Evaluation of Functionalized Porous Titanium Implants for Enhancing Angiogenesis in Vitro

Abstract

Implant constructs supporting angiogenesis are favorable for treating critically-sized bone defects, as ingrowth of capillaries towards the center of large defects is often insufficient. Consequently, the insufficient nutritional supply of these regions leads to impaired bone healing. Implants with specially designed angiogenic supporting geometry and functionalized with proangiogenic cytokines can enhance angiogenesis. In this study, Vascular Endothelial Growth Factor (VEGF) and High Mobility Group Box 1 (HMGB1) were used for incorporation into poly-ε-caprolactone (PCL)-coated porous titanium implants. Bioactivity of released factors and influence on angiogenesis of functionalized implants were evaluated using a migration assay and angiogenesis assays. Both implants released angiogenic factors, inducing migration of endothelial cells. Also, VEGF-functionalized PCL-coated titanium implants enhanced angiogenesis in vitro. Both factors were rapidly released in high doses from the implant coating during the first 72 h.

Keywords: HMGB1; PCL; VEGF; angiogenesis; functionalized implants; titanium.

Figure 1

ESEM (environmental scanning electron microscopy) pictures (Quanta FEG 250, FEI, Eindhoven, The Netherlands) of PCL-coated titanium implant. The whole implant (A) was imaged as well as different parts of the implant (B–D). After fixing the implants, the scanning electron micrographs were performed at 50 Pa pressure, with moisturized atmosphere and an accelerating voltage of 5 kV (HV = high voltage; det = detector; LFD = large field detector; WD = working distance, HFE = horizontal field width, mag = magnification).

Figure 2

Migration Assay with GM7373 and supernatants from functionalized implants. Comparison of chemotactic behavior of the endothelial cell line (GM7373) using supernatants from implants functionalized with VEGF (vascular endothelial growth factor), HMGB1 (high mobility group box 1) and a combination of HMGB1/VEGF. All of the functionalized implants showed significantly higher chemotaxis than DMEM with 20% FCS or 0.1% FCS. VEGF was significantly more chemotactic than the combination of VEGF + HMGB1. F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test were used (p ≤ 0.05).

Figure 3

Tubuli and Nets visible after Angiogenesis Assay. After staining with BCIP/NBT-Substrate, tubuli and net-structures became visible. (A) Titanium implant functionalized with VEGF; (B) titanium implant functionalized with HMGB1; and (C) titanium implant functionalized with a combination of VEGF + HMGB1.

Figure 4

Number of Junctions built due to the investigated implant. VEGF-functionalized titanium-PCL implants showed significantly more junctions than all of the other implants. VEGF + HMGB1-functionalized titanium-PCL implants built significantly more junctions than pure titanium implants, titanium implants coated with PCL and HMGB1-functionalized titanium-PCL implants. Significantly more junctions could be seen in wells with pure titanium implants than in wells with titanium-PCL implants. F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test were used (p ≤ 0.05).

Figure 5

Number of Tubules built by investigated implants. VEGF-functionalized titanium-PCL implants built significantly more tubules than all of the other implants. VEGF + HMGB1-functionalized titanium-PCL implants showed significantly more tubules than titanium-PCL implants and HMGB1 functionalized titanium-PCL implants. Pure titanium implants showed better results than titanium-PCL implants. F-test from the analysis of variance followed by pairwise multiple means comparisons with the Least Significant Difference test were used (p ≤ 0.05).

Figure 6

Total Tubule Length built by investigated implants. VEGF-functionalized titanium-PCL implants showed significantly the best results for the characteristic Total Tubule Length. VEGF + HMGB1-functionalized titanium-PCL implants showed a significantly higher Total Tubule Length than titanium-PCL implants and HMGB1-functionalized titanium-PCL implants, but comparable results to pure titanium implants. Pure titanium implants were significantly better than titanium-PCL implants and HMGB1-functionalized titanium-PCL implants. F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test were used (p ≤ 0.05).

Figure 7

Number of Nets built by investigated implants. VEGF-functionalized titanium-PCL implants lead to significantly more building of net-like structures than all of the other titanium implants with or without cytokines in the assay. VEGF + HMGB1-functionalized titanium-PCL implants built significantly more nets than pure titanium implants, titanium-PCL implants and HMGB1-functionalized titanium-PCL implants. F-test from the analysis of variance followed by pairwise multiple means comparisons with the Least Significant Difference test were used (p ≤ 0.05).

Figure 8

Results of Angiogenesis Assay with proangiogenic cytokines VEGF and HMGB1. Angiogenesis Assay with VEGF at a steady concentration of 10 ng/mL (n = 4) and a declining concentration of 117 ng/mL on day 2, 16 ng/mL on day 5, 7 ng/mL on day 8 and 6 ng/mL on day 11 (n = 4), respectively. HMGB1 was used at a steady concentration of 100 ng/mL (n = 4) and a declining concentration of 924 ng/mL at day 2, 130 ng/mL at day 5, 76 ng/mL at day 8 and 24 ng/mL at day 11, respectively. Results for Total Number of Junctions (A); Total Number of Tubules (B); Total Tubule Length (µm) (C); and Total Number of Nets (D) were analyzed using F-test from the analysis of variance followed by pairwise multiple means comparisons with the use of the Least Significant Difference (p ≤ 0.05). Both, VEGF using a concentration of 10 ng/mL and the declining concentrations, showed an angiogenesis stimulating effect. The steady concentration of 10 ng/mL showed significantly more tubules and junction formation than the declining concentrations. Neither the constant concentration of 100 ng/mL HMGB1 nor the declining concentrations of HMGB1 showed an angiogenesis stimulating effect.

Figure 8

Results of Angiogenesis Assay with proangiogenic cytokines VEGF and HMGB1. Angiogenesis Assay with VEGF at a steady concentration of 10 ng/mL (n = 4) and a declining concentration of 117 ng/mL on day 2, 16 ng/mL on day 5, 7 ng/mL on day 8 and 6 ng/mL on day 11 (n = 4), respectively. HMGB1 was used at a steady concentration of 100 ng/mL (n = 4) and a declining concentration of 924 ng/mL at day 2, 130 ng/mL at day 5, 76 ng/mL at day 8 and 24 ng/mL at day 11, respectively. Results for Total Number of Junctions (A); Total Number of Tubules (B); Total Tubule Length (µm) (C); and Total Number of Nets (D) were analyzed using F-test from the analysis of variance followed by pairwise multiple means comparisons with the use of the Least Significant Difference (p ≤ 0.05). Both, VEGF using a concentration of 10 ng/mL and the declining concentrations, showed an angiogenesis stimulating effect. The steady concentration of 10 ng/mL showed significantly more tubules and junction formation than the declining concentrations. Neither the constant concentration of 100 ng/mL HMGB1 nor the declining concentrations of HMGB1 showed an angiogenesis stimulating effect.

Figure 9

Released amounts of HMGB1. Releasing kinetics of HMGB1 from titanium implants coated with PCL and functionalized with HMGB1 and from titanium implants coated with PCL and functionalized with VEGF and HMGB1 (dashed lines). The concentration of HMGB1 released from titanium PCL scaffold HMGB1 3 (green line) was above the detection limit of 1668 ng/mL at day 5. Therefore, no result for this day can be shown.

Figure 10

Released amounts of VEGF. Releasing kinetics of VEGF from titanium implants coated with PCL and functionalized with VEGF and from titanium implants coated with PCL and functionalized with VEGF and HMGB1 (dashed lines).