Sourabh Ghosh

Three-Dimensional Culture of Melanoma Cells Profoundly Affects Gene Expression Profile: A High Density Oligonucleotide Array Study

Growth in three‐dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10‐30,000 cells were generated upon overnight culture in poly‐Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long‐term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with ≥threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, ≥threefold, upregulation of the expression of >100 transcripts while 73 were ≥threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO‐α, ‐β, and γ), IL‐8, CCL20 (MIP‐3α), and Angiopoietin‐like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real‐time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.

Bioprintable, cell-laden silk fibroin-gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs.

Bioprinting has exciting prospects for printing three-dimensional (3-D) tissue constructs by delivering living cells with appropriate matrix materials. However, progress in this field is currently extremely slow due to limited choices of bioink for cell encapsulation and cytocompatible gelation mechanisms. Here we report the development of clinically relevant sized tissue analogs by 3-D bioprinting, delivering human nasal inferior turbinate tissue-derived mesenchymal progenitor cells encapsulated in silk fibroin-gelatin (SF-G) bioink. Gelation in this bioink was induced via in situ cytocompatible gelation mechanisms, namely enzymatic crosslinking by mushroom tyrosinase and physical crosslinking via sonication. Mechanistically, tyrosinases oxidize the accessible tyrosine residues of silk and/or gelatin into reactive o-quinone moieties that can either condense with each other or undergo nonenzymatic reactions with available amines of both silk and gelatin. Sonication alters the hydrophobic interaction and accelerates self-assembly of silk fibroin macromolecules to form β-sheet crystals, which physically crosslink the hydrogel. However, sonication has no effect on the conformation of gelatin. The effect of optimized rheology, secondary conformations of silk-gelatin bioink, temporally controllable gelation strategies and printing parameters were assessed to achieve maximum cell viability and multilineage differentiation of the encapsulated human nasal inferior turbinate tissue-derived mesenchymal progenitor cells. This strategy offers a unique path forward in the direction of direct printing of spatially customized anatomical architecture in a patient-specific manner.

Silk fibroin as biomaterial for bone tissue engineering

UNLABELLED Silk fibroin (SF) is a fibrous protein which is produced mainly by silkworms and spiders. Its unique mechanical properties, tunable biodegradation rate and the ability to support the differentiation of mesenchymal stem cells along the osteogenic lineage, have made SF a favorable scaffold material for bone tissue engineering. SF can be processed into various scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified, which provides an impressive toolbox and allows SF scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing SF, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted. STATEMENT OF SIGNIFICANCE Silk fibroin is a natural biomaterial with remarkable biomedical and mechanical properties which make it favorable for a broad range of bone tissue engineering applications. It can be processed into different scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified which provides a unique toolbox and allows silk fibroin scaffolds to be tailored to specific applications. This review discusses and summarizes recent advancements in processing silk fibroin, focusing on different fabrication and functionalization methods and their application to grow bone tissue in vitro and in vivo. Potential areas for future research, current challenges, uncertainties and gaps in knowledge are highlighted.

Comparative Chondrogenesis of Human Cell Sources in 3D Scaffolds

Cartilage tissue can be engineered by starting from a diversity of cell sources, including stem‐cell based and primary cell‐based platforms. Selecting an appropriate cell source for the process of cartilage tissue engineering or repair is critical and challenging, due to the variety of cell options available. In this study, cellular responses of isolated human chondrocytes, human embryonic stem cells and mesenchymal stem cells (MSCs) derived from three sources, human embryonic stem cells, bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture. All cell sources were characterized by FACS analysis to compare expression of some surface markers. The cells were differentiated in two different biomaterial matrices, silk and chitosan scaffolds, in the presence and absence of bone morphogenetic protein 6 (BMP6), along with the standard chondrogenic differentiating factors. Embryonic stem cells‐derived MSCs showed unique characteristics, with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis, as determined by real time RT‐PCR, histological and microscopical analyses. After 4 weeks of cultivation, embryonic stem cells‐derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP6. The results suggest that cell source differences are important to consider with regard to chondrogenic outcomes, and among the variables addressed here the human embryonic stem cells‐derived MSCs were the preferred cell source. Copyright

New dimensions in tumor immunology: what does 3D culture reveal?

Experimental models indicate that tumor cells in suspension, unlike solid tumor fragments, might be unable to produce life-threatening cancer outgrowth when transferred to animal models, irrespective of the number of cells transferred, although they induce specific immune responses. Human tumor cells cultured in three dimensions display increased pro-angiogenic capacities and resistance to interferons, chemotherapeutic agents or irradiation, as compared with cells cultured in two-dimensional (2D) monolayers. Tumor cells cultured in three dimensions were also shown to be characterized by defective immune recognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) and by a capacity to inhibit CTL proliferation and dendritic cell (DC) functions. Downregulation of human leukocyte antigen (HLA) or TAA expression and high production of lactic acid might play a role in the elicitation of these effects. Here, we propose that growth in 3D architectures might provide new insights into tumor immunology and could represent an integral missing component in pathophysiological tumor immune escape mechanisms.

In vitro model of mesenchymal condensation during chondrogenic development

Mesenchymal condensation is a pre-requisite of chondrogenesis during embryonic development. The current understanding of chondrogenesis is limited in terms of chondrogenic condensation mechanisms. In particular, the role of matrix stiffness on homotypic cell-cell interactions leading to the establishment of distinct aggregated chondrogenic morphology from mesenchymal cells is unclear. An in vitro biomaterials-based model to assess the interactions of matrix stiffness on chondrogensis is described herein, where by sensing subtle variation in morphology and stiffness of nanofibrous silk protein matrixes human mesenchymal stem cells migrated and assumed aggregated morphologies, mimicking early stage chondrogenesis. This simple in vitro model system has potential to play a significant role to gain insight into underlying mechanisms of mesenchymal condensation steps during chondrogenesis, integrating concepts of developmental biology, biomaterials and tissue engineering.

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes.

Cancer cells’ growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells’ recognition by tumour-associated antigen (TAA)-specific HLA-A*0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA−) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-γ) production by HLA-A*0201-restricted Melan-A/MART-127–35 or gp100280–288-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-γ production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

Oriented lamellar silk fibrous scaffolds to drive cartilage matrix orientation: Towards annulus fibrosus tissue engineering

A novel design of silk-based scaffold is developed using a custom-made winding machine, with fiber alignment resembling the anatomical criss-cross lamellar fibrous orientation features of the annulus fibrosus of the intervertebral disc. Crosslinking of silk fibroin fibers with chondroitin sulphate (CS) was introduced to impart superior biological functionality. The scaffolds, with or without CS, instructed alignment of expanded human chondrocytes and of the deposited extracellular matrix while supporting their chondrogenic redifferentiation. The presence of CS crosslinking could not induce statistically significant changes in the measured collagen or glycosaminoglycan content, but resulted in an increased construct stiffness. By offering the combined effect of cell/matrix alignment and chondrogenic support, the silk fibroin scaffolds developed with precise fiber orientation in lamellar form represent a suitable substrate for tissue engineering of the annulus fibrosus part of the intervertebral disc.

The role of 3D structure and protein conformation on the innate and adaptive immune responses to silk-based biomaterials

We have investigated monocyte and T cell responsiveness to silk based biomaterials of different physico-chemical characteristics. Here we report that untransformed CD14+ human monocytes respond to overnight exposure to silk fibroin-based biomaterials in tridimensional form by IL-1β and IL-6, but not IL-10 gene expression and protein production. In contrast, fibroin based materials in bidimensional form are unable to stimulate monocyte responsiveness. The elicitation of these effects critically requires contact between biomaterials and responding cells, is not sustained and becomes undetectable in longer term cultures. We also observed that NF-κβ and p38 MAP kinase play key roles in monocyte activation by silk-based biomaterials. On the other hand, fibroin based materials, irrespective of their physico-chemical characteristics appeared to be unable to induce the activation of peripheral blood T cells from healthy donors, as evaluated by the expression of activation markers and IFN-γ gene.

Enhanced Redifferentiation of Chondrocytes on Microperiodic Silk/ Gelatin Scaffolds: Toward Tailor-Made Tissue Engineering

Direct-write assembly allows rapid fabrication of complex three-dimensional (3D) architectures, such as scaffolds simulating anatomical shapes, avoiding the need for expensive lithographic masks. However, proper selection of polymeric ink composition and tailor-made viscoelastic properties are critically important for smooth deposition of ink and shape retention. Deposition of only silk solution leads to frequent clogging due to shear-induced β-sheet crystallization, whereas optimized viscoelastic property of silk-gelatin blends facilitate the flow of these blends through microcapillary nozzles of varying diameter. This study demonstrates that induction of controlled changes in scaffold surface chemistry, by optimizing silk-gelatin ratio, can govern cell proliferation and maintenance of chondrocyte morphology. Microperiodic silk-gelatin scaffolds can influence postexpansion redifferentiation of goat chondrocytes by enhancing Sox-9 gene expression, aggregation, and driving cartilage matrix production, as evidenced by upregulation of collagen type II and aggrecan expression. The strategy for optimizing redifferentiation of chondrocytes can offer valuable consideration in scaffold-based cartilage repair strategies.