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Perfusion-based co-culture model system for bone tissue engineering.

ABSTRACT: In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic media for up to two weeks in order to induce mineral deposition. Mineral deposition in constructs containing only HUVECs, only Saos-2 cells, or a combination thereof was quantified by microCT to determine if the combination of endothelial cells and bone-like cells increased mineral deposition. Histological and fluorescent staining was used to verify mineral deposition and cellular function both along and between the perfused channels. While there was not a quantifiable difference in the amount of mineral deposited in Saos-2 only versus Saos-2 plus HUVEC samples, the location of the deposited mineral differed dramatically between the groups and indicated that the addition of HUVECs within the GelMA matrix allowed Saos-2 cells, in diffusion limited regions of the construct, to deposit bone mineral. This work serves as a model on how to create perfusable bone tissue engineering constructs using a combination of 3D printing and cellular co-cultures.

SUBMITTER: Sawyer SW

PROVIDER: S-EPMC7643915 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Perfusion-based co-culture model system for bone tissue engineering.

Sawyer Stephen W SW Zhang Kairui K Horton Jason A JA Soman Pranav P

AIMS bioengineering 20200529 2

In this work, we report on a perfusion-based co-culture system that could be used for bone tissue engineering applications. The model system is created using a combination of Primary Human Umbilical Vein Endothelial Cells (HUVECs) and osteoblast-like Saos-2 cells encapsulated within a Gelatin Methacrylate (GelMA)-collagen hydrogel blend contained within 3D printed, perfusable constructs. The constructs contain dual channels, within a custom-built bioreactor, that were perfused with osteogenic me ...[more]

PMID: 33163623

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Project description:Cell-derived extracellular matrix (ECM) has been employed as scaffolds for tissue engineering, creating a biomimetic microenvironment that provides physical, chemical and mechanical cues for cells and supports cell adhesion, proliferation, migration and differentiation by mimicking their in vivo microenvironment. Despite the enhanced bioactivity of cell-derived ECM, its application as a scaffold to regenerate hard tissues such as bone is still hampered by its insufficient mechanical properties. The combination of cell-derived ECM with synthetic biomaterials might result in an effective strategy to enhance scaffold mechanical properties and structural support. Electrospinning has been used in bone tissue engineering to fabricate fibrous and porous scaffolds, mimicking the hierarchical organized fibrillar structure and architecture found in the ECM. Although the structure of the scaffold might be similar to ECM architecture, most of these electrospun scaffolds have failed to achieve functionality due to a lack of bioactivity and osteoinductive factors. In this study, we developed bioactive cell-derived ECM electrospun polycaprolactone (PCL) scaffolds produced from ECM derived from human mesenchymal stem/stromal cells (MSC), human umbilical vein endothelial cells (HUVEC) and their combination based on the hypothesis that the cell-derived ECM incorporated into the PCL fibers would enhance the biofunctionality of the scaffold. The aims of this study were to fabricate and characterize cell-derived ECM electrospun PCL scaffolds and assess their ability to enhance osteogenic differentiation of MSCs, envisaging bone tissue engineering applications. Our findings demonstrate that all cell-derived ECM electrospun scaffolds promoted significant cell proliferation compared to PCL alone, while presenting similar physical/mechanical properties. Additionally, MSC:HUVEC-ECM electrospun scaffolds significantly enhanced osteogenic differentiation of MSCs as verified by increased ALP activity and osteogenic gene expression levels. To our knowledge, these results describe the first study suggesting that MSC:HUVEC-ECM might be developed as a biomimetic electrospun scaffold for bone tissue engineering applications.

Project description:White adipose tissue (WAT) is becoming widely used in regenerative medicine/cell therapy applications, and its physiological and pathological importance is increasingly appreciated. WAT is a complex organ composed of differentiated adipocytes, stromal mesenchymal progenitors known as adipose stromal cells (ASC), as well as endothelial vascular cells and infiltrating leukocytes. Two-dimensional (2D) culture that has been typically used for studying adipose cells does not adequately recapitulate WAT complexity. Improved methods for reconstruction of functional WAT ex vivo are instrumental for understanding of physiological interactions between the composing cell populations. Here, we used a three-dimensional (3D) levitation tissue culture system based on magnetic nanoparticle assembly to model WAT development and growth in organoids termed adipospheres. We show that 3T3-L1 preadipocytes remain viable in spheroids for a long period of time, while in 2D culture, they lose adherence and die after reaching confluence. Upon adipogenesis induction in 3T3-L1 adipospheres, cells efficiently formed large lipid droplets typical of white adipocytes in vivo, while only smaller lipid droplet formation is achievable in 2D. Adiposphere-based coculture of 3T3-L1 preadipocytes with murine endothelial bEND.3 cells led to a vascular-like network assembly concomitantly with lipogenesis in perivascular cells. Adipocyte-depleted stromal vascular fraction (SVF) of mouse WAT cultured in 3D underwent assembly into organoids with vascular-like structures containing luminal endothelial and perivascular stromal cell layers. Adipospheres made from primary WAT cells displayed robust proliferation and complex hierarchical organization reflected by a matricellular gradient incorporating ASC, endothelial cells, and leukocytes, while ASC quickly outgrew other cell types in adherent culture. Upon adipogenesis induction, adipospheres derived from the SVF displayed more efficient lipid droplet accumulation than 2D cultures. This indicates that 3D intercellular signaling better recapitulates WAT organogenesis. Combined, our studies show that adipospheres are appropriate for WAT modeling ex vivo and provide a new platform for functional screens to identify molecules bioactive toward individual adipose cell populations. This 3D methodology could be adopted for WAT transplantation applications and aid approaches to WAT-based cell therapy.

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Perfusion-based co-culture model system for bone tissue engineering.

Publications

Perfusion-based co-culture model system for bone tissue engineering.

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