Remote regulation of magnetic particle targeted Wnt signaling for bone tissue engineering.

Wnt signaling is critically involved in the differentiation of human Mesenchymal Stem Cells (hMSC). Wnt proteins therefore have considerable therapeutic value, but are expensive and difficult to produce. UM206 is a synthetic peptide and ligand for the Wnt receptor Frizzled. Attachment of UM206 to magnetic nanoparticles (MNP) enables the ligand-MNP complex to be manipulated using magnetic fields, allowing control of Frizzled stimulation. Using this approach, Wnt signaling was activated in hMSC which resulted in Frizzled clustering, ß-catenin translocalization and activation of TCF/LEF responsive transcription. During osteogenesis, UM206-MNP initiated localized mineralized matrix formation. Injection and magnetic stimulation of UM206-MNP-labeled MSC in ex vivo chick femurs resulted in increased mineralization which acted synergistically with addition of bone morphogenic protein 2 (BMP2) releasing micro-particles. As this facilitates external control over signal transduction, conjugated MNP technology has applications both as a research tool and for regulating tissue formation in clinical cell therapies.

Dynamic 3D culture: models of chondrogenesis and endochondral ossification.

The formation of cartilage from stem cells during development is a complex process which is regulated by both local growth factors and biomechanical cues, and results in the differentiation of chondrocytes into a range of subtypes in specific regions of the tissue. In fetal development cartilage also acts as a precursor scaffold for many bones, and mineralization of this cartilaginous bone precursor occurs through the process of endochondral ossification. In the endochondral formation of bones during fetal development the interplay between cell signalling, growth factors, and biomechanics regulates the formation of load bearing bone, in addition to the joint capsule containing articular cartilage and synovium, generating complex, functional joints from a single precursor anlagen. These joint tissues are subsequently prone to degeneration in adult life and have poor regenerative capabilities, and so understanding how they are created during development may provide useful insights into therapies for diseases, such as osteoarthritis, and restoring bone and cartilage lost in adulthood. Of particular interest is how these tissues regenerate in the mechanically dynamic environment of a living joint, and so experiments performed using 3D models of cartilage development and endochondral ossification are proving insightful. In this review, we discuss some of the interesting models of cartilage development, such as the chick femur which can be observed in ovo, or isolated at a specific developmental stage and cultured organotypically in vitro. Biomaterial and hydrogel-based strategies which have emerged from regenerative medicine are also covered, allowing researchers to make informed choices on the characteristics of the materials used for both original research and clinical translation. In all of these models, we illustrate the essential importance of mechanical forces and mechanotransduction as a regulator of cell behavior and ultimate structural function in cartilage.

A 4D printed self-assembling PEGDA microscaffold fabricated by digital light processing for arthroscopic articular cartilage tissue engineering.

Articular cartilage in synovial joints such as the knee has limited capability to regenerate independently, and most clinical options for focal cartilage repair merely delay total joint replacement. Tissue engineering presents a repair strategy in which an injectable cell-laden scaffold material is used to reconstruct the joint in situ through mechanical stabilisation and cell-mediated regeneration. In this study, we designed and 3D-printed millimetre-scale micro-patterned PEGDA biomaterial microscaffolds which self-assemble through tessellation at a scale relevant for applications in osteochondral cartilage reconstruction. Using simulated chondral lesions in an in vitro model, a series of scaffold designs and viscous delivery solutions were assessed. Hexagonal microscaffolds (750 µm x 300 µm) demonstrated the best coverage of a model cartilage lesion (at 73.3%) when injected with a 1% methyl cellulose solution. When chondrocytes were introduced to the biomaterial via a collagen hydrogel, they successfully engrafted with the printed microscaffolds and survived for at least 14 days in vitro, showing the feasibility of reconstructing stratified cartilaginous tissue using this strategy. Our study demonstrates a promising application of this 4D-printed injectable technique for future clinical applications in osteochondral tissue engineering. Supplementary Information: The online version contains supplementary material available at 10.1007/s40964-022-00360-0.

Multimodal analysis of the differential effects of cyclic strain on collagen isoform composition, fibril architecture and biomechanics of tissue engineered tendon.

Tendon is predominantly composed of aligned type I collagen, but additional isoforms are known to influence fibril architecture and maturation, which contribute to the tendon's overall biomechanical performance. The role of the less well-studied collagen isoforms on fibrillogenesis in tissue engineered tendons is currently unknown, and correlating their relative abundance with biomechanical changes in response to cyclic strain is a promising method for characterising optimised bioengineered tendon grafts. In this study, human mesenchymal stem cells (MSCs) were cultured in a fibrin scaffold with 3%, 5% or 10% cyclic strain at 0.5 Hz for 3 weeks, and a comprehensive multimodal analysis comprising qPCR, western blotting, histology, mechanical testing, fluorescent probe CLSM, TEM and label-free second-harmonic imaging was performed. Molecular data indicated complex transcriptional and translational regulation of collagen isoforms I, II, III, V XI, XII and XIV in response to cyclic strain. Isoforms (XII and XIV) associated with embryonic tenogenesis were deposited in the formation of neo-tendons from hMSCs, suggesting that these engineered tendons form through some recapitulation of a developmental pathway. Tendons cultured with 3% strain had the smallest median fibril diameter but highest resistance to stress, whilst at 10% strain tendons had the highest median fibril diameter and the highest rate of stress relaxation. Second harmonic generation exposed distinct structural arrangements of collagen fibres in each strain group. Fluorescent probe images correlated increasing cyclic strain with increased fibril alignment from 40% (static strain) to 61.5% alignment (10% cyclic strain). These results indicate that cyclic strain rates stimulate differential cell responses via complex regulation of collagen isoforms which influence the structural organisation of developing fibril architectures.

Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization.

OBJECTIVE: Vasculogenic progenitor cell therapy for ischemic diseases bears great potential but still requires further optimization for justifying its clinical application. Here, we investigated the effects of in vivo tissue engineering by combining vasculogenic progenitors with injectable scaffolds releasing controlled amounts of proangiogenic growth factors. METHODS AND RESULTS: We produced biodegradable, injectable polylactic coglycolic acid-based scaffolds releasing single factors or combinations of vascular endothelial growth factor, hepatocyte growth factor, and angiopoietin-1. Dual and triple combinations of scaffold-released growth factors were superior to single release. In murine hindlimb ischemia models, scaffolds releasing dual (vascular endothelial growth factor and hepatocyte growth factor) or triple combinations improved effects of cord blood-derived vasculogenic progenitors. Increased migration, homing, and incorporation of vasculogenic progenitors into the vasculature augmented capillary density, translating into improved blood perfusion. Most importantly, scaffold-released triple combinations including the vessel stabilizer angiopoietin-1 enhanced the number of perivascular smooth muscle actin(+) vascular smooth muscle cells, indicating more efficient vessel stabilization. CONCLUSIONS: Vasculogenic progenitor cell therapy is significantly enhanced by in vivo tissue engineering providing a proangiogenic and provasculogenic growth factor-enriched microenvironment. Therefore, combined use of scaffold-released growth factors and cell therapy improves neovascularization in ischemic diseases and may translate into more pronounced clinical effects.

A universal multi-platform 3D printed bioreactor chamber for tendon tissue engineering.

A range of bioreactors use linear actuators to apply tensile forces in vitro, but differences in their culture environments can limit a direct comparison between studies. The widespread availability of 3D printing now provides an opportunity to develop a 'universal' bioreactor chamber that, with minimal exterior editing can be coupled to a wide range of commonly used linear actuator platforms, for example, the EBERS-TC3 and CellScale MCT6, resulting in a greater comparability between results and consistent testing of potential therapeutics. We designed a bioreactor chamber with six independent wells that was 3D printed in polylactic acid using an Ultimaker 2+ and waterproofed using a commercially available coating (XTC-3D), an oxirane resin. The cell culture wells were further coated with Sylgard-184 polydimethylsiloxane (PDMS) to produce a low-adhesion well surface. With appropriate coating and washing steps, all materials were shown to be non-cytotoxic by lactate dehydrogenase assay, and the bioreactor was waterproof, sterilisable and reusable. Tissue-engineered tendons were generated from human mesenchymal stem cells in a fibrin hydrogel and responded to 5% cyclic strain (0.5 Hz, 5 h/day, 21 days) in the bioreactor by increased production of collagen-Iα1 and decreased production of collagen-IIIα1. Calcification of the extracellular matrix was observed in unstretched tendon controls indicating abnormal differentiation, while tendons cultured under cyclic strain did not calcify and exhibited a tenogenic phenotype. The ease of manufacturing this bioreactor chamber enables researchers to quickly and cheaply reproduce this culture environment for use with many existing bioreactor actuator platforms by downloading the editable CAD files from a public database and following the manufacturing steps we describe.

Magnetic ion channel activation of TREK1 in human mesenchymal stem cells using nanoparticles promotes osteogenesis in surrounding cells.

Magnetic ion channel activation technology uses superparamagnetic nanoparticles conjugated with targeting antibodies to apply mechanical force directly to stretch-activated ion channels on the cell surface, stimulating mechanotransduction and downstream processes. This technique has been reported to promote differentiation towards musculoskeletal cell types and enhance mineralisation. Previous studies have shown how mesenchymal stem cells injected into a pre-mineralised environment such as a foetal chick epiphysis, results in large-scale osteogenesis at the target site. However, the relative contributions of stem cells and surrounding host tissue has not been resolved, that is, are the mesenchymal stem cells solely responsible for the observed mineralisation or do mechanically stimulated mesenchymal stem cells also promote a host-tissue mineralisation response? To address this, we established a novel two-dimensional co-culture assay, which indicated that magnetic ion channel activation stimulation of human mesenchymal stem cells does not significantly promote migration but does enhance collagen deposition and mineralisation in the surrounding cells. We conclude that one of the important functions of injected human mesenchymal stem cells is to release biological factors (e.g., cytokines and microvesicles) which guide the surrounding tissue response, and that remote control of this signalling process using magnetic ion channel activation technology may be a useful way to both drive and regulate tissue regeneration and healing.

Fluorescence-Based Nano-Oxygen Particles for Spatiometric Monitoring of Cell Physiological Conditions.

Closed-loop artificial pancreas systems have recently been proposed as a solution for treating stage I diabetes by reproducing the function of the pancreas. However, there are many unresolved issues associated with their development, including monitoring and controlling oxygen, immune responses, and the optimization of glucose, all of which need to be monitored and controlled to produce an efficient and viable artificial organ that can become integrated in the patient and maintain homeostasis. This research focused on monitoring the oxygen concentration, specifically achieving this kinetically as the oxygen gradient in an artificial pancreas made of alginate spheres containing islet cells. Functional nanoparticles (NPs) for measuring the oxygen gradient in different hydrogel cellular environments using fluorescence-based (F) microscopy were developed and tested. By the ester bond, a linker Pluronic F127 was conjugated with a carboxylic acid-modified polystyrene NP (510 nm). A hydrophilic/hydrophobic interaction between the commercially available oxygen-sensitive fluorophore and F127 results in fluorescence-based nano-oxygen particles (FNOPs). The in-house synthesized FNOP was calibrated inside electrosprayed alginate-filled hydrogels and demonstrated a good broad dynamic range (2.73-22.23) mg/L as well as a resolution of -0.01 mg/L with an accuracy of ±4%. The calibrated FNOP was utilized for continuous measuring of the oxygen concentration gradient for cell lines RIN-m5F/HeLa for more than 5 days in alginate hydrogel spheres in vitro.

Photoperiod-Induced Increases in Bone Mineral Apposition Rate in Siberian Hamsters and the Involvement of Seasonal Leptin Changes.

The adipokine leptin regulates energy balance, appetite, and reproductive maturation. Leptin also acts on bone growth and remodeling, but both osteogenic and anti-osteogenic effects have been reported depending on experimental conditions. Siberian hamsters (Phodopus sungorus) have natural variation in circulating leptin concentrations, where serum leptin is significantly decreased during the short day (SD)-induced winter state. In summer long day (LD) photoperiods, appetite and body adiposity increase with associated central leptin insensitivity. This natural change in leptin secretion was exploited to investigate leptin's effect on bone growth. Hamsters were injected with calcium-chelating fluorescent dyes to measure bone mineral apposition rate (MAR). Measurements were initially obtained from 5-week and 6-month-old animals maintained in low leptin (SD) or high leptin (LD) states. A further study investigated effects of chronic administration of recombinant mouse leptin to hamsters housed in SD and LD conditions; growth plate thickness and bone density were also assessed. As expected, a reduction in body mass was seen in hamsters exposed to SD, confirming the phenotype change in all studies. Serum leptin concentrations were significantly reduced in SD animals in all studies. MAR was reproducibly and significantly increased in the femurs of SD animals in all studies. Vitamin D and growth plate thickness were significantly increased in SD animals at 6 months. No effect on bone density was observed in any study. Taken together these data suggest that bone growth is associated with the low leptin, winter, lean state. In leptin-treated animals, there was a significant interaction effect of leptin and photoperiod. In comparison to their vehicle counterparts, SD animals had decreased and LD animals had increased MAR, which was not apparent prior to leptin administration. In conclusion, increased MAR was associated with low serum leptin levels in early life and sustained over 6 months, implying that leptin has a negative effect on bone growth in this model. The unexpected finding that MAR increased after peripheral leptin administration in LD suggests that leptin exerts different effects on bone growth dependent on initial leptin status. This adds further weight to the hypothesis that leptin-treated LD animals display central leptin resistance.

Evaluation of the growth environment of a hydrostatic force bioreactor for preconditioning of tissue-engineered constructs.

Bioreactors have been widely acknowledged as valuable tools to provide a growth environment for engineering tissues and to investigate the effect of physical forces on cells and cell-scaffold constructs. However, evaluation of the bioreactor environment during culture is critical to defining outcomes. In this study, the performance of a hydrostatic force bioreactor was examined by experimental measurements of changes in dissolved oxygen (O2), carbon dioxide (CO2), and pH after mechanical stimulation and the determination of physical forces (pressure and stress) in the bioreactor through mathematical modeling and numerical simulation. To determine the effect of hydrostatic pressure on bone formation, chick femur skeletal cell-seeded hydrogels were subjected to cyclic hydrostatic pressure at 0-270 kPa and 1 Hz for 1 h daily (5 days per week) over a period of 14 days. At the start of mechanical stimulation, dissolved O2 and CO2 in the medium increased and the pH of the medium decreased, but remained within human physiological ranges. Changes in physiological parameters (O2, CO2, and pH) were reversible when medium samples were placed in a standard cell culture incubator. In addition, computational modeling showed that the distribution and magnitude of physical forces depends on the shape and position of the cell-hydrogel constructs in the tissue culture format. Finally, hydrostatic pressure was seen to enhance mineralization of chick femur skeletal cell-seeded hydrogels.

Remotely Activated Mechanotransduction via Magnetic Nanoparticles Promotes Mineralization Synergistically With Bone Morphogenetic Protein 2: Applications for Injectable Cell Therapy.

Bone requires dynamic mechanical stimulation to form and maintain functional tissue, yet mechanical stimuli are often lacking in many therapeutic approaches for bone regeneration. Magnetic nanoparticles provide a method for delivering these stimuli by directly targeting cell-surface mechanosensors and transducing forces from an external magnetic field, resulting in remotely controllable mechanotransduction. In this investigation, functionalized magnetic nanoparticles were attached to either the mechanically gated TREK1 K+ channel or the (integrin) RGD-binding domains of human mesenchymal stem cells. These cells were microinjected into an ex vivo chick fetal femur (embryonic day 11) that was cultured organotypically in vitro as a model for endochondral bone formation. An oscillating 25-mT magnetic field delivering a force of 4 pN per nanoparticle directly against the mechanoreceptor induced mechanotransduction in the injected mesenchymal stem cells. It was found that cells that received mechanical stimuli via the nanoparticles mineralized the epiphyseal injection site more extensively than unlabeled control cells. The nanoparticle-tagged cells were also seeded into collagen hydrogels to evaluate osteogenesis in tissue-engineered constructs: in this case, inducing mechanotransduction by targeting TREK1 resulted in a 2.4-fold increase in mineralization and significant increases in matrix density. In both models, the combination of mechanical stimulation and sustained release of bone morphogenetic protein 2 (BMP2) from polymer microspheres showed a significant additive effect on mineralization, increasing the effectiveness of BMP2 delivery and demonstrating that nanoparticle-mediated mechanotransduction can be used synergistically with pharmacological approaches for orthopedic tissue engineering to maximize bone formation.