Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s42242-023-00265-z.

A single-cell atlas of conventional central chondrosarcoma reveals the role of endoplasmic reticulum stress in malignant transformation.

The transformation of benign lesions to malignant tumours is a crucial aspect of understanding chondrosarcomas, which are malignant cartilage tumours that could develop from benign chondroid lesions. However, the process of malignant transformation for chondroid lesions remains poorly understood, and no reliable markers are available to aid clinical decision-making. To address this issue, we conducted a study analysing 11 primary cartilage tumours and controls using single-cell RNA sequencing. By creating a single-cell atlas, we were able to identify the role of endoplasmic reticulum (ER) stress in the malignant transformation of conventional central chondrosarcomas (CCCS). Our research revealed that lower levels of ER stress promote chondrosarcoma growth in a patient-derived xenograft mouse model, while intensive ER stress reduces primary chondrosarcoma cell viability. Furthermore, we discovered that the NF-κB pathway alleviates ER stress-induced apoptosis during chondrosarcoma progression. Our single-cell signatures and large public data support the use of key ER stress regulators, such as DNA Damage Inducible Transcript 3 (DDIT3; also known as CHOP), as malignant markers for overall patient survival. Ultimately, our study highlights the significant role that ER stress plays in the malignant transformation of cartilaginous tumours and provides a valuable resource for future diagnostic markers and therapeutic strategies.

Embracing ethical research: Implementing the 3R principles into fracture healing research for sustainable scientific progress.

As scientific advancements continue to reshape the world, it becomes increasingly crucial to uphold ethical standards and minimize the potentially adverse impact of research activities. In this context, the implementation of the 3R principles-Replacement, Reduction, and Refinement-has emerged as a prominent framework for promoting ethical research practices in the use of animals. This article aims to explore recent advances in integrating the 3R principles into fracture healing research, highlighting their potential to enhance animal welfare, scientific validity, and societal trust. The review focuses on in vitro, in silico, ex vivo, and refined in vivo methods, which have the potential to replace, reduce, and refine animal experiments in musculoskeletal, bone, and fracture healing research. Here, we review material that was presented at the workshop "Implementing 3R Principles into Fracture Healing Research" at the 2023 Orthopedic Research Society (ORS) Annual Meeting in Dallas, Texas.

Tracking cellular uptake, intracellular trafficking and fate of nanoclay particles in human bone marrow stromal cells.

Clay nanoparticles, in particular synthetic smectites, have generated interest in the field of tissue engineering and regenerative medicine due to their utility as cross-linkers for polymers in biomaterial design and as protein release modifiers for growth factor delivery. In addition, recent studies have suggested a direct influence on the osteogenic differentiation of responsive stem and progenitor cell populations. Relatively little is known however about the mechanisms underlying nanoclay bioactivity and in particular the cellular processes involved in nanoclay-stem cell interactions. In this study we employed confocal microscopy, inductively coupled plasma mass spectrometry and transmission electron microscopy to track the interactions between clay nanoparticles and human bone marrow stromal cells (hBMSCs). In particular we studied nanoparticle cellular uptake mechanisms and uptake kinetics, intracellular trafficking pathways and the fate of endocytosed nanoclay. We found that nanoclay particles present on the cell surface as µm-sized aggregates, enter hBMSCs through clathrin-mediated endocytosis, and their uptake kinetics follow a linear increase with time during the first week of nanoclay addition. The endocytosed particles were observed within the endosomal/lysosomal compartments and we found evidence for both intracellular degradation of nanoclay and exocytosis as well as an increase in autophagosomal activity. Inhibitor studies indicated that endocytosis was required for nanoclay upregulation of alkaline phosphatase activity but a similar dependency was not observed for autophagy. This study into the nature of nanoclay-stem cell interactions, in particular the intracellular processing of nanosilicate, may provide insights into the mechanisms underlying nanoclay bioactivity and inform the successful utilisation of clay nanoparticles in biomaterial design.

All-fiberized 1840-nm femtosecond thulium fiber laser for label-free nonlinear microscopy.

We report an all-fiberized 1840-nm thulium-fiber-laser source, comprising a dissipative-soliton mode-locked seed laser and a chirped-pulse-amplification system for label-free biological imaging through nonlinear microscopy. The mode-locked thulium fiber laser generated dissipative-soliton pulses with a pre-chirped duration of 7 ps and pulse energy of 1 nJ. A chirped-pulse fiber-amplification system employing an in-house-fabricated, short-length, single-mode, high-absorption, thulium fiber delivered pulses with energies up to 105 nJ. The pulses were capable of being compressed to 416 fs by passing through a grating pair. Imaging of mouse tissue and human bone samples was demonstrated using this source via third-harmonic generation microscopy.

Self-Assembly of Structured Colloidal Gels for High-Resolution 3D Micropatterning of Proteins at Scale.

Self-assembly, the spontaneous ordering of components into patterns, is widespread in nature and fundamental to generating function across length scales. Morphogen gradients in biological development are paradigmatic as both products and effectors of self-assembly and various attempts have been made to reproduce such gradients in biomaterial design. To date, approaches have typically utilized top-down fabrication techniques that, while allowing high-resolution control, are limited by scale and require chemical cross-linking steps to stabilize morphogen patterns in time. Here, a bottom-up approach to protein patterning is developed based on a novel binary reaction-diffusion process where proteins function as diffusive reactants to assemble a nanoclay-protein composite hydrogel. Using this approach, it is possible to generate scalable and highly stable 3D patterns of target proteins down to sub-cellular resolution through only physical interactions between clay nanoparticles and the proteins and ions present in blood. Patterned nanoclay gels are able to guide cell behavior to precisely template bone tissue formation in vivo. These results demonstrate the feasibility of stabilizing 3D gradients of biological signals through self-assembly processes and open up new possibilities for morphogen-based therapeutic strategies and models of biological development and repair.

Nanotopography reveals metabolites that maintain the immunomodulatory phenotype of mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells that are of considerable clinical potential in transplantation and anti-inflammatory therapies due to their capacity for tissue repair and immunomodulation. However, MSCs rapidly differentiate once in culture, making their large-scale expansion for use in immunomodulatory therapies challenging. Although the differentiation mechanisms of MSCs have been extensively investigated using materials, little is known about how materials can influence paracrine activities of MSCs. Here, we show that nanotopography can control the immunomodulatory capacity of MSCs through decreased intracellular tension and increasing oxidative glycolysis. We use nanotopography to identify bioactive metabolites that modulate intracellular tension, growth and immunomodulatory phenotype of MSCs in standard culture and during larger scale cell manufacture. Our findings demonstrate an effective route to support large-scale expansion of functional MSCs for therapeutic purposes.

Rapid fabrication and screening of tailored functional 3D biomaterials: Validation in bone tissue repair - Part II.

Regenerative medicine strategies place increasingly sophisticated demands on 3D biomaterials to promote tissue formation at sites where tissue would otherwise not form. Ideally, the discovery/fabrication of the 3D scaffolds needs to be high-throughput and uniform to ensure quick and in-depth analysis in order to pinpoint appropriate chemical and mechanical properties of a biomaterial. Herein we present a versatile technique to screen new potential biocompatible acrylate-based 3D scaffolds with the ultimate aim of application in tissue repair. As part of this process, we identified an acrylate-based 3D porous scaffold that promoted cell proliferation followed by accelerated tissue formation, pre-requisites for tissue repair. Scaffolds were fabricated by a facile freeze-casting and an in-situ photo-polymerization route, embracing a high-throughput synthesis, screening and characterization protocol. The current studies demonstrate the dependence of cellular growth and vascularization on the porosity and intrinsic chemical nature of the scaffolds, with tuneable 3D scaffolds generated with large, interconnected pores suitable for cellular growth applied to skeletal reparation. Our studies showed increased cell proliferation, collagen and ALP expression, while chorioallantoic membrane assays indicated biocompatibility and demonstrated the angiogenic nature of the scaffolds. VEGRF2 expression in vivo observed throughout the 3D scaffolds in the absence of growth factor supplementation demonstrates a potential for angiogenesis. This novel platform provides an innovative approach to 3D scanning of synthetic biomaterials for tissue regeneration.

Modelling skeletal pain harnessing tissue engineering.

Bone pain typically occurs immediately following skeletal damage with mechanical distortion or rupture of nociceptive fibres. The pain mechanism is also associated with chronic pain conditions where the healing process is impaired. Any load impacting on the area of the fractured bone will stimulate the nociceptive response, necessitating rapid clinical intervention to relieve pain associated with the bone damage and appropriate mitigation of any processes involved with the loss of bone mass, muscle, and mobility and to prevent death. The following review has examined the mechanisms of pain associated with trauma or cancer-related skeletal damage focusing on new approaches for the development of innovative therapeutic interventions. In particular, the review highlights tissue engineering approaches that offer considerable promise in the application of functional biomimetic fabrication of bone and nerve tissues. The strategic combination of bone and nerve tissue engineered models provides significant potential to develop a new class of in vitro platforms, capable of replacing in vivo models and testing the safety and efficacy of novel drug treatments aimed at the resolution of bone-associated pain. To date, the field of bone pain research has centred on animal models, with a paucity of data correlating to the human physiological response. This review explores the evident gap in pain drug development research and suggests a step change in approach to harness tissue engineering technologies to recapitulate the complex pathophysiological environment of the damaged bone tissue enabling evaluation of the associated pain-mimicking mechanism with significant therapeutic potential therein for improved patient quality of life. Graphical abstract: Rationale underlying novel drug testing platform development. Pain detected by the central nervous system and following bone fracture cannot be treated or exclusively alleviated using standardised methods. The pain mechanism and specificity/efficacy of pain reduction drugs remain poorly understood. In vivo and ex vivo models are not yet able to recapitulate the various pain events associated with skeletal damage. In vitro models are currently limited by their inability to fully mimic the complex physiological mechanisms at play between nervous and skeletal tissue and any disruption in pathological states. Robust innovative tissue engineering models are needed to better understand pain events and to investigate therapeutic regimes.

Ontogenetic changes in cortical bone vascular microstructure in the domestic duck (Anas platyrhynchos) and ring-necked pheasant (Phasianus colchicus).

Age-related changes in bone microstructure can inform our understanding the biology of both extant and fossil birds, but to date, histological work in birds, and particularly work using high-resolution 3D imaging, has largely been restricted to limited growth stages. We used minimally destructive synchrotron radiation-based X-ray computed tomography to visualise and measure key morphological and histological traits in 3D across development in the domestic duck and ring-necked pheasant. We use these measurements to build on the database of key reference material for interpreting bone histology. We found that growth patterns differed between the two species, with the ducks showing rapid growth in their lower limbs and early lower limb maturation, while pheasants grew more slowly, reflecting their later age at maturity. In the pheasant, both walking and flight occur early and their upper and lower limbs grew at similar rates. In the duck, flight and wing development are delayed until the bird is almost at full body mass. Through juvenile development, the second moment of area for the duck wing was low but increased rapidly towards the age of flight, at which point it became significantly greater than that of the lower limb, or the pheasant. On a microstructural level, both cortical porosity and canal diameter were related to cortical bone deposition rate. In terms of orientation, vascular canals in the bone cortex were more laminar in the humerus and femur compared with the tibiotarsus, and laminarity increased through juvenile development in the humerus, but not the tibiotarsus, possibly reflecting torsional vs compressive loading. These age-related changes in cortical bone vascular microstructure of the domestic duck and pheasant will help understanding the biology of both extant and fossil birds, including age estimation, growth rate and growth patterns, and limb function.

Osteoclast-derived extracellular vesicles are implicated in sensory neurons sprouting through the activation of epidermal growth factor signaling.

BACKGROUND: Different pathologies, affecting the skeletal system, were reported to display altered bone and/or cartilage innervation profiles leading to the deregulation of the tissue homeostasis. The patterning of peripheral innervation is achieved through the tissue-specific expression of attractive or repulsive axonal guidance cues in specific space and time frames. During the last decade, emerging findings attributed to the extracellular vesicles (EV) trading a central role in peripheral tissue innervation. However, to date, the contribution of EV in controlling bone innervation is totally unknown. RESULTS: Here we show that sensory neurons outgrowth induced by the bone resorbing cells-osteoclasts-is promoted by osteoclast-derived EV. The EV induced axonal growth is achieved by targeting epidermal growth factor receptor (EGFR)/ErbB2 signaling/protein kinase C phosphorylation in sensory neurons. In addition, our data also indicate that osteoclasts promote sensory neurons electrophysiological activity reflecting a possible pathway in nerve sensitization in the bone microenvironment, however this effect is EV independent. CONCLUSIONS: Overall, these results identify a new mechanism of sensory bone innervation regulation and shed the light on the role of osteoclast-derived EV in shaping/guiding bone sensory innervation. These findings provide opportunities for exploitation of osteoclast-derived EV based strategies to prevent and/or mitigate pathological uncontrolled bone innervation.

Gelatin Methacryloyl Hydrogels for Musculoskeletal Tissue Regeneration.

Musculoskeletal disorders are a significant burden on the global economy and public health. Hydrogels have significant potential for enhancing the repair of damaged and injured musculoskeletal tissues as cell or drug delivery systems. Hydrogels have unique physicochemical properties which make them promising platforms for controlling cell functions. Gelatin methacryloyl (GelMA) hydrogel in particular has been extensively investigated as a promising biomaterial due to its tuneable and beneficial properties and has been widely used in different biomedical applications. In this review, a detailed overview of GelMA synthesis, hydrogel design and applications in regenerative medicine is provided. After summarising recent progress in hydrogels more broadly, we highlight recent advances of GelMA hydrogels in the emerging fields of musculoskeletal drug delivery, involving therapeutic drugs (e.g., growth factors, antimicrobial molecules, immunomodulatory drugs and cells), delivery approaches (e.g., single-, dual-release system), and material design (e.g., addition of organic or inorganic materials, 3D printing). The review concludes with future perspectives and associated challenges for developing local drug delivery for musculoskeletal applications.

Cell-controlled dynamic surfaces for skeletal stem cell growth and differentiation.

Skeletal stem cells (SSCs, or mesenchymal stromal cells typically referred to as mesenchymal stem cells from the bone marrow) are a dynamic progenitor population that can enter quiescence, self-renew or differentiate depending on regenerative demand and cues from their niche environment. However, ex vivo, in culture, they are grown typically on hard polystyrene surfaces, and this leads to rapid loss of the SSC phenotype. While materials are being developed that can control SSC growth and differentiation, very few examples of dynamic interfaces that reflect the plastic nature of the stem cells have, to date, been developed. Achieving such interfaces is challenging because of competing needs: growing SSCs require lower cell adhesion and intracellular tension while differentiation to, for example, bone-forming osteoblasts requires increased adhesion and intracellular tension. We previously reported a dynamic interface where the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD) was presented to the cells upon activation by user-added elastase that cleaved a bulky blocking group hiding RGD from the cells. This allowed for a growth phase while the blocking group was in place and the cells could only form smaller adhesions, followed by an osteoblast differentiation phase that was induced after elastase was added which triggered exposure of RGD and subsequent cell adhesion and contraction. Here, we aimed to develop an autonomous system where the surface is activated according to the need of the cell by using matrix metalloprotease (MMP) cleavable peptide sequences to remove the blocking group with the hypothesis that the SSCs would produce higher levels of MMP as the cells reached confluence. The current studies demonstrate that SSCs produce active MMP-2 that can cleave functional groups on a surface. We also demonstrate that SSCs can grow on the uncleaved surface and, with time, produce osteogenic marker proteins on the MMP-responsive surface. These studies demonstrate the concept for cell-controlled surfaces that can modulate adhesion and phenotype with significant implications for stem cell phenotype modulation.

From hurdle to springboard: The macrophage as target in biomaterial-based bone regeneration strategies.

The past decade has seen a growing appreciation for the role of the innate immune response in mediating repair and biomaterial directed tissue regeneration. The long-held view of the host immune/inflammatory response as an obstacle limiting stem cell regenerative activity, has given way to a fresh appreciation of the pivotal role the macrophage plays in orchestrating the resolution of inflammation and launching the process of remodelling and repair. In the context of bone, work over the past decade has established an essential coordinating role for macrophages in supporting bone repair and sustaining biomaterial driven osteogenesis. In this review evidence for the role of the macrophage in bone regeneration and repair is surveyed before discussing recent biomaterial and drug-delivery based approaches that target macrophage modulation with the goal of accelerating and enhancing bone tissue regeneration.

Materials-driven fibronectin assembly on nanoscale topography enhances mesenchymal stem cell adhesion, protecting cells from bacterial virulence factors and preventing biofilm formation.

Post-operative infection is a major complication in patients recovering from orthopaedic surgery. As such, there is a clinical need to develop biomaterials for use in regenerative surgery that can promote mesenchymal stem cell (MSC) osteospecific differentiation and that can prevent infection caused by biofilm-forming pathogens. Nanotopographical approaches to pathogen control are being identified, including in orthopaedic materials such as titanium and its alloys. These topographies use high aspect ratio nanospikes or nanowires to prevent bacterial adhesion but these features also significantly reduce MSC adhesion and activity. Here, we use a poly (ethyl acrylate) (PEA) polymer coating on titanium nanowires to spontaneously organise fibronectin (FN) and to deliver bone morphogenetic protein 2 (BMP2) to enhance MSC adhesion and osteospecific signalling. Using a novel MSC-Pseudomonas aeruginosa co-culture, we show that the coated nanotopographies protect MSCs from cytotoxic quorum sensing and signalling molecules, enhance MSC adhesion and osteoblast differentiation and reduce biofilm formation. We conclude that the PEA polymer-coated nanotopography can both support MSCs and prevent pathogens from adhering to a biomaterial surface, thus protecting from biofilm formation and bacterial infection, and supporting osteogenic repair.

Synthesis of scaffold-free, three dimensional, osteogenic constructs following culture of skeletal osteoprogenitor cells on glass surfaces.

BACKGROUND: Efficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge. These constructs can be crucial for patients with bone defects due to congenital or traumatic reasons. The modulation of cell fate and function as a consequence of interaction with the physical and chemical properties of materials is well known. METHODS: The current study has examined the osteogenic differentiation potential of human skeletal populations following culture on glass surfaces, as a monolayer, or in glass tubes as a pellet culture. The 3D prosperities were assessed morphometrically and the differentiation was evaluated through molecular characterization as well as matrix formation. RESULTS: Early temporal expression of alkaline phosphatase expression of skeletal populations was observed following culture on glass surfaces. Skeletal populations seeded on glass tubes, adhered as a monolayer to the tube base and subsequently formed 3D pellets at the air -media interface. The pellets cultured on glass displayed 4.9 ± 1.3 times the weight and 2.9 ± 0.1 the diameter of their counterpart cultured in plastic tubes and displayed enhanced production of osteogenic matrix proteins, such a collagen I and osteonectin. The size and weight of the pellets correlated with surface area in contrast to cell numbers seeded. Global DNA methylation level was decreased in pellets cultured on glass. In contrast, gene expression analysis confirmed upregulation extracellular matrix proteins and osteogenesis-related growth factors. CONCLUSION: This simple approach to the culture of skeletal cells on glass tubes provides a scaffold-free, 3D construct platform for generating pellets enabling analysis and evaluation of tissue development and integration of multiple constructs with implications for tissue repair and regenerative application on scale-up.

Custom 3D-Printed Triflange Implants for Treatment of Severe Acetabular Defects, with and without Pelvic Discontinuity: Early Results of Our First 19 Consecutive Cases.

Treatment of massive acetabular defects, both with and without pelvic discontinuity, is challenging. The implants utilized in the surgical procedure need to be stable and integrate with poor host bone stock. In the present study, we describe our experience addressing this challenge. METHODS: We identified all patients who underwent surgical implantation of a custom 3D-printed triflange prosthesis with dual-mobility bearings for the treatment of Paprosky 3B acetabular defects between 2014 and 2020. Operative, functional, and radiographic outcomes were assessed. RESULTS: A total of 19 patients were identified, including 11 women. The mean age was 77 years (range, 53 to 91 years), and 8 patients (42%) had proven or likely pelvic discontinuity. The mean follow-up was 53 months (range, 17 to 88 months; mode, 57 months). The cumulative implant survivorship was 100%. Two patients suffered notable sciatic nerve palsy, with 1 case being recurrent. There were no dislocations or fractures. The mean Oxford Hip Score improved significantly, from a mean of 8.6 (range, 0 to 22) preoperatively to 35 (range, 10 to 48) postoperatively (p < 0.0001). Radiographically, there was excellent correlation between implant position and the preoperative plan (p > 0.05). There were no cases of implant loosening or migration, which suggests that stabilization was achieved even among cases with pelvic discontinuity. CONCLUSIONS: These early results suggest that the use of a custom 3D-printed triflange implant has potential advantages over traditional constructs in the treatment of massive acetabular defects, with and without pelvic discontinuity. Excellent implant survivorship and functional improvement were demonstrated in this challenging patient cohort. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Pancreas deficiency modifies bone development in the ovine fetus near term.

Hormones have an important role in the regulation of fetal growth and development, especially in response to nutrient availability in utero. Using micro-CT and an electromagnetic three-point bend test, this study examined the effect of pancreas removal at 0.8 fraction of gestation on the developing bone structure and mechanical strength in fetal sheep. When fetuses were studied at 10 and 25 days after surgery, pancreatectomy caused hypoinsulinaemia, hyperglycaemia and growth retardation which was associated with low plasma concentrations of leptin and a marker of osteoclast activity and collagen degradation. In pancreatectomized fetuses compared to control fetuses, limb lengths were shorter, and trabecular (Tb) bone in the metatarsi showed greater bone volume fraction, Tb thickness, degree of anisotropy and porosity, and lower fractional bone surface area and Tb spacing. Mechanical strength testing showed that pancreas deficiency was associated with increased stiffness and a greater maximal weight load at fracture in a subset of fetuses studied near term. Overall, pancreas deficiency in utero slowed the growth of the fetal skeleton and adapted the developing bone to generate a more compact and connected structure. Maintenance of bone strength in growth-retarded limbs is especially important in a precocial species in preparation for skeletal loading and locomotion at birth.

Short-Term Evaluation of Cellular Fate in an Ovine Bone Formation Model.

The ovine critical-sized defect model provides a robust preclinical model for testing tissue-engineered constructs for use in the treatment of non-union bone fractures and severe trauma. A critical question in cell-based therapies is understanding the optimal therapeutic cell dose. Key to defining the dose and ensuring successful outcomes is understanding the fate of implanted cells, e.g., viability, bio-distribution and exogenous infiltration post-implantation. This study evaluates such parameters in an ovine critical-sized defect model 2 and 7 days post-implantation. The fate of cell dose and behaviour post-implantation when combined with nanomedicine approaches for multi-model tracking and remote control using external magnetic fields is also addressed. Autologous STRO-4 selected mesenchymal stromal cells (MSCs) were labelled with a fluorescent lipophilic dye (CM-Dil), functionalised magnetic nanoparticles (MNPs) and delivered to the site within a naturally derived bone extracellular matrix (ECM) gel. Encapsulated cells were implanted within a critical-sized defect in an ovine medial femoral condyle and exposed to dynamic gradients of external magnetic fields for 1 h per day. Sheep were sacrificed at 2 and 7 days post-initial surgery where ECM was harvested. STRO-4-positive (STRO-4+) stromal cells expressed osteocalcin and survived within the harvested gels at day 2 and day 7 with a 50% loss at day 2 and a further 45% loss at 7 days. CD45-positive leucocytes were also observed in addition to endogenous stromal cells. No elevation in serum C-reactive protein (CRP) or non-haem iron levels was observed following implantation in groups containing MNPs with or without magnetic field gradients. The current study demonstrates how numbers of therapeutic cells reduce substantially after implantation in the repair site. Cell death is accompanied by enhanced leucocyte invasion, but not by inflammatory blood marker levels. Crucially, a proportion of implanted STRO-4+ stromal cells expressed osteocalcin, which is indicative of osteogenic differentiation. Furthermore, MNP labelling did not alter cell number or result in a further deleterious impact on stromal cells following implantation.