Polymeric Nanoparticles Enable mRNA Transfection and Its Translation in Intervertebral Disc and Human Joint Cells, Except for M1 Macrophages.

Chronic lower back pain caused by intervertebral disc degeneration and osteoarthritis (OA) are highly prevalent chronic diseases. Although pain management and surgery can alleviate symptoms, no disease-modifying treatments are available. mRNA delivery could halt inflammation and degeneration and induce regeneration by overexpressing anti-inflammatory cytokines or growth factors involved in cartilage regeneration. Here, we investigated poly(amidoamine)-based polymeric nanoparticles to deliver mRNA to human joint and intervertebral disc cells. Human OA chondrocytes, human nucleus pulposus (NP) cells, human annulus fibrosus (AF) cells, fibroblast-like synoviocytes (FLS) and M1-like macrophages were cultured and transfected with uncoated or PGA-PEG-coated nanoparticles loaded with EGFP-encoding mRNA. Cell viability and transfection efficiency were analyzed for all cell types. Nanoparticle internalization was investigated in FLS and M1-like macrophages. No significant decrease in cell viability was observed in most conditions. Only macrophages showed a dose-dependent reduction of viability. Transfection with either nanoparticle version resulted in EGFP expression in NP cells, AF cells, OA chondrocytes and FLS. Macrophages showed internalization of nanoparticles by particle-cell co-localization, but no detectable expression of EGFP. Taken together, our data show that poly (amidoamine)-based nanoparticles can be used for mRNA delivery into cells of the human joint and intervertebral disc, indicating its potential future use as an mRNA delivery system in OA and IVDD, except for macrophages.

PLGA Nanoparticles Grafted with Hyaluronic Acid to Improve Site-Specificity and Drug Dose Delivery in Osteoarthritis Nanotherapy.

Nanoparticles (NPs) have a tremendous potential in medicinal applications, and recent studies have pushed the boundaries in nanotherapy, including in osteoarthritis treatments. The aim of this study was to develop new poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) surfaces decorated with hyaluronic acid (HA) to enhance targeted drug specificity to the osteoarthritic knee joint. HA was selected since it binds to specific receptors expressed in many cells, such as the cluster determinant 44 (CD44), a major receptor of chondrocytes, and because of its function in the synovial fluid (SF), such as maintenance of high fluid viscosity. The PLGA polymer was grafted to sodium hyaluronate using dimethoxy-PEG (PLGA-HA) and compared with control PLGA NPs (not grafted). NPs were characterized by 1H-NMR and IR spectroscopy. Then, near-infrared (NIR) dye and gold (20 nm) were encapsulated in the formulated NPs and used to access NPs' performance in in vitro, in vivo, and ex vivo experiments. To test the NPs' CD44 receptor specificity, an antibody assay was performed. All NPs presented a size in the range viable for cell-uptake, no cytotoxicity to chondrocytes was registered. Although all the NPs had a high capacity to be absorbed by the cells, PLGA-HA NPs showed significantly higher affinity towards the chondrocytic C28/I2 cell line. In conclusion, PLGA NPs grafted to sodium hyaluronate showed increased binding to cartilage cells and tissue and enhanced accumulation at the target site. Thus, this study presents a safe drug-delivery system with improved receptor specificity, which may represent an advantageous alternative to current nanotherapies.

The Added Value of the "Co" in Co-Culture Systems in Research on Osteoarthritis Pathology and Treatment Development.

Osteoarthritis (OA) is a highly prevalent disease and a major health burden. Its development and progression are influenced by factors such as age, obesity or joint overuse. As a whole organ disease OA affects not only cartilage, bone and synovium but also ligaments, fatty or nervous tissue surrounding the joint. These joint tissues interact with each other and understanding this interaction is important in developing novel treatments. To incorporate and study these interactions in OA research, several co-culture models have evolved. They combine two or more cell types or tissues and investigate the influence of amongst others inflammatory or degenerative stimuli seen in OA. This review focuses on co-cultures and the differential processes occurring in a given tissue or cell as a consequence of being combined with another joint cell type or tissue, and/or the extent to which a co-culture mimics the in vivo processes. Most co-culture models depart from synovial lining and cartilage culture, but also fat pad and bone have been included. Not all of the models appear to reflect the postulated in vivo OA pathophysiology, although some of the discrepancies may indicate current assumptions on this process are not entirely valid. Systematic analysis of the mutual influence the separate compartments in a given model exert on each other and validation against in vivo or ex vivo observation is still largely lacking and would increase their added value as in vitro OA models.

Inferior Turbinate Reduction during Rhinoplasty: Is There Any Effect on Rhinitis Symptoms?

Abstract Introduction Inferior turbinate surgery is often performed concomitantly with rhinoseptoplasty. As inferior turbinates play a major role in allergic rhinitis, it seems reasonable to suggest that inferior turbinate surgery reduces allergy. Objective To assess the impact of nasal turbinate surgery on non-obstructive allergic symptoms (nasal discharge, sneezing, pruritus, and allergic conjunctivitis) and on the use of allergic medication in patients with allergic rhinitis undergoing rhinoseptoplasty. Methods Secondary analysis of aggregated data from two randomized controlled trials. Participants with allergic rhinitis aged 2: 16 years were recruited. Data from two groups were analyzed: patients with rhinoseptoplasty and concomitant turbinate reduction (intervention group) and patients with rhinoseptoplasty only (control group). The 90-day postoperative frequency of non-obstructive allergic symptoms and of nasal steroid and oral antihistamine use were analyzed. Results A total of 100 patients were studied. The groups were similar in terms of allergic symptom intensity and mean age. The frequency of non-obstructive allergic symptoms decreased 90 days postoperative in both groups (p < 0.01). There was no difference between the groups in the frequency of non-obstructive allergic symptoms at 90 days (p = 0.835). Topical nasal steroid and oral histamine antagonist use decreased in the intervention group at 90 days (p < 0.05). Conclusions Ninety days after the surgery, turbinate reduction performed in association with rhinoseptoplasty did not reduce the frequency of non-obstructive allergic symptoms more than rhinoplasty alone. However, the observed decrease in nasal steroid and oral antihistamine use suggests an impact of turbinate reduction on medication use in patients with allergic rhinitis undergoing rhinoseptoplasty. Trial Registration ClinicalTrials.gov database (NCT01457638 and NCT02231216).

Inferior Turbinate Reduction during Rhinoplasty: Is There Any Effect on Rhinitis Symptoms?

Introduction Inferior turbinate surgery is often performed concomitantly with rhinoseptoplasty. As inferior turbinates play a major role in allergic rhinitis, it seems reasonable to suggest that inferior turbinate surgery reduces allergy. Objective To assess the impact of nasal turbinate surgery on non-obstructive allergic symptoms (nasal discharge, sneezing, pruritus, and allergic conjunctivitis) and on the use of allergic medication in patients with allergic rhinitis undergoing rhinoseptoplasty. Methods Secondary analysis of aggregated data from two randomized controlled trials. Participants with allergic rhinitis aged ≥ 16 years were recruited. Data from two groups were analyzed: patients with rhinoseptoplasty and concomitant turbinate reduction (intervention group) and patients with rhinoseptoplasty only (control group). The 90-day postoperative frequency of non-obstructive allergic symptoms and of nasal steroid and oral antihistamine use were analyzed. Results A total of 100 patients were studied. The groups were similar in terms of allergic symptom intensity and mean age. The frequency of non-obstructive allergic symptoms decreased 90 days postoperative in both groups ( p < 0.01). There was no difference between the groups in the frequency of non-obstructive allergic symptoms at 90 days ( p = 0.835). Topical nasal steroid and oral histamine antagonist use decreased in the intervention group at 90 days ( p < 0.05). Conclusions Ninety days after the surgery, turbinate reduction performed in association with rhinoseptoplasty did not reduce the frequency of non-obstructive allergic symptoms more than rhinoplasty alone. However, the observed decrease in nasal steroid and oral antihistamine use suggests an impact of turbinate reduction on medication use in patients with allergic rhinitis undergoing rhinoseptoplasty. Trial Registration ClinicalTrials.gov database (NCT01457638 and NCT02231216).

Association between Oncostatin M Expression and Inflammatory Phenotype in Experimental Arthritis Models and Osteoarthritis Patients.

Pro-inflammatory cytokines are considered to play a major role in osteoarthritis (OA), yet so far, the specific cytokines involved in the pathology of OA have not been identified. Oncostatin M (OSM) is a cytokine from the interleukin 6 (IL-6) family that has been shown to be elevated in synovial fluid of most rheumatoid arthritis (RA) patients, but only in a limited subset of OA patients. Little is known about OSM in the different joint tissues during OA and how its expression correlates with hallmarks of disease. Here, we mapped OSM expression in the joint tissues of two rat models of arthritis: an acute inflammatory model and an instability-induced osteoarthritic model. OSM expression was correlated with hallmarks of OA, namely cartilage damage, synovitis, and osteophyte formation. Reanalysis of an existing dataset on cytokine profiling of OA synovial fluid was performed to assess pattern differences between patients positive and negative for OSM. In the inflammatory model, OSM expression correlated with synovitis and osteophyte formation but not with cartilage damage. On the contrary, in the instability model of OA, an increase in synovitis, cartilage damage, and osteophyte formation was observed without changes in OSM expression. In line with these findings, synovial fluid of OA patients with detectable OSM contained higher levels of other inflammatory cytokines, namely interferon gamma (IFN-γ), IL-1α and tumor necrosis factor alpha (TNF-α), likely indicating a more inflammatory state. Taken together these data indicate OSM might play a prominent role in inflammatory phenotypes of OA.

Drug delivery in intervertebral disc degeneration and osteoarthritis: Selecting the optimal platform for the delivery of disease-modifying agents.

Osteoarthritis (OA) and intervertebral disc degeneration (IVDD) as major cause of chronic low back pain represent the most common degenerative joint pathologies and are leading causes of pain and disability in adults. Articular cartilage (AC) and intervertebral discs are cartilaginous tissues with a similar biochemical composition and pathophysiological aspects of degeneration. Although treatments directed at reversing these conditions are yet to be developed, many promising disease-modifying drug candidates are currently under investigation. Given the localized nature of these chronic diseases, drug delivery systems have the potential to enhance therapeutic outcomes by providing controlled and targeted release of bioactives, minimizing the number of injections needed and increasing drug concentration in the affected areas. This review provides a comprehensive overview of the currently most promising disease-modifying drugs as well as potential drug delivery systems for OA and IVDD therapy.

Fibrin-hyaluronic acid hydrogel-based delivery of antisense oligonucleotides for ADAMTS5 inhibition in co-delivered and resident joint cells in osteoarthritis.

To date no disease-modifying drugs for osteoarthritis (OA) are available, with treatment limited to the use of pain killers and prosthetic replacement. The ADAMTS (A Disintegrin and Metallo Proteinase with Thrombospondin Motifs) enzyme family is thought to be instrumental in the loss of proteoglycans during cartilage degeneration in OA, and their inhibition was shown to reverse osteoarthritic cartilage degeneration. Locked Nucleic Acid (LNA)-modified antisense oligonucleotides (gapmers) released from biomaterial scaffolds for specific and prolonged ADAMTS inhibition in co-delivered and resident chondrocytes, is an attractive therapeutic strategy. Here, a gapmer sequence identified from a gapmer screen showed 90% ADAMTS5 silencing in a monolayer culture of human OA chondrocytes. Incorporation of the gapmer in a fibrin-hyaluronic acid hydrogel exhibited a sustained release profile up to 14 days. Gapmers loaded in hydrogels were able to transfect both co-embedded chondrocytes and chondrocytes in a neighboring gapmer-free hydrogel, as demonstrated by flow cytometry and confocal microscopy. Efficient knockdown of ADAMTS5 was shown up to 14 days in both cell populations, i.e. the gapmer-loaded and gapmer-free hydrogel. This work demonstrates the use applicability of a hydrogel as a platform for combined local delivery of chondrocytes and an ADAMTS-targeting gapmer for catabolic gene modulation in OA.

Biodegradable PEG-dendritic block copolymers: synthesis and biofunctionality assessment as vectors of siRNA.

One important drawback of most of the currently used dendrimers for biomedical applications is their high stability under physiological conditions that can result in cytotoxicity or complications induced by the accumulation of non-degradable synthetic materials in the organism. Particularly in the gene therapy field, vector stability can further hinder the intracellular release of the nucleic acid from the dendriplex, consequently leading to low transfection efficiencies. Therefore, biodegradable cationic dendritic structures have been eagerly awaited. However, the development of these dendritic nanocarriers is challenging because of the undesired and/or premature degradation observed during their synthesis and/or application. Here, we report new hybrid-biodegradable, biocompatible, non-toxic, and water-soluble azide-terminated PEG-GATGE dendritic block copolymers, based on a gallic acid (GA) core and triethylene glycol (TG) butanoate arms, incorporating ester bonds (E) at the dendritic arms/shell. Their successful functionalization by "click" chemistry with unprotected alkynated amines allowed complexation and delivery of siRNA. The hydrophobic character of the GATGE building unit confers to these hydrolyzable dendritic bionanomaterials a great ability to complex, protect and mediate the cellular internalization of siRNA. Moreover, the localization of the degradation points at the dendritic periphery, close to the complexed siRNA, was found to be important for nucleic acid release from the nanoparticles, rendering a significant improvement of the transfection efficiency compared to their hydrolytically stable PEG-GATG copolymer counterparts. The present study puts forward these biodegradable PEG-dendritic block copolymers not only as suitable vectors for nucleic acids, but also as new avenues for further developments exploring their use in theranostics.

Comparison of two analgesia protocols for the treatment of pediatric orthopedic emergencies.

OBJECTIVE: to compare the efficacy of two analgesia protocols (ketamine versus morphine) associated with midazolam for the reduction of dislocations or closed fractures in children. METHODS: randomized clinical trial comparing morphine (0.1mg/kg; max 5mg) and ketamine (2.0mg/kg, max 70mg) associated with midazolam (0.2mg/kg; max 10mg) in the reduction of dislocations or closed fractures in children treated at the pediatrics emergency room (October 2010 and September 2011). The groups were compared in terms of the times to perform the procedures, analgesia, parent satisfaction and orthopedic team. RESULTS: 13 patients were allocated to ketamine and 12 to morphine, without differences in relation to age, weight, gender, type of injury, and pain scale before the intervention. There was no failure in any of the groups, no differences in time to start the intervention and overall procedure time. The average hospital stay time was similar (ketamine = 10.8+5.1h versus morphine = 12.3+4.4hs; p=0.447). The median pain (faces pain scale) scores after the procedure was 2 in both groups. Amnesia was noted in 92.3% (ketamine) and 83.3% (morphine) (p=0.904). Parents said they were very satisfied in relation to the analgesic intervention (84.6% in the ketamine group and 66.6% in the morphine group; p=0.296). The satisfaction of the orthopedist regarding the intervention was 92.3% in the ketamine group and 75% in the morphine group (p=0.222). CONCLUSION: by producing results similar to morphine, ketamine can be considered as an excellent option in pain management and helps in the reduction of dislocations and closed fractures in pediatric emergency rooms.

Comparison of two analgesia protocols for the treatment of pediatric orthopedic emergencies / Comparação de dois protocolos de analgesia para tratamento de emergências ortopédicas pediátricas

SummaryObjective:to compare the efficacy of two analgesia protocols (ketamine versus morphine) associated with midazolam for the reduction of dislocations or closed fractures in children.Methods:randomized clinical trial comparing morphine (0.1mg/kg; max 5mg) and ketamine (2.0mg/kg, max 70mg) associated with midazolam (0.2mg/kg; max 10mg) in the reduction of dislocations or closed fractures in children treated at the pediatrics emergency room (October 2010 and September 2011). The groups were compared in terms of the times to perform the procedures, analgesia, parent satisfaction and orthopedic team.Results:13 patients were allocated to ketamine and 12 to morphine, without differences in relation to age, weight, gender, type of injury, and pain scale before the intervention. There was no failure in any of the groups, no differences in time to start the intervention and overall procedure time. The average hospital stay time was similar (ketamine = 10.8+5.1h versus morphine = 12.3+4.4hs; p=0.447). The median pain (faces pain scale) scores after the procedure was 2 in both groups. Amnesia was noted in 92.3% (ketamine) and 83.3% (morphine) (p=0.904). Parents said they were very satisfied in relation to the analgesic intervention (84.6% in the ketamine group and 66.6% in the morphine group; p=0.296). The satisfaction of the orthopedist regarding the intervention was 92.3% in the ketamine group and 75% in the morphine group (p=0.222).Conclusion:by producing results similar to morphine, ketamine can be considered as an excellent option in pain management and helps in the reduction of dislocations and closed fractures in pediatric emergency rooms.

ResumoObjetivo:comparar a eficácia de dois protocolos de analgesia (cetamina versus morfina) associados ao midazolam para a redução de luxações ou fraturas fechadas em crianças.Métodos:ensaio clínico randomizado comparando morfina (0,1 mg/kg; máx. 5 mg) e cetamina (2,0 mg/kg; máx. 70 mg) associados a midazolam (0,2 mg/kg; máx. 10 mg) na redução de luxações ou fraturas fechadas em crianças atendidas em emergência pediátrica, no período de outubro de 2010 a setembro de 2011. Os grupos foram comparados segundo os seguintes indicadores: tempo para realizar os procedimentos, analgesia, satisfação de pais e da equipe ortopédica.Resultados:treze pacientes foram alocados para cetamina e 12 para morfina, sem diferenças em relação a idade, peso, gênero, tipo de lesão e escala da dor antes da intervenção. Não houve falha em nenhum dos grupos, sem diferenças no tempo para iniciar a intervenção e no tempo total de procedimento. O tempo médio de hospitalização foi similar (cetamina=10,8±5,1 h versus morfina=12,3±4,4 h; p=0,447). A mediana de dor (escala de faces da dor) após o procedimento foi de 2 em ambos os grupos. Amnésia foi observada em 92,3% (cetamina) e 83,3% (morfina) (p=0,904). Os pais declararam estar muito satisfeitos em relação à intervenção analgésica (84,6% no grupo cetamina e 66,6% no grupo morfina; p=0,296). A satisfação do ortopedista em relação à intervenção foi de 92,3% no grupo cetamina e 75% no grupo da morfina (p=0,222).Conclusão:a cetamina, ao apresentar resultados semelhantes à morfina, pode ser considerada uma excelente opção no manejo da dor e no auxílio da redução de luxações e fraturas fechadas em salas de emergência pediátrica.

The Present and the Future of Degradable Dendrimers and Derivatives in Theranostics.

Interest in dendrimer-based nanomedicines has been growing recently, as it is possible to precisely manipulate the molecular weight, chemical composition, and surface functionality of dendrimers, tuning their properties according to the desired biomedical application. However, one important concern about dendrimer-based therapeutics remains-the nondegradability under physiological conditions of the most commonly used dendrimers. Therefore, biodegradable dendrimers represent an attractive class of nanomaterials, since they present advantages over conventional nondegradable dendrimers regarding the release of the loaded molecules and the prevention of bioaccumulation of synthetic materials and subsequent cytotoxicity. Here, we present an overview of the state-of-the-art of the design of biodegradable dendritic structures, with particular focus on the hurdles regarding the use of these as vectors of drugs and nucleic acids, as well as macromolecular contrast agents.