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 (ECM) 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 nondestructive staining, quantitation, and imaging of mineralizing bone-like nodules in live cultures of human bone marrow mesenchymal stem cells 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 mineralized 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

Fast UPLC/PDA determination of squalene in Sicilian P.D.O. pistachio from Bronte: optimization of oil extraction method and analytical characterization

A fast reversed-phase UPLC method was developed for squalene determination in Sicilian pistachio samples that entry in the European register of the products with P.D.O. In the present study the SPE procedure was optimized for the squalene extraction prior to the UPLC/PDA analysis. The precision of the full analytical procedure was satisfactory and the mean recoveries were 92.8 + 0.3 % and 96.6 + 0.1 % for 25 and 50 mg L-1 level of addition, respectively. Selected chromatographic conditions allowed a very fast squalene determination; in fact it was well separated in ~ 0.54 min with good resolution. Squalene was detected in all the pistachio samples analyzed and the levels ranged from 55.45 to 226.34 mg . kg-1. Comparing our results with those of other studies it emerges that squalene contents in P.D.O. Sicilian pistachio samples, generally, were higher than those measured for other samples of different geographic origins

Live simultaneous monitoring of mineral deposition and lipid accumulation in differentiating stem cells

Mesenchymal stem cells (MSCs) are progenitors for bone-forming osteoblasts and lipid-storing adipocytes, two major lineages co-existing in bone marrow. When isolated in vitro, these stem cells recapitulate osteoblast or adipocyte formation if treated with specialised media,modelling how these lineages interact in vivo. Osteogenic differentiation is characterised by mineral deposits accumulating in the extracellular matrix, typically assessed using histological techniques. Adipogenesis occurs with accumulation of intracellular lipids that can be routinely visualised by Oil Red O staining. In both cases, staining requires cell fixation and is thus limited to end-point assessments. Here, a vital staining approach was developed to simultaneously detect mineral deposits and lipid droplets in differentiating cultures. Stem cells induced to differentiate produced mixed cultures containing adipocytes and bone-like nodules, and after two weeks live cultures were incubated with tetracycline hydrochloride and Bodipy to label mineral- and lipid-containing structures, respectively. Fluorescence microscopy showed the simultaneous visualisation of mineralised areas and lipid-filled adipocytes in live cultures. Combined with the nuclear stain Hoechst 33258, this approach further enabled live confocal imaging of adipogenic cells interspersed within the mineralised matrix. This multiplex labelling was repeated at subsequent time-points, demonstrating the potential of this new approach for the real-time high-precision imaging of live stem cells

Porous calcium phosphate glass microspheres for orthobiologic applications

Orthobiologics is a rapidly advancing field utilising cell-based therapies and biomaterials to enable the body to repair and regenerate musculoskeletal tissues. This paper reports on a cost-effective flame spheroidisation process for production of novel porous glass microspheres from calcium phosphate-based glasses to encapsulate and deliver stem cells. Careful selection of the glass and pore-forming agent, along with a manufacturing method with the required processing window enabled the production of porous glass microspheres via a single-stage manufacturing process. The morphological and physical characterisation revealed porous microspheres with tailored surface and interconnected porosity (up to 76 ± 5%) with average pore size of 55 ± 8 µm and surface areas ranging from 0.34 to 0.9 m 2 g −1 . Furthermore, simple alteration of the processing parameters produced microspheres with alternate unique morphologies, such as with solid cores and surface porosity only. The tuneable porosity enabled control over their surface area, degradation profiles and hence ion release rates. Furthermore, cytocompatibility of the microspheres was assessed using human mesenchymal stem cells via direct cell culture experiments and analysis confirmed that they had migrated to within the centre of the microspheres. The novel microspheres developed have huge potential for tissue engineering and regenerative medicine applications. Statement of Significance: This manuscript highlights a simple cost-effective one-step process for manufacturing porous calcium phosphate-based glass microspheres with varying control over surface pores and fully interconnected porosity via a flame spheroidisation process. Moreover, a simple alteration of the processing parameters can produce microspheres which have a solid core with surface pores only. The tuneable porosity enabled control over their surface area, degradation profiles and hence ion release rates. The paper also shows that stem cells not only attach and proliferate but more importantly migrate to within the core of the porous microspheres, highlighting applications for bone tissue engineering and regenerative medicine

Functional performance of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold: a pre-clinical <i>in vitro</i> tribological study

Osteochondral grafts are used for repair of focal osteochondral lesions. Autologous grafts are the gold standard treatment; however, limited graft availability and donor site morbidity restrict use. Therefore, there is a clinical need for different graft sources/materials which replicate natural cartilage function. Chitosan has been proposed for this application. The aim of this study was to assess the biomechanics and biotribology of a bioresorbable chitosan/chitosan-nano-hydroxyapatite osteochondral construct (OCC), implanted in an in vitro porcine knee experimental simulation model. The OCC implanted in different surgical positions (flush, proud and inverted) was compared to predicate grafts in current clinical use and a positive control consisting of a stainless steel graft implanted proud of the cartilage surface. After 3 h (10 800 cycles) wear simulation under a walking gait, subsidence occurred in all OCC samples irrespective of surgical positioning, but with no apparent loss of material and low meniscus wear. Half the predicate grafts exhibited delamination and scratching of the cartilage surfaces. No graft subsidence occurred in the positive controls but wear and deformation of the meniscus were apparent. Implanting a new chitosan-based OCC either optimally (flush), inverted or proud of the cartilage surface resulted in minimal wear, damage and deformation of the meniscus