Novel Acellular Scaffold Made from Decellularized Schwann Cell Sheets for Peripheral Nerve Regeneration

Abstract

Extracellular matrix surrounding Schwann cells and neurons provides critical determinants of cellular phenotype during development as well as essential cues in stimulating and guiding regrowth. Using cell sheet technology, we developed a novel scaffold enriched with native extracellular matrix from Schwann cells. Schwann cells were grown into sheets and layered onto polycaprolactone fibers for support. Upon decellularization of these constructs, extracellular matrix remained with few traces of nucleic acids. This method of deposition of extracellular matrix provided more protein than traditional seeding method after decellularization. Additionally, the isolated matrix supported proliferation of Schwann cells better than covalently bound laminin. The proliferation and differentiation of Schwann cells grown on decellularized sheets were complemented by upregulation of Erbb2 and myelin protein zero. Laminin expression of β1 and γ1 chains was also elevated. PC12 cells grown on decellularized sheets produced longer neurite extensions than aligned polycaprolactone fibers alone, proving potential of these scaffolds to be used in future peripheral nerve regenerative studies.

Lay summary: Peripheral nerve injuries present a serious clinical need with approximately 50 % of surgical cases achieving only some restoration of function. In order to better guide regenerating nerves, supporting cells of the nerve tissue were grown into sheets and subsequently decellularized, leaving a myriad of surrounding protein as a scaffold. Constructs have been shown to support cell growth and neurite extension in vitro. Future projects will combine various cell types present in the nerve tissue as well as stem cells to fully support and reconstruct architecture of the peripheral nerves.

Keywords: Cell sheet; Decellularization; Extracellular matrix; Nerve tissue engineering; Schwann cell.

Fig. 1

a–b Intense purple staining of protein on decellularized Schwann cell sheets using Coomassie Blue stain. c–d Faint blue staining of nucleic acids showing lack of nuclei structures remaining on decellularized Schwann cell sheets using Methylene blue stain. e–f Intense purple staining using Coomassie Blue for protein remaining after decellularization of traditionally seeded Schwann cells. g–h Very diffuse Methylene blue staining showing small traces of nucleic acids on decellularized scaffolds traditionally seeded with Schwann cells. Scale bars represent 1 mm (a, c, e, g) and 100 μm (b, d, f, h). MB Methylene blue, CB Coomassie Blue

Fig. 2

a Quantification of protein remaining on a scaffold after decellularization protocol using BCA assay. Scaffolds were either seeded using traditional method, layered with a single decellularized sheet, or layered with three decellularized sheets (*p=0.0003). b Quantification of nucleic acids remaining on a scaffold after decellularization protocol using QuantiT assay

Fig. 3

MTS proliferation assay of Schwann cells seeded on various surfaced. PCL/CHIT + LAM fibers indicate covalent modification of fibers using EDC/NHS chemistry. PCL + SHEET indicate a single decellularized sheet attached to random polycaprolactone fibers. (*p<0.0001)

Fig. 4

Genetic expression of Schwann cells seeded on decellularized Schwann cell sheets: a laminin chain α2 (*p=0.5322), b receptor tyrosine-protein kinase erbB-2 (*p<0.0001), c laminin chain β1 (*p=0.0007), d myelin protein zero (*p=0.0015), e laminin chain γ1 (*p=0.0020), and f β1 integrin (*p=0.0193)

Fig. 5

PC12 cell culture and neurite extension on a aligned PCL fibers with a single decellularized cell sheet, b random PCL fibers with a single decellularized cell sheet, and c aligned PCL fibers without decellularized sheet. Scale bars indicate distance of 200 μm. d Graph shows average measurement of neurite extensions for each condition. (*p<0.0001)