Engineering a Microphysiological Model for Regenerative Endodontic Studies.

Dental pulp infections are common buccal diseases. When this happens, endodontic treatments are needed to disinfect and prepare the root canal for subsequent procedures. However, the lack of suitable in vitro models representing the anatomy of an immature root canal hinders research on regenerative events crucial in endodontics, such as regenerative procedures. This study aimed to develop a 3D microphysiological system (MPS) to mimic an immature root canal and assess the cytotoxicity of various irrigating solutions on stem cells. Utilizing the Dental Stem Cells SV40 (DSCS) cell line derived from human apical papilla stem cells, we analyzed the effects of different irrigants, including etidronic acid. The results indicated that irrigating solutions diminished cell viability in 2D cultures and influenced cell adhesion within the microphysiological device. Notably, in our 3D studies in the MPS, 17% EDTA and 9% 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) irrigating solutions demonstrated superior outcomes in terms of DSCS viability and adherence compared to the control. This study highlights the utility of the developed MPS for translational studies in root canal treatments and suggests comparable efficacy between 9% HEBP and 17% EDTA irrigating solutions, offering potential alternatives for clinical applications.

Microphysiological model reveals the promise of memory-like natural killer cell immunotherapy for HIV± cancer.

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

Inhibition of C5AR1 impairs osteoclast mobilization and prevents bone loss.

Age-related and chemotherapy-induced bone loss depends on cellular senescence and the cell secretory phenotype. However, the factors secreted in the senescent microenvironment that contribute to bone loss remain elusive. Here, we report a central role for the inflammatory alternative complement system in skeletal bone loss. Through transcriptomic analysis of bone samples, we identified complement factor D, a rate-limiting factor of the alternative pathway of complement, which is among the most responsive factors to chemotherapy or estrogen deficiency. We show that osteoblasts and osteocytes are major inducers of complement activation, while monocytes and osteoclasts are their primary targets. Genetic deletion of C5ar1, the receptor of the anaphylatoxin C5a, or treatment with a C5AR1 inhibitor reduced monocyte chemotaxis and osteoclast differentiation. Moreover, genetic deficiency or inhibition of C5AR1 partially prevented bone loss and osteoclastogenesis upon chemotherapy or ovariectomy. Altogether, these lines of evidence support the idea that inhibition of alternative complement pathways may have some therapeutic benefit in osteopenic disorders.

Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure.

The aging-protective gene α-Klotho (KL) produces two main transcripts. The full-length mRNA generates a transmembrane protein that after proteolytic ectodomain shedding can be detected in serum as processed Klotho (p-KL), and a shorter transcript which codes for a putatively secreted protein (s-KL). Both isoforms exhibit potent pleiotropic beneficial properties, although previous reports showed negative side effects on mineral homeostasis after increasing p-KL concentration exogenously. Here, we expressed independently both isoforms using gene transfer vectors, to assess s-KL effects on mineral metabolism. While mice treated with p-KL presented altered expression of several kidney ion channels, as well as altered levels of Pi and Ca2+ in blood, s-KL treated mice had levels comparable to Null-treated control mice. Besides, bone gene expression of Fgf23 showed a fourfold increase after p-KL treatment, effects not observed with the s-KL isoform. Similarly, bone microstructure parameters of p-KL-treated mice were significantly worse than in control animals, while this was not observed for s-KL, which showed an unexpected increase in trabecular thickness and cortical mineral density. As a conclusion, s-KL (but not p-KL) is a safe therapeutic strategy to exploit KL anti-aging protective effects, presenting no apparent negative effects over mineral metabolism and bone microstructure.

HERC1 deficiency causes osteopenia through transcriptional program dysregulation during bone remodeling.

Bone remodeling is a continuous process between bone-forming osteoblasts and bone-resorbing osteoclasts, with any imbalance resulting in metabolic bone disease, including osteopenia. The HERC1 gene encodes an E3 ubiquitin ligase that affects cellular processes by regulating the ubiquitination of target proteins, such as C-RAF. Of interest, an association exists between biallelic pathogenic sequence variants in the HERC1 gene and the neurodevelopmental disorder MDFPMR syndrome (macrocephaly, dysmorphic facies, and psychomotor retardation). Most pathogenic variants cause loss of HERC1 function, and the affected individuals present with features related to altered bone homeostasis. Herc1-knockout mice offer an excellent model in which to study the role of HERC1 in bone remodeling and to understand its role in disease. In this study, we show that HERC1 regulates osteoblastogenesis and osteoclastogenesis, proving that its depletion increases gene expression of osteoblastic makers during the osteogenic differentiation of mesenchymal stem cells. During this process, HERC1 deficiency increases the levels of C-RAF and of phosphorylated ERK and p38. The Herc1-knockout adult mice developed imbalanced bone homeostasis that presented as osteopenia in both sexes of the adult mice. By contrast, only young female knockout mice had osteopenia and increased number of osteoclasts, with the changes associated with reductions in testosterone and dihydrotestosterone levels. Finally, osteocytes isolated from knockout mice showed a higher expression of osteocytic genes and an increase in the Rankl/Opg ratio, indicating a relevant cell-autonomous role of HERC1 when regulating the transcriptional program of bone formation. Overall, these findings present HERC1 as a modulator of bone homeostasis and highlight potential therapeutic targets for individuals affected by pathological HERC1 variants.

Dental Stem Cells SV40, a new cell line developed in vitro from human stem cells of the apical papilla.

AIM: To establish and fully characterize a new cell line from human stem cells of the apical papilla (SCAPs) through immortalization with an SV40 large T antigen. METHODOLOGY: Human SCAPs were isolated and transfected with an SV40 large T antigen and treated with puromycin to select the infected population. Expression of human mesenchymal surface markers CD73, CD90 and CD105 was assessed in the new cell line named Dental Stem Cells SV40 (DSCS) by flow cytometry at early and late passages. Cell contact inhibition and proliferation were also analysed. To evaluate trilineage differentiation, quantitative polymerase chain reaction and histological staining were performed. RESULTS: DSCS cell flow cytometry confirmed the expression of mesenchymal surface markers even in late passages [100% positive for CD73 and CD90 and 98.9% for CD105 at passage (P) 25]. Fewer than 0.5% were positive for haematopoietic cell markers (CD45 and CD34). DSCS cells also showed increased proliferation when compared to the primary culture after 48 h, with a doubling time of 23.46 h for DSCS cells and 40.31 h for SCAPs, and retained the capacity to grow for >45 passages (150 population doubling) and their spindle-shaped morphology. Trilineage differentiation potential was confirmed through histochemical staining and gene expression of the chondrogenic markers SOX9 and COL2A1, adipogenic markers CEBPA and LPL, and osteogenic markers COL1A1 and ALPL. CONCLUSIONS: The new cell line derived from human SCAPs has multipotency, retains its morphology and expression of mesenchymal surface markers and shows higher proliferative capacity even at late passages (P45). DSCS cells can be used for in vitro study of root development and to achieve a better understanding of the regenerative mechanisms.

Microfluidics in vascular biology research: a critical review for engineers, biologists, and clinicians.

Neovascularization, the formation of new blood vessels, has received much research attention due to its implications for physiological processes and diseases. Most studies using traditional in vitro and in vivo platforms find challenges in recapitulating key cellular and mechanical cues of the neovascularization processes. Microfluidic in vitro models have been presented as an alternative to these limitations due to their capacity to leverage microscale physics to control cell organization and integrate biochemical and mechanical cues, such as shear stress, cell-cell interactions, or nutrient gradients, making them an ideal option for recapitulating organ physiology. Much has been written about the use of microfluidics in vascular biology models from an engineering perspective. However, a review introducing the different models, components and progress for new potential adopters of these technologies was absent in the literature. Therefore, this paper aims to approach the use of microfluidic technologies in vascular biology from a perspective of biological hallmarks to be studied and written for a wide audience ranging from clinicians to engineers. Here we review applications of microfluidics in vascular biology research, starting with design considerations and fabrication techniques. After that, we review the state of the art in recapitulating angiogenesis and vasculogenesis, according to the hallmarks recapitulated and complexity of the models. Finally, we discuss emerging research areas in neovascularization, such as drug discovery, and potential future directions.

Advancing Treatment of Bone Metastases through Novel Translational Approaches Targeting the Bone Microenvironment.

Bone metastases represent a lethal condition that frequently occurs in solid tumors such as prostate, breast, lung, and renal cell carcinomas, and increase the risk of skeletal-related events (SREs) including pain, pathologic fractures, and spinal cord compression. This unique metastatic niche consists of a multicellular complex that cancer cells co-opt to engender bone remodeling, immune suppression, and stromal-mediated therapeutic resistance. This review comprehensively discusses clinical challenges of bone metastases, novel preclinical models of the bone and bone marrow microenviroment, and crucial signaling pathways active in bone homeostasis and metastatic niche. These studies establish the context to summarize the current state of investigational agents targeting BM, and approaches to improve BM-targeting therapies. Finally, we discuss opportunities to advance research in bone and bone marrow microenvironments by increasing complexity of humanized preclinical models and fostering interdisciplinary collaborations to translational research in this challenging metastatic niche.

The Expression of TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Can Be Controlled by the Antioxidant Orchestrator NRF2 in Human Carcinoma Cells.

Hyperactivation of the KEAP1-NRF2 axis is a common molecular trait in carcinomas from different origin. The transcriptional program induced by NRF2 involves antioxidant and metabolic genes that render cancer cells more capable of dealing with oxidative stress. The TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) is an important regulator of glycolysis and the pentose phosphate pathway that was described as a p53 response gene, yet TIGAR expression is detected in p53-null tumors. In this study we investigated the role of NRF2 in the regulation of TIGAR in human carcinoma cell lines. Exposure of carcinoma cells to electrophilic molecules or overexpression of NRF2 significantly increased expression of TIGAR, in parallel to the known NRF2 target genes NQO1 and G6PD. The same was observed in TP53KO cells, indicating that NRF2-mediated regulation of TIGAR is p53-independent. Accordingly, downregulation of NRF2 decreased the expression of TIGAR in carcinoma cell lines from different origin. As NRF2 is essential in the bone, we used mouse primary osteoblasts to corroborate our findings. The antioxidant response elements for NRF2 binding to the promoter of human and mouse TIGAR were described. This study provides the first evidence that NRF2 controls the expression of TIGAR at the transcriptional level.

NRF2 function in osteocytes is required for bone homeostasis and drives osteocytic gene expression.

Osteocytes, the most abundant bone cell type, are derived from osteoblasts through a process in which they are embedded in an osteoid. We previously showed that nutrient restriction promotes the osteocyte transcriptional program and is associated with increased mitochondrial biogenesis. Here, we show that increased mitochondrial biogenesis increase reactive oxygen species (ROS) levels and consequently, NRF2 activity during osteocytogenesis. NRF2 activity promotes osteocyte-specific expression of Dmp1, Mepe, and Sost in IDG-SW3 cells, primary osteocytes, and osteoblasts, and in murine models with Nfe2l2 deficiency in osteocytes or osteoblasts. Moreover, ablation of Nfe2l2 in osteocytes or osteoblasts generates osteopenia and increases osteoclast numbers with marked sexual dimorphism. Finally, treatment with dimethyl fumarate prevented the deleterious effects of ovariectomy in trabecular bone masses of mice and restored osteocytic gene expression. Altogether, we uncovered the role of NRF2 activity in osteocytes during the regulation of osteocyte gene expression and maintenance of bone homeostasis.

ACVR1 Function in Health and Disease.

Activin A receptor type I (ACVR1) encodes for a bone morphogenetic protein type I receptor of the TGFß receptor superfamily. It is involved in a wide variety of biological processes, including bone, heart, cartilage, nervous, and reproductive system development and regulation. Moreover, ACVR1 has been extensively studied for its causal role in fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterised by progressive heterotopic ossification. ACVR1 is linked to different pathologies, including cardiac malformations and alterations in the reproductive system. More recently, ACVR1 has been experimentally validated as a cancer driver gene in diffuse intrinsic pontine glioma (DIPG), a malignant childhood brainstem glioma, and its function is being studied in other cancer types. Here, we review ACVR1 receptor function and signalling in physiological and pathological processes and its regulation according to cell type and mutational status. Learning from different functions and alterations linked to ACVR1 is a key step in the development of interdisciplinary research towards the identification of novel treatments for these pathologies.

Interplay between BMPs and Reactive Oxygen Species in Cell Signaling and Pathology.

The integration of cell extrinsic and intrinsic signals is required to maintain appropriate cell physiology and homeostasis. Bone morphogenetic proteins (BMPs) are cytokines that belong to the transforming growth factor-ß (TGF-ß) superfamily, which play a key role in embryogenesis, organogenesis and regulation of whole-body homeostasis. BMPs interact with membrane receptors that transduce information to the nucleus through SMAD-dependent and independent pathways, including PI3K-AKT and MAPKs. Reactive oxygen species (ROS) are intracellular molecules derived from the partial reduction of oxygen. ROS are highly reactive and govern cellular processes by their capacity to regulate signaling pathways (e.g., NF-κB, MAPKs, KEAP1-NRF2 and PI3K-AKT). Emerging evidence indicates that BMPs and ROS interplay in a number of ways. BMPs stimulate ROS production by inducing NOX expression, while ROS regulate the expression of several BMPs. Moreover, BMPs and ROS influence common signaling pathways, including PI3K/AKT and MAPK. Additionally, dysregulation of BMPs and ROS occurs in several pathologies, including vascular and musculoskeletal diseases, obesity, diabetes and kidney injury. Here, we review the current knowledge on the integration between BMP and ROS signals and its potential applications in the development of new therapeutic strategies.

Inhibition of phosphatidylinositol 3-kinase α (PI3Kα) prevents heterotopic ossification.

Heterotopic ossification (HO) is the pathological formation of ectopic endochondral bone within soft tissues. HO occurs following mechanical trauma, burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). FOP patients carry a conserved mutation in ACVR1 that becomes neomorphic for activin A responses. Here, we demonstrate the efficacy of BYL719, a PI3Kα inhibitor, in preventing HO in mice. We found that PI3Kα inhibitors reduce SMAD, AKT, and mTOR/S6K activities. Inhibition of PI3Kα also impairs skeletogenic responsiveness to BMPs and the acquired response to activin A of the Acvr1R206H allele. Further, the efficacy of PI3Kα inhibitors was evaluated in transgenic mice expressing Acvr1Q207D . Mice treated daily or intermittently with BYL719 did not show ectopic bone or cartilage formation. Furthermore, the intermittent treatment with BYL719 was not associated with any substantial side effects. Therefore, this work provides evidence supporting PI3Kα inhibition as a therapeutic strategy for HO.

LPS-squalene interaction on D-galactose intestinal absorption.

The dynamic and complex interactions between enteric pathogens and the intestinal epithelium often lead to disturbances in the intestinal barrier, altered fluid, electrolyte, and nutrient transport and can produce an inflammatory response. Lipopolysaccharide (LPS) is a complex polymer forming part of the outer membrane of Gram-negative bacteria. On the other hand, squalene is a triterpene present in high levels in the extra-virgin olive oil that has beneficial effects against several diseases and it has also anti-oxidant and anti-inflammatory properties. The aim of this work was to study whether the squalene could eliminate the LPS effect on D-galactose intestinal absorption in rabbits and Caco-2 cells. The results have shown that squalene reduced the effects of LPS on sugar absorption. High LPS doses increased D-galactose uptake through via paracellular but also decreased the active sugar transport because the SGLT1 levels were diminished. However, the endotoxin effect on the paracellular way seemed to be more important than on the transcellular route. At the same time, an increased in RELM-ß expression was observed. This event could be related to inflammation and cause a decrease in SGLT1 levels. In addition, MLCK protein is also increased by LPS which could lead to an increase in sugar transport through tight junctions. At low doses, the LPS could inhibit SGLT1 intrinsic activity. Bioinformatic studies by docking confirm the interaction between LPS-squalene as well as occur through MLCK and SGLT-1 proteins.

Glucose Restriction Promotes Osteocyte Specification by Activating a PGC-1α-Dependent Transcriptional Program.

Osteocytes, the most abundant of bone cells, differentiate while they remain buried within the bone matrix. This encasement limits their access to nutrients and likely affects their differentiation, a process that remains poorly defined. Here, we show that restriction in glucose supply promotes the osteocyte transcriptional program while also being associated with increased mitochondrial DNA levels. Glucose deprivation triggered the activation of the AMPK/PGC-1 pathway. AMPK and SIRT1 activators or PGC-1α overexpression are sufficient to enhance osteocyte gene expression in IDG-SW3 cells, murine primary osteoblasts, osteocytes, and organotypic/ex vivo bone cultures. Conversely, osteoblasts and osteocytes deficient in Ppargc1a and b were refractory to the effects of glucose restriction. Finally, conditional ablation of both genes in osteoblasts and osteocytes generate osteopenia and reduce osteocytic gene expression in mice. Altogether, we uncovered a role for PGC-1 in the regulation of osteocyte gene expression.

Alkynyl gold(I) complex triggers necroptosis via ROS generation in colorectal carcinoma cells.

Given the rise of apoptosis-resistant tumors, there exist a growing interest in developing new drugs capable of inducing different types of cell death to reduce colorectal cancer-related death rates. As apoptosis and necroptosis do not share cellular machinery, necroptosis induction may have a great therapeutic potential on those apoptosis-resistant cancers, despite the inflammatory effects associated with it. We have synthesized an alkynyl gold(I) complex [Au(CC-2-NC5H4)(PTA)] whose anticancer effect was tested on the colorectal adenocarcinoma Caco-2 cell line. With regard to its mechanism of action, this gold complex enters the mitochondria and disrupts its normal function, leading to an increase in ROS production, which triggers necroptosis. Necroptosis induction has been found dependent of TNF-α (Tumor necrosisfactor α) and TNFR1(Tumor necrosisfactor receptor 1) binding, RIP1(Receptor-Interacting Protein 1) activation and NF-κB (Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells) signaling. Moreover, the antitumor potential of [Au(CC-2-NC5H4)(PTA)] has also been confirmed on the 3D cancer model spheroid. Overall, the obtained data show firstly that gold complexes might have the ability of inducing necroptosis, and secondarily that our compound [Au(CC-2-NC5H4)(PTA)] is an interesting alternative to current chemotherapy drugs in cases of apoptosis resistance.

p53 inhibits SP7/Osterix activity in the transcriptional program of osteoblast differentiation.

Osteoblast differentiation is achieved by activating a transcriptional network in which Dlx5, Runx2 and Osx/SP7 have fundamental roles. The tumour suppressor p53 exerts a repressive effect on bone development and remodelling through an unknown mechanism that inhibits the osteoblast differentiation programme. Here we report a physical and functional interaction between Osx and p53 gene products. Physical interaction was found between overexpressed proteins and involved a region adjacent to the OSX zinc fingers and the DNA-binding domain of p53. This interaction results in a p53-mediated repression of OSX transcriptional activity leading to a downregulation of the osteogenic programme. Moreover, we show that p53 is also able to repress key osteoblastic genes in Runx2-deficient osteoblasts. The ability of p53 to suppress osteogenesis is independent of its DNA recognition ability but requires a native conformation of p53, as a conformational missense mutant failed to inhibit OSX. Our data further demonstrates that p53 inhibits OSX binding to their responsive Sp1/GC-rich sites in the promoters of their osteogenic target genes, such as IBSP or COL1A1. Moreover, p53 interaction to OSX sequesters OSX from binding to DLX5. This competition blocks the ability of OSX to act as a cofactor of DLX5 to activate homeodomain-containing promoters. Altogether, our data support a model wherein p53 represses OSX-DNA binding and DLX5-OSX interaction, and thereby deregulates the osteogenic transcriptional network. This mechanism might have relevant roles in bone pathologies associated to osteosarcomas and ageing.

Therapeutic Applications of Rose Hips from Different Rosa Species.

Rosa species, rose hips, are widespread wild plants that have been traditionally used as medicinal compounds for the treatment of a wide variety of diseases. The therapeutic potential of these plants is based on its antioxidant effects caused by or associated with its phytochemical composition, which includes ascorbic acid, phenolic compounds and healthy fatty acids among others. Over the last few years, medicinal interest in rose hips has increased as a consequence of recent research that has studied its potential application as a treatment for several diseases including skin disorders, hepatotoxicity, renal disturbances, diarrhoea, inflammatory disorders, arthritis, diabetes, hyperlipidaemia, obesity and cancer. In this review, the role of different species of Rosa in the prevention of treatment of various disorders related to oxidative stress, is examined, focusing on new therapeutic approaches from a molecular point of view.