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It is not waste if it is therapy: cellular, secretory and functional properties of reamer–irrigator–aspirator (RIA)-derived autologous bone grafts
No full textBackground: Large bone defects resulting from trauma, disease, or resection often exceed the intrinsic capacity of bones to heal. The current gold standard addressing these defects is autologous bone grafting (ABG). Procedures such as reamer–irrigator–aspirator (RIA) and conventional bone grafting from the iliac crest are widely recognized as highly effective interventions for critical-size bone defects. The early phase of fracture healing is particularly crucial, as it can determine whether a complete bony union occurs, or if delayed healing or non-unions develop. The initial composition of the bone marrow (BM)-rich ABG transplant, with its unique cellular (e.g., leukocytes, monocytes, and granulocytes) and acellular (e.g., growth factors and extracellular proteins) components, plays a key role in this process. However, despite many successful case reports, the role of ABG cells, growth factors, and their precise contributions to bone healing remain largely elusive. Materials and methods: We characterized the native cellularity of both solid and liquid RIA-derived ABG by analyzing primary, minimally manipulated populations of monocytes, macrophages, and T cells, as well as hematopoietic, endothelial, and mesenchymal progenitor cells by flow cytometry. Growth factor and cytokine contents were assessed through antibody arrays. Possible functional and immunomodulatory properties of RIA liquid were evaluated in functional in vitro assays. Results: Growth factor and protein arrays revealed a plethora of soluble factors that can be linked to specific immunomodulatory and angiogenic properties, which were evaluated for their potency using functional in vitro assays. We could demonstrate a strong M2-macrophage phenotype inducing the effect of RIA liquid on macrophages. Additionally, we observed an increase in anti-inflammatory T cell subsets generated from peripheral blood mononuclear cells and BM mononuclear cells upon stimulation with RIA liquid. Finally, in vitro endothelial tube formation assays revealed highly significant angiogenic properties of RIA liquid, even at further dilutions. Conclusion: The cytokine and protein content of RIA liquid exhibits potent immunomodulatory and angiogenic properties. These findings suggest significant therapeutic potential for RIA liquid in modulating immune responses and promoting angiogenesis. Anti-inflammatory and angiogenic properties demonstrated in this study might also help to further define and understand its particular mode of action while also providing explanations to the excellent bone-healing properties of ABG in general. Level of evidence: Case-series (Level 4)
Impact of physical activity on red blood cell osmotic stability and deformability
No full textIntroduction
The membrane of red blood cells (RBCs) plays a crucial role in determining their functional characteristics. During
exercise, RBCs undergo various mechanical and biochemical adaptations that affect their membrane characteristics
and can influence whole blood rheology and perfusion dynamics [1]. In this pilot study, we investigated the effects
of exercise on RBC membrane characteristics in athletes compared to healthy sedentary individuals, using deformability
and osmotic fragility tests.
Methods
Red blood cells were isolated from the venous blood of five basketball players and age-matched healthy sedentary
controls. The athletes’ blood was collected at three different time points: immediately after training (IAT), 24 hours after
training (24h), and 48 hours after training (48h). Osmotic fragility was determined by exposing RBCs to decreasing
concentrations of NaCl solution and measuring the optical density (OD540) of the released hemoglobin. Using
ektacytometry measurements, the RBC deformability was obtained as a function of shear stress and elongation
index, and the data was fitted using Hill’s function.
Results and Discussion
The osmotic fragility test revealed a lower half-maximal hemolytic (H50) value (% of NaCl) for athletes (IAT= 0.35, 24h
= 0.34, and 48h = 0.37) compared to control (0.41). Additionally, we found that athletes’ RBCs exhibited greater
responsiveness to deformation under increasing shear stress, confirmed by the higher Hill’s coefficient (slope) value
and lower half-maximal shear-stress values (K coefficient) of the deformability curves (IAE = 3.09, 24h = 2, 48h =
2.17 and CTRL = 9.35). These results reflect RBC specific adaptative response in athletes at three different time
points and between the athletes and sedentary controls [2].
Conclusions
These results highlight differences in osmotic stability and deformation response of RBCs between athletes and
controls in favor of exercise in maintaining optimal RBC function, oxygen delivery, and overall circulatory health,
offering potential therapeutic benefits
Nitrogen-doped carbon dots as biocompatible fluorescent agents for labelling human red blood cells
No full textEvaluating the biocompatibility of nanoparticles with blood is essential to demonstrate their biosafety, reduce potential adverse effects, and enable their application in nanomedicine. Although many studies have explored interactions between blood and nanomaterials, only limited number have specifically addressed the compatibility of nitrogen-doped carbon dots (N-CD) with red blood cells (RBC), the most abundant cells in blood, that are essential for healthy functioning of all vertebrates through their role in oxygen transport. This study investigated the biological properties of several concentrations (25, 50, 100, 200 μg/mL) of negatively charged, green fluorescent N-CD, synthesized using environmentally friendly precursors through a hydrothermal method, on healthy human RBC in vitro. Scanning electron microscopy and atomic force microscopy revealed that the treatment with N-CD, even at the highest concentration, did not significantly affect RBC morphology. Interfacial interaction between N-CD and RBC was demonstrated by photoluminescence spectroscopy, fluorescence microscopy, and synchronous fluorescence spectroscopy analysis. The treatment with N-CD at the highest concentration had no effects on the RBC osmotic fragility, slightly increased the RBC deformability, and demonstrated a noticeable protective effect on the RBC hemolysis after 24 h. Flow cytometry analysis confirmed that N-CD did not alter the RBC morphology and did not result in an increase in the production of reactive oxygen species or reactive nitrogen species, indicating that their interaction did not lead to oxidative stress induction in human RBC. These findings suggest that fluorescent N-CD are biocompatible, fluorescent RBC imaging agents and hold promise as candidates for developing novel RBC-based drug delivery systems
Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia
No full textIntroduction:Besides skeletal symptoms, dental abnormalities are a typical feature of the rare inherited disorder hypophosphatasia (HPP), which is caused by loss of function mutations in the ALPL gene (alkaline phosphatase, biomineralization associated) coding for tissue-nonspecific alkaline phosphatase (TNAP). Dental symptoms include premature loss of deciduous teeth, disturbance in dentin and cementum mineralization, and an increased risk for periodontitis. However, the underlying molecular mechanisms are not fully understood and experimental cell lines for in vitro analyses of these processes are missing.MethodsWe aimed to develop a physiologically relevant cellular model of dental origin with genetic ALPL variants to investigate the molecular consequences of TNAP deficiencies in vitro. For this purpose, we used immortalized periodontal ligament stem cells (PDL-hTERT cells) to establish five independent clonal cell lines via CRISPR/Cas9, harboring different ALPL genetic variants.ResultsDetailed investigation of their genetic properties revealed that four different genotypes were successfully established at two different positions within the ALPL gene locus. The detected variants either result in mis-splicing of ALPL mRNAs or in frameshift mutations. All determined variants implied severe consequences on TNAP function, as indicated by in silico modeling and comparison to reported human pathogenic variants. Subsequent detailed cell culture experiments demonstrated TNAP residual gene expression and altered TNAP activity in the newly established ALPLtg PDL-hTERT lines. Further assessment of cell line features showed significantly reduced cell growth, partly lower levels of intracellular ATP as well as mitochondrial function proteins. TNAP activity was furthermore investigated during in vitro osteogenic differentiation and strong suppression during this process in nearly all newly established lines was observed.DiscussionWe report the generation of a new set of immortalized ALPLtg PDL-hTERT cells for investigation of TNAP cellular function in PDL cells, which can be used in subsequent studies for deciphering molecular processes in dental cells affected by reduction of TNAP function
Factorial analysis of zinc serum levels, fatty acids, oxidative stress parameters and supplementation on assisted reproductive technology outcome
No full textBackground: Infertility remains a prevalent global reproductive challenge, significantly affecting the lives of couples worldwide. The aetiology of infertility can be affected by various factors that exhibit possible relationships with one another. The study aimed to investigate factors that can interact with and influence the pregnancy outcome in couples undergoing assisted reproductive technology procedures. Methods: This retrospective study included 64 couples (64 men and their female partners) undergoing assisted reproductive technology procedures, having different pregnancy outcomes and lifestyle habits. Biomarkers of antioxidative and fatty acid status in the serum of both male and female partners, as well as the concentration of zinc in serum and seminal plasma of men, and their impact on assisted reproductive technology outcome were examined. Results: We grouped the parameters using principal component analysis and identified the three most contributing factors to the pregnancy achievement - seminogram parameters in males and redox status scores in female participants; supplementation with vitamin D, magnesium, and zinc; and serum levels of omega-3 and omega-6 fatty acids. Conclusions: The study concluded that seminogram parameters, intake of micronutrients, and levels of zinc, omega-3, and omega-6 fatty acids are contributing factors to the success of assisted reproductive technology. Further studies on a larger cohort are needed to confirm the predictive role of these factors on the success of assisted reproductive technology
Behavioral barriers in the inclusion of neglected and underutilized species into mainstream diets
No full textIn recent years, there has been a notable trend toward a global demand for sustainable nutrition, coupled with increasing concerns regarding biodiversity. Consequently, this has prompted the reintroduction of underutilized and neglected species (NUS) into the agri-food market. NUS refers to previously cultivated species no longer grown due to economic, cultural, agronomic, or genetic factors. The primary reason for their neglect often lies in widespread agricultural practices such as monoculture and the intense competition inherent in the current global trade environment. These crops often embody cultural heritage and possess a diverse genetic profile specific to their region, thereby contributing to efforts aimed at preserving biodiversity. However, the revitalization of NUS faces various hindrances, including behavioral barriers that influence their inclusion into mainstream diets. This review aims to identify the behavioral factors that restrict or limit the inclusion of NUS in mainstream diets, utilizing the principles of the Prospect Theory. Peer-reviewed studies focusing on the consumer perspective of NUS were analyzed using an inductive approach. Given the limited number of studies conducted to date, the findings reveal a significant gap in our understanding of NUS consumption. As a practical contribution, we propose a design that reinterprets the MINDSPACE tool through the lens of the Fogg Behavioral Model, offering guidance for policy interventions and marketing strategies
Experimental analysis of bone marrow adipose tissue and bone marrow adipocytes: An update from the bone marrow adiposity society (BMAS)
No full textBone marrow adipose tissue (BMAT) is physiologically linked to bone and energy metabolism, endocrine regulation, hematopoiesis and cancer-related processes. A key challenge in the field is that methods for isolating BMAT or bone marrow adipocytes (BMAds) are variable because there are no widely adopted standardized protocols. To generate awareness of this challenge and to establish uniformity in experimental approaches requiring isolation, storage and characterization of BMAT and BMAds, the Biobanking Working Group of the international Bone Marrow Adiposity Society (BMAS) has previously recommended experimental standards. This paper provides an update on this effort and presents current state-of-the-art methods and technical considerations for isolation and characterization of BMAT and BMAds, including currently available high-throughput omics approaches. This review provides a reference point based on the consensus view of BMAS investigators to support studies on biomedical, biological, biochemical and biophysical questions associated with bone marrow adiposity
Multi-session tDCS over the posterior parietal cortex and associative memory
No full textAssociative memory (AM) plays a crucial role in our ability to link disparate elements of our experiences, yet it is especially vulnerable to age-related decline and pathological conditions. Non-invasive brain stimulation (NIBS), particularly transcranial direct current stimulation (tDCS), has been investigated as a potential intervention to enhance cognitive functions, including AM. Previous tDCS studies yielded inconsistent results, often due to variations in stimulation sites and protocols. Nonetheless, enough evidence suggests that tDCS over the posterior parietal cortex (PPC) can improve AM performance. This study aimed to investigate the cumulative effects of multiple anodal tDCS over the PPC on AM performance alongside item memory and verbal fluency. In a randomized sham-controlled trial, 59 healthy young adults were assigned to either anodal or sham stimulation group, receiving tDCS (1.5 mA, for 20 minutes, at P3) over three consecutive days. Memory performance was assessed at four timepoints: pretest, immediately after the first session, posttest (Day 5), and follow-up (Day 9). Although tDCS was well tolerated, the anticipated enhancement of memory performance was not observed. We interpret these findings in the light of methodological considerations and propose potential explanations for the observed results emphasizing the large between-participants variability in memory performance as a significant factor that may have hindered the detection of tDCS effects
Organ-Specific Responses to Chronic High-Fat Diets in Mice: Insights into Phospholipid Fatty Acid Distribution
No full textBackground/Objectives: This research aimed to investigate phospholipid fatty acid (PLFA) distribution in the brain, kidneys, and white adipose tissue (WAT) and lipid profiles in response to high-fat diets. Methods: Adult female C57BL/6 mice were fed high-fat diets containing 25% linseed, palm, or sunflower oil for 100 days. The fatty acid composition of dietary oils and tissue PL were analyzed using gas–liquid chromatography. Results: Linseed oil increased n-3 polyunsaturated fatty acids (PUFAs) with subsequent conversion into long-chain n-3 PUFAs in the brain and kidney PL, while only alpha-linolenic acid was elevated in WAT. Palm and sunflower oils resulted in unique PLFA distributions in the kidneys and WAT. Palm oil raised linoleic acid without conversion to pro-inflammatory n-6 PUFAs. Sunflower oil increased saturated palmitic acid, as opposed to the rise in monounsaturated oleic acid. Linseed oil also significantly improved lipid profiles, reducing LDL and increasing HDL levels while enhancing cardiovascular indices. Conclusions: This study demonstrates that dietary oils significantly impact organ-specific PLFA profiles, with linseed oil enriching brain and renal n-3 PUFAs, while palm and sunflower oils induce distinct modifications in the kidney and WAT. Moreover, linseed oil offers notable cardioprotective benefits due to the favorable lipid profile changes. These findings highlight the importance of dietary fat selection in achieving balanced lipid metabolism and suggest that diverse oil combinations may be essential for optimizing health outcomes
Engineering probiotic biohydrogen micro-factories to initiate reductive stress for boosting tumor vulnerability
No full textDisruption of redox homeostasis profoundly affects cellular metabolism and activities. While oxidative stress is extensively studied in cancer therapies, research on reductive stress remains in its infancy. Molecular hydrogen (H2), a well-known antioxidant, holds significant potential to induce reductive stress due to its strong antioxidative properties, making it a promising candidate for cancer therapy. However, it remains a major challenge to develop a sustainable H2 delivery system in vivo. Herein, we designed a micro-factory by engineering a gel-based microcapsule that encapsulates Enterobacter aerogenes, a.k.a. probiotic biohydrogen microcapsules (PBMCs), enabling the sustained H2 generation within tumor microenvironment. Notably, PBMCs effectively suppressed the proliferation of eight tumor cell lines as well as drug-resistant cancer cells. The prolonged H2 release from PBMCs induced reductive stress, as evidenced by a significant increase in the GSH/GSSG ratio in 4T1 cells. Moreover, PBMCs displayed significant antitumor effects in breast, melanoma and liver cancer models. The inhibition of PI3K-AKT pathway and the activation of MAPK pathway were identified as key mechanisms responsible for inducing tumor cell cycle arrest and apoptosis. The PBMCs also exhibited synergistic effects in combination with chemotherapeutics, resulting in robust inhibitions of preinvasive carcinoma growth and commonly associated pulmonary metastasis. Overall, our study introduces an innovative strategy to manipulate reductive stress in the tumor microenvironment through in situ H2 generation, thereby enhancing tumor vulnerability