Delivery of miRNAs Using Porous Silicon Nanoparticles Incorporated into 3D Hydrogels Enhances MSC Osteogenesis by Modulation of Fatty Acid Signaling and Silicon Degradation.

Strategies incorporating mesenchymal stromal cells (MSC), hydrogels and osteoinductive signals offer promise for bone repair. Osteoinductive signals such as growth factors face challenges in clinical translation due to their high cost, low stability and immunogenicity leading to interest in microRNAs as a simple, inexpensive and powerful alternative. The selection of appropriate miRNA candidates and their efficient delivery must be optimised to make this a reality. This study evaluated pro-osteogenic miRNAs and used porous silicon nanoparticles modified with polyamidoamine dendrimers (PAMAM-pSiNP) to deliver these to MSC encapsulated within gelatin-PEG hydrogels. miR-29b-3p, miR-101-3p and miR-125b-5p are strongly pro-osteogenic and are shown to target FASN and ELOVL4 in the fatty acid biosynthesis pathway to modulate MSC osteogenesis. Hydrogel delivery of miRNA:PAMAM-pSiNP complexes enhanced transfection compared to 2D. The osteogenic potential of hBMSC in hydrogels with miR125b:PAMAM-pSiNP complexes is evaluated. Importantly, a dual-effect on osteogenesis occurred, with miRNAs increasing expression of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2) whilst the pSiNPs enhanced mineralisation, likely via degradation into silicic acid. Overall, this work presents insights into the role of miRNAs and fatty acid signalling in osteogenesis, providing future targets to improve bone formation and a promising system to enhance bone tissue engineering.

Cell-laden injectable microgels: Current status and future prospects for cartilage regeneration.

Injectable hydrogels have been employed extensively as versatile materials for cartilage regeneration due to their excellent biocompatibility, tunable structure, and ability to accommodate bioactive factors, as well as their ability to be locally delivered via minimally invasive injection to fill irregular defects. More recently, in vitro and in vivo studies have revealed that processing these materials to produce cell-laden microgels can enhance cell-cell and cell-matrix interactions and boost nutrient and metabolite exchange. Moreover, these studies have demonstrated gene expression profiles and matrix regeneration that are superior compared to conventional injectable bulk hydrogels. As cell-laden microgels and their application in cartilage repair are moving closer to clinical translation, this review aims to present an overview of the recent developments in this field. Here we focus on the currently used biomaterials and crosslinking strategies, the innovative fabrication techniques being used for the production of microgels, the cell sources used, the signals used for induction of chondrogenic differentiation and the resultant biological responses, and the ability to create three-dimensional, functional cartilage tissues. In addition, this review also covers the current clinical approaches for repairing cartilage as well as specific challenges faced when attempting the regeneration of damaged cartilage tissue. New findings related to the macroporous nature of the structures formed by the assembled microgel building blocks and the novel use of microgels in 3D printing for cartilage tissue engineering are also highlighted. Finally, we outline the challenges and future opportunities for employing cell-laden microgels in clinical applications.

Interplay of Hydrogel Composition and Geometry on Human Mesenchymal Stem Cell Osteogenesis.

Microgels are emerging as an outstanding platform for tissue regeneration because they overcome issues associated with conventional bulk/macroscopic hydrogels such as limited cell-cell contact and cell communication and low diffusion rates. Owing to the enhanced mass transfer and injectability via a minimally invasive procedure, these microgels are becoming a promising approach for bone regeneration applications. Nevertheless, there still remains a huge gap between the understanding of how the hydrogel matrix composition can influence cell response and overall tissue formation when switching from bulk formats to microgel format, which is often neglected or rarely studied. Here, we fabricated polyethylene glycol-based microgels and bulk hydrogels incorporating gelatin and hyaluronic acid (HA), either individually or together, and assessed the impact of both hydrogel composition and format upon the osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs). Osteogenesis was significantly greater in microgels than bulk hydrogels for both gelatin alone (Gel) and gelatin HA composite (Gel:HA) hydrogels, as determined by the expression of Runt-related transcription factor (Runx2) and alkaline phosphatase (ALP) genes and mineral deposition. Interestingly, Gel and Gel:HA hydrogels behaved differently between bulk and microgel format. In bulk format, overall osteogenic outcomes were better in Gel:HA hydrogels, but in microgel format, while the level of osteogenic gene expression was equivalent between both compositions, the degree of mineralization was reduced in Gel:HA microgels. Investigation into the affinity of hydroxyapatite for the different matrix compositions indicated that the decreased mineralization of Gel:HA microgels was likely due to a low affinity of hydroxyapatite to bind to HA and support mineral deposition, which has a greater impact on microgels than bulk hydrogels. Together, these findings suggest that both hydrogel composition and format can determine the success of tissue formation and that there is a complex interplay of these two factors on both cell behavior and matrix deposition. This has important implications for tissue engineering, showing that hydrogel composition and geometry must be evaluated together when optimizing conditions for cell differentiation and tissue formation.

Amino acid-modified chitosan nanoparticles for Cu2+ chelation to suppress CuO nanoparticle cytotoxicity.

The extensive development and application of engineered nanoparticles (NPs) in various fields worldwide have been subjected to increasing concern due to their potential hazards to human health and the environment. Therefore, a simple, economical, and effective method for suppressing the toxicity of metal-based nanomaterials is needed. In this study, glutaraldehyde-crosslinked chitosan nanoparticles (CS NPs) were prepared and further modified with lysine (Ly-CS), glutamic acid (Glu-CS), or sodium borohydride reduction (R-CS), and used to suppress cytotoxicity induced by copper oxide NPs (CuO NPs) through chelation with intracellularly released copper ions. All three kinds of CS NPs had similar sizes of ∼100 nm in a dry state and ∼200 nm in cell culture medium, as determined by scanning electron microscopy, transmission electron microscopy, and dynamic light scattering. The chelating efficiency of different CS NPs followed the order Ly-CS > Glu-CS > R-CS. The CS NPs showed minimal or no toxicity to three different cell lines (HepG2, A549, and RAW264.7 cells) at 100 µg mL-1 with similar cell internalization and exocytosis processes. Comparatively, RAW264.7 cells exhibited higher endocytosis and exocytosis rates, as revealed by flow cytometry and confocal laser scanning microscopy. CS NPs were found as agglomerates inside A549 cells and RAW264.7 cells, with the amount of agglomerates inside RAW264.7 cells decreasing significantly with prolonged incubation. All three CS NPs, especially Ly-CS and Glu-CS NPs, efficiently suppressed the cytotoxicity induced by CuO NPs, and reduced the intracellular level of reactive oxygen species.

Citrate-capped iron oxide nanoparticles impair the osteogenic differentiation potential of rat mesenchymal stem cells.

Surface modification of iron oxide nanoparticles may cause unexpected impact upon interaction with cells, such as cytotoxicity and change in the differentiation potential of stem cells. In this study, two kinds of iron oxide nanoparticles with different surface chemistries, i.e. one in its pristine form (P-NPs) without extra capping molecules and the other coated with citrate (C-NPs), and with similar sizes, ∼10 nm, as measured by transmission electron microscopy and X-ray diffractometry, were prepared. Both P-NPs and C-NPs aggregated to some extent in water, with hydrodynamic diameters of 211.4 ± 29 and 128.6 ± 6.3 nm, and surface zeta potential values of +23.5 ± 0.3 and -49.6 ± 0.5 mV, respectively. However, both NPs further aggregated to a similar extent with hydrodynamic diameters of 260 ± 5.5 and 214 ± 6.4 nm and with a slightly negative surface charge (∼-10 mV) in cell differentiation media. After being incubated with rat mesenchymal stem cells (MSCs) for 14 d, both types of NPs showed similar cell uptake kinetics and final intracellular iron content, i.e. 53.3 pg per cell for P-NPs and 59.9 pg per cell for C-NPs, and minimal cytotoxicity at a concentration below 100 µg mL-1. The adipogenic differentiation potential of MSCs was unaltered regardless of the NP types, and the P-NPs did not have an obvious impact on the osteogenic differentiation potential of MSCs. The osteogenic differentiation potential of the MSCs, however, was significantly impaired by incubation with the C-NPs, as evidenced by significantly reduced expression of osteogenic markers, namely collagen type I (COL) and osteocalcin (OCN) and calcium deposition. The uptake of C-NPs and surface-anchored citrate molecules were found to have a synergistic effect.

Influence of titanium dioxide nanorods with different surface chemistry on the differentiation of rat bone marrow mesenchymal stem cells.

In this study, four kinds of TiO2 nanorods (TiO2 NRs), with similar aspect ratios but different surface functional groups, i.e. amines (-NH2), carboxyl groups (-COOH) and poly(ethylene glycol) (-PEG), were used to study their interaction with rat bone marrow stem cells (MSCs). The aspect ratios of the TiO2 NRs were measured (50 to 65 nm in length and 8 nm in width) under transmission electron microscopy (TEM). The cellular uptake of the TiO2 NRs was qualitatively studied by TEM and then quantified by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the MSCs ingested larger amounts of TiO2-core NRs and TiO2-NH2 NRs than those of TiO2-COOH NRs and TiO2-PEG NRs, with similar intracellular distribution patterns. TiO2-core NRs induced the highest cytotoxicity, as a result of the highest intracellular level of reactive oxygen species (ROS), which was lowered upon surface functionalization. The genotoxicity of the TiO2 NRs was neglectable at tested concentrations, studied by the comet assay. The adipogenic and osteogenic differentiation potentials of the MSCs were firstly evaluated in terms of lipid droplet formation and calcium deposition respectively in the presence of the TiO2 NRs. All of the TiO2 NRs did not show an obvious influence on the adipogenic differentiation potential of the MSCs. But TiO2-COOH NRs showed a significant impairment on the osteogenic differentiation behaviors. The influence of TiO2 NRs on the osteogenic differentiation of the MSCs was further quantitatively studied by the expression of osteogenic markers (collagen type I and osteocalcein), at both gene and protein levels. The results confirmed the strongest hindrance of the osteogenic differentiation of the MSCs by TiO2-COOH NRs, due to the up-regulation of transforming growth factor beta 1 (TGF-ß1) and fibroblast growth factor (FGF-2). The results provide new information that the differentiation potential of the MSCs can be influenced by the presence of TiO2 NRs with different surface functionalities, suggesting a careful analysis of the biological impact of nanomaterials.