The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres

Abstract

It is advantageous to incorporate controlled growth factor delivery into tissue engineering strategies. The objective of this study was to develop a three-dimensional (3D) porous tissue engineering scaffold with the capability of controlled releasing recombinant human bone morphogenetic protein-7 (rhBMP-7) for enhancement of bone regeneration. RhBMP-7 was first encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanospheres (NS) with an average diameter of 300nm. Poly(l-lactic acid) (PLLA) scaffolds with interconnected macroporous and nano-fibrous architectures were prepared using a combined sugar sphere template leaching and phase separation technique. A post-seeding technique was then utilized to immobilize rhBMP-7 containing PLGA nanospheres onto prefabricated nano-fibrous PLLA scaffolds with well-maintained 3D structures. In vitro release kinetics indicated that nanosphere immobilized scaffold (NS-scaffold) could release rhBMP-7 in a temporally controlled manner, depending on the chemical and degradation properties of the NS which were immobilized onto the scaffold. In vivo, rhBMP-7 delivered from NS-scaffolds induced significant ectopic bone formation throughout the scaffold while passive adsorption of rhBMP-7 into the scaffold resulted in failure of bone induction due to either the loss of rhBMP-7 biological function or insufficient duration within the scaffold. We conclude that the interconnected macroporous architecture and the sustained, prolonged delivery of bioactive rhBMP-7 from NS immobilized nano-fibrous scaffolds actively induced new bone formation throughout the scaffold. The approach offers a new delivery method of BMPs and a novel scaffold design for bone regeneration.

Figure 1

Characterization of PLGA50–64K nanospheres (NS) and nanosphere incorporated PLLA nano-fibrous scaffolds (NS-scaffolds). (A) Scanning electron micrograph of rhBMP-7 containing PLGA50–64K nanospheres; (B) Macroscopic photographs of PLLA scaffolds before (left) and after (right) nanosphere incorporation; (C, D) Scanning electron micrographs of PLLA nano-fibrous scaffolds before nanosphere incorporation at 100x (C) and 10,000x (D); (E, F) Scanning electron micrographs of PLLA nano-fibrous scaffolds after PLGA50–64K nanosphere incorporation at 100x (E) and 10,000x (F).

Figure 2

In vitro release kinetics of rhBMP-7 from nanospheres immobilized on nano-fibrous scaffolds: In 10 mM PBS with a BMP-7 loading of 200 ng/scaffold. Each data point represents a mean ± standard deviation (n=3).

Figure 3

Radiographic results of retrieved scaffold samples: (A,B) 3 weeks and (C,D) 6 weeks after implantation. In Figures A & C, I: control scaffolds; II: scaffolds with 5 μg adsorbed rhBMP-7; and III: scaffolds with PLGA50–64K nanospheres containing 5 μg rhBMP-7. In Figures B & D, Each data point represents a mean ± standard deviation (n=3). *, p<0.05; **, p<0.01.

Figure 4

Microscopic observations of the H & E stained tissue sections of scaffolds retrieved 3 weeks after implantation. (A, B) Control scaffold; (C, D) 5 μg rhBMP-7 adsorbed to scaffold; (E, F) 5 μg rhBMP-7 incorporated in NS-scaffold. Original magnifications: (A, C, E) 40x for full cross sections, and (B, D, F) 200x for high magnification views of selected representative areas (arrows point to the selected areas in A, C, and E).

Figure 5

Microscopic observations of the H & E stained tissue sections of scaffolds retrieved 6 weeks after implantation. (A, B) Control scaffold; (C, D) 5 μg rhBMP-7 adsorbed to scaffold; (E, F) 5 μg rhBMP-7 incorporated in NS-scaffold. Original magnifications: (A, C, E) 40x full cross sections, and (B, D, F) 200x for high magnification views of selected representative areas (arrows point to the selected areas in A, C, and E). Note: In order to conduct von Kossa staining (Figure 6), the engineered tissue samples were not decalcified and the sectioning resulted in some artifacts, which appeared in the H&E histology (multiple cracks in the highly mineralized constructs, i.e., E & F).

Figure 6

Microscopic observations of the von Kossa stained tissue sections of scaffolds retrieved 6 weeks after implantation. (A, B) Control scaffold; (C, D) 5 μg rhBMP-7 adsorbed to scaffold; (E, F) 5 μg rhBMP-7 incorporated in NS-scaffold. Original magnifications: (A, C, E) 40x and (B, D, F) 100x (arrows point to the selected areas in A, C, and E).