Hareklea Markides

Biocompatibility and toxicity of magnetic nanoparticles in regenerative medicine

Regenerative medicine is a pioneering field aimed at restoring and regenerating the function of damaged cells, organs and tissues in order to establish normal function. It demands the cross communication of disciplines to develop effective therapeutic stem cell based therapies. Nanotechnology has been instrumental in the development and translation of basic research to the clinically relevant therapies. In particular, magnetic nanoparticles (MNPs) have been applied to tag, track and activate stem cells offering an effective means of monitoring in vitro and in vivo behaviour. MNPs are comprised of an iron oxide core with a biocompatible biological polymer. Safety is an issue of constant concern and emphasises on the importance of investigating the issue of toxicity. Any indication of toxicity can ultimately limit the therapeutic efficiency of the therapy. Toxicity is highly dependent on the physical, chemical and structural properties of the MNP itself as well as dose and intended use. Few in vitro studies have reported adverse effects of MNP on cells at in vitro in therapeutic doses. However, long term in vivo studies have not been studied as extensively. This review aims to summarise current research in this topic highlighting commonly used toxicity assays to investigate this.

Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

Significance Efficient delivery of therapeutic molecules inside cells by nontransgenic approaches is key as gene editing/correction, directed differentiation, and in vivo cell modulation/tracking are translated for regenerative medicine applications. Here we describe a peptide-based system engineered to enhance the activity of cell-penetrating peptides to achieve exceptional intracellular transduction. Glycosaminoglycan-binding enhanced transduction (GET) uses peptides that interact with cell membrane heparan sulfates and promote cell-penetrating peptide-mediated endocytosis into cells. The system is not dependent on extensive positive charge and can be tailored to deliver peptides, recombinant proteins, nucleic acids, nanoparticles, and antibodies. Importantly, this approach does not affect cell proliferation and viability and can be used to deliver a plethora of functional cargoes. Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.

Orthopaedic applications of nanoparticle-based stem cell therapies

Stem cells have tremendous applications in the field of regenerative medicine and tissue engineering. These are pioneering fields that aim to create new treatments for disease that currently have limited therapies or cures. A particularly popular avenue of research has been the regeneration of bone and cartilage to combat various orthopaedic diseases. Magnetic nanoparticles (MNPs) have been applied to aid the development and translation of these therapies from research to the clinic. This review highlights contemporary research for the applications of iron-oxide-based MNPs for the therapeutic implementation of stem cells in orthopaedics. These MNPs comprise of an iron oxide core, coated with a choice of biological polymers that can facilitate the uptake of MNPs by cells through improving endocytic activity. The combined use of these oxides and the biological polymer coatings meet biological requirements, effectively encouraging the use of MNPs in regenerative medicine. The association of MNPs with stem cells can be achieved via the process of endocytosis resulting in the internalisation of these particles or the attachment to cell surface receptors. This allows for the investigation of migratory patterns through various tracking studies, the targeting of particle-labelled cells to desired locations via the application of an external magnetic field and, finally, for activation stem cells to initiate various cellular responses to induce the differentiation. Characterisation of cell localisation and associated tissue regeneration can therefore be enhanced, particularly for in vivo applications. MNPs have been shown to have the potential to stimulate differentiation of stem cells for orthopaedic applications, without limiting proliferation. However, careful consideration of the use of active agents associated with the MNP is suggested, for differentiation towards specific lineages. This review aims to broaden the knowledge of current applications, paving the way to translate the in vitro and in vivo work into further orthopaedic clinical studies.

Autonomous magnetic labelling of functional mesenchymal stem cells for improved traceability and spatial control in cell therapy applications.

Mesenchymal stem cells (MSCs) represent a valuable resource for regenerative medicine treatments for orthopaedic repair and beyond. Following developments in isolation, expansion and differentiation protocols, efforts to promote clinical translation of emerging cellular strategies now seek to improve cell delivery and targeting. This study shows efficient live MSC labelling using silica‐coated magnetic particles (MPs), which enables 3D tracking and guidance of stem cells. A procedure developed for the efficient and unassisted particle uptake was shown to support MSC viability and integrity, while surface marker expression and MSC differentiation capability were also maintained. In vitro, MSCs showed a progressive decrease in labelling over increasing culture time, which appeared to be linked to the dilution effect of cell division, rather than to particle release, and did not lead to detectable secondary particle uptake. Labelled MSC populations demonstrated magnetic responsiveness in vitro through directed migration in culture and, when seeded onto a scaffold, supporting MP‐based approaches to cell targeting. The potential of these silica‐coated MPs for MRI cell tracking of MSC populations was validated in 2D and in a cartilage repair model following cell delivery. These results highlight silica‐coated magnetic particles as a simple, safe and effective resource to enhance MSC targeting for therapeutic applications and improve patient outcomes.

Therapeutic Benefit for Late, but Not Early, Passage Mesenchymal Stem Cells on Pain Behaviour in an Animal Model of Osteoarthritis

Background Mesenchymal stem cells (MSCs) have a therapeutic potential for the treatment of osteoarthritic (OA) joint pathology and pain. The aims of this study were to determine the influence of a passage number on the effects of MSCs on pain behaviour and cartilage and bone features in a rodent model of OA. Methods Rats underwent either medial meniscal transection (MNX) or sham surgery under anaesthesia. Rats received intra-articular injection of either 1.5 × 106 late passage MSCs labelled with 10 μg/ml SiMAG, 1.5 × 106 late passage mesenchymal stem cells, the steroid Kenalog (200 μg/20 μL), 1.5 × 106 early passage MSCs, or serum-free media (SFM). Sham-operated rats received intra-articular injection of SFM. Pain behaviour was quantified until day 42 postmodel induction. Magnetic resonance imaging (MRI) was used to localise the labelled cells within the knee joint. Results Late passage MSCs and Kenalog attenuated established pain behaviour in MNX rats, but did not alter MNX-induced joint pathology at the end of the study period. Early passage MSCs exacerbated MNX-induced pain behaviour for up to one week postinjection and did not alter joint pathology. Conclusion Our data demonstrate for the first time the role of a passage number in influencing the therapeutic effects of MSCs in a model of OA pain.

SAT0554 Intra-Articular Injection of Mesenchymal Stem Cells Improves Pain Behaviour in A Model of OA Pain

Background Osteoarthritis (OA) is a degenerative joint disease affecting around 8 million people in the UK. Pain is a prominent and often disabling feature of OA, the relationship between pain and histopathological features of OA are poorly understood. Currently, there are no reparative or disease-modifying treatments for OA and thus many patients will undergo joint replacement surgery for pain relief. Objectives To explore the effect for intra-articular mesenchymal stem cells (MSC) treatment on pain behaviour in a rodent surgical model of OA. Methods Under isoflurane anaesthesia (1.5L/min O2, 2.5% isoflurane) joint pathology was induced in adult male Sprague Dawley Rats (160-200g) by transecting the medial collateral ligament and making a full thickness cut through the meniscus (day 0) of the left knee (Mapp et al., 2008). Baseline pain behaviour were taken immediately prior to surgery (day 0) and then from 3 until 42 days post-surgery. Pain measures were weight bearing asymmetry, assessed using an incapacitance tester, and the lowering of hindpaw mechanical withdrawal thresholds, quantified using von Frey monofilaments (1-15 g). 14 days post-surgery, rats were stratified according to behavioural pain responses and under brief isoflurane anaesthesia (3% 1L/min O2), received an intra-articular injection (through the infrapatellar ligament of the left knee) 50μl of 1.5x106 mesenchymal stem cells (n=7 rats) or serum free media (SFM; n=4 rats). Pain behaviour was quantified for a further four weeks post intra-articular injection and then the experiment was terminated. Data are expressed as mean weight bearing % (weight on ipsilateral paw – weight on contralateral paw/total weight on hindpaws) x 100% ± SEM. Statistical analysis between groups was performed using a Mann Whitney test. Results Consistent with previous studies, from 3 days following medial meniscal transection (MNX) surgery rats exhibited increased weight bearing asymmetry and decreased ipsilateral paw withdrawal thresholds, compared to pre-surgery values. MNX rats which received the control treatment (intra-articular injection of SFM) exhibited a gradual increase in pain behaviour up to day 42. Intra-articular injection of MSCs at day 14 prevented a further increase in weight bearing asymmetry, which was significant when compared to OA rats treated with SFM, up to day 42 (weight bearing % SFM =19.5±0.6, MSC =9.5±3.5; p<0.05). MSC treatment had no effect on the hindpaw mechanical withdrawal thresholds in MNX treated rats. Conclusions Intra-articular injection of MSCs attenuated the further development of weight bearing asymmetry in rats with established joint pathology in the MNX model of OA. There was no effect of this treatment on the development of hindpaw mechanical withdrawal thresholds. These data suggest that intra-articular injection of MSCs may alter peripherally-driven pain, but may not affect centrally-mediated pain responses in established OA. References Mapp PI, Avery PS, McWilliams DF, Bowyer J, Day C, Moores S, Webster R, Walsh DA (2008). Angiogenesis in two animal models of osteoarthritis. Osteoarthritis Cartilage 16(1): 61-69 Acknowledgements This work was funded by EPSRC and ARUK Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3731

Translation of remote control regenerative technologies for bone repair

AbstractThe role of biomechanical stimuli, or mechanotransduction, in normal bone homeostasis and repair is understood to facilitate effective osteogenesis of mesenchymal stem cells (MSCs) in vitro. Mechanotransduction has been integrated into a multitude of in vitro bone tissue engineering strategies and provides an effective means of controlling cell behaviour towards therapeutic outcomes. However, the delivery of mechanical stimuli to exogenous MSC populations, post implantation, poses a significant translational hurdle. Here, we describe an innovative bio-magnetic strategy, MICA, where magnetic nanoparticles (MNPs) are used to remotely deliver mechanical stimuli to the mechano-receptor, TREK-1, resulting in activation and downstream signalling via an external magnetic array. In these studies, we have translated MICA to a pre-clinical ovine model of bone injury to evaluate functional bone repair. We describe the development of a magnetic array capable of in vivo MNP manipulation and subsequent osteogenesis at equivalent field strengths in vitro. We further demonstrate that the viability of MICA-activated MSCs in vivo is unaffected 48 h post implantation. We present evidence to support early accelerated repair and preliminary enhanced bone growth in MICA-activated defects within individuals compared to internal controls. The variability in donor responses to MICA-activation was evaluated in vitro revealing that donors with poor osteogenic potential were most improved by MICA-activation. Our results demonstrate a clear relationship between responders to MICA in vitro and in vivo. These unique experiments offer exciting clinical applications for cell-based therapies as a practical in vivo source of dynamic loading, in real-time, in the absence of pharmacological agents.Bone repair: Magnetic nanoparticle therapy helps fix bone injuries in sheepA biomagnetic therapy that stimulates adult stem cells helps promote repair in a sheep model of bone injury. Alicia El Haj from Keele University, UK, and colleagues previously developed a technique for activating specific ion channel receptors on stem cells through the use of targeted magnetic nanoparticles and a small magnetic field, but they had not tried the method on anything larger than a mouse. Here, the scientists tested the technique on sheep with injuries to their leg bones. They designed a magnetic array compatible with a sheep leg which could stimulate the cells for repair. They then tagged bone marrow stem cells with the nanoparticles, implanted the tagged cells at the site of injury, and applied an external magnetic array around the leg. The therapy accelerated repair and enhanced bone growth
 compared to non-magnetically enhanced stem-cell treatments.

Triggering the activation of Activin A type II receptor in human adipose stem cells towards tenogenic commitment using mechanomagnetic stimulation

Stem cell therapies hold potential to stimulate tendon regeneration and homeostasis, which is maintained in response to the native mechanical environment. Activins are members of the mechano-responsive TGF-β superfamily that participates in the regulation of several downstream biological processes. Mechanosensitive membrane receptors such as activin can be activated in different types of stem cells via magnetic nanoparticles (MNPs) through remote magnetic actuation resulting in cell differentiation. In this work, we target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), using anti-ActRIIA functionalized MNPs, externally activated through a oscillating magnetic bioreactor. Upon activation, the phosphorylation of Smad2/3 is induced allowing translocation of the complex to the nucleus, regulating tenogenic transcriptional responses. Our study demonstrates the potential remote activation of MNPs tagged hASCs to trigger the Activin receptor leading to tenogenic differentiation. These results may provide insights toward tendon regeneration therapies.