This grant provides funding for research and development of a scientific and engineering knowledge base for studying the effect of bio-deposition process on living cells. The project activities involve: 1) studying the feasibility of depositing living cells and constructing cell-embedded structures; 2) developing an engineering model as well as a multi-scale modeling approach to predict the deposition process-induced mechanical forces and the effect of the process and the mechanical forces on the biological behavior of cells; and 3) quantifying the biological responses of cells, its viability, recovery, proliferation, and damage under varying processing conditions. This project can lead to developing new techniques and tools to help the advancement of emerging cell-based bio-fabrication for broad applications in therapeutic products, biochips and biosensors, diagnostic arrays, microfluidic systems, and pharmaceutical methods.
If successful, this research will foster the convergence of engineering and life sciences, and provide viable manufacturing tools to biology and life sciences community. This project will actively engage industry, government and medical institution. The outcome of research can be applied to help the mission of safe exploration of space, the development of biofabrication product standardizations, and the prioritization of the future cell-based products for tissue engineering manufacturer, thus have broad impacts on the technological development, economic growth, and improvement of the quality of life. This project will also develop a new interdisciplinary course spanning Design/Manufacturing, Biomaterials, and Bio/Cell Mechanics to educate the next generation of scientists and engineers.
For millions of years, cells have been thought of as Nature’s building blocks that make us what we are. Convergence of engineering and life sciences has evolved an emerging field of biofabrication which applies mechanical means to manipulate cells as building blocks to manufacture cell-based devices and products, such as engineered tissue constructs, microfluidic biochips and biosensors for drug delivery and pharmacokinetic studies, "tissue-on-a-chip"/"lab-on-a-chip" devices, and bioprinting cells and organs for regenerative medicine. Employing cells or other bioactive compounds as basic building blocks to "bio-manufacture" cell-integrated biological models offers tremendous opportunities for regenerative tissue substitutes, physiological and pharmacokinetic models, and provides biologists with sophisticated engineering and manufacturing tools to make in vitro biological models for better studying fundamental biological problems. In addition, direct cell deposition/printing can place cells in precise spatial patterns to enhance cell-cell communication, reduce the reliance on cell migration to populate tissue constructs, and create artificial tissue structures that more closely resemble their in vivo state. Bio-deposition is one of the enabling biomanufacturing processes that assemblies living cells through mechanical means. Typically, the process consists of simultaneous deposition of cells, biomaterial and/or growth factors under pressure through a micro-scale size nozzle. However, bioprinting living cells, such as dispensing, extruding, or depositing, can induce damage to cells. It is essential to understand the cell responses to process-induced mechanical disturbances because mechanical forces alter cell morphology and function. This NSF funded project has provided an opportunity to study the effect of bio-deposition process on living cells. The project major outcomes are: 1) Developed a science-based knowledge and engineering model for bio-fabrication of cells-based structures, including a feasible study on bio-deposition of cells and cell-embedded structures, a development of an engineering model to predict the deposition shear force associated with the bio-deposition process parameters, and a development of a multi-scale modeling method to study the shear force and process effect to cells. 2) Quantified the biological responses of living cells to the bio-deposition process under varying processing parameters, including the assessment of cell survival during deposition (viability), cell recovery or proliferation, and cell damage as well as its possible correlation with process associated mechanical force. 3) Statistical analysis showed that different cell survival responses and the average numbers representing the percentage of live, dead and apoptotic cells under the combined effects of pressure and nozzle diameter were significantly different at statistically significant levels P<0.05. The experimental results further showed different cell survival responses as a result of altering dispensing pressure compared to altering the printing nozzle diameter. 4) Developed educational components to training REU/RET participants and high school students to the proposed research, and developed the lecturing modulus on bioprinting as well as the associated lab modulus for teaching curriculum. 5) Published peer-reviewed technical articles and conducted invited seminars and conference presentations to broadly disseminate the latest research finding on bioprinting. The project major findings are: 1) Percentage live cells as a function of dispensing pressure and nozzle diameter was observed. 2) Significant effect of pressure on cell viability was observed and the probability distributions of live cells for test samples printed with 150m nozzle diameter at different pressures were obtained. 3) The experimental results reveal that the effect of pressure is significantly larger than the effect of the nozzle diameter. At higher pressures, there is an increase in the number of injured cells as well as necrotic cells. This is because normal cells respond to stress and injurious stimuli by undergoing adaptation. Cells which are unable to adapt, undergo cell injury followed by cell death. 4) Statistical analysis proved that all the average numbers representing the percentage of live, dead and apoptotic cells taking into account the combined effects of pressure and nozzle diameter were significantly different at statistically significant level P <0.05. 5) Varying degrees of nuclear damage resulting from the printing process were observed and can be detected by using the DNA stain along with the membrane stain for visualizing the morphological changes in the samples. 6) Compared to the control samples and samples printed under moderate process parameters (400microns nozzle tip diameter and dispensing pressure of 5psi), samples exposed to extreme conditions (nozzle tip diameter of 150microns and high dispensing pressure of 40psi) indicated pyknosis as well as karyolysis (Figures 2a-c) which lead to cell death and low cell viability. Clearly, high pressure and small nozzle diameters induce more damage to the cells as reflected quantitatively in the Annexin V kit and qualitatively in the DNA staining. 7) A phenomenological model was developed to correlate the percentage of live, apoptotic and necrotic cells to the process parameters. 8) An analytical formulation to predict the cell viability through the system as a function of the maximum shear stress in the system. 9) Study shows dispensing pressure has significant effect on cell viability compared to nozzle diameter.
