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Clinical implementation of endochondral bone regeneration (EBR) would benefit from the engineering of devitalized cartilaginous constructs of allogeneic origins. Nevertheless, development of effective devitalization strategies that preserves extracellular matrix (ECM) is still challenging. The aim of this study is to investigate EBR induced by devitalized, soft callus‐mimetic spheroids. To challenge the translatability of this approach, the constructs are generated using an allogeneic cell source. Neo‐bone formation is evaluated in an immunocompetent rat model, subcutaneously and in a critical size femur defect. Living spheroids are used as controls. Also, the effect of spheroid maturation towards hypertrophy is evaluated. The devitalization procedure successfully induces cell death without affecting ECM composition or bioactivity. In vivo, a larger amount of neo‐bone formation is observed for the devitalized chondrogenic group both ectopically and orthotopically. In the femur defect, accelerated bone regeneration is observed in the devitalized chondrogenic group, where defect bridging is observed 4 weeks post‐implantation. The authors' results show, for the first time, a dramatic increase in the rate of bone formation induced by devitalized soft callus‐mimetics. These findings pave the way for the development of a new generation of allogeneic, “off‐the‐shelf” products for EBR, which are suitable for the treatment of every patient. A devitalized soft‐callus mimetic derived from allogeneic non‐immunologically matched donor mesenchymal stem cells is engineered. In vitro, the devitalized constructs present the same characteristics of their living counterparts. In vivo the devitalized soft‐callus mimetic outperforms their living equivalent, in terms of accelerated bone regeneration and the quality of the newly formed bone.

Visible light‐mediated cross‐linking has utility for enhancing the structural capacity and shape fidelity of laboratory‐based polymers. With increased light penetration and cross‐linking speed, there is opportunity to extend future applications into clinical spheres. This study evaluated the utility of a ruthenium/sodium persulfate photocross‐linking system for increasing structural control in heterogeneous living tissues as an example, focusing on unmodified patient‐derived lipoaspirate for soft tissue reconstruction. Freshly‐isolated tissue is photocross‐linked, then the molar abundance of dityrosine bonds is measured using liquid chromatography tandem mass spectrometry and the resulting structural integrity assessed. The cell function and tissue survival of photocross‐linked grafts is evaluated ex vivo and in vivo, with tissue integration and vascularization assessed using histology and microcomputed tomography. The photocross‐linking strategy is tailorable, allowing progressive increases in the structural fidelity of lipoaspirate, as measured by a stepwise reduction in fiber diameter, increased graft porosity and reduced variation in graft resorption. There is an increase in dityrosine bond formation with increasing photoinitiator concentration, and tissue homeostasis is achieved ex vivo, with vascular cell infiltration and vessel formation in vivo. These data demonstrate the capability and applicability of photocrosslinking strategies for improving structural control in clinically‐relevant settings, potentially achieving more desirable patient outcomes using minimal manipulation in surgical procedures.

Mimicking endochondral bone formation is a promising strategy for bone regeneration. To become a successful therapy, the cell source is a crucial translational aspect. Typically, autologous cells are used. The use of non-autologous mesenchymal stromal cells (MSCs) represents an interesting alternative. Nevertheless, non-autologous, differentiated MSCs may trigger an undesired immune response, hampering bone regeneration. The aim of this study was to unravel the influence of the immune response on endochondral bone regeneration, when using xenogeneic (human) or allogeneic (Dark Agouti) MSCs. To this end, chondrogenically differentiated MSCs embedded in a collagen carrier were implanted in critical size femoral defects of immunocompetent Brown Norway rats. Control groups were included with syngeneic/autologous (Brown Norway) MSCs or a cell-free carrier. The amount of neo-bone formation was proportional to the degree of host-donor relatedness, as no full bridging of the defect was observed in the xenogeneic group whereas 2/8 and 7/7 bridges occurred in the allogeneic and the syngeneic group, respectively. One week post-implantation, the xenogeneic grafts were invaded by pro-inflammatory macrophages, T lymphocytes, which persisted after 12 weeks, and anti-human antibodies were developed. The immune response toward the allogeneic graft was comparable to the one evoked by the syngeneic implants, aside from an increased production of alloantibodies, which might be responsible for the more heterogeneous bone formation. Our results demonstrate for the first time the feasibility of using non-autologous MSC-derived chondrocytes to elicit endochondral bone regeneration in vivo . Nevertheless, the pronounced immune response and the limited bone formation observed in the xenogeneic group undermine the clinical relevance of this group. On the contrary, although further research on how to achieve robust bone formation with allogeneic cells is needed, they may represent an alternative to autologous transplantation.

The current limitations of commercial microcarriers for mesenchymal stromal cell (MSC) expansion are driving the demand for novel tuneable microcarriers.Novel microcarriers are effective in improving MSC expansion under static and dynamic culture conditions without compromising their stemness and differentiation potential.Integrating microcarrier-based cultures within dynamic bioreactors provides an effective scalable approach for MSC expansion and harvest for downstream clinical translation.The development of good manufacturing practice (GMP)-grade systems, non-xenogenic culture media, and optimised bioreactors, which are compatible with novel microcarriers, is essential to improve cell yield and reproducibility. Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs. Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs.

Light‐Activated Injectable Fat Grafts The artwork highlights an injectable fat graft for soft tissue reconstruction. In article number 2300538, Khoon S Lim and co‐workers develop and apply a light‐activated macromolecular chemistry to facilitate crosslinking of patient‐derived lipoaspirate, resulting in better control over the shape fidelity and mechanics of the fat grafts. This light‐activated injectable fat grafts are easily administered to the siteof‐interest with excellent integration and host vessel infiltration.

Abstract Endochondral bone regeneration is a promising approach in regenerative medicine. Callus mimics (CMs) are engineered and remodeled into bone tissue upon implantation. The long-term objective is to fabricate a sustainable off-the-shelf treatment option for patients. Devitalization was introduced to facilitate storage and using allogeneic (donor) cells would further propel the off-the-shelf approach. However, allogeneic CMs for bone regeneration pose a potential antigenicity concern. Here, we explored the impact of devitalization on antigenicity and osteoinductive bone formation when implanting syngeneic or allogeneic CM in a vital or devitalized state. For this, we implanted chondrogenically differentiated rat-derived mesenchymal stromal cells using an allogeneic immunocompetent ectopic rat model. Vital syngeneic CMs demonstrated the highest bone formation, and vital allogeneic CMs showed the lowest bone formation, while both devitalized CMs showed comparable intermediate levels of bone formation. Preceding bone formation, the level of tartrate-resistant acid phosphatase staining at 7 and 14 days was proportional to the level of eventual bone formation. No differences were observed for local innate immune responses at any time point before or after bone formation. In contrast, allogeneic CMs elicit a mild adaptive immune response, which still permits bone formation in an immunocompetent environment, albeit at a reduced rate compared to the autologous living counterpart. Overall, devitalization delays bone formation when autologous CMs are implanted, whereas it accelerates bone formation in allogeneic CMs, highlighting the potential of this approach for achieving off-the-shelf treatment. Graphical Abstract Graphical Abstract

We introduce Generative, Adaptive, Context-Aware 3D Printing (GRACE), a novel approach combining 3D imaging, computer vision, and parametric modelling to create tailored, context-aware geometries using volumetric additive manufacturing. GRACE rapidly and automatically generates complex structures capable of conforming directly around features ranging from cellular to macroscopic scales with minimal user intervention. We demonstrate its versatility in applications ranging from synthetic objects to biofabrication, including adaptive vascular-like geometries around cell-laden bioinks, resulting in improved functionality. GRACE also enables precise alignment of sequential prints, in addition to the detection and overprinting of opaque surfaces through shadow correction. Compatible with various printing modalities, GRACE transcends traditional additive manufacturing limitations, opening new avenues in tissue engineering and regenerative medicine.

We introduce Generative, Adaptive, Context-Aware 3D Printing (GRACE), a novel approach combining 3D imaging, computer vision, and parametric modelling to create tailored, context-aware geometries using volumetric additive manufacturing. GRACE rapidly and automatically generates complex structures capable of conforming directly around features ranging from cellular to macroscopic scales with minimal user intervention. We demonstrate its versatility in applications ranging from synthetic objects to biofabrication, including adaptive vascular-like geometries around cell-laden bioinks, resulting in improved functionality. GRACE also enables precise alignment of sequential prints, in addition to the detection and overprinting of opaque surfaces through shadow correction. Compatible with various printing modalities, GRACE transcends traditional additive manufacturing limitations, opening new avenues in tissue engineering and regenerative medicine.

There is an increasing need for novel biomaterials compatible with advanced biofabrication technologies, which also permit cells to remodel their microenvironment. This remodelling is crucial for maturing tissue constructs into fully functional tissue replacements. Recent progress in supramolecular chemistries has allowed for the production of dynamic biomaterials. Their properties enable bonds to be reversibly broken by cells, facilitating processes requiring morphological changes or migration, crucial for tissue development and homeostasis. Here, we present a one-of-its-kind gelatin-based hybrid covalent/supramolecular biomaterial. We demonstrate the advantage of adding supramolecular-reactive moieties on covalent materials, over covalent bonds alone, in facilitating processes such as cell growth, migration, spreading and organoid proliferation. This is exemplified by enhanced MSC and T cell migration and improved vascular network formation in hybrid hydrogels over covalent-only materials. The combination of supramolecular and covalent bonds further enabled control over photocrosslinking, allowing the use of the material in volumetric bioprinting of complex structures with high shape fidelity. As a proof-of-concept we bioprinted complex breast-like structures from encapsulated normal breast cell lines with a tumor organoid core. We demonstrated that engineered T cells can migrate large distances into the breast tissue, specifically targeting tumor cells.Competing Interest StatementM.F, P.N.B, M.B, A.R., and R.L. are inventors on a provisional patent application that covers the hydrogel reported in this manuscript and its application for bioprinting, and cell and organoid culture. R.L. is scientific advisor for Readily3D SA. The other authors declare no competing interests.