Adipose-derived stem cells and BMP-2 delivery in chitosan-based 3D constructs to enhance bone regeneration in a rat mandibular defect model

Abstract

Reconstructing segmental mandiblular defects remains a challenge in the clinic. Tissue engineering strategies provide an alternative option to resolve this problem. The objective of the present study was to determine the effects of adipose-derived stem cells (ASCs) and bone morphogenetic proteins-2 (BMP-2) in three-dimensional (3D) scaffolds on mandibular repair in a small animal model. Noggin expression levels in ASCs were downregulated by a lentiviral short hairpin RNA strategy to enhance ASC osteogenesis (ASCs(Nog-)). Chitosan (CH) and chondroitin sulfate (CS), natural polysaccharides, were fabricated into 3D porous scaffolds, which were further modified with apatite coatings for enhanced cellular responses and efficient delivery of BMP-2. The efficacy of 3D apatite-coated CH/CS scaffolds supplemented with ASCs(Nog-) and BMP-2 were evaluated in a rat critical-sized mandibular defect model. After 8 weeks postimplantation, the scaffolds treated with ASCs(Nog-) and BMP-2 significantly promoted rat mandibular regeneration as demonstrated by micro-computerized tomography, histology, and immunohistochemistry, compared with the groups treated with ASCs(Nog-) or BMP-2 alone. These results suggest that our combinatorial strategy of ASCs(Nog-)+BMP-2 in 3D apatite microenvironments can significantly promote mandibular regeneration, and these may provide a potential tissue engineering approach to repair large bony defects.

Figures

FIG. 1.

Scanning electron microscopy images of chitosan (CH)/chondroitin sulfate (CS) scaffolds before (a, c) and after (b, d) apatite coating: (a, b) low magnification. Scale bar=200 μm; (c, d) high magnification. Scale bar=10 μm.

FIG. 2.

(a) Live/dead fluorescent staining of noggin short hairpin RNA treated adipose-derived stem cells (ASCsNog−) seeded on noncoated (CH/CS) and apatite-coated (Ap-CH/CS) scaffolds after 1, 7, and 21 days in culture. Scale bar=500 μm. (b) AlamarBlue assay showing proliferation of ASCsNog− seeded on CH/CS and Ap-CH/CS scaffolds. Color images available online at www.liebertpub.com/tea

FIG. 3.

In vitro release of bone morphogenetic proteins-2 (BMP-2) from noncoated (CH/CS) and apatite-coated (Ap-CH/CS) scaffolds evaluated by ELISA assay. Color images available online at www.liebertpub.com/tea

FIG. 4.

Mandibular defect model. (a) The critical-sized defect (5×5 mm) was created in the left mandible of a rat. (b) Ap-CH/CS scaffold loaded with BMP-2, ASCsNog−, or BMP-2+ASCsNog− was placed into the defect. Photographs of sacrifice mandibular defects treated with Ap-CH/CS scaffold (c), Ap-CH/CS scaffold loaded with BMP-2 (d), ASCsNog−(e), or BMP-2+ASCsNog−(f ) at 8 weeks postsurgery. Color images available online at www.liebertpub.com/tea

FIG. 5.

Micro-computed tomography images of mandibular defects treated with Ap-CH/CS scaffold (a), Ap-CH/CS scaffold loaded with BMP-2 (b), ASCsNog−(c), or BMP-2+ASCsNog−(d) at 8 weeks postsurgery. (e) Quantification of bone volume/total volume (BV/TV) in mandibular defects.*p<0.05.

FIG. 6.

Hematoxylin-eosin staining of bone formation in mandibular defects treated with Ap-CH/CS scaffold (Blank), Ap-CH/CS scaffold loaded with BMP-2, ASCsNog−, or BMP-2+ASCsNog− at 8 weeks postsurgery. Dashed lines indicate approximate margin of defect. Areas within the black boxes are magnified below. Original magnifications: 40× (a); 200× (b); 400× (c). Color images available online at www.liebertpub.com/tea

FIG. 7.

Histological analysis of bone formation in mandibular defects treated with Ap-CH/CS scaffold (Blank), Ap-CH/CS scaffold loaded with BMP-2, ASCsNog−, or BMP-2+ASCsNog− at 8 weeks postsurgery by Masson's trichrome staining (a) and sirius red (SR) staining at bright filed (b) and polarized light (c). (d) Osteocalcin (OCN) immmuofluorescent staining of defect sites. Color images available online at www.liebertpub.com/tea

FIG. 8.

Relative expression of collagen (a) and OCN (b) determined by image analysis of sirius red staining and OCN immunofluorescent staining samples. *p<0.05. Color images available online at www.liebertpub.com/tea