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Spontaneous Formation of 3D Breast Cancer Tissues on Electrospun Chitosan/Poly(ethylene oxide) Nanofibrous Scaffolds.

ABSTRACT: Three-dimensional (3D) tissue culture has attracted a great deal of attention as a result of the need to replace the conventional two-dimensional cell cultures with more meaningful methods, especially for understanding the sophisticated nature of native tumor microenvironments. However, most techniques for 3D tissue culture are laborious, expensive, and limited to spheroid formation. In this study, a low-cost and highly effective nanofibrous scaffold is presented for spontaneous formation of reproducible 3D breast cancer microtissues. Experimentally, aligned and non-aligned chitosan/poly(ethylene oxide) nanofibrous scaffolds were prepared at one of two chitosan concentrations (2 and 4 wt %) and various electrospinning parameters. The resulting fabricated scaffolds (C2P1 and C4P1) were structurally and morphologically characterized, as well as analyzed in silico. The obtained data suggest that the fiber diameter, surface roughness, and scaffold wettability are tunable and can be influenced based on the chitosan concentration, electrospinning conditions, and alignment mode. To test the usefulness of the fabricated scaffolds for 3D cell culture, a breast cancer cell line (MCF-7) was cultured on their surfaces and evaluated morphologically and biochemically. The obtained data showed a higher proliferation rate for cells grown on scaffolds compared to cells grown on two-dimensional adherent plates (tissue culture plate). The MTT assay revealed that the rate of cell proliferation on nanofibrous scaffolds is statistically significantly higher compared to tissue culture plate (P ≤ 0.001) after 14 days of culture. The formation of spheroids within the first few days of culture shows that the scaffolds effectively support 3D tissue culture from the outset of the experiment. Furthermore, 3D breast cancer tissues were spontaneously formed within 10 days of culture on aligned and non-aligned nanofibrous scaffolds, which suggests that the scaffolds imitate the in vivo extracellular matrix in the tumor microenvironment. Detailed mechanisms for the spontaneous formation of the 3D microtissues have been proposed. Our results suggest that scaffold surface topography significantly influences tissue formation and behavior of the cells.

SUBMITTER: Rabie AMI

PROVIDER: S-EPMC8771982 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Our long-term goal is to develop smart biomaterials that can facilitate regeneration of critical-size craniofacial lesions. In this study, we tested the hypothesis that biomimetic scaffolds electrospun from chitosan (CTS) will promote tissue repair and regeneration in a critical size calvarial defect. To test this hypothesis, we first compared in vitro ability of electrospun CTS scaffolds crosslinked with genipin (CTS-GP) to those of mineralized CTS-GP scaffolds containing hydroxyapatite (CTS-HA-GP), by assessing proliferation/metabolic activity and alkaline phosphatase (ALP) levels of murine mesenchymal stem cells (mMSCs). The cells' metabolic activity exhibited a biphasic behavior, indicative of initial proliferation followed by subsequent differentiation for all scaffolds. ALP activity of mMSCs, a surrogate measure of osteogenic differentiation, increased over time in culture. After 3 weeks in maintenance medium, ALP activity of mMSCs seeded onto CTS-HA-GP scaffolds was approximately two times higher than that of cells cultured on CTS-GP scaffolds. The mineralized CTS-HA-GP scaffolds were also osseointegrative in vivo, as inferred from the enhanced bone regeneration in a murine model of critical size calvarial defects. Tissue regeneration was evaluated over a 3 month period by microCT and histology (Hematoxylin and Eosin and Masson's Trichrome). Treatment of the lesions with CTS-HA-GP scaffolds induced a 38% increase in the area of de novo generated mineralized tissue area after 3 months, whereas CTS-GP scaffolds only led to a 10% increase. Preseeding with mMSCs significantly enhanced the regenerative capacity of CTS-GP scaffolds (by ?3-fold), to 35% increase in mineralized tissue area after 3 months. CTS-HA-GP scaffolds preseeded with mMSCs yielded 45% new mineralized tissue formation in the defects. We conclude that the presence of HA in the CTS-GP scaffolds significantly enhances their osseointegrative capacity and that mineralized chitosan-based scaffolds crosslinked with genipin may represent a unique biomaterial with possible clinical relevance for the repair of critical calvarial bone defects.

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Spontaneous Formation of 3D Breast Cancer Tissues on Electrospun Chitosan/Poly(ethylene oxide) Nanofibrous Scaffolds.

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