Biology subjects

Ozbolat, I. T.

Publications and source records attributed to Ozbolat, I. T..

15 recordsLinked to original sources

bioRxiv2026

Bioinstructive Orthogonally-crosslinked Ovoprotein Microgels for Modular Bioprinting

Bioprinting increasingly requires biomaterials that are not only printable, but structurally adaptive and biologically instructive. Here we establish an ovoprotein-derived microgel platform that couples intrinsic protein bioactivity with orthogonal interparticle photocrosslinking for modular bioprinting. Methacrylated ovoproteins yielded a photoresponsive protein-rich hydrogel matrix with retained proteomic complexity, tunable mechanics, and cell-regulatory biofunction. Endogenous tyrosine chemistry drove interparticle dityrosine coupling between ovoprotein microgels, producing cohesive, microporous, and intrinsically autofluorescent granular networks. The resulting systems displayed programmable rheology and broad compatibility across digital light processing, extrusion-based and aspiration-assisted bioprinting. Functionally, the ovoprotein microgel matrices attenuated sustained pro-inflammatory macrophage activation, promoted endothelial organization and host angiogenic invasion, and supported spheroid-mediated vascular morphogenesis with progressive sprouting, lumenization, branching and inosculation. It further enabled bioprinted osteogenic constructs with long-term maturation into bone-like mineralized tissues in vitro. These findings establish ovoprotein microgel-spheroid bioassembly as an adaptive, bioinstructive strategy for engineering vascularized and mineralized tissue constructs.

bioengineering↗

bioRxiv2025

Interparticle Crosslinked Ion-responsive Microgels for 3D and 4D (Bio)printing Applications

AbstractMicrogels offer unique advantages over bulk hydrogels due to their improved diffusion limits for oxygen and nutrients. Particularly, stimuli-responsive microgels with inherently bioactive and self-supporting properties emerge as highly promising biomaterials. This study unveils the development of interparticle-crosslinked, self-supporting, ion-responsive microgels tailored for 3D and 4D (bio)printing applications. A novel strategy was proposed to develop microgels that enabled interparticle crosslinking, eliminating the need for filler hydrogels and preserving essential microscale void spaces to support cell migration and vascularization. Additionally, these microgels possessed unique, ion-responsive shrinking behavior primarily by the Hofmeister effect, reversible upon the removal of the stimulus. Two types of microgels, spherical ({micro}S) and random-shaped ({micro}R), were fabricated, with {micro}R exhibiting superior mechanical properties and higher packing density. Fabricated microgel-based constructs supported angiogenesis with tunable vessel size based on interstitial void spaces while demonstrating excellent shear-thinning and self-healing properties and high print fidelity. Various bioprinting techniques were employed and validated using these microgels, including extrusion-based, embedded, intraembedded, and aspiration-assisted bioprinting, facilitating the biofabrication of scalable constructs. Multi-material 4D printing was achieved by combining ion-responsive microgels with non-responsive microgels, enabling programmable shape transformations upon exposure to ionic solutions. Utilizing 4D printing, complex, dynamic structures were generated such as coiling filaments, grippers, and folding sheets, providing a foundation for the development of advanced tissue models and devices for regenerative medicine and soft robotics, respectively.

bioengineering↗

bioRxiv2024

High-Throughput Bioprinting of Spheroids for Scalable Tissue Fabrication

Tissue biofabrication that replicates an organ-specific architecture and function requires physiologically-relevant cell densities. Bioprinting using spheroids has the potential to create constructs with native cell densities, but its application is limited due to the lack of practical, scalable techniques. This study presents HITS-Bio (High-throughput Integrated Tissue Fabrication System for Bioprinting), a novel multiarray spheroid bioprinting technology enabling scalable tissue fabrication by rapidly positioning a number of spheroids simultaneously using a digitally-controlled nozzle array (DCNA) platform. HITS-Bio achieves an unprecedented speed, an order of magnitude faster compared to existing techniques while maintaining high cell viability (>90%). The platforms ability to pattern multiple spheroids simultaneously enhances fabrication rates proportionally to the size of DCNA used. The utility of HITS-Bio was exemplified in multiple applications, including intraoperative bioprinting with microRNA transfected spheroids for calvarial bone regeneration ([~]30 mm3) in a rat model achieving a near-complete defect closure ([~]91% in 3 weeks and [~]96% in 6 weeks). Additionally, the successful fabrication of scalable cartilage constructs (1 cm3) containing [~]600 chondrogenic spheroids highlights its high-throughput efficiency (under 40 min per construct) and potential for repairing volumetric tissue defects.

bioengineering↗

bioRxiv2023

Intraoperative Bioprinting of Human Adipose-derived Stem cells and Extra-cellular Matrix Induces Hair Follicle-Like Downgrowths and Adipose Tissue Formation during Full-thickness Craniomaxillofacial Skin Reconstruction

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2% or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo. Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

bioengineering↗

bioRxiv2023

Nested biofabrication: Matryoshka-inspired Intra-embedded Bioprinting

Engineering functional tissues and organs remains a fundamental pursuit in biofabrication. However, the accurate constitution of complex shapes and internal anatomical features of specific organs, including their intricate blood vessels and nerves, remains a significant challenge. Inspired by the Matryoshka doll, we here introduce a new method called Intra-Embedded Bioprinting (IEB), building upon existing embedded bioprinting methods. We used a xanthan gum-based material, which served a dual role as both a bioprintable ink and a support bath, due to its unique shear-thinning and self-healing properties. We demonstrated IEBs capabilities in organ modelling, creating a miniaturized replica of a pancreas using a photocrosslinkable silicone composite. Further, a head phantom and a Matryoshka doll were 3D printed, exemplifying IEBs capability to manufacture intricate, nested structures. Towards the use case of IEB and employing innovative coupling strategy between extrusion-based and aspiration-assisted bioprinting, we developed a breast tumor model that included a central channel mimicking a blood vessel, with tumor spheroids bioprinted in proximity. Validation using a clinically-available chemotherapeutic drug illustrated its efficacy in reducing the tumor volume via perfusion over time. This method opens a new way of bioprinting enabling the creation of complex-shaped organs with internal anatomical features.

bioengineering↗

bioRxiv2023

A 3D Printed Ventilated Perfused Lung Model Platform to Dissect the Lung's Response to Viral Infection in the Presence of Respiration

In this study, we developed a three-dimensionally (3D) printed lung model that faithfully recapitulates the intricate lung environment. This 3D model incorporated alveolar and vascular components that allow for a comprehensive exploration of lung physiology and responses to infection in vitro. In particular, we investigated the intricate role of ventilation on formation of the alveolar epithelial layer and its response to viral infections. In this regard, we subjected our 3D printed, perfused lung model to a continuous respiratory cycle at the air-liquid interface (ALI) for up to 10 days followed by infection with two viruses: influenza virus (Pr8) and respiratory syncytial virus (RSV), at two different concentrations for 24 or 48 h. The results revealed that ventilation induced increased tight-junction formation with better epithelial barrier function over time, facilitated higher expression of alveolar epithelial specific genes, enabled higher level of infection with an increased progression of viral spread and replication over time, and modulated the production of pro-inflammatory cytokines and chemokines. Our findings represent a critical step forward in advancing our understanding of lung-specific viral responses and respiratory infections in response to ventilation, which sheds light on vital aspects of pulmonary physiology and pathobiology.

bioengineering↗

bioRxiv2023

A Versatile Photocrosslinkable Silicone Composite for 3D Printing Applications

Embedded printing has emerged as a valuable tool for fabricating complex structures and microfluidic devices. Currently, an ample of amount of research is going on to develop new materials to advance its capabilities and increase its potential applications. Here, we demonstrate a novel, transparent, 3D printable, photocrosslinkable, and tuneable silicone composite that can be utilized as a support bath or an extrudable ink for embedded printing. The proposed silicone composite can be tuned to achieve ideal rheological properties, such as optimal self-recovery and yield stress, for use in 3D printing. When used as a support bath, it facilitated the generation microfluidic devices with circular channels of diameter up to 30 {micro}m. To demonstrate its utility, flow focusing microfluidic devices were fabricated for generation of Janus microrods, which can be easily modified for multitude of applications. When used as an extrudable ink, 3D printing of complex-shaped micro- and macro-constructs were achieved with integrated electronics, which greatly extends its potential applications towards developing complex flexible parts for soft robotics and prosthetics. Further, its biocompatibility was tested with multiple cell types to validate its applicability for medical and tissue engineering use. Altogether, this material offers a myriad of potential applications in material and medical fields by providing a facile approach to develop complicated 3D structures and interconnected channels that can further advance microfluidics and soft-robotics research.

bioengineering↗

bioRxiv2023

High-Throughput Bioprinting of the Nasal Epithelium using Patient-derived Nasal Epithelial Cells

Human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models. However, the manual approach is slow, low-throughput and has limitations in terms of achieving the intricate 3D structure of the natural nasal epithelium in a uniform manner. 3D Bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of nasal progenitor cells ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4-week air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes such as disease modeling, immunological studies, and drug screening.

bioengineering↗

bioRxiv2022

High-throughput microgel biofabrication via air-assisted co-axial jetting for cell encapsulation, 3D bioprinting, and scaffolding applications

Microgels have recently received widespread attention for their applications in a wide array of domains such as tissue engineering, regenerative medicine, and cell and tissue transplantation because of their properties like injectability, modularity, porosity, and the ability to be customized in terms of size, form, and mechanical properties. However, it is still challenging to mass produce microgels with diverse sizes and tunable properties. Herein, we developed an air-assisted co-axial device (ACAD) for continuous production of microgels in a high-throughput manner. To test its robustness, microgels of multiple hydrogels and their combination, including alginate (Alg), gelatin methacrylate (GelMA) and Alg-GelMA, were formed at a maximum production rate of 65,000 microgels per sec while retaining circularity and a size range of 50-500 m based on varying air pressure levels. The ACAD platform allowed single and multiple cell encapsulation with around 75% efficiency. These microgels illustrated appealing rheological properties such as yield stress, viscosity, and shear modulus for bioprinting applications. Specifically, Alg microgels have the potential to be used as a sacrificial support bath while GelMA microgels have potential for direct extrusion both on their own or when loaded in a bulk GelMA hydrogel. Generated microgels showed high cell viability (>90%) and proliferation over 7 days with their increased interactions with cells, particularly for GelMA microgels. The developed strategy provides a facile and rapid approach without any complex or expensive consumables and accessories for scalable high-throughput microgel production for cell therapy, tissue regeneration and 3D bioprinting applications.

bioengineering↗

bioRxiv2022

3D Bioprinted perfusable and vascularized breast tumor model for dynamic screening of chemotherapeutics and CAR-T cells

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the rapid and early screening of anti-cancer agents. A major barrier in this endeavor, is the complex interplay between the tumor microenvironment and host immune response and lack of predictive biomarkers for clinical benefit. To tackle this challenge, we have developed a dynamic-flow based three-dimensionally (3D) bioprinted vascularized breast tumor model, responding to chemo and immunotherapeutic treatments. Heterotypic tumor spheroids, comprising metastatic breast cancer cells (MDA-MB-231), human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs), precisely bioprinted at pre-defined distances from a perfused vasculature, exhibited tumor angiogenesis and cancer invasion. Proximally bioprinted tumors ([~]100 m) exhibited enhanced capillary sprouting, anastomosis to perfused vasculature and increased cancer cell migration as compared to distally bioprinted spheroids ([~]500 m). Proximally bioprinted tumors treated with varying dosages of doxorubicin for 72 h enabled functional analysis of drug response, wherein, tumors portrayed a dose-dependent drug response behavior with [~]70% decrease in tumor volume for 1 M dose. Additionally, a cell based immune therapy approach was explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8+ T cells for 24 or 72 h through the central vasculature. Extensive CAR-T cell recruitment to the endothelium and substantial T cell activation and infiltration in the tumor site, resulted in [~]70% reduction in tumor growth for high CAR treatment densities, after 72 h of treatment. The presented 3D model paves the way for a robust, precisely fabricated and physiologically-relevant 3D tumor microenvironment platform for future translation of anti-cancer therapies to personalized medicine for cancer patients. One Sentence SummaryA physiologically-relevant 3D bioprinted perfusable vascularized tumor model capable of dynamic screening of chemo and immunotherapeutics.

bioengineering↗

bioRxiv2022

Development of 3D breast cancer models with human T cells expressing engineered MAIT cell receptors

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we developed proof-of-concept 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+ T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast (HDF) tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3-day culture period, demonstrating that the engineered TCR recognized MR1 expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as IFN{gamma}, IL-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

immunology↗

bioRxiv2021

Aspiration-assisted freeform bioprinting of mesenchymal stem cell spheroids within alginate microgels

Aspiration-assisted freeform bioprinting (AAfB) has emerged as a promising technique for precise placement of tissue spheroids in three-dimensional (3D) space for fabrication of tissues. For successful embedded bioprinting using AAfB, an ideal support bath should possess shear-thinning behavior and yield-stress to obtain tightly fused assembly of bioprinted spheroids. Several studies have demonstrated support baths for embedded bioprinting, but these materials pose major challenges due to their low biocompatibility, opaqueness, complex and prolonged preparation procedures, and limited spheroid fusion efficacy. In this study, to circumvent the aforementioned limitations, we present the feasibility of AAfB of human mesenchymal stem cell (hMSC) spheroids in alginate microgels as a support bath. First, alginate microgels were prepared with different particle sizes modulated by blending time and concentration, followed by determination of the optimal bioprinting conditions by the assessment of rheological properties, bioprintability, and spheroid fusion efficiency. The bioprinted and consequently self-assembled tissue structures made of hMSC spheroids were osteogenically induced for bone tissue formation. Alongside, we investigated the effects of peripheral blood monocyte-derived osteoclast incorporation into the hMSC spheroids in heterotypic bone tissue formation. We demonstrated that alginate microgels enabled unprecedented positional accuracy (~5%), transparency for visualization, and improved fusion efficiency (~97%) of bioprinted hMSC spheroids for bone fabrication. This study demonstrates the feasibility of using alginate microgels as a support bath for many different applications including but not limited to freeform bioprinting of spheroids, cell-laden hydrogels, and fugitive inks to form viable tissue constructs.

bioengineering↗

bioRxiv2020

The role of concentration on drop formation and breakup of collagen, fibrinogen, and thrombin solutions during inkjet bioprinting

The influence of protein concentration on drop formation and breakup of aqueous solutions of fibrous proteins collagen, fibrinogen, and globular protein thrombin in different concentration regimes is investigated during drop-on-demand (DOD) inkjet bioprinting. The capillary-driven thinning and breakup of dilute (c/c* < 1, where c is the concentration and c* is the overlap concentration) collagen, fibrinogen, and thrombin solutions is predominantly resisted by inertial force on the initial onset of necking. The minimum diameter (Dfmin(t)) of the necked fluid up to the critical pinch-off time (tc) scales with time as Dfmin(t) [~] (tc - t)2/3, a characteristic of potential flows. Although the capillary-driven thinning and breakup of semidilute unentangled collagen (1 [≤] c/c* [≤] 4) and fibrinogen (1 [≤] c/c* [≤] 1.3) solutions is predominantly resisted by inertial force on the initial onset of necking, the breakup of droplets is delayed beyond tc, where the minimum diameter of the necked fluid decreases exponentially with time because of the resistance of elastic force. The resistance of viscous force to the necking of both the dilute and semidilute untangled protein solutions is negligible. Aggregates or subvisible particles (between 1 and 100 m) constantly disrupt the formation of droplets for the semidilute unentangled protein solutions, even when their inverse Ohnesorge number (Z) is within the printability range of 4 [≤] Z [≤] 14. Although aggregates are present in the dilute protein solutions, they do not disrupt the formation of droplets.

bioengineering↗

bioRxiv2020

3D bioprinting of co-cultured osteogenic spheroids for bone tissue fabrication

Conventional top-down approaches in tissue engineering involving cell seeding on scaffolds have been widely used in bone engineering applications. However, scaffold-based bone tissue constructs have had limited clinical translation due to constrains in supporting scaffolds, minimal flexibility in tuning scaffold degradation, and low achievable cell seeding density as compared with native bone tissue. Here, we demonstrate a pragmatic and scalable bottom-up method, inspired from embryonic developmental biology, to build three-dimensional (3D) scaffold-free constructs using spheroids as building blocks. Human umbilical vein endothelial cells (HUVECs) were introduced to human mesenchymal stem cells (hMSCs) (hMSC/HUVEC) and spheroids were fabricated by an aggregate culture system. Bone tissue was generated by induction of osteogenic differentiation in hMSC/HUVEC spheroids for 10 days, with enhanced osteogenic differentiation and cell viability in the core of the spheroids compared to hMSC-only spheroids. Aspiration-assisted bioprinting (AAB) is a new bioprinting technique which allows precise positioning of spheroids (11% with respect to the spheroid diameter) by employing aspiration to lift individual spheroids and bioprint them onto a hydrogel. AAB facilitated bioprinting of scaffold-free bone tissue constructs using the pre-differentiated hMSC/HUVEC spheroids. These constructs demonstrated negligible changes in their shape for two days after bioprinting owing to the reduced proliferative potential of differentiated stem cells. Bioprinted bone tissues showed interconnectivity with actin-filament formation and high expression of osteogenic and endothelial-specific gene factors. This study thus presents a viable approach for 3D bioprinting of complex-shaped geometries using spheroids as building blocks, which can be used for various applications including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, drug screening and, disease modeling.

bioengineering↗

bioRxiv2020

Aspiration-assisted Freeform Bioprinting of Tissue Spheroids in a Yield-stress Gel

Bioprinting of cellular aggregates, such as tissue spheroids or organoids, in complex three-dimensional (3D) arrangements has been a major obstacle for scaffold-free fabrication of tissues and organs. In this research, we unveiled a new approach to the bioprinting of tissue spheroids in a yield stress granular gel, which exhibited unprecedented capabilities in freeform positioning of spheroids in 3D. Due to its Herschel-Bulkley and self-healing properties as well as its biological inertness, the granular gel supported both the positioning and self-assembly of tissue spheroids. We studied the underlying physical mechanism of the approach to elucidate the interactions between the aspirated spheroids and the gels yield-stress during the transfer of spheroids from cell media to the gel. We demonstrate the application of the proposed approach in the realization of various freeform shapes and self-assembly of human mesenchymal stem cell spheroids for the construction of cartilage and bone tissues.

bioengineering↗