Optimized bacterial community characterization through full-length 16S rRNA gene sequencing utilizing MinION nanopore technology.

BACKGROUND: Accurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1-V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control. RESULTS: Based on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92. CONCLUSIONS: These findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification.

The Swiss Spinal Cord Injury Cohort Study (SwiSCI) biobank: from concept to reality.

OBJECTIVES: To describe the concept, establishment and the operationalization of the biobank of the Swiss Spinal Cord Injury Cohort Study (SwiSCI), the available biosamples, and demographic and clinical characteristics of study participants. SETTING: The SwiSCI biobank is a platform for research within SwiSCI. It collects and processes serum, plasma, PBMCs, RNA, DNA, and urine from three rehabilitation centers. Samples are collected at admission to first rehabilitation and at discharge. Additionly, the biobank provides services to projects nested in SwiSCI or otherclinical trials among Spinal Cord Injury population. METHODS: Descriptive statistics were used for an overview of available biosamples, study participant characteristics, and comparison of the participating centers. RESULTS: Between the SwiSCI biobank establishment on June 27th, 2016, and October 19th, 2023, the SwiSCI Study has obtained informed consent from 524 individuals. Of these, 315 (60.1%) have agreed to donate biospecimens to the biobank. The average age of the contributors was 54 years (range: 38-65), with the majority being male (80%). Most participants suffered from traumatic injuries (66%) and were classified as paraplegic (64%). Approximately 80% presented with motor and sensory-incomplete SCI. The median Spinal Cord Independence Measure (SCIM) score was 31 (Interquartile Range: 19-58). The proportion of individuals providing paired biosamples at two distinct time points ranged from 63% (for RNA) to 65% (for urine and urine sediment). CONCLUSIONS: The SwiSCI biobank is a unique platform designed to serve as a basis for collaborative SCI research, including multi-omics approaches. The longitudinal collection of biospecimens and cryopreservation of multiple aliquots for each participant are fundamental for scrutinizing the temporal associations, ensuring research reproducibility, and achieving an adequate sample size for future investigations.

Endocrinological and inflammatory markers in individuals with spinal cord injury: A systematic review and meta-analysis.

Spinal cord injury (SCI) can lead to dramatic physiological changes which can be a factor in developing secondary health conditions and might be reflected in biomarker changes in this elevated risk group. We focused specifically on the endocrine and inflammation profile differences between SCI and able-bodied individuals (ABI). Our aim was to determine the differences in inflammatory markers and endocrine profiles between SCI and ABI. We systematically searched 4 electronic databases for relevant studies. Human observational (cross-sectional, cohort, case-control) studies that compared biomarkers of interest between SCI and ABI population were included. Weighted mean difference between SCI and ABI was calculated using random-effects models. Heterogeneity was computed using I2 statistic and chi-squared test. Study quality was evaluated through the Newcastle-Ottawa Scale. The search strategy yielded a total of 2,603 studies from which 256 articles were selected for full-text assessment. Sixty-two studies were included in the meta-analysis. SCI individuals had higher levels of pro-inflammatory C-reactive protein and IL-6 than ABI. Creatinine and 25-hydroxyvitamin D3 levels were lower in SCI than ABI. Total testosterone levels and IGF-1 were also found to be lower, while cortisol and leptin levels were higher in SCI when compared to ABI. Accordingly, meta-regression, subgroup analysis, and leave-one-out analysis were performed, however, they were only able to partially explain the high levels of heterogeneity. Individuals with SCI show higher levels of inflammatory markers and present significant endocrinological changes when compared to ABI. Moreover, higher incidence of obesity, diabetes, osteoporosis, and hypogonadism in SCI individuals, together with decreased creatinine levels reflect some of the readily measurable aspects of the phenotype changes in the SCI group. These findings need to be considered in anticipating medically related complications and personalizing SCI medical care.

Systematic review of the changes in the microbiome following spinal cord injury: animal and human evidence.

STUDY DESIGN: Systematic review. OBJECTIVES: To investigate the changes in the microbiome among human and animal populations with spinal cord injury (SCI). METHODS: Four databases (EMBASE, Medline (Ovid), Web of Science, Cochrane Central Register of Trials (CENTRAL)) and Google Scholar were searched. No language restrictions were applied. Data extraction was done in parallel and independently by two reviewers. The search was last conducted on 07 April 2021. RESULTS: There were 6869 studies retrieved, 43 full-text studies reviewed, and 19 studies included. There were seven animal gut studies, six human gut studies, and six urinary tract studies identified. There were no publications found on other body sites. Among the included studies, we observed a consistent and significant difference in gut microbiome composition between populations with SCI and able-bodied populations. This is characterized by a decrease in beneficial butyrate-producing bacteria (Faecalbacterium, Megamonas, Roseburia) and an increase in inflammation-associated bacteria (Alistipes, Anaerotruncus, and Lachnoclostridium). On the other hand, the urine of individuals with SCI was polymicrobial and members of Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) were frequently observed. Probiotics were shown to induce a significant but transient shift in the urinary tract microbiome. The studies had low to moderate risks of bias. CONCLUSIONS: There are limited studies on the changes in microbiome among SCI populations. The gut microbiome was characterized by bacterial profiles associated with chronic inflammation and metabolic disorder while the studies of the urinary tract microbiome show the dominance of bacterial genera associated with urinary tract infection.

Systematic Review of the Effects of Oat Intake on Gastrointestinal Health.

BACKGROUND: Oats are a food source with multiple health benefits that could support beneficial bacterial groups and provide important bioactive compounds for the gut. OBJECTIVES: This review explores the association between oat intake, gastrointestinal (GI) symptoms, and microbial community changes in individuals with celiac disease (CeD), irritable bowel syndrome (IBS), and inflammatory bowel disease (IBD) and without GI disease. METHODS: Four databases and Google Scholar were systematically searched from inception until April 29, 2021. Clinical trials, observational studies, and in vitro studies with human gut-derived samples were included. RESULTS: There were 84 articles [23 randomized controlled trials (RCTs), 21 nonrandomized trials, 8 observational studies, and 32 in vitro studies] included. Oat intake increased total bacterial count, Lactobacilli spp., and Bifidobacterium spp. in healthy individuals and those with CeD. There was an increased concentration of short-chain fatty acids and improved gut permeability with oat intake but with no significant quality-of-life difference. In some individuals with CeD, consumption of certain oat types was associated with worsening of GI symptoms. We found no studies reporting on IBS and only 3 for IBD. The quality of RCTs showed some concerns mostly in domains of randomization (73.9%), whereas the quality of evidence of non-RCTs, observational studies, and in vitro studies was satisfactory. CONCLUSIONS: Oat intake was associated with the increase of beneficial bacterial groups in individuals without GI disease and those with CeD. Most studies showed no changes in GI symptoms with oat consumption. In vitro studies in CeD provide insight to oat-sensitive individuals and their GI mucosa, but the clinical studies remain limited, precluding our ability to draw firm conclusions. The prevalence of oat sensitivity in individuals with CeD should be further explored as this could improve clinical management and facilitate inclusion of oat in the diet for this population.

The neurological level of spinal cord injury and cardiovascular risk factors: a systematic review and meta-analysis.

STUDY DESIGN: Systematic review and meta-analysis. OBJECTIVE: To determine the difference in cardiovascular risk factors (blood pressure, lipid profile, and markers of glucose metabolism and inflammation) according to the neurological level of spinal cord injury (SCI). METHODS: We searched 5 electronic databases from inception until July 4, 2020. Data were extracted by two independent reviewers using a pre-defined data collection form. The pooled effect estimate was computed using random-effects models, and heterogeneity was calculated using I2 statistic and chi-squared test (CRD42020166162). RESULTS: We screened 4863 abstracts, of which 47 studies with 3878 participants (3280 males, 526 females, 72 sex unknown) were included in the meta-analysis. Compared to paraplegia, individuals with tetraplegia had lower systolic and diastolic blood pressure (unadjusted weighted mean difference, -14.5 mmHg, 95% CI -19.2, -9.9; -7.0 mmHg 95% CI -9.2, -4.8, respectively), lower triglycerides (-10.9 mg/dL, 95% CI -19.7, -2.1), total cholesterol (-9.9 mg/dL, 95% CI -14.5, -5.4), high-density lipoprotein (-1.7 mg/dL, 95% CI -3.3, -0.2) and low-density lipoprotein (-5.8 mg/dL, 95% CI -9.0, -2.5). Comparing individuals with high- vs. low-thoracic SCI, persons with higher injury had lower systolic and diastolic blood pressure (-10.3 mmHg, 95% CI -13.4, -7.1; -5.3 mmHg 95% CI -7.5, -3.2, respectively), while no differences were found for low-density lipoprotein, serum glucose, insulin, and inflammation markers. High heterogeneity was partially explained by age, prevalent cardiovascular diseases and medication use, body mass index, sample size, and quality of studies. CONCLUSION: In SCI individuals, the level of injury may be an additional non-modifiable cardiovascular risk factor. Future well-designed longitudinal studies with sufficient follow-up and providing sex-stratified analyses should confirm our findings and explore the role of SCI level in cardiovascular health and overall prognosis and survival.

Immunosenescence in persons with spinal cord injury in relation to urinary tract infections -a cross-sectional study.

BACKGROUND: Individuals with a spinal cord injury (SCI), despite specialized rehabilitation and good health care, have a reduced life expectancy. Infectious diseases, such as pneumonias, infected pressure sores and urinary tract infections (UTI) have been identified as the leading causes of mortality. We hypothesise that a premature onset of immune frailty occurs in SCI, possibly caused also by recurrent urinary tract infections.A cross sectional study was performed comparing blood and urine samples between able bodied controls (n = 84) and persons with spinal cord injury (n = 85). The results were grouped according to age (below and above 60 years). Assessed were the abundancies of immune cells, the concentration of soluble biomarkers, the in vitro functioning of lymphocytes as well as phenotypic exhaustion of T-cells in blood and urine. Further, the leucocyte telomere length and the cytomegalovirus (CMV) serological status were compared between the groups. RESULTS: We observed in people with SCI lower proportions of naïve T-cells, more memory T-cells, reduced T-cell proliferation and higher CMV prevalence compared to age-matched controls. SCI participants older than 60 years had a higher prevalence of UTI compared with SCI persons younger than 60 years. CONCLUSION: The immune system of people with SCI shows traits of an increased immunological strain and a premature onset of immune frailty. The role of UTI in the onset of immune frailty remains to be elucidated as we did not see significantly higher abundancies of circulating UTI-bacteria specific T-cell clones in persons with SCI. We assume that any impact of UTI on the immune system might be compartmentalized and locally restricted to the urinary tract.

Characteristics of the gut microbiome of Swiss elite athletes with a spinal cord injury: An exploratory study.

OBJECTIVES: To illustrate and explore associations between the gut microbiome and spinal cord injury (SCI) characteristics, physical training, dietary intake, body composition, and blood biomarkers of elite Swiss athletes. DESIGN AND SETTING: Baseline data analysis of athletes with SCI who participated in a pilot trial (NCT04659408) in the Swiss Paraplegic Center, Nottwil, Switzerland. PARTICIPANTS: Elite athletes, five males, and six females, with SCI who competed internationally. OUTCOME MEASURES: We conducted a differential abundance analysis and measured the alpha and beta diversity of the gut microbiome. RESULTS: The athletes' median age was 34.5 years. Six had traumatic SCI and five had a spina bifida. The athletes competed in para-cycling (5), wheelchair athletics (3), and wheelchair tennis (3). A higher duration of training per week was positively associated with Akkermansia and Akkermansiaceae but negatively associated with Prevotellaceae. Muribaculaceae was negatively associated with the average number of trainings per week. Waist circumference is negatively associated with Butyricimonas. Significant differences in the alpha diversity were found with sex, gastrointestinal quality of life index (GIQLI) scores, total caloric intake, total fat intake, total carbohydrate intake, and high-sensitivity C-reactive protein (hs-CRP). Beta diversity differences were found with impairment of the sympathetic nervous system of the gut at the genus level and HbA1c at the family level. CONCLUSIONS: This study provides insight into the gut microbiome of athletes with SCI. Our results were similar to those found in athletes without SCI. Further replication is needed to confirm the relationships of organisms observed in the gut of athletes with SCI.

Gut microbiome and inflammation among athletes in wheelchair in a crossover randomized pilot trial of probiotic and prebiotic interventions.

Disorders related to gut health are a significant cause of morbidity among athletes in wheelchair. This pilot feasibility trial aims to investigate whether probiotics compared to prebiotics can improve inflammatory status and gut microbiome composition in elite athletes in wheelchair. We conducted a 12-week, randomized, cross-over controlled trial involving 14 elite Swiss athletes in wheelchair. Participants were given a multispecies-multistrain probiotic or prebiotic (oat bran) daily for 4 weeks (Clinical trials.gov NCT04659408 09/12/2020). This was followed by a 4-week washout and then crossed over. Thirty inflammatory markers were assessed using bead-based multiplex immunoassays (LegendPlex) from serum samples. The gut microbiome was characterized via 16S rRNA sequencing of stool DNA samples. Statistical analyses were conducted using linear mixed-effect models (LMM). At baseline, most athletes (10/14) exhibited low levels of inflammation which associated with higher gut microbiome alpha diversity indices compared to those with high inflammation levels. The use of probiotic had higher decrease in 25 (83%) inflammatory markers measured compared to prebiotic use. Probiotic has the potential in lowering inflammation status and improving the gut microbiome diversity. The future trial should focus on having sufficient sample sizes, population with higher inflammation status, longer intervention exposure and use of differential abundance analysis.

Growth defects and impaired cognitive-behavioral abilities in mice with knockout for Eif4h, a gene located in the mouse homolog of the Williams-Beuren syndrome critical region.

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.

Understanding the impact of spinal cord injury on the microbiota of healthy skin and pressure injuries.

Pressure injuries (PI) are a common issue among individuals with spinal cord injury (SCI), especially in the sitting areas of the body. Considering the risk of infections occurring to PI during the wound healing process, the skin microbiome is likely to be a source of bacteria. We investigated the relationship between skin and PI microbiomes, and assessed any correlation with clinically relevant outcomes related to PI. Samples were isolated from SCI patients undergoing reconstructive surgery of PI, severity grades III and IV. DNA samples from skin and PI were analysed using 16S rRNA gene sequencing. Our results showed disparities in microbiome composition between skin and PI. The skin had lower diversity, while PI showed increased bacterial homogeneity as the severity grade progressed. The skin bacterial composition varied based on its location, influenced by Cutibacterium. Compositional differences were identified between PI grades III and IV, with clusters of bacteria colonizing PI, characterized by Pseudomonas, Proteus and Peptoniphilus. The skin and PI microbiomes were not affected by the level of the SCI. Our study highlights the differences in the microbiome of skin and PI in SCI patients. These findings could be used to target specific bacteria for PI treatment in clinical practice.

Advancements in cell-based therapies for the treatment of pressure injuries: A systematic review of interventional studies.

The high recurrence and complications associated with severe pressure injuries (PI) necessitate the exploration of advanced treatments, such as cell-based therapies, to facilitate wound healing. Such techniques harness the ability of different cell types to promote angiogenesis, re-epithelialization of the skin, and tissue regeneration. This systematic review explores the efficacy of cell-based therapies and tissue engineering in treating deep PI. We searched for interventional studies using cells in the treatment of PI in adults in four online libraries (PubMed, Embase, Ovid Medline, and Cochrane; latest search 10th June 2023). We found one randomized clinical trial (RCT), two non-RCT, and three pre-post studies, comprising 481 study participants with PI (253 intervention/228 controls). The risk of bias was categorized as moderate due to minimal bias in outcome measurements, or high owing to unclear patient randomization methods, as assessed by the ROBINS-I, NIH, and RoB-2 tools. Four cell types were identified in the context of cell-based therapies of PI: bone marrow mononuclear stem cells (BM-MNCs, n = 2); hematopoietic derived stem cells (HSC, n = 1); macrophages and activated macrophage suspensions (AMS, n = 2); and cryopreserved placental membrane containing viable cells (vCPM, n = 1). Wound healing outcomes were observed in patients undergoing cell-based therapies, including complete wound closure (AMS, vCPM; n = 142), faster healing rate (BM-MNCs, AMS; n = 146), improved granulation tissue formation (HSC, n = 3) and shorter hospitalization time (BM-MNCs; n = 108) compared to standard of care, with no adverse reactions. PI healing rate decreased only in one study with BM-MNC therapy, compared to control (n = 86). Based on the available data, though with limited evidence, it seems that macrophage deployment showed the most favorable outcomes. The results indicate that cell-based therapies offer a potential avenue for enhancing wound healing and tissue repair in PI; however, more extensive research is needed in this domain.

Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells.

INTRODUCTION: Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells. MATERIALS AND METHODS: Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry. RESULTS: MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower. CONCLUSIONS: Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.

The feasibility of a crossover, randomized controlled trial design to assess the effect of probiotics and prebiotics on health of elite Swiss para-athletes: a study protocol.

BACKGROUND: Spinal cord injury (SCI) may cause an autonomic imbalance in the gastrointestinal tract, leading to deficits in colonic motility, mucosal secretions, vascular tone, and an increase of intestinal barrier permeability. Autonomic denervation and factors such as age, physical activity, antibiotic use and stress may cause intestinal bacterial translocation, decreased microbiota diversity, known as gut dysbiosis and thus increase susceptibility to experiencing gastrointestinal discomfort. Probiotic treatment in individuals with SCI may normalize the gut microbiota and improve overall health. We aim to assess the feasibility of probiotic and prebiotic intervention in athletes with SCI and collect information necessary for sample size calculation of a definite trial on improving health outcomes in para-athletes. METHODS AND ANALYSIS: Elite Swiss para-athletes (aged> 18 years), being shortlisted for the Paralympic Games 2021 in Tokyo or a member of a national team (n = 43), will be invited to participate in this single-center randomized crossover trial. Athletes suffering from chronic inflammatory bowel diseases, those currently taking antibiotics or other medication to alleviate gastro-intestinal complaints will not be eligible to be included in the study. Athletes will be randomized (1:1) to receive for 4 weeks a daily dose of either 3 g of probiotic preparation or 5 g of prebiotic (organic oat bran) supplementation in addition to usual diet, followed by a 4-week washout period or vice versa. The primary outcome is the feasibility of the study, measured by recruitment and dropout rates, feasibility of the measurements, acceptability and adherence to the intervention. Secondary outcomes include gastrointestinal health assessment, diet and training information, handgrip strength, blood diagnostic parameters, and intestinal microbiome characterization. The changes in clinically relevant secondary outcome values will be used to make a power calculation for definite trial. DISCUSSION: This pilot trial will address two common challenges in SCI research: the difficulty to recruit enough participants for a sufficiently powered study and the ability to collect data within the limits of a realistic budget and time frame. Upon demonstrated feasibility of the intervention and study procedures, the intervention will be evaluated in a definitive controlled trial comprising a larger sample of para-athletes (elite, engaged, or recreationally active) individuals with a SCI. TRIAL REGISTRATION: NCT04659408.

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions.

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules--dexamethasone, triiodothyronine (T3) and insulin--on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.

Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro.

The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5 µg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-ß1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1ß and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n = 13) were divided in two groups: responders (n = 9), where inflammation was inhibited by MSCs and non-responders (n = 4), where MSCs did not decrease inflammation. At 1 week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-ß1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1ß expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissue.

Body Composition According to Spinal Cord Injury Level: A Systematic Review and Meta-Analysis.

The level of injury is linked with biochemical alterations and limitations in physical activity among individuals with spinal cord injury (SCI), which are crucial determinants of body composition. We searched five electronic databases from inception until 22 July 2021. The pooled effect estimates were computed using random-effects models, and heterogeneity was calculated using I2 statistics and the chi-squared test. Study quality was assessed using the Newcastle-Ottawa Scale. We pooled 40 studies comprising 4872 individuals with SCI (3991 males, 825 females, and 56 sex-unknown) in addition to chronic SCI (median injury duration 12.3 y, IQR 8.03-14.8). Individuals with tetraplegia had a higher fat percentage (weighted mean difference (WMD) 1.9%, 95% CI 0.6, 3.1) and lower lean mass (WMD -3.0 kg, 95% CI -5.9, -0.2) compared to those with paraplegia. Those with tetraplegia also had higher indicators of central adiposity (WMD, visceral adipose tissue area 0.24 dm2 95% CI 0.05, 0.43 and volume 1.05 L 95% CI 0.14, 1.95), whereas body mass index was lower in individuals with tetraplegia than paraplegia (WMD -0.9 kg/mg2, 95% CI -1.4, -0.5). Sex, age, and injury characteristics were observed to be sources of heterogeneity. Thus, individuals with tetraplegia have higher fat composition compared to paraplegia. Anthropometric measures, such as body mass index, may be inaccurate in describing adiposity in SCI individuals.

Autofluorescence-based sorting removes senescent cells from mesenchymal stromal cell cultures.

Mesenchymal stromal cells (MSC) are used in cell therapy, but results depend on the unknown quality of cell populations. Extended culture time of MSC increases their senescent levels, leading to a critical loss of cell fitness. Here, we tested the suitability of MSC-sorting based on their FACS autofluorescence profile, for a rapid and non-invasive method of senescent cell elimination. Cells were classified in low- (LA) and high- (HA) autofluorescence groups, and results compared to the original MSC population (control). Three days after sorting, cells were screened by replicative senescence markers (cell volume, SA-ß-Gal assay and gene/protein expression) and MSC differentiation assays. The transcriptional profiles of sorted MSC were also analyzed by RNA-Seq. Compared to control, LA cells had 10% lower cell volume and autofluorescence, and 50% less SA-ß-Gal + cells. Instead, HA cells had 20% higher cell volume and autofluorescence, and 120% more SA-ß-Gal + cells. No changes in replicative senescence and differentiation potentials were observed between all groups. However, 68 genes (16 related to senescence) were significantly differentially expressed (DEG) between LA and other groups. Biological network of DEG identified CXCL12 as topological bottleneck. In summary, MSC sorting may have practical clinical implications to enhance the results of MSC-based therapies.

Novel Fast and Reliable Method for Nano-Erythrosome Production Using Shear Force.

PURPOSE: The production of nano-erythrosomes (NEs) by extrusion, which is considered the "gold standard", has several disadvantages such as difficult equipment assembly, long procedure time, variable pressure, and problems with sterility. An alternative approach, using ultrasound probe, has been shown to overheat the sample and have suboptimal results compared to the extrusion method. In our study, we propose, develop, and test a new method for the fabrication of NEs based on shear force and then compare it to the "gold standard" extrusion approach. METHODS: The new method consists of mechanical shear force disruption of the hemoglobin-depleted erythrocyte ghost membranes, with the aid of a rotor stator based tissue homogenizer. Using the same batches of erythrocyte ghost membranes, we compared NEs produced by shear force to NEs produced by the well-established extrusion approach. NEs were characterized for yield, size, encapsulation efficiency, morphology, and stability by flow cytometry (FC), transmission electron microscopy (TEM), and zeta potential analysis. RESULTS: The shear force based process was easier to set up, significantly faster, had better sterility control, and decreased variability between batches. The shear force method generated NEs with the desired size distribution (particles diameter ~125 nm), which were morphologically and functionally equivalent to the NEs produced by extrusion. NEs produced by shear force were stable in terms of counts, size, and fluorescence intensity for 3 weeks at +4°C. Moreover, they showed colloidal stability and minimal influence to centrifugal stress, turbulence shock, and hemolytic potential. CONCLUSION: The newly proposed shear force method allows faster, easier, and highly reproducible NEs production when compared to the conventional extrusion approach. The new setup allows simultaneous production of sterile batches of NEs, which have homogenous size distribution, good stability, and improved shelf life storage. The ability of the shear force method to process also high concentration samples indicates a future potential development of large-scale NEs production and industrial application, which has been a challenge for the extrusion method.

Autofluorescence is a Reliable in vitro Marker of Cellular Senescence in Human Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) are used in cell therapies, however cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies' outcomes. The determination of cellular senescence is time consuming and logistically intensive. Here, we propose the use of endogenous autofluorescence as real-time quantification of cellular senescence in human MSC, based on label-free flow cytometry analysis. We correlated cell autofluorescence to senescence using senescence-associated beta-galactosidase assay (SA-ß-Gal) with chromogenic (X-GAL) and fluorescent (C12FDG) substrates, gene expression of senescence markers (such as p16INK4A, p18INK4C, CCND2 and CDCA7) and telomere length. Autofluorescence was further correlated to MSC differentiation assays (adipogenesis, chondrogenesis and osteogenesis), MSC stemness markers (CD90/CD106) and cytokine secretion (IL-6 and MCP-1). Increased cell autofluorescence significantly correlated with increased SA-ß-Gal signal (both X-GAL and C12FDG substrates), cell volume and cell granularity, IL-6/MCP-1 secretion and with increased p16INK4A and CCND2 gene expression. Increased cell autofluorescence was negatively associated with the expression of the CD90/CD106 markers, osteogenic and chondrogenic differentiation potentials and p18INK4C and CDCA7 gene expression. Cell autofluorescence correlated neither with telomere length nor with adipogenic differentiation potential. We conclude that autofluorescence can be used as fast and non-invasive senescence assay for comparing MSC populations under controlled culture conditions.