Biogenerated Oxygen-Related Environmental Stressed Apoptotic Vesicle Targets Endothelial Cells.

The dynamic balance between hypoxia and oxidative stress constitutes the oxygen-related microenvironment in injured tissues. Due to variability, oxygen homeostasis is usually not a therapeutic target for injured tissues. It is found that when administered intravenously, mesenchymal stem cells (MSCs) and in vitro induced apoptotic vesicles (ApoVs) exhibit similar apoptotic markers in the wound microenvironment where hypoxia and oxidative stress co-existed, but MSCs exhibited better effects in promoting angiogenesis and wound healing. The derivation pathway of ApoVs by inducing hypoxia or oxidative stress in MSCs to simulate oxygen homeostasis in injured tissues is improved. Two types of oxygen-related environmental stressed ApoVs are identified that directly target endothelial cells (ECs) for the accurate regulation of vascularization. Compared to normoxic and hypoxic ones, oxidatively stressed ApoVs (Oxi-ApoVs) showed the strongest tube formation capacity. Different oxygen-stressed ApoVs deliver similar miRNAs, which leads to the broad upregulation of EC phosphokinase activity. Finally, local delivery of Oxi-ApoVs-loaded hydrogel microspheres promotes wound healing. Oxi-ApoV-loaded microspheres achieve controlled ApoV release, targeting ECs by reducing the consumption of inflammatory cells and adapting to the proliferative phase of wound healing. Thus, the biogenerated apoptotic vesicles responding to oxygen-related environmental stress can target ECs to promote vascularization.

Balancing macrophage polarization via stem cell-derived apoptotic bodies for diabetic wound healing.

BACKGROUND: Adipose tissue-derived stem cell-derived apoptotic bodies (ADSC-ABs) have shown great potential for immunomodulation and regeneration, particularly in diabetic wound therapy. However, their local application has been limited by unclear regulatory mechanisms, rapid clearance, and short tissue retention times. METHODS: We analyzed the key role molecules and regulatory pathways of ADSC-ABs in regulating inflammatory macrophages by mRNA sequencing and microRNA (miRNA) sequencing and then verified them by gene knockdown. To prevent rapid clearance, we employed microfluidics technology to prepare methacrylate-anhydride gelatin (GelMA) microspheres (GMS) for controlled release of ABs. Finally, we evaluated the effectiveness of ADSC-AB-laden GMSs (ABs@GMSs) in a diabetic rat wound model. FINDINGS: Our results demonstrated that ADSC-ABs effectively balanced macrophage inflammatory polarization through the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, mediated by miR-20a-5p. Furthermore, we showed that AB@GMSs had good biocompatibility, significantly delayed local clearance of ABs, and ameliorated diabetic wound inflammation and promoted vascularization, thus facilitating its healing. CONCLUSIONS: Our study reveals the regulatory mechanism of ADSC-ABs in balancing macrophage inflammatory polarization and highlightsthe importance of delaying their local clearance by GMSs. These findings have important implications for the development of novel therapies for diabetic wound healing. FUNDING: This research was supported by the National Key Research and Development Program of China (2020YFA0908200), National Natural Science Foundation of China (82272263, 82002053, 32000937, and 82202467), Shanghai "Rising Stars of Medical Talents" Youth Development Program (22MC1940300), Shanghai Municipal Health Commission (20204Y0354), and Shanghai Science and Technology Development Funds (22YF1421400).

Transcriptome network analysis of inflammation and fibrosis in keloids.

BACKGROUND: Keloid (KL) is a common benign skin tumor. KL is typically characterized by significant fibrosis and an intensive inflammatory response. Therefore, a comprehensive understanding of the interactions between cellular inflammation and fibrotic cells is essential to elucidate the mechanisms driving the progression of KL and to develop therapeutics. OBJECTIVE: Investigate the transcriptome landscape of inflammation and fibrosis in keloid scars. METHODS: In this paper, we performed transcriptome sequencing and microRNA (miRNA) sequencing on unselected live cells from six human keloid tissues and normal skin tissues to elucidate a comprehensive transcriptome landscape. In addition, we used single-cell RNA sequencing (scRNA-seq) analysis to analyze intercellular communication networks and enrich fibroblast populations in two additional keloid and normal skin samples to study fibroblast diversity. RESULTS: By RNA sequencing and a miRNA-mRNA-PPI network analysis, we identified miR-615-5p and miR-122b-3p as possible miRNAs associated with keloids, as they differed most significantly in keloids. Similarly, COL3A1, COL1A2, THBS2, TNC, IGTA, THBS4, TGFB3 as genes with significant differences in keloid may be associated with keloid development. Using single-cell RNA sequencing data from 24,086 cells collected from normal or keloid, we report reconstructed intercellular signaling network analysis and aggregation to modules associated with specific cell subpopulations at the cellular level for keloid alterations. CONCLUSIONS: Our multitranscriptomic dataset delineates inflammatory and fibro heterogeneity of human keloids, underlining the importance of intercellular crosstalk between inflammatory cells and fibro cells and revealing potential therapeutic targets.

Regulation of Glucose Metabolism for Cell Energy Supply In Situ via High-Energy Intermediate Fructose Hydrogels.

The cellular functions, such as tissue-rebuilding ability, can be directly affected by the metabolism of cells. Moreover, the glucose metabolism is one of the most important processes of the metabolism. However, glucose cannot be efficiently converted into energy in cells under ischemia hypoxia conditions. In this study, a high-energy intermediate fructose hydrogel (HIFH) is developed by the dynamic coordination between sulfhydryl-functionalized bovine serum albumin (BSA-SH), the high-energy intermediate in glucose metabolism (fructose-1,6-bisphosphate, FBP), and copper ion (Cu2+ ). This hydrogel system is injectable, self-healing, and biocompatible, which can intracellularly convert energy with high efficacy by regulating the glucose metabolism in situ. Additionally, the HIFH can greatly boost cell antioxidant capacity and increase adenosine triphosphate (ATP) in the ischemia anoxic milieu by roughly 1.3 times, improving cell survival, proliferation and physiological functions in vitro. Furthermore, the ischemic skin tissue model is established in rats. The HIFH can speed up the healing of damaged tissue by promoting angiogenesis, lowering reactive oxygen species (ROS), and eventually expanding the healing area of the damaged tissue by roughly 1.4 times in vivo. Therefore, the HIFH can provide an impressive perspective on efficient in situ cell energy supply of damaged tissue.

Reconstruction of Nasal Tip Using "Seagull-shaped" Cavum Concha Cartilage Composite Scaffold.

BACKGROUND: The nasal tip plays a crucial role both esthetically and functionally. The application of nasal tip grafts is an effective method for improving nasal tip form. Ear cartilage is a common choice for nasal tip grafts, but it still presents several challenges in clinical application that need to be addressed. This study aims to address the issues associated with the use of ear cartilage in clinical rhinoplasty applications through the development of a novel septal extension graft using ear cartilage for nasal tip reconstruction. METHODS: From May 2018 to April 2022, a total of 132 cases of nasal tip reconstruction surgeries were performed using a seagull-shaped nasal septum extension graft, constructed with bilateral cavum concha cartilage. Among these cases, 25 patients had previously undergone rhinoplasty using silicone implant, 7 patients had undergone augmentation rhinoplasty using expanded polytetrafluoroethylene, whereas the rest were primary rhinoplasty cases. All patients were followed up for a period ranging from 3 months to 4 years postoperatively, with photographs taken to assess the nasal tip morphology. RESULTS: In this study, all patients exhibited good healing of the incisions made at the posterior aspect of the auricular concha, with no occurrences of hematoma and inconspicuous scarring. In 116 cases, significant improvement in nasal appearance and a realistic nasal tip form were achieved postoperatively, yielding satisfactory outcomes. Only 16 patients experienced minor issues with nasal tip morphology, which were subsequently improved through further surgical procedures. CONCLUSION: This study reports a surgical technique for nasal tip refinement using bilaterally harvested cavum concha cartilage to construct a seagull-shaped nasal septal extension graft. The procedure has achieved satisfactory outcomes, and its application is worth extending to clinical practice.

"Find-eat" strategy targeting endothelial cells via receptor functionalized apoptotic body nanovesicle.

Endothelial cell (EC) injury plays a key role in the chronic wound process. A long-term hypoxic microenvironment hinders the vascularization of ECs, thus delaying wound healing. In this study, CX3CL1-functionalized apoptotic body nanovesicles (nABs) were constructed. The "Find-eat" strategy was implemented through a receptor-ligand combination to target ECs that highly express CX3CR1 in the hypoxic microenvironment, therefore amplifying the "Find-eat" signal and promoting angiogenesis. Apoptotic bodies (ABs) were obtained by chemically inducing apoptosis of adipose-derived stem cells (ADSCs), and then functionalized nABs containing deferoxamine (DFO-nABs) were obtained through a series of steps, including optimized hypotonic treatment, mild ultrasound, drug mixing and extrusion treatment. In vitro experiments showed that nABs had good biocompatibility and an effective "Find-eat" signal via CX3CL1/CX3CR1 to induce ECs in the hypoxic microenvironment, thereby promoting cell proliferation, cell migration, and tube formation. In vivo experiments showed that nABs could promote the rapid closure of wounds, release the "Find-eat" signal to target ECs and realize the sustained release of angiogenic drugs to promote new blood vessel formation in diabetic wounds. These receptor-functionalized nABs, which can target ECs by releasing dual signals and achieve the sustained release of angiogenic drugs, may provide a novel strategy for chronic diabetic wound healing.

Regulated extravascular microenvironment via reversible thermosensitive hydrogel for inhibiting calcium influx and vasospasm.

Arterial vasospasm after microsurgery can cause severe obstruction of blood flow manifested as low tissue temperature, leading to tissue necrosis. The timely discovery and synchronized treatment become pivotal. In this study, a reversible, intelligent, responsive thermosensitive hydrogel system is constructed employing both the gel-sol transition and the sol-gel transition. The "reversible thermosensitive (RTS)" hydrogel loaded with verapamil hydrochloride is designed to dynamically and continuously regulate the extravascular microenvironment by inhibiting extracellular calcium influx. After accurate implantation and following in situ gelation, the RTS hydrogel reverses to the sol state causing massive drug release to inhibit vasospasm when the tissue temperature drops to the predetermined transition temperature. Subsequent restoration of the blood supply alleviates further tissue injury. Before the temperature drops, the RTS hydrogel maintains the gel state as a sustained-release reservoir to prevent vasospasm. The inhibition of calcium influx and vasospasm in vitro and in vivo is demonstrated using vascular smooth muscle cells, mice mesenteric arterial rings, and vascular ultrasonic Doppler detection. Subsequent animal experiments demonstrate that RTS hydrogel can promote tissue survival and alleviate tissue injury responding to temperature change. Therefore, this RTS hydrogel holds therapeutic potential for diseases requiring timely detection of temperature change.

The Roles of Podoplanin-Positive/Podoplanin-Negative Cells from Adipose-Derived Stem Cells in Lymphatic Regeneration.

BACKGROUND: Secondary lymphedema is a refractory disease, for which adipose-derived stem cells have shown some therapeutic potential. However, the mechanism of this action remains poorly understood. METHODS: The authors identified podoplanin-expressing adipose-derived stem cells, which allowed them to divide adipose-derived stem cells into podoplanin-positive and podoplanin-negative groups that they characterized in vitro. The authors then used a mouse hindlimb model for lymphedema to trace the fate of podoplanin-positive, podoplanin-negative, and unsorted adipose-derived stem cells in vivo. RESULTS: When induced in culture, podoplanin-positive cells were noted to up-regulate the expression of lymphatic endothelial cell markers, including LYVE-1, and assumed a cobblestone morphology. In addition, a substantial increase in lymphangiogenic cytokines was detected in the podoplanin-positive supernatant. The above findings were largely absent from the podoplanin-negative and unsorted groups. In the mouse model, the implanted cells relieved the limb lymphedema by promoting lymphangiogenesis, with the podoplanin-positive group showing the most significant effect. Immunocolocalization further revealed that the podoplanin-positive cells incorporated into lymphatic vessels were positive for LYVE-1. CONCLUSIONS: These data demonstrated that actions by means of both paracrine and differentiation pathways were involved in the adipose-derived stem cell-mediated therapeutic effects. The podoplanin-positive cells possessed lymphatic paracrine and differentiation abilities and may represent lymphatic endothelial cell precursor cells. The podoplanin-negative cells, which constitute a considerable proportion of the adipose-derived stem cells, may play an important paracrine role by secreting mesenchymal stem cell-related factors.

Novel Hybrid Dextran-Gadolinium Nanoparticles as High-relaxivity T1 Magnetic Resonance Imaging Contrast Agent for Mapping the Sentinel Lymph Node.

OBJECTIVES: To assess the applicability of a novel hybrid dextran-gadolinium nanoparticles (NPs) as high-relaxivity T1 magnetic resonance imaging (MRI) contrast agent for mapping the sentinel lymph node (SLN). METHODS: Dextran-bis-acrylamide-polyacrylic acid (Dex-MBA-PAA) NPs were synthesized through a self-assembly assisted approach and complexed with multiple chelated gadolinium (Gd) (III) ions. After their characterization was validated, they were used to mapping SLNs by MRI in Wistar rats, and their biosafety was evaluated. RESULTS: Dextran-MBA-polyacrylic acid-Gd NPs have suitable particle size and much higher longitudinal relaxivity (r1) than that of commonly used clinical MRI contrast agents (eg, gadopentetic acid dimeglumine salt injection). The in vivo T1-weighted MRI results revealed their effectiveness at mapping SLNs. And their biological safety was also verified. CONCLUSIONS: Dextran-MBA-polyacrylic acid-Gd NPs were synthesized and validated by in vitro and in vivo experiments for their ability to visualize SLNs by MRI with accurate positioning and excellent biosafety, and they have great potential for clinical SLN mapping.

IL-7 enhances the differentiation of adipose-derived stem cells toward lymphatic endothelial cells through AKT signaling.

Our study was designed to investigate the effects of IL-7 during the differentiation process of adipose-derived stem cells (ADSCs) toward lymphatic endothelial cells (LECs). IL-7 was added to the traditional induced medium, which was called the IL-7 (+) group, while the group that used traditional induced medium was called the IL-7 (-) group. After 7 days of induction of ADSCs, a comprehensive analysis was conducted between these two groups. We examined the changes in Prox1, LYVE-1, Podoplanin and VEGFR-3 on the RNA and protein level and found that the expression of LEC markers in the IL-7 (+) group was higher than in the IL-7 (-) group. The characteristics of differentiated cells were confirmed by flow cytometry and immunofluorescence. At the same time, we detected the MAPK/ERK and PI3K/AKT pathway involved in the differentiation process, and we found that the phosphorylation of AKT increased, however the expression of ERK was not significantly changed. In conclusion, our study found that IL-7 could improve the differentiation efficiency of ADSCs toward LECs through AKT signaling pathways.