Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles.

BACKGROUND AIMS: As evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect of EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are rarely reported. METHODS: The authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, -20°C, -80°C) for various durations as well as after lyophilization. RESULTS: Mesenchymal stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4-6 weeks at -20°C and -80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs. CONCLUSIONS: These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes.

Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing.

Wound therapies involving gene delivery to the skin have significant potential due to the advantage and ease of local treatment. However, choosing the appropriate vector to enable successful gene expression while also ensuring that the treatment's immediate material components are conducive to healing itself is critical. In this study, we utilized a particulate formulation of the polymer chitosan (chitosan particles, CPs) as a non-viral vector for the delivery of a plasmid encoding human CA5-HIF-1α, a degradation resistant form of HIF-1α, to enhance wound healing. We also compared the angiogenic potential of our treatment (HIF/CPs) to that of chitosan particles containing only the plasmid backbone (bb/CPs) and the chitosan particle vector alone (CPs). Our results indicate that chitosan particles exert angiogenic effects that are enhanced with the human CA5-HIF-1α-encoded plasmid. Moreover, HIF/CPs enhanced wound healing in diabetic db/db mice (p < 0.01), and healed tissue was found to contain a significantly increased number of blood vessels compared to bb/CPs (p < 0.01), CPs (p < 0.05) and no-treatment groups (p < 0.01). Thus, this study represents a method of gene delivery to the skin that utilizes an inherently pro-wound-healing polymer as a vector for plasmid DNA that has broad application for the expression of other therapeutic genes.

Potential Role of Silencing Ribonucleic Acid for Esophageal Cancer Treatment.

INTRODUCTION: Esophageal cancer is an aggressive malignancy with high mortality. Optimal treatment of esophageal cancer remains an elusive goal. Ribonucleic acid (RNA) interference is a novel potential targeted approach to treat esophageal cancer. Targeting oncogenes that can alter critical cellular functions with silencing RNA molecules is a promising approach. The silencing of specific oncogenes in esophageal cancer cells in the experimental setting has been shown to decrease the expression of oncogenic proteins. This has resulted in cell apoptosis, reduction in cell proliferation, reduced invasion, migration, epithelial-mesenchymal transition, decrease in tumor angiogenesis and metastasis, and overcoming drug resistance. The Hedgehog (Hh) signaling pathway has been shown to be involved in esophageal adenocarcinoma formation in a reflux animal model. In addition to Hh, we will focus on other targets with clinical potential in the treatment of esophageal cancer. MATERIALS AND METHODS: We searched for articles published from 2005 to August 2020 that studied the siRNA effects on inhibiting esophageal cancer formation in experimental settings. We used combinations of the following terms for searching: "esophageal cancer," "RNA interference," "small interfering RNA," "siRNA," "silencing RNA," "Smoothened (Smo)," "Gli," "Bcl-2," "Bcl-XL," "Bcl-W,″ "Mcl-1," "Bfl-1," "STAT3,"and "Hypoxia inducible factor (HIF)". A total of 21 relevant articles were found. RESULTS AND CONCLUSIONS: Several proto-oncogenes/oncogenes including Hh pathway mediators, glioma-associated oncogene homolog 1 (Gli-1), Smoothened (Smo), and antiapoptotic Bcl-2 have potential as targets for silencing RNA in the treatment of esophageal cancer.

Preconditioning of surgical pedicle flaps with DNA plasmid expressing hypoxia-inducible factor-1α (HIF-1α) promotes tissue viability.

Ischemic necrosis of surgical flaps after reconstruction is a major clinical problem. Hypoxia-inducible factor-1α (HIF-1α) is considered the master regulator of the adaptive response to hypoxia. Among its many properties, it regulates the expression of genes encoding angiogenic growth factors, which have a short half-life in vivo. To achieve a continuous application of the therapeutic, we utilized DNA plasmid delivery. Transcription of the DNA plasmid confirmed by qRT-PCR showed significantly increased mRNA for HIF-1α in the transfected tissue compared to saline control tissue. Rats were preconditioned by injecting with either HIF-1α DNA plasmid or saline intradermally in the designated flap region on each flank. Seven days after preconditioning, each rat had two isolated pedicle flaps raised with a sterile silicone sheet implanted between the skin flap and muscle layer. The flaps preconditioned with HIF-1α DNA plasmid had significantly less necrotic area. Angiogenesis measured by CD31 staining showed a significant increase in the number of vessels per high powered field in the HIF-1α group (p < 0.05). Our findings offer a potential therapeutic strategy for significantly promoting the viability of surgical pedicle flaps by ischemic preconditioning with HIF-1α DNA plasmid.

Robot-assisted versus laparoscopic Roux-en-Y gastric bypass and sleeve gastrectomy: a propensity score-matched comparative analysis using the 2015-2016 MBSAQIP database.

BACKGROUND: Robotic-assisted bariatric surgery is part of the armamentarium in many bariatric centers. However, limited data correlate the robotic benefits to with clinical outcomes. This study compares 30-day outcomes between robotic-assisted and laparoscopic procedures for Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). METHODS: Using the 2015-2016 Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (MBSAQIP) database, patients between18- and 65-year-old were included. To adjust for potential confounders, 1:1 propensity-score matching (PSM) was performed using 22 preoperative characteristics. Second PSM analysis was performed adding operative time and conversion rate. RESULTS: 269,923 patients underwent SG (n = 190,494) or RYGB (n = 79,429). The operative time was significantly longer in the Robotic-assisted compared to laparoscopic approach either for SG (102.58 ± 46 vs. 73.38 ± 36; P < 0.001) or for RYGB (158.29 ± 65 vs. 120.17 ± 56; P < 0.001). In the SG cohort (12,877 matched cases), the robotic approach showed significant reduction of postoperative bleeding (0.16% vs. 0.43%; P < 0.001) and strictures (0.19% vs. 0.33%; P = 0.04) with similar results in the other 30-day outcomes in both analyses. Similarly, for the RYGB cohort (5780 matched cases), the robotic approach showed significantly fewer requirements for blood transfusions (0.64% vs. 1.16%; P = 0.004) with no statistically different results for the other's outcomes. Conversely, when adding operative time and conversion rate to the PSM analysis, the robotic platform showed significantly shorter length of stay (2.12 ± 1.9 vs. 2.30 ± 3.1 days; P < 0.001), reduction of anastomotic leak (0.52% vs. 0.92%; P = 0.01), renal complications (0.16% vs. 0.38%; P = 0.004), and venous thromboembolism (0.24% vs. 0.52%; P = 0.02). CONCLUSIONS: Our findings show that postoperative bleeding and blood transfusion are significantly reduced with the robotic platform, and after correcting for all factors including operative time, the robotic-assisted approach is associated with better postoperative outcomes especially for RYGB.

Free vascularized fibular flap for clavicle reconstruction: A systematic review with a new case.

BACKGROUND/OBJECTIVES: Due to the rarity of the need for claviculectomy and the subsequent clavicle reconstruction, currently there is no consensus on the reconstructive approach for the clavicle. The clavicle is an essential bony structure that is necessary for optimal upper limb anatomical and physiological functionalities. OBJECTIVE: This study analyzes the reconstructive approach, vascular anastomosis, complications, and long-term outcome of clavicle reconstruction using a free vascularized fibular flap through a systematic review of the literature and a case report from our institution. METHODS: A comprehensive literature search was executed in the Ovid MEDLINE, Embase, and Google Scholar databases. The search strategy was designed to capture the concept of cases that underwent clavicle reconstruction after necessary claviculectomy with sufficient clinical information for detailed analysis. Using the final included articles, we analyzed and summarized the outcomes associated with clavicle reconstruction using free fibular osteocutaneous flap. RESULTS: A review of 179 articles yielded 11 publications with 26 cases that had detailed clinical information. We presented an additional case from our institution database. The systematic review of 27 cases revealed that clavicle nonunion due to various causes accounted for 73.08% of the cases for claviculectomy and the eventual reconstruction with a free fibular flap. The mean follow-up period in this study is 29.54 months with the range of 3 to 120 months. A total of 92.31% of the cases showed evidence of complete osseous consolidation. CONCLUSION: When claviculectomy is necessary, a free fibular flap can be utilized for the subsequent clavicle reconstruction to provide optimal anatomical and physiological functionality of the clavicle.

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

HOTAIR-Loaded Mesenchymal Stem/Stromal Cell Extracellular Vesicles Enhance Angiogenesis and Wound Healing.

Chronic wounds remain a substantial source of morbidity worldwide. An emergent approach that may be well-suited to induce the complex, multicellular processes such as angiogenesis that are required for wound repair is the use of extracellular vesicles (EVs). EVs contain a wide variety of proteins and nucleic acids that enable multifactorial signaling. Here, the capability of EVs is leveraged to be engineered via producer cell modification to investigate the therapeutic potential of EVs from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR). HOTAIR is previously shown by the authors' group to be critical in mediating angiogenic effects of endothelial cell EVs, and MSCs are chosen as EV producer cells for this study due to their widely reported intrinsic angiogenic properties. The results indicate that MSCs overexpressing HOTAIR (HOTAIR-MSCs) produce EVs with increased HOTAIR content that promote angiogenesis and wound healing in diabetic (db/db) mice. Further, endothelial cells exposed to HOTAIR-MSC EVs exhibit increased HOTAIR content correlated with upregulation of the angiogenic protein vascular endothelial growth factor. Thus, this study supports EV-mediated HOTAIR delivery as a strategy for further exploration toward healing of chronic wounds.