Mitochondrial Transplantation Modulates Inflammation and Apoptosis, Alleviating Tendinopathy Both In Vivo and In Vitro.

Tendinopathy is a common musculoskeletal condition causing pain and dysfunction. Conventional treatment and surgical procedures for tendinopathy are insufficient; accordingly, recent research has focused on tendon-healing regenerative approaches. Tendon injuries usually occur in the hypoxic critical zone, characterized by increased oxidative stress and mitochondrial dysfunction; thus, exogenous intact mitochondria may be therapeutic. We aimed to assess whether mitochondrial transplantation could induce anti-inflammatory activity and modulate the metabolic state of a tendinopathy model. Exogenous mitochondria were successfully delivered into damaged tenocytes by centrifugation. Levels of Tenomodulin and Collagen I in damaged tenocytes were restored with reductions in nuclear factor-κB and matrix metalloproteinase 1. The dysregulation of oxidative stress and mitochondrial membrane potential was attenuated by mitochondrial transplantation. Activated mitochondrial fission markers, such as fission 1 and dynamin-related protein 1, were dose-dependently downregulated. Apoptosis signaling pathway proteins were restored to the pre-damage levels. Similar changes were observed in a collagenase injection-induced rat model of tendinopathy. Exogenous mitochondria incorporated into the Achilles tendon reduced inflammatory and fission marker levels. Notably, collagen production was restored. Our results demonstrate the therapeutic effects of direct mitochondrial transplantation in tendinopathy. These effects may be explained by alterations in anti-inflammatory and apoptotic processes via changes in mitochondrial dynamics.

The immune modulatory effects of mitochondrial transplantation on cecal slurry model in rat.

BACKGROUND: Sepsis has a high mortality rate, but no specific drug has been proven effective, prompting the development of new drugs. Immunologically, sepsis can involve hyperinflammation, immune paralysis, or both, which might pose challenges during drug development. Recently, mitochondrial transplantation has emerged as a treatment modality for various diseases involving mitochondrial dysfunction, but it has never been tested for sepsis. METHODS: We isolated mitochondria from L6 muscle cells and umbilical cord mesenchymal stem cells and tested the quality of the isolated mitochondria. We conducted both in vivo and in vitro sepsis studies. We investigated the effects of intravenous mitochondrial transplantation on cecal slurry model in rats in terms of survival rate, bacterial clearance rate, and the immune response. Furthermore, we observed the effects of mitochondrial transplantation on the immune reaction regarding both hyperinflammation and immune paralysis. To do this, we studied early- and late-phase cytokine production in spleens from cecal slurry model in rats. We also used a lipopolysaccharide (LPS)-stimulated human PBMC monocyte model to confirm the immunological effects of mitochondrial transplantation. Apoptosis and the intrinsic apoptotic pathway were investigated in septic spleens. RESULTS: Mitochondrial transplantation improved survival and bacterial clearance. It also mitigated mitochondrial dysfunction and apoptosis in septic spleens and attenuated both hyperinflammation and immune paralysis in the spleens of cecal slurry model in rats. This effect was confirmed with an LPS-stimulated human PBMC study. CONCLUSIONS: In rat polymicrobial cecal slurry model, the outcome is improved by mitochondrial transplantation, which might have an immunomodulatory effect.

Umbilical Cord-Mesenchymal Stem Cell-Conditioned Medium Improves Insulin Resistance in C2C12 Cell.

Background: Umbilical cord-mesenchymal stem cell-conditioned medium (UC-MSC-CM) has emerged as a promising cell-free therapy. The aim of this study was to explore the therapeutic effects of UC-MSC-CM on insulin resistance in C2C12 cell. Methods: Insulin resistance was induced by palmitate. Effects of UC-MSC-CM on insulin resistance were evaluated using glucose uptake, glucose transporter type 4 (GLUT4) translocation, the insulin-signaling pathway, and mitochondrial contents and functions in C2C12 cell. Results: Glucose uptake was improved by UC-MSC-CM. UC-MSC-CM treatment increased only in membranous GLUT4 expression, not in cytosolic GLUT4 expression. It restored the insulin-signaling pathway in insulin receptor substrate 1 and protein kinase B. Mitochondrial contents evaluated by mitochondrial transcription factor A, mitochondrial DNA copy number, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha were increased by UC-MSC-CM. In addition, UC-MSC-CM significantly decreased mitochondrial reactive oxygen species and increased fatty acid oxidation and mitochondrial membrane potential. There was no improvement in adenosine triphosphate (ATP) contents, but ATP synthesis was improved by UC-MSC-CM. Cytokine and active factor analysis of UC-MSC-CM showed that it contained many regulators inhibiting insulin resistance. Conclusion: UC-MSC-CM improves insulin resistance with multiple mechanisms in C2C12 cell.

Lifetime and diffusion distance of singlet oxygen in air under everyday atmospheric conditions.

Singlet oxygen is a toxic chemical but powerful oxidant, exploited in many chemical and biological applications. However, the lifetime of singlet oxygen in air under atmospheric conditions is yet to be known. This has limited safe usage of singlet oxygen in air, despite being a strong antimicrobial agent with the unique property of relaxing to breathable oxygen after serving its purpose. Here, we solve this long-standing problem by combining experimental and theoretical research efforts; we generate singlet oxygen using a photosensitizer at a local source and monitor the time-dependent extent of singlet oxygen reaction with probe molecules at a detector, precisely controlling the detector distance from the source. To explain our experimental results, we employ a theoretical model that fully accounts for singlet oxygen diffusion, radiative and nonradiative relaxations, and the bimolecular reaction with probe molecules at the detector. For all cases investigated, our model, with only two adjustable parameters, provides an excellent quantitative explanation of the experiment. From this analysis, we extract the lifetime of singlet oxygen in the air to be 2.80 s at 23 °C under 1 atm, during which time singlet oxygen diffuses about 0.992 cm. The correctness of this estimation is confirmed by a simple mean-first-passage time analysis of the maximum distance singlet oxygen can reach from the source. We also confirm the sterilization effects of singlet oxygen for distances up to 0.6-0.8 cm, depending on the bacteria strain in question, between the bacteria and the singlet oxygen source.

Nanoinjection system for precise direct delivery of biomolecules into single cells.

Intracellular delivery of functional molecules such as proteins, transcription factors and DNA is effective and promising in cell biology. However, existing transfection methods are often unsuitable to deliver big molecules into cells or require carriers such as viruses and peptides specific to the target molecules. In addition, the nature of bulk processing does not generally provide accurate dose control of individual cells. The concept of single-cell-based material injection based on electrokinetic pumping through nanocapillaries could overcome these problems, yet the fabrication and operation of nanoscale 3-dimensional structures have remained unsolved. In this research, a hybrid (PDMS/glass) microfluidic chip with a true 3-dimensional nanoinjection structure (called "nanoinjection system") is presented. The nanoinjection structure was fabricated by femtosecond-laser (fs-laser) ablation in a single solid glass, which showed very successful delivery of red fluorescent protein (RFP) and expression of plasmid DNA in several different types of cells. This system is promising in that the amount of molecules to be delivered is controllable and the processed cells are systematically separated into a harvesting chamber, which can radically improve the purity of the processed cells. In addition, it was confirmed that the cells were healthy even after the molecule injection for a few seconds, indicating that the injection time can be significantly elongated, further improving the delivery efficiency of biomolecules without affecting the cell viability. We envision that the nanoinjection system having the major features of being carrier-free and dose-controllable, having an unlimited injection period, and ease of harvesting will greatly contribute to the next-generation research studies in the fields of cell biology and cell therapeutics.

Delivery of exogenous mitochondria via centrifugation enhances cellular metabolic function.

Mitochondria are essential organelles involved in the maintenance of cell growth and function, and have been investigated as therapeutic targets in various diseases. Recent studies have demonstrated that direct mitochondrial transfer can restore cellular functions of cells with inherited or acquired mitochondrial dysfunction. However, previous mitochondrial transfer methods are inefficient and time-consuming. Here, we developed a simple and easy mitochondrial transfer protocol using centrifugation, which can be applied to any cell type. By our simple centrifugation method, we found that the isolated mitochondria could be successfully transferred into target cells, including mitochondrial DNA-deleted Rho0 cells and dexamethasone-treated atrophic muscle cells. We found that mitochondrial transfer normalised ATP production, mitochondrial membrane potential, mitochondrial reactive oxygen species level, and the oxygen consumption rate of the target cells. Furthermore, delivery of intact mitochondria blocked the AMPK/FoxO3/Atrogene pathway underlying muscle atrophy in atrophic muscle cells. Taken together, this simple and rapid mitochondrial transfer method can be used to treat mitochondrial dysfunction-related diseases.

A Crucial Role of Rh Substituent Ion in Photoinduced Internal Electron Transfer and Enhanced Photocatalytic Activity of CdS-Ti(5.2-x)/6 Rhx /2 O2 Nanohybrids.

The photocatalytic activity and photostability of CdS quantum dot (QD) can be remarkably enhanced by hybridization with Rh-substituted layered titanate nanosheet even at very low Rh substitution rate (<1%). Mesoporous CdS-Ti(5.2-x)/6 Rhx/2O2 nanohybrids are synthesized by a self-assembly of exfoliated Ti(5.2-x)/6 Rhx/2O2 nanosheets with CdS QDs. The partial substitution of Rh(3+)/Rh(4+) ions for Ti(4+) ions in layered titanate is quite effective in enhancing an electronic coupling between hybridized CdS and titanate components via the formation of interband Rh 4d states. A crucial role of Rh substituent ion in the internal electron transfer is obviously evidenced from in situ X-ray absorption spectroscopy showing the elongation of (RhO) bond under visible light irradiation. This is the first spectroscopic evidence for the important role of substituent ion in the photoinduced electron transfer of hybrid-type photocatalyst. The CdS-Ti(5.2-x)/6 Rhx/2O2 nanohybrids show much higher photocatalytic activity for H2 production and better photostability than do CdS and unsubstituted CdS-TiO2 nanohybrid. This result is ascribable to the enhancement of visible light absorptivity, the depression of electron-hole recombination, and the enhanced hole curing of CdS upon Rh substitution. The present study underscores that the hybridization with composition-controlled inorganic nanosheet provides a novel efficient methodology to optimize the photo-related functionalities of semiconductor nanocrystal.

Cell therapy with embryonic stem cell-derived cardiomyocytes encapsulated in injectable nanomatrix gel enhances cell engraftment and promotes cardiac repair.

A significant barrier to the therapeutic use of stem cells is poor cell retention in vivo. Here, we evaluate the therapeutic potential and long-term engraftment of cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) encapsulated in an injectable nanomatrix gel consisting of peptide amphiphiles incorporating cell adhesive ligand Arg-Gly-Asp-Ser (PA-RGDS) in experimental myocardial infarction (MI). We cultured rat neonatal CMs in PA-RGDS for 7 days and found that more than 90% of the CMs survived. Next, we intramyocardially injected mouse CM cell line HL-1 CMs with or without PA-RGDS into uninjured hearts. Histologic examination and flow cytometry analysis of digested heart tissues showed approximately 3-fold higher engraftment in the mice that received CMs with PA-RGDS compared to those without PA-RGDS. We further investigated the therapeutic effects and long-term engraftment of mESC-CMs with PA-RGDS on MI in comparison with PBS control, CM-only, and PA-RGDS only. Echocardiography demonstrated that the CM-only and CM+PA-RGDS groups showed higher cardiac function at week 2 compared to other groups. However, from 3 weeks, higher cardiac function was maintained only in the CM+PA-RGDS group; this was sustained for 12 weeks. Confocal microscopic examination of the cardiac tissues harvested at 14 weeks demonstrated sustained engraftment and integration of mESC-CMs into host myocardium in the CM+PA-RGDS group only. This study for the first time demonstrated that PA-RGDS encapsulation can enhance survival of mESC-derived CMs and improve cardiac function post-MI. This nanomatrix gel-mediated stem cell therapy can be a promising option for treating MI.

Wound healing properties of a 3-D scaffold comprising soluble silkworm gland hydrolysate and human collagen.

Biomaterials that serve as scaffolds for cell proliferation and differentiation are increasingly being used in wound repair. In this study, the potential regenerative properties of a 3-D scaffold containing soluble silkworm gland hydrolysate (SSGH) and human collagen were evaluated. The scaffold was generated by solid-liquid phase separation and a freeze-drying method using a homogeneous aqueous solution. The porosity, swelling behavior, protein release, cytotoxicity, and antioxidative properties of scaffolds containing various ratios of SSGH and collagen were evaluated. SSGH/collagen scaffolds had a high porosity of 61-81% and swelling behavior studies demonstrated a 50-75% increase in swelling, along with complete protein release in the presence of phosphate-buffered saline. Cytocompatibility of the SSGH/collagen scaffold was demonstrated using mesenchymal stem cells from human umbilical cord. Furthermore, SSGH/collagen efficiently attenuated oxidative stress-induced cell damage. In an in vivo mouse model of wound healing, the SSGH/collagen scaffold accelerated wound re-epithelialization over a 15-day period. Overall, the microporous SSGH/collagen 3-D scaffold maintained optimal hydration of the exposed tissues and decreased wound healing time. These results contribute to the generation of advanced wound healing materials and may have future therapeutic implications.