archives@tulane.edu / 1 / Benjamin Vinson

Tissue engineering is an emerging field aimed to combine biological, engineering and material methods to create a biomimetic three dimensional (3D) environment to control cells proliferation and functional tissue formation. In such an artificial structural environment, a scaffold, made from biomaterial(s), plays an essential role by providing a mechanical support and biological guidance platform. Hence, fabrication of tissue scaffolds is of a fundamental importance, yet a challenging task, in tissue engineering. This task becomes more challenging if living cells need to be encapsulated in the scaffolds so as to fabricate scaffolds with structures to mimic the native ones, mainly due to the issue of process-induced cell damage. This research aims to develop novel methods to model the process of fabricating cell-encapsulated scaffolds and process-induced cell damage. Particularly, this research focuses on the scaffold fabrication process based on the dispensing-based rapid prototyping technique - one of the most promising scaffold fabrication methods nowadays, by which a 3D scaffold is fabricated by laying down multiple, precisely formed layers in succession. In the dispensing-based scaffold fabrication process, the flow behavior of biomaterials solution can significantly affect the flow rate of material dispensed, thus the structure of scaffold fabricated. In this research, characterization of flow behavior of materials was studied; and models to represent the flow behaviour and its influence on the scaffold structure were developed. The resultant models were shown able to greatly improve the scaffold fabrication in terms of process parameter determination. If cells are encapsulated in hydrogel for scaffold fabrication, cell density can affect the mechanical properties of hydrogel scaffolds formed. In this research, the influence of cell density on mechanical properties of hydrogel scaffolds was investigated. Furthermore, finite element analysis (FEA) of mechanical properties of scaffolds with varying cell densities was performed.The results show that the local stress and strain energy on cells varies at different cell densities. The method developed may greatly facilitate hydrogel scaffolds design to minimize cell damage in scaffold and promote tissue regeneration. . In the cell-encapsulated scaffold fabrication process, cells inevitably suffer from mechanical forces and other process-induced hazards. In such a harsh environment, cells deform and may be injured, even damaged due to mechanical breakage of cell membrane. In this research, three primary physical variables: shear stress, exposure time, and temperature were examined and investigated with regard to their effects on cell damage. Cell damage laws through the development phenomenal models and computational fluidic dynamic (CFD) models were established; and their applications to the cell-encapsulated scaffold fabrication process were pursued. The results obtained show these models and modeling methods not only allow one to optimize process parameters to preserve cell viability but also provide a novel strategy to probe cell damage mechanism in microscopic view.

We have developed a new biofabrication process in which the precise control of bacterial motion is used to fabricate customizable networks of cellulose nanofibrils. This work describes how the motion of Acetobacter xylinum can be controlled by electric fields while the bacteria simultaneously produce nanocellulose, resulting in networks with aligned fibers. Since the electrolysis of water due to the application of electric fields produces the oxygen in the culture media far from the liquid-air boundary, aerobic cellulose production in 3D structures is readily achievable. Five separate sets of experiments were conducted to demonstrate the assembly of nanocellulose by Acetobacter xylinum in the presence of electric fields in micro and macro environments. This work demonstrates a new concept of bottom up material synthesis by control of a biological assembly process. / Master of Science

The vascular system, responsible for the transport of nutrients, oxygen, and waste removal, overcomes the limitations of oxygen diffusion in solid tissues through blood perfusion, thereby preventing necrosis. The mechanical stimuli from blood flow are pivotal for vascular development and engineering, influencing endothelial cell morphology and vessel remodeling via mechanosensing. Current organ-on-chip systems, while successful in applying dynamic flow to endothelial cells, have limitations, including dependency on pumps and confinement to closed microfluidic channels. Additionally, the interaction between immune cells and these systems under long-term recirculating flow conditions has not been adequately demonstrated. This thesis introduces a novel biofabrication and device manufacturing technique that utilizes a flexible, patternable sacrificial material on a 2D surface. This material morphs in response to an aqueous hydrogel and then degrades, forming perfusable vascular networks within a natural hydrogel matrix. We achieved perfusion using a rocker mechanism that periodically changes tilt direction, while the open-well design facilitates the visualization of perfusable tubular tissues via clinical ultrasound imaging and the construction of complex, vascularized hepatic tissues embedded in gel matrices (Chapter 2). To mimic the unidirectional recirculating flow characteristic of blood vessels, we created the UniPlate platform, combining injection molding with 3D printing (Chapter 3). This innovation allows for the perfusion and recirculation of monocytes through vascular channels without compromising cell viability or eliciting an inflammatory response. Furthermore, by integrating cancer spheroids into the vascular tissues on UniPlate, we developed a vascularized cancer spheroid model that exhibited temporally dependent and tissue-specific macrophage recruitment toward tumor sites with continuous monocyte recirculation (Chapter 4). Collectively, this series of research work introduces a versatile and robust platform capable of replicating vascular functions and immune responses, offering a substantial advancement in the investigation of vascular biology and the mechanism of disease progression. / Thesis / Doctor of Philosophy (PhD) / Vascular networks of the circulatory system are crucial organs in the body, determining the life and death of tissues and organisms by distributing nutrients and oxygen throughout the body. Dysfunction in blood vessel systems is closely related to clinical diseases such as stroke, atherosclerosis, tumor angiogenesis, and cancer metastasis. Mechanical stimuli in blood vessels play a crucial role in regulating the structure and function of endothelial cells during in vivo embryonic development and in vitro vascular tissue formation. Understanding and mimicking the complex environment of blood vessels is vital for studying diseases related to endothelial dysfunction. In this thesis, we introduce a novel subtractive manufacturing method to create three-dimensional (3D) perfusable tubular tissues within a hydrogel. Unidirectional recirculating flow, stromal cells and spheroids, as well as circulating immune cells, were then introduced to the engineered vascular tissues to develop more complex tissue models. These models reproduce the cell diversity, 3D structure, mechanical stimuli, and immune components found in the native tissue microenvironment, providing a valuable tool for the study of vascular diseases and the development of potential treatments.

Over the past three decades, the field of tissue engineering has witnessed significant advancements. However, a persistent challenge is the development of an approach to generate rapidly perfused vascular networks at scale to support engineered tissues of appreciable size and able to adapt to changing needs. Current techniques able to create perfusable channels such as 3D printing are resource intensive and have not overcome the inherent tradeoff between vessel resolution and assembly time, limiting their utility and scalability for engineering tissues. Here we present two sacrificial self-assembly techniques that collectively develop microvascular networks and can anastomose to a variety of engineered forms. The first is vasculogenic cellular self-assembly, which leverages the innate ability of endothelial and sacrificial support cells to spontaneously form a capillary network, which we term CAMEO, or Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis. By varying the removal timing of the support cells, we determine fibroblasts are necessary for the initial vascular morphogenesis in our engineered system, and that this initial support period is sufficient for the endothelial cells to form a perfusable vasculogenic network and enhance the function of primary hepatocyte aggregates. The second is a flexible and scalable technique we term SPAN – Sacrificial Percolation of Anisotropic Networks. It uses microvascular-scale sacrificial fibers that make contacts to span a volume above a percolation density threshold and are then degraded. The resulting interconnected anisotropic voids form a perfusable fluidic network within minutes. We show that SPAN relieves hypoxia compared to bulk gels only, and the resulting voids created by SPAN can be endothelialized in a scalable way. These simple platforms can generate conduits with length scales spanning arterioles to capillaries within constructs. We show that both techniques can be used in combination with common tissue engineering processes, paving the way for rapid assembly of adaptable and perfusable tissues. / 2026-01-17T00:00:00Z

<p dir="ltr">Approximately 2.39 million Americans suffer from inflammatory bowel disease (IBD), an autoimmune disorder that is characterized by chronic inflammation of the gastrointestinal (GI) tract. Current treatment options for IBD, which are limited, include oral medications, surgery, and supportive care. These therapeutics often times are not effective and are associated with high toxicity. Thus, there is a pressing clinical need for a therapy that can be delivered both locally and precisely, while also having an improvement in efficacy and lower toxicity.</p><p dir="ltr">This study introduces three novel microrobot designs fabricated using stereolithography (SLA) 3D printing, which aims to address the challenges seen in IBD treatment. The microrobots utilize a reservoir design to encapsulate the drug for an on-demand release, allowing for improved control and precision. The SLA microrobots were evaluated for cytotoxicity as well as drug release capabilities. We were able to demonstrate a local release of a protein on-demand at a biologically relevant temperature. The integration of microrobots in IBD therapy has the capability to significantly improve patient outcomes and quality of life, offering a more efficient and less toxic treatment approach.</p>

L’Ingénierie tissulaire (IT) est un domaine interdisciplinaire qui applique les principes de l'ingénierie et des sciences de la vie au développement de substituts biologiques afin de restaurer, maintenir ou améliorer la fonction tissulaire. Sa première application consiste à remplacer les tissus endommagés par des produits cellulaires artificiels. Une autre application de l’IT est basée sur la production des modèles en 2 et 3 dimensions (2D et 3D) pour des études biologiques et pharmacologiques in vitro. Ces modèles ou remplacements de tissus peuvent être fabriqués en utilisant des différentes méthodes de médecine, biologie, chimie, physique, informatique et mécanique, fournissant un micro-environnement spécifique avec différents types de cellules, facteurs de croissance et matrice. L'un des principaux défis de l'IT la pénétration cellulaire limitée dans les parties internes des biomatériaux poreux. Une faible viabilité cellulaire au centre du produit d'IT est la conséquence de la diffusion limitée d'oxygène et de nutriments du fait d’un réseau vasculaire insuffisant dans l'ensemble de la construction 3D. Le BioAssembage couche-par-couche est une nouvelle approche basée sur l'assemblage de petites constructions cellularisées permettant une distribution cellulaire homogène et une vascularisation plus efficace dans des produits d’IT.Notre hypothèse est que l'approche couche-par-couche est plus adaptée à la régénération osseuse que l'approche conventionnelle de l'IT. L'objectif principal de cette thèse était d'évaluer les avantages de l'approche couche-par-couche en utilisant des membranes de polymères imprimées en 3D et ensemencées avec des cellules primaires humaines. Nous avons évalué l'efficacité de la formation du réseau vasculaire in vivo dans toute la construction 3D en utilisant cette approche et en la comparant à l'approche conventionnelle basée sur l'ensemencement des cellules sur la surface des scaffolds massives. Il n'y avait pas de différence significative dans le nombre de vaisseaux sanguins formés en 3D au niveau des parties externes des constructions implantées en site souscutanée chez des souris. Mais dans les parties internes des implants qui n'étaient pas en contact direct avec un tissu hôte, nous avons pu observer une formation des vaisseaux sanguins statistiquement plus efficace lorsque l'approche du bio-assemblage couche-par-couche a été utilisée. Cette formation de réseau vasculaire était plus importante dans le cas de co-cultures que de mono-cultures.Il y avait plusieurs objectifs secondaires dans ce travail. Le premier était de fabriquer des constructions 3D cellularisées pour l'IT en utilisant des membranes d'acide polylactique (PLA) et des cellules primaires humaines : des cellules de stroma de moelle osseuse humaine (HBMSCs) isolées de la moelle osseuse et des cellules progénitrices endothéliales (EPCs) isolées du sang du cordon ombilical. Ensuite, nous avons comparé différentes technologies de fabrication des scaffolds: impression 3D directe à partir de poudre de PLA et impression par fil fondu en utilisant une imprimante commerciale et une autre fabriquée sur mesure. L'imprimante sur mesure a permis le plus haut niveau de résolution d'impression spécialement adaptée à la forme et la taille des pores. Par ailleurs, nous avons évalué différents systèmes de stabilisation pour l'assemblage couche par couche : l’utilisation de clips en PLA imprimés en 3D a fourni une stabilisation plus efficace pour empiler les membranes PLA couche par couche. Un autre avantage de ce système de stabilisation est qu'il peut être implanté avec des implants. Ensuite, nous avons observé une prolifération et une différenciation cellulaire plus efficaces lorsque le système de co-culture était utilisé, en comparaison avec des mono-cultures.L'approche du bioassemblage couche-par-couche semble être une solution appropriée pour une vascularisation efficace dans des structures 3D entières d'ingénierie tissulaire. / Tissue Engineering (TE) is “an interdisciplinary field that applies principles of engineering and the life sciences toward development of biological substitutes that restore, maintain, or improve tissue function”. The First application of TE is to replace damaged tissues by artificial cell-materials products of tissue engineering (TE). Another TE application is to produce 2 or 3 dimensional (2D and 3D) models for biological and pharmacological in vitro studies. These models or tissue replacements can be fabricated using a combination of different interdisciplinary methods of medicine, biology, chemistry, physics, informatics and mechanics, providing specific micro-environment with different cell types, growth factors and matrix.One of the major challenges of tissue engineering is related to limited cell penetration in the inner parts of porous biomaterials. Poor cell viability in the center of engineered tissue is a consequence of limited oxygen and nutrients diffusion due to insufficient vascular network within the entire construct. Layer-by-layer (LBL) BioAssembly is a new approach based on assembly of small cellularized constructs that may lead to homogenous cell distribution and more efficient three dimensional vascularization of large tissue engineering constructs.Our hypothesis is that LBL Bioassembly approach is more suitable for bone regeneration than conventional tissue engineering approach. The primary objective of this thesis was to evaluate the advantages of LBL Bioassembly approach using 3D-printed polymer membranes seeded with human primary cells. We have evaluated the efficiency of vascular network formation in vivo within entire 3D tissue engineering construct using LBL bioassembly approach and comparing it to the conventional approach based on seeding of cells on the surface of massive 3D scaffolds. There was no significant difference in number of formed blood vessels in 3D at the outer parts of constructs implanted subcutaneously in mice 8 weeks post-implantation. But in the inner parts of implants which were not in direct contact with a host tissue, we could observe statistically more blood vessel formation when LBL bioassembly approach was used. This vascular network formation was more important in the case of co-cultures than mono-vultures of HBMSCs.There were several secondary objectives in this work. The first was to fabricate cellularized 3D constructs for bone tissue engineering using poly(lactic) acid (PLA) membranes and human primary cells: human bone marrow stroma cells (HBMSCs) isolated from the bone marrow, and endothelial progenitor cells (EPCs) isolated from the umbilical cord blood. Then, we have compared different Additive manufacturing technologies to fabricate scaffolds: direct 3D printing (3DP) starting from PLA powder dissolved in chloroform and fused deposition modelling (FDM) using a commercial or a custom-made printer with different resolutions.The custom-made printer equipped with 100 μm nozzle allowed the highest level of printing resolution concerning pores shape and size. In the meantime we evaluated different stabilization systems for layer-by-layer assembling of PLA membranes with human primary cells: the use of 3D printed PLA clips provided the most efficient stabilization to stack PLA membranes in 3D. Another advantage of this stabilization system is that it could be implanted together with LBL constructs. Then we investigated the most suitable cell culture system for such constructs and we observed more efficient cell proliferation and differentiation when co-culture system is used, comparing to mono-cultures.LBL bioassembly approach seems to be suitable solution for efficient vascularization within entire large 3D tissue engineering constructs especially when co-cultures of mesenchymal and endothelial cells are used.

Des membranes biodégradables en polycaprolactone pour la régénération osseuse guidée, obtenues par electrospinning, incorporés avec différents rapports de nanocomposites de nanocristaux de cellulose et du Biosilicate®, ont été fabriquées, avec propriétés mécaniques et ostéogéniques améliorés. En tant que stratégie de vascularisation rapide, un greffon biomimétique suturable obtenue par fusion de membranes électrofilées a été fabriqué, avec des motifs poreux obtenus par micro- usinage au laser pour permettre la migration des cellules endothéliales vers le greffon osseux. Les motifs poreux créés sur les greffes suturables ont permis aux cellules endothéliales migrer vers la culture 3D des ostéoblastes dans des hydrogels en gélatine méthacryloyl (GelMA), et des structures 3D ont été observées. Par conséquent, cette stratégie peut être utilisée pour améliorer la taille et la survie des implants osseux biofabriqués, en accélérant la traduction clinique de l'ingénierie du tissu osseux. / Biodegradable membranes for guided bone regeneration, made of polycaprolactone, obtained by electrospinning, incorporated with different nanocomposite ratios of cellulose nanocrystals and Biosilicate®, have been manufactured, with improved mechanical and osteogenic properties. As fast vascularization strategy, a suturable biomimetic graft obtained by fusion of electrospun membranes was fabricated, with porous patterns obtained by laser micromachining to allow migration of endothelial cells to the bone graft. The porous patterns created on the suturable grafts allowed the endothelial cells to migrate to the 3D culture of the osteoblasts in gelatin methacryloyl (GelMA), and 3D structures were observed. Therefore, this strategy can be used to improve the size and survival of biofabricated bone implants, accelerating the clinical translation of bone tissue engineering.

Living tissue consists primarily of cells and extracellular matrix. Cells perform functions, communicate, respire and remodel extracellular matrix. Likewise, diffusive chemical conditions and extracellular matrix exhibit their own effects on cellular and intracellular processes, depending on the consistency of the matrix and phenotype of the cell. These interactions produce the emergent phenomena of tissue function, repair and morphology. Computational modeling seeks to quantify these processes for the purposes of fundamental study and predictive capability in various applications, including wound healing, tumor vascularization and biofabrication of living tissue. Hybrid kinetic Monte Carlo models are well known to be capable of predicting observed behaviors like cell sorting and spheroid fusion due to differential adhesion and energy minimization. However, no hybrid model sufficiently provides a formal treatment of full cell, chemical and matrix interactivity in a dynamic environment, including heterogeneous matrix conditions, advecting materials, and intracellular processes. In this work, hybrid kinetic Monte Carlo models are developed to describe full interactivity of cells, soluble signals and insoluble signals in a complex, dynamic microenvironment at the cellular level. Modeling of intracellular chemical dynamics and effects on the cellular state is developed as stochastic processes, and cell perform metabolic and matrix remodeling activities. Computational models of select \textit{in vivo} and \textit{in vitro} phenomena are developed and simulated, showing the ability to simulate new phenomena concerning cell viability, growth dynamics, highly heterogeneous cellular distributions, and complex tissue structures resulting from phenomena like intercellular signaling, matrix remodeling, and cell polarity.

Recreating organ-specific microenvironments of the extracellular matrix (ECM) in vitro has been an ongoing challenge in biofabrication. In this study, I present a biofunctional ECM-mimicking protein scaffold with tunable biochemical, mechanical and topographical properties. This scaffold, formed by microfibres, displays three favorable characteristics as a cell culture platform: high-loading of key ECM proteins, single-layered mesh membrane with controllable mesh size, and flexibility for supporting a range of cell culture configurations. Decellularized extracellular matrix (dECM) powder was used to fabricate this protein scaffold, as a close replicate of the chemical composition of physiological ECM. The highest dECM concentration in the solidified protein scaffold was 50 wt%, with gelatin consisting the rest. In practice, a high density of dECM-laden nano- to microfibres was directly patterned on a variety of substrates to form a single layer of mesh membrane, using the low-voltage electrospinning patterning (LEP) method. The smallest fibre diameter was measured at 450 nm, the smallest mesh size of the membrane was below 1 μm, and the thickness of the membrane was estimated to be less than 2 μm. This fabrication method demonstrated a good preservation of the key ECM proteins and growth factors, including collagen IV, laminin, fibronectin, VEGF and b-FGF. The integrated fibrous mesh exhibited robust mechanical properties, with tunable fibril Young’s modulus for over two orders of magnitude in the physiological range (depending on the dECM concentration). Combining this mesh membrane with 3D printing, a cell culture device was constructed. Co-culture of human glomerulus endothelial cells and podocytes was performed on this device, to simulate the blood-to-urine interface in vitro. Good cell attachment and viability were demonstrated, and specific cell differentiation and fibronectin secretion were observed. This dECM-laden protein scaffold sees the potential to be incorporated into a glomerulus-on-chip model, to further improve the physiological relevance of in vitro pathological models.