COMPUTER SIMULATION OF A HOLLOW-FIBER BIOREACTOR: HEPARAN REGULATED GROWTH FACTORS-RECEPTORS BINDING AND DISSOCIATION ANALYSIS

This thesis demonstrates the use of numerical simulation in predicting the behavior of proteins in a flow environment. A novel convection-diffusion-reaction computational model is first introduced to simulate fibroblast growth factor (FGF-2) binding to its receptor (FGFR) on cell surfaces and regulated by heparan sulfate proteoglycan (HSPG) under flow in a bioreactor. The model includes three parts: (1) the flow of medium using incompressible Navier-Stokes equations; (2) the mass transport of FGF-2 using convection-diffusion equations; and (3) the cell surface binding using chemical kinetics. The model consists of a set of coupled nonlinear partial differential equations (PDEs) for flow and mass transport, and a set of coupled nonlinear ordinary differential equations (ODEs) for binding kinetics. To handle pulsatile flow, several assumptions are made including neglecting the entrance effects and an approximate analytical solution for axial velocity within the fibers is obtained. To solve the time-dependent mass transport PDEs, the second order implicit Euler method by finite volume discretization is used. The binding kinetics ODEs are stiff and solved by an ODE solver (CVODE) using Newton’s backward differencing formula. To obtain a reasonable accuracy of the biochemical reactions on cell surfaces, a uniform mesh is used. This basic model can be used to simulate any growth factor-receptor binding on cell surfaces on the wall of fibers in a bioreactor, simply by replacing binding kinetics ODEs. Circulation is an important delivery method for natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule’s fate, are difficult to interpret using traditional approaches. Growth factor capture under flow is analyzed and predicted using computer modeling mentioned above and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process is desired. The experimental module consists of a bioreactor with synthetic endotheliallined hollow fibers under flow. The physical design of the system is incorporated into the model parameters. FGF-2 is used for both the experiments and simulations. The computational model is based on the flow and reactions within a single hollow fiber and is scaled linearly by the total number of fibers for comparison with experimental results. The model predicts, and experiments confirm, that removal of heparan sulfate (HS) from the system will result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicts a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters are investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biological or drug delivery investigations. For some complicated binding systems, where there are more growth factors or proteins with competing binding among them moving through hollow fibers of a bioreactor coupled with biochemical reactions on cell surfaces on the wall of fibers, a complex model is deduced from the basic model mentioned above. The fluid flow is also modeled by incompressible Navier-Stokes equations as mentioned in the basic model, the biochemical reactions in the fluid and on the cell surfaces are modeled by two distinctive sets of coupled nonlinear ordinary differential equations, and the mass transports of different growth factors or complexes are modeled separately by different sets of coupled nonlinear partial differential equations. To solve this computationally intensive system, parallel algorithms are devised, in which all the numerical computations are solved in parallel, including the discretization of mass transport equations and the linear system solver Stone’s Implicit Procedure (SIP). A parallel SIP solver is designed, in which pipeline technique is used for LU factorization and an overlapped Jacobi iteration technique is chosen for forward and backward substitutions. For solving binding equations ODEs in the fluid and on cell surfaces, a parallel scheme combined with a sequential CVODE solver is used. The simulation results are obtained to demonstrate the computational efficiency of the algorithms and further experiments need to be conducted to verify the predictions

Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow

Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biologicals or drug delivery investigations

USE OF 3D PRINTED POLY(PROPYLENE FUMARATE) SCAFFOLDS FOR THE DELIVERY OF DYNAMICALLY CULTURED HUMAN MESENCHYMAL STEM CELLS AS A MODEL METHOD TO TREAT BONE DEFECTS

This project investigates the use of a tissue engineering approach of an absorbable polymer, poly(propylene fumarate) (PPF) to provide long term mechanical stability while delivering a bioactive material, precultured human mesenchymal stem cells (hMSC) encapsulated in hydrogel, to repair bone defects. Annually over 2.2 million bone grafting procedures are performed worldwide; however, current treatment options are limited for critically sized and load bearing bone defects. Much progress has been made in development of bone tissue replacements within the field of bone tissue engineering. The combination of a polymer scaffold seeded with cells for the eventual replacement by host tissue has shown significant promise. One such polymer is PPF, a synthetic linear polyester. PPF has been shown to be biocompatible, biodegradable and provide sufficient mechanical strength for bone tissue engineering applications. Additionally PPF is able to be photocrosslinked and therefore can be fabricated into specific geometries using advanced three-dimensional (3-D) rapid prototyping. Current technology to culture and differentiate hMSCs into osteoblasts has been enhanced with the development of the tubular perfusion system (TPS). The TPS bioreactor has been shown to enhance osteoblastic differentiation in hMSCs when encapsulated in alginate beads. Although this system is effective in differentiating hMSCs it lacks the sufficient mechanical strength for the treatment of bone defects. Therefore this work suggests a combination strategy of harnessing the ability of the TPS bioreactor to enhance osteoblastic differentiation with the mechanical properties of poly(propylene fumarate) to develop a porous PPF sleeve scaffold for the treatment of bone defects. This is accomplished through four steps. The first step investigates the cytotoxicity of the polymer PPF. Concurrently the second step focuses on designing, fabricating and characterizing PPF scaffolds. The third step investigates the degradation properties of 3D printed porous PPF scaffolds. The fourth step characterizes alginate bead size and composition for use within the PPF sleeve scaffolds. The successful completion of these aims will develop a functional biodegradable bone tissue engineering strategy that utilizes PPF fabricated scaffolds for use with the TPS bioreactor

Mechanobiology of Stem Cells: Implications for Vascular Tissue Engineering

Current challenges in vascular medicine (e.g., bypass grafting, stenting, and angioplasty.) have driven the field of vascular regenerative medicine. Bone marrow-derived mesenchymal stem cells (BMMSCs) are adult stem cells which may be a suitable cell source for vascular regenerative medicine applications. While it is well known that BMMSCs readily differentiate into musculoskeletal cells, recent studies have provided evidence for their differentiation into smooth muscle cells (SMCs) and endothelial cells (ECs). We and others have demonstrated the ability of the mechanical stimulus of cyclic stretch to drive BMMSC differentiation towards SMCs in vitro, but a rigorous, systematic analysis of other relevant forces is lacking. The working hypothesis that this work addressed is that mechanical stimuli relevant to the vasculature will guide BMMSC differentiation towards SMCs and ECs. To test this hypothesis, rat BMMSCs were exposed to physiologically relevant magnitudes and frequencies of a Mechanical Panel, which consisted of cyclic stretch, cyclic pressure, and shear stress, each applied in parallel to subcultures of BMMSCs. Quantitative changes in morphology, proliferation, and gene and protein expression were assessed to determine the differential effect of each stimulus in a dose- and frequency-dependant manner. Next, we investigated the importance of the duration of applied stimulation to BMMSC differentiation as well as tissue commitment (i.e., cell plasticity) following mechanical stimulation.Our results demonstrate that mechanical stimulation differentially altered BMMSC morphology, proliferation, and gene and protein expression towards the cardiovascular lineage while limiting expression for other lineages including bone, fat, and chondrocyte. This was particularly evident for cyclic stretch, which caused an elongated, spindle-shape and expression of the SMC proteins alpha-actin, calponin, and myosin heavy chain. Furthermore, we found that cyclic pressure and shear stress tended to increase endothelial gene expression when these stimuli are applied to confluent BMMSCs. While our findings as a whole tended to support our hypothesis, our data indicate that SMC protein expression is more readily increased by mechanical stimulation, and is highly variable, even without associated changes in gene expression. Future work employing systems biology approaches that take into consideration the resulting transcriptional and proteomic changes in BMMSCs from these mechanical stimuli will be necessary to more accurately identify how mechanical stimulation can be used as a tool for regenerative medicine

Scaffolds for 3D in vitro culture of neural lineage cells.

Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions for this exciting and important field of biomaterials research

Development of small-scale fluidised bed bioreactor for 3D cell culture

Three-dimensional cell culture has gained significant importance by producing physiologically relevant in vitro models with complex cell-cell and cell-matrix interactions. However, current constructs lack vasculature, efficient mass transport and tend to reproduce static or short-term conditions. The work presented aimed to design a benchtop fluidised bed bioreactor (sFBB) for hydrogel encapsulated cells to generate perfusion for homogenous diffusion of nutrients and, host substantial biomass for long-term evolution of tissue-like structures and “per cell” performance analysis. The sFBB induced consistent fluidisation of hydrogel spheres while maintaining their shape and integrity. Moreover, this system expanded into a multiple parallel units’ setup with equivalent performances enabling simultaneous comparisons. Long term culture of alginate encapsulated hepatoblastoma cells under dynamic environment led to proliferation of highly viable cell spheroids with a 2-fold increase in cellular density over static (27.3 vs 13.4 million cells/mL beads). Upregulation of hepatic phenotype markers (transcription factor C/EBP-α and drug-metabolism CYP3A4) was observed from an early stage in dynamic culture. This environment also affected ERK1/2 signalling pathway, progressively reducing its activation while increasing it in static conditions. Furthermore, culture of primary human mesenchymal stem cells was evaluated. Cell proliferation was not observed but continuous perfusion sustained their viability and undifferentiated phenotype, enabling differentiation into chondrogenic and adipogenic lineages after de-encapsulation. These biological readouts validated the sFBB as a robust dynamic platform and the prototype design was optimised using computer-aided design and computational fluid dynamics, followed by experimental tests. This thesis proved that dynamic environment promoted by fluidisation sustains biomass viability in long-term cell culture and leads 3D cell constructs with physiologically relevant phenotype. Therefore, this bioreactor would constitute a simple and versatile tool to generate in vitro tissue models and test their response to different agents, potentially increasing the complexity of the system by modifying the scaffold or co-culturing relevant cell types

3D Bioprinting Hydrogel for Tissue Engineering an Ascending Aortic Scaffold

The gold standard in 2016 for thoracic aortic grafts is Dacron®, polyethylene terephthalate, due to the durability over time, the low immune response elicited and the propensity for endothelialization of the graft lumen over time. These synthetic grafts provide reliable materials that show remarkable long term patency. Despite the acceptable performance of Dacron® grafts, it is noted that autographs still outperform other types of vascular grafts when available due to recognition of the host\u27s cells and adaptive mechanical properties of a living graft. 3-D bioprinting patient-specific scaffolds for tissue engineering (TE) brings the benefits of non-degrading synthetic grafts and autologous grafts together by constructing a synthetic scaffold that supports cell infiltration, adhesion, and development in order to promote the cells to build the native extracellular matrix in response to biochemical and physical cues. Using the BioBots 3-D bioprinter, scaffold materials we tested non-Newtonian photosensitive hydrogel that formed a crosslinked matrix under 365 nm UV light with appropriate water content and mechanical properties for cell infiltration and adhesion to the bioprinted scaffold. Viscometry data on the PEGDA-HPMC 15%-2% w/v hydrogel (non-Newtonian behavior) informed CFD simulation of the extrusion system in order to exact the pressure-flow rate relationship for every hydrogel and geometry combination. Surface tension data and mechanical properties were obtained from material testing and provide content to further characterize each hydrogel and resulting crosslinked scaffold. The goal of this work was to create a basis to build a database of hydrogels with corresponding print settings and resulting mechanical properties in order to progress the field of tissue engineered vascular grafts fabricated by nozzle-based rapid prototyping

Modern Approaches in Cardiovascular Disease Therapeutics: From Molecular Genetics to Tissue Engineering

Cardiovascular disease (CVD) currently represents one of the leading causes of death worldwide. Each year, more than 17.9 million people die due to CVD manifestations. To reverse these manifestations, the transplantation of secondary vessels or the use of synthetic vascular grafts represents the gold standard procedure. However, significant adverse reactions have been described in the literature regarding the use of these type of grafts. In this regard, modern therapeutic strategies focused on CVD therapeutics must be proposed and evaluated. As alternative therapies, advanced tissue engineering approaches, including decellularization procedures and the 3D additive bio-printing methods, are currently being investigated. In this Special Issue of Bioengineering, we aimed to highlight modern approaches regarding CVD. This Special Issue, entitled “Modern Approaches in Cardiovascular Disease Therapeutics: From Molecular Genetics to Tissue Engineering”, includes 5 articles. These articles are related to the efficient production of small-diameter vascular grafts, vascular graft development with 3D printing approaches, and in vitro models for the improved assessment of atherosclerosis mechanisms. The Guest Editors of this Special Issue wish to express their gratitude to all contributors for their unique and outstanding articles. Additionally, special credit is given to all reviewers for their comprehensive analysis and overall effort in improving the quality of the published articles

Novel Low Shear 3D Bioreactor for the Scaled Production of High Purity Human Mesenchymal Stem Cells

Human mesenchymal stem cells are an ideal candidate for stem cell therapies. They have been researched since the 1960’s and can differentiate into many desired functional cell types without undergoing teratogenesis. However, higher yields are needed for a marketable, successful stem cell therapy. To accomplish this, cells will have to be cultured to expand them to therapeutically relevant dosages for multiple patients. Bioreactor production is an ideal method to solve this problem. The aim of this thesis is to test and validate a novel bioreactor for the cultivation of human mesenchymal stem cells. In this work, we investigate a novel suspended matrix for the culture on human mesenchymal stem cells (hMSCs). Initially we investigated various fiber meshes, both random and structured, for stem cell growth and morphology. We also investigated hMSC proliferation on rigid polymers commonly used in 3D printing. We then took the conditions that worked best in 2D culture and tested them in a small-scale model of the Express bioreactor from Sepragen. We have assessed cell growth on 3D printed Polylactic Acid (PLA) matrices and developed a scale down model bioreactor for development and characterization. Computational Fluid Dynamic (CFD) modeling was used in parallel with the described in-vitro experimentation to characterize shear profiles. From the CFD we were also able to predict a flow rate which resulted in almost zero shear. What we found was that hMSCs readily form confluent monolayers on the PLA lattice, and retain their surface marker expression and stemness. When combined with a short hypoxic treatment, the cells performed better than control flasks, resulting in a four-fold increase from seed with no impact on biomarker profile and differentiation ability

Regulation of mesenchymal stromal cell culture in 3D collagen and NiTi scaffolds by inflammatory and biomechanical factors

The processes of bone fracture healing and bone development share certain similarities and are affected by mechanical loads, the local microenvironment and other factors. In this thesis, an established in vitro fracture callus model was further developed through the introduction of mechanical loading. This system allows for the investigation of the effects of physiological mechanical loads on fracture calluses (engineered endochondral constructs), NiTi-reinforced endochondral constructs and native tissues. Exploring the benefits of rapid-prototyping, shape-memory-alloys and mechanical loading the introduction of a novel, in vitro model for mechanically modulated endochondral ossification is intended. Inflammatory cytokines, which are present in the environment of the fracture site, are important modulators of fracture healing. Thus, the effect of IL-1β on glycosaminoglycan (GAG) production and BMP-2 expression during chondrogenesis and ECM calcification during the hypertrophic phase of in vitro cultures was investigated. These constructs depict an in vitro model for fracture calluses and are therefore used to investigate the effect of IL-1β on the remodeling process, which occurs upon in vivo implantation. It has been demonstrated that IL-1β finely modulates early and late events of the endochondral bone formation by MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC as well as improve the engineering of implantable tissues. Secondary bone fracture healing is a physiological process, which leads to functional tissue regeneration recapitulating endochondral bone formation. Besides other factors, mechanical loading is known to modulate the process of fracture healing. Therefore, a novel perfused compression bioreactor system (PCB) is demonstrated for the investigation of the effect of dynamic mechanical loading on the mineralization process of engineered, hypertrophic constructs. The results obtained demonstrate that dynamic mechanical loading enhances the maturation process of MSC towards late hypertrophic chondrocytes and the mineralization of the extracellular matrix. Moreover, the system possibly allows for the identification of suitable loading regimes to accelerate the process of fracture healing. In order to improve primary implant stability and to upscale endochondral constructs, selective laser melting (SLM)-based NiTi constructs are foreseen to be utilized as a backbone for hypertrophic cartilage templates. NiTi alloys possess a unique combination of mechanical properties including a relatively low elastic modulus, pseudoelasticity, and high damping capacity, matching the properties of bone. Hence, we demonstrated biocompatibility of NiTi-based constructs. Moreover, MSC adhesion, proliferation and differentiation along the osteogenic lineage were similar to MSC cultured on clinically used Ti. When seeded and cultured on porous 3D SLM-NiTi scaffolds, MSC homogeneously colonized the scaffold, and following osteogenic induction, filled the scaffold’s pore volume with extracellular matrix. The combination of bone-related mechanical properties of SLM-NiTi with its cytocompatibility and support of osteogenic properties by MSC highlights its potential as a superior bone substitute as compared to Ti. In conclusion, MSC based chondrogenic and hypertrophic constructs depict in vitro models for soft and hard fracture calluses, respectively. This constructs are responsive to both inflammatory cytokines (IL-1β modulating early and late events of the endochondral bone formation) and dynamic mechanical loading (increased degree of maturation of both MSC and ECM). Moreover, it has been shown that the PCB serves as a promising tool for further systematic studies in an in vitro setting leading to a reduction of animal experiments within the field. Nevertheless, the established models (including mechanically loaded constructs) are not capable of supporting load-bearing fracture sites. Therefore, to overcome the lack of mechanical stability a NiTi-based approach is intended. SLM-NiTi was shown to be biocompatible and MSC do colonize these constructs and differentiate along the osteogenic lineage. Using SLM-NiTi scaffolds as a backbone supporting initial load-bearing, MSC could be used to colonize it and fill the scaffolds pores with a chondrogenic and/or hypertrophic ECM. This construct depicts a NiTi-reinforced, mechanically stable endochondral implant intended for orthotopic implantation