Porcine mammary fatty tissues represent an abundant source of natural biomaterial for generation of breast-specific extracellular matrix (ECM). Here we report the extraction of total ECM proteins from pig breast fatty tissues, the fabrication of hydrogel and porous scaffolds from the extracted ECM proteins, the structural properties of the scaffolds (tissue matrix scaffold, TMS), and the applications of the hydrogel in human mammary epithelial cell spatial cultures for cell surface receptor expression, metabolomics characterization, acini formation, proliferation, migration between different scaffolding compartments, and in vivo tumor formation. This model system provides an additional option for studying human breast diseases such as breast cancer.
Background: Breast cancer cells invading the connective tissues outside the mammary lobule or duct immerse in a reservoir of extracellular matrix (ECM) that is structurally and biochemically distinct from that of their site of origin. The ECM is a spatial network of matrix proteins, which not only provide physical support but also serve as bioactive ligands to the cells. It becomes evident that the dimensional, mechanical, structural, and biochemical properties of ECM are all essential mediators of many cellular functions. To better understand breast cancer development and cancer cell biology in native tissue environment, various tissue-mimicking culture models such as hydrogel have been developed. Collagen I (Col I) and Matrigel are the most common hydrogels used in cancer research and have opened opportunities for addressing biological questions beyond the two-dimensional (2D) cell cultures. Yet, it remains unclear whether these broadly used hydrogels can recapitulate the environmental properties of tissue ECM, and whether breast cancer cells grown on CoI I or Matrigel display similar phenotypes as they would on their native ECM. Methods: We investigated mammary epithelial cell phenotypes and metabolic profiles on animal breast ECMderived tissue matrix gel (TMG), Col I, and Matrigel. Atomic force microscopy (AFM), fluorescence microscopy, acini formation assay, differentiation experiments, spatial migration/invasion assays, proliferation assay, and nuclear magnetic resonance (NMR) spectroscopy were used to examine biological phenotypes and metabolic changes. Student's t test was applied for statistical analyses. Results: Our data showed that under a similar physiological stiffness, the three types of hydrogels exhibited distinct microstructures. Breast cancer cells grown on TMG displayed quite different morphologies, surface receptor expression, differentiation status, migration and invasion, and metabolic profiles compared to those cultured on Col I and Matrigel. Depleting lactate produced by glycolytic metabolism of cancer cells abolished the cell proliferation promoted by the non-tissue-specific hydrogel. Conclusion: The full ECM protein-based hydrogel system may serve as a biologically relevant model system to study tissue-and disease-specific pathological questions. This work provides insights into tissue matrix regulation of cancer cell biomarker expression and identification of novel therapeutic targets for the treatment of human cancers based on tissue-specific disease modeling.
Developing a high-efficiency manufacturing system for personalized medicine plays an important role in increasing the feasibility of personalized medication. The purpose of this study is to investigate the feasibility of a new extrusion-based fabrication process for personalized drugs with a faster production rate. This process uses two syringe pumps with a coaxial needle as an extruder, which extrudes two materials with varying ratios into a capsule. The mixture of hydrogel, polyethylene glycol (PEG), hydroxypropyl methylcellulose, poly acrylic acid and the simulated active pharmaceutical ingredient, Aspirin, was used. To validate the method, samples with different ratios of immediate release (IR) and sustained release (SR) mixtures were fabricated. The results of a dissolution test show that it is feasible to control the release profile by changing the IR and SR ratio using this fabrication setup. The fabrication time for each capsule is about 20 seconds, which is significantly faster than the current 3D printing methods. In conclusion, the proposed fabrication method shows a clear potential to step toward the feasibility of personalized medication.
Tubular structures of hydrogel are used in a variety of applications such as 3D cell culturing for delivery of nutrient supplies. The wall thickness of the tube determines the speed of diffusion or delivery rate. In this study, we aimed to fabricate tubular structures with varying of wall thicknesses using a thermal-crosslinking hydrogel, gellan gum, with the coaxial needle approach. The wall thickness is controlled by changing the flow rate ratio between the inner (phosphate-buffered saline) and outer needles (gellan gum). A simulation model was developed to estimate the proper extrusion speed to allow the gellan gum to be extruded around its glass transition temperature. While keeping the extrusion rate of gellan gum fixed, different PBS extrusion rates were tested to investigate the printability to form continuous tubular structures, range of printable wall thickness, and possibility to form tubes with closed ends to encapsulate fluid or drug inside the tube. The ranges of printable wall thickness with two pairs of coaxial needle were identified. It was found that at about 200% of the baseline PBS extrusion speed, a maximum of 20% difference in wall thickness can be achieved, while a close end can still be formed.
Coaxial extrusion is a commonly used process to manufacture tubular structures to mimic vascular systems in 3D bioprinting. In this study, the stability of coaxial extrusion of a non-Newtonian material, Pluronic F127, is investigated. The extrusion process is considered stable when the extrudate form a core-annular structure. When it is unstable, dripping or jetting of the inner fluid is observed. In this study, the effects of the viscosity ratio, flow rate ratio, and the non-Newtonian behaviors on the stability of the coaxial extrusion process are investigated experimentally and numerically. The results show that all three factors can affect the stability of the process. When the ratio of viscosities increases, the process becomes unstable. The extrusion process tends to be stable when the flow rate of the outer fluid is much higher than that of the inner fluid. When the overall flow rate decreases, due to the non-Newtonian fluid behavior, the extrusion process can become unstable. This study shows the interconnected relationship between viscosity, flow rate, and non-Newtonian fluid behaviors and their effects on the stability of the coaxial extrusion process. The non-Newtonian flow behavior needs to be considered when studying or using coaxial extrusion. This study also provides a guiding principle on how to alter extrusion parameters in order to achieve the desired flow pattern.
Tubular structures of the hydrogel are used in a variety of applications such as delivering nutrient supplies for 3D cell culturing. The wall thickness of the tube determines the delivery rate. In this study, we used the coaxial extrusion process to fabricate tubular structures with varying wall thicknesses using a thermal-crosslinking hydrogel, gellan gum (GG). The objectives of this study are to investigate the thermal extrusion process of GG to form tubular structures, the range of achievable wall thickness, and a possibility to form tubular structures with closed ends to encapsulate fluid or drug inside the tube. The wall thickness is controlled by changing the relative flow velocity of the inner needle (phosphate-buffered saline, PBS) to the outer needle, while keeping the velocity of outer needles (GG) constant. Two pairs of coaxial needles were used which are 18-12 gauge (G) and 20-12G. The controllable wall thickness ranges from 0.618 mm (100% relative velocity) to 0.499 mm (250%) for 18-12G and from 0.77 mm (80%) to 0.69 (200%) for 20-12G. Encapsulation is possible in a smaller range of flow velocities in both needle combinations. A finite element model was developed to estimate the temperature distribution and the wall thickness. The model is found to be accurate. The dynamic viscosity of GG determines the pressure equilibrium and the range of achievable wall thickness. Changing the inner needle size or the flow velocity both affect the heat exchange and thus the temperature-dependent dynamic viscosity.
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