Injuries to the meniscus of the knee commonly lead to osteoarthritis. Current therapies for meniscus regeneration, including meniscectomies and scaffold implantation, fail to achieve complete functional regeneration of the tissue. This has led to increased interest in cell and gene therapies and tissue engineering approaches to meniscus regeneration. The implantation of a biomimetic implant, incorporating cells, growth factors, and extracellular matrix (ECM)‐derived proteins, represents a promising approach to functional meniscus regeneration. The objective of this study was to develop a range of ECM‐functionalised bioinks suitable for 3D bioprinting of meniscal tissue. To this end, alginate hydrogels were functionalised with ECM derived from the inner and outer regions of the meniscus and loaded with infrapatellar fat pad‐derived stem cells. In the absence of exogenously supplied growth factors, inner meniscus ECM promoted chondrogenesis of fat pad‐derived stem cells, whereas outer meniscus ECM promoted a more elongated cell morphology and the development of a more fibroblastic phenotype. With exogenous growth factors supplementation, a more fibrogenic phenotype was observed in outer ECM‐functionalised hydrogels supplemented with connective tissue growth factor, whereas inner ECM‐functionalised hydrogels supplemented with TGFβ3 supported the highest levels of Sox‐9 and type II collagen gene expression and sulfated glycosaminoglycans (sGAG) deposition. The final phase of the study demonstrated the printability of these ECM‐functionalised hydrogels, demonstrating that their codeposition with polycaprolactone microfibres dramatically improved the mechanical properties of the 3D bioprinted constructs with no noticeable loss in cell viability. These bioprinted constructs represent an exciting new approach to tissue engineering of functional meniscal grafts.

Metastasis is responsible for most cancer-related deaths. However, only a fraction of circulating cancer cells succeed in forming secondary tumours, indicating that adaptive mechanisms during circulation play a part in dissemination. Here, we report that constriction during microcapillary transit triggers reprogramming of melanoma cells to a tumorigenic cancer stem cell-like state. Using a microfluidic device mimicking physiological flow rates and gradual capillary narrowing, we show that compression through narrow channels causes cell and nuclear deformation, rapid chromatin remodelling and increased calcium signalling via mechanosensor PIEZO1. Within minutes, cells upregulate transcripts associated with metabolic reprogramming and metastatic processes. Over time, this results in the stable adoption of a cancer stem cell-like state. Squeezed cells express elevated melanoma stem cell markers, exhibit increased trans-endothelium invasion and display enhanced tumorigenicity in vitro and in vivo. Pharmacological inhibition of PIEZO1 blocks this transition, while activation with Yoda1 induces the stem cell-like state irrespective of constriction. Deletion of PIEZO1 completely abolishes the constriction-induced phenotype. Together, these findings demonstrate that compressive forces during circulation reprogram circulating cancer cells into tumorigenic, stem cell-like states, primed for extravasation and metastatic colonization.

Mesenchymal stem cell therapy has suffered from wide variability in clinical efficacy, largely due to heterogeneous starting cell populations and large‐scale cell death during and after implantation. Optimizing the manufacturing process has led to reproducible cell populations that can be cryopreserved for clinical applications. Nevertheless, ensuring a reproducible cell state that persists after cryopreservation remains a significant challenge, and is necessary to ensure reproducible clinical outcomes. Here we demonstrate how matrix‐conjugated hydrogel cell culture materials can normalize a population of induced pluripotent stem cell derived mesenchymal stem cells (iPSC‐MSCs) to display a defined secretory profile that promotes enhanced neovascularization in vitro and in vivo. Using a protein‐conjugated biomaterials screen we identified two conditions—1 kPa collagen and 10 kPa fibronectin coated polyacrylamide gels—that promote reproducible secretion of pro‐angiogenic and immunomodulatory cytokines from iPSC‐MSCs that enhance tubulogenesis of endothelial cells in Geltrex and neovascularization in chick chorioallantoic membranes. Using defined culture substrates alone, we demonstrate maintenance of secretory activity after cryopreservation for the first time. This advance provides a simple and scalable approach for cell engineering and subsequent manufacturing, toward normalizing and priming a desired cell activity for clinical regenerative medicine.

Breast cancer progression is a consequence of intricate dynamics between cells and their matrix in the tumor microenvironment. However, most in vitro models are not amenable to studying the behavior of multiple cell types within a defined matrix architecture. In this study, we demonstrate a microporous matrix where breast cancer cells and adipose derived stromal cells are integrated to evaluate crosstalk between matrix parameters and heterotypic cell populations. To do this, we leveraged two biofabrication techniques, granular suspension matrices and drop-on-demand bioprinting, to deposit multiple cell types in a reproducible format amenable to high-throughput screening. 3D gelatin-methacryloyl microgels were used to create a yield stress granular suspension bath with tunable interstitial volume to mimic the porosity and densities of healthy and fibrotic microenvironments. Invasive and non-invasive breast cancer cells (MCF-7 and MDA-MB-231) were bioprinted at the interface of the ADSC-laden granular matrix to probe invasive processes and heterotypic crosstalk as a tumor–stroma model. We focused on cancer cell migration through model fibrotic tissue and ADSC transformations into cancer associated fibroblasts. α-smooth muscle actin expression indicated that the high density microgel matrices are more conducive to ADSC-CAF transformations, which in turn influenced the expression of molecular markers associated with tumorigenicity and chemoresistance in the resident cancer cells. Treatment with doxorubicin supported increased tumorigenicity in the co-cultures. Together, this work demonstrates how defined microengineered matrices can serve as platforms to evaluate cell behavior, with scope for translation to in vitro assays for biological discovery and drug development.

Purpose/aim of study: Menisectomies account for over 1.5 million surgical interventions in Europe annually, and there is a growing interest in regenerative strategies to improve outcomes in meniscal replacement. The overall objective of this study was to evaluate the role of intraoperatively applied fresh chondrocyte (FC) isolates compared to minced cartilage (MC) fragments, used without cell isolation, to improve bioactivity and tissue integration when combined with a polyurethane replacement. Materials and methods: First, to optimize the intraoperative cell isolation protocol, caprine articular cartilage biopsies were digested with 750 U/ml or 3000 U/ml collagenase type II (ratio of 10 ml per g of tissue) for 30 min, 1 h or 12 h with constant agitation and compared to culture-expanded chondrocytes in terms of matrix deposition when cultured on polyurethane scaffolds. Finally, FCs and MC-augmented polyurethane scaffolds were evaluated in a caprine meniscal explant model to assess the potential enhancements on tissue integration strength. Results: Adequate numbers of FCs were harvested using a 30 min chondrocyte isolation protocol and were found to demonstrate improved matrix deposition compared to standard culture-expanded cells in vitro. Upon evaluation in a meniscus explant defect model, both FCs and MC showed improved matrix deposition at the tissue-scaffold interface and enhanced push-out strength, fourfold and 2.5-fold, respectively, compared with the acellular implant. Conclusions: Herein, we have demonstrated a novel approach that could be applied intraoperatively, using FCs or MC for improved tissue integration with a polyurethane meniscal replacement.

Periosteum is a highly vascularized membrane lining the surface of bones. It plays essential roles in bone repair following injury and reconstruction following invasive surgeries. To broaden the use of periosteum, including for augmenting in vitro bone engineering and/or in vivo bone repair, we have developed an ex vivo perfusion bioreactor system to maintain the cellular viability and metabolism of surgically resected periosteal flaps. Each specimen was placed in a 3D printed bioreactor connected to a peristaltic pump designed for the optimal flow rates of tissue perfusate. Nutrients and oxygen were perfused via the periosteal arteries to mimic physiological conditions. Biochemical assays and histological staining indicate component cell viability after perfusion for almost 4 weeks. Our work provides the proof-of-concept of ex vivo periosteum perfusion for long-term tissue preservation, paving the way for innovative bone engineering approaches that use autotransplanted periosteum to enhance in vivo bone repair.