Tissue Formation and Remodeling in Tissue Engineered Pulmonary Conduits

Over the past decade, the tissue engineering paradigm has gained attention as a potential means to restore native tissue functionality. Although attractive, the wide variety of scaffold materials, cell sources, and mechanical conditioning regimes coupled with the paucity of structurally-based, finite deformation framework constitutive models found in the literature hinders the elucidation of extracellular matrix (ECM) formation and remodeling in engineered tissues. Therefore, the overall objective of this work is to develop structurally guided generalized finite deformation based constitutive models than can be used to gain an understanding of tissue formation and remodeling in tissue engineering applications. Further, it is the intent of this work to apply such an approach to investigate tissue formation and remodeling in tissue engineered pulmonary arteries.In the first part of this work, a novel technique for acquiring and quantifying high resolution three dimensional structural data was used on bone-marrow stem cell-seeded polymeric scaffold composites, and it was shown that the continuous anisotropic scaffold phase transitioned to a highly discontinuous isotropic scaffold phase after twelve weeks in vivo. Next, structural constitutive models were developed based on the scaffold continuity. For continuous scaffold composites, scaffold-ECM interactions were included in the model as extensional and shearing terms, while it was shown that such effects were negligible in the discontinuous scaffold composites. A parameter estimation and model validation procedure was described using a tunable tissue-analog system of polyacrylamide (PAM) gel. It was found that the scaffold-ECM interaction due to fiber extension was highly non-linear, showing a reinforcing effect larger than from rule of mixtures predictions. Experimental validation with PAM gel supported the models. Finally, both models were used to investigate tissue formation and remodeling in in vivo engineered pulmonary arteries. At early timepoints (7 days), little change in ECM mechanical properties was observed. In later timepoints (42 to 140 days), the collagen effective modulus and collagen recruitment parameters changed substantially, suggesting collagen maturation via increased cross-linking and crimp organization. Ultimately, a methodical approach to understanding tissue formation and remodeling via structural constitutive models was presented and successfully applied to a clinically-relevant tissue engineering system

The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering.

The permeability of scaffolds and other three-dimensional constructs used for tissue engineering applications is important as it controls the diffusion of nutrients in and waste out of the scaffold as well as influencing the pressure fields within the construct. The objective of this study was to characterize the permeability/fluid mobility of collagen-GAG scaffolds as a function of pore size and compressive strain using both experimental and mathematical modeling techniques. Scaffolds containing four distinct mean pore sizes (151, 121, 110, 96 microns) were fabricated using a freeze-drying process. An experimental device was constructed to measure the permeability of the scaffold variants at different levels of compressive strain (0, 14, 29 and 40% while a low-density open-cell foam cellular solids model utilizing a tetrakaidecahedral unit cell was used to accurately model the permeability of each scaffold variant at all level of applied strain. The results of both the experimental and the mathematical analysis revealed that scaffold permeability increases with increasing pore size and decreases with increasing compressive strain. The excellent comparison between experimentally measured and predicted scaffold permeability suggests that cellular solids modelling techniques can be utilized to predict scaffold permeability under a variety of physiological loading conditions as well as to predict the permeability of future scaffolds with a wide variety of pore microstructures

Evaluating the Effect of Non-cellular Bioactive Glass-Containing Scaffolds on Osteogenesis and Angiogenesis in in vivo Animal Bone Defect Models

The use of bone scaffolds to replace injured or diseased bone has many advantages over the currently used autologous and allogeneic options in clinical practice. This systematic review evaluates the current evidence for non-cellular scaffolds containing bioactive glass on osteogenesis and angiogenesis in animal bone defect models. Studies that reported results of osteogenesis via micro-CT and results of angiogenesis via Microfil perfusion or immunohistochemistry were included in the review. A literature search of PubMed, EMBASE and Scopus was carried out in November 2019 from which nine studies met the inclusion and exclusion criteria. Despite the significant heterogeneity in the composition of the scaffolds used in each study, it could be concluded that scaffolds containing bioactive glass improve bone regeneration in these models, both by osteogenic and angiogenic measures. Incorporation of additional elements into the glass network, using additives, and using biochemical factors generally had a beneficial effect. Comparing the different compositions of non-cellular bioactive glass containing scaffolds is however difficult due to the heterogeneity in bioactive glass compositions, fabrication methods and biochemical additives used

Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites

Engineering molecular recognition and catalysis - examples of designed peptides and stimuli responsive materials

Biomimetics are a growing field with several applications in Bioengineering. This work focuses on two examples of biomimetic systems applied to the fields of Biocatalysis and Biosensing. Enzymes are highly versatile catalysts present in biological systems with a coveted technological potential. Enzymes tend to be large and complex proteins and the development of simpler Biomimetic catalysts has been a long-term goal. In this work, two computationally designed protein-based peptides, RD01v2 and RD02, mimicking metalloprotease activity, were synthesized by SPPS and purified by Preparative HPLC Chromatography. The characterization process was divided in folding studies by far-UV CD spectroscopy and hydrolytic activity studies. All the assays were accomplished for a range of pH from 7 to 10, with and without added Zinc (II) at constant temperature. A Zinc titration at all these pH values, with the mentioned conditions, was performed to calculate the apparent dissociation constant (KpepZn,App in the range of 105 M-1). RD peptides presented low catalytic hydrolytic activity towards 4-nPA with second order rate constant (k2 <1 M-1s-1). The second exampled studied included the development of stimuli responsive materials mimicking the sense of olfaction

Engineering PNIPAAm Biomaterial Scaffolds to Model Microenvironmental Regulation of Glioblastoma Stem-Like Cells

abstract: Following diagnosis of a glioblastoma (GBM) brain tumor, surgical resection, chemotherapy and radiation together yield a median patient survival of only 15 months. Importantly, standard treatments fail to address the dynamic regulation of the brain tumor microenvironment that actively supports tumor progression and treatment resistance. Moreover, specialized niches within the tumor microenvironment maintain a population of highly malignant glioblastoma stem-like cells (GSCs). GSCs are resistant to traditional chemotherapy and radiation therapy and are likely responsible for near universal rates of tumor recurrence and associated morbidity. Thus, disrupting microenvironmental support for GSCs could be critical to more effective GBM therapies. Three-dimensional (3D) culture models of the tumor microenvironment are powerful tools for identifying key biochemical and biophysical inputs that may support or inhibit malignant behaviors. Here, we developed synthetic poly(N-isopropylacrylamide-co-Jeffamine M-1000® acrylamide) or PNJ copolymers as a model 3D system for culturing GBM cell lines and low-passage patient-derived GSCs in vitro. These temperature responsive scaffolds reversibly transition from soluble to insoluble in aqueous solution by heating from room temperature to body temperature, thereby enabling easy encapsulation and release of cells in a 3D scaffold. We also designed this system with the capacity for presenting the cell-adhesion peptide sequence RGD for adherent culture conditions. Using this system, we identified conditions that promoted GBM proliferation, invasion, GSC phenotypes, and radiation resistance. In particular, using two separate patient-derived GSC models, we observed that PNJ scaffolds regulated self-renewal, provided protection from radiation induced cell death, and may promote stem cell plasticity in response to radiation. Furthermore, PNJ scaffolds produced de novo activation of the transcription factor HIF2α, which is critical to GSC tumorigenicity and stem plasticity. All together, these studies establish the robust utility of PNJ biomaterials as in vitro models for studying microenvironmental regulation of GSC behaviors and treatment resistance.Dissertation/ThesisDoctoral Dissertation Biomedical Engineering 201

Introducing monitoring and automation in cartilage tissue engineering, toward controlled clinical translation

The clinical application of tissue engineered products requires to be tightly connected with the possibility to control the process, assess graft quality and define suitable release criteria for implantation. The aim of this work is to establish techniques to standardize and control the in vitro engineering of cartilage grafts. The work is organized in three sub-projects: first a method to predict cell proliferation capacity was studied, then an in line technique to monitor the draft during in vitro culture was developed and, finally, a culture system for the reproducible production of engineered cartilage was designed and validated. Real-time measurements of human chondrocyte heat production during in vitro proliferation. Isothermal microcalorimetry (IMC) is an on-line, non-destructive and high resolution technique. In this project we aimed to verify the possibility to apply IMC to monitor the metabolic activity of primary human articular chondrocytes (HAC) during their in vitro proliferation. Indeed, currently, many clinically available cell therapy products for the repair of cartilage lesions involve a process of in vitro cell expansion. Establishing a model system able to predict the efficiency of this lengthy, labor-intensive, and challenging to standardize step could have a great potential impact on the manufacturing process. In this study an optimized experimental set up was first established, to reproducible acquire heat flow data; then it was demonstrated that the HAC proliferation within the IMC-based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. Finally, based on the results from 12 independent donors, the possible predictive potential of this technique was assessed. Online monitoring of oxygen as a non-destructive method to quantify cells in engineered 3D tissue constructs. This project aimed at assessing a technique to monitor graft quality during production and/or at release. A quantitative method to monitor the cells number in a 3D construct, based on the on-line measurement of the oxygen consumption in a perfusion based bioreactor system was developed. Oxygen levels dissolved in the medium were monitored on line, by two chemo-optic flow-through micro-oxygen sensors connected at the inlet and the outlet of the bioreactor scaffold chamber. A destructive DNA assay served to quantify the number of cells at the end of the culture. Thus the oxygen consumption per cell could be calculated as the oxygen drop across the perfused constructs at the end of the culture period and the number of cells quantified by DNA. The method developed would allow to non-invasively monitoring in real time the number of chondrocytes on the scaffold. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing. The aim of this project was to upscale the size of engineered human cartilage grafts. The main aim of this project consisted in the design and prototyping of a direct perfusion bioreactor system, based on fluidodynamic models (realized in collaboration with the Institute for Bioengineering of Catalonia, Spain), able to guarantee homogeneous seeding and culture conditions trough the entire scaffold surface. The system was then validated and the capability to reproducibly support the process of tissue development was tested by histological, biochemical and biomechanical assays. Within the same project the automation of the designed scaled up bioreactor system, thought as a stand alone system, was proposed. A prototype was realized in collaboration with Applikon Biotechnology BV, The Netherlands. The developed system allows to achieve within a closed environment both cell seeding and culture, controlling some important environmental parameters (e.g. temperature, CO2 and O2 tension), coordinating the medium flow and tracking culture development. The system represents a relevant step toward process automation in tissue engineering and, as previously discussed, enhancing the automation level is an important requirement in order to move towards standardized protocols of graft generation for the clinical practice. These techniques will be critical towards a controlled and standardized procedure for clinical implementation of tissue engineering products and will provide the basis for controlled in vitro studies on cartilage development. Indeed the resulting methods have already been integrated in a streamlined, controlled, bioreactor based protocol for the in vitro production of up scaled engineered cartilage drafts. Moreover the techniques described will serve as the foundation for a recently approved Collaborative Project funded by the European Union, having the goal to produce cartilage tissue grafts. In order to reach this goal the research based technologies and processes described in this dissertation will be adapted for GMP compliance and conformance to regulatory guidelines for the production of engineered tissues for clinical use, which will be tested in a clinical trial