Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration

Abstract

Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by imageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within 3 days of incubation, fibroblast spheroids interacted with the fibers and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for engineering soft and dense connective tissues with the required structural characteristics and functions needed for wound healing applications. Rapid regeneration of these layers should enhance healing of open wounds in a harsh oral environment.

Engineering principles for guiding spheroid function in the regeneration of bone, cartilage, and skin

“…PLGA nanofibers coated with fibrin, collagen or no protein were placed on top of fibroblast spheroids for gingiva connective tissue engineering. (104) Fibers with collagen and fibrin prompted cell extensions protruding out of the spheroid, while uncoated fibers induced no spheroid migration. Further experiments showed collagen coated nanofibers allowed fibroblasts to migrate deeper into the scaffold and fuse into larger microtissues.…”

Poly(caprolactone)‐aligned nanofibers associated with fibronectin‐loaded collagen hydrogel as a potent bioactive scaffold for cell‐free regenerative endodontics

“…However, Kaufman et al (2018) seeded fibroblast monolayers and 3D spheroids on poly‐(DL‐lactide‐co‐glycolide; PLGA) nanofibres coated with collagen. The authors showed that the collagen‐coated scaffolds induced higher levels of cell migration towards fibres and supported cellular growth within them.…”

“…The biomaterial also induced higher gene expression values of key components of the extracellular matrix (ECM; type I collagen, FN and MMP2) than uncoated scaffolds. Therefore, collagen-coated scaffolds have a strong potential for use in connective tissue engineering applications, as they have the structural characteristics and functions required for wound healing applications (Kaufman et al, 2018). In another study, different formulations of 3D electrospun PLGA scaffolds, either modified with collagen-I and/or FN or not, were developed to assess the synergistic effects of these proteins on supporting the viability and functional properties of Huh-7.5 cells (Das et al, 2020).…”

Porphyromonas gingivalis lipopolysaccharide and gingival fibroblast augment MMP‐9 expression of monocytic U937 cells through cyclophilin A

“…14 At the same time, the cells also release the catabolic ingredients, including MMPs, to make the constituting periodontal tissue balanced. 15 During the inflammation, however, the fibroblasts are also involved in part of the disease progression. Studies have shown that the bacteria themselves or the bacterial products, such as lipopolysaccharide (LPS), could induce the expression or release of certain inflammatory mediators from the gingival or periodontal fibroblasts.…”

“…These fibroblasts produce and secret the metabolic substances, such as the collagen, in structuring the gingival and periodontal extracellular matrix 14 . At the same time, the cells also release the catabolic ingredients, including MMPs, to make the constituting periodontal tissue balanced 15 . During the inflammation, however, the fibroblasts are also involved in part of the disease progression.…”