The Enderstruder: An accessible open-source syringe extruder compatible with Ender series 3D printers.

Bioprinting has enabled the precise spatiotemporal deposition of cells and biomaterials, opening new avenues of research in tissue engineering and regenerative medicine. Although several open-source syringe extruder adaptations for bioprinters have been published and adopted by end users, only one has been specifically adapted for the Ender series, an affordable and open-source line of thermoplastic 3D printers. Here, we introduce the Enderstruder, a cost-effective extruder attachment that uses a standard 10 mL BD syringe, positions the stepper motor at the level of the gantry, enhances x-axis stability with a linear rail, and uses the originally included stepper motor, resulting in reduced cost and simplified assembly. Furthermore, we present an iterative process to fine-tune printing profiles for high-viscosity biomaterial inks. To facilitate the implementation of our work by other researchers, we provide fully editable Cura profiles for five commonly used biomaterials. Using these five materials to validate and characterize our design, we employ the Enderstruder to print established calibration patterns and complex shapes. By presenting the Enderstruder and its iterative development process, this study contributes to the growing repository of open-source bioprinting solutions, fostering greater accessibility and affordability for researchers in tissue engineering.

Fabrication and Characterization of Quad-Component Bioinspired Hydrogels to Model Elevated Fibrin Levels in Central Nervous Tissue Scaffolds.

Multicomponent interpenetrating polymer network (mIPN) hydrogels are promising tissue-engineering scaffolds that could closely resemble key characteristics of native tissues. The mechanical and biochemical properties of mIPNs can be finely controlled to mimic key features of target cellular microenvironments, regulating cell-matrix interactions. In this work, we fabricated hydrogels made of collagen type I (Col I), fibrin, hyaluronic acid (HA), and poly (ethylene glycol) diacrylate (PEGDA) using a network-by-network fabrication approach. With these mIPNs, we aimed to develop a biomaterial platform that supports the in vitro culture of human astrocytes and potentially serves to assess the effects of the abnormal deposition of fibrin in cortex tissue and simulate key aspects in the progression of neuroinflammation typically found in human pathologies such as Alzheimer's disease (AD), Parkinson's disease (PD), and tissue trauma. Our resulting hydrogels closely resembled the complex modulus of AD human brain cortex tissue (~7.35 kPa), promoting cell spreading while allowing for the modulation of fibrin and hyaluronic acid levels. The individual networks and their microarchitecture were evaluated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Human astrocytes were encapsulated in mIPNs, and negligible cytotoxicity was observed 24 h after the cell encapsulation.

Interpenetrating polymer network hydrogels as bioactive scaffolds for tissue engineering.

Tissue engineering, after decades of exciting progress and monumental breakthroughs, has yet to make a significant impact on patient health. It has become apparent that a dearth of biomaterial scaffolds that possess the material properties of human tissue while remaining bioactive and cytocompatible has been partly responsible for this lack of clinical translation. Herein, we propose the development of interpenetrating polymer network hydrogels as materials that can provide cells with an adhesive extracellular matrix-like 3D microenvironment while possessing the mechanical integrity to withstand physiological forces. These hydrogels can be synthesized from biologically-derived or synthetic polymers, the former polymer offering preservation of adhesion, degradability, and microstructure and the latter polymer offering tunability and superior mechanical properties. We review critical advances in the enhancement of mechanical strength, substrate-scale stiffness, electrical conductivity, and degradation in IPN hydrogels intended as bioactive scaffolds in the past five years. We also highlight the exciting incorporation of IPN hydrogels into state-of-the-art tissue engineering technologies, such as organ-on-a-chip and bioprinting platforms. These materials will be critical in the engineering of functional tissue for transplant, disease modeling, and drug screening.

Phototunable interpenetrating polymer network hydrogels to stimulate the vasculogenesis of stem cell-derived endothelial progenitors.

Vascularization of engineered scaffolds remains a critical obstacle hindering the translation of tissue engineering from the bench to the clinic. We previously demonstrated the robust micro-vascularization of collagen hydrogels with induced pluripotent stem cell (iPSC)-derived endothelial progenitors; however, physically cross-linked collagen hydrogels compact rapidly and exhibit limited strength. We have synthesized an interpenetrating polymer network (IPN) hydrogel comprised of collagen and norbornene-modified hyaluronic acid (NorHA) to address these challenges. This dual-network hydrogel combines the natural cues presented by collagen's binding sites and extracellular matrix (ECM)-mimicking fibrous architecture with the in situ modularity and chemical cross-linking of NorHA. We modulated the IPN hydrogel's stiffness and degradability by varying the concentration and sequence, respectively, of the NorHA peptide cross-linker. Rheological characterization of the photo-mediated gelation process revealed that the IPN hydrogel's stiffness increased with cross-linker concentration and was decoupled from the bulk NorHA content. Conversely, the swelling of the IPN hydrogel decreased linearly with increasing cross-linker concentration. Collagen microarchitecture remained relatively unchanged across cross-linking conditions, although the addition of NorHA delayed collagen fibrillogenesis. Upon iPSC-derived endothelial progenitor encapsulation, robust, lumenized microvascular networks developed in IPN hydrogels over two weeks. Subsequent computational analysis showed that an initial rise in stiffness increased the number of branch points and vessels, but vascular growth was suppressed in high stiffness IPN hydrogels. These results suggest that an IPN hydrogel consisting of collagen and NorHA is highly tunable, compaction resistant, and capable of supporting vasculogenesis.

Mimicking the physical cues of the ECM in angiogenic biomaterials.

A functional microvascular system is imperative to build and maintain healthy tissue. Impaired microvasculature results in ischemia, thereby limiting the tissue's intrinsic regeneration capacity. Therefore, the ability to regenerate microvascular networks is key to the development of effective cardiovascular therapies. To stimulate the formation of new microvasculature, researchers have focused on fabricating materials that mimic the angiogenic properties of the native extracellular matrix (ECM). Here, we will review biomaterials that seek to imitate the physical cues that are natively provided by the ECM to encourage the formation of microvasculature in engineered constructs and ischemic tissue in the body.

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

The Role of Reactive Oxygen Species in In Vitro Cardiac Maturation.

Recent advances in developmental biology and biomedical engineering have significantly improved the efficiency and purity of cardiomyocytes (CMs) generated from pluripotent stem cells (PSCs). Regardless of the protocol used to derive CMs, these cells exhibit hallmarks of functional immaturity. In this Opinion, we focus on reactive oxygen species (ROS), signaling molecules that can potentially modulate cardiac maturation. We outline how ROS impacts nearly every aspect associated with cardiac maturation, including contractility, calcium handling, metabolism, and hypertrophy. Though the precise role of ROS in cardiac maturation has yet to be elucidated, ROS may provide a valuable perspective for understanding the molecular mechanisms for cardiac maturation under various conditions.

Temporal Impact of Substrate Anisotropy on Differentiating Cardiomyocyte Alignment and Functionality.

Anisotropic biomaterials can affect cell function by driving cell alignment, which is critical for cardiac engineered tissues. Recent work, however, has shown that pluripotent stem cell-derived cardiomyocytes may self-align over long periods of time. To determine how the degree of biomaterial substrate anisotropy impacts differentiating cardiomyocyte structure and function, we differentiated mouse embryonic stem cells to cardiomyocytes on nonaligned, semialigned, and aligned fibrous substrates and evaluated cell alignment, contractile displacement, and calcium transient synchronicity over time. Although cardiomyocyte gene expression was not affected by fiber alignment, we observed gradient- and threshold-based differences in cardiomyocyte alignment and function. Cardiomyocyte alignment increased with the degree of fiber alignment in a gradient-based manner at early time points and in a threshold-based manner at later time points. Calcium transient synchronization tightly followed cardiomyocyte alignment behavior, allowing highly anisotropic biomaterials to drive calcium transient synchronization within 8 days, while such synchronized cardiomyocyte behavior required 20 days of culture on nonaligned biomaterials. In contrast, cardiomyocyte contractile displacement had no directional preference on day 8 yet became anisotropic in the direction of fiber alignment on aligned fibers by day 20. Biomaterial anisotropy impact on differentiating cardiomyocyte structure and function is temporally dependent. Impact Statement This work demonstrates that biomaterial anisotropy can quickly drive desired pluripotent stem cell-derived cardiomyocyte structure and function. Such an understanding of matrix anisotropy's time-dependent influence on stem cell-derived cardiomyocyte function will have future applications in the development of cardiac cell therapies and in vitro cardiac tissues for drug testing. Furthermore, our work has broader implications concerning biomaterial anisotropy effects on other cell types in which function relies on alignment, such as myocytes and neurons.

Quantifying the Vasculogenic Potential of Induced Pluripotent Stem Cell-Derived Endothelial Progenitors in Collagen Hydrogels.

IMPACT STATEMENT: Our work reinforces the role of extracellular matrix (ECM) density and matrix metalloprotease activity on the formation of microvasculature from induced pluripotent stem cell (iPSC)-derived vascular cells. The cell-matrix interactions discussed in this study underscore the importance of understanding the role of mechanoregulation and matrix degradation on vasculogenesis and can potentially drive the development of ECM-mimicking angiogenic biomaterials. Furthermore, our work has broader implications concerning the response of iPSC-derived cells to the mechanics of engineered microenvironments. An understanding of these interactions will be critical to creating physiologically relevant transplantable tissue replacements.

Electrospun poly(N-isopropyl acrylamide)/poly(caprolactone) fibers for the generation of anisotropic cell sheets.

Cell alignment in muscle, nervous tissue, and cartilage is requisite for proper tissue function; however, cell sheeting techniques using the thermosensitive polymer poly(N-isopropyl acrylamide) (PNIPAAm) can only produce anisotropic cell sheets with delicate and resource-intensive modifications. We hypothesized that electrospinning, a relatively simple and inexpensive technique to generate aligned polymer fibers, could be used to fabricate anisotropic PNIPAAm and poly(caprolactone) (PCL) blended surfaces that both support cell viability and permit cell sheet detachment via PNIPAAm dissolution. Aligned electrospun PNIPAAm/PCL fibers (0%, 25%, 50%, 75%, 90%, and 100% PNIPAAm) were electrospun and characterized. Fibers ranged in diameter from 1-3 µm, and all fibers had an orientation index greater than 0.65. Fourier transform infrared spectroscopy was used to confirm the relative content of PNIPAAm and PCL. For advancing water contact angle and mass loss studies, only high PNIPAAm-content fibers (75% and greater) exhibited, temperature-dependent properties like 100% PNIPAAm fibers, whereas 25% and 50% PNIPAAm fibers behaved similarly to PCL-only fibers. 3T3 fibroblasts seeded on all PNIPAAm/PCL fibers had high cell viability and spreading except for the 100% PNIPAAm fibers. Cell sheet detachment by incubation with cold medium was successful only for 90% PNIPAAm fibers, which had a sufficient amount of PCL to allow cell attachment and spreading but not enough to prevent detachment upon PNIPAAm dissolution. This study demonstrates the feasibility of using anisotropic electrospun PNIPAAm/PCL fibers to generate aligned cell sheets that can potentially better recapitulate anisotropic architecture to achieve proper tissue function.