3D bioprinted small extracellular vesicles from periodontal cells enhance mesenchymal stromal cell function.

Recent research indicates that combining 3D bioprinting and small extracellular vesicles (sEVs) offers a promising 'cell-free' regenerative medicine approach for various tissue engineering applications. Nonetheless, the majority of existing research has focused on bioprinting of sEVs sourced from cell lines. There remains a notable gap in research regarding the bioprinting of sEVs derived from primary human periodontal cells and their potential impact on ligamentous and osteogenic differentiation. Here, we investigated the effect of 3D bioprinted periodontal cell sEVs constructs on the differentiation potential of human buccal fat pad-derived mesenchymal stromal cells (hBFP-MSCs). Periodontal cell-derived sEVs were enriched by size exclusion chromatography (SEC) with particle-shaped morphology, and characterized by being smaller than 200 nm in size and CD9/CD63/CD81 positive, from primary human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). The sEVs were then 3D bioprinted in 10 % gelatin methacryloyl (GelMA) via microextrusion bioprinting. Release of sEVs from bioprinted constructs was determined by DiO-labelling and confocal imaging, and CD9 ELISA. Attachment and ligament/osteogenic/cementogenic differentiation of hBFP-MSCs was assessed on bioprinted GelMA, without and with sEVs (GelMA/hPDLCs-sEVs and GelMA/hGFs-sEVs), scaffolds. hBFP-MSCs seeded on the bioprinted sEVs constructs spread well with significantly enhanced focal adhesion, mechanotransduction associated gene expression, and ligament and osteogenesis/cementogenesis differentiation markers in GelMA/hPDLCs-sEVs, compared to GelMA/hGFs-sEVs and GelMA groups. A 2-week osteogenic and ligamentous differentiation showed enhanced ALP staining, calcium formation and toluidine blue stained cells in hBFP-MSCs on bioprinted GelMA/hPDLCs-sEVs constructs compared to the other two groups. The proof-of-concept data from this study supports the notion that 3D bioprinted GelMA/hPDLCs-sEVs scaffolds promote cell attachment, as well as ligamentous, osteogenic and cementogenic differentiation, of hBFP-MSCs in vitro.

The effect of culture conditions on the bone regeneration potential of osteoblast-laden 3D bioprinted constructs.

Three Dimensional (3D) bioprinting is one of the most recent additive manufacturing technologies and enables the direct incorporation of cells within a highly porous 3D-bioprinted construct. While the field has mainly focused on developing methods for enhancing printing resolution and shape fidelity, little is understood about the biological impact of bioprinting on cells. To address this shortcoming, this study investigated the in vitro and in vivo response of human osteoblasts subsequent to bioprinting using gelatin methacryloyl (GelMA) as the hydrogel precursor. First, bioprinted and two-dimensional (2D) cultured osteoblasts were compared, demonstrating that the 3D microenvironment from bioprinting enhanced bone-related gene expression. Second, differentiation regimens of 2-week osteogenic pre-induction in 2D before bioprinting and/or 3-week post-printing osteogenic differentiation were assessed for their capacity to increase the bioprinted construct's biofunctionality towards bone regeneration. The combination of pre-and post-induction regimens showed superior osteogenic gene expression and mineralisation in vitro. Moreover, a rat calvarial model using microtomography and histology demonstrated bone regeneration potential for the pre-and post-differentiation procedure. This study shows the positive impact of bioprinting on cells for osteogenic differentiation and the increased in vivo osteogenic potential of bioprinted constructs via a pre-induction method. STATEMENT OF SIGNIFICANCE: 3D bioprinting, one of the most recent technologies for tissue engineering has mostly focussed on developing methods for enhancing printing properties, little is understood on the biological impact of bioprinting and /or subsequent in vitro maturation methods on cells. Therefore, we addressed these fundamental questions by investigating osteoblast gene expression in bioprinted construct and assessed the efficacy of several induction regimen towards osteogenic differentiation in vitro and in vivo. Osteogenic induction of cells prior to seeding in scaffolds used in conventional tissue engineering applications has been demonstrated to increase the osteogenic potential of the resulting construct. However, to the best of our knowledge, pre-induction methods have not been investigated in 3D bioprinting.

Optimization of 3D bioprinting of periodontal ligament cells.

Three-dimensional (3D) bioprinting of cells is an emerging area of research but has been not explored yet in the context of periodontal tissue engineering. OBJECTIVE: This study reports on the optimisation of the 3D bioprinting of periodontal ligament cells for potential application in periodontal regeneration. METHODS: We systematically investigated the printability of various concentrations of gelatin methacryloyl (GelMA) hydrogel precursor using a microextrusion based three-dimensional (3D) printer. The influence of different printing parameters such as photoinitiator concentration, UV exposure, pressure and dispensing needle diameter on the viability of periodontal ligament cells encapsulated within the 3D bioprinted construct were subsequently assessed. RESULTS: This systematic evaluation enabled the selection of the most suited printing conditions for achieving high printing resolution, dimensional stability and cell viability for 3D bioprinting of periodontal ligament cells. SIGNIFICANCE: The optimised bioprinting system is the first step towards to the reproducible manufacturing of cell laden, space maintaining scaffolds for the treatment of periodontal lesions.

Ciprofloxacin- and Fluconazole-Containing Fibrin-Nanoparticle-Incorporated Chitosan Bandages for the Treatment of Polymicrobial Wound Infections.

Polymicrobial wound infections often require high dosages of antibiotics and fungicides. However, prolonged antimicrobial therapies are associated with potential systemic side effects and an increased risk of the development of drug-resistant microbes. With this focus, we aimed at developing chitosan bandages loaded with antimicrobial drug (ciprofloxacin and fluconazole) nanoparticles for a sustained slow release of drugs. The particle sizes of the prepared ciprofloxacin- and flucanazole-loaded fibrin nanoparticles were observed to be 132 ± 16 and 175 ± 17 nm, respectively. The chitosan bandages with drug-containing nanoparticles were flexible and had adequate tensile strength and porosity of 80-85%, which would favor excess exudate absorption in an infectious wound. The in vitro toxicity of the bandages studied against the human dermal fibroblast cell line proved its cytocompatibility. Ciprofloxacin and fluconazole were released from bandages for up to 14 days in a sustained manner. The antimicrobial-drug-loaded bandages showed significant antimicrobial activity toward polymicrobial cultures of Candida albicans, Escherichia coli, and Staphylococcus aureus in vitro and ex vivo. In vivo studies were conducted on a polymicrobially infected rat wound model. A significant reduction in microbial load was obtained upon application of antimicrobial-drug-loaded chitosan bandages in vivo.

Evolution of Synonymous Codon Usage Bias in West African and Central African Strains of Monkeypox Virus.

The evolution of bias in synonymous codon usage in chosen monkeypox viral genomes and the factors influencing its diversification have not been reported so far. In this study, various trends associated with synonymous codon usage in chosen monkeypox viral genomes were investigated, and the results are reported. Identification of factors that influence codon usage in chosen monkeypox viral genomes was done using various codon usage indices, such as the relative synonymous codon usage, the effective number of codons, and the codon adaptation index. The Spearman rank correlation analysis and a correspondence analysis were used for correlating various factors with codon usage. The results revealed that mutational pressure due to compositional constraints, gene expression level, and selection at the codon level for utilization of putative optimal codons are major factors influencing synonymous codon usage bias in monkeypox viral genomes. A cluster analysis of relative synonymous codon usage values revealed a grouping of more virulent strains as one major cluster (Central African strains) and a grouping of less virulent strains (West African strains) as another major cluster, indicating a relationship between virulence and synonymous codon usage bias. This study concluded that a balance between the mutational pressure acting at the base composition level and the selection pressure acting at the amino acid level frames synonymous codon usage bias in the chosen monkeypox viruses. The natural selection from the host does not seem to have influenced the synonymous codon usage bias in the analyzed monkeypox viral genomes.