Advances in Human Mitochondria-Based Therapies

Mitochondria are the key biological generators of eukaryotic cells, controlling the energy supply while providing many important biosynthetic intermediates. Mitochondria act as a dynamic, functionally and structurally interconnected network hub closely integrated with other cellular compartments via biomembrane systems, transmitting biological information by shuttling between cells and tissues. Defects and dysregulation of mitochondrial functions are critically involved in pathological mechanisms contributing to aging, cancer, inflammation, neurodegenerative diseases, and other severe human diseases. Mediating and rejuvenating the mitochondria may therefore be of significant benefit to prevent, reverse, and even treat such pathological conditions in patients. The goal of this review is to present the most advanced strategies using mitochondria to manage such disorders and to further explore innovative approaches in the field of human mitochondriabased therapies

Is Extracellular Vesicle-Based Therapy the Next Answer for Cartilage Regeneration?

“Extracellular vesicles” (EVs) is a term gathering biological particles released from cells that act as messengers for cell-to-cell communication. Like cells, EVs have a membrane with a lipid bilayer, but unlike these latter, they have no nucleus and consequently cannot replicate. Several EV subtypes (e.g., exosomes, microvesicles) are described in the literature. However, the remaining lack of consensus on their specific markers prevents sometimes the full knowledge of their biogenesis pathway, causing the authors to focus on their biological effects and not their origins. EV signals depend on their cargo, which can be naturally sourced or altered (e.g., cell engineering). The ability for regeneration of adult articular cartilage is limited because this avascular tissue is partly made of chondrocytes with a poor proliferation rate and migration capacity. Mesenchymal stem cells (MSCs) had been extensively used in numerous in vitro and preclinical animal models for cartilage regeneration, and it has been demonstrated that their therapeutic effects are due to paracrine mechanisms involving EVs. Hence, using MSC-derived EVs as cell-free therapy tools has become a new therapeutic approach to improve regenerative medicine. EV-based therapy seems to show similar cartilage regenerative potential compared with stem cell transplantation without the associated hindrances (e.g., chromosomal aberrations, immunogenicity). The aim of this short review is to take stock of occurring EV-based treatments for cartilage regeneration according to their healing effects. The article focuses on cartilage regeneration through various sources used to isolate EVs (mature or stem cells among others) and beneficial effects depending on cargos produced from natural or tuned EVs

Effective remodelling of human osteoarthritic cartilage by sox9 gene transfer and overexpression upon delivery of rAAV vectors in polymeric micelles

[Abstract] Recombinant adeno-associated virus (rAAV) vectors are well suited carriers to provide durable treatments for human osteoarthritis (OA). Controlled release of rAAV from polymeric micelles was already shown to increase both the stability and bioactivity of the vectors while overcoming barriers, precluding effective gene transfer. In the present study, we examined the convenience of delivering rAAV vectors via poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) polymeric (PEO–PPO–PEO) micelles to transfer and overexpress the transcription factor SOX9 in monolayers of human OA chondrocytes and in experimentally created human osteochondral defects. Human osteoarthritic (OA) chondrocytes and human osteochondral defect models were produced using human OA cartilage obtained from patients subjected to total knee arthroplasty. Samples were genetically modified by adding a rAAV-FLAG-hsox9 vector in its free form or via polymeric micelles for 10 days relative to control conditions (unmodified cells). The effects of sox9 overexpression in human OA cartilage samples were monitored by biochemical, histological, and immunohistochemical analyses. Delivery of rAAV-FLAG-hsox9 via polymeric micelles enhanced the levels of sox9 expression compared with free vector administration, resulting in increased proteoglycan deposition and in a stimulated cell proliferation index in OA chondrocytes. Moreover, higher production of type II collagen and decreased hypertrophic events were noted in osteochondral defect cultures when compared with control conditions. Controlled therapeutic rAAV sox9 gene delivery using PEO–PPO–PEO micelles is a promising, efficient tool to promote the remodelling of human OA cartilage

Effects of rAAV-Mediated Overexpression of sox9 and TGF-ß via Alginate Hydrogel-Guided Vector Delivery on the Chondroreparative Activities of Human Bone Marrow-Derived Mesenchymal Stromal Cells

Recombinant adeno-associated virus (rAAV) vectors have a strong potential to promote the healing of traumatic cartilage defects and osteoarthritic lesions upon delivery and overexpression of therapeutic genes from suitable biomaterials that support a controlled release of the candidate constructs. Te goal of the present work is to examine whether the administration of chondrogenic rAAV sox9 and rAAV TGF-ß gene vehicles via alginate hydrogel-guided vector delivery stimulates the biological and chondroreparative activities of human bone marrow-derived mesenchymal stromal cells (hMSCs) as a source of improved reparative cells for future implantation in sites of cartilage damage. Te delivery of rAAV using an alginate (AlgPH155) hydrogel system is successfully achieved in hMSCs over time (21 days), leading to the efective overexpression of sox9 and TGF-ß that signifcantly increases the proliferation and chondrogenic diferentiation activities of the cells relative to control (marker lacZ) gene transfer while advantageously preventing premature hypertrophy, osteogenesis, and mineralization. Tis study reveals the potential of alginate hydrogel-based systems to control the delivery of rAAV (sox9 and TGF-ß) gene vectors to adeptly trigger the chondroreparative activities of hMSCs for future applications that aim at improving cartilage repair

Cytotoxic effects of different mouthwash solutions on primary human articular chondrocytes and normal human articular cartilage : an in vitro study

Objectives To compare the cytotoxicity of octenidine dihydrochloride and chlorhexidine gluconate at diferent concentrations on primary human articular chondrocytes and cartilage. Materials and methods Primary cultures of human normal adult articular chondrocytes were exposed to octenidine dihydrochloride (0.001562%, 0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, and 0.1%), chlorhexidine gluconate (0.003125%, 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, and 0.2%), and control (Dulbecco’s modifed Eagle medium or phosphate-bufered saline) for 30 s. Normal human articular cartilage explants were exposed to octenidine dihydrochloride (0.1% versus control) and chlorhexidine gluconate (0.1% versus control) for 30 s. The viability of human articular chondrocytes was measured by Trypan blue staining, Cell Proliferation Reagent WST-1, and Live/Dead staining. The proliferation of human chondrocytes was measured using the Cell Proliferation Reagent WST-1. The viability of human articular cartilage explants was measured by using Live/Dead staining. Results Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability and proliferation in a dose-dependent manner in primary human articular chondrocytes. Octenidine dihydrochloride and chlorhexidine gluconate exposure decreased cell viability in human articular cartilage explant cultures. Conclusion The degree of toxicity varied between octenidine dihydrochloride and chlorhexidine gluconate, with chlorhexidine gluconate being less toxic than octenidine dihydrochloride at the same concentration. Additionally, both octenidine dihydrochloride and chlorhexidine gluconate evaluation had cytotoxic efects on human articular cartilage. Therefore, dosing for the antimicrobial mouthwash ingredients administration would ideally be determined to remain below IC50. Clinical relevance These data support the in vitro safety of antimicrobial mouthwashes on primary adult human articular chondrocytes

Advanced Gene Therapy Strategies for the Repair of ACL Injuries

The anterior cruciate ligament (ACL), the principal ligament for stabilization of the knee, is highly predisposed to injury in the human population. As a result of its poor intrinsic healing capacities, surgical intervention is generally necessary to repair ACL lesions, yet the outcomes are never fully satisfactory in terms of long-lasting, complete, and safe repair. Gene therapy, based on the transfer of therapeutic genetic sequences via a gene vector, is a potent tool to durably and adeptly enhance the processes of ACL repair and has been reported for its workability in various experimental models relevant to ACL injuries in vitro, in situ, and in vivo. As critical hurdles to the effective and safe translation of gene therapy for clinical applications still remain, including physiological barriers and host immune responses, biomaterial-guided gene therapy inspired by drug delivery systems has been further developed to protect and improve the classical procedures of gene transfer in the future treatment of ACL injuries in patients, as critically presented here

rAAV TGF-β and FGF-2 Overexpression via pNaSS-Grafted PCL Films Stimulates the Reparative Activities of Human ACL Fibroblasts

Lesions in the human anterior cruciate ligament (ACL) are frequent, unsolved clinical issues due to the limited self-healing ability of the ACL and lack of treatments supporting full, durable ACL repair. Gene therapy guided through the use of biomaterials may steadily activate the processes of repair in sites of ACL injury. The goal of the present study was to test the hypothesis that functionalized poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can effectively deliver recombinant adeno-associated virus (rAAV) vectors as a means of overexpressing two reparative factors (transforming growth factor beta-TGF-β and basic fibroblast growth factor-FGF-2) in primary human ACL fibroblasts. Effective, durable rAAV reporter red fluorescent protein and candidate TGF-β and FGF-2 gene overexpression was achieved in the cells for at least 21 days, especially when pNaSS-grafted PCL films were used versus control conditions, such as ungrafted films and systems lacking vectors or films (between 1.8- and 5.2-fold differences), showing interactive regulation of growth factor production. The expression of TGF-β and FGF-2 from rAAV via PCL films safely enhanced extracellular matrix depositions of type-I/-III collagen, proteoglycans/decorin, and tenascin-C (between 1.4- and 4.5-fold differences) in the cells over time with increased levels of expression of the specific transcription factors Mohawk and scleraxis (between 1.7- and 3.7-fold differences) and without the activation of the inflammatory mediators IL-1β and TNF-α, most particularly with pNaSS-grafted PCL films relative to the controls. This work shows the value of combining rAAV gene therapy with functionalized PCL films to enhance ACL repair

Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair

Subjective self-assessment of physical activity is negatively affected by monitoring awareness in subjects with mild cognitive impairment : A crossover randomised controlled trial

Publisher Copyright: © 2021 Verduci Editore s.r.l. All rights reserved.OBJECTIVE: Physical activity plays an important role in maintaining mental and physical health. This study assessed the effect of physical activity monitoring awareness on the physical activity level and subjective self-assessment of physical activity in middle-aged subjects with normal cognitive function (NCF) and mild cognitive impairment (MCI). PATIENTS AND METHODS: Thirty-five subjects aged 50-65 years with NCF and MCI were randomised into two experimental groups, each taking part in two one-week intervention periods. Subjects in group A were not aware that their physical activity was monitored in the first week (phase I) and were aware of the monitoring in the second week (phase II), whereas it was the opposite order for group B. Physical activity was assessed using the ActiGraph GT9X accelerometer and International Physical Activity Questionnaire (IPAQ). RESULTS: A total of 32 subjects (MCI: n = 12, NCF: n = 20) completed both intervention periods, with MCI subjects having significantly lower objectively assessed physical activity than NCF participants. Moreover, subjectively assessed physical activity in the MCI group was significantly higher when the participants were unaware of physical activity monitoring. A significant phase-group interaction was found in total (MET-min/d: P = 0.0072; min/d: P = 0.0194) and moderate (MET-min/d: P = 0.0015; min/d: P = 0.0020) physical activity as well as energy expenditure (p = 0.0366) assessed by the IPAQ and in the percentage of sedentary behaviour (p = 0.0330) and the average number of steps (p = 0.0342) assessed by ActiGraph. CONCLUSIONS: The awareness of physical activity assessment might decrease the ability to subjectively assess physical activity in subjects with MCI.Peer reviewe