarin Red S; (c) morphology of C3H10T1/2 cells cultured on surface modified Ca–P/PHBV nanocomposite scaffold for 21 days in osteogenic medium; (d) high magnification view showing the interaction between C3H10T1/2 cells and Ca–P/PHBV microspheres in the strut of nanocomposite scaffold; (e) ALP activity of C3H10T1/2 cells cultured on different scaffolds in osteogenic medium for 21 days. White bars, Ca–P/PHBV nanocomposite scaffolds; grey bars, Ca–P/PHBV nanocomposite scaffolds with simply adsorbed rhBMP-2; black bars, surface modified Ca–P/PHBV nanocomposite scaffolds loaded with rhBMP-2 (*p < 0.05; **p < 0.01); (f) gene expression of different osteogenic markers after 21-day cell culture for different scaffolds: (i) Ca–P/PHBV nanocomposite scaffolds; (ii) Ca–P/PHBV nanocomposite scaffolds with simply adsorbed rhBMP-2; (iii) surface-modified Ca–P/PHBV nanocomposite scaffolds loaded with rhBMP-2. (Collagen IA1 (Col1A1); alkaline phosphatase (ALP); osteocalcin (OCN); glyceraldehyde-3-phosphatedehydrogenase (GAPDH).)