In vitroosteogenesis of hMSCs on collagen membranes embedded within LEGO®-inspired 3D printed PCL constructs for mandibular bone repair.

The field of bone tissue engineering aims to develop an effective and aesthetical bone graft substitute capable of repairing large mandibular defects. However, graft failure resulting from necrosis and insufficient integration with native tissue due to lack of oxygen and nutrient transportation remains a concern. To overcome these drawbacks, this study aims to develop a 3D printed polycaprolactone layered construct with a LEGO®-inspired interlocking mechanism enabling spatial distribution of biological components. To highlight itsin vitroosteogenic potential, human mesenchymal stromal cells are cultured onto Bio-Gide®Compressed collagen (Col) membranes, which are embedded within the layered construct for 28 d. The osteogenic response is assessed through the measurement of proliferation, relevant markers for osteogenesis including alkaline phosphatase (ALP) activity, expression of transcriptional genes (SP7, RUNX2/SOX9) as well matrix-related genes (COL1A1, ALPL IBSP, SPP1), osteoprotegerin secretion.In vitroosteogenic differentiation results showed increased levels of these osteogenic markers, indicating the layered construct's potential to support osteogenesis. In this study, a novel workflow of 3D printing a patient-specific LEGO®-inspired layered construct that can spatially deliver biological elements was successfully demonstrated. These layered constructs have the potential to be employed as a bone tissue engineering strategy, with particular focus on the repair of large mandibular defects.

Prognostic and therapeutic potential of microRNAs for fracture healing processes and non-union fractures: A systematic review.

BACKGROUND: Approximately 10% of all bone fractures result in delayed fracture healing or non-union; thus, the identification of biomarkers and prognostic factors is of great clinical interest. MicroRNAs (miRNAs) are known to be involved in the regulation of the bone healing process and may serve as functional markers for fracture healing. AIMS AND METHODS: This systematic review aimed to identify common miRNAs involved in fracture healing or non-union fractures using a qualitative approach. A systematic literature search was performed with the keywords 'miRNA and fracture healing' and 'miRNA and non-union fracture'. Any original article investigating miRNAs in fracture healing or non-union fractures was screened. Eventually, 82 studies were included in the qualitative analysis for 'miRNA and fracture healing', while 19 were selected for the 'miRNA and fracture non-union' category. RESULTS AND CONCLUSIONS: Out of 151 miRNAs, miR-21, miR-140 and miR-214 were the most investigated miRNAs in fracture healing in general. miR-31-5p, miR-221 and miR-451-5p were identified to be regulated specifically in non-union fractures. Large heterogeneity was detected between studies investigating the role of miRNAs in fracture healing or non-union in terms of patient population, sample types and models used. Nonetheless, our approach identified some miRNAs with the potential to serve as biomarkers for non-union fractures, including miR-31-5p, miR-221 and miR-451-5p. We provide a discussion of involved pathways and suggest on alignment of future research in the field.

Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results.