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Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts.

ABSTRACT: Background:Cell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise micro pore sizes via predictable fibre deposition. The effect of the scaffold's architecture on cellular gene expression however has not been fully elucidated. Methods:The design and fabrication of three different uniform pore structures (250, 500 and 750??m), as well as two offset scaffolds with different layout of fibres (30 and 50%) and one complex scaffold with three gradient pore sizes of 250-500 - 750??m, was performed by using MEW. Calcium phosphate modification was applied to enhance the PCL scaffold hydrophilicity and bone inductivity prior to seeding with osteoblasts which were then maintained in culture for up to 30?days. Over this time, osteoblast cell morphology, matrix mineralisation, osteogenic gene expression and collagen production were assessed. Results:The in vitro findings revealed that the gradient scaffold significantly increased alkaline phosphatase activity in the attached osteoblasts while matrix mineralization was higher in the 50% offset scaffolds. The expression of osteocalcin and osteopontin genes were also upregulated compared to other osteogenic genes following 30?days culture, particularly in offset and gradient scaffold structures. Immunostaining showed significant expression of osteocalcin in offset and gradient scaffold structures. Conclusions:This study demonstrated that the heterogenous pore sizes in gradient and fibre offset PCL scaffolds prepared using MEW significantly improved the osteogenic potential of osteoblasts and hence may provide superior outcomes in bone regeneration applications.

SUBMITTER: Abbasi N

PROVIDER: S-EPMC6942301 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts.

Abbasi Naghmeh N Ivanovski Saso S Gulati Karan K Love Robert M RM Hamlet Stephen S

Biomaterials research 20200103

<h4>Background</h4>Cell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise mi ...[more]

PMID: 31911842

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Project description:Introduction: To date, tubular tissue engineering relies on large, non-porous tubular scaffolds (Ø > 2 mm) for mechanical self-support, or smaller (Ø 150-500 μm) tubes within bulk hydrogels for studying renal transport phenomena. To advance the engineering of kidney tubules for future implantation, constructs should be both self-supportive and yet small-sized and highly porous. Here, we hypothesize that the fabrication of small-sized porous tubular scaffolds with a highly organized fibrous microstructure by means of melt-electrowriting (MEW) allows the development of self-supported kidney proximal tubules with enhanced properties. Materials and Methods: A custom-built melt-electrowriting (MEW) device was used to fabricate tubular fibrous scaffolds with small diameter sizes (Ø = 0.5, 1, 3 mm) and well-defined, porous microarchitectures (rhombus, square, and random). Human umbilical vein endothelial cells (HUVEC) and human conditionally immortalized proximal tubular epithelial cells (ciPTEC) were seeded into the tubular scaffolds and tested for monolayer formation, integrity, and organization, as well as for extracellular matrix (ECM) production and renal transport functionality. Results: Tubular fibrous scaffolds were successfully manufactured by fine control of MEW instrument parameters. A minimum inner diameter of 1 mm and pore sizes of 0.2 mm were achieved and used for subsequent cell experiments. While HUVEC were unable to bridge the pores, ciPTEC formed tight monolayers in all scaffold microarchitectures tested. Well-defined rhombus-shaped pores outperformed and facilitated unidirectional cell orientation, increased collagen type IV deposition, and expression of the renal transporters and differentiation markers organic cation transporter 2 (OCT2) and P-glycoprotein (P-gp). Discussion and Conclusion: Here, we present smaller diameter engineered kidney tubules with microgeometry-directed cell functionality. Due to the well-organized tubular fiber scaffold microstructure, the tubes are mechanically self-supported, and the self-produced ECM constitutes the only barrier between the inner and outer compartment, facilitating rapid and active solute transport.

Project description:Tuning cell shape by altering the biophysical properties of biomaterial substrates on which cells operate would provide a potential shape-driven pathway to control cell phenotype. However, there is an unexplored dimensional scale window of three-dimensional (3D) substrates with precisely tunable porous microarchitectures and geometrical feature sizes at the cell's operating length scales (10-100 μm). This paper demonstrates the fabrication of such high-fidelity fibrous substrates using a melt electrowriting (MEW) technique. This advanced manufacturing approach is biologically qualified with a metrology framework that models and classifies cell confinement states under various substrate dimensionalities and architectures. Using fibroblasts as a model cell system, the mechanosensing response of adherent cells is investigated as a function of variable substrate dimensionality (2D vs. 3D) and porous microarchitecture (randomly oriented, "non-woven" vs. precision-stacked, "woven"). Single-cell confinement states are modeled using confocal fluorescence microscopy in conjunction with an automated single-cell bioimage data analysis workflow that extracts quantitative metrics of the whole cell and sub-cellular focal adhesion protein features measured. The extracted multidimensional dataset is employed to train a machine learning algorithm to classify cell shape phenotypes. The results show that cells assume distinct confinement states that are enforced by the prescribed substrate dimensionalities and porous microarchitectures with the woven MEW substrates promoting the highest cell shape homogeneity compared to non-woven fibrous substrates. The technology platform established here constitutes a significant step towards the development of integrated additive manufacturing-metrology platforms for a wide range of applications including fundamental mechanobiology studies and 3D bioprinting of tissue constructs to yield specific biological designs qualified at the single-cell level.

Dataset Information

Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts.

Publications

Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts.

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