title: Developing a novel 3D platform for investigating the osteogenic differentiation and the anti-inflammatory potential of human mesenchymal stem cells and their secretome
Creators: name: Bicer, Mesude
Publisher: name: University of Reading
Year: 2023

description: Osteoporosis and osteoporosis-associated fractures are one of the most prevalent global public health problems due to the rising population age. These diseases result in a large economic burden. The use of autologous mesenchymal stem cells (MSCs) is a potential therapeutic approach in in orthopaedics and dentistry as well in treatment of osteoporosis and osteoporotic fractures. However, both the number and the osteogenic potential of MSCs decrease with age. Recent research has suggested that osteogenic potential of MSCs can be positively affected by combining electrical stimulation (ES) and cultivation in 3D. Moreover, 3D cell culture has been shown to increase viability of MSCs and reduce cellular senescence. Adipose-derived stem cells (ASCs) are an easily accessible and readily available type of MSCs and represent promising candidates for cell-based therapies for bone regeneration. This thesis investigated how cultivation of MSCs in different 3D scaffolds in combination with ES affects proliferation, osteogenic potential, as well as the anti�inflammatory potential of ASCs. To assess the optical properties of different scaffolds, absorbance spectra of different concentrations of anionic nanofibrillar cellulose (aNFC) were compared to other commonly used scaffolds, such as alginate and fibrin. Biocompatibility was studied by assessment of cellular viability using XTT and live/dead assays. Data analysis revealed that aNFC is highly biocompatible with ASCs whilst having low autofluorescence and light absorption allowing for easy monitoring of osteogenic differentiation using colorimetric and fluorescence-based methods. In subsequent experiments, the osteogenic potential of ASCs in aNFC was evaluated using RT-PCT, assessment of alkaline phosphatase (ALP) activity, analysis of calcification by Alizarin Red S staining and immunocytochemical staining against the osteogenic markers osteopontin (OPN) and osteocalcin (OCN). As an additional read�out, osteogenic differentiation was assessed by analysing intracellular calcium oscillation patterns. Osteogenic differentiation of ASCs in 3D aNFC resulted in a robust induction of osteogenesis related transcripts and down-regulation of stem cell marker expression. Moreover, ASCs subjected to differentiation in 3D showed high levels of mineralisation and an increased expression of OPN and OCN at protein level. To study the impact of a combination of 3D cell culture and ES, ASCs in osteogenic and standard media were exposed to electric fields for up to 21 days followed by analysis of osteogenic differentiation. When exposed to ES in 3D, ASCs showed high ALP activity and an increased calcium deposition evidenced by Alizarin Red S staining. Furthermore, exposure of ASCs to ES in 3D aNFC resulted in an increased expression of the osteogenic markers OPN and OCN and a rearrangement and alignment of the actin cytoskeleton. Since the regenerative potential of ASCs at least partly depends on paracrine factors, the impact of the combination of ES and 3D cell culture on the anti-inflammatory potential of ASC-secretomes was assessed. The anti-inflammatory potential of ASCs secretomes in 3D was investigated by analysing the nuclear translocation of p65 in human fibroblasts exposed to tumour necrosis factor (TNF-) using immunocytochemistry and confocal imaging. Additionally, luciferase reporter assays were performed to quantify TNF- induced NF-B activity in an established reporter cell line exposed to secretomes from 3D cultured ASCs with and without ES. 3D cell culture combined with ES resulted in an increase on the anti-inflammatory potential of ASCs secretomes from 3D aNFC. Taken together, this thesis suggests that cultivation of ASCs in 3D aNFC can increase their viability without interfering with their osteogenic potential. Moreover, levels of osteogenic differentiation can be increased if 3D cultivation is combined with ES. Finally, both cultivation of ASCs and ES has positive effects on the anti-inflammatory potential of the ASC-secretome. The results presented in this thesis could pave the way for developing improved MSC-based strategies for bone regeneration in multiple clinical scenarios including but not limited to osteoporosis, osteoporotic fractures, regenerative dentistry, and regenerative orthopaedics. Future studies should investigate optimal experimental parameters (e.g., ES prior or after differentiation, alternative hydrogels) and include in vivo experiments (e.g., critical size defects in rodents and large animal models) to validate the promising in vitro findings presented in this thesis.”