Delivery of S1P receptor-targeted drugs via biodegradable polymer scaffolds enhances bone regeneration in a critical size cranial defect

Abstract

Biodegradable polymer scaffolds can be used to deliver soluble factors to enhance osseous remodeling in bone defects. To this end, we designed a poly(lactic-co-glycolic acid) (PLAGA) microsphere scaffold to sustain the release of FTY720, a selective agonist for sphingosine 1-phosphate (S1P) receptors. The microsphere scaffolds were created from fast degrading 50:50 PLAGA and/or from slow-degrading 85:15 PLAGA. Temporal and spatial regulation of bone remodeling depended on the use of appropriate scaffolds for drug delivery. The release profiles from the scaffolds were used to design an optimal delivery system to treat critical size cranial defects in a rodent model. The ability of local FTY720 delivery to maximize bone regeneration was evaluated with micro-computed tomography (microCT) and histology. Following 4 weeks of defect healing, FTY720 delivery from 85:15 PLAGA scaffolds resulted in a significant increase in bone volumes in the defect region compared to the controls. A 85:15 microsphere scaffolds maintain their structural integrity over a longer period of time, and cause an initial burst release of FTY720 due to surface localization of the drug. This encourages cellular in-growth and an increase in new bone formation.

Keywords: biodegradable polymer scaffolds; bone tissue engineering; drug delivery.

Figure 1

Release profiles of FTY720 from PLAGA microspheres of different compositions in 1:1 DMF:MeCl₂. (A) Cumulative FTY720 release from 85:15 (○), and 50:50 (■) microsphere scaffolds dissolved in 1:1 DMF:MeCl₂. (B) Mass loss of 85:15 (○), and 50:50 (■) microsphere scaffolds dissolved in 1:1 DMF:MeCl₂ at each time point. (C) Cumulative ratio of fraction of FTY720 released to the fraction mass loss of microspheres made of different PLAGA composition. SEM images of FTY720 loaded 50:50 microsphere scaffolds at (D) day 0 and (E) week 3. Confocal imaging of (F) 85:15 microspheres loaded with labeled FTY720 confirms surface localization of the drug. No signal is seen in (G) unloaded microspheres. A second method of visualizing FTY720 was implemented by using fluorescently tagged drug. It also shows (H) more uniform distribution in 50:50 microspheres and (I) drug accumulation at the surface in 85:15 microspheres. No signal is seen in (J) unloaded microspheres. (K) The fluorescent intensity at different regions normalized to the average intensity of the microsphere confirms this observation.

Figure 2

Release profiles of FTY720 from PLAGA microspheres of different compositions. (A) Mathematical prediction of cumulative release of FTY720 from 85:15 (-) and 50:50 (---) microsphere scaffolds up to 15 days based on experimental values obtained for the first 7 days (B) Cumulative in vitro FTY720 release from 85:15 (●), and 50:50 (■) microsphere scaffolds over a period of 15 days.

Figure 3

¹H NOESY NMR scan of a 1:200 mixture of FTY720 and PLAGA at combined concentration of 0.7 M in d-chloroform. Inset: Focuses on the downfield region at 6.0 – 9.0 ppm. While there is a single amide proton in FTY720, there is none in PLAGA and the peak at 8 ppm and the weak cross peak at 8.3 ppm may be attributed to intermolecular relationships.

Figure 4

85:15 microsphere scaffolds develop a more stable scaffold that results in enhanced bone formation over longer time periods. (A) MicroCT imaging shows that the amount of bone formed is higher in the half of the defect region that is treated with 85:15 FTY720 loaded microsphere scaffold compared to the 50:50 loaded scaffolds. Yellow outline shows the image of the original defect region at day 0, the blue outline shows the image at week 6 with the new bone formation (white arrows). (B) Change in bone volume between weeks 0–2, 0–4, 0–6, and 0–9 for the empty defect ( ), the 50:50 side of the 50:50–85:15 scaffold ( ), and the 85:15 side of the 50:50–85:15 scaffold ( ). *Statistical significance, where p<0.05. This result is reflected in the histology. (C) Untreated empty defects have minimal bone formation. (D) The side treated with 50:50 microsphere scaffold swells and loses pore volume and has less bone formation (red arrows) compared to the side treated with (E) 85:15 microsphere scaffold. All histological analysis was done 9 weeks after treatment.

Figure 5

FTY720 loading increases the amount of new bone formation in the defect region. (A) Microct imaging shows that the amount of bone formed is higher in the half of the defect region that is treated with a FTY720 loaded microsphere scaffold. Yellow outline shows the image of the original defect region at day 0, the blue outline shows the image at week 6 after the new bone formation at the periphery (white arrows) and away from the periphery (while arrowheads). (B) Change in bone volume between weeks 0–2, 0–4, and 0–6 for the empty defect ( ), the FTY720-loaded side of the 85:15 (L)( ) scaffold, and the unloaded side of the 85:15 (U) scaffold ( ). *Statistical significance, where p<0.05. This result is reflected in the histology. (C) The loaded side of the scaffold (left) shows more bone formation (red arrows) compared to the (D) unloaded side (right). All histological analysis was done 6 weeks after treatment.