Rapid conversion of highly porous borate glass microspheres into hydroxyapatite

This paper reports on the rapid development of porous hydroxyapatite (HA) microspheres with large external pores and fully interconnected porosity. These porous microspheres were produced by converting borates glasses (namely 45B5, B53P4 and 13-93B) into HA by immersing them in potassium phosphate media and simulated body fluid (SBF). Solid (SGMS) non-porous and highly porous (PGMS) microspheres were prepared from borate glasses via a novel flame spheroidisation process and their physicochemical properties including in vitro biological response were investigated. Morphological and physical characterisation of the PGMS showed interconnected porosity (up to 75 ± 5%) with average external pore sizes of 50 ± 5 μm. Mass loss, ion release, X-ray diffraction (XRD) and Scanning electron microscopy (SEM) analysis confirmed complete conversion to HA in 0.02 M K2HPO4 solution for the PGMS (with exception of 13-93B glass) and at significantly faster rates compared to their SGMS counterparts. However, 13-93B microspheres only converted to HA in Na2HPO4 solution. The in vitro SBF bioactivity studies for all the borate compositions showed HA formation and much earlier for PGMS compared to SGMS. Direct cell culture studies using hMSCs revealed that the converted porous HA microspheres showed enhanced pro-osteogenic properties compared to their unconverted counterparts and such are considered as highly promising candidate materials for bone repair (and orthobiological) applications

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

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

Effect of varying the Mg with Ca content in highly porous phosphate-based glass microspheres

Natural ventilation is a low energy strategy used in many building types. Design approaches are mature but are dependent on variables with high uncertainty, such as the aerodynamic behaviour of purpose provided openings (PPOs), which need improved characterisation. An analytical framework is used to define different types of flow through openings based on the balance of environmental forces that drive flow, and the different flow structures they create. This allows a comprehensive literature review to be made, where different studies and descriptive equations can be compared on a like-for-like basis, and from which clear gaps in knowledge, technical standards, and design data are identified. Phenomena whose understanding could be improved by analysis of existing data are identified and explored. A Statistical Effective Area Model (SEAM) is developed from academic data to estimate the performance of butt hinged openings during the design stage, that accounts for the impact of aspect ratio and opening angle. Its predictions are compared against available empirical data and are found to have a standard error of 1.2%, which is substantially lower than the 15–25% prediction errors of free area models commonly used in practice. An analytical model is made based on entrainment theory to explain the increase in flow rate that occurs through two aligned openings. This model defines characteristic design parameters and predicts a detrimental impact on the ventilation of the wider space. Finally, an analytical model is created to explain the reduction in discharge coefficient that occurs when a large temperature difference exists across an opening. This model defines novel dimensionless parameters that characterise the flow, and predicts empirical data well, suggesting that it should be integrated into design equations

Porous phosphate-based glass microspheres show biocompatibility, tissue infiltration and osteogenic onset in an ovine bone defect model

Phosphate-based glasses (PBG) are bioactive and fully degradable materials with tailorable degradation rates. PBGs can be produced as porous microspheres through a single-step process, using changes in their formulation and geometry to produce varying pore sizes and interconnectivity for use in a range of applications, including biomedical use. Calcium phosphate PBG have recently been proposed as orthobiologics, based on their in vitro cytocompatibility and ion release profile.In this study, porous microspheres made of two PBG formulations either containing TiO2 (P40Ti) or without (P40) were implanted in vivo in a large animal model of bone defect. The biocompatibility and osteogenic potential of these porous materials were assessed 13 weeks post-implantation in sheep, and compared to empty defects and autologous bone grafts used as negative and positive controls. Histological analysis showed marked differences between the two formulations, as lower trabeculae-like interconnection and higher fatty bone marrow content were observed in the faster degrading P40-implanted defects, whilst the slower degrading P40Ti material promoted dense interconnected tissue. Autologous bone marrow concentrate (BMC) was also incorporated within the P40 and P40Ti microspheres in some defects, however no significant differences were observed in comparison to microspheres implanted alone. Both formulations induced the formation of a collagen-enriched matrix, from 20 % to 40 % for P40 and P40Ti2.5 groups, suggesting commitment towards the bone lineage. With the faster degrading P40 formulation, mineralisation of the tissue matrix was observed both with and without BMC. Some lymphocyte-like cells and foreign body multinucleated giant cells were observed with P40Ti2.5, suggesting this more durable formulation might be linked to an inflammatory response. In conclusion, these first in vivo results indicate that PBG microspheres could be useful candidates for bone repair and regenerative medicine strategies, and highlight the role of material degradation in the process of tissue formation and maturation

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 highly 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 m2g-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, biocompatibility of the microspheres was assessed using human mesenchymal stem cells (hMSCs) 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

Development and in vitro assessment of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold

An innovative approach was developed to engineer a multi-layered chitosan scaffold for osteochondral defect repair. A combination of freeze drying and porogen-leaching out methods produced a porous, bioresorbable scaffold with a distinct gradient of pore size (mean = 160–275 μm). Incorporation of 70 wt% nano-hydroxyapatite (nHA) provided additional strength to the bone-like layer. The scaffold showed instantaneous mechanical recovery under compressive loading and did not delaminate under tensile loading. The scaffold supported the attachment and proliferation of human mesenchymal stem cells (MSCs), with typical adherent cell morphology found on the bone layer compared to a rounded cell morphology on the chondrogenic layer. Osteogenic and chondrogenic differentiation of MSCs preferentially occurred in selected layers of the scaffold in vitro, driven by the distinct pore gradient and material composition. This scaffold is a suitable candidate for minimally invasive arthroscopic delivery in the clinic with potential to regenerate damaged cartilage and bone

Functional performance of a bi-layered chitosan-nano-hydroxyapatite osteochondral scaffold : a pre-clinical in vitro 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

Role of substrate biomechanics in controlling (stem) cell fate: Implications in regenerative medicine

Tissue-specific stem cells reside in a specialized environment known as the niche. The niche plays a central role in the regulation of cell behavior and, through the concerted action of soluble molecules, supportive somatic cells and extracellular matrix-components, directs stem cells to proliferate, differentiate or remain quiescent. Great efforts have been done to decompose and separately analyze the contribution of these cues in the in vivo environment. Specifically, the mechanical properties of the extracellular matrix influence many aspects of cell behavior, including self-renewal and differentiation. Deciphering the role of biomechanics could thereby provide important insights to control the stem cells responses in a more effective way with the aim to promote their therapeutic potential. In this review, we provide a wide overview of the effect that the microenvironment stiffness exerts on the control of cell behavior with a particular focus on the induction of stem cells differentiation. We also describe the process of mechanotransduction and the molecular effectors involved. Finally, we critically discuss the potential involvement of tissue biomechanics in the design of novel tissue engineering strategies