Painful Terminal Neuroma Prevention by Capping PRGD/PDLLA Conduit in Rat Sciatic Nerves

Abstract

Neuroma formation after amputation as a long-term deficiency leads to spontaneous neuropathic pain that reduces quality of life of patients. To prevent neuroma formation, capping techniques are implemented as effective treatments. However, an ideal, biocompatible material covering the nerves is an unmet clinical need. In this study, biocompatible characteristics presented by the poly(D,L-lactic acid)/arginylglycylaspartic acid (RGD peptide) modification of poly{(lactic acid)-co- [(glycolic acid)-alt-(L-lysine)]} (PRGD/PDLLA) are evaluated as a nerve conduit. After being capped on the rat sciatic nerve stump in vivo, rodent behaviors and tissue structures are compared via autotomy scoring and histological analyses. The PRGD/PDLLA capped group gains lower autotomy score and improves the recovery, where inflammatory infiltrations and excessive collagen deposition are defeated. Transmission electron microscopy images of the regeneration of myelin sheath in both groups show that abnormal myelination is only present in the uncapped rats. Changes in related genes (MPZ, MBP, MAG, and Krox20) are monitored quantitative real-time polymerase chain reaction (qRT-PCR) for mechanism investigation. The PRGD/PDLLA capping conduits not only act as physical barriers to inhibit the invasion of inflammatory infiltration in the scar tissue but also provide a suitable microenvironment for promoting nerve repairing and avoiding neuroma formation during nerve recovery.

Keywords: inflammation; nerve conduits; neuroma prevention; painful scar neuropathy; scar deposition.

Figure 1

Surgical procedures of the transected rat model and autotomy assessment. A) PRGD/PDLLA conduit and B) surface morphology observation using scanning electron microscopy. C,D) Transected rat model, the right sciatic nerve was transected into proximal and distal segments at the center of the right thigh, the proximal stump was capped by a PRGD/PDLLA capping conduit and the distal stump was removed for 10 mm to avoid spontaneous nerve regeneration. E) The assessment of weekly average autotomy score from 1 to 8 weeks postoperatively (n = 6, *P < 0.01, compared to a noncapped group). F) The expression level of α‐SMA mRNA in different groups at 2 and 8 weeks postoperatively, noncapped group as the control (n = 3, *p < 0.01).

Figure 2

Cross‐sections of the proximal nerve stumps with immunohistochemical staining at both 2 and 8 weeks postoperatively. A2–B8) Labeled with immunohistochemical antibodies of CD3 (black arrow). A2'–B8') Labeled with immunohistochemical antibodies of CD68 (red arrow). B,C) The statistics of T cells (CD3) and macrophages (CD68) in different groups. D,E) The mRNA expression of TNF‐α and IL‐β in different groups (n = 6, *P < 0.01, # p < 0.05, compared to noncapped group).

Figure 3

Cross‐ sections of the proximal nerve stump with Mason's trichrome masons staining at 2 and 8 weeks postoperatively. The collagen deposition, clotted blood, and disorganized nerve fibers are indicated by the thin arrow, triangle, and thick arrow, respectively. (A2–B8, scale bar: 500 µm; A2'–B8' is the partial magnified view of A2–A8, scale bar: 50 µm.)

Figure 4

Sirius red staining and analysis of proximal nerve stumps at 2 and 8 weeks postoperatively. A) Images of sirius red staining, collagen I (thick fibers) presents yellow orange; collagen III (thin fibers) presents green. (A2''–B8'' is the partial magnified view of A2'–A8'.) B) The average area of collagen I in different groups. C) The average area of collagen III in different groups (n = 5, *P < 0.01, compared to noncapped group).

Figure 5

Observation and assay of cross‐sections of the proximal nerve stump at both 2nd and 8th weeks. A) Morphology of myelin sheath in different groups (A2'–B8' is the partial magnified view of A2–A8) observed by TEM. B–D) Myelinated nerve fiber diameter, myelin sheath thickness, and the ratio of unmyelinated and myelinated nerve fiber were quantitatively evaluated and statistically analyzed. The red arrows show the collagenous fibers with larger particles (*P < 0.01, ^# P < 0.05, compared to noncapped group).

Figure 6

Quantification analyses of the myelin maturation relative genes in different groups. A–D) MBP, Krox20, MPZ, and MAG, respectively (*P < 0.01, ^# P < 0.05, compared to noncapped group).

Figure 7

Schematic of PRGD/PDLLA conduit in preventing traumatic neuroma. A) As the consequence of peripheral nerve injury without capping, inflammatory cells (such as macrophages) accumulate at the injured site and secrete inflammatory mediators, which are able to develop hyperalgesia and induce adjacent fibroblasts to differentiate into myofibroblasts, which has the ability of spontaneous contraction. Then, prolonged inflammation response will lead to over expression of Type I collagen, which further produces mechanical stress to axons. This disturbs the recovery progress from Wallerian degeneration to remyelination; the exposed nerve fibers also attribute the intractable painfulness, leading to neuroma. B) With the covering of PRGD/PDLLA tube, a suitable microenvironment can be established for coordinating the apoptosis and tissue reconstruction, which directly isolates ectopic stimulation and prevents neuroma formation.