Bioprinting is currently the most promising method to biofabricate complex tissuesin vitrowith the potential to transform the future of organ transplantation and drug discovery. Efforts to create such tissues are, however, almost exclusively based on animal-derived materials, such as gelatin methacryloyl, which have demonstrated efficacy in bioprinting of complex tissues. While these materials are already used in clinical applications, uncertainty about their safety still remains due to their animal origin. Alternatively, synthetic bioinks have been developed that match the printability of natural bioinks but lack their biological complexity, and thereby often fail to support cell growth and facilitate tissue formation. Additionally, most synthetic materials do not meet the mechanical demands of bioprint stable constructs while providing a suitable environment for cells to grow, limiting the number of available bioinks. To bridge this gap and synergize bioprinting and 3D cell culture, we developed a polyethylene glycol-based bioink system to promote the growth and spreading of cell spheroids that consist of human primary endothelial cells and fibroblasts. The 3D bioprinted centimeter-scale constructs have a high shape fidelity and accelerated softening to provide sufficient space for cells to grow. Adjusting the rate of degradability, induced by the integration of ester-functionalized crosslinkers in addition to protease cleavable crosslinkers into the hydrogel network, improves the growth of spheroids in larger printed hydrogel constructs containing an interconnected channel structure. The perfusable constructs enable extensive spheroid sprouting and the formation of a cellular network upon fusion of sprouts as initial steps toward tissue formation with the potential for clinical translation.

Despite significant advances in bioprinting technology, current hardware platforms lack the capability for process monitoring and quality control. This limitation hampers the translation of the technology into industrial GMP-compliant manufacturing settings. As a key step towards a solution, we developed a novel bioprinting platform integrating a high-resolution camera forin-situmonitoring of extrusion outcomes during embedded bioprinting. Leveraging classical computer vision and image analysis techniques, we then created a custom software module for assessing print quality. This module enables quantitative comparison of printer outputs to input points of the computer-aided design model's 2D projections, measuring area and positional accuracy. To showcase the platform's capabilities, we then investigated compatibility with various bioinks, dyes, and support bath materials for both 2D and 3D print path trajectories. In addition, we performed a detailed study on how the rheological properties of granular support hydrogels impact print quality during embedded bioprinting, illustrating a practical application of the platform. Our results demonstrated that lower viscosity, faster thixotropy recovery, and smaller particle sizes significantly enhance print fidelity. This novel bioprinting platform, equipped with integrated process monitoring, holds great potential for establishing auditable and more reproducible biofabrication processes for industrial applications.

A functional bioink with potential in bone tissue engineering must be subjected to critical investigation throughout its intended lifespan. The aim of this study was to develop alginate-gelatin-based (Alg-Gel) multicomponent bioinks systematically and to assess the short- and long-term exposure responses of human bone marrow stromal cells (hBMSCs) printed within these bioinks with and without crosslinking.The first generation of bioinkswas established by incorporating a range of cellulose nanofibrils (CNFs), to evaluate their effect on viscosity, printability and cell viability. Adding CNFs to Alg-Gel solution increased viscosity and printability without compromising cell viability. Inthe second generation of bioinks, the influence of nano-hydroxyapatite (nHA) on the performance of the optimized Alg-Gel-CNF formulation was investigated. The addition of nHA increased the viscosity and improved printability, and an adjustment in alginate concentration improved the stability of the structures in long-term culture. The third generation bioink incorporated RGD-functionalized alginate to support cell attachment and osteogenic differentiation. The optimized bioink composition exhibited improved printability, structural integrity in long-term culture and high hBMSC viability. In addition, the final bioink composition, RGD-Alg-Gel-CNF-nHA, showed osteogenic potential: production of the osteogenic marker proteins (Runx2, OCN), enzyme (ALP), and gene expression (Runx2,OCN). A further aim of the study was to evaluate the osteogenic functionality of cells released from the structures after bioprinting. Cells were printed in two bioinks with different viscosities and incubated at 37 °C in growth medium without additional CaCl2. This caused gelatin to dissolve, releasing the cells to attach to tissue culture plates. The results demonstrated differences in hBMSC osteogenic differentiation. Moreover, the osteogenic differentiation of the released cells was different from that of the embedded cells cultured in 3D. Thus, this systematic investigation into bioink development shows improved results through the generations and sheds light on the biological effects of the bioprinting process.

Background.Recent studies have shown promising results using decellularized extracellular matrix (dECM) matrikines-based hydrogel as attractive strategies for preventing and alleviating fibrosis.Methods & Results.Porcine lung decellularization and pepsin digestion were used to prepare the lung dECM hydrogel. Proteomic analysis revealed that the lung dECM hydrogel was enriched in glycoproteins, collagens, laminins, fibrinogen, held receptors, and bound growth factors. With porous structures and good mechanical properties and stability, the lung dECM hydrogel showed low cytotoxicity and good biocompatibility bothin vitroandin vivo. The lung dECM hydrogel was further administered to verify the safety and effectiveness of reversing pulmonary fibrosis in a bleomycin induced rat model. The results revealed a relatively complete alveolar structure with less inflammatory infiltration and a reduced amount of collagen fiber deposition. TMT quantification proteomic analyses revealed significant downregulation of proteins, pathways, and interactions involved in the regulation of ECM components, tissue remodeling, inflammation, and the cytoskeleton and indicated that fibrosis-related proteins were obviously downregulated and inflammation-related proteins were significantly changed, particularly in macrophages, after administration of the lung dECM hydrogel. Opal multiplex immunohistochemistry (mIHC) staining of lung tissue revealed that the inflammatory response was regulated by the lung dECM hydrogel, as indicated by a decrease in the number of CD3+ T cells and macrophages and the suppression of M2 macrophage polarization. Gene set enrichment analysis revealed that downregulated ficolin signaling was enriched in macrophages after lung dECM hydrogel administration, and the findings were verified in lung tissue by mIHC. Additionally, the effects of ficolin B proteins on macrophage polarization were provedin vitro. Conclusion.This study suggested that the lung dECM hydrogel can reverse pulmonary fibrosis by suppressing M2 macrophage polarization through downregulation of the ficolin signaling pathway. Thus, the dECM hydrogel represent a promising class of biological materials for use in regenerative medicine.

In this research, carboxymethyl cellulose (CMC)/gelatin (Gel)/graphene oxide (GO)-based scaffolds were produced by using extrusion-based 3D printing for cardiac tissue regeneration. Rheological studies were conducted to evaluate the printability of CMC/Gel/GO inks, which revealed that CMC increased viscosity and enhanced printability. The 3D-printed cardiac patches were crosslinked with N-(3-dimethylaminopropyl)-n'-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) (100:20 mM, 50:10 mM, 25:5 mM) and then characterized by mechanical analysis, electrical conductivity testing, contact angle measurements and degradation studies. Subsequently, cell culture studies were conducted to evaluate the viability of H9C2 cardiomyoblast cells by using the Alamar Blue assay and fluorescence imaging. A high concentration of EDC/NHS (100:20 mM) led to the stability of the patches; however, it drastically reduced the flexibility of the scaffolds. Conversely, a concentration of 25:5 mM resulted in flexible but unstable scaffolds in phosphate buffer saline solution. The suitable EDC/NHS concentration was found to be 50:10 mM, as it produced flexible, stable, and stiff cardiac scaffolds with high ultimate tensile strength. Mechanical characterization revealed that % strain at break of C15/G7.5/GO1 exhibited a remarkable increase of 61.03% compared to C15/G7.5 samples. The improvement of flexibility was attributed to the hydrogen bonding between CMC, Gel and GO. The electrical conductivity of 3D printed CMC/Gel/GO cardiac patches was 7.0 × 10-3S cm-1, demonstrating suitability for mimicking the desired electrical conductivity of human myocardium. The incorporation of 1 wt% of GO and addition of CMC concentration from 7.5 wt% to 15 wt% significantly enhanced relative % cell viability. Overall, although this research is at its infancy, CMC/Gel/GO cardiac patches have potential to improve the physiological function of cardiac tissue.

Intervertebral disc (IVD) function is achieved through integration of its two component regions: the nucleus pulposus (NP) and the annulus fibrosus (AF). The NP is soft (0.3-5 kPa), gelatinous and populated by spherical NP cells in a polysaccharide-rich extracellular matrix (ECM). The AF is much stiffer (∼100 kPa) and contains layers of elongated AF cells in an aligned, fibrous ECM. Degeneration of the disc is a common problem with age being a major risk factor. Progression of IVD degeneration leads to chronic pain and can result in permanent disability. The development of therapeutic solutions for IVD degeneration is impaired by a lack ofin vitromodels of the disc that are capable of replicating the fundamental structure and biology of the tissue. This study aims to investigate if a newly developed suspended hydrogel bioprinting system (termed SLAM) could be employed to fabricate IVD analogues with integrated structural and compositional features similar to native tissue. Bioprinted IVD analogues were fabricated to recapitulate structural, morphological and biological components present in the native tissue. The constructs replicated key structural components of native tissue with the presence of a central, polysaccharide-rich NP surrounded by organised, aligned collagen fibres in the AF. Cell tracking, actin and matrix staining demonstrated that embedded NP and AF cells exhibited morphologies and phenotypes analogous to what is observedin vivowith elongated, aligned AF cells and spherical NP cells that deposited HA into the surrounding environment. Critically, it was also observed that the NP and AF regions contained a defined cellular and material interface and segregated regions of the two cell types, thus mimicking the highly regulated structure of the IVD.