Sustained release of sphingosine 1-phosphate for therapeutic arteriogenesis and bone tissue engineering

Abstract

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that impacts migration, proliferation, and survival in diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. In this study, we investigated the effects of sustained release of S1P on microvascular remodeling and associated bone defect healing in vivo. The murine dorsal skinfold window chamber model was used to evaluate the structural remodeling response of the microvasculature. Our results demonstrated that 1:400 (w/w) loading and subsequent sustained release of S1P from poly(lactic-co-glycolic acid) (PLAGA) significantly enhanced lumenal diameter expansion of arterioles and venules after 3 and 7 days. Incorporation of 5-bromo-2-deoxyuridine (BrdU) at day 7 revealed significant increases in mural cell proliferation in response to S1P delivery. Additionally, three-dimensional (3D) scaffolds loaded with S1P (1:400) were implanted into critical-size rat calvarial defects, and healing of bony defects was assessed by radiograph X-ray, microcomputed tomography (muCT), and histology. Sustained release of S1P significantly increased the formation of new bone after 2 and 6 weeks of healing and histological results suggest increased numbers of blood vessels in the defect site. Taken together, these experiments support the use of S1P delivery for promoting microvessel diameter expansion and improving the healing outcomes of tissue-engineered therapies.

Figure 1

Mathematical diffusion model of S1P release from PLAGA film. With an initial loading of 1:400 (S1P:PLAGA), the concentration profile is displayed. S1P concentration at a distance of 1mm from the film is approximately 461nM (day 3) and 264nM (day 7). At a distance of 2mm, the maximum distance used in quantitative analysis, the [S1P] is about 71nM (day 3) and 53nM (day 7).

Figure 2

Intravital microscopy images of control PLAGA films (top) or S1P loaded films (bottom) in the dorsal skinfold window chamber at 0, 3, and 7 days post-implantation. Substantial lumenal expansion of both arterioles (black) and venules (white) is induced by S1P over the course of 7 days (arrows). Changes in arteriole diameter in response to unloaded PLAGA control are negligible (arrowheads). Scale bar = 500 μm.

Figure 3

Changes in microvascular length density following 3 (A) and 7 (B) days post-implantation of either 1mm PLAGA control films or PLAGA films loaded with S1P. Release of S1P enhanced vascular length density (a measure of total vessel length), although not significant.

Figure 4

Changes in arteriolar diameter following 3 (A) and 7 (B) days post-implantation of either 1mm PLAGA control films or PLAGA films loaded with S1P. S1P significantly stimulated lumenal expansion of arterioles in the first three days of treatment (*p<0.05). Arterioles were grouped by the initial diameter measurement at day 0 (<50 μm, 50–100 μm, and >100 μm) and changes in microvessel diameter were tracked after 3 (C) and 7 (D) days.

Figure 5

Changes in venular diameter following 3 (A) or 7 (B) days post-implantation of either 1mm PLAGA control films or PLAGA films loaded with S1P. Venules were grouped by the initial diameter measurement at day 0 (<50 μm, 50–100 μm, and >100 μm) and changes in diameter were assessed after 3 (C) and 7 (D) days. S1P stimulated the lumenal expansion of venules <50 μm in the first 3 days of treatment. A reduction in lumenal venular diameter occurred in vessels >100 μm in the first three days of treatment with S1P and was maintained over 7 days. (*p<0.05)

Figure 6

Assessment of mural cell proliferation on expanding microvessels. A) Ratio of BrdU-positive nuclei per total nuclei stained with smooth muscle α-actin. S1P significantly enhances the number of proliferating cells found on microvessels, suggestive of an arteriogenic effect. B) Number of microvessels with smooth muscle α-actin-positive cells per tissue section. S1P did not significantly enhance the number of microvessels per area, consistent with length density analysis. C) Representative confocal microscopy images of smooth muscle α-actin-postive mural cells (red), cell nuclei (blue), or proliferating, BrdU-postive cell nuclei (green). S1P enhances the number of proliferating cells per vessel, demonstrated by colocalization of blue and green labeled nuclei (arrows). Scale bar = 25μm.

Figure 7

A) Following 2 and 6 weeks of cranial bone healing, the defect and surrounding native bone were excised from the cranial defect site. X-ray imaging analysis was performed on each ex vivo sample. Representative images from each experimental group are shown (A). Scale bar = 4mm. New bone volume formed within defect area following 2 weeks (B) and 6 weeks (C) of healing. Values calculated from high resolution 3D reconstructed images acquired using ex vivo microCT scans of each sample. (*p<0.05)

Figure 8

H&E staining of cranial defect histological sections after 6 weeks. Substantial bone healing and increased number of vessels observed in S1P-loaded scaffold groups (PLAGA + S1P), compared to PLAGA controls. Scale bar = 100μm.