A composite poly-hydroxybutyrate-glial growth factor conduit for long nerve gap repairs

Abstract

There is considerable evidence that peripheral nerves have the potential to regenerate in an appropriate microenvironment. We have developed a novel artificial nerve guide composed of poly 3-hydroxybutyrate (PHB) filled with glial growth factor (GGF) suspended in alginate hydrogel. Gaps of 2-4 cm in rabbit common peroneal nerve were bridged using a PHB conduit containing either GGF in alginate hydrogel (GGF) or alginate alone (Alginate), or with an empty PHB conduit (Empty). Tissues were harvested 21, 42 and 63 days post-operatively. Schwann cell and axonal regeneration were assessed using quantitative immunohistochemistry. At 21 days, addition of GGF increased significantly the distance of axonal and Schwann cells regeneration in comparison with that observed in Alginate and Empty conduits for both gap lengths. The axons bridged the 2-cm GGF conduits gap by 63 days, with a comparable rate of regeneration seen in 4-cm conduits. Schwann cells and axonal regeneration quantity was similar for both gap lengths in each group. However, at all time points the quantity of axonal and Schwann cells regeneration in GGF grafts was significantly greater than in both Alginate and Empty conduits, the latter showing better regeneration than Alginate conduits. The results indicate an inhibitory effect of alginate on regeneration, which is partially reversed by the addition of GGF to the conduits. In conclusion, GGF stimulates a progressive and sustainable regeneration increase in long nerve gap conduits.

Fig. 1

PanNF staining at 21 days post-operatively in (A) Empty and (B) GGF conduits. The dotted line indicates a distance of 5 mm distal to the proximal end of the conduit, where the area of immunostaining (regeneration area) was quantified.

Fig. 2

2-cm gap – Axonal (A) and Schwann cell (B) regeneration distance in Empty, Alginate and GGF conduits at 21, 42 and 63 days. Raw data expressed with median values highlighted (N= 5 for each group). (A) Pan NF staining: *P ≤ 0.03 GGF vs. Empty and Alginate at 21 days (Kruskal–Wallis test); GGF vs. Alginate and Empty vs. Alginate at 42 days (Kruskal–Wallis test). *P = 0.02 GGF vs. Alginate and Empty vs. Alginate at 63 days (Kruskal–Wallis test). (B) S100 staining: *P ≤ 0.05 GGF vs. Empty and Alginate at 21 days (Kruskal–Wallis test). *P = 0.02 GGF vs. Alginate and Empty vs. Alginate at 42 days (Kruskal–Wallis test).

Fig. 3

4-cm gap – Axonal (A) and Schwann cell (B) regeneration distance in Empty, Alginate and GGF conduits at 21, 42 and 63 days. Raw data expressed with median value highlighted (N = 5 for each group). (A) PanNF staining: *P = 0.03 GGF vs. Empty and Alginate at 21 days (Kruskal–Wallis test). #P < 0.05 GGF vs. Alginate and *P = 0.02 Empty vs. GGF and Alginate at 42 days (Kruskal–Wallis test). *P = 0.02 Empty vs. GGF and Alginate at 63 days (Kruskal–Wallis test). (B) S100 staining: *P = 0.03 GGF vs. Empty and Alginate at 21 days (Kruskal–Wallis test). #P = 0.04 GGF vs. Alginate and *P = 0.02 Empty vs. GGF and Alginate at 42 days (Kruskal–Wallis test). #P = 0.03 GGF vs. Alginate and *P = 0.02 Empty vs. GGF and Alginate at 63 days (Kruskal–Wallis test).

Fig. 4

Percentage area of PanNF staining in Empty, Alginate and GGF conduits at 21, 42 and 63 days. Square root transformed data expressed as group mean values with 95% confidence intervals (N = 10 for each group). ***P < 0.001 GGF vs. Empty at 21 and 63 days, GGF vs. Alginate at 21, 42 and 63 days and Empty vs. Alginate at 63 days (post hoc t-test). **P = 0.004 Empty vs. Alginate at 21 days and *P = 0.017 Empty vs. Alginate at 42 days.

Fig. 5

Percentage area of S100 staining in Empty, Alginate and GGF conduits at 21, 42 and 63 days. Square root transformed data expressed as group mean values with 95% confidence intervals (N = 10 for each group). **P < 0.001 GGF vs. Empty and Alginate at 21, 42 and 63 days (post hoc t-test).