Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical analysis showed no signs of collagen denaturation during the formation process. The values of thermal characteristics indicated that crosslinking occurred and that its efficiency was enhanced by the presence of HA. Although the crosslinking reduced the swelling of the strut material in water, the collagen-HA matrix as a whole tended to swell more and show higher dissolution resistance than pure collagen samples. The compressive modulus and elastic collapse stress were higher for collagen-HA composites. All the scaffolds were shown to support the proliferation and differentiation 3T3-L1 preadipocytes while collagen-HA samples maintained a significantly increased proportion of cycling cells (Ki-67+). Furthermore, collagen-HA composites displayed significantly raised Adipsin gene expression with adipogenic culture supplementation for 8 days vs. control conditions. These results indicate that collagen-HA scaffolds may offer robust, freely permeable 3-D matrices that enhance mammary stromal tissue development in vitro.

Cancer is characterized by cell heterogeneity and the development of 3D in vitro assays that can distinguish more invasive or migratory phenotypes could enhance diagnosis or drug discovery. 3D collagen scaffolds have been used to develop analogues of complex tissues in vitro and are suited to routine biochemical and immunological assays. We sought to increase 3D model tractability and modulate the migration rate of seeded cells using an ice-templating technique to create either directional/anisotropic or non-directional/isotropic porous architectures within cross-linked collagen scaffolds. Anisotropic scaffolds supported the enhanced migration of an invasive breast cancer cell line MDA-MB-231 with an altered spatial distribution of proliferative cells in contrast to invasive MDA-MB-468 and non-invasive MCF-7 cells lines. In addition, MDA-MB-468 showed increased migration upon epithelial-to-mesenchymal transition (EMT) in anisotropic scaffolds. The provision of controlled architecture in this system may act both to increase assay robustness and as a tuneable parameter to capture detection of a migrated population within a set time, with consequences for primary tumour migration analysis. The separation of invasive clones from a cancer biomass with in vitro platforms could enhance drug development and diagnosis testing by contributing assay metrics including migration rate, as well as modelling cell-cell and cell-matrix interaction in a system compatible with routine histopathological testing.

Sponge-like matrices with a specific three-dimensional structural design resembling the actual extracellular matrix of a particular tissue show significant potential for the regeneration and repair of a broad range of damaged anisotropic tissues. The manipulation of the structure of collagen scaffolds using a freeze-drying technique was explored in this work as an intrinsically biocompatible way of tailoring the inner architecture of the scaffold. The research focused on the influence of temperature gradients, imposed during the phase of crystallisation of collagen suspensions, upon the degree of anisotropy in the microstructures of the scaffolds produced. Moulding technology was employed to achieve differences in heat transfer rates during the freezing processes. For this purpose various moulds with different configurations were developed with a view to producing uniaxial and multi-directional temperature gradients across the sample during this process. Scanning electron microscopy analysis of different cross-sections (longitudinal and horizontal) of scaffolds revealed that highly aligned matrices with axially directed pore architectures were obtained where single unidirectional temperature gradients were induced. Altering the freezing conditions by the introduction of multiple temperature gradients allowed collagen scaffolds to be produced with complex pore orientations, and anisotropy in pore size and alignment.

Stem cells are undifferentiated cells that are capable of proliferation, self-maintenance and differentiation towards specific cell phenotypes. These processes are controlled by a variety of cues including physicochemical factors associated with the specific mechanical environment in which the cells reside. The control of stem cell biology through mechanical factors remains poorly understood and is the focus of the developing field of mechanobiology. This review provides an insight into the current knowledge of the role of mechanical forces in the induction of differentiation of stem cells. While the details associated with individual studies are complex and typically associated with the stem cell type studied and model system adopted, certain key themes emerge. First, the differentiation process affects the mechanical properties of the cells and of specific subcellular components. Secondly, that stem cells are able to detect and respond to alterations in the stiffness of their surrounding microenvironment via induction of lineage-specific differentiation. Finally, the application of external mechanical forces to stem cells, transduced through a variety of mechanisms, can initiate and drive differentiation processes. The coalescence of these three key concepts permit the introduction of a new theory for the maintenance of stem cells and alternatively their differentiation via the concept of a stem cell 'mechano-niche', defined as a specific combination of cell mechanical properties, extracellular matrix stiffness and external mechanical cues conducive to the maintenance of the stem cell population.

Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.