Surface modification of lipid nanoparticles for gene therapy.

Gene therapies have the potential to target and effectively treat a variety of diseases including cancer as well as genetic, neurological, and autoimmune disorders. Although we have made significant advances in identifying non-viral strategies to deliver genetic cargo, certain limitations remain. In general, gene delivery is challenging for several reasons including the instabilities of nucleic acids to enzymatic and chemical degradation and the presence of restrictive biological barriers such as cell, endosomal and nuclear membranes. The emergence of lipid nanoparticles (LNPs) helped overcome many of these challenges. Despite its success, further optimization is required for LNPs to yield efficient gene delivery to extrahepatic tissues, as LNPs favor accumulation in the liver after systemic administration. In this mini-review, we provide an overview of current preclinical approaches in that LNP surface modification was leveraged for cell and tissue targeting by conjugating aptamers, antibodies, and peptides among others. In addition to their cell uptake and efficiency-enhancing effects, we outline the (dis-)advantages of the different targeting moieties and commonly used conjugation strategies.

Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin.

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10-16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%-72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%-12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.

Bioprinting of alginate-carboxymethyl chitosan scaffolds for enamel tissue engineeringin vitro.

Tissue engineering offers a great potential in regenerative dentistry and to this end, three dimensional (3D) bioprinting has been emerging nowadays to enable the incorporation of living cells into the biomaterials (such a mixture is referred as a bioink in the literature) to create scaffolds. However, the bioinks available for scaffold bioprinting are limited, particularly for dental tissue engineering, due to the complicated, yet compromised, printability, mechanical and biological properties simultaneously imposed on the bioinks. This paper presents our study on the development of a novel bioink from carboxymethyl chitosan (CMC) and alginate (Alg) for bioprinting scaffolds for enamel tissue regeneration. CMC was used due to its antibacterial ability and superior cell interaction properties, while Alg was added to enhance the printability and mechanical properties as well as to regulate the degradation rate. The bioinks with three mixture ratios of Alg and CMC (2-4, 3-3 and 4-2) were prepared, and then printed into the calcium chloride crosslinker solution (100 mM) to form a 3D structure of scaffolds. The printed scaffolds were characterized in terms of structural, swelling, degradation, and mechanical properties, followed by theirin vitrocharacterization for enamel tissue regeneration. The results showed that the bioinks with higher concentrations of Alg were more viscous and needed higher pressure for printing; while the printed scaffolds were highly porous and showed a high degree of printability and structural integrity. The hydrogels with higher CMC ratios had higher swelling ratios, faster degradation rates, and lower compressive modulus. Dental epithelial cell line, HAT-7, could maintain high viability in the printed constructs after 1, 7 and 14 d of culture. HAT-7 cells were also able to maintain their morphology and secrete alkaline phosphatase after 14 d of culture in the 3D printed scaffolds, suggesting the capacity of these cells for mineral deposition and enamel-like tissue formation. Among all combinations Alg4%-CMC2% and in a less degree 2%Alg-4%CMC showed the higher potential to promote ameloblast differentiation, Ca and P deposition and matrix mineralizationin vitro. Taken together, Alg-CMC has been illustrated to be suitable to print scaffolds with dental epithelial cells for enamel tissue regeneration.

Gemini surfactant-based nanoparticles T-box1 gene delivery as a novel approach to promote epithelial stem cells differentiation and dental enamel formation.

Enamel is the highest mineralized tissue in the body protecting teeth from external stimuli, infections, and injuries. Enamel lacks the ability to self-repair due to the absence of enamel-producing cells in the erupted teeth. Here, we reported a novel approach to promote enamel-like tissue formation via the delivery of a key ameloblast inducer, T-box1 gene, into a rat dental epithelial stem cell line, HAT-7, using non-viral gene delivery systems based on cationic lipids. We comparatively assessed the lipoplexes prepared from glycyl-lysine-modified gemini surfactants and commercially available 1,2-dioleoyl-3-trimethylammonium-propane lipids at three nitrogen-to phosphate (N/P) ratios of 2.5, 5 and 10. Our findings revealed that physico-chemical characteristics and biological activities of the gemini surfactant-based lipoplexes with a N/P ratio of 5 provide the most optimal outcomes among those examined. HAT-7 cells were transfected with T-box1 gene using the optimal formulation then cultured in conventional 2D cell culture systems. Ameloblast differentiation, mineralization, bio-enamel interface and structure were assessed at different time points over 28 days. Our results showed that our gemini transfection system provides superior gene expression compared to the benchmark agent, while keeping low cytotoxicity levels. T-box1-transfected HAT-7 cells strongly expressed markers of secretory and maturation stages of the ameloblasts, deposited minerals, and produced enamel-like crystals when compared to control cells. Taken together, our gemini surfactant-based T-box1 gene delivery system is effective to accelerate and guide ameloblastic differentiation of dental epithelial stem cells and promote enamel-like tissue formation. This study would represent a significant advance towards the tissue engineering and regeneration of dental enamel.

Self-Crosslinkable Oxidized Alginate-Carboxymethyl Chitosan Hydrogels as an Injectable Cell Carrier for In Vitro Dental Enamel Regeneration.

Injectable hydrogels, as carriers, offer great potential to incorporate cells or growth factors for dental tissue regeneration. Notably, the development of injectable hydrogels with appropriate structures and properties has been a challenging task, leaving much to be desired in terms of cytocompatibility, antibacterial and self-healing properties, as well as the ability to support dental stem cell functions. This paper presents our study on the development of a novel self-cross-linkable hydrogel composed of oxidized alginate and carboxymethyl chitosan and its characterization as a cell carrier for dental enamel regeneration in vitro. Oxidized alginate was synthesized with 60% theoretical oxidation degree using periodate oxidation and characterized by Fourier Transform Infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and Ultraviolet-visible absorption spectroscopy. Then, hydrogels were prepared at three varying weight ratios of oxidized alginate to carboxymethyl chitosan (4:1, 3:1, and 2:1) through Schiff base reactions, which was confirmed by Fourier Transform Infrared spectroscopy. The hydrogels were characterized in terms of gelation time, swelling ratio, structure, injectability, self-healing, antibacterial properties, and in vitro characterization for enamel regeneration. The results demonstrated that, among the three hydrogels examined, the one with the highest ratio of oxidized alginate (i.e., 4:1) had the fastest gelation time and the lowest swelling ability, and that all hydrogels were formed with highly porous structures and were able to be injected through a 20-gauge needle without clogging. The injected hydrogels could be rapidly reformed with the self-healing property. The hydrogels also showed antibacterial properties against two cariogenic bacteria: Streptococcus mutans and Streptococcus sobrinus. For in vitro enamel regeneration, a dental epithelial cell line, HAT-7, was examined, demonstrating a high cell viability in the hydrogels during injection. Furthermore, HAT-7 cells encapsulated in the hydrogels showed alkaline phosphatase production and mineral deposition, as well as maintaining their round morphology, after 14 days of in vitro culture. Taken together, this study has provided evidence that the oxidized alginate-carboxymethyl chitosan hydrogels could be used as an injectable cell carrier for dental enamel tissue engineering applications.

Novel trends, challenges and new perspectives for enamel repair and regeneration to treat dental defects.

Dental enamel is the hardest tissue in the human body, providing external protection for the tooth against masticatory forces, temperature changes and chemical stimuli. Once enamel is damaged/altered by genetic defects, dental caries, trauma, and/or dental wear, it cannot repair itself due to the loss of enamel producing cells following the tooth eruption. The current restorative dental materials are unable to replicate physico-mechanical, esthetic features and crystal structures of the native enamel. Thus, development of alternative approaches to repair and regenerate enamel defects is much needed but remains challenging due to the structural and functional complexities involved. This review paper summarizes the clinical aspects to be taken into consideration for the development of optimal therapeutic approaches to tackle dental enamel defects. It also provides a comprehensive overview of the emerging acellular and cellular approaches proposed for enamel remineralization and regeneration. Acellular approaches aim to artificially synthesize or re-mineralize enamel, whereas cell-based strategies aim to mimic the natural process of enamel development given that epithelial cells can be stimulated to produce enamel postnatally during the adult life. The key issues and current challenges are also discussed here, along with new perspectives for future research to advance the field of regenerative dentistry.

Bioprinting and In Vitro Characterization of an Eggwhite-Based Cell-Laden Patch for Endothelialized Tissue Engineering Applications.

Three-dimensional (3D) bioprinting is an emerging fabrication technique to create 3D constructs with living cells. Notably, bioprinting bioinks are limited due to the mechanical weakness of natural biomaterials and the low bioactivity of synthetic peers. This paper presents the development of a natural bioink from chicken eggwhite and sodium alginate for bioprinting cell-laden patches to be used in endothelialized tissue engineering applications. Eggwhite was utilized for enhanced biological properties, while sodium alginate was used to improve bioink printability. The rheological properties of bioinks with varying amounts of sodium alginate were examined with the results illustrating that 2.0-3.0% (w/v) sodium alginate was suitable for printing patch constructs. The printed patches were then characterized mechanically and biologically, and the results showed that the printed patches exhibited elastic moduli close to that of natural heart tissue (20-27 kPa) and more than 94% of the vascular endothelial cells survived in the examination period of one week post 3D bioprinting. Our research also illustrated the printed patches appropriate water uptake ability (>1800%).

Noninvasive Three-Dimensional In Situ and In Vivo Characterization of Bioprinted Hydrogel Scaffolds Using the X-ray Propagation-Based Imaging Technique.

Hydrogel-based three-dimensional (3D) bioprinting has been illustrated as promising to fabricate tissue scaffolds for regenerative medicine. Notably, bioprinting of hydrated and soft 3D hydrogel scaffolds with desired structural properties has not been fully achieved so far. Moreover, due to the limitations of current imaging techniques, assessment of bioprinted hydrogel scaffolds is still challenging, yet still essential for scaffold design, fabrication, and longitudinal studies. This paper presents our study on the bioprinting of hydrogel scaffolds and on the development of a novel noninvasive imaging method, based on synchrotron propagation-based imaging with computed tomography (SR-PBI-CT), to study the structural properties of hydrogel scaffolds and their responses to environmental stimuli both in situ and in vivo. Hydrogel scaffolds designed with varying structural patterns were successfully bioprinted through rigorous printing process regulations and then imaged by SR-PBI-CT within physiological environments. Subjective to controllable compressive loadings, the structural responses of scaffolds were visualized and characterized in terms of the structural deformation caused by the compressive loadings. Hydrogel scaffolds were later implanted in rats as nerve conduits for SR-PBI-CT imaging, and the obtained images illustrated their high phase contrast and were further processed for the 3D structure reconstruction and quantitative characterization. Our results show that the scaffold design and printing conditions play important roles in the printed scaffold structure and mechanical properties. More importantly, our obtained images from SR-PBI-CT allow us to visualize the details of hydrogel 3D structures with high imaging resolution. It demonstrates unique capability of this imaging technique for noninvasive, in situ characterization of 3D hydrogel structures pre- and post-implantation in diverse physiological milieus. The established imaging platform can therefore be utilized as a robust, high-precision tool for the design and longitudinal studies of hydrogel scaffold in tissue engineering.

Process-induced cell damage: pneumatic versus screw-driven bioprinting.

During the bioprinting processes that employ either pneumatic or screw-driven mechanisms, living cells are subject to process-induced forces, which may cause cell injury or damage. However, the similarities and differences between these two mechanisms have not been discovered and documented in terms of process-induced forces and cell damage. In this paper, we examined the process-induced forces, including hydrostatic pressure, shear stress, extensional stress, and tensile/compressive forces that the cells experienced during the bioprinting processes by means of these two mechanisms; we also experimentally investigated the process-induced cell damage (featured by the rupture of the cell membrane) under various printing conditions or factors, including the volumetric flow rates, cell types, bioink solutions, needle types and sizes, and printing head-movement speeds. On this basis, we correlated the percent of cell damage to the process-induced forces, which were considered mainly responsible for the rupture of the cell membrane. Our results illustrate that compared to the pneumatic bioprinting process, the screw-driven bioprinting process generally induces more cell damage, varying with the printing conditions. This study, for the first time, discovers the similarities and differences between the pneumatic and screw-driven bioprinting processes and further demonstrates their merits and demerits for bioprinting in terms of printing-process control, process-induced forces, and cell damage.

Controlled Drug Delivery Systems for Oral Cancer Treatment-Current Status and Future Perspectives.

Oral squamous cell carcinoma (OSCC), which encompasses the oral cavity-derived malignancies, is a devastating disease causing substantial morbidity and mortality in both men and women. It is the most common subtype of the head and neck squamous cell carcinoma (HNSCC), which is ranked the sixth most common malignancy worldwide. Despite promising advancements in the conventional therapeutic approaches currently available for patients with oral cancer, many drawbacks are still to be addressed; surgical resection leads to permanent disfigurement, altered sense of self and debilitating physiological consequences, while chemo- and radio-therapies result in significant toxicities, all affecting patient wellbeing and quality of life. Thus, the development of novel therapeutic approaches or modifications of current strategies is paramount to improve individual health outcomes and survival, while early tumour detection remains a priority and significant challenge. In recent years, drug delivery systems and chronotherapy have been developed as alternative methods aiming to enhance the benefits of the current anticancer therapies, while minimizing their undesirable toxic effects on the healthy non-cancerous cells. Targeted drug delivery systems have the potential to increase drug bioavailability and bio-distribution at the site of the primary tumour. This review confers current knowledge on the diverse drug delivery methods, potential carriers (e.g., polymeric, inorganic, and combinational nanoparticles; nanolipids; hydrogels; exosomes) and anticancer targeted approaches for oral squamous cell carcinoma treatment, with an emphasis on their clinical relevance in the era of precision medicine, circadian chronobiology and patient-centred health care.

Dental Pulp Stem Cells: Isolation, Characterization, Expansion, and Odontoblast Differentiation for Tissue Engineering.

Tissue engineering is an interdisciplinary area offering a promising approach by the use of stem cells combined with scaffolds and signaling factors for regeneration of damaged or lost tissues. Incorporation of a sufficient number of cells which do not elicit the immunoreaction in the body is a pivotal element for successful tissue formation using this method. Stem cells exhibiting strong capacity to self-renew and differentiate into different cell types are considered as a potent cell source. Among various cell sources, dental pulp stem cells (DPSCs) are widely under investigation due to the fact that they are simply obtainable from extracted third molars or orthodontically extracted teeth and show an excellent potential for clinical application and also their harvesting method is minimally invasive. DPSCs are odontogenic progenitor cells with clonogenic abilities, rapid proliferation rates, and multiple differentiation potentials. Here, we describe protocols that allow 1) the isolation of DPSCs from a single tooth; 2) the characterization of human mesenchymal stem cells markers of DPSCs by flow cytometry; 3) the culture growth of DPSCs in 2D (in cell culture flasks) and 3D (by 3D printing of cell-laden constructs); and 4) the in vivo evaluation of differentiation potential of DPSCs.

A Review on Bioengineering Approaches to Insulin Delivery: A Pharmaceutical and Engineering Perspective.

Diabetes mellitus (DM) is the most prevalent non-contagious disease, which has affected a large number of people all over the world. Among all treatments known to have a positive influence in the control of DM, insulin therapy is the most common and effective one. Nowadays, various methods of insulin delivery are under investigation, which are able to reach a plausible bioavailability with ignorable side effects instead of insulin injection. This article presents a comprehensive review of the insulin therapy approach with a focus on modified methods in insulin delivery strategies and current advances in engineered insulin delivery systems.

Printability and Cell Viability in Bioprinting Alginate Dialdehyde-Gelatin Scaffolds.

Three-dimensional (3D) bioprinting is a promising technique used to fabricate scaffolds from hydrogels with living cells. However, the printability of hydrogels in bioprinting has not been adequately studied. The aim of this study was to quantitatively characterize the printability and cell viability of alginate dialdehyde (ADA)-gelatin (Gel) hydrogels for bioprinting. ADA-Gel hydrogels of various concentrations were synthesized and characterized using Fourier transform infrared spectroscopy, along with rheological tests for measuring storage and loss moduli. Scaffolds (with an area of 11 × 11 mm) of 1, 2, and 13 layers were fabricated from ADA-Gel hydrogels using a 3D-bioplotter under printing conditions with and without the use of cross-linker, respectively, at room temperature and at 4 °C. Scaffolds were then quantitatively assessed in terms of the minimum printing pressure, quality of strands and pores, and structural integrity, which were combined together for the characterization of ADA-Gel printability. For the assessment of cell viability, scaffolds were bioprinted from ADA-Gel hydrogels with human umbilical vein endothelial cells (HUVECs) and rat Schwann cells and were then examined at day 7 with live/dead assay. HUVECs and Schwann cells were used as models to demonstrate biocompatibility for potential angiogenesis and nerve repair applications, respectively. Our results illustrated that ADA-Gel hydrogels with a loss tangent (ratio of loss modulus over storage modulus) between 0.24 and 0.28 could be printed in cross-linker with the best printability featured by uniform strands, square pores, and good structural integrity. Additionally, our results revealed that ADA-Gel hydrogels with an appropriate printability could maintain cell viability over 7 days. Combined together, this study presents a novel method to characterize the printability of hydrogels in bioprinting and illustrates that ADA-Gel hydrogels can be synthesized and bioprinted with good printability and cell viability, thus demonstrating their suitability for bioprinting scaffolds in tissue engineering applications.