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Primary ciliopathies are rare inherited disorders caused by structural or functional defects in the primary cilium, a subcellular organelle present on the surface of most cells. Primary ciliopathies show considerable clinical and genetic heterogeneity, with disruption of over 100 genes causing the variable involvement of several organs, including the central nervous system, kidneys, retina, skeleton and liver. Pathogenic variants in one and the same gene may associate with a wide range of ciliopathy phenotypes, supporting the hypothesis that the individual genetic background, with potential additional variants in other ciliary genes, may contribute to a mutational load eventually determining the phenotypic manifestations of each patient. Functional studies in animal models have uncovered some of the pathophysiological mechanisms linking ciliary gene mutations to the observed phenotypes; yet, the lack of reliable human cell models has previously limited preclinical research and the development of new therapeutic strategies for primary ciliopathies. Recent technical advances in the generation of patient-derived two-dimensional (2D) and three-dimensional (3D) cellular models give a new spur to this research, allowing the study of pathomechanisms while maintaining the complexity of the genetic background of each patient, and enabling the development of innovative treatments to target specific pathways. This review provides an overview of available models for primary ciliopathies, from existing in vivo models to more recent patient-derived 2D and 3D in vitro models. We highlight the advantages of each model in understanding the functional basis of primary ciliopathies and facilitating novel regenerative medicine, gene therapy and drug testing strategies for these disorders.

Adipocytes and osteoblasts derive from a common mesenchymal progenitor present in a range of connective tissues. Differentiation of the progenitors toward the two cell lineages can be induced in vitro through well-established protocols, and leads to the appearance of lipid-laden adipocytes and osteoblasts embedded in a mineralized matrix. The formation of these two lineages in cell cultures can be monitored using lipophilic dyes such as Oil Red O and substances binding to mineral deposits such as Alizarin Red S, respectively. However, these common staining techniques require cell fixation and are thus incompatible with live analyses. Recently, alternative approaches using vital stains have allowed the dual visualization and fluorescence imaging of adipogenic and osteogenic lineages in live cultures. Here we present the concomitant analysis of cultures containing adipogenic and osteogenic cell types using live staining, combining LipidTox Red and tetracycline with NucRed nuclear counterstain for confocal imaging. This approach can be applied to visualize the kinetics and 3D structure of differentiating mesenchymal cultures over time and highlights the interaction of adipose and mineralized compartments associated with bone marrow stroma.

Beige adipocytes possess the morphological and biochemical characteristics of brown adipocytes, including the mitochondrial uncoupling protein (UCP)1. Mesenchymal stem cells (MSCs) are somatic multipotent progenitors which differentiate into lipid‐laden adipocytes. Induction of MSC adipogenesis under hypothermic culture conditions (ie 32°C) promotes the appearance of a beige adipogenic phenotype, but the stability of this phenotypic switch after cells are returned to normothermic conditions of 37°C has not been fully examined. Here, cells transferred from 32°C to 37°C retained their multilocular beige‐like morphology and exhibited an intermediate gene expression profile, with both beige‐like and white adipocyte characteristics while maintaining UCP1 protein expression. Metabolic profile analysis indicated that the bioenergetic status of cells initially differentiated at 32°C adapted post‐transfer to 37°C, showing an increase in mitochondrial respiration and glycolysis. The ability of the transferred cells to respond under stress conditions (eg carbonyl cyanide‐4‐phenylhydrazone (FCCP) treatment) demonstrated higher functional capacity of enzymes involved in the electron transport chain and capability to supply substrate to the mitochondria. Overall, MSC‐derived adipocytes incubated at 32°C were able to remain metabolically active and retain brown‐like features after 3 weeks of acclimatization at 37°C, indicating these phenotypic characteristics acquired in response to environmental conditions are not fully reversible.

Phosphate-based glasses (PBGs) are biomaterials that degrade under physiological conditions and can be modified to release various ions depending on end applications. This study utilized slow-degrading (P45:45P2O5-16CaO-24MgO-11Na2O10 4Fe2O3, mol %) and comparatively faster degrading (P40:40P2O5-16CaO-24MgO-20Na2O, mol %) PBG microspheres with or without porosity, to evaluate the combined effect of chemical formulation and geometry on human mesenchymal stem cells (MSCs), a clinically relevant cell source for orthopedic applications. Scanning electron microscopy showed 2, 46, and 29% of P45 bulk (P45-B), P40 bulk (P40-B), and P40 porous (P40-P) microspheres, respectively, that had cracks or peeling off surfaces after 42 days of incubation in culture medium. Cytotoxicity assessment showed that glass debris released into the culture medium may interact with cells and affect their survival. Direct-contact cell experiments up to 42 days showed that P45-B microspheres did not sustain viable long-term cell cultures and did not facilitate extracellular matrix formation. On the other hand, P40-B microspheres enhanced alkaline phosphatase activity, calcium deposition, and collagen and osteocalcin production in MSCs. Introduction of porosity in P40 glass further enhanced these parameters and proliferation at later time points. The small pore windows (

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.

Adult neurogenesis is well-described in the subventricular and subgranular zones of the mammalian brain. Recent observations that resident glia express stem cell markers in some areas of the brain not traditionally associated with neurogenesis hint to a possible role in tissue repair. The Bergmann glia (BG) population in the cerebellum displays markers and in vitro features associated with neural stem cells (NSC), however the physiological relevance of this phenotypic overlap remains unclear in the absence of established in vivo evidence of tissue regeneration in the adult cerebellum. Here, this BG population was analysed in the adult cerebellum of different species and showed conservation of NSC-associated marker expression including Sox1, Sox2 and Sox9, in chick, primate and mouse cerebellum tissue. NSC-like cells isolated from adult mouse cerebellum showed slower growth when compared to lateral ventricle NSC, as well as differences upon differentiation. In a mouse model of cerebellar degeneration, progressive Purkinje cell loss was linked to cerebellar cortex disorganisation and a significant increase in Sox-positive cells compared to matching controls. These results show that this Sox-positive population responds to cerebellar tissue disruption, suggesting it may represent a mobilisable cellular resource for targeted strategies to promote tissue repair.

Temperature is a key regulator of brown adipose tissue (BAT) function, acting through central sensory inputs to influence metabolism and energy storage. Although animal models have produced a wealth of information on the pathways, effectors and responses mediating the physiological response of adipose tissue to temperature in vivo, the use of cell culture models now offers evidence of an additional cell-autonomous response to temperature changes, in the absence of neural input. In particular, stem cell models provide new insight into the regulation of adipogenic differentiation and the induction of browning features in vitro. Here the basis for adipogenic responsiveness to low temperature is discussed, together with different human cell models available to outline the benefits of cell-based approaches for future BAT research.

Brown adipose tissue (BAT) is able to rapidly generate heat and metabolise macronutrients, such as glucose and lipids, through activation of mitochondrial uncoupling protein 1 (UCP1). Diet can modulate UCP1 function but the capacity of individual nutrients to promote the abundance and activity of UCP1 is not well established. Caffeine consumption has been associated with loss of body weight and increased energy expenditure, but whether it can activate UCP1 is unknown. This study examined the effect of caffeine on BAT thermogenesis in vitro and in vivo. Stem cell-derived adipocytes exposed to caffeine (1 mM) showed increased UCP1 protein abundance and cell metabolism with enhanced oxygen consumption and proton leak. These functional responses were associated with browning-like structural changes in mitochondrial and lipid droplet content. Caffeine also increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial biogenesis, together with a number of BAT selective and beige gene markers. In vivo, drinking coffee (but not water) stimulated the temperature of the supraclavicular region, which co-locates to the main region of BAT in adult humans, and is indicative of thermogenesis. Taken together, these results demonstrate that caffeine can promote BAT function at thermoneutrality and may have the potential to be used therapeutically in adult humans.

Low-power sonication is widely used to disaggregate extracellular vesicles (EVs) after isolation, however, the effects of sonication on EV samples beyond dispersion are unclear. The present study analysed the characteristics of EVs collected from mesenchymal stem cells (MSCs) after sonication, using a combination of transmission electron microscopy, direct stochastic optical reconstruction microscopy, and flow cytometry techniques. Results showed that beyond the intended disaggregation effect, sonication using the lowest power setting available was enough to alter the size distribution, membrane integrity, and uptake of EVs in cultured cells. These results point to the need for a more systematic analysis of sonication procedures to improve reproducibility in EV-based cellular experiments.

International audience; Background/Aims: The skeleton is a metabolically active organ undergoing continuous remodelling initiated by mesenchymal progenitors present in bone and bone marrow. Under certain pathological conditions this remodelling balance shifts towards increased resorption resulting in weaker bone microarchitecture, and there is consequently a therapeutic need to identify pathways that could inversely enhance bone formation from stem cells. Metabolomics approaches recently applied to stem cell characterisation could help identify new biochemical markers involved in osteogenic differentiation. Methods: Combined intra- and extracellular metabolite profiling was performed by liquid chromatography-mass spectrometry (LC-MS) on human mesenchymal stem cells (MSCs) undergoing osteogenic differentiation in vitro. Using a combination of univariate and multivariate analyses, changes in metabolite and nutrient concentration were monitored in cultures under osteogenic treatment over 10 days. Results: A subset of differentially detected compounds was identified in differentiating cells, suggesting a direct link to metabolic processes involved in osteogenic response. Conclusion: These results highlight new metabolite candidates as potential biomarkers to monitor stem cell differentiation towards the bone lineage.

Cancer has been traditionally viewed as a disease characterised by excessive and uncontrolled proliferation, leading to the development of cytotoxic therapies against highly proliferating malignant cells. However, tumours frequently relapse due to the presence of slow-cycling cancer stem cells eluding chemo and radiotherapy. Since these malignant stem cells are largely undifferentiated, inducing their lineage commitment has been proposed as a potential intervention strategy to deplete tumours from their most resistant components. Pro-differentiation approaches have thus far yielded clinical success in the reversion of acute promyelocytic leukaemia (APL), and new developments are fast widening their therapeutic applicability to solid carcinomas. Recent advances in cancer differentiation discussed here highlight the potential and outstanding challenges of differentiation-based approaches.

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.

Phosphate-based glasses (PBGs) offer significant therapeutic potential due to their bioactivity, controllable compositions, and degradation rates. Several PBGs have already demonstrated their ability to support direct cell growth and in vivo cytocompatibility for bone repair applications. This study investigated development of PBG formulations with pyro- and orthophosphate species within the glass system (40 &minus, x)P2O5&middot, (16 + x)CaO&middot, 20Na2O&middot, 24MgO (x = 0, 5, 10 mol%) and their effect on stem cell adhesion properties. Substitution of phosphate for calcium revealed a gradual transition within the glass structure from Q2 to Q0 phosphate species. Human mesenchymal stem cells were cultured directly onto discs made from three PBG compositions. Analysis of cells seeded onto the discs revealed that PBG with higher concentration of pyro- and orthophosphate content (61% Q1 and 39% Q0) supported a 4.3-fold increase in adhered cells compared to glasses with metaphosphate connectivity (49% Q2 and 51% Q1). This study highlights that tuning the composition of PBGs to possess pyro- and orthophosphate species only, enables the possibility to control cell adhesion performance. PBGs with superior cell adhesion profiles represent ideal candidates for biomedical applications, where cell recruitment and support for tissue ingrowth are of critical importance for orthopaedic interventions.

Background/aims Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on the differentiation of adipocytes and lipid droplet accumulation. Methods Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine - MSO) during differentiation and their response was compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). Results Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration, and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. Conclusion These results suggest that the glutamine pathway may have a previously over-looked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity.

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 m2 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.

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.

A major challenge in orthopedics is the repair of large non-union bone fractures. A promising therapy for this indication is the use of biodegradable bioinspired biomaterials that stabilize the fracture site, relieve pain and initiate bone formation and healing. This study uses a multidisciplinary evaluation strategy to assess immunogenicity, allergenicity, bone responses and physicochemical properties of a novel biomaterial scaffold. Two-photon stereolithography generated personalized custom-built scaffolds with a repeating 3D structure of Schwarz Primitive minimal surface unit cell with a specific pore size of ?400 ?m from three different methacrylated poly(D,L-lactide-co-?-caprolactone) copolymers with lactide to caprolactone monomer ratios of 16?:?4, 18?:?2 and 9?:?1. Using in vitro and in vivo assays for bone responses, immunological reactions and degradation dynamics, we found that copolymer composition influenced the scaffold physicochemical and biological properties. The scaffolds with the fastest degradation rate correlated with adverse cellular effects and mechanical stiffness correlated with in vitro osteoblast mineralization. The physicochemical properties also correlated with in vivo bone healing and immune responses. Overall these observations provide compelling support for these scaffolds for bone repair and illustrate the effectiveness of a promising multidisciplinary strategy with great potential for the preclinical evaluation of biomaterials.

Human mesenchymal stem cells (MSCs) show promise for musculoskeletal repair applications. Animal-derived serum is extensively used for MSC culture as a source of nutrients, extracellular matrix proteins and growth factors. However, the routine use of fetal calf serum (FCS) is not innocuous due to its animal antigens and ill-defined composition, driving the development of alternatives protocols. The present study sought to reduce exposure to FCS via the transient use of human serum. Transient exposure to animal serum had previously proved successful for the osteogenic differentiation of MSCs but had not yet been tested with alternative serum sources. Here, human serum was used to support the proliferation of MSCs, which retained surface marker expression and presented higher alkaline phosphatase activity than those in FCS-based medium. Addition of osteogenic supplements supported strong mineralisation over a 3-week treatment. When limiting serum exposure to the first five days of treatment, MSCs achieved higher differentiation with human serum than with FCS. Finally, human serum analysis revealed significantly higher levels of osteogenic components such as alkaline phosphatase and 25-Hydroxyvitamin D, consistent with the enhanced osteogenic effect. These results indicate that human serum used at the start of the culture offers an efficient replacement for continuous FCS treatment and could enable short-term exposure to patient-derived serum in the future.

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.

Phosphate-based glasses (PBGs) 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 PBGs 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 postimplantation 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, while 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 toward the bone lineage. With the faster degrading P40 formulation, mineralization 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 that 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.

This paper reports on the role of phosphate-based glass (PBG) microspheres and their physicochemical properties including in vitro biological response to human mesenchymal stem cells (hMSCs). Solid and porous microspheres were prepared via a flame spheroidisation process. The Mg content in the PBG formulations explored was reduced from 24 to 2 mol% with a subsequent increase in Ca content. A small quantity of TiO2 (1 mol%) was added to the lower Mg-content glass (2 mol%) to avoid crystallisation. Morphological and physical characterisation of porous microspheres revealed interconnected porosity (up to 76 ± 5 %), average external pore sizes of 55 ± 5 μm with surface areas ranging from 0.38 to 0.43 m2 g−1. Degradation and ion release studies conducted compared the solid (non-porous) and porous microspheres and revealed 1.5 to 2.5 times higher degradation rate for porous microspheres. Also, in vitro bioactivity studies using simulated body fluid (SBF) revealed Ca/P ratios for porous microspheres of all three glass formulations were between 0.75 and 0.92 which were within the range suggested for precipitated amorphous calcium phosphate. Direct cell seeding and indirect cell culture studies (via incubation with microsphere degradation products) revealed hMSCs were able to grow and undergo osteogenic differentiation in vitro, confirming cytocompatibility of the formulations tested. However, the higher Mg content (24 mol%) porous microsphere showed the most potent osteogenic response and is therefore considered as a promising candidate for bone repair applications.

Development of a biomimetic tubular scaffold capable of recreating developmental neurogenesis using pluripotent stem cells offers a novel strategy for the repair of spinal cord tissues. Recent advances in 3D printing technology have facilitated biofabrication of complex biomimetic environments by precisely controlling the 3D arrangement of various acellular and cellular components (biomaterials, cells and growth factors). Here, we present a 3D printing method to fabricate a complex, patterned and embryoid body (EB)-laden tubular scaffold composed of polycaprolactone (PCL) and hydrogel (alginate or gelatine methacrylate (GelMA)). Our results revealed 3D printing of a strong, macro-porous PCL/hydrogel tubular scaffold with a high capacity to control the porosity of the PCL scaffold, wherein the maximum porosity in the PCL wall was 15%. The method was equally employed to create spatiotemporal protein concentration within the scaffold, demonstrating its ability to generate linear and opposite gradients of model molecules (fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) and rhodamine). 3D bioprinting of EBs-laden GelMA was introduced as a novel 3D printing strategy to incorporate EBs in a hydrogel matrix. Cell viability and proliferation were measured post-printing. Following the bioprinting of EBs-laden 5% GelMA hydrogel, neural differentiation of EBs was induced using 1 μM retinoic acid (RA). The differentiated EBs contained βIII-tubulin positive neurons displaying axonal extensions and cells migration. Finally, 3D bioprinting of EBs-laden PCL/GelMA tubular scaffold successfully supported EBs neural differentiation and patterning in response to co-printing with 1 μM RA. 3D printing of a complex heterogeneous tubular scaffold that can encapsulate EBs, spatially controlled protein concentration and promote neuronal patterning will help in developing more biomimetic scaffolds capable of replicating the neural patterning which occurs during neural tube development.

Characterization of the elasticity of biological cells is growing as a new way to gain insight into cell biology. Cell mechanics are related to most aspects of cellular behavior, and applications in research and medicine are broad. Current methods are often limited since they require physical contact or lack resolution. From the methods available for the characterization of elasticity, those relying on high frequency ultrasound (phonons) are the most promising because they offer label-free, high (even super-optical) resolution and compatibility with conventional optical microscopes. In this Perspective contribution, we review the state of the art of picosecond ultrasonics for cell imaging and characterization, particularly for Brillouin scattering-based methods, offering an opinion for the challenges faced by the technology. The challenges are separated into biocompatibility, acquisition speed, resolution, and data interpretation and are discussed in detail along with new results.

Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of transforming growth factor beta (TGF-&beta, ) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens.

Multiphase hybrids were fabricated from poly(d,l-lactide-co-ɛ-caprolactone) (PLCL) copolymer scaffolds impregnated with silk-elastin-like recombinamers (SELRs) hydrogel containing 2 wt% hydroxyapatite nanoparticles (nHA). The PLCL scaffolds, triply-periodic minimal surface geometry, were manufactured using two-photon stereolithography. In vitro degradation studies were conducted on PLCL scaffolds in inflamed tissue mimic media (pH ~ 4.5–6.5) or phosphate buffered saline (PBS) at 37 °C. Compression test revealed instant shape recovery of PLCL scaffolds after compression to 70% strain, ideal for arthroscopic delivery. Degradation of these scaffolds was accelerated in acidic media, where mass loss and compressive properties at day 56 were about 2–6 times lower than the scaffolds degraded in PBS. No significant difference was seen in the compressive properties between PLCL scaffolds and the hybrids due to the order of magnitude difference between the hydrogels and the PLCL scaffolds. Moreover, degradation properties of the hybrids did not significantly change by inclusion of SELR+/−nHA nanocomposite hydrogels. The hybrids lost approximately 40% and 84% of their initial weight and mechanical properties, respectively after 112 days of degradation. Cytotoxicity assessment revealed no cytotoxic effects of PLCL or PLCL-SELR+/−2%nHA scaffolds on bone marrow-derived human Mesenchymal Stem Cells. These findings highlight the potential of these hybrid constructs for bone and cartilage repair. Keywords: Poly(d,l-lactide-co-ɛ-caprolactone) copolymer, Multiphase hybrids, In vitro degradation, Mechanical recovery, human Mesenchymal Stem Cells

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication., Graphical abstract Unlabelled Image, Highlights • Direct Stochastic Optical Reconstruction Microscopy (d-STORM) can be used to characterise Extracellular Vesicles (EVs). • d-STORM imaging can detect EVs below 50 nm in size. • The d-STORM approach results in highly reproducible EV size and concentration measurements. • DiD-labelled EVs allow for live analysis of EV trafficking.

The chemical formulation of phosphate-based glasses (PBGs) can be tailored to fit particular end applications such as bone tissue engineering. While most reports to date have evaluated the effect of PBG chemical formulation on bone cells, this study specifically explored the manufacturing process, the changes in physical and chemical properties of PBG particles after flame spheroidization, and subsequent effects on human mesenchymal stem cells (hMSCs), a prime cell type for regenerative medicine applications. Flame spheroidization involves feeding irregular PBG particles (microparticles, MP) into a hot flame, causing them to melt and mold into solid spherical PBG particles (microspheres, MS). The laser diffraction analysis showed an increase in the volume-weighted mean diameter of particles from 48 to 139 μm after spheroidization and also revealed changes in the chemical composition of smaller MS (45 μm in size), whereas MS in other size ranges did not show significantly different chemical composition compared to MP. Additionally, some air bubbles were entrapped inside particles during spheroidization, causing a 2% drop in relative density of MS. However, the packing density of MS was 30% higher than that of MP. Culture of hMSCs on the particles showed significant improvement in cell spreading on MS compared to that on MP and nearly 2 times higher cell metabolic activity after 7 days of culture, suggesting that MS provided a more favorable support and geometry for hMSC attachment and growth for tissue engineering.

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 40P2O5·(16‐x)CaO·20Na2O·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.

Bone regeneration is a complex biological process where major cellular changes take place to support the osteogenic differentiation of mesenchymal bone progenitors. To characterise these biological changes and better understand the pathways regulating the formation of mature bone cells, the metabolic profile of mesenchymal stem cell (MSC) differentiation in vitro has been assessed non-invasively during osteogenic (OS) treatment using a footprinting technique. Liquid chromatography (LC)-mass spectrometry (MS)-based metabolite profiling of the culture medium was carried out in parallel to mineral deposition and alkaline phosphatase activity which are two hallmarks of osteogenesis in vitro. Metabolic profiles of spent culture media with a combination of univariate and multivariate analyses investigated concentration changes of extracellular metabolites and nutrients linked to the presence of MSCs in culture media. This non-invasive LC-MS-based analytical approach revealed significant metabolic changes between the media from control and OS-treated cells showing distinct effects of MSC differentiation on the environmental footprint of the cells in different conditions (control vs. OS treatment). A subset of compounds was directly linked to the osteogenic time-course of differentiation, and represent interesting metabolite candidates as non-invasive biomarkers for characterising the differentiation of MSCs in a culture medium.

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.\ud \ud 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.

Brown and beige adipocytes are characterised as expressing the unique mitochondrial uncoupling protein (UCP)1 for which the primary stimulus in vivo is cold exposure. The extent to which cold-induced UCP1 activation can also be achieved in vitro, and therefore perform a comparable cellular function, is unknown. We report an in vitro model to induce adipocyte browning using bone marrow (BM) derived mesenchymal stem cells (MSC), which relies on differentiation at 32 °C instead of 37 °C. The low temperature promoted browning in adipogenic cultures, with increased adipocyte differentiation and upregulation of adipogenic and thermogenic factors, especially UCP1. Cells exhibited enhanced uncoupled respiration and metabolic adaptation. Cold-exposed differentiated cells showed a marked translocation of leptin to adipocyte nuclei, suggesting a previously unknown role for leptin in the browning process. These results indicate that BM-MSC can be driven to forming beige-like adipocytes in vitro by exposure to a reduced temperature. This in vitro model will provide a powerful tool to elucidate the precise role of leptin and related hormones in hitherto functions in the browning process.