The impact of the extracellular matrix environment on sost expression by the MLO-Y4 osteocyte cell line

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model

The impact of the extracellular matrix environment on sost expression by the MLO-Y4 osteocyte cell line

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model

The impact of the extracellular matrix environment on sost expression by the MLO-Y4 osteocyte cell line

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model

Nano sized gallium oxide surface features for enhanced antimicrobial and osteo-integrative responses

Gallium oxide has known beneficial osteo-integrative properties. This may have importance for improving the osteointegration of orthopedic implants. At high concentrations gallium is cytotoxic. Therefore, integration of gallium into implant devices must be carefully controlled to limit its concentration and release. A strategy based on surface doping of gallium although challenging seems an appropriate approach to limit dose amounts to minimize cytotoxicity and maximize osteointegration benefits. In this work we develop a novel form of patterned surface doping via a block copolymer-based surface chemistry that enables very low gallium content but enhanced osteointegration as proven by comprehensive bioassays. Polystyrene-b-poly 4vinyl pyridine (PS-b-P4VP) BCP (block copolymer) films were produced on surfaces. Selective infiltration of the BCP pattern with a gallium salt precursor solution and subsequent UV-ozone treatment produced a surface pattern of gallium oxide nanodots as evidenced by atomic force and scanning electron microscopy. A comprehensive study of the bioactivity was carried out, including antimicrobial and sterility testing, gallium ion release kinetics and the interaction with human marrow mesenchymal stomal cells and mononuclear cells. Comparing the data from osteogenesis media assay tests with osteoclastogenesis tests demonstrated the potential for the gallium oxide nanodot doping to improve osteointegration properties of a surface. </p

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

Background: Human mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source. Methods: In this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality-two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred. Results: During osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism. Conclusion: In summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.</p

Nano sized gallium oxide surface features for enhanced antimicrobial and osteo-integrative responses

Gallium oxide has known beneficial osteo-integrative properties. This may have importance for improving the osteointegration of orthopedic implants. At high concentrations gallium is cytotoxic. Therefore, integration of gallium into implant devices must be carefully controlled to limit its concentration and release. A strategy based on surface doping of gallium although challenging seems an appropriate approach to limit dose amounts to minimize cytotoxicity and maximize osteointegration benefits. In this work we develop a novel form of patterned surface doping via a block copolymer-based surface chemistry that enables very low gallium content but enhanced osteointegration as proven by comprehensive bioassays. Polystyrene-b-poly 4vinyl pyridine (PS-b-P4VP) BCP (block copolymer) films were produced on surfaces. Selective infiltration of the BCP pattern with a gallium salt precursor solution and subsequent UV-ozone treatment produced a surface pattern of gallium oxide nanodots as evidenced by atomic force and scanning electron microscopy. A comprehensive study of the bioactivity was carried out, including antimicrobial and sterility testing, gallium ion release kinetics and the interaction with human marrow mesenchymal stomal cells and mononuclear cells. Comparing the data from osteogenesis media assay tests with osteoclastogenesis tests demonstrated the potential for the gallium oxide nanodot doping to improve osteointegration properties of a surface. </p

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

Background: Human mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source. Methods: In this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality-two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred. Results: During osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism. Conclusion: In summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.</p

A microphysiological model of bone development and regeneration

Endochondral ossification (EO) is an essential biological process than underpins how human bones develop, grow, and heal in the event of a fracture. So much is unknown about this process, thus clinical manifestations of dysregulated EO cannot be adequately treated. This can be partially attributed to the absence of predictivein vitromodels of musculoskeletal tissue development and healing, which are integral to the development and preclinical evaluation of novel therapeutics. Microphysiological systems, or organ-on-chip devices, are advancedin vitromodels designed for improved biological relevance compared to traditionalin vitroculture models. Here we develop a microphysiological model of vascular invasion into developing/regenerating bone, thereby mimicking the process of EO. This is achieved by integrating endothelial cells and organoids mimicking different stages of endochondral bone development within a microfluidic chip. This microphysiological model is able to recreate key events in EO, such as the changing angiogenic profile of a maturing cartilage analogue, and vascular induced expression of the pluripotent transcription factors SOX2 and OCT4 in the cartilage analogue. This system represents an advancedin vitroplatform to further EO research, and may also serve as a modular unit to monitor drug responses on such processes as part of a multi-organ system. </p

A microphysiological model of bone development and regeneration

Endochondral ossification (EO) is an essential biological process than underpins how human bones develop, grow, and heal in the event of a fracture. So much is unknown about this process, thus clinical manifestations of dysregulated EO cannot be adequately treated. This can be partially attributed to the absence of predictivein vitromodels of musculoskeletal tissue development and healing, which are integral to the development and preclinical evaluation of novel therapeutics. Microphysiological systems, or organ-on-chip devices, are advancedin vitromodels designed for improved biological relevance compared to traditionalin vitroculture models. Here we develop a microphysiological model of vascular invasion into developing/regenerating bone, thereby mimicking the process of EO. This is achieved by integrating endothelial cells and organoids mimicking different stages of endochondral bone development within a microfluidic chip. This microphysiological model is able to recreate key events in EO, such as the changing angiogenic profile of a maturing cartilage analogue, and vascular induced expression of the pluripotent transcription factors SOX2 and OCT4 in the cartilage analogue. This system represents an advancedin vitroplatform to further EO research, and may also serve as a modular unit to monitor drug responses on such processes as part of a multi-organ system. </p

Mechanoregulatory role of TRPV4 in prenatal skeletal development

Biophysical cues are essential for guiding skeletal development, but the mechanisms underlying the mechanical regulation of cartilage and bone formation are unknown. TRPV4 is a mechanically sensitive ion channel involved in cartilage and bone cell mechanosensing, mutations of which lead to skeletal developmental pathologies. We tested the hypothesis that loading-driven prenatal skeletal development is dependent on TRPV4 activity. We first establish that mechanically stimulating mouse embryo hindlimbs cultured ex vivo stimulates knee cartilage growth, morphogenesis, and expression of TRPV4, which localizes to areas of high biophysical stimuli. We then demonstrate that loading-driven joint cartilage growth and shape are dependent on TRPV4 activity, mediated via control of cell proliferation and matrix biosynthesis, indicating a mechanism by which mechanical loading could direct growth and morphogenesis during joint formation. We conclude that mechanoregulatory pathways initiated by TRPV4 guide skeletal development; therefore, TRPV4 is a valuable target for the development of skeletal regenerative and repair strategies. </p