Abstract
Achieving a satisfactory functional recovery after severe peripheral nerve injuries (PNI) remains one of the major clinical challenges despite advances in microsurgical techniques. Nerve autografting is currently the gold standard for the treatment of PNI, but there exist several major limitations. Accumulating evidence has shown that various types of nerve guidance conduits (NGCs) combined with post-natal stem cells as the supportive cells may represent a promising alternative to nerve autografts. In this study, gingiva-derived mesenchymal stem cells (GMSCs) under 3D-culture in soft collagen hydrogel showed significantly increased expression of a panel of genes related to development/differentiation of neural crest stem-like cells (NCSC) and/or Schwann cell precursor-like (SCP) cells and associated with NOTCH3 signaling pathway activation as compared to their 2D-cultured counterparts. The upregulation of NCSC-related genes induced by 3D-collagen hydrogel was abrogated by the presence of a specific NOTCH inhibitor. Further study showed that GMSCs encapsulated in 3D-collagen hydrogel were capable of transmigrating into multilayered extracellular matrix (ECM) wall of natural NGCs and integrating well with the aligned matrix structure, thus leading to biofabrication of functionalized NGCs. In vivo, implantation of functionalized NGCs laden with GMSC-derived NCSC/SCP-like cells (designated as GiSCs), significantly improved the functional recovery and axonal regeneration in the segmental facial nerve defect model in rats. Together, our study has identified an approach for rapid biofabrication of functionalized NGCs through harnessing 3D collagen hydrogel-directed conversion of GMSCs into GiSCs.
Highlights
Mesenchymal stromal/stem cells (MSCs), a subpopulation of postnatal stem cells existing in almost all mesodermal connective tissues[1], possess multipotent differentiation capabilities, potent immunomodulatory/anti-inflammatory functions, and pleiotropic effects through the secretion of a large panel of growth factors, making them a good candidate for cell-based therapies to treat a large spectrum of diseases or pathological conditions[2,3], including peripheral nerve regeneration[4,5]
3D-collagen hydrogel drives the conversion of GMSCs into neural crest stem-like cell (NCSC)/Schwann cell precursor-like (SCP)-like cells According to our previous studies[35], human gingiva-derived mesenchymal stem cells (GMSCs) were routinely isolated and characterized by the expression of several MSC-associated cell surface markers, e.g., CD44, CD73, and CD90, but negative for hematopoietic cell markers, e.g., CD45 (Supplementary Fig. 1a, b) as well as their multipotent differentiation capacities into adipocytes (Supplementary Fig. 1c, d) and osteocytes (Supplementary Fig. 1e, f)
We initially cultured GMSCs for 48 h in methacrylated 3D-collagen hydrogel with different matrix densities or stiffness achieved by varying collagen concentrations (2, 3, 4, 6 mg/mL) with regular MSC culture medium (α-MEM + 10% FBS) (Fig. 1a), and mRNA expression of NCSC/SCP-related genes was determined by Quantitative real-time polymerase chain reaction (qRT-PCR)
Summary
Mesenchymal stromal/stem cells (MSCs), a subpopulation of postnatal stem cells existing in almost all mesodermal connective tissues[1], possess multipotent differentiation capabilities, potent immunomodulatory/anti-inflammatory functions, and pleiotropic effects through the secretion of a large panel of growth factors, making them a good candidate for cell-based therapies to treat a large spectrum of diseases or pathological conditions[2,3], including peripheral nerve regeneration[4,5]. Special culture conditions (e.g., crestosphere), are required to maintain their NCSC properties[6,7,8,13] Due to their wide existence and multipotency, adult NCSCs, those from the accessible orofacial tissues[14], represent an attractive source of stem cells for cell-based regenerative therapy of various diseases, for nerve regeneration because of their intrinsic propensity to differentiate into glial and Schwann-like cells[15,16,17].
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