Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone

The bone marrow-derived multipotent mesenchymal cells (MSCs) have captured scientific interest due to their multi-purpose features and clinical applications. The operational dimension of MSCs is not limited to the bone marrow reservoir, which exerts bone-building and niche anabolic tasks; they also meet the needs of quenching inflammation and restoring inflamed tissues. Thus, the range of MSC activities extends to conditions such as neurodegenerative diseases, immune disorders and various forms of osteopenia. Steering these cells towards becoming an effective therapeutic tool has become mandatory. Many laboratories have employed distinct strategies to improve the plasticity and secretome of MSCs. We aimed to present how photobiomodulation therapy (PBM-t) can manipulate MSCs to render them an extraordinary anti-inflammatory and osteogenic instrument. Moreover, we discuss the outcomes of different PBM-t protocols on MSCs, concluding with some perplexities and complexities of PBM-t in vivo but encouraging and feasible in vitro solutions

Multipotent Mesenchymal Cells Homing and Differentiation on Poly(ε-caprolactone) Blended with 20% Tricalcium Phosphate and Polylactic Acid Incorporating 10% Hydroxyapatite 3D-Printed Scaffolds via a Commercial Fused Deposition Modeling 3D Device

Simple Summary As highlighted by the 'Global Burden of Disease Study 2019' conducted by the World Health Organization, ensuring fair access to medical care through affordable and targeted treatments remains crucial for an ethical global healthcare system. Versatile polymers show promise, particularly in 3D printing, aiming to reduce costs and enhance healthcare accessibility, such as meeting dentistry's demand for standardized osteoconductive products. It is essential to bridge biomaterial innovation with commercial printing technology. Our study emphasizes the metabolic behavior and lineage commitment of bone marrow-derived cells on two types of substrates: poly(epsilon-caprolactone) + 20% tricalcium phosphate (PCL + 20% beta-TCP) and L-polylactic acid + 10% hydroxyapatite (PLLA + 10% HA). Despite the limitations of these polymers, these biomaterials effectively promoted osteoinductivity. Both substrates proved optimal for the commitment of bone marrow-derived multipotent mesenchymal cells (MSCs) to mature bone cells across different temporal sequences.Abstract As highlighted by the 'Global Burden of Disease Study 2019' conducted by the World Health Organization, ensuring fair access to medical care through affordable and targeted treatments remains crucial for an ethical global healthcare system. Given the escalating demand for advanced and urgently needed solutions in regenerative bone procedures, the critical role of biopolymers emerges as a paramount necessity, offering a groundbreaking avenue to address pressing medical needs and revolutionize the landscape of bone regeneration therapies. Polymers emerge as excellent solutions due to their versatility, making them reliable materials for 3D printing. The development and widespread adoption of this technology would impact production costs and enhance access to related healthcare services. For instance, in dentistry, the use of commercial polymers blended with beta-tricalcium phosphate (TCP) is driven by the need to print a standardized product with osteoconductive features. However, modernization is required to bridge the gap between biomaterial innovation and the ability to print them through commercial printing devices. Here we showed, for the first time, the metabolic behavior and the lineage commitment of bone marrow-derived multipotent mesenchymal cells (MSCs) on the 3D-printed substrates poly(e-caprolactone) combined with 20% tricalcium phosphate (PCL + 20% beta-TCP) and L-polylactic acid (PLLA) combined with 10% hydroxyapatite (PLLA + 10% HA). Although there are limitations in printing additive-enriched polymers with a predictable and short half-life, the tested 3D-printed biomaterials were highly efficient in supporting osteoinductivity. Indeed, considering different temporal sequences, both 3D-printed biomaterials resulted as optimal scaffolds for MSCs' commitment toward mature bone cells. Of interest, PLLA + 10% HA substrates hold the confirmation as the finest material for osteoinduction of MSCs

Interpenetrating Hydrogel Networks Enhance Mechanical Stability, Rheological Properties, Release Behavior and Adhesiveness of Platelet-Rich Plasma

Platelet‐rich plasma (PRP) has attracted much attention for the treatment of articular cartilage defects or wounds due to its intrinsic content of growth factors relevant for tissue repair. However, the short residence time of PRP in vivo, due to the action of lytic enzymes, its weak mechanical properties and the consequent short‐term release of bioactive factors has restricted its application and efficacy. The present work aimed at designing new formulation strategies for PRP, based on the use of platelet concentrate (PC)‐loaded hydrogels or interpenetrating polymer networks, directed at improving mechanical stability and sustaining the release of bioactive growth factors over a prolonged time‐span. The interpenetrating hydrogels comprised two polymer networks interlaced on a molecular scale: (a) a first covalent network of thermosensitive and biodegradable vinyl sulfone bearing p(hydroxypropyl methacrylamide‐lacate)‐polyethylene glycol triblock copolymers, tandem cross‐linked by thermal gelation and Michael addition when combined with thiolated hyaluronic acid, and (b) a second network composed of cross‐linked fibrin. The PC‐loaded hydrogels, instead, was formed only by network (a). All the designed and successfully synthesized formulations greatly increased the stability of PRP in vitro, leading to significant increase in degradation time and storage modulus of PRP gel. The resulting viscoelastic networks showed the ability to controllably release platelet derived growth factor and transforming growth factr β1, and to improve the tissue adhesiveness of PRP. The newly developed hydrogels show great potential for application in the field of wound healing, cartilage repair and beyond

Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved

Removal of the endocrine disrupter butyl benzyl phthalate from the environment

Butyl benzyl phthalate (BBP), an aryl alkyl ester of 1,2-benzene dicarboxylic acid, is extensively used in vinyl tiles and as a plasticizer in PVC in many commonly used products. BBP, which readily leaches from these products, is one of the most important environmental contaminants, and the increased awareness of its adverse effects on human health has led to a dramatic increase in research aimed at removing BBP from the environment via bioremediation. This review highlights recent progress in the degradation of BBP by pure and mixed bacterial cultures, fungi, and in sludge, sediment, and wastewater. Sonochemical degradation, a unique abiotic remediation technique, and photocatalytic degradation are also discussed. The degradation pathways for BBP are described, and future research directions are considered

Archetypal autophagic players through new lenses for bone marrow stem/mature cells regulation

The bone marrow landscape consists of specialized and stem/progenitor cells, which coordinate important tissue-related and systemic physiological features. Within the marrow cavity, stem/progenitor and differentiated hematopoietic and skeletal cells congregate into dynamic functional assemblies throughout specific anatomical regions, termed niches. There is a need for better understanding of the bone marrow microareas, through exploration of the intramural physical and molecular interactions of the distinctive cell populations. The elective liaisons established among the mesenchymal/stromal stem cell and hematopoietic stem cell lineage trees play a key role in orchestrating the stem/mature cell behavior and customized hierarchies within bone marrow cell populations. Recently, the autophagic apparatus has been discovered to be an important feature of bone marrow homeostasis. Autophagy-related factors involved in the labyrinthic and highly dynamic bone marrow workshop redesign the niche framework by coordinating the operational schedule of pluripotent stem and mature cells. The following report summarizes the most recent breakthroughs in our understanding of the intramural relationships between bone marrow cells and key autophagic mediators. Doubtless, the consideration of the autophagy-related and unrelated functions of main players, such as p62, Atg7, Atg5, and Beclin-1 remains a compelling task to thoroughly understand the complex relations between the heterogenic cell types that populate bone marrow

Phthalate esters: Bioaccumulation and intracellular signal modifications in in vivo and in vitro models

Phthalates, a class of chemicals used in a whole range of industrial applications, are considered to be ubiquitous global contaminants and endocrine disruptors (EDs). These hormone-mimicking compounds bioaccumulate within organ tissues in several animals, including fishes, amphibians and mammals with different and selective patterns of distribution. The endocrine disrupting effects are probably related to the absorption of these chemicals via the alimentary canal. In particular, phthalates have been seen to be stored in the oxyntic cells of the gastric tubular glands in fishes and amphibians. A relation between bioconcentration and organ functions was also observed. Moreover, in vitro studies on rat osteoblastic cultures demonstrated that benzyl butyl phthalate and di-n-butyl phthalate bioaccumulate, modify actin cytoarchitecture and exert mitogenic effects involving microfilament disruption and nuclear actin/lamin A regulation. On the other hand, primary mouse calvarial osteoblasts, treated with the above chemicals showed DNA base lesions with an increase of apoptotic markers induced by the p53-related pathway. These data strongly suggest that chronic exposure to phthalates could probably affect new bone formation and matrix deposition with clinical implications on bone homeostasis and mineral density. Understanding the way of action of phthalates through the study of autochthon or laboratory animals could reveal the mechanism by which these compounds, as EDs, regulate the fate of the organism

Assessing the Effects of Curcumin and 450 nm Photodynamic Therapy on Oxidative Metabolism and Cell Cycle in Head and Neck Squamous Cell Carcinoma: An In Vitro Study

Simple Summary The study investigates the effects of curcumin in native conditions and after irradiation with 450 nm light on the energy metabolism, redox balance, and cellular growth of head and neck cancer cells and human primary fibroblasts. Although curcumin in native conditions already shows an anti-cancer effect, affecting energy metabolism and limiting the growth of tumor cells and cells that inhabit the surrounding microenvironment, irradiation with 450 nm light (photodynamic) enhances its effect by acting on antioxidant defenses. This study, therefore, opens up new perspectives on specific wavelengths of light that appear to be able to improve the drug's action on cancer growth and proliferation, offering hope for better patient outcomes in the future.Abstract Oral cancer is the 16th most common malignant tumor worldwide. The risk of recurrence and mortality is high, and the survival rate is low over the following five years. Recent studies have shown that curcumin causes apoptosis in tumor cells by affecting FoF1-ATP synthase (ATP synthase) activity, which, in turn, hinders cell energy production, leading to a loss of cell viability. Additionally, irradiation of curcumin within cells can intensify its detrimental effects on cancer cell viability and proliferation (photodynamic therapy). We treated the OHSU-974 cell line, a model for human head and neck squamous cell carcinoma (HNSCC), and primary human fibroblasts. The treatment involved a 1 h exposure of cells to 0.1, 1.0, and 10 mu M curcumin, followed or not by irradiation or the addition of the same concentration of pre-irradiated curcumin. Both instances involved a diode laser with a wavelength of 450 nm (0.25 W, 15 J, 60 s, 1 cm2, continuous wave mode). The treatment with non-irradiated 1 and 10 mu M curcumin caused ATP synthase inhibition and a consequent reduction in the oxygen consumption rate (OCR) and the ATP/AMP ratio, which was associated with a decrement in lipid peroxidation accumulation and a slight increase in glutathione reductase and catalase activity. By contrast, 60 s curcumin irradiation with 0.25 W-450 nm caused a further oxidative phosphorylation (OxPhos) metabolism impairment that induced an uncoupling between respiration and energy production, leading to increased oxidative damage, a cellular growth and viability reduction, and a cell cycle block in the G1 phase. These effects appeared to be more evident when the curcumin was irradiated after cell incubation. Since cells belonging to the HNSCC microenvironment support tumor development, curcumin's effects have been analyzed on primary human fibroblasts, and a decrease in cell energy status has been observed with both irradiated and non-irradiated curcumin and an increase in oxidative lipid damage and a slowing of cell growth were observed when the curcumin was irradiated before or after cellular administration. Thus, although curcumin displays an anti-cancer role on OHSU-974 in its native form, photoactivation seems to enhance its effects, making it effective even at low dosages

P62/SQSTM1 enhances osteogenesis and attenuates inflammatory signals in bone marrow microenvironment

Bone marrow-derived mesenchymal/stromal stem cells (MSCs) became a major focus of research since the anti-inflammatory features and the osteogenic commitment of these cells can prevent the inflamm-aging and various form of osteopenia in humans and animals. We previously showed that p62/SQSTM1 plasmid can prompt release of anti-inflammatory cytokines/chemokines by MSC when injected in adult mice. Furthermore, it can enhance osteoblastogenesis at the expense of adipogenesis and ameliorate bone density and bone remodeling. On the other hand, absence of p62 partially exhausted MSC pool caused expansion of fat cells within bone marrow and pro-inflammatory mediator's accumulation. Given the critical function of p62 as molecular hub of MSC dynamics, here, using MSCs from p62 knockout adult mice, we investigated the effect of this protein on MSC survival and bone-forming molecule cascades. We found that the main osteogenic routes are impaired in absence of p62. In particular, lack of p62 can suppress Smads activation, and Osterix and CREBs expression, thus significantly modifying the schedule of MSCs differentiation. MSCs obtained from p62-/- mice have also demonstrate an amplified NFκB/ Smad1/5/8 colocalization along with NFκB activation in the nucleus, which precludes Smads binding to target promoters. Considering the “teamwork” of TGFβ, PTH and BMP2 on MSC homeostatic behavior, we consider that p62 exerts an essential role as a hub protein. Lastly, ex vivo pulsing p62-deficient MSCs, which then will be administered to a patient as a cell therapy, may be considered as a treatment for bone and bone marrow disorders

Runx/Smads interaction is impaired in osteoblasts from Fgf2-/- mice

Bone morphogenetic protein 2 (BMP2) is one of the most potent regulator of osteoblast differentiation and bone formation. R-Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they translocated in the nucleus regulating transcription target genes by interacting with various transcription factors (1). Runx2 proteins have been shown to interact through their C-terminal segment with Smads and this interaction is required for in vivo osteogenesis (2). In particular, recruitment of Smads to intranuclear sites is Runx2 dependent, and Runx2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites (3). Previously, we have shown, by in vitro and in vivo experiments, that BMP2 up-regulated FGF-2 which is important for the maximal responses of BMP-2 in bone (4). Now, by biochemical, immunofluorescence and immunoelectron microscopy approaches, we found that BPM2 was also able to induce nuclear accumulation and colocalization of Runx2 and Smads1/5/8 in presence of endogenous FGF-2, while Runx/Smads nuclear interaction was markedly reduced in Fgf2-/- osteoblasts. Based on these preliminary data, we hypothesize that the impaired nuclear accumulation of Runx2 in Fgf2-/- osteoblasts could reduce R-Smads sub-nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice