3D Bioprinting Knee, Aneurysm Model, 3D Organization Using Microfluidics

Category: Blog,From Academia
Nov 26, 2020

In this issue, three articles related to recent advancements in 3D bioprinting is presented. The first article highlights new ways to bioprint fibrocartilaginous tissues, such as those found in the knee joint, with improvements in both mechanical and structural integrity. The second article features an in vitro aneurysm simulation model, with improved simulation to the cellular environment and components of the in vivo version. The final publication features a novel tissue manufacturing process (3D Organization) using open volume microfluidics and multiple cell lines. “From Academia” features recent, relevant, close to commercialization academic publications. Subjects include but not limited to healthcare 3D printing, 3D bioprinting, and related emerging technologies.

Email: Rance Tino ([email protected]) if you want to share relevant academic publications with us.

3D Bioprinted Highly Elastic Hybrid Constructs for Advanced Fibrocartilaginous Tissue Regeneration

– Authored by João B. Costa, Jihoon Park, Adam M. Jorgensen, Joana Silva-Correia, Rui L. Reis, Joaquim M. Oliveira, Anthony Atala, James J. Yoo, and Sang Jin Lee. Chemistry of Materials. 25 September 2020

Schematic illustration of bioink formulations for bioprinted hybrid tissue construct. (A) GG and FB composite bioink, forming a stable hydrogel construct by a combination of ionic and enzymatic cross-linking. (B) Chemical modification of SF with methacrylate groups by reaction with MA, yielding Sil-MA. Copyright Chemistry of Materials
Schematic illustration of bioink formulations for bioprinted hybrid tissue construct. (A) GG and FB composite bioink, forming a stable hydrogel construct by a combination of ionic and enzymatic cross-linking. (B) Chemical modification of SF with methacrylate groups by reaction with MA, yielding Sil-MA. Copyright Chemistry of Materials 

Abstract: 

Advanced strategies to bioengineer a fibrocartilaginous tissue to restore the function of the meniscus are necessary. Currently, 3D bioprinting technologies have been employed to fabricate clinically relevant patient-specific complex constructs to address unmet clinical needs.

In this study, a highly elastic hybrid construct for fibrocartilaginous regeneration is produced by co-printing a cell-laden gellan gum/fibrinogen (GG/FB) composite bioink together with a silk fibroin methacrylate (Sil-MA) bioink in an interleaved crosshatch pattern. We characterize each bioink formulation by measuring the rheological properties, swelling ratio, and compressive mechanical behavior. For in vitro biological evaluations, porcine primary meniscus cells (pMCs) are isolated and suspended in the GG/FB bioink for the printing process.

The results show that the GG/FB bioink provides a proper cellular microenvironment for maintaining the cell viability and proliferation capacity, as well as the maturation of the pMCs in the bioprinted constructs, while the Sil-MA bioink offers excellent biomechanical behavior and structural integrity. More importantly, this bioprinted hybrid system shows the fibrocartilaginous tissue formation without a dimensional change in a mouse subcutaneous implantation model during the 10-week postimplantation. Especially, the alignment of collagen fibers is achieved in the bioprinted hybrid constructs. The results demonstrate that this bioprinted mechanically reinforced hybrid construct offers a versatile and promising alternative for the production of advanced fibrocartilaginous tissue. 

In vivo biological characterization of 3D bioprinted hybrid constructs. (A) Scheme of the printed patterning used for the production of 3D bioprinted constructs and gross appearance of the explants at 2, 5, and 10 weeks after implantation. (B) Dimensional changes and (C) compressive elastic modulus of 3D bioprinted constructs at 2, 5, and 10 weeks after implantation (*p < 0.05, **N.S.: not significant). Copyright Chemistry of Materials
In vivo biological characterization of 3D bioprinted hybrid constructs. (A) Scheme of the printed patterning used for the production of 3D bioprinted constructs and gross appearance of the explants at 2, 5, and 10 weeks after implantation. (B) Dimensional changes and (C) compressive elastic modulus of 3D bioprinted constructs at 2, 5, and 10 weeks after implantation (*p < 0.05, **N.S.: not significant). Copyright Chemistry of Materials

Three-dimensional bioprinting of aneurysm-bearing tissue structure for endovascular deployment of embolization coils

– Authored by Lindy K Jang, Javier A Alvarado, Marianna Pepona, Elisa M Wasson, Landon D Nash, Jason M Ortega, Amanda Randles, Duncan J Maitland, Monica L Moya and William F Hynes, Biofabrication. 16 October 2020 

The in vitro living cerebral aneurysm. (a) Illustration of the 3D printed aneurysm bioreactor. (b) The in vitro aneurysm vessel structure perfused with red fluorescent beads, demonstrating the formation of patent vessels post-evacuation of sacrificial ink. Copyright. Biofabrication
The in vitro living cerebral aneurysm. (a) Illustration of the 3D printed aneurysm bioreactor. (b) The in vitro aneurysm vessel structure perfused with red fluorescent beads, demonstrating the formation of patent vessels post-evacuation of sacrificial ink. Copyright. Biofabrication

Abstract: 

Various types of embolization devices have been developed for the treatment of cerebral aneurysms. However, it is challenging to properly evaluate device performance and train medical personnel for device deployment without the aid of functionally relevant models. Current in vitro aneurysm models suffer from a lack of key functional and morphological features of brain vasculature that limit their applicability for these purposes. These features include the physiologically relevant mechanical properties and the dynamic cellular environment of blood vessels subjected to constant fluid flow.

Herein, we developed three-dimensionally (3D) printed aneurysm-bearing vascularized tissue structures using gelatin-fibrin hydrogel of which the inner vessel walls were seeded with human cerebral microvascular endothelial cells (hCMECs). The hCMECs readily exhibited cellular attachment, spreading, and confluency all around the vessel walls, including the aneurysm walls.

Additionally, the in vitro platform was directly amenable to flow measurements via particle image velocimetry, enabling the direct assessment of the vascular flow dynamics for comparison to a 3D computational fluid dynamics model.

Detachable coils were delivered into the printed aneurysm sac through the vessel using a microcatheter and static blood plasma clotting was monitored inside the aneurysm sac and around the coils. This biomimetic in vitro aneurysm model is a promising method for examining the biocompatibility and hemostatic efficiency of embolization devices and for providing hemodynamic information that would aid in predicting aneurysm rupture or healing response after treatment.

Plasma clot formation in response to BPC deployment within in vitro living aneurysm dome. Maximum projection confocal image stack of the complete in vitro aneurysm after BPC deployment and injection with bovine plasma. Clot formation is visualized via accumulation of trace fluorescently labeled red human fibrinogen included with the plasma mixture. Endothelial cells are fluorescently stained green for actin. Imaging reveals clot formation and occlusion of the aneurysm sac, with no major clot formation present elsewhere in the vessel structures. Copyright. Biofabrication
Plasma clot formation in response to BPC deployment within in vitro living aneurysm dome. Maximum projection confocal image stack of the complete in vitro aneurysm after BPC deployment and injection with bovine plasma. Clot formation is visualized via accumulation of trace fluorescently labeled red human fibrinogen included with the plasma mixture. Endothelial cells are fluorescently stained green for actin. Imaging reveals clot formation and occlusion of the aneurysm sac, with no major clot formation present elsewhere in the vessel structures. Copyright. Biofabrication

3D micro-organisation printing of mammalian cells to generate biological tissues

– Authored by Gavin D. M. Jeffries, Shijun Xu, Tatsiana Lobovkina, Vladimir Kirejev, Florian Tusseau, Christoffer Gyllensten, Avadhesh Kumar Singh, Paul Karila, Lydia Moll & Owe Orwar. Nature, Scientific Reports. 10 November 2020

Introduction to the direct cell bioprinting approach, highlighting the key components. (a–c) illustrates the core concept of printing cells from a recirculating fluid flow. (d,e) illustrate the method of pattern formation using multiple cell types. (f–h) Experimental brightfield images demonstrate the formation of a structured pattern composed of alternating stripes of HaCaT and A431 cells onto a petri dish surface immersed in DMEM growth media. (i) presents a three-line structure composed of alternating printed stripes of HaCaT and A431 cells, labelled with cytotracker-green and cytotracker-red respectively. This structure was post-print nuclei stained with Hoechst 33342 and fixed with paraformaldehyde. A demonstration of the printing precision is shown in (j), whereby a single cell array was constructed using A431 cells. The scale bars represent 100 µm. Copyright. Nature Scientific Reports
Introduction to the direct cell bioprinting approach, highlighting the key components. (a–c) illustrates the core concept of printing cells from a recirculating fluid flow. (d,e) illustrate the method of pattern formation using multiple cell types. (f–h) Experimental brightfield images demonstrate the formation of a structured pattern composed of alternating stripes of HaCaT and A431 cells onto a petri dish surface immersed in DMEM growth media. (i) presents a three-line structure composed of alternating printed stripes of HaCaT and A431 cells, labelled with cytotracker-green and cytotracker-red respectively. This structure was post-print nuclei stained with Hoechst 33342 and fixed with paraformaldehyde. A demonstration of the printing precision is shown in (j), whereby a single cell array was constructed using A431 cells. The scale bars represent 100 µm. Copyright. Nature Scientific Reports

Abstract: 

Significant strides have been made in the development of in vitro systems for disease modelling. However, the requirement of microenvironment control has placed limitations on the generation of relevant models.

Herein, we present a biological tissue printing approach that employs open-volume microfluidics to position individual cells in complex 2D and 3D patterns, as well as in single-cell arrays. The variety of bioprinted cell types employed, including skin epithelial (HaCaT), skin cancer (A431), liver cancer (Hep G2), and fibroblast (3T3-J2) cells, all of which exhibited excellent viability and survivability, allowing printed structures to rapidly develop into confluent tissues.

To demonstrate a simple 2D oncology model, A431 and HaCaT cells were printed and grown into tissues. Furthermore, a basic skin model was established to probe drug response. 3D tissue formation was demonstrated by co-printing Hep G2 and 3T3-J2 cells onto an established fibroblast layer, the functionality of which was probed by measuring albumin production, and was found to be higher in comparison to both 2D and monoculture approaches. Bioprinting of primary cells was tested using acutely isolated primary rat dorsal root ganglia neurons, which survived and established processes. The presented technique offers a novel open-volume microfluidics approach to bioprint cells for the generation of biological tissues.

Post-printing model development and cell growth. (a–d) present experimental examples of an oncology model, highlighting before and after a 20 h incubation. (e,f) present examples of the basic skin model, composed of a two-printed stripe structure of differentiated and non-differentiated HaCaT cells expressing different levels of CK10. The green and white bands at the top of the panels indicate the approximate printed stripe width of differentiated and non-differentiated cells, respectively. A summary plot of the RA treatment is shown in (g), presenting the mean values of CK10 expression for both the controls (81.8%, n = 10) and the treated samples (61.4%, n = 16), demonstrating a statistically significant reduction of approximately 25% in CK10 expression. Error bars represent standard error. (h,i) Show acutely isolated primary rat dorsal root ganglia (DRG) neurons, which were patterned and allowed to form processes. Post printing, the sample was allowed to mature and grow for 9 days in standard neuronal culture conditions, where network establishment could be monitored. The scale bar in all panels represents 200 µm. Copyright. Nature Scientific Reports
Post-printing model development and cell growth. (a–d) present experimental examples of an oncology model, highlighting before and after a 20 h incubation. (e,f) present examples of the basic skin model, composed of a two-printed stripe structure of differentiated and non-differentiated HaCaT cells expressing different levels of CK10. The green and white bands at the top of the panels indicate the approximate printed stripe width of differentiated and non-differentiated cells, respectively. A summary plot of the RA treatment is shown in (g), presenting the mean values of CK10 expression for both the controls (81.8%, n = 10) and the treated samples (61.4%, n = 16), demonstrating a statistically significant reduction of approximately 25% in CK10 expression. Error bars represent standard error. (h,i) Show acutely isolated primary rat dorsal root ganglia (DRG) neurons, which were patterned and allowed to form processes. Post printing, the sample was allowed to mature and grow for 9 days in standard neuronal culture conditions, where network establishment could be monitored. The scale bar in all panels represents 200 µm. Copyright. Nature Scientific Reports

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