Chronic oral methylphenidate treatment increases microglial activation in rats.

Methylphenidate (MP) is a widely prescribed psychostimulant used to treat attention deficit hyperactivity disorder. Previously, we established a drinking paradigm to deliver MP to rats at doses that result in pharmacokinetic profiles similar to treated patients. In the present study, adolescent male rats were assigned to one of three groups: control (water), low-dose MP (LD; 4/10 mg/kg), and high dose MP (HD; 30/60 mg/kg). Following 3 months of treatment, half of the rats in each group were euthanized, and the remaining rats received only water throughout a 1-month-long abstinence phase. In vitro autoradiography using [3H] PK 11195 was performed to measure microglial activation. HD MP rats showed increased [3H] PK 11195 binding compared to control rats in several cerebral cortical areas: primary somatosensory cortex including jaw (68.6%), upper lip (80.1%), barrel field (88.9%), and trunk (78%) regions, forelimb sensorimotor area (87.3%), secondary somatosensory cortex (72.5%), motor cortices 1 (73.2%) and 2 (69.3%), insular cortex (59.9%); as well as subcortical regions including the thalamus (62.9%), globus pallidus (79.4%) and substantia nigra (22.7%). Additionally, HD MP rats showed greater binding compared to LD MP rats in the hippocampus (60.6%), thalamus (59.6%), substantia nigra (38.5%), and motor 2 cortex (55.3%). Following abstinence, HD MP rats showed no significant differences compared to water controls; however, LD MP rats showed increased binding in pre-limbic cortex (78.1%) and ventromedial caudate putamen (113.8%). These findings indicate that chronic MP results in widespread microglial activation immediately after treatment and following the cessation of treatment in some brain regions.

Medication-related osteonecrosis of the jaw: A disease of significant importance for older patients.

BACKGROUND: Medication-related osteonecrosis of the jaw (MRONJ) is clinically defined as a non-healing jawbone ulcerative-necrotic lesion appearing after dental therapy or minor trauma in patients treated previously with anti-resorptive, anti-angiogenic or immunomodulators. Older patients with osteoporosis and cancer receive these pharmacological agents regularly. As these patients are long-term survivors, efficient treatment is of paramount importance for their quality of life. METHODS: Literature searches via PubMed were conducted to identify relevant MRONJ studies. Basic information on MRONJ classification, clinical features, and pathosphysiology is presented herein as well as various clinical studies dealing with MRONJ in patients with osteoporosis and cancer. Lastly, we discuss current managment of patients and new trends in treatment of MRONJ. RESULTS: Although close follow-up and local hygiene have been advocated by some authors, severe forms of MRONJ are not responsive to conservative therapy. At present, there is no "gold standard" therapy for this condition. However, as the physiopathological basis of MRONJ is represented by the anti-angiogenic action of various pharmacological agents, new methods to increase and promote local angiogenesis and vascularization have recently been successfully tested in vitro, limited preclinical studies, and in a pilot clinical study. CONCLUSIONS: It appears that the best method implies application on the lesion of endothelial progenitor cells as well as pro-angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) and other related molecules. More recently, scaffolds in which these factors have been incorporated have shown positive results in limited trials. However, these studies must be replicated to include a large number of cases before any official therapeutic protocol is adopted.

What Do COVID-19 Vaccines Tell Us About Nucleic Acid Delivery In Vivo?

The utilization of the mRNA-based Pfizer-BioNTech and Moderna coronavirus disease 2019 (COVID-19) vaccines represents the culmination of many years of nonviral nucleic acid delivery, but more importantly, they signify a massive clinical scientific success. Scientists working in the area of nucleic acid delivery using lipid nanoparticles will undoubtedly be energized by the success of these vaccines and begin to collect much needed data in the realm of nonviral-based RNA and DNA delivery, specifically, the use of lipid nanoparticles, the immune response, safety, and efficacy. It is easily conceivable that in the future we can utilize these data to help streamline our approach for the delivery of DNA for gene therapy and regulatory RNAs for therapeutic and regenerative medicine (ie, wound repair) applications.

Enhanced composite electrospun nanofiber scaffolds for use in drug delivery.

The utility of nanofibrous electrospun composite scaffolds has greatly expanded over the last decade, so that they now serve as viable drug delivery vehicles for a host of different biomedical applications. The material properties of electrospun scaffolds are extremely advantageous for drug delivery, in which site-specificity and lower overall medicinal dosages lead to a potential industry-altering mechanism of delivering therapeutics. Different drugs used to predominantly treat infections and cancers can easily be incorporated and released at therapeutic dosages. Further, the inherent high porosity of these electrospun scaffolds allows for a more precisely controlled degradation which is tunable by polymer composition and fiber morphology, leading to sustained drug release. This review examines the current research and breakthrough discoveries that have elevated electrospun scaffolds to a cutting-edge technology that will dramatically alter the landscape of drug delivery.

Functionalization of poly(L-lactide) nanofibrous scaffolds with bioactive collagen molecules.

The utilization of electrospun biodegradable scaffolds by fine-tuning their biofunctionalities through a simple mixing method was demonstrated in this study. Poly(L-lactide) (PLLA)-based scaffolds containing small amounts of bioactive collagen type I molecules were investigated for enhancements in cellular behavior. Electron microscopy revealed no topological alterations of the fibers in the collagen/PLLA scaffolds when compared with pure PLLA scaffolds. Cell attachment after 24 h was robust on collagen/PLLA scaffolds, with cytoskeletal analysis showing that the attached cells were aligned along the fibers assuming a spindle-shape appearance. Despite these morphological differences, gene expression analyses revealed no apparent alterations in mRNA levels of four genes involved in cell attachment across the various scaffolds. Although cell proliferation was not adversely affected, there were clear differences in cell penetration; after 1 week, cells migrated through 32 and 85% of PLLA and collagen/PLLA scaffolds, respectively. Mineralization of primary calvaria osteoblasts provided further evidence that collagen-containing electrospun PLLA scaffolds could sustain cell differentiation. Overall, the inclusion of collagen type I in even miniscule amounts (<1 wt %) within electrospun PLLA scaffolds could effectively modulate certain aspects of cellular behavior.

In vitro non-viral gene delivery with nanofibrous scaffolds.

Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (beta-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1x TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L8E78L8), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro.

Control of degradation rate and hydrophilicity in electrospun non-woven poly(D,L-lactide) nanofiber scaffolds for biomedical applications.

Typical properties of poly(D,L-lactide) (PLA)-based scaffolds (films and foams), such as long degradation time, mechanical stiffness and hydrophobicity, are sometimes not suitable for biomedical applications. These properties can be substantially altered by electrospinning of PLA blends with miscible poly(lactide-co-glycolide) (PLGA) random copolymers, poly(lactide-b-ethylene glycol-b-lactide) (PLA-b-PEG-b-PLA) triblock copolymers, and a lactide (used as a hydrolytic catalyst). Electrospun scaffolds based on the multi-component PLA blends, comprised of randomly interconnected webs of sub-micron sized fibers, have a bulk density of 0.3-0.4 g/cm3. In this study, the concentration effects of PLA-b-PEG-b-PLA triblock copolymer and lactide on the cell proliferation and the hydrophilicity of electrospun scaffolds were investigated. Based on in vitro degradation study, we found that the electrospun scaffold having PLA (40 wt%), PLGA (LA/GA=50/50, 25 wt%), PLA-b-PEG-b-PLA (20 wt%), and lactide (15 wt%) underwent a rapid weight loss of approximately 65% in 7 weeks. The hydrophobicity of this membrane, as determined by contact angle measurements in a cell buffer solution, decreased by approximately 50% from 105 degrees (of an electrospun PLA scaffold) to 50 degrees. The selection of suitable chemical compositions in conjunction with the non-invasive electrospinning process is useful in the production of a new kind of biodegradable scaffolds suitable for different biomedical applications such as cell storage and delivery as well as prevention of post-surgical adhesion because of their porosity, mechanical flexibility and tunable biodegradability.

Incorporation and controlled release of a hydrophilic antibiotic using poly(lactide-co-glycolide)-based electrospun nanofibrous scaffolds.

The successful incorporation and sustained release of a hydrophilic antibiotic drug (Mefoxin, cefoxitin sodium) from electrospun poly(lactide-co-glycolide) (PLGA)-based nanofibrous scaffolds without the loss of structure and bioactivity was demonstrated. The morphology and density of the electrospun scaffold was found to be dependent on the drug concentration, which could be attributed to the effect of ionic salt on the electrospinning process. The drug release behavior from the electrospun scaffolds and its antimicrobial effects on Staphylococcus aureus cultures were also investigated. In all tested scaffolds, the maximum dosage of drug was released after 1 h of incubation in water at 37 degrees C. The usage of the amphiphilic block copolymer (PEG-b-PLA) reduced the cumulative amount of the released drug at earlier time points and prolonged the drug release rate at longer times (up to a 1-week period). The antibiotic drug released from these electrospun scaffolds was effective in their ability to inhibit Staphylococcus aureus growth (>90%). The combination of mechanical barriers based on non-woven nanofibrous biodegradable scaffolds and their capability for local delivery of antibiotics increases their desired utility in biomedical applications, particularly in the prevention of post-surgical adhesions and infections.

Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

RNA interference (RNAi) is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh) RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL) alone (Control) and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i) and EGFP (EGFPi, also served as a control) shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing) and 0.2-3 µm (Control). While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release) of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

Induction of cell migration in vitro by an electrospun PDGF-BB/PLGA/PEG-PLA nanofibrous scaffold.

Electrospinning is a versatile technique used to fabricate potential tissue engineering scaffolds with a structure similar to the native extracellular matrix (ECM). In this study, Platelet-Derived Growth Factor (PDGF)-BB with BSA as a carrier protein was incorporated into an electrospun PLGA/PEG-PLA composite scaffold for induction of cell migration, an early process necessary for tissue regeneration and wound healing. Incorporating PDGF-BB into the fibers did not change the overall morphology of the scaffold, with the exception of a slight increase (approximately 12%) in the number of fibers with diameters ranging from 1-100 nm. Following a strong burst of release during the initial 24 hours, approximately 20% of the total incorporated PDGF-BB was released from the scaffold over 5 days, as determined by ELISA. The presence of the released PDGF-BB was also confirmed via SDS-PAGE. Using an in vitro agarose-cell migration assay with MC3T3 pre-osteoblastic cells, the preserved bioactivity of the released PDGF-BB was demonstrated via its ability to stimulate robust cell migration, equivalent to that of pure unincorporated (control) PDGF-BB. Overall, this study demonstrates that it is feasible to incorporate and deliver bioactive PDGF-BB via an electrospun scaffold for potential tissue repair applications, especially as a potent inducer of cell migration.

The role of moderate static magnetic fields on biomineralization of osteoblasts on sulfonated polystyrene films.

We have investigated the effects of moderate static magnetic fields (SMFs) on murine MC3T3-E1 osteoblasts, and found that they enhance proliferations and promote differentiation. The increase in proliferation rates in response to SMFs was greater in cultures grown on partially sulfonated polytstyrene (SPS, degree of sulfonation: 33%) than in cultures grown on tissue culture plastic. We have previously shown that when the degree of sulfonation exceeded a critical value (12%) [1], spontaneous fibrillogenesis occured which allowed for direct observation of the ECM fibrillar organization under the influence of external fields. We found that the ECM produced in cultures grown on the SPS in the presence of the SMFs assembled into a lattice with larger dimensions than the ECM of the cultures grown in the absence of SMFs. During the early stages of the biomineralization process (day 7), the SMF exposed cultures also templated mineral deposition more rapidly than the control cultures. The rapid response is attributed to orientation of diamagnetic ECM proteins already present in the serum, which could then initiate further cellular signaling. SMFs also influenced late stage osteoblast differentiation as measured by the increased rate of osteocalcin secretion and gene expression beginning 15 days after SFM exposure. This correlated with a large increase in mineral deposition, and in cell modulus. GIXD and EDXS analysis confirmed early deposition of crystalline hydroxyapatite. Previous studies on the effects of moderate SMF had focused on cellular gene and protein expression, but did not consider the organization of the ECM fibers. Our ability to form these fibers has allowed us explore this additional effect and highlight its significance in the initiation of the biomineralization process.