Harnessing 3D collagen hydrogel-directed conversion of human GMSCs into SCP-like cells to generate functionalized nerve conduits

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.

Implantation of a nerve protector embedded with human GMSC-derived Schwann-like cells accelerates regeneration of crush-injured rat sciatic nerves

“…Gingival tissues were obtained as discarded tissues from healthy human subjects (aged from 20 to 40 years) who underwent a dental procedure, which was approved by the Institutional Review Board (IRB) at the University of Pennsylvania, while informed consent forms were obtained from the subjects. Primary GMSCs were routinely isolated, characterized, and maintained in our laboratory as described previously [ 32 , 37 ]. Briefly, GMSCs were cultured in the complete culture medium: α-minimum essential medium (α-MEM: Invitrogen) supplemented with 10% fetal bovine serum (FBS: Zen-Bio, Inc., Durham, NC), 1% antibiotics (100U/ml penicillin/100 µg/ml streptomycin; Invitrogen), 2 mM L-glutamine, 100 mM non-essential amino acid (NEAA), and 550 μM 2-mercaptoethanol (2-ME; Sigma-Aldrich) and cultured at 37 °C in a humidified tissue culture incubator with 5% CO 2 .…”

“…A purified methacrylated Type I bovine collagen (> 98%) was purchased from Advanced BioMatrix, Inc. (Carlsbad, CA). A stock solution of collagen hydrogel at a 6 mg/mL concentration was prepared according to the manufacturer’s instructions and our recent studies [ 37 ]. Briefly, a calculated volume of the chilled neutralization solution (NS) and collagen stock solution was mixed thoroughly by pipetting, followed by adding GMSCs resuspended in a calculated volume of chilled PBS into the mixture.…”

“…Briefly, a calculated volume of the chilled neutralization solution (NS) and collagen stock solution was mixed thoroughly by pipetting, followed by adding GMSCs resuspended in a calculated volume of chilled PBS into the mixture. According to our recent study, an optimized final concentration of the collagen hydrogel at 4 mg/mL and a cell density at 2 × 10 6 /mL were selected throughout our present study [ 37 ]. Then, the mixture was aliquoted into a Petri culture dish and incubated at 37 °C for 20 min allowing for hydrogel formation, followed by culturing for 48 h in the complete culture medium (α-MEM supplemented with 10% FBS, 1% antibiotics, 2 mM L-glutamine, 100 mM NEAA, and 550 μM 2-ME) in the absence of extra mitogens, e.g., basic fibroblast growth factor (bFGF) and platelet-derived growth factor AA (PDGF-AA), neurotrophic factors, e.g., heregulin-β-1, and chemicals, e.g., all trans-retinoic acid (RA) and forskolin, all of which are commonly used for the induction of Schwann-like cells from MSCs [ 33 , 34 ].…”

“…Our recent study showed that GMSCs could directly convert into NCSC-like cells under defined culture conditions [ 34 ] possibly due to their neural crest origin [ 18 , 35 , 36 ]. Most recently, we demonstrated the feasibility to generate functionalized neural guidance conduits through harnessing 3D collagen hydrogel-directed rapid conversion of GMSCs into Schwann-like cells (GiSCs), which displayed significant therapeutic potentials to facilitate the functional recovery and axonal regeneration of transected rat facial nerves [ 37 ]. In the present study, we aim to further explore the immunomodulatory effect of GiSCs encapsulated in 3D-collagen hydrogel on macrophages and their therapeutic effects in a sciatic nerve crush injury model in rats.…”

Chitosan Hydrogel Supplemented with Metformin Promotes Neuron–like Cell Differentiation of Gingival Mesenchymal Stem Cells

“…Hsu et al [ 12 ] used Neurobasal A medium and B27 supplement, in addition, Hsu et al used a 0.1% gelatin–coating as a culture substrate. Recently, 3D–collagen hydrogel drives the conversion of GMSCs into NCSC/SCP–like cells [ 13 ]. Metformin is a drug commonly used to treat diabetes, but recently, its role in neurodevelopment has also been confirmed.…”