Sciatic nerve repair using poly(ε-caprolactone) tubular prosthesis associated with nanoparticles of carbon and graphene

Abstract

Introduction: Injuries to peripheral nerves generate disconnection between spinal neurons and the target organ. Due to retraction of the nerve stumps, end-to-end neurorrhaphy is usually unfeasible. In such cases, autologous grafts are widely used, nonetheless with some disadvantages, such as mismatching of donor nerve dimensions and formation of painful neuromas at the donor area. Tubulization, using bioresorbable polymers, can potentially replace nerve grafting, although improvements are still necessary. Among promising bioresorbable synthetic polymers, poly(l-lactic acid) (PLLA) and poly(ε-caprolactone) (PCL) are the most studied. Carbon nanotubes and graphene sheets have been proposed, however, as adjuvants to improve mechanical and regenerative properties of tubular prostheses. Thus, the present work evaluated nerve tubulization repair following association of PCL with nanoparticles of carbon (NPC) and graphene (NPG).

Methods: For that, adult Lewis rats were subjected to unilateral sciatic nerve tubulization and allowed to survive for up to 8 and 12 weeks postsurgery.

Results: Nanocomposites mechanical/chemical evaluation showed that nanoparticles do not alter PCL crystallinity, yet providing reinforcement of polymer matrix. Thus, there was a decrease in the enthalpy of melting when the mixture of PCL + NPC + NPG was used. Nanocomposites displayed positive changes in molecular mobility in the amorphous phase of the polymer. Also, the loss modulus (E") and the glass transition exhibited highest values for PCL + NPC + NPG. Scanning electron microscopy analysis revealed that PCL + NPC + NPG prostheses showed improved cell adhesion as compared to PCL alone. Surgical procedures with PCL + NPC + NPG were facilitated due to improved flexibility of the prosthesis, resulting in better stump positioning accuracy. In turn, a twofold increased number of myelinated axons was found in such repaired nerves. Consistent with that, target muscle atrophy protection has been observed.

Conclusion: Overall, the present data show that nanocomposite PCL tubes facilitate nerve repair and result in a better regenerative outcome, what may, in turn, represent a new alternative to pure PCL or PLLA prostheses.

Keywords: PCL; carbon nanotubes; graphene; peripheral nerves; tubulization.

Figure 1

Tubulization of the sciatic nerve by using a PCL + NPC + NPG prosthesis. (a) Exposure of the left sciatic nerve. (b) Sutured distal stump to the end of the tube. (c) Sutured stumps into the tube. (d) Animal skin sutures at the incision site. Note the tube transparency, allowing correct alignment of the stumps, leaving a 3‐ to 4‐mm gap. PCL, poly(ε‐caprolactone); NPC, nanoparticles of carbon; NPG, nanoparticles of graphene

Figure 2

Micrographs of the tubular prosthesis inner surface before implantation: (a) PCL, (b) PCL + NPC, (c) PCL + NPG, and (d) PCL + NPC + NPG. Scale bar: 2 μm, 5,000×. PCL, poly(ε‐caprolactone); NPC, nanoparticles of carbon; NPG, nanoparticles of graphene

Figure 3

Differential scanning calorimetry (DSC) curves. (a) First heating, (b) cooling, and (c) second heating

Figure 4

Dynamic mechanical analysis (DMA) curves. (a) E' (storage modulus) and (b) E” (loss modulus)

Figure 5

Scanning electron microscopy of the prosthesis before implantation, showing the presence of globular structures and pores. (a) PCL, (b) NPC, (c) NPG, and (d) mixture. The use of nanoparticles greatly decreased the size of such globular structures. Scale bar: 100 μm, 250×. PCL, poly(ε‐caprolactone)

Figure 6

Scanning electron microscopy of the inner surface of tubular prostheses 8 and 12 weeks postimplantation. (a, b) PCL, (c, d) PCL + NPC, (e, f) PCL + NPG, and (g, h) PCL + NPC + NPG. After 12 weeks, matrix deposition was greater, but with few cells. Magnification: 800×. P, area not covered by matrix; M, area covered by matrix; arrow, cells; asterisk, marks indicative of blood vessels. Scale bar: 10 μm, 1,500×. PCL, poly(ε‐caprolactone); NPC, nanoparticles of carbon; NPG, nanoparticles of graphene

Figure 7

Panoramic view of regenerated nerves from the different experimental groups observed under light microscopy. Eight weeks postimplantation: (a) contralateral, (b) PCL, (c) PCL + NPC, (d) PCL + NPG, and (e) PCL + NPC + NPG. Sciatic nerve area (f) and estimated number of axons (g) found in cross sections of the regenerated nerves at tube midpoint, 8 weeks postsurgery. Overview of regenerated nerves, 12 weeks postlesion and post‐tubulization: (h) contralateral, (i) PCL, (j) PCL + NPC, (k) PCL + NPG, and (l) PCL + NPC + NPG. Sciatic nerve area (m) and estimated number of axons (n) found in cross sections of the regenerated nerves at tube midpoint, 12 weeks postsurgery. Staining: Sudan Black. Magnification: 200× and 1,000×. Scale: 50 μm and 100 μm, respectively. PCL, poly(ε‐caprolactone); NPC, nanoparticles of carbon; NPG, nanoparticles of graphene. p< 0.05 (*), p< 0.01 (**), and p< 0.001 (***)

Figure 8

Comparison of the ratio of the muscle masses of the ipsilateral and contralateral sides. (a) Soleus and (b) tibialis cranialis