Extracellular vesicles from apoptotic BMSCs ameliorate osteoporosis via transporting regenerative signals.

Rationale: Mesenchymal stromal cells (MSCs) are considered a promising resource for cell therapy, exhibiting efficacy in ameliorating diverse bone diseases. However, most MSCs undergo apoptosis shortly after transplantation and produce apoptotic extracellular vesicles (ApoEVs). This study aims to clarify the potential role of ApoEVs from apoptotic MSCs in ameliorating osteoporosis and molecular mechanism. Methods: In this study, Dio-labeled bone marrow mesenchymal stem cells (BMSCs) were injected into mice to track BMSCs apoptosis and ApoEVs production. ApoEVs were isolated from BMSCs after inducing apoptosis, the morphology, size distribution, marker proteins expression of ApoEVs were characterized. Protein mass spectrometry analysis revealed functional differences in proteins between ApoEVs and BMSCs. BMSCs were adopted to test the cellular response to ApoEVs. Ovariectomy mice were used to further compare the ability of ApoEVs in promoting bone formation. SiRNA and lentivirus were used for gain and loss-of-function assay. Results: The results showed that BMSCs underwent apoptosis within 2 days after being injected into mice and produce a substantial quantity of ApoEVs. Proteomic analysis revealed that ApoEVs carried a diverse functional array of proteins, and easily traversed the circulation to reach the bone. After being phagocytized by endogenous BMSCs, ApoEVs efficiently promoted the proliferation, migration, and osteogenic differentiation of BMSCs. In an osteoporosis mouse model, treatment of ApoEVs alleviated bone loss and promoted bone formation. Mechanistically, ApoEVs carried Ras protein and activated the Ras/Raf1/Mek/Erk pathway to promote osteogenesis and bone formation in vitro and in vivo. Conclusion: Given that BMSC-derived ApoEVs are high-yield and easily obtained, our data underscore the substantive role of ApoEVs from dying BMSCs to treat bone loss, presenting broad implications for cell-free therapeutic modalities.

Notch Signaling Hydrogels Enable Rapid Vascularization and Promote Dental Pulp Tissue Regeneration.

Successful dental pulp regeneration is closely associated with rapid revascularization and angiogenesis, processes driven by the Jagged1(JAG1)/Notch signaling pathway. However, soluble Notch ligands have proven ineffective in activating this pathway. To overcome this limitation, a Notch signaling hydrogel is developed by indirectly immobilizing JAG1, aimed at precisely directing the regeneration of vascularized pulp tissue. This hydrogel displays favorable mechanical properties and biocompatibility. Cultivating dental pulp stem cells (DPSCs) and endothelial cells (ECs) on this hydrogel significantly upregulate Notch target genes and key proangiogenic markers expression. Three-dimensional (3D) culture assays demonstrate Notch signaling hydrogels improve effectiveness by facilitating encapsulated cell differentiation, enhancing their paracrine functions, and promoting capillary lumen formation. Furthermore, it effectively communicates with the Wnt signaling pathway, creating an odontoinductive microenvironment for pulp-dentin complex formation. In vivo studies show that short-term transplantation of the Notch signaling hydrogel accelerates angiogenesis, stabilizes capillary-like structures, and improves cell survival. Long-term transplantation further confirms its capability to promote the formation of pulp-like tissues rich in blood vessels and peripheral nerve-like structures. In conclusion, this study introduces a feasible and effective hydrogel tailored to specifically regulate the JAG1/Notch signaling pathway, showing potential in advancing regenerative strategies for dental pulp tissue.

Recent adavances of functional modules for tooth regeneration.

Dental diseases, such as dental caries and periodontal disorders, constitute a major global health challenge, affecting millions worldwide and often resulting in tooth loss. Traditional dental treatments, though beneficial, typically cannot fully restore the natural functions and structures of teeth. This limitation has prompted growing interest in innovative strategies for tooth regeneration methods. Among these, the use of dental stem cells to generate functional tooth modules represents an emerging and promising approach in dental tissue engineering. These modules aim to closely replicate the intricate morphology and essential physiological functions of dental tissues. Recent advancements in regenerative research have not only enhanced the assembly techniques for these modules but also highlighted their therapeutic potential in addressing various dental diseases. In this review, we discuss the latest progress in the construction of functional tooth modules, especially on regenerating dental pulp, periodontal tissue, and tooth roots.

Nanotube topography rejuvenates the senescence of mesenchymal stem cells by activating YAP signalling.

Improving the regenerative ability of senescent stem cells is a critical issue in combating aging. The destiny and function of senescent stem cells are controlled by the niche, including the physical architecture of the surface of the extracellular matrix (ECM). In this study, we explored the functions of TiO2 nanotube topography on mesenchymal stem cells (MSCs) under senescence, as well as its mechanical effects on senescence. First, we created different nanotube topographies on the titanium samples. Next, we cultured senescent mesenchymal stem cells (S-MSCs) on samples with various nanotube topographies to determine suitable parameters. We found nanotube with a diameter of 10 nm significantly alleviated the cellular senescence of S-MSCs and improved the osteogenic differentiation of S-MSCs in vitro. Using an ectopic periodontium regeneration model, we confirmed that specific nanotube topography could promote tissue regeneration of S-MSCs in vivo. Moreover, we demonstrated that nanotube topography activated YAP in S-MSCs and reformed nuclear-cytoskeletal morphology to inhibit senescence. Taken together, our study establishes a bridge linking between nano-topography, mechanics, and senescence, suggesting a potential strategy to improve tissue regeneration in aged individuals by providing optimized surface topography on biomaterials.

[Research progress in regulation of hair growth by dermal adipose tissue].

Objective: To summarize the dynamic and synchronized changes between the hair cycle and dermal adipose tissue as well as the impact of dermal adipose tissue on hair growth, and to provide a new research idea for the clinical treatment of hair loss. Methods: An extensive review of relevant literature both domestic and international was conducted, analyzing and summarizing the impact of dermal adipose precursor cells, mature dermal adipocytes, and the processes of adipogenesis in dermal adipose tissue on the transition of hair cycle phases. Results: Dermal adipose tissue is anatomically adjacent to hair follicles and closely related to the changes in the hair cycle. The proliferation and differentiation of dermal adipose precursor cells promote the transition of hair cycle from telogen to anagen, while mature adipocytes can accelerate the transition from anagen to catagen of the hair cycle by expressing signaling molecules, with adipogenesis in dermal adipose tissue and hair cycle transition signaling coexistence. Conclusion: Dermal adipose tissue affects the transition of the hair cycle and regulates hair growth by secreting various signaling molecules. However, the quantity and depth of existing literature are far from sufficient to fully elucidate its prominent role in regulating the hair cycle, and the specific regulatory mechanisms needs to be further studied.

Small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells inhibit cell apoptosis and alveolar bone loss in periodontitis.

OBJECTIVE: This study aimed to explore the effects of small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells (L-D-sEV) on periodontal ligament cells from periodontitis affected teeth (p-PDLCs) in vitro and experimental periodontitis in mice. DESIGN: In vitro, the biological function of p-PDLCs and the underlying molecular mechanism were investigated by flow cytometry, Western blot, and quantitative real-time PCR (qRT-PCR) analysis. Eighteen-eight-week-old male C57BL/6 mice were randomly divided into three groups: control (Con), periodontitis (Peri), and L-D-sEV groups. Mice periodontitis model was induced by placing the 5-0 silk thread (around the maxillary second molar) and P.gingivalis (1 ×107 CFUs per mouse). In vivo, the alveolar bone loss, osteoclast activity, and macrophage polarization were measured by micro-computed tomography and histological analysis. RESULTS: In vitro, the RANKL/OPG ratio and phosphorylation of JNK and P38 protein levels of p-PDLCs were significantly decreased after L-D-sEV administration. Besides, flow cytometry and qRT-PCR analysis showed that L-D-sEV reduced apoptosis of p-PDLCs, down-regulated apoptosis-related genes Caspase-3 and BCL-2-Associated X expression, and up-regulated B-cell lymphoma-2 gene levels. In vivo, L-D-sEV administration significantly reduced alveolar bone loss, inhibited osteoclast activity, and induced M2 polarization. The histological analysis showed that iNOS/CD206, RANKL/OPG, p-JNK/JNK, and p-P38/P38 ratios were significantly lower in the L-D-sEV group than in the Peri group. CONCLUSIONS: L-D-sEV administration alleviated alveolar bone loss by mediating RANKL/OPG-related osteoclast activity and M2 macrophage polarization, alleviating p-PDLCs apoptosis and proliferation via the JNK and P38 pathways.

Functional diversity of apoptotic vesicle subpopulations from bone marrow mesenchymal stem cells in tissue regeneration.

Apoptosis releases numerous apoptotic vesicles that regulate processes such as cell proliferation, immunity, and tissue regeneration and repair. Now, it has also emerged as an attractive candidate for biotherapeutics. However, apoptotic vesicles encompass a diverse range of subtypes, and it remains unclear which specific subtypes play a pivotal role. In this study, we successfully isolated different apoptotic vesicle subtypes based on their sizes and characterized them using NTA and TEM techniques, respectively. We compared the functional variances among the distinct subtypes of apoptotic vesicles in terms of stem cell proliferation, migration, and differentiation, as well as for endothelial cell and macrophage function, effectively identifying subtypes that exhibit discernible functional differences. ApoSEV (with diameter <1000 nm) promoted stem cell proliferation, migration, and multi-potent differentiation, and accelerated skin wound healing of diabetes mouse model, while apoBD (with diameter >1000 nm) played the opposite effect on cell function and tissue regeneration. Lastly, employing protein analysis and gene sequencing techniques, we elucidated the intrinsic mechanisms underlying these differences between different subtypes of apoEVs. Collectively, this study identified that apoptotic vesicle subtypes possessed distinct bio-functions in regulating stem cell function and behaviour and modulating tissue regeneration, which primarily attribute to the distinct profiling of protein and mRNA in different subtypes. This comprehensive analysis of specific subtypes of apoEVs would provide novel insights for potential therapeutic applications in cell biology and tissue regeneration.

Sustained free chlorine-releasing polydimethylsiloxane/Ca(ClO)2 materials with long-lasting disinfection efficacy.

A novel sustained chlorine-releasing polydimethylsiloxane/Ca(ClO)2 (PDMS/Ca(ClO)2) material was fabricated by encapsulating Ca(ClO)2 in a PDMS matrix due to its high hydrophobicity and high chemical stability, which showed immediate-responsive and long-lasting antibacterial capabilities in aqueous conditions. Free chlorine could be released from the PDMS/Ca(ClO)2 after immersion in water for 2 min and could also be sustainedly released for 2 weeks, while the released concentration is negatively related to the duration time and positively with the initial Ca(ClO)2 contents. Additionally, Ca(ClO)2 powder as a filler significantly affects the crosslinking and pore size of PDMS. The PDMS/Ca(ClO)2 materials exhibited enduring antibacterial performance against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in both planktonic and multispecies-biofilm status. It is expected that this PDMS/Ca(ClO)2 material and its similar composite would be promising candidates for wide sustainable disinfection applications in biomedical and industrial fields.

Neuritin promotes autophagic flux by inhibiting the cGAS-STING pathway to alleviate brain injury after subarachnoid haemorrhage.

BACKGROUND: Early brain injury (EBI) is closely associated with poor prognosis in patients with subarachnoid haemorrhage (SAH), with autophagy playing a pivotal role in EBI. However, research has shown that the stimulator of interferon genes (STING) pathway impacts autophagic flux. While the regulatory impact of neuritin on EBI and autophagic flux has been established previously, the underlying mechanism remains unclear. This study aimed to determine the role of the cGAS-STING pathway in neuritin-mediated regulation of autophagic flux following SAH. METHODS: A SAH model was established in male Sprague-Dawley rats via intravascular perforation. Neuritin overexpressions using adeno-associated virus, the STING antagonist "C-176," and the activator, "CMA," were determined to investigate the cGAS-STING pathway's influence on autophagic flux and brain injury post-SAH, along with the neuritin's regulatory effect on STING. In this study, SAH grade, neurological score, haematoxylin and eosin (H&E) staining, brain water content (BWC), sandwich enzyme-linked immunosorbent assay, Evans blue staining, immunofluorescence staining, western blot analysis, and transmission electron microscopy (TEM) were examined. RESULTS: Neuritin overexpression significantly ameliorated neurobehavioural scores, blood-brain barrier injury, brain oedema, and impaired autophagic flux in SAH-induced rats. STING expression remarkably increased post-SAH. C-176 and CMA mitigated and aggravated autophagic flux injury and brain injury, respectively, while inhibiting and enhancing STING, respectively. Particularly, CMA treatment nullified the protective effects of neuritin against autophagic flux and mitigated brain injury. CONCLUSION: Neuritin alleviated EBI by restoring impaired autophagic flux after SAH through the regulation of the cGAS-STING pathway.

Eliminating senescent cells by white adipose tissue-targeted senotherapy alleviates age-related hepatic steatosis through decreasing lipolysis.

Cellular senescence is an important risk factor in the development of hepatic steatosis. Senolytics present therapeutic effects on age-related hepatic steatosis without eliminating senescent hepatocytes directly. Therefore, it highlights the need to find senolytics' therapeutic targets. Dysfunction of adipose tissue underlies the critical pathogenesis of lipotoxicity in the liver. However, the correlation between adipose tissue and hepatic steatosis during aging and its underlying molecular mechanism remains poorly understood. We explored the correlation between white adipose tissue (WAT) and the liver during aging and evaluated the effect of lipolysis of aged WAT on hepatic steatosis and hepatocyte senescence. We screened out the ideal senolytics for WAT and developed a WAT-targeted delivery system for senotherapy. We assessed senescence and lipolysis of WAT and hepatic lipid accumulation after treatment. The results displayed that aging accelerated cellular senescence and facilitated lipolysis of WAT. Free fatty acids (FFAs) generated by WAT during aging enhanced hepatic steatosis and induced hepatocyte senescence. The combined usage of dasatinib and quercetin was screened out as the ideal senolytics to eliminate senescent cells in WAT. To minimize non-specific distribution and enhance the effectiveness of senolytics, liposomes decorated with WAT affinity peptide P3 were constructed for senotherapy in vivo. In vivo study, WAT-targeted treatment eliminated senescent cells in WAT and reduced lipolysis, resulting in the alleviation of hepatic lipid accumulation and hepatocyte senescence when compared to non-targeted treatment, providing a novel tissue-targeted, effective and safe senotherapy for age-related hepatic steatosis.

Cellular Senescence in Craniofacial Tissue Regeneration: Inducers, Biomarkers, and Interventions.

Craniofacial defects and dental tissue loss have significant negative impacts on the structure and function of jaws and face, often resulting in psychological issues in patients, emphasizing the urgent need for effective craniofacial tissue reconstruction. Unfortunately, natural regeneration of these tissues is limited. Dental-derived mesenchymal stem cells (MSCs) have emerged as a promising resource for tissue engineering-based therapeutic approaches. However, the clinical outcomes of MSC-based transplantation have not met expectations due to various complex reasons, and cellular senescence is recognized as one of the potential mechanisms contributing to the suboptimal results. The quality of MSC decreases during large-scale in vitro expansion, and it is also influenced by the age and the health status of donors. To address these challenges, extensive efforts have been made to developing strategies to combat senescence in tissue engineering, leveraging on current knowledge of underlying mechanisms. This review aims to elucidate the impact of cell senescence in craniofacial and dental regeneration and provides an overview of state-of-the-art antisenescence strategies. We first discuss the potential factors that trigger cell senescence in craniofacial tissue engineering. Then we describe senescence biomarkers, monitoring methods for senescent MSCs, and their underlying molecular mechanisms. The primary focus of this review is on current strategies to inhibit and alleviate cell senescence in tissue engineering. We summarize the strategies concerning the prevention of cell senescence, senolysis, modulation of the senescent associated secretory phenotype, and reversal of senescent MSCs, offering promising opportunities to overcome the challenges associated with cell senescence in craniofacial tissue engineering.

Hierarchically patterned triple-layered gelatin-based electrospun membrane functionalized by cell-specific extracellular matrix for periodontal regeneration.

OBJECTIVES: Regenerating the periodontium poses a critical challenge in oral medicine. To repair various periodontal defects, it is necessary to adopt a bio-scaffold that provides both the architecture and bioactive cues for local stem cells to migrate, reside, proliferate, and differentiate. The objective of this study is to combine a cell-specific decellularized extracellular matrix (ECM) and a biomimetic electrospinning scaffold to regenerate severely destructed periodontium. METHODS: SEM, water contact angle (WCA), live/dead staining, swelling ratio, tensile test and immune-fluorescent staining were used to define the suitable topography for certain dental stem cells seeding and culturing. Transwell assay, CCK-8, Alizarin Red staining and PCR immune-fluorescent staining were used to determine ideal cell-specific ECM for PDLSCs/BMSCs migration, viability, and oriented differentiation. A biodegradable triple-layered electrospun scaffold (TLS) was fabricated by electrospinning with aligned fibers on both surfaces and a polyporous structure in the middle. The morphology and inter-porous structure of the TLS were characterized by SEM and mercury intrusion porosimetry (MIP). The surface of the TLS was functionalized with cell-specific ECM (Bi-ECM-TLS) through decellularization of the cell sheets cultured on the scaffold. The regenerative outcome of Bi-ECM-TLS was assessed by an in-situ rat periodontal defect model. Micro-CT, HE-staining, Masson's trichome staining, Sirius Red staining and Immunofluorescent staining were used for histological analysis. RESULTS: Aligned Gelatin/PCL fibrous membrane (GPA) was most effective for both PDLSCs and BMSCs in culture with WCA around 50 degrees and better mechanical strength than the rest. MSCs favored the same type of ECM (cell-specific ECM), and their regenerative properties were effectively induced with better chemotaxis, proliferative and differentiating behaviors. TLS characterization showed that TLS possessed aligned-random-aligned structure and inter-porous structure. In a rat model of periodontal defects, the TLS functionalized by BMSC-specific ECM for bone regeneration and PDLSC-specific ECM demonstrated highest BV/TV ratio, best bone structure and ligament fiber orientation and blood vessel formation, suggesting optimal performance in regenerating both alveolar bone and periodontal ligaments over TLS, single-ECM loaded TLS and r-Bi-ECM-TLS. SIGNIFICANCE: This study highlights the importance of combining a cell-specific decellularized ECM and a biomimetic electrospinning scaffold for targeted periodontal tissue regeneration, with potential implications for periodontal tissue engineering and improved patient outcomes.

Biomimetic crystallization for long-pursued -COOH-functionalized gold nanocluster with near-infrared phosphorescence.

As an interdisciplinary product, water-soluble gold nanoclusters (AuNCs) stabilized by ligands containing carboxyl (-COOH) group have garnered significant attention from synthetic chemists and biologists due to their immense potential for biomedical applications. However, revealing the crystallographic structures of -COOH-functionalized AuNCs remains a bottleneck. Herein, we successfully applied the salting-out method to obtain a series of high-quality single crystals of -COOH-functionalized Au25 nanoclusters and revealed their crystallographic structures. Particularly, K3Au25(2-Hmna)9(mna)6]- (Au25a) protected by 2-mercaptonicotinic acid features an unprecedented tetrameric Au4(SRS)3(SRS,N)2 staple motifs surrounding the icosahedral Au13 kernel, breaking the traditional perception on the structure of Au25(SR)18. Au25a exhibits a distinct near-infrared emission at 970 nm with long lifetime of 8690 ns, which have been studied by transient absorption spectroscopy and time-dependent density functional theory. This work compensates for the research gap in the experimental structure of -COOH-functionalized AuNCs and opens up a new avenue to explore their structure-property correlations.

Injectable Xenogeneic Dental Pulp Decellularized Extracellular Matrix Hydrogel Promotes Functional Dental Pulp Regeneration.

The present challenge in dental pulp tissue engineering scaffold materials lies in the development of tissue-specific scaffolds that are conducive to an optimal regenerative microenvironment and capable of accommodating intricate root canal systems. This study utilized porcine dental pulp to derive the decellularized extracellular matrix (dECM) via appropriate decellularization protocols. The resultant dECM was dissolved in an acid pepsin solution to form dECM hydrogels. The analysis encompassed evaluating the microstructure and rheological properties of dECM hydrogels and evaluated their biological properties, including in vitro cell viability, proliferation, migration, tube formation, odontogenic, and neurogenic differentiation. Gelatin methacrylate (GelMA) hydrogel served as the control. Subsequently, hydrogels were injected into treated dentin matrix tubes and transplanted subcutaneously into nude mice to regenerate dental pulp tissue in vivo. The results showed that dECM hydrogels exhibited exceptional injectability and responsiveness to physiological temperature. It supported the survival, odontogenic, and neurogenic differentiation of dental pulp stem cells in a 3D culture setting. Moreover, it exhibited a superior ability to promote cell migration and angiogenesis compared to GelMA hydrogel in vitro. Additionally, the dECM hydrogel demonstrated the capability to regenerate pulp-like tissue with abundant blood vessels and a fully formed odontoblast-like cell layer in vivo. These findings highlight the potential of porcine dental pulp dECM hydrogel as a specialized scaffold material for dental pulp regeneration.

Skeletal muscle-derived extracellular vesicles transport glycolytic enzymes to mediate muscle-to-bone crosstalk.

Identification of cues originating from skeletal muscle that govern bone formation is essential for understanding the crosstalk between muscle and bone and for developing therapies for degenerative bone diseases. Here, we identified that skeletal muscle secreted multiple extracellular vesicles (Mu-EVs). These Mu-EVs traveled through the bloodstream to reach bone, where they were phagocytized by bone marrow mesenchymal stem/stromal cells (BMSCs). Mu-EVs promoted osteogenic differentiation of BMSCs and protected against disuse osteoporosis in mice. The quantity and bioactivity of Mu-EVs were tightly correlated with the function of skeletal muscle. Proteomic analysis revealed numerous proteins in Mu-EVs, some potentially regulating bone metabolism, especially glycolysis. Subsequent investigations indicated that Mu-EVs promoted the glycolysis of BMSCs by delivering lactate dehydrogenase A into these cells. In summary, these findings reveal that Mu-EVs play a vital role in BMSC metabolism regulation and bone formation stimulation, offering a promising approach for treating disuse osteoporosis.

ApoSEVs-Mediated Modulation of Versatile Target Cells Promotes Diabetic Wound Healing: Unveiling a Promising Strategy.

Background: Diabetic chronic wounds present a formidable challenge in clinical management, lacking effective treatment options. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for tissue repair and regeneration. However, transplanted MSCs often undergo rapid apoptosis, giving rise to heterogeneous extracellular vesicles (EVs), including apoptotic bodies (apoBDs) and apoptotic small extracellular vesicles (apoSEVs). The potential stimulatory role of these EVs in diabetic wound healing remains unknown. Methods: In this study, we investigated the effects of apoSEVs derived from adipose-derived mesenchymal/stromal cells (ADSCs) on the recovery of diabetic wounds by modulating the function of versatile target cells. First, we characterized the apoSEVs and apoBDs derived from apoptotic ADSCs. Subsequently, we evaluated the effects of apoSEVs and apoBDs on macrophages, endothelial cells, and fibroblasts, three essential cell types in wound healing, under high-glucose conditions. Furthermore, we developed a gelatin methacryloyl (GelMA) hydrogel for the sustained release of apoSEVs and investigated its therapeutic effects on wound healing in type 2 diabetic mice in vivo. Results: apoSEVs facilitated the polarization of M1 phenotype macrophages to M2 phenotype, promoted proliferation, migration, and tube formation of endothelial cells, and enhanced fibroblast proliferation and migration. However, apoBDs failed to improve the function of endothelial cells and fibroblasts. In vivo, the apoSEVs-loaded GelMA effectively promoted wound healing by facilitating collagen fiber deposition, angiogenesis, and immune regulation. Conclusion: Our study elucidates the beneficial effects of apoSEVs on wound recovery in diabetes and introduces a novel strategy for diabetic wound treatment based on apoSEVs.

Kinase-independent role of mTOR and on-/off-target effects of an mTOR kinase inhibitor.

mTOR, as a serine/threonine kinase, is a widely pursued anticancer target. Multiple clinical trials of mTOR kinase inhibitors are ongoing, but their specificity and safety features remain lacking. Here, we have employed an inducible kinase-inactive D2338A mTOR knock-in mouse model (mTOR-/KI) together with a mTOR conditional knockout model (mTOR-/-) to assess the kinase-dependent/-independent function of mTOR in hematopoiesis and the on-/off-target effects of mTOR kinase inhibitor AZD2014. Despite exhibiting many similar phenotypes to mTOR-/- mice in hematopoiesis, the mTOR-/KI mice survived longer and showed differences in hematopoietic progenitor cells compared to mTOR-/- mice, suggesting a kinase-independent function of mTOR in hematopoiesis. Gene expression signatures in hematopoietic stem cells (HSCs) further revealed both kinase-dependent and independent effects of mTOR. AZD2014, a lead mTOR kinase inhibitor, appeared to work mostly on-target in suppressing mTOR kinase activity, mimicking that of mTOR-/KI HSCs in transcriptome analysis, but it also induced a small set of off-target responses in mTOR-/KI HSCs. In murine and human myeloid leukemia, besides kinase-inhibitory on-target effects, AZD2014 displayed similar off-target and growth-inhibitory cytostatic effects. These studies provide new insights into kinase-dependent/-independent effects of mTOR in hematopoiesis and present a genetic means for precisely assessing the specificity of mTOR kinase inhibitors.

A Novel Magnetic Manipulation Promotes Directional Growth of Periodontal Ligament Stem Cells.

Periodontium is the rally of soft and hard tissues, which will be devastated continuously by the compromise of periodontitis. Current periodontal therapeutic methods cannot effectively reconstruct periodontal ligament (PDL), which is oriented at an angle with tooth root and combined hard tissues to form cementum-PDL-alveolar bone complex. Hence, it is urgent to find new techniques for PDL reconstruction to achieve functional regeneration of periodontium. Herein, we developed a novel method to manipulate the distribution and growth of periodontal ligament stem cells (PDLSCs) by utilizing highly paralleled static magnetic field (SMF) and magnetic nanoparticles (MNPs). PDLSCs were incubated with MNPs in vitro to label with them. Meanwhile, CCK8 and live/dead cell staining assay were used to detect the impact of SMF and MNPs on cell viability. The directional migration and growth of PDLSCs were visualized under microscope. Furthermore, real-time quantitative PCR and western blot were utilized to calculate the expression level of PDL-related genes. The results showed that PDLSCs could rapidly take up MNPs without compromising cell proliferation and viability, consequently endowed with the ability to respond via magnetic force. The cell migration analysis indicated that PDLSCs could move along the magnetic induction line, testifying that SMF exerted forces on PDLSCs that labeled with MNPs. It was demonstrated that collective application of SMF and MNPs not only induced PDLSCs organized and grew directionally, but also initiated elongation of cells and nucleus. Furthermore, the morphological alteration of the nucleus could also effectively enhance the gene and protein expression of Collagen Ⅰα2, Collagen Ⅲ, and Periostin, suggesting the capability of PDLSCs to differentiate into PDL. In conclusion, this study exhibits a new approach for directional reconstruction of PDL to obtain physiological and functional regeneration of periodontium. The Clinical Trial Registration number: WCHSIRB-D-2022-458.

Lipoaspirate fluid derived factors and extracellular vesicles accelerate wound healing in a rat burn model.

Background: The regenerative capabilities of derivatives derived from the fat layer of lipoaspirate have been demonstrated. However, the large volume of lipoaspirate fluid has not attracted extensive attention in clinical applications. In this study, we aimed to isolate the factors and extracellular vesicles from human lipoaspirate fluid and evaluate their potential therapeutic efficacy. Methods: Lipoaspirate fluid derived factors and extracellular vesicles (LF-FVs) were prepared from human lipoaspirate and characterized by nanoparticle tracking analysis, size-exclusion chromatography and adipokine antibody arrays. The therapeutic potential of LF-FVs was evaluated on fibroblasts in vitro and rat burn model in vivo. Wound healing process was recorded on days 2, 4, 8, 10, 12 and 16 post-treatment. The scar formation was analyzed by histology, immunofluorescent staining and scar-related gene expression at day 35 post-treatment. Results: The results of nanoparticle tracking analysis and size-exclusion chromatography indicated that LF-FVs were enriched with proteins and extracellular vesicles. Specific adipokines (adiponectin and IGF-1) were detected in LF-FVs. In vitro, LF-FVs augmented the proliferation and migration of fibroblasts in a dose-dependent manner. In vivo, the results showed that LF-FVs significantly accelerated burn wound healing. Moreover, LF-FVs improved the quality of wound healing, including regenerating cutaneous appendages (hair follicles and sebaceous glands) and decreasing scar formation in the healed skin. Conclusion: LF-FVs were successfully prepared from lipoaspirate liquid, which were cell-free and enriched with extracellular vesicles. Additionally, they were found to improve wound healing in a rat burn model, suggesting that LF-FVs could be potentially used for wound regeneration in clinical settings.

AtacAnnoR: a reference-based annotation tool for single cell ATAC-seq data.

Here, we present AtacAnnoR, a two-round annotation method for scATAC-seq data using well-annotated scRNA-seq data as reference. We evaluate AtacAnnoR's performance against six competing methods on 11 benchmark datasets. Our results show that AtacAnnoR achieves the highest mean accuracy and the highest mean balanced accuracy and performs particularly well when unpaired scRNA-seq data are used as the reference. Furthermore, AtacAnnoR implements a 'Combine and Discard' strategy to further improve annotation accuracy when annotations of multiple references are available. AtacAnnoR has been implemented in an R package and can be directly integrated into currently popular scATAC-seq analysis pipelines.