RESOURCES

Abstract
Deciphering breast cancer treatment resistance remains hindered by the lack of models that can successfully capture the four-dimensional dynamics of the tumor microenvironment. Here, we show that microextrusion bioprinting can reproducibly generate distinct cancer and stromal compartments integrating cells relevant to human pathology. Our findings unveil the functional maturation of this millimeter-sized model, showcasing the development of a hypoxic cancer core and an increased surface proliferation. Maturation was also driven by the presence of cancer-associated fibroblasts (CAF) that induced elevated microvascular-like structures complexity. Such modulation was concomitant to extracellular matrix remodeling, with high levels of collagen and matricellular proteins deposition by CAF, simultaneously increasing tumor stiffness and recapitulating breast cancer fibrotic development. Importantly, our bioprinted model faithfully reproduced response to treatment, further modulated by CAF. Notably, CAF played a protective role for cancer cells against radiotherapy, facilitating increased paracrine communications. This model holds promise as a platform to decipher interactions within the microenvironment and evaluate stroma-targeted drugs in a context relevant to human pathology.

Abstract
Bioprinting technologies are powerful new bioengineering tools that can spatially reproduce multiple microenvironmental cues in a highly controlled, tunable, and precise manner. In this study, microvalve bioprinting technology was successfully used to print in close proximity endothelial and tumor cells at higher concentrations than previously thought possible, while preserving their viability. We propose that the resulting multicellular models, bioprinted in a controlled extracellular matrix microenvironment, are well-suited to study endothelial and cancer cell crosstalk within a cancer niche. As proof of concept, microvalve bioprinting was applied to the bioengineering of a simplified glioblastoma model in which biological processes involved in tumor expansion, such as tumor cell invasion patterns, cell proliferation, and senescence could be easily visualized and quantified. In this model, U251 glioblastoma cells and primary human umbilical vein endothelial cells (HUVECs) exhibited good printability and high viability after printing. U251 cells formed physiologically relevant clusters and invasion margins, while HUVECs generated vascular-like networks when primary fibroblasts were added to the model. An oxidative stress mimicking the one encountered within a tumor microenvironment during radiotherapy or genotoxic chemotherapy was shown to both diminish endothelial cells proliferation and to increase their senescence. Results also suggested that stressed glioblastoma cells may alter normal endothelial cell proliferation but not impact their senescence. This data demonstrates the potential of microvalve bioprinting to fabricate in vitro models that can help decipher endothelial and tumor cell crosstalk, within controlled and modulable microenvironments, and can then be used to address critical questions in the context of cancer recurrence.

Abstract
Producing oral soft tissues using tissue engineering could compensate for the disadvantages of autologous grafts (limited availability and increased patient morbidity) and currently available substitutes (shrinkage). However, there is a lack of in vitro-engineered oral tissues due to the difficulty of obtaining stable pre-vessels that connect to the host and enable graft success. The main objective was to assess the connection of pre-vascularised 3D-bioprinted gingival substitutes to the host vasculature when subcutaneously implanted in immunodeficient mice. This study produced vascularised connective tissue substitutes using extrusion-based 3D-bioprinting of primary human gingival fibroblasts (hGFs) and fluorescent human endothelial cells (RFP-HUVEC) co-cultures. Pre-vascularised (hGF + RFP-HUVEC-CC grids) and control (hGF only -HG grids) grids were bioprinted and pre-cultivated for 14 d to enable pre-vessels formation. In vitro vessel formation follow-up was performed. Eight-week-old female NOG mice were used for in vivo experiments. One grid per mouse was subcutaneously implanted in 20 mice (10HG/10CC). The fluorescent activity of RFP-HUVEC was monitored. Samples were retrieved at 7, 14 and 21 d. Histological, immunohistochemical, and immunofluorescent staining was performed. CC-grids formed efficient and stable pre-vessel networks within 14 d of static pre-culture. HG-grids did not contain any vessel, while CC-grids successfully connected to the host vasculature by presenting erythrocytes within the vessel lumen inside the grids starting day 7. From days 7–21, vessel density was stable. Human pre-vessels were present at 7 d and were progressively replaced by murine ECs. This study showed that primary hGF-HUVEC co-cultures can be successfully 3D-bioprinted within biomimetic hydrogels having a close composition to the gingival connective tissue, and HUVEC organise themselves into pre-vessel networks that connect to the murine vasculature when implanted in vivo. This approach represents a promising strategy to enhance current and future oral soft tissue substitutes for prospective clinical applications.

Abstract
Abstract Bioprinting applications in the clinical field generate great interest, but developing suitable biomaterial inks for medical settings is a challenge. Placental tissues offer a promising solution due to their abundance, stability, and status as medical waste. They contain basement membrane components, have a clinical history, and support angiogenesis. This study formulates bioinks from two placental tissues, amnion (AM) and chorion (CHO), and compares their unique extracellular matrix (ECM) and growth factor compositions. Rheological properties of the bioinks are evaluated for bioprinting and maturation of human endothelial cells. Both AM and Cho-derived bioinks sustained human endothelial cell viability, proliferation, and maturation, promoting optimal vasculogenesis. These bioinks derived from human sources have significant potential for tissue engineering applications, particularly in supporting vasculogenesis. This research contributes to the advancement of tissue engineering and regenerative medicine, bringing everyone closer to clinically viable bioprinting solutions using placental tissues as valuable biomaterials.

Abstract
When considering regenerative approaches, the efficient creation of a functional vasculature, that can support the metabolic needs of bioengineered tissues, is essential for their survival after implantation. However, it is widely recognized that the post-implantation microenvironment of the engineered tissues is often hypoxic due to insufficient vascularization, resulting in ischemia injury and necrosis. This is one of the main limitations of current tissue engineering applications aiming at replacing significant tissue volumes. Here, we have explored the use of a new biomaterial, the cell-assembled extracellular matrix (CAM), as a biopaper to biofabricate a vascular system. CAM sheets are a unique, fully biological and fully human material that has already shown stable long-term implantation in humans. We demonstrated, for the first time, the use of this unprocessed human ECM as a microperforated biopaper. Using microvalve dispensing bioprinting, concentrated human endothelial cells (30 millions ml−1) were deposited in a controlled geometry in CAM sheets and cocultured with HSFs. Following multilayer assembly, thick ECM-based constructs fused and supported the survival and maturation of capillary-like structures for up to 26 d of culture. Following 3 weeks of subcutaneous implantation in a mice model, constructs showed limited degradative response and the pre-formed vasculature successfully connected with the host circulatory system to establish active perfusion.This mechanically resilient tissue equivalent has great potential for the creation of more complex implantable tissues, where rapid anastomosis is sine qua non for cell survival and efficient tissue integration.

Abstract
Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.

Abstract
The field of bioprinting has shown a tremendous development in recent years, focusing on the development of advanced in vitro models and on regeneration approaches. In this scope, the lack of suitable biomaterials that can be efficiently formulated as printable bioinks, while supporting specific cellular events, is currently considered as one of the main limitations in the field. Indeed, extracellular matrix (ECM)-derived biomaterials formulated to enable printability and support cellular response, for instance via integrin binding, are eagerly awaited in the field of bioprinting. Several bioactive laminin sequences, including peptides such as YIGSR and IKVAV, have been identified to promote endothelial cell attachment and/or neurite outgrowth and guidance, respectively. Here, we show the development of two distinct bioinks, designed to foster vasculogenesis or neurogenesis, based on methacrylated collagen and hyaluronic acid (CollMA and HAMA, respectively), both relevant ECM-derived polymers, and on their combination with cysteine-flanked laminin-derived peptides. Using this strategy, it was possible to optimize the bioink printability, by tuning CollMA and HAMA concentration and ratio, and modulate their bioactivity, through adjustments in the cell-active peptide sequence spatial density, without compromising cell viability. We demonstrated that cell-specific bioinks could be customized for the bioprinting of both human umbilical vein cord endothelial cells (HUVECs) or adult rat sensory neurons from the dorsal root ganglia, and could stimulate both vasculogenesis and neurite outgrowth, respectively. This approach holds great potential as it can be tailored to other cellular models, due to its inherent capacity to accommodate different peptide compositions and to generate complex peptide mixtures and/or gradients.