Engineering Functional Cardiac Tissues for Regenerative Medicine Applications.

PURPOSE OF REVIEW: Tissue engineering has expanded into a highly versatile manufacturing landscape that holds great promise for advancing cardiovascular regenerative medicine. In this review, we provide a summary of the current state-of-the-art bioengineering technologies used to create functional cardiac tissues for a variety of applications in vitro and in vivo. RECENT FINDINGS: Studies over the past few years have made a strong case that tissue engineering is one of the major driving forces behind the accelerating fields of patient-specific regenerative medicine, precision medicine, compound screening, and disease modeling. To date, a variety of approaches have been used to bioengineer functional cardiac constructs, including biomaterial-based, cell-based, and hybrid (using cells and biomaterials) approaches. While some major progress has been made using cellular approaches, with multiple ongoing clinical trials, cell-free cardiac tissue engineering approaches have also accomplished multiple breakthroughs, although drawbacks remain. This review summarizes the most promising methods that have been employed to generate cardiovascular tissue constructs for basic science or clinical applications. Further, we outline the strengths and challenges that are inherent to this field as a whole and for each highlighted technology.

Extrusion-Based 3D Bioprinting of Adhesive Tissue Engineering Scaffolds Using Hybrid Functionalized Hydrogel Bioinks.

Adhesive tissue engineering scaffolds (ATESs) have emerged as an innovative alternative means, replacing sutures and bioglues, to secure the implants onto target tissues. Relying on their intrinsic tissue adhesion characteristics, ATES systems enable minimally invasive delivery of various scaffolds. This study investigates development of the first class of 3D bioprinted ATES constructs using functionalized hydrogel bioinks. Two ATES delivery strategies, in situ printing onto the adherend versus printing and then transferring to the target surface, are tested using two bioprinting methods, embedded versus air printing. Dopamine-modified methacrylated hyaluronic acid (HAMA-Dopa) and gelatin methacrylate (GelMA) are used as the main bioink components, enabling fabrication of scaffolds with enhanced adhesion and crosslinking properties. Results demonstrate that dopamine modification improved adhesive properties of the HAMA-Dopa/GelMA constructs under various loading conditions, while maintaining their structural fidelity, stability, mechanical properties, and biocompatibility. While directly printing onto the adherend yields superior adhesive strength, embedded printing followed by transfer to the target tissue demonstrates greater potential for translational applications. Together, these results demonstrate the potential of bioprinted ATESs as off-the-shelf medical devices for diverse biomedical applications.

Microbial Changes occurring during oronasal fistula wound healing.

The oral microbiome is a complex community that matures with dental development while oral health is also a recognized risk factor for systemic disease. Despite the oral cavity having a substantial microbial burden, healing of superficial oral wounds occurs quickly and with little scarring. By contrast, creation of an oro-nasal fistula (ONF), often occurring after surgery to correct a cleft palate, is a significant wound healing challenge that is further complicated by a connection of the oral and nasal microbiome. In this study, we characterized the changes in the oral microbiome of mice following a freshly inflicted wound in the oral palate that results in an open and unhealed ONF. Creation of an ONF in mice significantly lowered oral microbiome alpha diversity, with concurrent blooms of Enterococcus faecalis, Staphylococcus lentus, and Staphylococcus xylosus in the oral cavity. Treatment of mice with oral antibiotics one week prior to ONF infliction resulted in a reduction in the alpha diversity, prevented E. faecalis and S. lentus, and S. xylosus blooms, but did not impact ONF healing. Strikingly, delivery of the beneficial microbe Lactococcus lactis subsp. cremoris (LLC) to the wound bed of the freshly inflicted ONF via a PEG-MAL hydrogel vehicle resulted in rapid healing of the ONF. Healing of the ONF was associated with the maintenance of relatively high microbiome alpha diversity, and limited the abundance of E. faecalis and S. lentus, and S. xylosus in the oral cavity. These data demonstrate that a freshly inflicted ONF in the murine palate is associated with a dysbiotic oral microbiome state that may prevent ONF healing, and a bloom of opportunistic pathogens. The data also demonstrate that delivery of a specific beneficial microbe, LLC, to the ONF can boost wound healing, can restore and/or preserve oral microbiome diversity, and inhibit blooms of opportunistic pathogens.

Embedded 3D Bioprinting of Gelatin Methacryloyl-Based Constructs with Highly Tunable Structural Fidelity.

Three-dimensional (3D) bioprinting of hydrogel-based constructs at adequate consistency and reproducibility can be obtained through a compromise between the hydrogel's inherent instability and printing fidelity. There is an increasing demand to develop bioprinting modalities that enable high-fidelity fabrication of 3D hydrogel structures that closely correspond to the envisioned design. In this work, we performed a systematic, in-depth characterization and optimization of embedded 3D bioprinting to create 3D gelatin-methacryloyl (gelMA) structures with highly controlled fidelity using Carbopol as suspension bath. The role of various embedded printing process parameters in bioprinting fidelity was investigated using a combination of experimental and theoretical approaches. We examined the effect of rheological properties of gelMA and Carbopol at varying concentrations, as well as printing conditions on the volumetric flow rate of gelMA bioink. Printing speed was examined and optimized to successfully print gelMA into the support bath at varying Carbopol concentrations. Printing fidelity was characterized in terms of printed strand diameter, uniformity, angle, and area. The optimal Carbopol solution that retained filament shape at highest fidelity was determined. The efficacy of developed bioprinting approach was then demonstrated by fabricating 3D hydrogel constructs with varying geometries and visualized using an advanced synchrotron-based imaging technique. We also investigated the influence of the Carbopol medium on cross-linking and the resulting stiffness of gelMA constructs. Finally, in vitro cytotoxicity of the developed bioprinting approach was assessed by printing human umbilical vein endothelial cells encapsulated in the gelMA bioink. These results demonstrate the significance of the close interplay between bioink-support bath rheology and printing parameters and help to establish an optimized workflow for creating 3D hydrogel structures with high fidelity and cytocompatibility via embedded bioprinting techniques. This robust platform could further expand the application of bioprinted soft tissue constructs in a wide variety of biomedical applications.

The Human Embryoid Body Cystic Core Exhibits Architectural Complexity Revealed by use of High Throughput Polymer Microarrays.

In pluripotent stem cell differentiation, embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here, we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 µm diameter microarray wells by use of single cells, 2D clusters, or 3D early aggregates were compared. We observe that 200 µm EBs are monocystic versus 500 µm multicystic EBs that contain macro, meso and microsized cysts. In adherent differentiation of 500 µm EBs, the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation.