Comprehensive ex vivo and in vivo preclinical evaluation of novel chemo enzymatic decellularized peripheral nerve allografts

Abstract

As a reliable alternative to autografts, decellularized peripheral nerve allografts (DPNAs) should mimic the complex microstructure of native nerves and be immunogenically compatible. Nevertheless, there is a current lack of decellularization methods able to remove peripheral nerve cells without significantly altering the nerve extracellular matrix (ECM). The aims of this study are firstly to characterize ex vivo, in a histological, biochemical, biomechanical and ultrastructural way, three novel chemical-enzymatic decellularization protocols (P1, P2 and P3) in rat sciatic nerves and compared with the Sondell classic decellularization method and then, to select the most promising DPNAs to be tested in vivo. All the DPNAs generated present an efficient removal of the cellular material and myelin, while preserving the laminin and collagen network of the ECM (except P3) and were free from any significant alterations in the biomechanical parameters and biocompatibility properties. Then, P1 and P2 were selected to evaluate their regenerative effectivity and were compared with Sondell and autograft techniques in an in vivo model of sciatic defect with a 10-mm gap, after 15 weeks of follow-up. All study groups showed a partial motor and sensory recovery that were in correlation with the histological, histomorphometrical and ultrastructural analyses of nerve regeneration, being P2 the protocol showing the most similar results to the autograft control group.

Keywords: acellular graft; decellularization; decellularized nerve allograft; peripheral nerve repair; tissue engineering.

FIGURE 1

Histological, histochemical and immunohistochemical evaluation of cellular components remained in the different DPNA groups. (A) General morphology of the study groups was assessed by hematoxylin-eosin histochemical method. (B) Fluorescent microscopy images of nerves stained with the intercalant fluorochrome agent 4′,6-diamidino-2-phenylindole (DAPI) to detect DNA remnants. (C) Immunodetection of the Schwann cell marker S-100. (D) Immunohistochemical evaluation of the cytoskeletal protein neurofilament (NFL). (E) Immunodetection of the vimentin (VIM) as cytoskeletal fibroblast protein.

FIGURE 2

Histological, immunohistochemical and ultrastructural evaluation of the ECM and myelin content in the DPNAs. (A) Acid proteoglycans evaluation by Alcian blue (AB) histochemical method. (B) Histochemical identification of fibrillar collagens using Picrosirius staining (PS). (C) Immunohistochemistry for the basal membrane glycoprotein laminin (LAM); brown positive reaction contrasted by hematoxylin. (D) Simultaneous myelin and collagen network assessment by the myelin-collagen histochemical method (MCOLL); myelin (light-blue histochemical reaction) and collagen fibers (red positive reaction). (E) SEM and (F) TEM images of cross-sectional section of the DPNAs generated. Black arrows: Rest of myelin and cell membrane residues; * (white): well-organized collagen fibers; * (black): Disorganized collagen fibers; White arrows: Cytoplasmatic residues. Scale bar: (A–D) = 100 µm; (E) = 10 µm and (F) = 2 µm.

FIGURE 3

Quantitative biochemical analyses of DNA and sGAGs in DPNAs. (A) Quantification of extracted DNA. (B) Quantification of total sGAGs extracted. Graphical and numeric representation were expressed as mean values ± standard deviation values (error bars). Statistically significant differences (p < 0.05) were determined with the Mann–Whitney test and indicated as follows: a = vs. NAT group. b = vs. SD group. c = vs. P1. d = vs. P2 and e = vs. P3.

FIGURE 4

Macroscopic aspect and tensile properties of the DPNAs generated. (A) Macroscopic images of the different study groups. (B) Stress at fracture, (C) strain at fracture and (D) Young’s modulus biomechanical results were obtained after the tensile test of the DPNAs and native nerves. Statistically significant differences (p < 0.05) were determined with the Mann–Whitney test and indicated as follows: a = vs. NAT group. b = vs. SD group. c = vs. P1. d = vs. P2 and e = vs. P3.

FIGURE 5

Ex vivo cytocompatibility assessment of DPNAs. The behavior of ADMSC cultured in the endoneurial part of DPNAs is shown for (A) Live&Dead (L/D) analysis and (B) WST-1cell metabolic assay. For (A) L/D assay non-cell seeded DPNAs of each decellularization method used were included as technical controls in order to confirm that these analyses were conducted with cell-free biomaterials and no cells remained after the decellularization process. Scale bar = 200 µm. For (B) mean values with their respective error bars corresponding to standard deviations were graphed. Statistically significant differences (p < 0.05) were determined with Mann–Whitney test as follows: # = vs. 2D – CTR; a = vs. 2D + CTR; b = vs. SD group; c = vs. P1 group; d = vs. P2 group; e = vs. P3 group.

FIGURE 6

Macroscopic and graphical representation of muscular changes after 15 weeks of sciatic nerve repair by using DPNAs and autograft techniques. (A) Macroscopic analysis of the whole leg and the gastrocnemius and tibialis anterior muscles on the operated side (left) as compared to the contralateral legs (right) in all operated animals. Morphometric analyses comparing the lesioned side (gray) with the contralateral leg (black) of the whole leg (B), the gastrocnemius (C) and tibialis anterior muscles (D) in all operated animals (AUTO, SD, P1, P2). The lost percentage is summarized in the upper part of each column group. Statistically significant differences (p < 0.05) were determined with Mann–Whitney test as follows: a = vs. CTR.

FIGURE 7

Peripheral nerve regeneration histological profile of the central region of the implanted grafts after 15 weeks of surgery. (A) General morphology of the operated and control nerves assessed by hematoxylin-eosin (HE) histochemical method. (B) Evaluation of myelin and collagen fibers content and distribution was performed by myelin-collagen (MCOLL) histochemical method. (C) Schwann cell identification by S-100 marker immunostaining. (D) Immunodetection of GAP-43 marker to assess the newly nerve fibers generated. (E) Mature nerve fibers generated were showed by neurofilament (NFL) immunohistochemical staining. Scale bar = 50 nm.

FIGURE 8

Quantitative and ultrastructural assessment of nerve regeneration in the distal region of the graft. (A-1) General morphology evaluation by toluidine blue (TB) staining. (A-2) Representative TEM images of cross-sectional section. Scale bar: (A-1) = 20 µm; (A-2) = 10 nm low magnification and 1 nm high magnification images. (B) Quantitative histomorphometry of the DPNAs were performed. (B-1) Density of fibers; (B-2) Total fiber number; (B-3) Axon diameter; (B-4) Fiber diameter; (B-5) Myelin thickness; (B-6) G-ratio (axon diameter/fiber diameter). Statistically significant differences (p < 0.05) were determined with Mann–Whitney test as follows: a = vs. CTR; b = vs. AUTO; c = vs. SD group; d = vs. P1 group; e = vs. P2 group.