Live quantitative monitoring of mineral deposition in stem cells using tetracycline hydrochloride

The final stage of in vitro osteogenic differentiation is characterized by the production of mineral deposits containing calcium cations and inorganic phosphates, which populate the extracellular matrix surrounding the cell monolayer. Conventional histological techniques for the assessment of mineralization, such as Von Kossa and Alizarin Red S staining, are end-point techniques requiring cell fixation. Moreover, in both cases staining quantitation requires dye extraction which irreversibly alters the ECM conformation and structure, therefore preventing the use of the sample for further analysis. In this study, the use of Tetracycline hydrochloride (TC) is proposed for the non-destructive staining, quantitation and imaging of mineralizing bone-like nodules in live cultures of human bone marrow mesenchymal stem cells (MSCs) cultured under osteogenic conditions. Overnight administration of TC to living cells was shown not to alter the metabolic activity or the progression of cell differentiation. When applied to differentiating cultures, cell exposure to serial doses of TC was found to produce quantifiable fluorescence emission specifically in osteogenic cultures. Incubation with TC enabled fluorescence imaging of mineralised areas in live cultures and the combination with other fluorophores using appropriate filters. These results demonstrate that serial TC administration over the differentiation time course provides a qualitative and quantitative tool for the monitoring and evaluation of the differentiation process in live cells

Degradation and characterization of resorbable phosphate-based glass thin-film coatings applied by radio-frequency magnetron sputtering

Quinternary phosphate-based glasses of up to 2.67 μm, deposited by radio-frequency magnetron sputtering, were degraded in distilled water and phosphate-buffered saline (PBS) to investigate their degradation characteristics. Magnetron- sputtered coatings have been structurally compared to their compositionally equivalent melt-quenched bulk glass counterparts. The coatings were found to have structurally variable surfaces to melt-quenched glass such that the respective bridging oxygen to nonbridging oxygen bonds were 34.2% to 65.8% versus 20.5% to 79.5%, forming metaphosphate (PO3)−(Q2) versus less soluble (P2O7)4− (Q1) and (PO4)3− (Q0), respectively. This factor led to highly soluble coatings, exhibiting a t1/2 degradation dependence in the first 2 h in distilled water, followed by a more characteristic linear profile because the subsequent layers were less soluble. Degradation was observed to preferentially occur, forming voids characteristic of pitting corrosion, which was confirmed by the use of a focused ion beam. Coating degradation in PBS precipitated a (PO3)−metaphosphate, an X-ray amorphous layer, which remained adherent to the substrate and seemingly formed a protective diffusion barrier, which inhibited further coating degradation. The implications are that while compositionally similar, sputter-deposited coatings and melt-quenched glasses are structurally dissimilar, most notably, with regard to the surface layer. This factor has been attributed to surface etching of the as-deposited coating layer during deposition and variation in the thermal history between the processes of magnetron sputtering and melt quenching

Wet and dry flexural high cycle fatigue behaviour of fully bioresorbable glass fibre composites: in-situ polymerisation versus laminate stacking

Fully bioresorbable phosphate based glass fibre reinforced polycaprolactone (PCL/PGF) composites are potentially excellent candidates to address current issues experienced with use of metal implants for hard tissue repair, such as stress shielding effects. It is therefore essential to investigate these materials under representative loading cases and to understand their fatigue behaviour (wet and dry) in order to predict their lifetime in service and their likely mechanisms of failure. This paper investigated the dry and wet flexural fatigue behaviour of PCL/PGF composites with 35% and 50% fibre volume fraction (Vf). Significantly longer flexural fatigue life (p < 0.0001) and superior fatigue damage resistance were observed for In-situ Polymerised (ISP) composites as compared to the Laminate Stacking (LS) composites in both dry and wet conditions, indicating that the ISP promoted considerably stronger interfacial bonding than the LS. Immersion in fluid (wet) during the flexural fatigue tests resulted in significant reduction (p < 0.0001) in the composites fatigue life, earlier onset of fatigue damage and faster damage propagation. Regardless of testing conditions, increasing fibre content led to shorter fatigue life for the PCL/PGF composites. Meanwhile, immersion in degradation media caused softening of both LS and ISP composites during the fatigue tests, which led to a more ductile failure mode. Among all the composites that were investigated, ISP35 (35% Vf) composites maintained at least 50% of their initial stiffness at the end of fatigue tests in both conditions, which is comparable to the flexural properties of human cortical bones. Consequently, ISP composites with 35% Vf maintained at least 50% of its flexural properties after the fatigue failure, which the mechanical retentions were well matched with the properties of human cortical bones

Role of geometrical cues in bone marrow-derived mesenchymal stem cell survival, growth and osteogenic differentiation

Mesenchymal stem cells play a vital role in bone formation process by differentiating into osteoblasts, in a tissue that offers not a flat but a discontinuous three-dimensional (3D) topography in vivo. In order to understand how geometry may be affecting mesenchymal stem cells, this study explored the influence of 3D geometry on mesenchymal stem cell-fate by comparing cell growth, viability and osteogenic potential using monolayer (two-dimensional, 2D) with microsphere (3D) culture systems normalised to surface area. The results suggested lower cell viability and reduced cell growth in 3D. Alkaline phosphatase activity was higher in 3D; however, both collagen and mineral deposition appeared significantly lower in 3D, even after osteogenic supplementation. Also, there were signs of patchy mineralisation in 3D with or without osteogenic supplementation as early as day 7. These results suggest that the convex surfaces on microspheres and inter-particulate porosity may have led to variable cell morphology and fate within the 3D culture. This study provides deeper insights into geometrical regulation of mesenchymal stem cell responses applicable for bone tissue engineering

In vitro degradation and mechanical properties of PLA-PCL copolymer unit cell scaffolds generated by two-photon polymerization

The manufacture of 3D scaffolds with specific controlled porous architecture, defined microstructure and an adjustable degradation profile was achieved using two-photon polymerization (TPP) with a size of 2  ×  4  ×  2 mm3. Scaffolds made from poly(D,L-lactide-co-ɛ-caprolactone) copolymer with varying lactic acid (LA) and ɛ -caprolactone (CL) ratios (LC16:4, 18:2 and 9:1) were generated via ring-opening-polymerization and photoactivation. The reactivity was quantified using photo-DSC, yielding a double bond conversion ranging from 70% to 90%. The pore sizes for all LC scaffolds were see 300 μm and throat sizes varied from 152 to 177 μm. In vitro degradation was conducted at different temperatures; 37, 50 and 65 °C. Change in compressive properties immersed at 37 °C over time was also measured. Variations in thermal, degradation and mechanical properties of the LC scaffolds were related to the LA/CL ratio. Scaffold LC16:4 showed significantly lower glass transition temperature (T g) (4.8 °C) in comparison with the LC 18:2 and 9:1 (see 32 °C). Rates of mass loss for the LC16:4 scaffolds at all temperatures were significantly lower than that for LC18:2 and 9:1. The degradation activation energies for scaffold materials ranged from 82.7 to 94.9 kJ mol−1. A prediction for degradation time was applied through a correlation between long-term degradation studies at 37 °C and short-term studies at elevated temperatures (50 and 65 °C) using the half-life of mass loss (Time (M1/2)) parameter. However, the initial compressive moduli for LC18:2 and 9:1 scaffolds were 7 to 14 times higher than LC16:4 (see 0.27) which was suggested to be due to its higher CL content (20%). All scaffolds showed a gradual loss in their compressive strength and modulus over time as a result of progressive mass loss over time. The manufacturing process utilized and the scaffolds produced have potential for use in tissue engineering and regenerative medicine applications

A phosphate glass reinforced composite acrylamide gradient scaffold for osteochondral interface regeneration

The authors would like to thank Dr. Joshua Price, Dr. Tina Dale, Dr Hamza Abu Owida and Dr Marwan Merkhan for help and advice with experimental lab work. Also, special thanks to Dr. Ann Canning for her help in the 3D printing design to support the current project.Peer reviewe

Rheological, Surface Tension and Conductivity Insights on the Electrospinnability of Poly(lactic-co-glycolic acid)-hyaluronic Acid Solutions and Their Correlations with the Nanofiber Morphological Characteristics

In this study, solutions were prepared with fixed concentrations of hyaluronic acid (HA) but varied concentrations of poly (lactic-co-glycolic acid) (PLGA) to emphasize the effects of PLGA concentration and HA addition on solution properties and to further evaluate their electrospinning performance. The dependence of specific viscosity on PLGA concentration was studied to determine the concentration regimes and evaluate the critical concentration (Ce) for successful fiber generation. The Ce of PLGA solutions is 12.07% compared to 10.09% for PLGA-HA solutions. Blending with HA results in a lower concentration dependence and better consistency to the theoretical scaling mechanisms due to the additional topological constrains, which thus result in more chain entanglements. Solutions in semi-dilute entangled regimes show the crossover of complex moduli, verifying the stable and reliable entanglement network. Higher concentrations and HA addition both led to lower crossover frequencies and, thus, a longer relaxation time. The effects of a higher PLGA concentration and HA addition on the surface tension were not evident. However, the HA addition significantly improved the solution conductivity up to three times in the pure PLGA solutions due to its polyelectrolyte nature. Defect-free and uniform nanofibers were generated from 35% to 40% of the PLGA-HA solutions, yet fibers with bead-on-string structures were produced from all studied pure PLGA solutions. Such solution characteristics and parametric correlations can provide predictive insights on tailoring the morphological characteristics of nanofibers for specific applications

The Use of Biomaterials in Internal Radiation Therapy

Radiotherapy has become one of the most prominent and effective modalities for cancer treatment and care. Ionising radiation, delivered either from external or internal sources, can be targeted to cancerous cells causing damage to DNA that can induce apoptosis. External beam radiotherapy delivers either photon radiation (x-rays or gamma rays) or particle radiation (neutrons or protons) in a targeted manner to specific tumour locations. Internal radiotherapy involves placing radioactive sources within the body to deliver localised doses of therapeutic radiation to tumours using short range radionuclides. Biomaterials have been developed to allow more precise targeting of radiotherapy in order to reduce toxicity to surrounding healthy tissues and increase treatment efficacy. These unique biomaterials have been developed from polymers, glasses and ceramics. Polymeric materials have been used to both displace healthy tissue from tumours receiving radiation, and to deliver radioactive sources into the body. These polymers can respond to various stimuli, such as radiation or reactive oxygen species, to deliver therapeutic payloads to target tissue during or post radiotherapy. Glass-based biomaterials doped with radionuclides have also been developed to provide in situ radiotherapy. Novel biomaterials that can enhance the synergistic effect of other treatment modalities, such as chemotherapy and immunotherapy, continue to be developed. Theranostic materials that are capable of providing diagnostic information whilst simultaneously delivering a therapeutic effect to enhance radiotherapy are also briefly reviewed

Developing unique geometries of phosphate-based glasses and their prospective biomedical applications

© 2019 Johnson Matthey Phosphate-based glasses are promising materials for a range of applications including biomedical, veterinary and optical. These glasses are distinguished by the presence of at least one terminal oxygen atom, which gives phosphate-based glasses unique properties of which the most interesting is their easily controllable degradation profiles. This article describes the main structural features and applications of phosphate-based glass materials focusing primarily on biomedical applications. The processes utilised for developing varying geometries such as fibres, solid and porous microspheres and coatings are also explored

Developing unique geometries of phosphate-based glasses and their prospective biomedical applications

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