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. 2024 Feb 22;18(1):16.
doi: 10.1186/s13036-024-00412-9.

Preparation of bilayer tissue-engineered polyurethane/poly-L-lactic acid nerve conduits and their in vitro characterization for use in peripheral nerve regeneration

Mehran Nabipour  1   2 , Amir Mellati  1   3 , Mozhgan Abasi  1   4 , Somayeh Ebrahimi Barough  5 , Ayoob Karimizade  1   2 , Parnian Banikarimi  1   2 , Elham Hasanzadeh  6   7   8
Affiliations

Preparation of bilayer tissue-engineered polyurethane/poly-L-lactic acid nerve conduits and their in vitro characterization for use in peripheral nerve regeneration

Mehran Nabipour et al. J Biol Eng. .

Abstract

Background: Due to loss of peripheral nerve structure and/or function resulting from trauma, accidents, and other causes, peripheral nerve injuries continue to be a major clinical problem. These injuries can cause partial or total loss of sensory, motor, and autonomic capabilities as well as neuropathic pain. PNI affects between 13 and 23 out of every 100,000 people annually in developed countries. Regeneration of damaged nerves and restoration of function after peripheral nerve injury remain significant therapeutic challenges. Although autologous nerve graft transplantation is a viable therapy option in several clinical conditions, donor site morbidity and a lack of donor tissue often hinder full functional recovery. Biomimetic conduits used in tissue engineering to encourage and direct peripheral nerve regeneration by providing a suitable microenvironment for nerve ingrowth are only one example of the cutting-edge methods made possible by this field. Many innate extracellular matrix (ECM) structures of different tissues can be successfully mimicked by nanofibrous scaffolds. Nanofibrous scaffolds can closely mimic the surface structure and morphology of native ECMs of many tissues.

Methods: In this study, we have produced bilayer nanofibrous nerve conduit based on poly-lactic acid/polyurethane/multiwall carbon nanotube (PLA/PU/MWCNT), for application as composite scaffolds for static nerve tissue engineering. The contact angle was indicated to show the hydrophilicity properties of electrospun nanofibers. The SEM images were analyzed to determine the fiber's diameters, scaffold morphology, and endometrial stem cell adhesion. Moreover, MTT assay and DAPI staining were used to show the viability and proliferation of endometrial stem cells.

Results: The constructed bilayer PLA/PU/MWCNT scaffolds demonstrated the capacity to support cell attachment, and the vitality of samples was assessed using SEM, MTT assay, and DAPI staining technique.

Conclusions: According to an in vitro study, electrospun bilayer PLA/PU/MWCNT scaffolds can encourage the adhesion and proliferation of human endometrial stem cells (hEnSCs) and create the ideal environment for increasing cell survival.

Keywords: Carbon nanotube; Human endometrial stem cells; Nanofibrous scaffolds; Nerve conduit; Neural differentiation; Poly-L-lactic acid; Polyurethane; Sciatic nerve injury; Tissue engineering.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Schematic diagram of (a) dual and (b) single electrospinning system for fabrication of PU/MWCNT and PLA fibers
Fig. 2
Fig. 2
Macrographs of nanofibrous nerve construct. a-d Bilayer nanofibre nerve conduit; (e, f) scanning electron micrographs (SEMs) of bilayer nanofibrous nerve conduit at different magnifications; (g-j) SEM images of the electrospun nanofibers at different magnifications demonstrating porosity and homogeneous fibrous structures in (g) PLA, (h) PU, and (i, j) PU/MWCNT nanofibrous mats. Multiwalled carbon nanotubes (MWCNTs) embedded in nanofibres are denoted by Red arrows
Fig. 3
Fig. 3
The mechanical attributes of the PLA, PU, PU/MWCNT, and PLA/PU/MWCNT conduits. Tensile properties measurement under 10 N tensile load and an extension rate of 1 mm/min at 37 °C. Compared to PLA/PU/MWCNT scaffolds, PLA (P < 0.05), PU (P > 0.05), and PU/MWCNT (P > 0.05) nanofibrous conduits had a lower tensile strength. UTS: ultimate tensile strength, Ɛb: elongation a break. Data are presented as mean SD, n = 3
Fig. 4
Fig. 4
Average water contact angle of the electrospun (A) PLA, (B) PU, and (C) PU/MWCNT nanofibrous mats. The addition of MWCNTs enhanced the hydrophilicity of PU nanofibers. Data are reported as mean SD, n = 3 (***p < 0.001, **p < 0.01)
Fig. 5
Fig. 5
The rate of degradation (a) and pH variation (b) of PLA, PU, PU/MWCNT, and PLA/PU/MWCNT conduits during 8 weeks of sucking in PBS (pH 7.4) at 37 °C. a In all groups, the degradation rates show a significant change during eight weeks (p < 0.05). b In all groups, we can see a little decrease in pH value over eight weeks but without statistical significance (P > 0.05). The data are shown as mean SD, (n = 3)
Fig. 6
Fig. 6
Swelling ratios of the PLA, PU, PU/MWCNT, and PLA/PU/MWCNT nanofibrous scaffolds after 48 hours of incubation at 37 °C with PBS (pH 7.4). Data are presented as mean SD, n = 3 (***p < 0.001, **p < 0.01, *p < 0.05)
Fig. 7
Fig. 7
The porosity of the PLA, PU, PU/MWCNT, and PLA/PU/MWCNT nanofibrous scaffolds. The PLA conduit to the PU, PU/MWCNT, and PLA/PU/MWCNT conduits, it was shown to have more porosity, but without statistical significance (P > 0.05). Data are presented as mean SD, n = 3
Fig. 8
Fig. 8
The FTIR-ATR spectroscopy. Chemical structure of PLA, PU, PU/MWCNT, and PLA/PU/MWCNT nanofibers. Remarkable functional groups in each sample are denoted by stars
Fig. 9
Fig. 9
SEM images of hEnSCs cultured on PLA, PU, PU/MWCNT, and PLA/PU/MWCNT nanofibrous scaffolds after 48 hours. (A–D) Scaffolds without cells; (E–H) scaffolds with cells (pseudo-colored) at different magnifications
Fig. 10
Fig. 10
MTT assay. The viability of hEnSCs cells seeded on tissue culture plate (TCP) and different nanofibrous scaffolds after 1, 3, and 5 days. Data are presented as mean SD, n = 3 (***p < 0.001, **p < 0.01, *p < 0.05)
Fig. 11
Fig. 11
Fluorescent images of hEnSCs stained with DAPI seeded on tissue culture plate (TCP) and scaffolds after 1, 3, and 5 days. The stained cell’s nuclei were blue under fluorescence microscopy
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