Serum amyloid A proteins reduce bone mass during mycobacterial infections.

Introduction: Osteopenia has been associated to several inflammatory conditions, including mycobacterial infections. How mycobacteria cause bone loss remains elusive, but direct bone infection may not be required. Methods: Genetically engineered mice and morphometric, transcriptomic, and functional analyses were used. Additionally, inflammatory mediators and bone turnover markers were measured in the serum of healthy controls, individuals with latent tuberculosis and patients with active tuberculosis. Results and discussion: We found that infection with Mycobacterium avium impacts bone turnover by decreasing bone formation and increasing bone resorption, in an IFNγ- and TNFα-dependent manner. IFNγ produced during infection enhanced macrophage TNFα secretion, which in turn increased the production of serum amyloid A (SAA) 3. Saa3 expression was upregulated in the bone of both M. avium- and M. tuberculosis-infected mice and SAA1 and 2 proteins (that share a high homology with murine SAA3 protein) were increased in the serum of patients with active tuberculosis. Furthermore, the increased SAA levels seen in active tuberculosis patients correlated with altered serum bone turnover markers. Additionally, human SAA proteins impaired bone matrix deposition and increased osteoclastogenesis in vitro. Overall, we report a novel crosstalk between the cytokine-SAA network operating in macrophages and bone homeostasis. These findings contribute to a better understanding of the mechanisms of bone loss during infection and open the way to pharmacological intervention. Additionally, our data and disclose SAA proteins as potential biomarkers of bone loss during infection by mycobacteria.

The Secretome of Parental and Bone Metastatic Breast Cancer Elicits Distinct Effects in Human Osteoclast Activity after Activation of ß2 Adrenergic Signaling.

The sympathetic nervous system (SNS), particularly through the ß2 adrenergic receptor (ß2-AR), has been linked with breast cancer (BC) and the development of metastatic BC, specifically in the bone. Nevertheless, the potential clinical benefits of exploiting ß2-AR antagonists as a treatment for BC and bone loss-associated symptoms remain controversial. In this work, we show that, when compared to control individuals, the epinephrine levels in a cohort of BC patients are augmented in both earlier and late stages of the disease. Furthermore, through a combination of proteomic profiling and functional in vitro studies with human osteoclasts and osteoblasts, we demonstrate that paracrine signaling from parental BC under ß2-AR activation causes a robust decrease in human osteoclast differentiation and resorption activity, which is rescued in the presence of human osteoblasts. Conversely, metastatic bone tropic BC does not display this anti-osteoclastogenic effect. In conclusion, the observed changes in the proteomic profile of BC cells under ß-AR activation that take place after metastatic dissemination, together with clinical data on epinephrine levels in BC patients, provided new insights on the sympathetic control of breast cancer and its implications on osteoclastic bone resorption.

Profiling the Adrenergic System in Breast Cancer and the Development of Metastasis.

Epidemiological studies and preclinical models suggest that chronic stress might accelerate breast cancer (BC) growth and the development of metastasis via sympathetic neural mechanisms. Nevertheless, the role of each adrenergic pathway (α1, α2, and ß) in human samples remains poorly depicted. Herein, we propose to characterize the profile of the sympathetic system (e.g., release of catecholamines, expression of catecholamine metabolic enzymes and adrenoreceptors) in BC patients, and ascertain its relevance in the development of distant metastasis. Our results demonstrated that BC patients exhibited increased plasma levels of catecholamines when compared with healthy donors, and this increase was more evident in BC patients with distant metastasis. Our analysis using the BC-TCGA database revealed that the genes coding the most expressed adrenoreceptors in breast tissues (ADRA2A, ADRA2C, and ADRB2, by order of expression) as well as the catecholamine synthesizing (PNMT) and degrading enzyme (MAO-A and MAO-B) genes were downregulated in BC tissues. Importantly, the expression of ADRA2A, ADRA2C, and ADRB2 was correlated with metastatic BC and BC subtypes, and thus the prognosis of the disease. Overall, we gathered evidence that under stressful conditions, both the α2- and ß2-signaling pathways might work on a synergetic matter, thus paving the way for the development of new therapeutic approaches.

Osteoclast-derived extracellular vesicles are implicated in sensory neurons sprouting through the activation of epidermal growth factor signaling.

BACKGROUND: Different pathologies, affecting the skeletal system, were reported to display altered bone and/or cartilage innervation profiles leading to the deregulation of the tissue homeostasis. The patterning of peripheral innervation is achieved through the tissue-specific expression of attractive or repulsive axonal guidance cues in specific space and time frames. During the last decade, emerging findings attributed to the extracellular vesicles (EV) trading a central role in peripheral tissue innervation. However, to date, the contribution of EV in controlling bone innervation is totally unknown. RESULTS: Here we show that sensory neurons outgrowth induced by the bone resorbing cells-osteoclasts-is promoted by osteoclast-derived EV. The EV induced axonal growth is achieved by targeting epidermal growth factor receptor (EGFR)/ErbB2 signaling/protein kinase C phosphorylation in sensory neurons. In addition, our data also indicate that osteoclasts promote sensory neurons electrophysiological activity reflecting a possible pathway in nerve sensitization in the bone microenvironment, however this effect is EV independent. CONCLUSIONS: Overall, these results identify a new mechanism of sensory bone innervation regulation and shed the light on the role of osteoclast-derived EV in shaping/guiding bone sensory innervation. These findings provide opportunities for exploitation of osteoclast-derived EV based strategies to prevent and/or mitigate pathological uncontrolled bone innervation.

Advances in nanoenabled 3D matrices for cartilage repair.

Cartilage repair strategies are evolving at a fast pace with technology development. Matrices that offer multifaceted functions and a full adaption to the cartilage defect are of pivotal interest. Current cartilage repair strategies face numerous challenges, mostly related to the development of highly biomimetic materials, non-invasive injectable solutions, and adequate degradation rates. These strategies often fail due to feeble mechanical properties, the inability to sustain cell adhesion, growth, and differentiation or by underestimating other players of cartilage degeneration, such as the installed pro-inflammatory microenvironment. The integration of nanomaterials (NMs) into 3D scaffolds, hydrogels and bioinks hold great potential in the improvement of key features of materials that are currently applied in cartilage tissue engineering. NMs offer a high surface to volume ratio and their multiple applications can be explored to enhance cartilage mechanical properties, biocompatibility, cell differentiation, inflammation modulation, infection prevention and even to function as diagnostic tools or as stimuli-responsive cues in these 3D structures. In this review, we have critically reviewed the latest advances in the development of nanoenabled 3D matrices - enhanced by means of NMs - in the context of cartilage regeneration. We have provided a wide perspective of the synergistic effect of combining 3D strategies with NMs, with emphasis on the benefits brought by NMs in achieving functional and enhanced therapeutic outcomes. STATEMENT OF SIGNIFICANCE: Cartilage is one of the most challenging tissues to treat owing to its limited self-regeneration potential. Novel strategies using nanoenabled 3D matrices have emerged from the need to design more efficient solutions for cartilage repair, that take into consideration its unique mechanical properties and can direct specific cell behaviours. Here we aim to provide a comprehensive review on the synergistic effects of 3D matrices nanoenrichment in the context of cartilage regeneration, with emphasis on the heightening brought by nanomaterials in achieving functional and enhanced therapeutic outcomes. We anticipate this review to provide a wide perspective on the past years' research on the field, demonstrating the great potential of these approaches in the treatment and diagnosis of cartilage-related disorders.

Stress in Metastatic Breast Cancer: To the Bone and Beyond.

Breast cancer (BRCA) remains as one the most prevalent cancers diagnosed in industrialised countries. Although the overall survival rate is high, the dissemination of BRCA cells to distant organs correlates with a significantly poor prognosis. This is due to the fact that there are no efficient therapeutic strategies designed to overcome the progression of the metastasis. Over the past decade, critical associations between stress and the prevalence of BRCA metastases were uncovered. Chronic stress and the concomitant sympathetic hyperactivation have been shown to accelerate the progression of the disease and the metastases incidence, specifically to the bone. In this review, we provide a summary of the sympathetic profile on BRCA. Additionally, the current knowledge regarding the sympathetic hyperactivity, and the underlying adrenergic signalling pathways, involved on the development of BRCA metastasis to distant organs (i.e., bone, lung, liver and brain) will be revealed. Since bone is a preferential target site for BRCA metastases, greater emphasis will be given to the contribution of α2- and ß-adrenergic signalling in BRCA bone tropism and the occurrence of osteolytic lesions.

Cutting-Edge Technologies for Inflamed Joints on Chip: How Close Are We?

Osteoarthritis (OA) is a painful and disabling musculoskeletal disorder, with a large impact on the global population, resulting in several limitations on daily activities. In OA, inflammation is frequent and mainly controlled through inflammatory cytokines released by immune cells. These outbalanced inflammatory cytokines cause cartilage extracellular matrix (ECM) degradation and possible growth of neuronal fibers into subchondral bone triggering pain. Even though pain is the major symptom of musculoskeletal diseases, there are still no effective treatments to counteract it and the mechanisms behind these pathologies are not fully understood. Thus, there is an urgent need to establish reliable models for assessing the molecular mechanisms and consequently new therapeutic targets. Models have been established to support this research field by providing reliable tools to replicate the joint tissue in vitro. Studies firstly started with simple 2D culture setups, followed by 3D culture focusing mainly on cell-cell interactions to mimic healthy and inflamed cartilage. Cellular approaches were improved by scaffold-based strategies to enhance cell-matrix interactions as well as contribute to developing mechanically more stable in vitro models. The progression of the cartilage tissue engineering would then profit from the integration of 3D bioprinting technologies as these provide 3D constructs with versatile structural arrangements of the 3D constructs. The upgrade of the available tools with dynamic conditions was then achieved using bioreactors and fluid systems. Finally, the organ-on-a-chip encloses all the state of the art on cartilage tissue engineering by incorporation of different microenvironments, cells and stimuli and pave the way to potentially simulate crucial biological, chemical, and mechanical features of arthritic joint. In this review, we describe the several available tools ranging from simple cartilage pellets to complex organ-on-a-chip platforms, including 3D tissue-engineered constructs and bioprinting tools. Moreover, we provide a fruitful discussion on the possible upgrades to enhance the in vitro systems making them more robust regarding the physiological and pathological modeling of the joint tissue/OA.

The Neuroimmune Interplay in Joint Pain: The Role of Macrophages.

Chronic pain associated with joint disorders, such as rheumatoid arthritis (RA), osteoarthritis (OA) and implant aseptic loosening (AL), is a highly debilitating symptom that impacts mobility and quality of life in affected patients. The neuroimmune crosstalk has been demonstrated to play a critical role in the onset and establishment of chronic pain conditions. Immune cells release cytokines and immune mediators that can activate and sensitize nociceptors evoking pain, through interaction with receptors in the sensory nerve terminals. On the other hand, sensory and sympathetic nerve fibers release neurotransmitters that bind to their specific receptor expressed on surface of immune cells, initiating an immunomodulatory role. Macrophages have been shown to be key players in the neuroimmune crosstalk. Moreover, macrophages constitute the dominant immune cell population in RA, OA and AL. Importantly, the targeting of macrophages can result in anti-nociceptive effects in chronic pain conditions. Therefore, the aim of this review is to discuss the nature and impact of the interaction between the inflammatory response and nerve fibers in these joint disorders regarding the genesis and maintenance of pain. The role of macrophages is highlighted. The alteration in the joint innervation pattern and the inflammatory response are also described. Additionally, the immunomodulatory role of sensory and sympathetic neurotransmitters is revised.

A metastasis-on-a-chip approach to explore the sympathetic modulation of breast cancer bone metastasis.

Organ-on-a-chip models have emerged as a powerful tool to model cancer metastasis and to decipher specific crosstalk between cancer cells and relevant regulators of this particular niche. Recently, the sympathetic nervous system (SNS) was proposed as an important modulator of breast cancer bone metastasis. However, epidemiological studies concerning the benefits of the SNS targeting drugs on breast cancer survival and recurrence remain controversial. Thus, the role of SNS signaling over bone metastatic cancer cellular processes still requires further clarification. Herein, we present a novel humanized organ-on-a-chip model recapitulating neuro-breast cancer crosstalk in a bone metastatic context. We developed and validated an innovative three-dimensional printing based multi-compartment microfluidic platform, allowing both selective and dynamic multicellular paracrine signaling between sympathetic neurons, bone tropic breast cancer cells and osteoclasts. The selective multicellular crosstalk in combination with biochemical, microscopic and proteomic profiling show that synergistic paracrine signaling from sympathetic neurons and osteoclasts increase breast cancer aggressiveness demonstrated by augmented levels of pro-inflammatory cytokines (e.g. interleukin-6 and macrophage inflammatory protein 1α). Overall, this work introduced a novel and versatile platform that could potentially be used to unravel new mechanisms involved in intracellular communication at the bone metastatic niche.

Micropathological Chip Modeling the Neurovascular Unit Response to Inflammatory Bone Condition.

Organ-on-a-chip in vitro platforms accurately mimic complex microenvironments offering the ability to recapitulate and dissect mechanisms of physiological and pathological settings, revealing their major importance to develop new therapeutic targets. Bone diseases, such as osteoarthritis, are extremely complex, comprising of the action of inflammatory mediators leading to unbalanced bone homeostasis and de-regulation of sensory innervation and angiogenesis. Although there are models to mimic bone vascularization or innervation, in vitro platforms merging the complexity of bone, vasculature, innervation, and inflammation are missing. Therefore, in this study a microfluidic-based neuro-vascularized bone chip (NVB chip) is proposed to 1) model the mechanistic interactions between innervation and angiogenesis in the inflammatory bone niche, and 2) explore, as a screening tool, novel strategies targeting inflammatory diseases, using a nano-based drug delivery system. It is possible to set the design of the platform and achieve the optimized conditions to address the neurovascular network under inflammation. Moreover, this system is validated by delivering anti-inflammatory drug-loaded nanoparticles to counteract the neuronal growth associated with pain perception. This reliable in vitro tool will allow understanding the bone neurovascular system, enlightening novel mechanisms behind the inflammatory bone diseases, bone destruction, and pain opening new avenues for new therapies discovery.

Calcium Signalling in Breast Cancer Associated Bone Pain.

Calcium (Ca2+) is involved as a signalling mediator in a broad variety of physiological processes. Some of the fastest responses in human body like neuronal action potential firing, to the slowest gene transcriptional regulation processes are controlled by pathways involving calcium signalling. Under pathological conditions these mechanisms are also involved in tumoral cells reprogramming, resulting in the altered expression of genes associated with cell proliferation, metastatisation and homing to the secondary metastatic site. On the other hand, calcium exerts a central function in nociception, from cues sensing in distal neurons, to signal modulation and interpretation in the central nervous system leading, in pathological conditions, to hyperalgesia, allodynia and pain chronicization. It is well known the relationship between cancer and pain when tumoral metastatic cells settle in the bones, especially in late breast cancer stage, where they alter the bone micro-environment leading to bone lesions and resulting in pain refractory to the conventional analgesic therapies. The purpose of this review is to address the Ca2+ signalling mechanisms involved in cancer cell metastatisation as well as the function of the same signalling tools in pain regulation and transmission. Finally, the possible interactions between these two cells types cohabiting the same Ca2+ rich environment will be further explored attempting to highlight new possible therapeutical targets.

Polymeric Microspheres/Cells/Extracellular Matrix Constructs Produced by Auto-Assembly for Bone Modular Tissue Engineering.

Modular tissue engineering (MTE) is a novel "bottom-up" approach to create engineered biological tissues from microscale repeating units. Our aim was to obtain microtissue constructs, based on polymer microspheres (MSs) populated with cells, which can be further assembled into larger tissue blocks and used in bone MTE. Poly(L-lactide-co-glycolide) MS of 165 ± 47 µm in diameter were produced by oil-in-water emulsification and treated with 0.1 M NaOH. To improve cell adhesion, MSs were coated with poly-L-lysine (PLL) or human recombinant collagen type I (COL). The presence of oxygenated functionalities and PLL/COL coating on MS was confirmed by X-ray photoelectron spectroscopy (XPS). To assess the influence of medium composition on adhesion, proliferation, and osteogenic differentiation, preosteoblast MC3T3-E1 cells were cultured on MS in minimal essential medium (MEM) and osteogenic differentiation medium (OSG). Moreover, to assess the potential osteoblast-osteoclast cross-talk phenomenon and the influence of signaling molecules released by osteoclasts on osteoblast cell culture, a medium obtained from osteoclast culture (OSC) was also used. To impel the cells to adhere and grow on the MS, anti-adhesive cell culture plates were utilized. The results show that MS coated with PLL and COL significantly favor the adhesion and growth of MC3T3-E1 cells on days 1 and 7, respectively, in all experimental conditions tested. On day 7, three-dimensional MS/cell/extracellular matrix constructs were created owing to auto-assembly. The cells grown in such constructs exhibited high activity of early osteogenic differentiation marker, namely, alkaline phosphatase. Superior cell growth on PLL- and COL-coated MS on day 14 was observed in the OSG medium. Interestingly, deposition of extracellular matrix and its mineralization was particularly enhanced on COL-coated MS in OSG medium on day 14. In our study, we developed a method of spontaneous formation of organoid-like MS-based cell/ECM constructs with a few millimeters in size. Such constructs may be regarded as building blocks in bone MTE.

Sympathetic activity in breast cancer and metastasis: partners in crime.

The vast majority of patients with advanced breast cancer present skeletal complications that severely compromise their quality of life. Breast cancer cells are characterized by a strong tropism to the bone niche. After engraftment and colonization of bone, breast cancer cells interact with native bone cells to hinder the normal bone remodeling process and establish an osteolytic "metastatic vicious cycle". The sympathetic nervous system has emerged in recent years as an important modulator of breast cancer progression and metastasis, potentiating and accelerating the onset of the vicious cycle and leading to extensive bone degradation. Furthermore, sympathetic neurotransmitters and their cognate receptors have been shown to promote several hallmarks of breast cancer, such as proliferation, angiogenesis, immune escape, and invasion of the extracellular matrix. In this review, we assembled the current knowledge concerning the complex interactions that take place in the tumor microenvironment, with a special emphasis on sympathetic modulation of breast cancer cells and stromal cells. Notably, the differential action of epinephrine and norepinephrine, through either α- or ß-adrenergic receptors, on breast cancer progression prompts careful consideration when designing new therapeutic options. In addition, the contribution of sympathetic innervation to the formation of bone metastatic foci is highlighted. In particular, we address the remarkable ability of adrenergic signaling to condition the native bone remodeling process and modulate the bone vasculature, driving breast cancer cell engraftment in the bone niche. Finally, clinical perspectives and developments on the use of ß-adrenergic receptor inhibitors for breast cancer management and treatment are discussed.

Microfluidic-based models to address the bone marrow metastatic niche complexity.

Bone marrow (BM) is a preferential metastatic site for solid cancers, contributing to higher morbidity and mortality among millions of oncologic patients worldwide. There are no current efficient therapies to minimize this health burden. Microfluidic based in vitro models emerge as powerful alternatives to animal testing, as well as promising tools for the development of personalized medicine solutions. The complexity associated with the BM metastatic niche originated a wide variety of microfluidic platforms designed to mimic this microenvironment. This review gathers the essential parameters to design an accurate in vitro microfluidic device, based on a comparative analysis of existing models created to address the different steps of the metastatic cascade.

Fluorescent H2 Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications.

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH2R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27) was synthesized and characterized. The highest hH2R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pK i: 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pK i: 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pK d values comparable to the pK i values. The fluorescent probes 21, 23, and 25 could be used to localize H2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds.

Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometry.

Neuropeptide Y (NPY) is a peptide widely distributed throughout the body that is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. 5-Carbamimidamido-2-(2,2-diphenylacetamido)-N-[(4-hydroxyphenyl)methyl]pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized application in bone regeneration studies, requiring quantification at picogram levels. Hence, BIBP 3226 determination is proposed here by a validated HPLC-MS/MS method, based on a reversed-phase Kinetex® core-shell C8 column (2.6 µm, 150 × 2.1 mm) at 30 °C, elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25 mL min-1, detection in positive ionization mode, and data acquisition in selected reaction monitoring mode. Calibration curves were linear for concentrations ranging from 0.25 to 30 ng mL-1 with LOD and LOQ values as low as 0.1 and 0.3 pg in cell extracts and 16 and 48 pg in supernatant culture media, respectively. BIBP 3226 was successfully determined in cell extracts and supernatants obtained from internalization assays. Using similar exposure conditions, the amount of BIBP 3226 found in breast cancer cells (MCF7) was 72 to 657 times higher than that found in bone marrow cells (Wt C57BL/6 mice), providing an indirect indicator of NPY Y1 receptor expression.

Evaluation of Nisin and LL-37 Antimicrobial Peptides as Tool to Preserve Articular Cartilage Healing in a Septic Environment.

Cartilage repair still represents a challenge for clinicians and only few effective therapies are nowadays available. In fact, surgery is limited by the tissue poor self-healing capacity while the autologous transplantation is often forsaken due to the poor in vitro expansion capacity of chondrocytes. Biomaterials science offers a unique alternative based on the replacement of the injured tissue with an artificial tissue-mimicking scaffold. However, the implantation surgical practices and the scaffold itself can be a source of bacterial infection that currently represents the first reason of implants failure due to the increasing antibiotics resistance of pathogens. So, alternative antibacterial tools to prevent infections and consequent device removal are urgently required. In this work, the role of Nisin and LL-37 peptides has been investigated as alternative to antibiotics to their antimicrobial performances for direct application at the surgical site or as doping chemicals for devices aimed at articular cartilage repair. First, peptides cytocompatibility was investigated toward human mesenchymal stem cells to determine safe concentrations; then, the broad-range antibacterial activity was verified toward the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis as well as the Gram-negative Escherichia coli and Aggregatibacter actinomycetemcomitans pathogens. The peptides selective antibacterial activity was verified by a cells-bacteria co-culture assay, while chondrogenesis was assayed to exclude any interference within the differentiation route to simulate the tissue repair. In the next phase, the experiments were repeated by moving from the cell monolayer model to 3D cartilage-like spheroids to revisit the peptides activity in a more physiologically relevant environment model. Finally, the spheroid model was applied in a perfusion bioreactor to simulate an infection in the presence of circulating peptides within a physiological environment. Results suggested that 75 µg/ml Nisin can be considered as a very promising candidate since it was shown to be more cytocompatible and potent against the investigated bacteria than LL-37 in all the tested models.

Osteoblasts are inherently programmed to repel sensory innervation.

Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation.

Human dental pulp stem cells exhibit enhanced properties in comparison to human bone marrow stem cells on neurites outgrowth.

Mesenchymal stem cells (MSCs) have the capacity to self-renew and differentiate into specific cell types and are, therefore, key players during tissue repair and regeneration. The use of MSCs for the regeneration of tissues in vivo is increasingly being explored and already constitutes a promising alternative to existing clinical treatments. MSCs also exert paracrine and trophic functions, including the promotion of innervation that plays fundamental roles in regeneration and in restoration of the function of organs. Human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been used in studies that aimed at the repair and/or regeneration of bone or other tissues of the craniofacial complex. However, the capabilities of hBMSCs and hDPSCs to elicit the growth of specific axons in order to reestablish functional innervation of the healing tissues are not known. Here, we compared the neurotrophic effects of hDPSCs and hBMSCs on trigeminal and dorsal root ganglia neurons using microfluidic organs-on-chips devices. We found that hDPSCs express significantly higher levels of neurotrophins than hBMSCs and consequently neurons cocultured with hDPSCs develop longer axons in the microfluidic co-culture system when compared to neurons cocultured with hBMSCs. Moreover, hDPSCs elicited the formation of extensive axonal networks and established close contacts with neurons, a phenomenon not observed in presence of hBMSCs. Taken together, these findings indicate that hDPSCs constitute a superior option for restoring the functionality of damaged craniofacial tissues, as they are able to support and promote extensive trigeminal innervation.

The lack of neuropeptide Y-Y1 receptor signaling modulates the chemical and mechanical properties of bone matrix.

Genetic and pharmacological functional studies have provided evidence that the lack of Neuropeptide Y-Y1  receptor (Y1 R) signaling pathway induces a high bone mass phenotype in mice. However, clinical observations have shown that drug or genetic mediated improvement of bone mass might be associated to alterations to bone extracellular matrix (ECM) properties, leading to bone fragility. Hence, in this study we propose to characterize the physical, chemical and biomechanical properties of mature bone ECM of germline NPY-Y1 R knockout (Y1 R-/- ) mice, and compare to their wild-type (WT) littermates. Our results demonstrated that the high bone mass phenotype observed in Y1 R-/- mice involves alterations in Y1 R-/-  bone ECM ultrastructure, as a result of accelerated deposition of organic and mineral fractions. In addition, Y1 R-/- bone ECM displays enhanced matrix maturation characterized by greater number of mature/highly packed collagen fibers without pathological accumulation of immature/mature collagen crosslinks nor compromise of mineral crystallinity. These unique features of Y1 R-/-  bone ECM improved the biochemical properties of Y1 R-/-  bones, reflected by mechanically robust bones with diminished propensity to fracture, contributing to greater bone strength. These findings support the future usage of drugs targeting Y1 R signaling as a promising therapeutic strategy to treat bone loss-related pathologies.