Delivery of PDGF-B and BMP-7 by mesoporous bioglass/silk fibrin scaffolds for the repair of osteoporotic defects

Abstract

Osteoporosis is a chronic disease affecting millions of people worldwide caused by an imbalance between bone-forming osteoblasts and bone-resorbing osteoclasts. Despite recent developments in pharmacological agents to prevent osteoporotic-related fractures, much less attention has been placed on the repair of bone defects following fracture. Critical to this process is the recruitment of mesenchymal stem cells (MSCs) to defect sites by growth factors. One method which has been effective for the sustained release of growth factors is that of gene therapy. The aim of the present study was to investigate newly developed mesoporous bioglass/silk fibrin scaffolds containing adPDGF-b and adBMP-7 into osteoporotic critical-sized femur defects in ovariectomised rats following treatment periods of 2 and 4 weeks. In vivo osteogenetic efficiency evaluated by μ-CT analysis, hematoxylin and eosin staining, and immunohistochemical (type I collagen, osteopontin and BSP) revealed significantly new bone formation in defects containing adenovirus for both PDGF-b and BMP-7 when compared to scaffolds alone and scaffolds containing BMP-7. TRAP-positive staining also demonstrated the ability for these scaffolds to be degraded over time and initiate bone turnover/remodeling. Although the use of gene therapy for clinical applications is still in its infancy, results from the present study demonstrate their potent ability to recruit mesenchymal progenitor cells through sustained release of PDGF-b and BMP-7 which may be beneficial for patients suffering from osteoporotic-related fractures.

Fig. 1

SEM micrographs of scaffolds (A), lower magnification of pure MBG/silk scaffolds (B), higher magnification of pure MBG/silk scaffolds (C), lower magnification of pure MBG/silk + BMP-7 scaffolds (D) higher magnification of pure MBG/silk + BMP-7 scaffolds.

Fig. 2

Establishment of rat osteoporotic model in femur head. 2D representation of normal bone (A) and osteoporotic bone (H), 3D visualization for subchondral trabecular regions of the normal bone (B) in (A) and the osteoporotic bone (I) in (H). 3D μ-CT images of normal bone (C, D) and osteoporotic bone (J, K) from axial section and cross section (bar = 1 mm). Representative H&E staining for the normal (E, F, G) and osteoporotic (L, M, N) femur in magnification of 2×, 10× and 40×, respectively.

Fig. 3

3D μ-CT images of mineralized bone formation in distal femur defects left alone (A, E) and filled with MBG/silk scaffold (B, F), MBG/silk + BMP-7 scaffold (C, G) and MBG/silk + BMP-7 + PDGF-b scaffold (D, H) after 2 and 4 weeks (bar = 1 mm). Representative H&E staining demonstrating new bone matrix deposition within defects left alone (a, e) and implanted with MBG/silk scaffold (b, f), MBG/silk + BMP-7 scaffold (c, g) and MBG/silk + BMP-7 + PDGF-b scaffold (d, h), respectively (bar = 200 μm).

Fig. 4

The 3D microarchitectural parameters analyzed by μ-CT. The regenerative potential of bone healing were compared following local implantation of MBG/silk scaffold alone or in combination with BMP-7 or BMP-7 + PDGF-b in femur defects. Data are presented as mean ± SD and analyzed by one-way ANOVA and SNK test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

Semiquantitative scores of bone regeneration within femur defects are present as box plots. The box represents the 25 and 75 percentiles and the horizontal line in the box represents median value. Lines outside the box represent the spread of values.

Fig. 6

Representative Saffarin O staining of subchondral bone formation within defects 2 and 4 weeks post-surgery at low (×10; bar = 200 μm) and high (×40; bar = 50 μm) magnification. Fat-rich bone marrow-like tissue gradually filled the defect in the drill control group (A, a, E, e). The degradation of remaining scaffolds (black arrow head) were accompanied with increased bony ingrowth (black star). A large quantity of stranded connective tissue surrounded by osteoblastic cells can be observed in MBG/silk scaffold group (B, b, F, f), while more mature bone matrix was detected in MBG/silk + BMP-7 scaffold group (C, c, G, g). Plate-like trabeculae with osteocytes deposition was observed in MBG/silk + BMP-7 + PDGF-b scaffold group (D, d, H, h). Dotted lines represent defect margin.

Fig. 7

Quantitative comparison of scaffold remnant fraction in MBG/silk scaffold, MBG/silk + BMP-7 scaffold and MBG/silk + BMP-7 + PDGF-b scaffold groups at two time points (Scaffold remnant = area of silk scaffolds/total area). Data are presented as mean ± SD and analyzed by one-way ANOVA and SNK test. *P < 0.05, **P < 0.01.

Fig. 8

Representative immunohistological detection of COL I, OPN and BSP in distal femur defects left alone and implanted with MBG/silk scaffold, MBG/silk + BMP-7 scaffold and MBG/silk + BMP-7 + PDGF-b scaffold. MBG/silk + BMP-7 + PDGF-b scaffold stimulates the positively strongest expression with the largest area of COL I, OPN and BSP within defects both at 2 and 4 weeks (bar = 200 μm). Dotted lines represent defect margin.

Fig. 9

Representative TRAP staining of osteoclasts (white arrow) in shuttle shape were lining around the osteoporotic trabecular surface in the drill control group at 2 weeks (A), abundant mononuclear macrophages gradually fused and differentiated into mature osteoclasts by 4 weeks (E); more osteoclasts co-existed with osteogenic cells was found especially surrounding remaining scaffolds after 4 weeks (F,G,H) compared with the corresponding scaffold groups at 2-week time point (B,C,D). bar = 50 μm.