The augmentative role of chitosan and platelet-rich fibrin in reducing epidural fibrosis after laminectomy in rats

Abstract

Background: Epidural fibrosis (EF) is the major complication that develops in the operative region of the spinal vertebrae. This fibrous scar connects the connective tissue around the lateral nerve roots and epidural, resulting in severe pain post-spinal operation and impairment of the nerves' function.

Purpose: This study was conducted to investigate the effect of using platelet-rich fibrin (PRF), chitosan, and their combination in reducing epidural fibrosis after laminectomy in rats.

Methods: Ninety male Sprague Dawley rats weighing 255 ± 55 g were randomly assigned to five groups, each consisting of 15 rats: the normal group (control), the laminectomy group, the PRF group, the chitosan group, the combination group (PRF/chitosan), and the donor group. All rats, except the control group, had lumbar laminectomy surgeries between L3 and L5. Macroscopic analysis, histological evaluations, and mRNA analysis for TGFβ-1 and IL6 were compared statistically after a 30-day follow-up.

Results: In comparison to the laminectomy group, the EF area was significantly decreased in the PRF, chitosan, and combination groups. Histological study, macroscopic inspection, and mRNA expression of TGFβ-1 (P < 0.0001) and IL6 (P < 0.0001) show that the use of PRF with chitosan topically in dura following laminectomy resulted in a decrease in scar tissue formation, inflammation, and EF post-laminectomy.

Conclusion: The combination of chitosan with PRF is a potential therapeutic approach for minimizing EF in rats after laminectomy.

Keywords: Chitosan; Epidural fibrosis; Laminectomy; Platelets-rich fibrin.

Fig. 1

A Platelet-rich fibrin (PRF) clot. B Chitosan film

Fig. 2

SEM images of different sizes of chitosan film at 100 nm magnifications

Fig. 3

Laminectomy process in rats. A Skin incision at the dorsal spinous processes of the L3 and L5 levels. B Dissection of muscle and subcutaneous tissue. C incision of ligamentum flavum. D Total laminectomy at the L3 vertebral level. E Chitosan/PRF application on the surgical site. F muscle suture. G Subcutaneous suture. H Skin suture

Fig. 4

Macroscopic assessment of epidural scar adhesion grades among groups. A The control one showed a grade of zero. B The laminectomy group showed a grade of three. C The PRF and chitosan groups showed a grade of two. D The combination PRF/chitosan group showed a grade of one

Fig. 5

Histopathological evaluation by H&E stain; X: 40 bar 200μm and X: 100 bar 100μm. The control group contains normal dura mater (black arrow), pia mater (dashed black arrow), bone (B), and spinal cord (S) components. The laminectomy group revealed an extensive epidural deposition of scar tissue (blue arrow), a granuloma formation (yellow arrow), and severe inflammation (thick black arrow) surrounding the spinal cord, which led to adhesion to the pia mater and increased dura mater thickness. Inflammation and the formation of epidural scar connective tissue showed a significant reduction in the PRF group (blue arrow). The chitosan group exhibited moderate inflammation and the production of epidural scar tissue (blue arrow), which resulted in increased dura mater thickness and partial adherence to the pia mater. The combined group showed mild epidural fibrosis (blue arrow)

Fig. 6

Microscopic pictures, MT stain; X: 40 bar 200μm. The control group does not exhibit fibrous tissue deposition. The laminectomy group exhibits excessive fibrous tissue epidural deposition (black arrow), granuloma formation (yellow arrow) surrounding the spinal cord, and thicker dura mater invasion of nervous tissue (*) in certain areas. The PRF group has significantly less epidural fibrous connective tissue formation (black arrow). The chitosan group exhibits a substantial quantity of epidural scar connective tissue (black arrow). The PRF/chitosan group, mild epidural fibrosis (black arrow) is observed. S: spinal cord, B: bone

Fig. 7

Histopathological analysis by MT stain; X: 400 bar 50 μm. Dura mater (thin black arrow); pia mater (dashed black arrow). The control, PRF, chitosan, and PRF/chitosan groups showed a reduction in the thickness of the dura mater compared to the laminectomy group

Fig. 8

Inflammation scores significantly decreased in the treatment groups (PRF, chitosan, and combined) compared to the laminectomy group. Scare tissue density score also showed a significant decrease in the treatment groups compared to the laminectomy group. In addition, Dura mater thickness significantly decreased in all the treated groups compared to the laminectomy group. Different small alphabetical letters indicate significance when the P value < 0.05

Fig. 9

The degree of TGF-β1 and IL-6 mRNA expression in each group’s epidural scar tissue. The relative mRNA expression level was assessed using Kruskal–Wallis statistical analysis and Dunn’s test RT-PCR testing in comparison to the laminectomy group. The IL-6 and TGF-β1 expression level in the combined group was more significantly decreased (P < 0.0001) compared to the other groups, data are presented as mean ± SD. Different superscript letters indicate significant change among groups