Melt electrowritten scaffold architectures to mimic tissue mechanics and guide neo-tissue orientation.

All human tissues present with unique mechanical properties critical to their function. This is achieved in part through the specific architecture of the extracellular matrix (ECM) fibres within each tissue. An example of this is seen in the walls of the vasculature where each layer presents with a unique ECM orientation critical to its functions. Current adopted vascular grafts to bypass a stenosed/damaged vessel fail to recapitulate this unique mechanical behaviour, particularly in the case of small diameter vessels (<6 mm), leading to failure. Therefore, in this study, melt-electrowriting (MEW) was adopted to produce a range of fibrous scaffolds to mimic the extracellular matrix (ECM) architecture of the tunica media of the vasculature, in an attempt to match the mechanical and biological behaviour of the native porcine tissue. Initially, the range of collagen architectures within the native vessel was determined, and subsequently replicated using MEW (winding angles (WA) 45°, 26.5°, 18.4°, 11.3°). These scaffolds recapitulated the anisotropic, non-linear mechanical behaviour of native carotid blood vessels. Moreover, these grafts facilitated human mesenchymal stem cell (hMSC) infiltration, differentiation, and ECM deposition that was independent of WA. The bioinspired MEW fibre architecture promoted cell alignment and preferential neo-tissue orientation in a manner similar to that seen in native tissue, particularly for WA 18.4° and 11.3°, which is a mandatory requirement for long-term survival of the regenerated tissue post-scaffold degradation. Lastly, the WA 18.4° was translated to a tubular graft and was shown to mirror the mechanical behaviour of small diameter vessels within physiological strain. Taken together, this study demonstrates the capacity to use MEW to fabricate bioinspired scaffolds to mimic the tunica media of vessels and recapitulate vascular mechanics which could act as a framework for small diameter graft development to guide tissue regeneration and orientation.

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.

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.

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.

Bioengineering extracellular vesicles: smart nanomaterials for bone regeneration.

In the past decade, extracellular vesicles (EVs) have emerged as key regulators of bone development, homeostasis and repair. EV-based therapies have the potential to circumnavigate key issues hindering the translation of cell-based therapies including functional tissue engraftment, uncontrolled differentiation and immunogenicity issues. Due to EVs' innate biocompatibility, low immunogenicity, and high physiochemical stability, these naturally-derived nanoparticles have garnered growing interest as potential acellular nanoscale therapeutics for a variety of diseases. Our increasing knowledge of the roles these cell-derived nanoparticles play, has made them an exciting focus in the development of novel pro-regenerative therapies for bone repair. Although these nano-sized vesicles have shown promise, their clinical translation is hindered due to several challenges in the EV supply chain, ultimately impacting therapeutic efficacy and yield. From the biochemical and biophysical stimulation of parental cells to the transition to scalable manufacture or maximising vesicles therapeutic response in vivo, a multitude of techniques have been employed to improve the clinical efficacy of EVs. This review explores state of the art bioengineering strategies to promote the therapeutic utility of vesicles beyond their native capacity, thus maximising the clinical potential of these pro-regenerative nanoscale therapeutics for bone repair.

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.

Surface Free Energy Dominates the Biological Interactions of Postprocessed Additively Manufactured Ti-6Al-4V.

Additive manufacturing (AM) has emerged as a disruptive technique within healthcare because of its ability to provide personalized devices; however, printed metal parts still present surface and microstructural defects, which may compromise mechanical and biological interactions. This has made physical and/or chemical postprocessing techniques essential for metal AM devices, although limited fundamental knowledge is available on how alterations in physicochemical properties influence AM biological outcomes. For this purpose, herein, powder bed fusion Ti-6Al-4V samples were postprocessed with three industrially relevant techniques: polishing, passivation, and vibratory finishing. These surfaces were thoroughly characterized in terms of roughness, chemistry, wettability, surface free energy, and surface ζ-potential. A significant increase in Staphylococcus epidermidis colonization was observed on both polished and passivated samples, which was linked to high surface free energy donor γ- values in the acid-base, γAB component. Early osteoblast attachment and proliferation (24 h) were not influenced by these properties, although increased mineralization was observed for both these samples. In contrast, osteoblast differentiation on stainless steel was driven by a combination of roughness and chemistry. Collectively, this study highlights that surface free energy is a key driver between AM surfaces and cell interactions. In particular, while low acid-base components resulted in a desired reduction in S. epidermidis colonization, this was followed by reduced mineralization. Thus, while surface free energy can be used as a guide to AM device development, optimization of bacterial and mammalian cell interactions should be attained through a combination of different postprocessing techniques.

Scaffold microarchitecture regulates angiogenesis and the regeneration of large bone defects.

Emerging 3D printing technologies can provide exquisite control over the external shape and internal architecture of scaffolds and tissue engineering (TE) constructs, enabling systematic studies to explore how geometric design features influence the regenerative process. Here we used fused deposition modelling (FDM) and melt electrowriting (MEW) to investigate how scaffold microarchitecture influences the healing of large bone defects. FDM was used to fabricate scaffolds with relatively large fibre diameters and low porosities, while MEW was used to fabricate scaffolds with smaller fibre diameters and higher porosities, with both scaffolds being designed to have comparable surface areas. Scaffold microarchitecture significantly influenced the healing response following implantation into critically sized femoral defects in rats, with the FDM scaffolds supporting the formation of larger bone spicules through its pores, while the MEW scaffolds supported the formation of a more round bone front during healing. After 12 weeksin vivo, both MEW and FDM scaffolds supported significantly higher levels of defect vascularisation compared to empty controls, while the MEW scaffolds supported higher levels of new bone formation. Somewhat surprisingly, this superior healing in the MEW group did not correlate with higher levels of angiogenesis, with the FDM scaffold supporting greater total vessel formation and the formation of larger vessels, while the MEW scaffold promoted the formation of a dense microvasculature with minimal evidence of larger vessels infiltrating the defect region. To conclude, the small fibre diameter, high porosity and high specific surface area of the MEW scaffold proved beneficial for osteogenesis and bone regeneration, demonstrating that changes in scaffold architecture enabled by this additive manufacturing technique can dramatically modulate angiogenesis and tissue regeneration without the need for complex exogenous growth factors. These results provide a valuable insight into the importance of 3D printed scaffold architecture when developing new bone TE strategies.

An ECM-Mimetic Hydrogel to Promote the Therapeutic Efficacy of Osteoblast-Derived Extracellular Vesicles for Bone Regeneration.

The use of extracellular vesicles (EVs) is emerging as a promising acellular approach for bone regeneration, overcoming translational hurdles associated with cell-based therapies. Despite their potential, EVs short half-life following systemic administration hinders their therapeutic efficacy. EVs have been reported to bind to extracellular matrix (ECM) proteins and play an essential role in matrix mineralisation. Chitosan and collagen type I are naturally-derived pro-osteogenic biomaterials, which have been demonstrated to control EV release kinetics. Therefore, this study aimed to develop an injectable ECM-mimetic hydrogel capable of controlling the release of osteoblast-derived EVs to promote bone repair. Pure chitosan hydrogels significantly enhanced compressive modulus (2.48-fold) and osteogenic differentiation (3.07-fold), whilst reducing gelation times (2.09-fold) and proliferation (2.7-fold) compared to pure collagen gels (p ≤ 0.001). EV release was strongly associated with collagen concentration (R2 > 0.94), where a significantly increased EV release profile was observed from chitosan containing gels using the CD63 ELISA (p ≤ 0.001). Hydrogel-released EVs enhanced human bone marrow stromal cells (hBMSCs) proliferation (1.12-fold), migration (2.55-fold), and mineralisation (3.25-fold) compared to untreated cells (p ≤ 0.001). Importantly, EV-functionalised chitosan-collagen composites significantly promoted hBMSCs extracellular matrix mineralisation when compared to the EV-free gels in a dose-dependent manner (p ≤ 0.001). Taken together, these findings demonstrate the development of a pro-osteogenic thermosensitive chitosan-collagen hydrogel capable of enhancing the therapeutic efficacy of osteoblast-derived EVs as a novel acellular tool for bone augmentation strategy.

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.

Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair.

Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs' potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.

Hydrostatic pressure promotes chondrogenic differentiation and microvesicle release from human embryonic and bone marrow stem cells.

Mechanical stimulation plays in an important role in regulating stem cell differentiation and their release of extracellular vesicles (EVs). In this study, effects of low magnitude hydrostatic pressure (HP) on the chondrogenic differentiation and microvesicle release from human embryonic stem cells (hESCs) and human bone marrow stem cells (hBMSCs) are examined. hESCs were differentiated into chondroprogenitors and then embedded in fibrin gels and subjected to HP (270 kPa, 1 Hz, 5 days per week). hBMSC pellets were differentiated in chondrogenic media and subjected to the same regime. HP significantly enhanced ACAN expression in hESCs. It also led to a significant increase in DNA content, sGAG content and total sGAG/DNA level in hBMSCs. Furthermore, HP significantly increased microvesicle protein content released from both cell types. These results highlight the benefit of HP bioreactor in promoting chondrogenesis and EV production for cartilage tissue engineering.

Development of a Bone-Mimetic 3D Printed Ti6Al4V Scaffold to Enhance Osteoblast-Derived Extracellular Vesicles' Therapeutic Efficacy for Bone Regeneration.

Extracellular Vesicles (EVs) are considered promising nanoscale therapeutics for bone regeneration. To date, EVs are typically procured from cells on 2D tissue culture plastic, an artificial environment that limits cell growth and does not replicate in situ biochemical or biophysical conditions. This study investigated the potential of 3D printed titanium scaffolds coated with hydroxyapatite to promote the therapeutic efficacy of osteoblast-derived EVs. Ti6Al4V titanium scaffolds with different pore sizes (500 and 1000 µm) and shapes (square and triangle) were fabricated by selective laser melting. A bone-mimetic nano-needle hydroxyapatite (nnHA) coating was then applied. EVs were procured from scaffold-cultured osteoblasts over 2 weeks and vesicle concentration was determined using the CD63 ELISA. Osteogenic differentiation of human bone marrow stromal cells (hBMSCs) following treatment with primed EVs was evaluated by assessing alkaline phosphatase activity, collagen production and calcium deposition. Triangle pore scaffolds significantly increased osteoblast mineralisation (1.5-fold) when compared to square architectures (P ≤ 0.001). Interestingly, EV yield was also significantly enhanced on these higher permeability structures (P ≤ 0.001), in particular (2.2-fold) for the larger pore structures (1000 µm). Furthermore osteoblast-derived EVs isolated from triangular pore scaffolds significantly increased hBMSCs mineralisation when compared to EVs acquired from square pore scaffolds (1.7-fold) and 2D culture (2.2-fold) (P ≤ 0.001). Coating with nnHA significantly improved osteoblast mineralisation (>2.6-fold) and EV production (4.5-fold) when compared to uncoated scaffolds (P ≤ 0.001). Together, these findings demonstrate the potential of harnessing bone-mimetic culture platforms to enhance the production of pro-regenerative EVs as an acellular tool for bone repair.

Functionalization of Electrospun Polycaprolactone Scaffolds with Matrix-Binding Osteocyte-Derived Extracellular Vesicles Promotes Osteoblastic Differentiation and Mineralization.

Synthetic polymeric materials have demonstrated great promise for bone tissue engineering based on their compatibility with a wide array of scaffold-manufacturing techniques, but are limited in terms of the bioactivity when compared to naturally occurring materials. To enhance the regenerative properties of these materials, they are commonly functionalised with bioactive factors to guide growth within the developing tissue. Extracellular matrix vesicles (EVs) play an important role in facilitating endochondral ossification during long bone development and have recently emerged as important mediators of cell-cell communication coordinating bone regeneration, and thus represent an ideal target to enhance the regenerative properties of synthetic scaffolds. Therefore, in this paper we developed tools and protocols to enable the attachment of MLO-Y4 osteocyte-derived EVs onto electrospun polycaprolactone (PCL) scaffolds for bone repair. Initially, we optimize a method for the functionalization of PCL materials with collagen type-1 and fibronectin, inspired by the behaviour of matrix vesicles during endochondral ossification, and demonstrate that this is an effective method for the adhesion of EVs to the material surface. We then used this functionalization process to attach osteogenic EVs, collected from mechanically stimulated MLO-Y4 osteocytes, to collagen-coated electrospun PCL scaffolds. The EV-functionalized scaffold promoted osteogenic differentiation (measured by increased ALP activity) and mineralization of the matrix. In particular, EV-functionalised scaffolds exhibited significant increases in matrix mineralization particularly at earlier time points compared to uncoated and collagen-coated controls. This approach to matrix-based adhesion of EVs provides a mechanism for incorporating vesicle signalling into polyester scaffolds and demonstrates the potential of osteocyte derived EVs to enhance the rate of bone tissue regeneration.

Biofabrication of vasculature in microphysiological models of bone.

Bone contains a dense network of blood vessels that are essential to its homoeostasis, endocrine function, mineral metabolism and regenerative functions. In addition, bone vasculature is implicated in a number of prominent skeletal diseases, and bone has high affinity for metastatic cancers. Despite vasculature being an integral part of bone physiology and pathophysiology, it is often ignored or oversimplified inin vitrobone models. However, 3D physiologically relevant vasculature can now be engineeredin vitro, with microphysiological systems (MPS) increasingly being used as platforms for engineering this physiologically relevant vasculature. In recent years, vascularised models of bone in MPSs systems have been reported in the literature, representing the beginning of a possible technological step change in how bone is modelledin vitro. Vascularised bone MPSs is a subfield of bone research in its nascency, however given the impact of MPSs has had inin vitroorgan modelling, and the crucial role of vasculature to bone physiology, these systems stand to have a substantial impact on bone research. However, engineering vasculature within the specific design restraints of the bone niche is significantly challenging given the different requirements for engineering bone and vasculature. With this in mind, this paper aims to serve as technical guidance for the biofabrication of vascularised bone tissue within MPS devices. We first discuss the key engineering and biological considerations for engineering more physiologically relevant vasculaturein vitrowithin the specific design constraints of the bone niche. We next explore emerging applications of vascularised bone MPSs, and conclude with a discussion on the current status of vascularised bone MPS biofabrication and suggest directions for development of next generation vascularised bone MPSs.

Estrogen withdrawal alters cytoskeletal and primary ciliary dynamics resulting in increased Hedgehog and osteoclastogenic paracrine signalling in osteocytes.

Estrogen deficiency during post-menopausal osteoporosis leads to osteoclastogenesis and bone loss. Increased pro-osteoclastogenic signalling (RANKL/OPG) by osteocytes occurs following estrogen withdrawal (EW) and is associated with impaired focal adhesions (FAs) and a disrupted actin cytoskeleton. RANKL production is mediated by Hedgehog signalling in osteocytes, a signalling pathway associated with the primary cilium, and the ciliary structure is tightly coupled to the cytoskeleton. Therefore, the objective of this study was to investigate the role of the cilium and associated signalling in EW-mediated osteoclastogenic signalling in osteocytes. We report that EW leads to an elongation of the cilium and increase in Hedgehog and osteoclastogenic signalling. Significant trends were identified linking cilia elongation with reductions in cell area and % FA area/cell area, indicating that cilia elongation is associated with disruption of FAs and actin contractility. To verify this, we inhibited FA assembly via αvß3 antagonism and inhibited actin contractility and demonstrated an elongated cilia and increased expression of Hh markers and Rankl expression. Therefore, our results suggest that the EW conditions associated with osteoporosis lead to a disorganisation of αvß3 integrins and reduced actin contractility, which were associated with an elongation of the cilium, activation of the Hh pathway and osteoclastogenic paracrine signalling.

A meta-analysis of the diagnostic accuracy of Hounsfield units on computed topography relative to dual-energy X-ray absorptiometry for the diagnosis of osteoporosis in the spine surgery population.

BACKGROUND: The preoperative identification of osteoporosis in the spine surgery population is of crucial importance. Limitations associated with dual-energy x-ray absorptiometry, such as access and reliability, have prompted the search for alternative methods to diagnose osteoporosis. The Hounsfield Unit(HU), a readily available measure on computed tomography, has garnered considerable attention in recent years as a potential diagnostic tool for reduced bone mineral density. However, the optimal threshold settings for diagnosing osteoporosis have yet to be determined. METHODS: We selected studies that included comparison of the HU(index test) with dual-energy x-ray absorptiometry evaluation(reference test). Data quality was assessed using the standardised QUADAS-2 criteria. Studies were characterised into 3 categories, based on the threshold of the index test used with the goal of obtaining a high sensitivity, high specificity or balanced sensitivity-specificity test. RESULTS: 9 studies were eligible for meta-analysis. In the high specificity group, the pooled sensitivity was 0.652 (95% CI 0.526 - 0.760), specificity 0.795 (95% CI 0.711 - 0.859) and diagnostic odds ratio was 6.652 (95% CI 4.367 - 10.133). In the high sensitivity group, the overall pooled sensitivity was 0.912 (95% CI 0.718 - 0.977), specificity was 0.67 (0.57 - 0.75) and diagnostic odds ratio was 19.424 (5.446 - 69.275). In the balanced sensitivity-specificity group, the overall pooled sensitivity was 0.625 (95% CI 0.504 - 0.732), specificity was 0.914 (0.823 - 0.960) and diagnostic odds ratio was 14.880 (7.521 - 29.440). Considerable heterogeneity existed throughout the analysis. CONCLUSION: In conclusion, the HU is a clinically useful tool to aide in the diagnosis of osteoporosis. However, the heterogeneity seen in this study warrants caution in the interpretation of results. We have demonstrated the impact of differing HU threshold values on the diagnostic ability of this test. We would propose a threshold of 135 HU to diagnose OP. Future work would investigate the optimal HU cut-off to differentiate normal from low bone mineral density.

Primary cilium-mediated MSC mechanotransduction is dependent on Gpr161 regulation of hedgehog signalling.

The benefits of physical loading to skeletal mass are well known. The primary cilium has emerged as an important organelle in bone mechanobiology/mechanotransduction, particularly in mesenchymal stem/stromal cells, yet the molecular mechanisms of cilium mechanotransduction are poorly understood. In this study, we demonstrate that Gpr161 is a mechanoresponsive GPCR, that localises to the cilium, and is involved in fluid shear-induced cAMP signalling and downstream osteogenesis. This Gpr161-mediated mechanotransduction is dependent on IFT88/cilium and may act through adenylyl cyclase 6 (AC6) to regulate cAMP and MSC osteogenesis. Moreover, we demonstrate that Hh signalling is positively associated with osteogenesis and that Hh gene expression is mechanically regulated and required for loading-induced osteogenic differentiation through a mechanism that involves IFT88, Gpr161, AC6, and cAMP. Therefore, we have delineated a molecular mechanism of MSC mechanotransduction which likely occurs at the cilium, leading to MSC osteogenesis, highlighting novel mechanotherapeutic targets to enhance osteogenesis.

Mechanically stimulated osteocytes promote the proliferation and migration of breast cancer cells via a potential CXCL1/2 mechanism.

Bone represents the most common site for breast cancer metastasis. Bone is a highly dynamic organ that is constantly adapting to its biophysical environment, orchestrated largely by the resident osteocyte network. Osteocytes subjected to physiologically relevant biophysical conditions may therefore represent a source of key factors mediating breast cancer cell metastasis to bone. Therefore, we investigated the potential proliferative and migratory capacity of soluble factors released by mechanically stimulated osteocytes on breast cancer cell behaviour. Interestingly the secretome of mechanically stimulated osteocytes enhanced both the proliferation and migration of cancer cells when compared to the secretome of statically cultured osteocytes, demonstrating that mechanical stimuli is an important physiological stimulus that should be considered when identifying potential targets. Using a cytokine array, we further identified a group of mechanically activated cytokines in the osteocyte secretome, which potentially drive breast cancer metastasis. In particular, CXCL1 and CXCL2 cytokines are highly expressed, mechanically regulated, and are known to interact with one another. Lastly, we demonstrate that these specific factors enhance breast cancer cell migration independently and in a synergistic manner, identifying potential osteocyte derived factors mediating breast cancer metastasis to bone.

Loss of Adenylyl Cyclase 6 in Leptin Receptor-Expressing Stromal Cells Attenuates Loading-Induced Endosteal Bone Formation.

Bone marrow stromal/stem cells represent a quiescent cell population that replenish the osteoblast bone-forming cell pool with age and in response to injury, maintaining bone mass and repair. A potent mediator of stromal/stem cell differentiation in vitro and bone formation in vivo is physical loading, yet it still remains unclear whether loading-induced bone formation requires the osteogenic differentiation of these resident stromal/stem cells. Therefore, in this study, we utilized the leptin receptor (LepR) to identify and trace the contribution of bone marrow stromal cells to mechanoadaptation of bone in vivo. Twelve-week-old Lepr-cre;tdTomato mice were subjected to compressive tibia loading with an 11 N peak load for 40 cycles, every other day for 2 weeks. Histological analysis revealed that Lepr-cre;tdTomato+ cells arise perinatally around blood vessels and populate bone surfaces as lining cells or osteoblasts before a percentage undergo osteocytogenesis. Lepr-cre;tdTomato+ stromal cells within the marrow increase in abundance with age, but not following the application of tibial compressive loading. Mechanical loading induces an increase in bone mass and bone formation parameters, yet does not evoke an increase in Lepr-cre;tdTomato+ osteoblasts or osteocytes. To investigate whether adenylyl cyclase-6 (AC6) in LepR cells contributes to this mechanoadaptive response, Lepr-cre;tdTomato mice were further crossed with AC6 fl/fl mice to generate a LepR+ cell-specific knockout of AC6. These Lepr-cre;tdTomato;AC6 fl/fl animals have an attenuated response to compressive tibia loading, characterized by a deficient load-induced osteogenic response on the endosteal bone surface. This, therefore, shows that Lepr-cre;tdTomato+ cells contribute to short-term bone mechanoadaptation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.