Poly-ε-caprolactone Coated and Functionalized Porous Titanium and Magnesium Implants for Enhancing Angiogenesis in Critically Sized Bone Defects

Abstract

For healing of critically sized bone defects, biocompatible and angiogenesis supporting implants are favorable. Murine osteoblasts showed equal proliferation behavior on the polymers poly-ε-caprolactone (PCL) and poly-(3-hydroxybutyrate)/poly-(4-hydroxybutyrate) (P(3HB)/P(4HB)). As vitality was significantly better for PCL, it was chosen as a suitable coating material for further experiments. Titanium implants with 600 µm pore size were evaluated and found to be a good implant material for bone, as primary osteoblasts showed a vitality and proliferation onto the implants comparable to well bottom (WB). Pure porous titanium implants and PCL coated porous titanium implants were compared using Live Cell Imaging (LCI) with Green fluorescent protein (GFP)-osteoblasts. Cell count and cell covered area did not differ between the implants after seven days. To improve ingrowth of blood vessels into porous implants, proangiogenic factors like Vascular Endothelial Growth Factor (VEGF) and High Mobility Group Box 1 (HMGB1) were incorporated into PCL coated, porous titanium and magnesium implants. An angiogenesis assay was performed to establish an in vitro method for evaluating the impact of metallic implants on angiogenesis to reduce and refine animal experiments in future. Incorporated concentrations of proangiogenic factors were probably too low, as they did not lead to any effect. Magnesium implants did not yield evaluable results, as they led to pH increase and subsequent cell death.

Keywords: HMGB1; VEGF; angiogenesis; poly-(3-hydroxybutyrate)/poly-(4-hydroxybutyrate); poly-ε-caprolactone; titanium implants.

Figure 1

Vitality (%) of murine osteoblasts settled on PCL (poly-ε-caprolactone), P(3HB)/P(4HB) (poly(3-hydroxy-butyrate)/poly(4-hydroxy-butyrate)) and WB (well bottom) for 96 h. Global F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test showed a significant difference between PCL and (P(3HB)/P(4HB)) meanwhile between WB and (P(3HB)/P(4HB)) (* = p ≤ 0.05, n = 8; circle = outlier; rhombus = mean; centered line = median).

Figure 2

Vitality (%) of murine osteoblasts settled on P(3HB)/P(4HB) (poly(3-hydroxy-butyrate)/poly(4-hydroxy-butyrate) for 48, 72 and 96 h. Global F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test showed a significant difference between the vitality after 48 and 72 h meanwhile between 48 and 96 h (* = p ≤ 0.05, n = 8; circle = outlier; rhombus = mean; centered line = median).

Figure 3

Proliferation index of murine osteoblasts for different materials PCL (poly-ε-caprolactone), P(3HB)/P(4HB) (poly(3-hydroxy-butyrate)/poly(4-hydroxy-butyrate)) and WB (well bottom). No significant difference between the materials could be observed using Global F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test, * = p ≤ 0.05, n = 9; circle = outlier; rhombus = mean; centered line = median.

Figure 4

Vitality (%) of murine osteoblasts settled on porous titanium implants with 600 µm pore-size compared to vitality (%) of murine osteoblasts settled on WB (well bottom) for 96 h. Although WB showed a slightly better vitality, Global F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test did not show a significant difference between WB and titanium implant (* = p ≤ 0.05, n = 8; rhombus = mean; centered line = median).

Figure 5

Vitality (%) of murine osteoblasts settled on porous titanium implants for 48, 72 and 96 h. Over time, increasing of the vitality of murine osteoblasts is observable. Global F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test showed a significant difference between the vitality after 48 and 96 h (* = p ≤ 0.05, n = 8; rhombus = mean; centered line = median).

Figure 6

Proliferation index of murine osteoblasts settled on well bottom (WB) compared to porous titanium implants did not show a significant difference between the materials (Global F-test from the analyses of variance and pairwise multiple means comparisons with the Least Significant Difference test, * = p ≤ 0.05, n = 9; rhombus = mean; centered line = median).

Figure 7

Live Cell Imaging (LCI) of GFP-Osteoblasts seeded on titanium implants on (a) Day 1; (b) Day 4 and (c) Day 7 compared to GFP-Osteoblasts seeded on PCL coated titanium implants on (d) Day 1; (e) Day 4 and (f) Day 7. Pictures were taken in 10-fold magnification using an exposure time of 6 ms, a gain of 5.8 and an intensity of 3 s. Scale bar: 250 µm.

Figure 8

LCI of murine osteoblasts seeded on titanium implants and titanium implants covered with PCL (poly-ε-caprolactone). (a) Cell count meaning cells counted on the implant and (b) cell spreading area development (Covered Area) over the implant surface over seven days were examined. Neither for cell count nor for cell spreading areas could significant difference between the two materials be observed. The homogeneity of the regression coefficients was tested using the F-test for interaction between time and implant materials (circle = single data for titanium implant; plus sign = single data for titanium PCL implant).

Figure 9

Tubule formation in the angiogenesis assay performed with (a) titanium implant; (b) titanium implant coated with PCL (poly-ε-caprolactone); (c) titanium implant coated with PCL functionalized with VEGF (Vascular Endothelial Growth Factor) and (d) HMGB1 (High Mobility Group Box 1) in a magnification of 40. Scale bar: 500 µm.

Figure 10

Results of the angiogenesis assay with titanium implants coated with poly-ε-caprolactone (PCL), pure titanium implants, titanium implant coated with PCL functionalized with Vascular Endothelial Growth Factor (VEGF) and High Mobility Group Box 1 (HMGB1) for (a) Number of Junctions; (b) Number of Tubules; (c) Total Tubule Length (µm); (d) Number of Nets. F-test from the analyses of variance followed by pairwise multiple means comparisons with the Least Significant Difference test were used (* = p ≤ 0.05; circle = outlier; rhombus = mean; centered line = median).

Figure 11

No viable cells or tubule formation could be observed in the angiogenesis assay with magnesium implants covered with PCL. Cell shed detached at Day 3. Scale bar: 500 µm, magnification of 40×.

Figure 12

Environmental Scanning Electron Microscopy (ESEM) (Quanta FEG 250, FEI, Eindhoven, The Netherlands) images of PCL coated titanium (A) and PCL coated magnesium implants (B). After fixing the implants, the scanning electron micrographs were performed at 50 Pa pressure, moisturized atmosphere and an accelerating voltage of 10 kV (HV = high voltage; det = detector; LFD = large field detector; WD = working distance, HFE = horizontal field width, mag = magnification).