Single-Cell RNA-Sequencing Reveals Heterogeneity and Transcriptional Dynamics in Porcine Circulating CD8+ T Cells.

Pigs are the most important source of meat and valuable biomedical models. However, the porcine immune system, especially the heterogeneity of CD8 T cell subtypes, has not been fully characterized. Here, using single-cell RNA sequencing, we identified 14 major cell types from peripheral blood circulating cells of pigs and observed remarkable heterogeneity among CD8 T cell types. Upon re-clustering of CD8+ T cells, we defined four CD8 T cell subtypes and revealed their potential differentiation trajectories and transcriptomic differences among them. Additionally, we identified transcription factors with potential regulatory roles in maintaining CD8 T cell differentiation. The cell-cell communication analysis inferred an extensive interaction between CD8 T cells and other immune cells. Finally, cross-species analysis further identified species-specific and conserved cell types across different species. Overall, our study provides the first insight into the extensive functional heterogeneity and state transitions among porcine CD8 T cell subtypes in pig peripheral blood, complements the knowledge of porcine immunity, and enhances its potential as a biomedical model.

Unveiling clinical applications of bacterial extracellular vesicles as natural nanomaterials in disease diagnosis and therapeutics.

Bacterial extracellular vesicles (BEVs) are naturally occurring bioactive membrane-bound nanoparticles released by both gram-negative and positive bacterial species, exhibiting a multifaceted role in mediating host-microbe interactions across various physiological conditions. Increasing evidence supports BEVs as essential mediators of intercellular exchange, influencing bacterial pathogenicity, disease mechanisms, and modulating the host immune response. However, the extent to which these BEV-mediated actions can be leveraged to predict disease onset, guide treatment strategies, and determine clinical outcomes remains uncertain, particularly in terms of their clinical translation potentials. This review briefly describes BEV biogenesis and their internalisation by recipient cells and summarises methods for isolation and characterization, essential for understanding their composition and cargo. Further, it discusses the potential of biofluid-associated BEVs as biomarkers for various diseases, spanning both cancer and non-cancerous conditions. Following this, we also outline the ongoing human clinical trials of using BEVs for vaccine development. In addition to disease diagnostics, this review explores the emerging research of using natural or engineered BEVs as smart nanomaterials for applications in anti-cancer therapy and bone regeneration. This discussion extends to key factors for unlocking the clinical potential of BEVs, such as standardization of BEVs isolation and characterisation, as well as other hurdles in translating these findings to the clinical setting. We propose that addressing these hurdles through collaborative research efforts and well-designed clinical trials holds the key to fully harnessing the clinical potential of BEVs. As this field advances, this review suggests that BEV-based nanomedicine has the potential to revolutionize disease management, paving the way for innovative diagnosis, therapeutics, and personalized medicine approaches. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) from both host cells and bacteria serve as multifunctional biomaterials and are emerging in the fields of biomedicine, bioengineering, and biomaterials. However, most of the current studies focus on host-derived EVs, leaving a gap in comprehensive research on bacteria-derived EVs (BEVs). Although BEVs offer an attractive option as nanomaterials for drug delivery systems, their unique nanostructure and easy-to-modify functions make them a potential method for disease diagnosis and treatment as well as vaccine development. Our work among the pioneering studies investigating the potential of BEVs as natural nanobiomaterials, plays a crucial role in both understanding the development of diseases and therapeutic interventions.

Tin-oxo nanoclusters for extreme ultraviolet photoresists: Effects of ligands, counterions, and doping.

The extreme ultraviolet (EUV) nanolithography technology is the keystone for developing the next-generation chips. As conventional chemically amplified resists are approaching the resolution limit, metal-containing photoresists, especially tin-oxo clusters, seize the opportunity to embrace this challenge owing to their small sizes, precise atomic structures, and strong EUV absorption. However, atomistic insights into the mechanism for regulating their photolithographic behavior are lacking. Herein, we systematically explored the effects of ligands, counterions, and endohedral doping on the photophysical properties of tin-oxo cage clusters by first-principles calculations combined with molecular dynamics simulations. Photoresists assembled by allyl-protected clusters with small-size OH- or Cl- counterions have a high absorption coefficient at the EUV wavelength of 13.5 nm and a low energy cost for ligand detachment and superior stability to ensure high sensitivity and strong etch resistance, respectively. The photoresist performance can further be improved by endohedral doping of the metal-oxo nanocage with Ag+ and Cd2+ ions, which exhibit superatomic characteristics and are likely to be synthesized in laboratory. These theoretical results provide useful guidance for modification of metal-oxo clusters for high-resolution EUV photolithography.

Exploration of decision aids to support advance care planning: A scoping review.

BACKGROUND: Advance care planning is a process through which people communicate their goals and preferences for future medical care. Due to the complexity of the decision-making process, decision aids can assist individuals in balancing potential benefits and risks of treatment options. OBJECTIVE: While decision aids have the potential to better promote advance care planning, their characteristics, content and application effectiveness are unclear and lack systematic review. Therefore, we aimed to explore these three aspects and establish a foundation for future research. DESIGN: Scoping review. METHODS: This scoping review adheres to the framework proposed by Arksey and O'Malley and the PRISMA-ScR list. Six English-language databases were systematically searched from the time of construction until 1 December 2023. Two researchers conducted the article screening and data extraction, and the extracted data was presented in written tables and narrative summaries. RESULTS: Of the 1479 titles and abstracts, 20 studies fulfilled the inclusion criteria. Types of decision aids were employed, mainly websites and videos. Decision aid's primary components center around 11 areas, such as furnishing information, exploring treatment and care preferences. The main manifestations were a significant increase in knowledge and improved recognition of patients' target value preferences. Among the aids, websites and videos for advance care planning have relatively high content acceptability and decision-making process satisfaction, but their feasibility has yet to be tested. CONCLUSIONS: Decision aids were varied, with content focused on describing key information and exploring treatment and care preferences. Regarding application effects, the aids successfully facilitated the advance care planning process and improved the quality of participants' decisions. Overall, decision aids are efficient in improving the decision-making process for implementing advance care planning in cancer and geriatric populations. In the future, personalised decision aids should be developed based on continuous optimization of tools' quality and promoted for clinical application. REPORTING METHOD: The paper has adhered to the EQUATOR guidelines and referenced the PRISMAg-ScR checklist. NO PATIENT OR PUBLIC CONTRIBUTION: This is a review without patient and public contribution. REGISTRATION: https://doi.org/10.17605/OSF.IO/YPHKF, Open Science DOI: 10.17605/OSF.IO/YPHKF.

Crosstalk between autophagy and ferroptosis mediate injury in ischemic stroke by generating reactive oxygen species.

Stroke represents a significant threat to global human health, characterized by high rates of morbidity, disability, and mortality. Predominantly, strokes are ischemic in nature. Ischemic stroke (IS) is influenced by various cell death pathways, notably autophagy and ferroptosis. Recent studies have increasingly highlighted the interplay between autophagy and ferroptosis, a process likely driven by the accumulation of reactive oxygen species (ROS). Post-IS, either the inhibition of autophagy or its excessive activation can escalate ROS levels. Concurrently, the interaction between ROS and lipids during ferroptosis further augments ROS accumulation. Elevated ROS levels can provoke endoplasmic reticulum stress-induced autophagy and, in conjunction with free iron (Fe2+), can trigger ferroptosis. Moreover, ROS contribute to protein and lipid oxidation, endothelial dysfunction, and an inflammatory response, all of which mediate secondary brain injury following IS. This review succinctly explores the mechanisms of ROS-mediated crosstalk between autophagy and ferroptosis and the detrimental impact of increased ROS on IS. It also offers novel perspectives for IS treatment strategies.

Mechanisms of intermittent theta-burst stimulation attenuating nerve injury after ischemic reperfusion in rats through endoplasmic reticulum stress and ferroptosis.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) exerts neuroprotective effects early in cerebral ischemia/reperfusion (I/R) injury. Intermittent theta-brust stimulation (iTBS), a more time-efficient modality of rTMS, improves the efficiency without at least decreasing the efficacy of the therapy. iTBS elevates cortical excitability, and in recent years it has become increasingly common to apply iTBS to patients in the early post-IS period. However, little is known about the neuroprotective mechanisms of iTBS. Endoplasmic reticulum stress (ERS), and ferroptosis have been shown to be involved in the development of I/R injury. We aimed to investigate the potential regulatory mechanisms by which iTBS attenuates neurological injury after I/R in rats. METHODS: Rats were randomly divided into three groups: sham-operated group, MCAO/R group, and MCAO/R + iTBS group, and were stimulated with iTBS 36 h after undergoing middle cerebral artery occlusion (MCAO) or sham-operated. The expression of ERS, ferroptosis, and apoptosis-related markers was subsequently detected by western blot assays. We also investigated the mechanism by which iTBS attenuates nerve injury after ischemic reperfusion in rats by using the modified Neurological Severity Score (mNSS) and the balance beam test to measure nerve function. RESULTS: iTBS performed early in I/R injury attenuated the levels of ERS, ferroptosis, and apoptosis, and improved neurological function, including mNSS and balance beam experiments. It is suggested that this mode of stimulation reduces the cost per treatment by several times without compromising the efficacy of the treatment and could be a practical and less costly intervention.

Evaluating the efficacy of balloon pulmonary angioplasty using quantitative analysis of SPECT pulmonary ventilation/perfusion scintigraphy in patients with chronic thromboembolic pulmonary hypertension.

Background: Single-photon emission computed tomography (SPECT) ventilation perfusion imaging is the main imaging method for the diagnosis of pulmonary embolism, and its application in the diagnosis and efficacy evaluation of chronic thromboembolic pulmonary hypertension (CTEPH) has been paid more and more attention. In recent years, with the development of computer software technology, ventilation/perfusion (V/Q) imaging quantitative analysis technology has become more and more mature. The objective of this study was to investigate the utility of quantitative analysis of pulmonary V/Q scintigraphy in evaluating the efficacy of balloon pulmonary angioplasty (BPA) in patients with CTEPH. Methods: In this retrospective analysis, we collected data of patients diagnosed with CTEPH who underwent BPA at the China-Japan Friendship Hospital from April 2018 to September 2020. The sample consisted of 23 males and 28 females, with an average age of 55.1±12.7 years. All patients underwent V/Q scintigraphy within one week before surgery, and we reviewed the pulmonary angiography within 1-3 months following the last BPA procedure. We repeated V/Q scintigraphy within 1 week before or after the pulmonary angiography, at the time of collecting clinical and hemodynamic parameters of these patients. We divided the patients into two groups based on the presence of residual pulmonary hypertension post-surgery and compared the pre- and post-operative quantitative pulmonary perfusion defect percentage scores (PPDs%) using the t-test. Results: In all, 102 V/Q scintigraphy scans were performed in 51 patients. The quantitative PPDs% were positively correlated with the hemodynamic indexes mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance (PVR), and mean right ventricular pressure (RVP) (r=0.605, 0.391, and 0.464, respectively, all P<0.001) and negatively correlated with the 6-minute walking distance (6MWD) (r=-0.254, P=0.010). The average preoperative quantitative PPDs% were (49.0±15.6)% which significantly decreased to (33.5±13.9)% after surgery (t=11.249, P<0.001). The preoperative quantitative PPDs% were (54.7±15.7)% and (44.0±13.8)% in the residual pulmonary hypertension group and the non-residual pulmonary hypertension group, respectively (t=2.599, P=0.012). The postoperative quantitative PPDs% were (41.5±12.5)% and (26.3±11.0)%, in the residual pulmonary hypertension group and the non-residual pulmonary hypertension group, respectively (t=4.647, P<0.001). Conclusions: In this study, we found that quantitative analysis of SPECT pulmonary V/Q scintigraphy adequately reflected the pulmonary artery pressure and clinical status in patients with CTEPH. Our results demonstrate its definite utility in predicting residual pulmonary hypertension and in evaluating the postoperative efficacy of BPA in patients with CTEPH.

Optical Activity and Excitonic Characteristics of Chiral CdSe Quantum Dots.

Introduction of chirality to colloidal semiconductor quantum dots (QDs) triggers a chiroptical effect. However, there remains a knowledge gap in the mechanism of chirality transfer and amplification from molecules to QDs. By time-dependent density functional theory calculations combined with a correlated electron-hole picture, we explored the chiroptical activity of CdSe QDs decorated with different chiral monocarboxylic acids from an excitonic perspective. Our calculations showed strong circular dichroism (CD) signals in the visible region for the chiral CdSe QDs. The excitonic states with large CD originate from QDs, while the chiral molecules break the orthogonality between electric and magnetic transition dipoles, which synergistically facilitates the prominent dissymmetric effect. The considered monocarboxylic acid chiral molecules all favor the bidentate adsorption configuration of the carboxyl group on the CdSe surface, endowing an identical CD signature but distinct excitonic characteristics. These findings are crucial for the regulation of chirality and excitons in semiconductor QDs to develop excitonic devices.

Effect of non-surgical periodontal therapy on salivary histone deacetylases expression: A prospective clinical study.

AIM: To evaluate the effect of non-surgical periodontal therapy (NSPT) on salivary histone deacetylases (HDACs) gene expression in patients with Stage III-IV periodontitis at baseline and at 3 and 6 months post NSPT treatment. MATERIALS AND METHODS: Twenty patients completed the study. Periodontitis (as well as the corresponding staging and grading) was diagnosed according to the 2017 World Workshop Classification. Clinical measures were recorded and whole unstimulated saliva was collected at baseline and at 3 and 6 months after NSPT. The expression of 11 HDACs was determined using reverse-transcription PCR, and the respective changes over time were evaluated. RESULTS: Six months after NSPT, significant improvements in all clinical periodontal parameters were observed, concomitant with significant up-regulation of HDAC2, 4, 6, 8, 9 and 11 expressions. Subgroup analyses of non-responders and responders revealed no significant differences in HDACs mRNA expression between groups at any time point. CONCLUSIONS: This prospective clinical study identified longitudinal changes in salivary HDACs expression in response to NSPT, which provides new insights into the epigenetic mechanisms underlying the pathobiology of periodontitis and creates avenues for the discovery of novel biomarkers.

Comparative transcriptome analysis of T lymphocyte subpopulations and identification of critical regulators defining porcine thymocyte identity.

Introduction: The development and migration of T cells in the thymus and peripheral tissues are crucial for maintaining adaptive immunity in mammals. However, the regulatory mechanisms underlying T cell development and thymocyte identity formation in pigs remain largely underexplored. Method: Here, by integrating bulk and single-cell RNA-sequencing data, we investigated regulatory signatures of porcine thymus and lymph node T cells. Results: The comparison of T cell subpopulations derived from porcine thymus and lymph nodes revealed that their transcriptomic differences were influenced more by tissue origin than by T cell phenotypes, and that lymph node cells exhibited greater transcriptional diversity than thymocytes. Through weighted gene co-expression network analysis (WGCNA), we identified the key modules and candidate hub genes regulating the heterogeneity of T cell subpopulations. Further, we integrated the porcine thymocyte dataset with peripheral blood mononuclear cell (PBMC) dataset to systematically compare transcriptomic differences between T cell types from different tissues. Based on single-cell datasets, we further identified the key transcription factors (TFs) responsible for maintaining porcine thymocyte identity and unveiled that these TFs coordinately regulated the entire T cell development process. Finally, we performed GWAS of cell type-specific differentially expressed genes (DEGs) and 30 complex traits, and found that the DEGs in thymus-related and peripheral blood-related cell types, especially CD4_SP cluster and CD8-related cluster, were significantly associated with pig productive and reproductive traits. Discussion: Our findings provide an insight into T cell development and lay a foundation for further exploring the porcine immune system and genetic mechanisms underlying complex traits in pigs.

Enriched environment treatment promotes neural functional recovery together with microglia polarization and remyelination after cerebral ischemia in rats.

BACKGROUND: Microglia activation and oligodendrocyte maturation are critical for remyelination after cerebral ischemia. Studies have shown that enriched environment (EE) can effectively alleviate stroke-induced neurological deficits. However, little is known about the mechanism associated with glial cells underlying the neuroprotection of EE. Therefore, this study focuses on investigating the effect of EE on activated microglia polarization as well as oligodendrogenesis in the progress of remyelination following cerebral ischemia. METHODS: The ischemia/reperfusion (I/R) injury model was established by middle cerebral artery occlusion (MCAO) in rats. Animals executed 4 weeks of environmental intervention after performing MCAO or sham surgery and were divided into sham, MCAO, and MCAO+EE groups. Cognitive function, myelin damage, microglia activation and polarization, inflammation, oligodendrogenesis, remyelination, and protein expression of the PI3K/AKT/GSK3ß signaling pathway were determined. RESULTS: The staining of NeuN indicated that the infarct size of MCAO rats was decreased under EE. EE intervention improved animal performance in the Morris water maze test and novel object recognition test, promoting the recovery of cognitive function after I/R injury. EE treatment alleviated myelin damage in MCAO rats, as evidenced by the lower fluorescence intensity ratio of SMI-32/MBP in MCAO+EE group. EE increased the fluorescence intensity ratio of NG2+/Ki67+/Olig2+, MBP, and MOG, enhancing the proliferation and differentiation of OPCs and oligodendrogenesis after MCAO. In terms of remyelination, more myelinated axons and lower G/ratio were detected in MCAO+EE rats compared with MCAO group. Moreover, EE treatment decreased the number of Iba1+/CD86+ M1 microglia, increased the number of Iba1+/CD206+ M2 microglia, and suppressed the inflammation response after I/R injury, which could be attributed to the augmented expression of PI3K/AKT/GSK3ß axis. CONCLUSION: EE improved long­term recovery of cognitive function after cerebral I/R injury, at least in part by promoting M2 microglia transformation through activation of the PI3K/AKT/GSK3ß signaling pathway, inhibiting inflammation to provide a favorable microenvironment for oligodendrocyte maturation and remyelination. The effect of the EE on myelin and inflammation could account for the neuroprotection provided by EE.

3D bioprinted small extracellular vesicles from periodontal cells enhance mesenchymal stromal cell function.

Recent research indicates that combining 3D bioprinting and small extracellular vesicles (sEVs) offers a promising 'cell-free' regenerative medicine approach for various tissue engineering applications. Nonetheless, the majority of existing research has focused on bioprinting of sEVs sourced from cell lines. There remains a notable gap in research regarding the bioprinting of sEVs derived from primary human periodontal cells and their potential impact on ligamentous and osteogenic differentiation. Here, we investigated the effect of 3D bioprinted periodontal cell sEVs constructs on the differentiation potential of human buccal fat pad-derived mesenchymal stromal cells (hBFP-MSCs). Periodontal cell-derived sEVs were enriched by size exclusion chromatography (SEC) with particle-shaped morphology, and characterized by being smaller than 200 nm in size and CD9/CD63/CD81 positive, from primary human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). The sEVs were then 3D bioprinted in 10 % gelatin methacryloyl (GelMA) via microextrusion bioprinting. Release of sEVs from bioprinted constructs was determined by DiO-labelling and confocal imaging, and CD9 ELISA. Attachment and ligament/osteogenic/cementogenic differentiation of hBFP-MSCs was assessed on bioprinted GelMA, without and with sEVs (GelMA/hPDLCs-sEVs and GelMA/hGFs-sEVs), scaffolds. hBFP-MSCs seeded on the bioprinted sEVs constructs spread well with significantly enhanced focal adhesion, mechanotransduction associated gene expression, and ligament and osteogenesis/cementogenesis differentiation markers in GelMA/hPDLCs-sEVs, compared to GelMA/hGFs-sEVs and GelMA groups. A 2-week osteogenic and ligamentous differentiation showed enhanced ALP staining, calcium formation and toluidine blue stained cells in hBFP-MSCs on bioprinted GelMA/hPDLCs-sEVs constructs compared to the other two groups. The proof-of-concept data from this study supports the notion that 3D bioprinted GelMA/hPDLCs-sEVs scaffolds promote cell attachment, as well as ligamentous, osteogenic and cementogenic differentiation, of hBFP-MSCs in vitro.

Saliva biofilm-derived outer membrane vesicles regulate biofilm formation and immune response of oral epithelial cells on titanium surfaces.

OBJECTIVES: While the significant roles of outer membrane vesicles (OMVs) from individual oral bacterial species in bacterial-host interactions are known, the involvement of saliva biofilm-derived OMVs in peri-implant disease pathogenesis remains unclear. This study aimed to investigate the effect of saliva biofilm-derived OMVs on regulating saliva biofilm formation and modulating the immune response of the epithelial cells on titanium surfaces. MATERIALS AND METHODS: Saliva derived biofilms were cultured on tissue culture plates (TCP) for 4 days using pooled saliva from four healthy donors. OMVs secreted from the TCP bound biofilm (referred to as OMVs or healthy saliva biofilm OMVs) were enriched using the size-exclusion chromatography method. We then evaluated the effects of these OMVs on the viability, metabolic activity, and the presence of oral pathogens in saliva biofilm grown on titanium discs for 24 h and 72 h. Furthermore, the impact of OMVs on the mRNA expression and inflammatory cytokines [interleukin (IL)-6, IL-1α, and monocyte chemoattractant protein-1 (MCP-1)] in human oral epithelial cells (OKF6/TERT-2) was investigated using RT-qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Healthy saliva biofilm OMVs improved the biomass and activity of saliva biofilm cultured on the titanium surfaces, with inhibited Porphyromonas gingivalis and Fusobacterium nucleatum, and enhanced Streptococcus mutans expression. Additionally, OMVs increased pro-inflammatory cytokine IL-6 mRNA and IL-6 cytokine expression in human oral epithelial cells. However, IL-1α and MCP-1 cytokines were inhibited 24-hour post-incubation with OMVs. CONCLUSION: Healthy saliva biofilm derived OMVs regulate the activity and pathogen composition of biofilms formed on titanium, while modulating the secretion of pro-inflammation factors of oral epithelial cells grown on titanium surfaces. CLINICAL RELEVANCE: Healthy saliva biofilm OMVs may regulate the early biofilm formation on abutment surfaces and modulate epithelial cell immune response, which may alter the peri-implant niche and participate in the pathogenesis of peri-implant disease.

Microvesicle-eluting nano-engineered implants influence inflammatory response of keratinocytes.

Besides enhancing osseo- and soft tissue integration, modulating inflammation at the implant site is also crucial for dental implant success. Uncontrolled peri-implant inflammation can cause significant loss of surrounding tissue and implant failure. It was recently shown that microvesicles (MVs), a less-studied type of extracellular vesicles, play a crucial role in cell-to-cell communication and may modulate angiogenesis and inflammatory response. The effect of MVs on regulating inflammation at an implant site, however, remains unexplored. In the current study, MVs were isolated and characterised from human primary gingival fibroblasts (hGFs) and loaded within titania nanotubes (TNTs, fabricated via anodisation on 3D Ti wire implants) towards their local release. The modified implants were characterised using SEM and confocal imaging to confirm the loading and local release of MVs from TNTs. In vitro studies demonstrated the internalisation of hGFs-MVs by human gingival keratinocytes (OKF6/TERT2 cell line), which caused a significant reduction in the production of pro-inflammatory cytokines. The results support MVs-releasing TNTs as a promising implant surface modification strategy to reduce inflammation, paving the way for further advancements in therapeutic dental implants.

Clinical usage of dental stem cells and their derived extracellular vesicles.

Stem cell-based therapies remain at the forefront of tissue engineering and regenerative medicine because stem cells are a unique cell source with enormous potential to treat incurable diseases and even extend lifespans. The search for the best stem cell candidates continues to evolve and in recent years, dental stem cells have received significant attention due to their easy accessibility, high plasticity, and multipotential properties. Dental stem cells have been the subject of extensive research in both animal models and human clinical trials over the past two decades, and have demonstrated significant potential in ocular therapy, bone tissue engineering, and, of course, therapeutic applications in dentistry such as regenerative endodontics and periodontal tissue regeneration. These new sources of cells may be advantageous for cellular therapy and the advancement of regenerative medicine strategies, such as allogeneic transplantation or therapy with extracellular vesicles (EVs), which are functional nanoscale membrane vesicles produced by cells. This chapter discusses the accumulating research findings on cell-based regenerative therapy utilizing dental stem cells and their derived EVs, which could be a viable tool for the treatment of a variety of diseases and hence extremely valuable to mankind in the long run.

Engineered adult stem cells: Current clinical trials status of disease treatment.

Regenerative medicine is an interdisciplinary field involving the process of replacing and regenerating cells/tissues or organs by integrating medicine, science, and engineering principles to enhance the intrinsic regenerative capacity of the host. Recently, engineered adult stem cells have gained attention for their potential use in regenerative medicine by reducing inflammation and modulating the immune system. This chapter introduces adult stem cell engineering and chimeric antigen receptor T cells (CAR T) gene therapy and summarises current engineered stem cell- and extracellular vesicles (EVs)-focused clinical trial studies that provide the basis for the proposal of a personalised medicine approach to diseases diagnosis and treatment.

An in vitro dynamic bioreactor model for evaluating antimicrobial effectiveness on periodontal polymicrobial biofilms: a proof-of-concept study.

BACKGROUND: The aim of this study was to investigate an in vitro dynamic bioreactor model by evaluating the antimicrobial effect of clinically relevant amoxicillin doses on polymicrobial microcosm biofilms derived from subgingival plaque. METHODS: Biofilms from pooled subgingival plaque were grown for 108  hours in control and experimental dynamic biofilm reactors. Amoxicillin was subsequently infused into the experimental reactor to simulate the pharmacokinetic profile of a standard 500 mg thrice-daily dosing regimen over 5 days and biofilms were assessed by live/dead staining, scanning electron microscopy, and quantitative polymerase chain reaction. RESULTS: Following establishment of the oral microcosm biofilms, confocal imaging analysis showed a significant increase in dead bacteria at 8 hours (p = 0.0095), 48 hours (p = 0.0070), 96 hours (p = 0.0140), and 120 hours (p < 0.0001) in the amoxicillin-treated biofilms compared to the control biofilms. Nevertheless, viable bacteria remained in the center of the biofilm at all timepoints. Significant reductions/elimination in Campylobacter rectus, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Peptostreptococcus anaerobius was observed among the amoxicillin-treated biofilms at the 96 and 120 hour timepoints. CONCLUSION: A novel in vitro dynamic model of oral microcosm biofilms was effective in modeling the antimicrobial effect of a pharmacokinetically simulated clinically relevant dose of amoxicillin.

Effects of periodontal cells-derived extracellular vesicles on mesenchymal stromal cell function.

OBJECTIVE: To enrich and compare three extracellular vesicles-EV subtypes (apoptotic bodies, microvesicles and small EV) from three periodontal cells (periodontal ligament cells-PDLCs, alveolar bone-derived osteoblasts-OBs and gingival fibroblasts-GFs), and assess uptake and cell function changes in buccal fat pad-derived mesenchymal stromal cells (BFP-MSCs). BACKGROUND: Periodontal cells such as PDLCs, OBs and GFs have the potential to enhance bone and periodontal regeneration, but face significant challenges, such as the regulatory and cost implications of in vitro cell culture and storage. To address these challenges, it is important to explore alternative 'cell-free' strategies, such as extracellular vesicles which have emerged as promising tools in regenerative medicine, to facilitate osteogenic differentiation and bone regeneration. METHODS AND MATERIALS: Serial centrifuges at 2600 and 16 000 g were used to isolate apoptotic bodies and microvesicles respectively. Small EV-sEV was enriched by our in-house size exclusion chromatography (SEC). The cellular uptake, proliferation, migration and osteogenic/adipogenic differentiation genes were analysed after EVs uptake in BFP-MSCs. RESULTS: Three EV subtypes were enriched and characterised by morphology, particle size and EV-associated protein expression-CD9. Cellular uptake of the three EVs subtypes was observed in BFP-MSCs for up to 7 days. sEV from the three periodontal cells promoted proliferation, migration and osteogenic gene expression. hOBs-sEV showed superior levels of osteogenesis markers compared to that hPDLCs-sEV and hGFs-sEV, while hOBs-16k EV promoted adipogenic gene expression compared to that from hPDLCs and hGFs. CONCLUSIONS: Our proof-of-concept data demonstrate that hOBs-sEV might be an alternative cell-free therapeutic for bone tissue engineering.

Evaluating models and assessment techniques for understanding oral biofilm complexity.

Oral biofilms are three-dimensional (3D) complex entities initiating dental diseases and have been evaluated extensively in the scientific literature using several biofilm models and assessment techniques. The list of biofilm models and assessment techniques may overwhelm a novice biofilm researcher. This narrative review aims to summarize the existing literature on biofilm models and assessment techniques, providing additional information on selecting an appropriate model and corresponding assessment techniques, which may be useful as a guide to the beginner biofilm investigator and as a refresher to experienced researchers. The review addresses previously established 2D models, outlining their advantages and limitations based on the growth environment, availability of nutrients, and the number of bacterial species, while also exploring novel 3D biofilm models. The growth of biofilms on clinically relevant 3D models, particularly melt electrowritten fibrous scaffolds, is discussed with a specific focus that has not been previously reported. Relevant studies on validated oral microcosm models that have recently gaining prominence are summarized. The review analyses the advantages and limitations of biofilm assessment methods, including colony forming unit culture, crystal violet, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt assays, confocal microscopy, fluorescence in situ hybridization, scanning electron microscopy, quantitative polymerase chain reaction, and next-generation sequencing. The use of more complex models with advanced assessment methodologies, subject to the availability of equipment/facilities, may help in developing clinically relevant biofilms and answering appropriate research questions.