3D Culture Modeling of Metastatic Breast Cancer Cells in Additive Manufactured Scaffolds.

Cancer biology research is increasingly moving toward innovative in vitro 3D culture models, as conventional and current 2D cell cultures fail to resemble in vivo cancer biology. In the current study, porous 3D scaffolds, designed with two different porosities along with 2D tissue culture polystyrene (TCP) plates were used with a model breast cancer human cell line. The 3D engineered system was evaluated for the optimal seeding method (dynamic versus static), adhesion, and proliferation rate of MDA-MB-231 breast cancer cells. The expression profiles of proliferation-, stemness-, and dormancy-associated cancer markers, namely, ki67, lamin A/C, SOX2, Oct3/4, stanniocalcin 1 (STC1), and stanniocalcin 2 (STC2), were evaluated in the 3D cultured cells and compared to the respective profiles of the cells cultured in the conventional 2D TCP. Our data suggested that static seeding was the optimal seeding method with porosity-dependent efficiency. Moreover, cells cultured in 3D scaffolds displayed a more dormant phenotype in comparison to 2D, which was manifested by the lower proliferation rate, reduced ki67 expression, increased lamin A/C expression, and overexpression of STCs. The possible relationship between the cell affinity to different extracellular matrix (ECM) proteins and the RANK expression levels was also addressed after deriving collagen type I (COL-I) and fibronectin (FN) MDA-MB-231 filial cell lines with enhanced capacity to attach to the respective ECM proteins. The new derivatives exhibited a more mesenchymal like phenotype and higher RANK levels in relation to the parental cells, suggesting a relationship between ECM cell affinity and RANK expression. Therefore, the present 3D cell culture model shows that cancer cells on printed scaffolds can work as better representatives in cancer biology and drug screening related studies.

Acrylic Acid Plasma Coated 3D Scaffolds for Cartilage tissue engineering applications.

The current generation of tissue engineered additive manufactured scaffolds for cartilage repair shows high potential for growing adult cartilage tissue. This study proposes two surface modification strategies based on non-thermal plasma technology for the modification of poly(ethylene oxide terephthalate/poly(butylene terephthalate) additive manufactured scaffolds to enhance their cell-material interactions. The first, plasma activation in a helium discharge, introduced non-specific polar functionalities. In the second approach, a carboxylic acid plasma polymer coating, using acrylic acid as precursor, was deposited throughout the scaffolds. Both surface modifications were characterized by significant changes in wettability, linked to the incorporation of new oxygen-containing functional groups. Their capacity for chondrogenesis was studied using ATDC5 chondroblasts as a model cell-line. The results demonstrate that the carboxylic acid-rich plasma coating had a positive effect on the generation of the glucoaminoglycans (GAG) matrix and stimulated the migration of cells throughout the scaffold. He plasma activation stimulated the formation of GAGs but did not stimulate the migration of chondroblasts throughout the scaffolds. Both plasma treatments spurred chondrogenesis by favoring GAG deposition. This leads to the overall conclusion that acrylic acid based plasma coatings exhibit potential as a surface modification technique for cartilage tissue engineering applications.

Tuning Cell Differentiation into a 3D Scaffold Presenting a Pore Shape Gradient for Osteochondral Regeneration.

Osteochondral regeneration remains nowadays a major problem since the outcome of current techniques is not satisfactory in terms of functional tissue formation and development. A possible solution is the combination of human mesenchymal stem cells (hMSCs) with additive manufacturing technologies to fabricate scaffolds with instructive properties. In this study, the differentiation of hMSCs within a scaffold presenting a gradient in pore shape is presented. The variation in pore shape is determined by varying the angle formed by the fibers of two consequent layers. The fiber deposition patterns are 0-90, which generate squared pores, 0-45, 0-30, and 0-15, that generate rhomboidal pores with an increasing major axis as the deposition angle decreases. Within the gradient construct, squared pores support a better chondrogenic differentiation whereas cells residing in the rhomboidal pores display a better osteogenic differentiation. When cultured under osteochondral conditions the trend in both osteogenic and chondrogenic markers is maintained. Engineering the pore shape, thus creating axial gradients in structural properties, seems to be an instructive strategy to fabricate functional 3D scaffolds that are able to influence hMSCs differentiation for osteochondral tissue regeneration.

Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds.

Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells.

Influencing chondrogenic differentiation of human mesenchymal stromal cells in scaffolds displaying a structural gradient in pore size.

UNLABELLED: Articular cartilage lesions have a limited ability to heal by themselves. Yet, golden standard treatments for cartilage repair such as drilling, microfracture and mosaicplasty provide further damage and an unstable solution that degenerates into fibrocartilage in time. Articular cartilage presents a number of gradients in cell number and size along with structural gradients in extra cellular matrix (ECM) composition. Therefore, creating scaffolds that display a structural gradient can be an appealing strategy for cartilage tissue regeneration treatments. In the present study, a scaffold with an in-built discrete gradient in pore size was produced by additive manufacturing. Human mesenchymal stromal cells (hMSCs) were seeded within the gradient scaffolds and their proliferation, differentiation and ECM deposition was evaluated with respect to 2 non-gradient scaffolds. Glycosaminoglycan (GAG) deposition was significantly higher in gradient scaffolds and non-gradient scaffolds with the smallest pore size compared to non-gradient scaffolds with the largest pore size. A gradual increase of chondrogenic markers was observed within the gradient structures with decreasing pore size, which was also accompanied by an increasingly compact ECM formation. Therefore, scaffolds displaying a structural gradient in pore size seem to be a promising strategy to aid in the process of hMSC chondrogenic differentiation and could be considered for improved cartilage tissue regeneration applications. STATEMENT OF SIGNIFICANCE: We present the development of a novel hierarchical scaffold obtained by additive manufacturing. Structural hierarchy is obtained by changing pore size within the pore network characterizing the fabricated scaffolds and proves to be a functional element in the scaffold to influence adult stem cell differentiation in the chondrogenic lineage. Specifically, in regions of the scaffolds presenting smaller pores an increasing differentiation of stem cells toward the chondrogenic differentiation is displayed. Taking inspiration from the zonal organization of articular cartilage tissue, pore size gradients could, therefore, be considered as a new and important element in designing 3D scaffolds for regenerative medicine applications, in particular for all those tissues where gradient physical properties are present.

Surface energy and stiffness discrete gradients in additive manufactured scaffolds for osteochondral regeneration.

Swift progress in biofabrication technologies has enabled unprecedented advances in the application of developmental biology design criteria in three-dimensional scaffolds for regenerative medicine. Considering that tissues and organs in the human body develop following specific physico-chemical gradients, in this study, we hypothesized that additive manufacturing (AM) technologies would significantly aid in the construction of 3D scaffolds encompassing such gradients. Specifically, we considered surface energy and stiffness gradients and analyzed their effect on adult bone marrow derived mesenchymal stem cell differentiation into skeletal lineages. Discrete step-wise macroscopic gradients were obtained by sequentially depositing different biodegradable biomaterials in the AM process, namely poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymers. At the bulk level, PEOT/PBT homogeneous scaffolds supported a higher alkaline phosphatase (ALP) activity compared to PCL, PLA, and gradient scaffolds, respectively. All homogeneous biomaterial scaffolds supported also a significantly higher amount of glycosaminoglycans (GAGs) production compared to discrete gradient scaffolds. Interestingly, the analysis of the different material compartments revealed a specific contribution of PCL, PLA, and PEOT/PBT to surface energy gradients. Whereas PEOT/PBT regions were associated to significantly higher ALP activity, PLA regions correlated with significantly higher GAG production. These results show that cell activity could be influenced by the specific spatial distribution of different biomaterial chemistries in a 3D scaffold and that engineering surface energy discrete gradients could be considered as an appealing criterion to design scaffolds for osteochondral regeneration.