Incorporation of a prolyl hydroxylase inhibitor into scaffolds: a strategy for stimulating vascularization

Abstract

Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity. To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy. PHIs are small molecule drugs that can stabilize the alpha subunit of hypoxia-inducible factor-1 (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms. In this study, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) through amide bonds to a gelatin sponge (Gelfoam(®)). Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor than cells grown on Gelfoam without PDCA. Reporter cells expressing green fluorescent protein-tagged HIF-1α exhibited dose-dependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs. Subsequently, we implanted PDCA-Gelfoam scaffolds into the perirenal fat tissue of Sprague Dawley rats for 8 days. Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner. Thus, the incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications.

FIG. 1.

Reaction scheme for the conjugation of pyridine-2,4-dicarboxylic acid (PDCA) to Gelfoam through amide bonds. 1,1′-Carbonyldiimidazole (CDI) was used to facilitate formation of the amide bonds. PDCA's carboxylic groups were first converted by CDI into acyl imidazole groups (activation). Imidazole and carbon dioxide were produced as by-products, with the imidazole remaining in solution and the carbon dioxide escaping as effervescence. When the activated PDCA was added to Gelfoam, the acyl imidazole groups reacted with the amine groups in Gelfoam to form amide bonds. Imidazole occurred again as a by-product, and was subsequently removed by repeated washing of scaffolds.

FIG. 2.

Ultraviolet absorbance spectra of PDCA-Gelfoam. (a) One millimolar PDCA, (b) untreated Gelfoam, and (c) PDCA-Gelfoam samples (0–15% w/w). Readings were taken between 230 and 330 nm at 5 nm intervals. PDCA's absorption maximum at 280 nm was evident in Gelfoam samples conjugated with PDCA, indicating that drug conjugation was successful. Drug loading measurements (expressed in % w/w) were calculated using absorbance values measured at 290 nm, where PDCA's absorbance relative to Gelfoam's was the highest. Color images available online at www.liebertpub.com/tea

FIG. 3.

Physical appearance of PDCA-Gelfoam and enzymatic drug release profiles. (a) Macroscopic appearance of Gelfoam samples in its original form, as purchased from Pfizer and after conjugation with ascending amounts of PDCA (b) corresponding scanning electron microscopy images. Compared with untreated Gelfoam, the treated PDCA-Gelfoam samples had a rougher texture, but remained highly porous. The morphology of PDCA-Gelfoam samples appeared similar across all dosages. (c) PDCA-Gelfoam samples were digested with 0.4 μg/mL papain as an accelerated simulation of PDCA release by cellular proteases. The approximately linear slopes of the release profiles showed that most of the conjugated PDCA (∼90%) was released at a sustained and nearly constant rate, without initial burst. (n=3. Error bars represent standard deviation.) Color images available online at www.liebertpub.com/tea

FIG. 4.

Assessment of PDCA-Gelfoam's effects on the proliferation and viability of IMR-90 fibroblasts. (a) Cytotoxicity was monitored by measuring leakage of glucose 6-phosphate dehydrogenase (G6PD) from cells using the Vybrant cytotoxicity assay. Results show that PDCA-Gelfoam has very low cytotoxicity (<10% compared with positive control) at all dosages tested. No significant differences were observed across the different dosages of PDCA-Gelfoam. (n=3. Error bars represent standard deviation.) (b) Cell proliferation was assessed by harvesting the scaffolds after 7 days of in vitro culture, digesting the scaffolds with papain, and measuring the DNA content using PicoGreen. Although PDCA slowed down cell proliferation in a dose-dependent manner, cell numbers at day 7 were still two to four times higher than the initially seeded number (250,000 cells, indicated by the blue dotted line). (*p<0.05, n=3. Error bars represent standard deviation.) (c) Cell infiltration and attachment was visualized by confocal imaging. Scaffolds were harvested after 7 days of in vitro culture, stained with 4′,6-diamidino-2-phenylindole (DAPI), and imaged under a confocal microscope. Three-dimensional reconstructions generated using the z-stacks showed that the scaffolds supported good cell infiltration and attachment at all dosages tested. Labels on the bounding box indicate the scale in μm (each unit=100 μm). Color images available online at www.liebertpub.com/tea

FIG. 5.

Assessing PDCA-Gelfoam ability to stabilize hypoxia-inducible factor-1α (HIF-1α) and stimulate vascular endothelial growth factor (VEGF) secretion. (a) Green fluorescent protein (GFP)-HIF-1α-transfected reporter cells were labeled with PKH26 membrane dye (orange), seeded on PDCA-Gelfoam scaffolds, and imaged 24 h later. The amount of HIF-1α present (visible as green GFP fluorescence localized to cell nuclei) increased as the PDCA content of the scaffolds increased, demonstrating a dose-dependent stabilization of HIF-1α by the PDCA-Gelfoam scaffolds. Scale bar=100 μm. (b) VEGF measurements performed on PDCA-Gelfoam showed that the higher dosages of PDCA (10% and 15% w/w) significantly increased VEGF secretion. (*p<0.05, n=3. Error bars represent standard deviation.) Color images available online at www.liebertpub.com/tea

FIG. 6.

Assessment of PDCA-Gelfoam's effects on vascular infiltration using a rat perirenal fat implantation model. (a) PDCA-Gelfoam samples were implanted into the perirenal fat of Sprague Dawley rats to assess their effects on vascular infiltration in vivo. (b) Appearance of PDCA-Gelfoam explants at day 8 postimplantation. The scaffolds were harvested with some of the surrounding perirenal fat intact to minimize damage to the scaffolds and the vasculature. Scale bar=5 mm. Color images available online at www.liebertpub.com/tea

FIG. 7.

Morphometric analysis of explant cryosections. (a) DAPI nuclei staining (blue) of explant cryosections shows that the samples were well-infiltrated by cells by 8 days postimplantation. Rat endothelial cell antigen-1 (RECA-1) immunofluorescence (red) shows the distribution of endothelial cells with the explants. White dotted lines delineate the edges of the scaffolds. The samples with higher PDCA dosages (10% and 15% w/w) have visibly higher densities of endothelial cells. It is also notable that while endothelial cells were present in the 0% and 5% w/w cells, they were mostly localized to the edges of the explants; by contrast, endothelial cells present in the 10% and 15% w/w samples were distributed throughout the explants, indicating a deeper depth of vascular infiltration. Scale bar=100 μm. (b) To quantitatively compare the degrees of vascular infiltration, the percentage of RECA-1-positive areas was quantified in ImageJ and plotted. Results show that at the higher dosages (10% and 15% w/w), vascular infiltration was substantially increased. (c) To rule out the influence of variations in cell density, the number of cell nuclei per section was also quantified in ImageJ, and the percentage of RECA-1-positive areas were normalized to cell density and plotted. Comparison of the normalized and un-normalized graphs shows that the trend remains similar after normalization to cell numbers, and the observed increase in the quantity of endothelial cells in the higher dosages is not due to differences in general cell density. (*p<0.05, n=2–4. Error bars represent standard deviation.) Color images available online at www.liebertpub.com/tea