\ counts Aare as Pio? Microrheology study on matrix remodelling by osteoblasts in 3D hydrogel in vitro culture lanne I. Koivisto’, Claude Oelschlaeger®, David Menne®, Norbert Willenbacher®, Tuomas Nireoja*® “Karolinska Institutet, Division of Pathology- Department of Laboratory Medicine, Stocktolm, Sweden Karlsruhe Institute of Technology, Institute of Mechanical Process Engineering and Meckanics, Karlsruhe, Germany “University of Turku, Department of Life Technologies, Turku, Finland Introduction: Development of in vitro bone disease models are useful in biomarker discovery and cell biology af bone. To study pre- osteoblast attachment phase in 3D culture, we focused on the changes in microstructural as well as local mechanical properties, primarily using multiple particle tracking (MPT) microrheology in the direct microenvironment of cells in collagen-illed scaffolds Methods: Mouse MC3T3-E1 pre-osteoblasts were cultured in ‘osteogenic differentiation medium, inside 3D constructs prepared from 3D printed beta-tricalcium phosphate ((-TCP) scaffold filled with TeloCollagen® (3mg/ml. & 6mg/mL) hydrogel and cells, Local viscoelastic properties (n=3) were measured on days 0, 4 and 7 using microrheology and bulk rotational cheometty. Remodelling was visualized using Voronoi diagrams based on microrheology. Results: At the start of the culture the hydrogel network is homogenous and highly elastic with very few viscous inclusions. On| day 4 in 3mgiml collagen, the structure has become highly heterogeneous and large viscous regions appear close to the cell, while bulk properties remain relatively unchanged. On day 4 bull stiffness of the 6mgiml and 3mgiml collagen filled scaffolds had declined to 61% and 78% respectively No further decrease was seen © om tsar 1 Bae ia Y i {- noe a 4oi:10.1016/}bonr 2022.101387 i = uJ 7a i : ee », betel fe ns aime tiptoe tin Oath ‘ctor rnd pene ptan Gonna seen vas mate {egoaocan rand ets. aren shan oof os at eds ts tees sim odes is Conclusions: Osteoblast attachment induced a rapid remodelling of collagen, without other cells involvement, seen at day 4 in ‘microscale, especially within P-TCP scaffolds. The microtheology36 Absa with Voronoi diagrams is shown to be powerful tool to visualize microstructural and micromechanical properties at cell-relevant scale during cell cultivation. Financial support: Osk. Huttunen Foundation. oi:10.1016/} ons. 2022.101388 Pros ‘Transcriptome analyses to study the molecular influence of Wnt ‘on osteoblasts Wenbo Zhae*, Michael Amling*, Timur Alexander Yorgan’, Thorsten Schinke” University Medical Center Homburg-Eppendorf; Department of Osteology and Biomechanics, Hamburg. Germany Background/Introduction: Mutations of WITT have been identified to cause either early-onset osteoporosis or osteogenesis imperfecta type XV. The key role of Wat as an osteoanabolic molecule was also confirmed by the analysis of corresponding mouse models. Importantly, however, the detailed mechanism of Watt action in bone anabolism is stil poorly understood. Purpose: Identification of transcriptional targets of Wnt in osteoblasts and responsible receptors mediating the response. Methods: A recombinant Wat1/Strp1 complex or Sirp1 alone was administered to either ST2 or MC3T3-EI cells to study the influence on differentiation and gene expression, Results: Long-term treatment (15 days) with reWntt/Sfep! during osteogenic differentiation significantly promoted osteogenesis of mesenchymal ST2 cells, while reSfipt did not Genome-wide transcriptional analysis after short-term treatment (6 hours) revealed that reWntl/Strpl, but not reSfrpt, induced the expression of known Wnt target genes (such as Apedd), but also of| Osteomodulin (Omd) and Periostin (Postn). This was confirmed by RT-PCR, where short-term treatment with reWtt Shp strongly induced the expression of Apeddt, Omd and Postn (expression increase compared to control group: Apeddl; 14,7-fold, p=0.0001; ‘Omd: 6.0-fold, p= 0.0009, Posen: 4.7-fold; p=0.0002). We also performed the same experiments with pre-osteoblastic MC3T3-£1 cells, where we did not observe a postive influence of reWntt/Sttp1 fon the formation of a mineralized matrix. Mozeover, only Omd expression was significantly increased toa small extent (1.7-fld, p= 0.0094). We therefore analyzed the expression of all known Fed genes encoding putative components of a Wat! receptor complex. Here we observed remarkably higher expression of Fd2, Frd¢ and Fed8 in Wntt-responsive ST2 cells, when compared to Watt unresponsive MC3T3-E1 cells Conclusion: Our data demonstrate that two established osteogenic cell lines display diferent responsiveness to Wat administration. A comparative molecular analysis of these influences may lead to novel insights with respect to relevant Wat! receptors and downstream targets, oi:10.1016/3onr.2022.101389 109 Proteomic analysis of caspase-9 deficient MC3T3-E1 cells Barbora Vesela’, Alice RameSovs", Kamila fthov®, Petr Laptik, Pet Bene®, Pavel Bouchal’, Eva Matalovi* “Institute of Animal Physiology and Genetcs- CAS- vx, Laboratory of Odontogenesis and Osteogenesis, Brno, Cech Republic "Masaryk University, Department of Experimental Biology- Faculty of Science, Brno, Czech Republic ‘Masaryk University, Department of Biochemistry- Faculty of Science, Bro, Czech Republic Background/Introduction: Caspase-9 represents the apical caspase of the intrinsic pathway of apoptosis. Notably, recent knowledge about functions of this cysteine protease keeps expanding beyond programmed cell death. This applies also for the bones, but the non-apoptotie mechanisms have not yet been elucidated Purpose: In this investigation, caspase-9 deficent osteoblastic cells (MC3T3-E1) were generated by the CRISPR/Cas9 approach to analyse their proteome. Methods: Cells were cultured, harvested and proteins were identified and quantified using LC-MS/MS analysis. Proteome of two independent caspase-9 deficient clones was evaluated and compared to controls Results; In total, 7659 proteins were identified and quantified (FDR-0.01). 283 and 187 proteins were significantly upregulated and downregulated in caspase-9 deficient cells. The lst included proteins participating in regulation of DNA replication, cell cycle, proliferation, migration, adhesion and extracellular matrix assembly. The top ones have been further examined. Serine/threonine-protein Kinase SgI3, and cytochrome P450/26B1, a regulator of the retinoic acid metabolism, belonged to the top 10 downregulated proteins ‘Among top 10 up-regulated proteins, gap junction beta-3 protein oF fibrlin-1, 2 protein with important structural and regulatory properties including interactions with bone morphogenetic proteins, were identife. Conclusion: The obtained results provide a robust dataset for further investigation of non-apoptotic caspase-9 functions in bone cells with focus on the osteoblastic population, The research was supported by the Czech Science Foundation, project GACR 15-147278. 40:10.1016/)bonr. 2022.101380 Pro Osteogenically differentiated human fibroblasts — an alternative ‘model to study bone diseases Sanda Piblstrim*®,Kirsi Maatta*®, Rilkka Makitie*®5, Mira Aronen® (Outi Makitie"®4*, Minna Pekkinen*™* “University of Helsinki, Faculty of Medicine, Helsnk, Finland ‘Folkhalsan Research Center, Institute of Genetics, Helsink, Finland Stelsink University Hospital and University of Helsinki, Department of (Otorhinolaryngology- Head and Neck Surgery, Helsinki, Finland "Karolinska Institute, Department of Molecular Medicine and Surgery and Center for Molecular Medicine, Stockholm, Sweden "Helsinki University Hospital and University of Helsinki, Chiléren's Hospital, Helsinki, Finland Several skeletal disorders exhibit abnormal osteoblast, evelopment and function along with mineralization defects, creating a demand for an osteoblastic in viero system. However, nalive osteoblasts ate difficult to isolate from affected patients and problematic to expand in vitro. Similar issue concerns bone marrow mesenchymal stem cells (MSCs), the original progenitors of osteoblasts. One potential alternative source of cells that are non- immunogenic, easily expandable and readily available through a ‘minimal invasive harvesting procedure are human dermal fibroblasts. Therefore, we developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We