Biology subjects

Oreffo, R. O. C.

Publications and source records attributed to Oreffo, R. O. C..

10 recordsLinked to original sources

bioRxiv2026

Decoupled ionic and particulate clearance pathways determine the in vivo fate of a synthetic nanoclay-BMP-2 biomaterial during ectopic bone induction.

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.

bioengineering↗

bioRxiv2025

Characterising Type I Collagen Architecture in Human Bone and Osteosarcoma with Second Harmonic Generation Microscopy

Aberrant collagen matrix production is a hallmark of many cancers, including osteosarcoma (OS), the most common primary cancer of bone which remains poorly diagnosed. Here, the phenotyping potential of second harmonic generation (SHG) microscopy has been demonstrated through label-free imaging of pathologically altered collagen matrices within clinical biopsies. This work examined SHG imaging in quantifying OS-specific collagen (type I) signatures - focused on fibre length-based parameters - across clinical biopsies. We describe a novel SHG analysis workflow, following systematic optimisation of image resolution and field of view (FOV) size to enable robust quantification of collagen fibre-length metrics in human bone and OS. This included analyses of SHG images with a FOV spanning 350 {micro}m x 350 {micro}m to 1400 {micro}m x 1400 {micro}m, with the latter enabling whole-biopsy visualisation via stitching of up to 64 regions. In normal cortical (rib) bone biopsies, average fibre lengths did not significantly differ across increasing FOV size (26.44 {+/-} 1.75 {micro}m, 26.34 {+/-} 2.01 {micro}m, 27.25 {+/-} 2.47 {micro}m, 28.78 {+/-} 3.58 {micro}m). However, analysis of the top 50th percentile of collagen fibres revealed significant differences between the smallest FOVs; 350 {micro}m x 350 {micro}m and 466 {micro}m x 466 {micro}m, versus the largest 1400 {micro}m x 1400 {micro}m (p = 0.03 and p = 0.04 respectively), highlighting the requirement for larger FOVs for optimal characterisation of human bone collagen. Comparison of normal bone and OS (stage IIB) biopsies, the largest FOV revealed the largest differences, with OS exhibiting significantly reduced collagen fibre lengths (female OS: 23.18 {+/-} 0.56 {micro}m vs. bone: 29.39 {+/-} 2.17 {micro}m, p < 0.001; male OS: 24.39 {+/-} 1.32 {micro}m vs. bone: 27.87 {+/-} 1.05 {micro}m, p = 0.02) versus normal bone. Subsequent analyses across OS stages (IB - IVB) showed further evidence of pathological collagen signatures amplified with OS progression. These findings demonstrate that SHG microscopy enables robust, quantitative phenotyping of human bone and osteosarcoma collagen matrices, offering new potential for improved diagnosis and disease staging.

cancer biology↗

bioRxiv2024

Bioactive coatings on 3D printed scaffolds for bone regeneration: Use of Laponite(R) to deliver BMP-2 in an ovine femoral condyle defect model

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(R) 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(R)/BMP-2 produced significant bone formation compared to the uncoated PCL-TMA 900 scaffold in vivo, assessed by micro-computed tomography ({micro}CT) and histology. This indicated the ability of Laponite(R) to deliver the bioactive BMP-2 on the PCL-TMA900 scaffold. Bone formed around the Laponite(R)/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.

bioengineering↗

bioRxiv2023

Bioactive coatings on 3D printed polycaprolactone scaffolds for bone regeneration: a novel murine femur defect model for examination of the biomaterial capacity for repair.

Bone tissue engineering is a rapidly advancing field that seeks to develop efficacious approaches for treating non-healing fractures and large bone defects. Healing complications arise due to trauma, disease, infection, aseptic loosening of orthopaedic implants or iatrogenic causes. An ideal biodegradable scaffold would induce and support bone formation until the bone matrix is sufficiently stable to facilitate healing. The current study has examined bone augmentation, using functionalised coated scaffolds, with the hypothesised potential to induce skeletal cell differentiation for the repair of critical-sized bone defects. However, challenges in clinical translation arise from the alterations in cellular microenvironment that are present in the translation from in vitro to in vivo with the application of animal models of progressively increasing size and complexity of the implantation site. 3D printed, porous poly(caprolactone) trimethacrylate (denoted PCL-TMA900) scaffolds were applied within a murine femur defect, stabilised by a polyimide intramedullary pin, to assess the efficacy of select coatings in inducing bone formation. The PCL-TMA900 scaffolds were coated with i) elastin-like polypeptide (ELP), ii) poly(ethyl acrylate)/fibronectin/bone morphogenetic protein-2 (PEA/FN/BMP-2), iii) both ELP and PEA/FN/BMP-2 concurrently, or iv) Laponite nanoclay binding BMP-2, as bioactive coatings. The murine femur defect model was refined to assess the coated PCL-TMA900 scaffolds in an osseous defect, with sequential microcomputed tomography ({micro}CT) and histological analysis of the new bone tissue. Overall, PCL-TMA900 was found to be an optimal robust, biocompatible, 3D printable scaffold material. All PCL-TMA900 scaffolds, uncoated and coated, showed integration with the femur. The PCL-TMA900 scaffold coated with the nanoclay material Laponite and BMP-2 induced consistent, significant bone formation compared to the uncoated PCL-TMA900 scaffold. Bone formation was observed within the pores of the Laponite/BMP-2 coated scaffold. Critically, no heterotopic bone formation was observed as the BMP-2 was retained around the scaffold and not released into the tissues, producing bone around the scaffold in the desired shape and volume, compared to bone formation observed with the positive control (collagen sponge/BMP-2 construct). In comparison, the ELP coated and PEA/FN/BMP-2 scaffolds did not demonstrate significant or consistent bone formation compared to uncoated PCL-TMA900 control scaffolds. In summary, nanoclay 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 translation to the clinic.

bioengineering↗

bioRxiv2023

Bioactive coatings on 3D printed scaffolds for bone regeneration: Use of Laponite™ to deliver BMP-2 for bone tissue engineering - progression through in vitro, chorioallantoic membrane assay and murine subcutaneous model validation.

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.

bioengineering↗

bioRxiv2023

Bioactive coatings on 3D printed scaffolds for bone regeneration: Translation from in vitro to in vivo models and the impact of material properties and growth factor concentration.

Bone tissue engineering is a rapidly advancing field that seeks to develop new functional bone tissue, harnessing materials for application in bone defects which may fail to heal without intervention, as seen in critical-sized bone defects. The material properties must be developed, tailored and optimised as the environment progresses, through increasing animal size and complexity, of the target bone defect site. This study has examined the potential of a poly(caprolactone) trimethacrylate (PCL-TMA) 3D-printable scaffold with select bioactive coatings to function as a scaffold to augment bone formation. Three bioactive coatings were examined, i) elastin-like protein (ELP), ii) poly (ethyl acrylate) (PEA), fibronectin (FN) and bone morphogenetic protein-2 (BMP-2) applied sequentially (PEA/FN/BMP-2) and iii) both ELP and PEA/FN/BMP-2 coatings applied concurrently. The PCL-TMA scaffold construct was observed to be a robust scaffold material and the bioactive coatings applied were found to be biocompatible, with a significant osteogenic response from human skeletal cell populations observed in vitro. The PCL-TMA scaffold and bioactive coatings supported angiogenesis and displayed excellent biocompatibility following evaluation on the chorioallantoic membrane (CAM) assay. Biocompatibility was confirmed, however, no significant bone formation was detected, following examination of heterotopic bone formation in the murine subcutaneous implantation model, whereas extensive mineralisation was observed in the positive control material of collagen sponge with BMP-2. The absence of bone formation on the PCL-TMA scaffolds, in vivo, was potentially a consequence of the method of action of the applied coatings, the surface area of the scaffold construct for BMP-2 binding and the necessity of an appropriate in vivo environment to facilitate skeletal cell ingress, warranting future examination in an orthotopic bone defect model of bone tissue repair. The current studies demonstrate the development of a range of innovative scaffold constructs with in vitro efficacy and clearly illustrate the importance of an appropriate in vivo environment to validate in vitro functionality prior to scale up and preclinical application.

bioengineering↗

bioRxiv2023

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 but can fail to provide a long-term clinical resolution, emphasising the need for a functional biomimetic tissue engineering alternative. An attractive sustainable opportunity for tissue regeneration would be the application of human bone waste tissue for the synthesis of a material ink for 3D bioprinting of skeletal tissue. The use of human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate micro-environment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. Extrusion-based deposition was mediated by the blending of human bone-ECM (B) with nanoclay (L, Laponite(R)) and alginate (A) polymer, to engineer a novel material ink (LAB). The inclusion of nanofiller and polymeric material increased the rheological, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularisation following implantation in an ex vivo chick chorioallantoic membrane (CAM) model. Addition of bone morphogenetic protein-2 (BMP-2) with HBMSCs further enhanced vascularisation together with mineralisation after only 7 days. The current 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 personalised skeletal tissue repair. Graphical AbstractEngineering nanoclay-based bone ECM novel bioink for bone regeneration. Human bone trabecular tissue was demineralised, decellularised and blended with nanoclay (Laponite(R)) and alginate after digestion. The resulting ink was investigated for printability following rheological and filament fusion investigation. The microstructural arrangement of the blends was examined together with viability and functionality of bioprinted HBMSCs. Finally, the ability of the novel blend to support drug release ex vivo in a CAM model was determined confirming the potential of the bone ECM ink to support bone formation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/536074v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@b9dc13org.highwire.dtl.DTLVardef@1f6f0dorg.highwire.dtl.DTLVardef@1b3c098org.highwire.dtl.DTLVardef@295192_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Single cell RNA sequence analysis of human bone marrow samples reveals new targets for isolation of skeletal stem cells using DNA-coated gold nanoparticles

There is a wealth of data indicating human bone marrow derived stromal cells (HBMSCs) contain the skeletal stem cell (SSC) with the potential to differentiate along the stromal osteogenic, adipogenic and chondrogenic lineages. However, despite these advances, current methods to isolate skeletal stem cells (SSCs) from human tissues have proved challenging as no single specific marker has been identified limiting understanding of SSC fate, immunophenotype and the widespread clinical application of these cells. While a number of cell surface markers can enrich for SSCs, none of the proposed markers, alone, provide a platform to isolate single cells with the ability to form bone, cartilage, and adipose tissue in humans. The current study details the application of oligonucleotide-coated nanoparticles, spherical nucleic acids (SNAs), to rapidly isolate human cells using mRNAs signatures detected in SSCs in real time, to identify stem and progenitor skeletal populations using single cell RNA sequencing. Based on scRNA-seq of samples from 11 patients, this method was able to identify novel targets for SSC enrichment, which were assessed in a total of 80 patients. This methodology was able to isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with a capacity for tri-lineage differentiation in vitro. The current approach provides new targets and a platform to advance SSC isolation, enrichment with significant therapeutic impact therein.

cell biology↗

bioRxiv2019

Optimising DNA-Coated Gold Nanoparticles to Enrich Skeletal Stem Cells from Human Bone Marrow Samples

Human bone marrow (BM) derived stromal cells contain a population of skeletal stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic and chondrogenic lineages enabling their application to clinical therapies. However, current methods, to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a specific SSC marker. Unfortunately, none of the current proposed markers, alone, can isolate a homogenous cell population with the ability to form bone, cartilage, and adipose tissue in humans. Here, we have designed DNA-gold nanoparticles able to identify and sort SSCs displaying specific mRNA signatures. The current approach demonstrates the significant enrichment attained in the isolation of SSCs, with potential therein to enhance our understanding of bone cell biology and translational applications. TABLE OF CONTENTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/882563v3_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@48c5acorg.highwire.dtl.DTLVardef@1a9d096org.highwire.dtl.DTLVardef@1bd4da2org.highwire.dtl.DTLVardef@133dd1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2018

Osteoclasts control sensory neurons axonal growth through epidermal growth factor receptor signaling

The patterning of peripheral innervation is accomplished through the tissue expression, in specific space and timeframe, of attractive or repulsive axonal guidance cues. At the bone microenvironment, neurotrophic factors such as nerve growth factor, brain-derived neurotrophic factor, vascular endothelial growth factor, netrin-1 and others were described to regulate the nerve ingrowth towards the bone compartment, by acting directly on receptors expressed at the nerve terminals. Interestingly, besides the gradient of soluble factors, neurons were described to be responsive to extracellular vesicles (EV) derived from myelinating cells and mesenchymal stem cells. Here we provide evidence on a new mechanism by which peripheral innervation can be coordinated. We show that sensory nerves outgrowth and electric signal propagation are dependent on the EV secreted by osteoclasts, the bone resorbing cells. Furthermore, we demonstrate that the axonal sprouting is achieved through the activation of epidermal-growth factor receptor (EGFR) family signaling pathway. We proved that the EV-depleted osteoclast secretome leads to a significant decrease of neurons firing rate and axonal sprouting, concomitant with a decrease of EGFR/ErbB2 activation levels. Excitingly, the proteomic analysis of the osteoclast-derived EV cargo shows a high correlation with synaptic components reinforcing the role on sensory neurons/osteoclast crosstalk. Our findings that osteoclast-derived EV hold effect in axonal outgrowth, contributing actively to the dynamics of the sensory neurons sprouting and electrophysiology, is a step toward unraveling target mechanisms to control electrical signal propagation and nerve fibers sprouting and consequently open new avenues for the development of innovative therapies to control bone pain. Significance StatementSensory nerve fibers sprouting in bone pathologies is highly associated with pain. Thus, understanding the mechanisms behind sensory nerves ingrowth, sprouting and electrical activity, within the bone compartment, is essential for improving the strategies to overcome pain in bone disorders. We provide a new mechanism on the sensory nerves sprouting, indicating that the effect is dependent on the extracellular vesicles (EV) released by osteoclasts, through the epidermal growth factor receptor family targeting, by integrin independent pathways. We show different electrophysiology patterns being triggered in the presence of osteoclasts secretome and the abolishment of sensory neurons firing rate in EV-depleted conditions. Overall, our results elucidate novel mechanisms on the peripheral nerves sprouting, essential for pursuing new targets for bone pain therapies.

cell biology↗