There is evidence that sorptive clays have been used since prehistory as materials to treat skin abrasions and wounds. Despite this, there is a paucity of studies that systematically address the efficacy of defined clays in improving skin wound healing. In this study, we tested the hypothesis that a well-defined synthetic smectite clay, Laponite, enhances healing in a delayed skin wound healing model. Full-thickness skin wounds were made in the back skin of db/db and wild-type male adult mice (8–10 weeks), and Laponite clay gels or controls of phosphate-buffered saline (PBS) or alginate were applied after 24 h and held in place with a semi-occlusive dressing. Although Laponite treatment did not accelerate macroscopic wound closure, it significantly improved healing quality, including re-epithelialisation, epithelial cell division, epithelial thickness and fibroblast invasion, compared with PBS- or alginate-treated db/db mice. Moreover, hair follicle anagen was stimulated in proximity to Laponite-treated wounds, but was absent in PBS- or alginate-treated wounds. Neutrophil infiltration at day 18 was also reduced. In contrast to alginate, in which VEGF improved wound healing, VEGF in Laponite showed negligible additional benefit. These data indicate a role for synthetic nanoclay gels in wound healing.

Foot and ankle arthrodesis remains a critical surgical approach for managing end-stage joint disease, yet its success is frequently hindered by the challenge of non-union. Despite advances in surgical techniques, complex cases - such as tibiotalocalcaneal fusion - still face non-union rates as high as 27 %, largely due to the region's unique biomechanical demands and compounded by patient comorbidities and surgical variability. In recent years, orthobiologics and advanced biomaterials have demonstrated significant promise in enhancing fusion outcomes, particularly in high-risk patients with bone defects, metabolic disorders, or a history of infection. However, much of the existing clinical evidence stems from other orthopaedic contexts, with limited high-quality data specific to foot and ankle applications. To bridge this gap and support translational innovation, the development of preclinical models that accurately replicate the physiological and pathological characteristics of the human foot and ankle is essential. This review provides a comprehensive overview of the latest advancements in biologics and biomaterials for foot and ankle arthrodesis, with particular emphasis on hydrogels as next-generation platforms for bone regeneration and targeted drug delivery. Additionally, we critically examine the limitations of current preclinical models in terms of biomechanical compatibility and pathological relevance, highlighting opportunities for future refinement. STATEMENT OF SIGNIFICANCE: Foot and ankle arthrodesis often fails due to non-union in complex, poorly vascularized regions. Emerging biomaterials, such as B2A-coated ceramics and peptide-enhanced grafts, show promise in enhancing fusion outcomes. However, a critical barrier to translation is the lack of preclinical models that reflect the foot and ankle's pathological and mechanical complexity. This review not only surveys advanced orthobiologics but also proposes strategies for developing "negative models" and integrating high-risk conditions to better evaluate fusion efficacy. By bridging biomaterial innovation with model development, it offers a structured path for future research and clinical translation in foot and ankle fusion.

Abstract Nanoclay-based biomaterials offer promise for localised growth factor presentation, yet their in vivo degradation, clearance, and systemic fate remain poorly defined. Here, we investigate the fate of a synthetic nanoclay-BMP-2 gel during ectopic bone induction using a combination of in vivo imaging, histology, and component-resolved elemental analysis. Fluorescent tracking confirmed prolonged localisation of BMP-2 within the nanoclay gel and robust bone induction despite negligible growth-factor release. Inductively coupled plasma mass spectrometry (ICP-MS) revealed divergent clearance kinetics for lithium and silicon, structurally distinct components of the clay crystalline lattice, indicating decoupled ionic and particulate degradation pathways. Early clearance was dominated by cell-mediated fragmentation and the transport of clay particulates, while later stages involved preferential lithium release associated with local clay dissolution as well as integration within newly formed bone. Systemic biodistribution analysis demonstrated rapid, transient lithium release into circulation with renal clearance, contrasted with initial hepatic and then later-phase renal handling of silicon species. Together, these findings define a multiphasic in vivo clearance model for nanoclay biomaterials consistent with progressive remodelling, localised BMP-2 activity and, importantly, safe systemic handling. This work provides mechanistic insight into the activity and clearance of nanoclay-based regenerative therapies and establishes the importance of component-resolved tracking for evaluating the biodistribution of degradable inorganic biomaterials.

Synthetic hectorite nanoclays, such as Laponite®, are widely explored as model anisotropic colloids and injectable biomaterials due to their high specific surface area, charge anisotropy and ability to form physically cross-linked gels in aqueous environments. Despite this interest, the role of nanoclay surface chemistry in governing application-specific properties such as gelation and protein adsorption remains largely unexplored. In this study, we systematically analyse how modulation of the Mg/Li ratio influences electrokinetic behaviour and gelation performance under physiologically relevant conditions, with a view to optimising clay-based materials for biomedical applications. Synthetic hectorite analogues (RENOVITE®) were engineered via Mg/Li ratio control during hydrothermal synthesis to tune permanent layer charge while preserving structural fidelity. Composition was validated by ICP-OES, while structural and morphological fidelity was confirmed by XRD, ATR-FTIR and SAXS. Electrokinetic measurements revealed a monotonic decrease in permanent basal charge with increasing Mg/Li ratio, accompanied by a systematic shift in agglomeration onset at higher pH. Rheometric characterisation in physiological solutions revealed a clear compositional optimum with an intermediate Mg/Li ratio, yielding maximal storage modulus, while both lower and higher Mg/Li formulations formed weaker networks. This optimum arises from a balance between sufficient platelet delamination (enabled by basal charge) and effective edge-mediated connectivity, positioning the system near a percolation threshold under physiological conditions. In summary, this work establishes a direct link between Mg/Li-mediated surface chemistry, electrokinetics, and diffusion-driven gel mechanics, providing a framework for rationale design of nanoclay biomaterials with tunable performance in biomedical applications.

AbstractBone tissue engineering seeks to develop treatment approaches for nonhealing and large bone defects. An ideal biodegradable scaffold will induce and support bone formation. The current study examines bone augmentation in critical‐sized bone defects, using functionalized scaffolds, with the hypothesized potential to induce skeletal cell differentiation. 3D printed, porous poly(caprolactone) trimethacrylate (PCL‐TMA900) scaffolds are applied within a murine femur defect, stabilized by a polyimide intramedullary (IM) pin. The PCL‐TMA900 scaffolds are coated with i) elastin‐like polypeptide (ELP), ii) poly(ethyl acrylate) (PEA)/fibronectin (FN)/bone morphogenetic protein‐2 (PEA/FN/BMP‐2), iii) both ELP and PEA/FN/BMP‐2, or iv) Laponite nanoclay binding BMP‐2. Sequential microcomputed tomography (µCT) and histological analysis are performed. PCL‐TMA900 is robust and biocompatible and when coated with the nanoclay material Laponite and BMP‐2 induce consistent, significant bone formation compared to the uncoated PCL‐TMA900 scaffold. Critically, the BMP‐2 is retained, due to the Laponite, producing bone around the scaffold in the desired shape and volume, compared to bone formation observed with the positive control (collagen sponge/BMP‐2). The ELP and/or PEA/FN/BMP‐2 scaffolds do not demonstrate significant or consistent bone formation. In summary, Laponite/BMP‐2 coated PCL‐TMA900 scaffolds offer a biodegradable, osteogenic construct for bone augmentation with potential for development into a large scale polymer scaffold for clinical translation.

The extracellular matrix (ECM) of tissues progressively changes its mechanical properties in processes such as tissue development, repair, and disease progression. While stiffness has become a key design parameter of biomaterials, most synthetic biomaterials employed in cell culture or tissue regeneration do not display these gradual changes in mechanical properties. Here, we report on a hydrogel platform with the capacity to exhibit progressive stiffening from 0.8 to 7.4 kPa within a ∼48 h time period. The material integrates the tyramine derivative of hyaluronic acid (HAT) and Laponite® (Lap) and harnesses the diffusion of cations from culture media to trigger gradual secondary Lap-HAT cross-linking, resulting in the progressive stiffening of the hydrogel. We assessed the applicability of the hydrogel by first using it as a substrate for in vitro culture to investigate cross-talk between human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs). The progressively stiffening hydrogel led to changes in cell morphology and enhanced differentiation and communication compared to control substrates. In addition, we also tested the potential of the progressively stiffening hydrogels for bone regeneration using a critical-size rat cranial defect model and found that the hydrogel construct promoted vascularized bone regeneration. The current study introduces a hydrogel material that offers a more physiologically relevant environment for in vitro and in vivo applications and provides insight into the mechanical complexity of the ECM and its role in tissue physiology. STATEMENT OF SIGNIFICANCE: This study presents a dynamic hydrogel platform that imitates the progressive mechanical changes of the native extracellular matrix (ECM), transitioning from soft (0.8 kPa) to stiff (7.4 kPa) over 48 h. By co-assembling HAT and Lap, the hydrogel achieves gradual stiffening through cation diffusion - mediated gradual secondary Lap-HAT cross-linking, offering a physiologically relevant microenvironment. In vitro, it enhances HBMSC and HUVEC cross-talk, improving differentiation and morphology. In vivo, it promotes vascularized bone regeneration in a critical-size cranial defect model. This innovation bridges the gap between static synthetic biomaterials and dynamic ECM mechanics, advancing applications in tissue engineering, disease modeling, and regenerative medicine.

Decellularized tissues offer significant potential as biological materials for tissue regeneration given their ability to preserve the complex compositions and architecture of the native extracellular matrix (ECM). However, the evaluation and derivation of decellularized matrices from human bone tissue remains largely unexplored. We examined how the physiochemical and biological properties of ECM hydrogels derived from human bone ECM could be controlled by manipulating bone powder size (45-250 mu m, 250-1000 mu m, and 1000-2000 mu m) and ECM composition through modulation of enzyme digestion time (3-5-7 days). A reduction in material bone powder size and an increase in ECM digestion time produced enhanced protein concentrations in the ECM hydrogels, accompanied by the presence of a diverse array of proteins and improved gelation strength. Human bone marrow-derived stromal cells (HBMSCs) cultured on ECM hydrogels from 45 to 250 mu m bone powder, over 7 days, demonstrated enhanced osteogenic differentiation compared to hydrogels derived from larger bone powders and collagen gels confirming the potential of the hydrogels as biologically active materials for bone regeneration. Digestion time and bone powder size modulation enabled the generation of hydrogels with enhanced release of ECM proteins and appropriate gelation and rheological properties, offering new opportunities for application in bone repair.

Bone morphogenetic protein 2 (BMP2) is clinically applied for treating intractable fractures and promoting spinal fusion because of its osteogenic potency. However, adverse effects following the release of supraphysiological doses of BMP2 from collagen carriers are widely reported. Nanoclay gel (NC) is attracting attention as a biomaterial, given the potential for localized efficacy of administered agents. However, the efficacy and mechanism of action of NC/BMP2 remain unclear. This study explored the efficacy of NC as a BMP2 carrier in bone regeneration and the enhancement mechanism. Subfascial implantation of NC containing BMP2 elicited superior bone formation compared with collagen sponge (CS). Cartilage was uniformly formed inside the NC, whereas CS formed cartilage only on the perimeter. Additionally, CS induced a dose-dependent inflammatory response around the implantation site, whereas NC induced a minor response, and inflammatory cells were observed inside the NC. In a rat spinal fusion model, NC promoted high-quality bony fusion compared to CS. In vitro, NC enhanced chondrogenic and osteogenic differentiation of hBMSCs and ATDC5 cells while inhibiting osteoclastogenesis. Overall, NC/BMP2 facilitates spatially controlled, high-quality endochondral bone formation without BMP2-induced inflammation and promotes high-density new bone, functioning as a next-generation BMP2 carrier.

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair.

Critical bone defects and fractures are typically treated using autologous bone grafts, which are limited by volume of bone that can be harvested, or allogeneic or synthetic bone grafts that lack osteoinductive properties. Human bone extracellular matrix (hbECM) is widely available and offers the potential to be used as a native material to synthesize functional injectable hydrogel systems. However, hbECM lacks the mechanical stability required for injectability and bone growth. We have explored the use of Laponite® (LAP) nanoclay and sodium polyacrylate to augment the mechanical and biological properties of hbECM gel. We demonstrated that the inclusion of LAP into ECM improved the physicochemical properties of hbECM and consequently promoted cell responses confirming that the nanoclay platelet-to-platelet interaction is key to sustain hbECM functionality. This novel hbECM detailed offers significant clinical promise for bone repair.

As we navigate the transition from the Fourth to the Fifth Industrial Revolution, the emerging fields of biomanufacturing and biofabrication are transforming life sciences and healthcare. These sectors are benefiting from a synergy of synthetic and engineering biology, sustainable manufacturing, and integrated design principles. Advanced techniques such as 3D bioprinting, tissue engineering, directed assembly, and self-assembly are instrumental in creating biomimetic scaffolds, tissues, organoids, medical devices, and biohybrid systems. The field of biofabrication in the United Kingdom and Ireland is emerging as a pivotal force in bioscience and healthcare, propelled by cutting-edge research and development. Concentrating on the production of biologically functional products for use in drug delivery, in vitro models, and tissue engineering, research institutions across these regions are dedicated to innovating healthcare solutions that adhere to ethical standards while prioritising sustainability, affordability, and healthcare system benefits.

AbstractFracture non‐union occurs due to various factors, leading to the development of potentially substantial bone defects. While autograft and allograft are the current gold standards for non‐union fractures, challenges related to availability and immune rejection highlight the need for improved treatments. A strategy in bone tissue engineering is to harness growth factors to induce an effect on cells to change their phenotype, behavior and initiate signaling pathways which lead to increased matrix deposition and tissue formation. Bone morphogenetic protein‐2 (BMP‐2) is a potent osteogenic growth factor however, given its rapid clearance time in vivo, there is a specific therapeutic window for efficacy while avoiding potential deleterious side‐effects. It is demonstrated that a Laponite nanoclay coating on a 3D printable and bioresorbable poly(caprolactone) trimethacrylate‐based resin enables binding of BMP‐2, decreases the rate of release, enabling reduced concentrations to be used while enhancing osteoinduction in both in vitro and in vivo models.

Biomaterial-based approaches for bone regeneration seek to explore alternative strategies to repair non-healing fractures and critical-sized bone defects. Fracture non-union occurs due to a number of factors resulting in the formation of bone defects. Rigorous evaluation of the biomaterials in relevant models and assessment of their potential to translate towards clinical use is vital. Large animal experimentation can be used to model fracture non-union while scaling-up materials for clinical use. Growth factors modulate cell phenotype, behaviour and initiate signalling pathways leading to changes in matrix deposition and tissue formation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic growth factor, with a rapid clearance time in vivo necessitating clinical use at a high dose, with potential deleterious side-effects. The current studies have examined the potential for Laponite® nanoclay coated poly(caprolactone) trimethacrylate (PCL-TMA900) scaffolds to bind BMP-2 for enhanced osteoinduction in a large animal critical-sized bone defect. An ovine femoral condyle defect model confirmed PCL-TMA900 scaffolds coated with Laponite®/BMP-2 produced significant bone formation compared to the uncoated PCL-TMA 900 scaffold in vivo, assessed by micro-computed tomography (μCT) and histology. This indicated the ability of Laponite® to deliver the bioactive BMP-2 on the PCL-TMA900 scaffold. Bone formed around the Laponite®/BMP-2 coated PCL-TMA900 scaffold, with no erroneous bone formation observed away from the scaffold material confirming localisation of BMP-2 delivery. The current studies demonstrate the ability of a nanoclay to localise and deliver bioactive BMP-2 within a tailored octet-truss scaffold for efficacious bone defect repair in a large animal model with significant implications for translation to the clinic.

This investigation seeks to integrate LAPONITE® clay gels with thermoresponsive branched copolymer surfactants (BCSs) to develop advanced functional materials with temperature-induced sol-gel behaviour. It is known that a diverse range of molecules adsorb strongly to clays which may be used to control liberation of the species in healthcare applications, and as such the development of polymer/clay hybrid materials which can add function to the native clay behaviour are of great interest. BCS were synthesised with a structure that encompasses poly(ethylene glycol)methacrylate (PEGMA), ethylene glycol dimethacrylate (EGDMA), and dodecanethiol (DDT), conferring versatile and tuneable thermoresponsive attributes. Systematic modulation of the monomer : DDT/initiator ratio was used to facilitate the synthesis of BCS architectures spanning a range of molecular weights. Through application of small-amplitude oscillatory shear (SAOS) rheology and small-angle neutron scattering (SANS) in conjunction with controlled temperature variations, the sol-gel transition dynamics of these nanocomposite materials were elucidated. Complementary insights into the mechanisms underpinning this transition and temperature-induced alterations in the constituents are gleaned through the utilization of SANS techniques employing contrast-matching methodologies to mitigate clay and polymer scattering interference. It is found that heating systems from room- to body- temperature induces self-assembly of BCS in the bulk aqueous phase with concurrent structuration of clay in gel-forming samples with lower number average molecular weight (Mn). SANS study unpicks this phenomenon to find that gelation occurs with concurrent aggregation of BCS in the bulk, inducing clay-clay interactions only in lower Mn BCS systems with large nanoaggregates.

Fracture non-union occurs as a consequence of various factors, leading to the development of potentially substantial bone defects. Biomaterial-based approaches for bone regeneration aim to explore alternative strategies to repair non-healing fractures and critical-sized bone defects. Thus, rigorous assessment of the ability to translate biomaterials towards clinical use is vital. Growth factors induce an effect on cells to change their phenotype, behaviour and initiate signalling pathways, leading to an effect on matrix deposition and tissue formation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic growth factor, with a rapid clearance time in vivo necessitating clinical use of high doses, with potential deleterious side-effects. This work explored the potential for Laponite™ nanoclay coating of poly(caprolactone) trimethacrylate (PCL-TMA900) scaffolds to bind BMP-2 for enhanced osteoinduction. In vitro experiments confirmed the cytocompatibility of the PCL-TMA900 scaffolds and effective osteogenic differentiation of C2C12 myoblast cells in response to the Laponite/BMP-2 coating. The chorioallantoic membrane (CAM) assay verified PCL-TMA900 scaffold material biocompatibility and ability to support angiogenesis. A murine subcutaneous implantation model assessed heterotopic bone formation in response to the Laponite/BMP-2 coating, when used immediately post-coating and after 24 hours of room temperature storage, to evaluate a delayed use manner. The Laponite/BMP-2 coated PCL-TMA900 scaffolds implanted showed consistent, significant bone formation over the study period compared to the uncoated PCL-TMA 900 scaffold and BMP-2 only coated control scaffolds in vivo , indicating the ability of Laponite to bind the BMP-2 to the PCL-TMA900 scaffold. Bone formed peripherally around the Laponite/BMP-2 coated scaffold, with no aberrant bone formation observed. The Laponite/BMP-2 coating was found to retain its bioactivity after storage for 24 hours prior to use in vivo , however this was not to the same volume or reliability of bone formation as when used immediately post-coating. To take these studies forward, the Laponite/BMP-2 coating warrants examination in a critical-sized bone defect model to assess efficacy in an osseous site.