Proteomic Analysis of Nucleus Pulposus Cell-derived Extracellular Matrix Niche and Its Effect on Phenotypic Alteration of Dermal Fibroblasts

Abstract

Reconstituting biomimetic matrix niche in vitro and culturing cells at the cell niche interface is necessary to understand the effect and function of the specific matrix niche. Here we attempted to reconstitute a biomimetic extracellular matrix (ECM) niche by culturing nucleus pulposus cells (NPCs) in a collagen microsphere system previously established and allowing them to remodel the template matrix. The reconstituted NPC-derived complex ECM was obtained after decellularization and the composition of such niche was evaluated by proteomic analysis. Results showed that a complex acellular matrix niche consisting of ECM proteins and cytoskeletal proteins by comparing with the template collagen matrix starting material. In order to study the significance of the NPC-derived matrix niche, dermal fibroblasts were repopulated in such niche and the phenotypes of these cells were changed, gene expression of collagen type II and CA12 increased significantly. A biomimetic NPC-derived cell niche consisting of complex ECM can be reconstituted in vitro, and repopulating such matrix niche with fibroblasts resulted in changes in phenotypic markers. This work reports a 3D in vitro model to study cell niche factors, contributing to future understanding of cellular interactions at the cell-niche interface and rationalized scaffold design for tissue engineering.

Engineering Cell‐Derived Matrices: From 3D Models to Advanced Personalized Therapies

“…Purpose Reference DAPI (4′,6-diamidino-2-phenylindole) Nuclei staining [122] Hoechst 33258 Nuclei staining [123] Quantitating double-stranded DNA (dsDNA) DNA quantification [124] Alexa Fluro 488-labeled phalloidin F-actin staining [14] DiI (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate) Cell membrane staining [14] Immunofluorescence Protein staining [46,[125][126][127][128][129] Immunohistochemistry Protein staining [46,[125][126][127][128][129] Immunocytochemistry Protein staining [46,[125][126][127][128][129] Alcian Blue staining Acidic polysaccharides staining [130] Picrosirius red Collagen types I and III staining [131] Blyscan Glycosaminoglycans staining [130] Colorimetric assays Collagen and elastin quantification [132] Enzyme-linked immunosorbent assay (ELISA) Protein quantification [133] SDS-PAGE (Coomassie, silver, Ponceau staining) Protein profile identification [134] Western blot Protein profile identification [135] Mass spectrometry Protein profile identification [136,137] Nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) Protein profile identification [46,135,138] adhesion through multiple focal adhesions, [151] as has been observed in vivo. [152,153] Focal, fibrillar, and nascent adhesions involve i...…”

“…[44] In several studies, microparticles have been used as carriers to encapsulate cells together with their own ECM or just pure CDM. [45,46] Cheng and co-workers [16] were able to encapsulate porcine chondrocytes within rat tail collagen type I microspheres, promoting the creation of chondrogenic ECM and preserving about 40% of the glycosaminoglycans (GAGs) compared to the control group. hMSCs seeded in this construct acquired a chondrogenic phenotype without supplementing media.…”

“…Similarly, Yuan et al also encapsulated primary nucleus pulposus cells (NPCs) in collagen microspheres to produce a CDM and evaluated its composition by proteomic analysis. [46] Human dermal fibroblasts (hDF) reseeded in NPC-derived matrices acquired a less fibroblastic phenotype and were characterized by an increased expression of collagen type II and the nonchondrogenic NPC marker CA12.…”

“…Collagen types I, II, III, IV, and X, fibronectin, biglycan, decorin, versican, and laminin have been reported to be present in CDMs after decellularization. [46,[125][126][127][128][129] Other techniques can also be used to examine specific protein contents. For example, Alcian blue staining visualizes proteoglycan deposition, [130] while Picrosirius red staining analyzes the presence and spatial distribution of collagen fibers, specifically collagen types I and III.…”

“…[136,137] Different studies have analyzed CDM proteins using nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS) after efficient decellularization and scaffold template removal. [46,135,138] The diverse characterization methods reviewed here highlight the heterogeneous nature of CDMs. This heterogeneity, marked by the enormous range of possibilities in CDM production, emphasizes the essential requirement of designing CDMs based on the tissue or structure being mimicked.…”