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

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

Development and In Vitro Assessment of a Bi-layered Chitosan-Nano-Hydroxyapatite Osteochondral Scaffold.

none10siopenPitrolino KA, Felfel RM, Macri Pellizzeri L, McLaren J, Popov AA, Sottile V, Scotchford CA, Scammell BE, Roberts GAF, Grant DMPitrolino, Ka; Felfel, Rm; Macri Pellizzeri, L; Mclaren, J; Popov, Aa; Sottile, V; Scotchford, Ca; Scammell, Be; Roberts, Gaf; Grant, D

In vitro cellular testing of strontium/calcium substituted phosphate glass discs and microspheres shows potential for bone regeneration

Phosphate-based glasses (PBGs) are ideal materials for regenerative medicine strategies because their composition, degradation rates, and ion release profiles can easily be controlled. Strontium has previously been found to simultaneously affect bone resorption and deposition. Therefore, by combining the inherent properties of resorbable PBG and therapeutic activity of strontium, these glasses could be used as a delivery device of therapeutic factors for the treatment of orthopaedic diseases such as osteoporosis. This study shows the cytocompatibility and osteogenic potential of PBGs where CaO is gradually replaced by SrO in the near invert glass system 40P 2 O 5 ·(16-x)CaO·20Na 2 O·24MgO·xSrO (x = 0, 4, 8, 12, and 16 mol%). Direct seeding of MG63 cells onto glass discs showed no significant difference in cell metabolic activity and DNA amount measurement across the different formulations studied. Cell attachment and spreading was confirmed via scanning electron microscopy (SEM) imaging at Days 3 and 14. Alkaline phosphatase (ALP) activity was similarly maintained across the glass compositions. Follow-on studies explored the effect of each glass composition in microsphere conformation (size: 63-125 μm) on human mesenchymal stem cells (hMSCs) in 3D cultures, and analysis of cell metabolic activity and ALP activity showed no significant differences at Day 14 over the compositional range investigated, in line with the observations from MG63 cell culture studies. Environmental SEM and live cell imaging at Day 14 of hMSCs seeded on the microspheres showed cell attachment and colonisation of the microsphere surfaces, confirming these formulations as promising candidates for regenerative medicine strategies addressing compromised musculoskeletal/orthopaedic diseases

In vitro cellular testing of strontium/calcium substituted phosphate glass discs and microspheres shows potential for bone regeneration

Phosphate-based glasses (PBGs) are ideal materials for regenerative medicine strategies because their composition, degradation rates, and ion release profiles can easily be controlled. Strontium has previously been found to simultaneously affect bone resorption and deposition. Therefore, by combining the inherent properties of resorbable PBG and therapeutic activity of strontium, these glasses could be used as a delivery device of therapeutic factors for the treatment of orthopaedic diseases such as osteoporosis. This study shows the cytocompatibility and osteogenic potential of PBGs where CaO is gradually replaced by SrO in the near invert glass system 40P 2 O 5 ·(16-x)CaO·20Na 2 O·24MgO·xSrO (x = 0, 4, 8, 12, and 16 mol%). Direct seeding of MG63 cells onto glass discs showed no significant difference in cell metabolic activity and DNA amount measurement across the different formulations studied. Cell attachment and spreading was confirmed via scanning electron microscopy (SEM) imaging at Days 3 and 14. Alkaline phosphatase (ALP) activity was similarly maintained across the glass compositions. Follow-on studies explored the effect of each glass composition in microsphere conformation (size: 63-125 μm) on human mesenchymal stem cells (hMSCs) in 3D cultures, and analysis of cell metabolic activity and ALP activity showed no significant differences at Day 14 over the compositional range investigated, in line with the observations from MG63 cell culture studies. Environmental SEM and live cell imaging at Day 14 of hMSCs seeded on the microspheres showed cell attachment and colonisation of the microsphere surfaces, confirming these formulations as promising candidates for regenerative medicine strategies addressing compromised musculoskeletal/orthopaedic diseases