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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220429	1/1	Mus musculus	Osteoclasts	(d)(U)C	Faqeer, Abdullah	2023	78%
Study summary Full title Cleaved SPP1-rich extracellular vesicles from osteoclasts promote bone regeneration via TGFβ1/SMAD3 signaling All authors Abdullah Faqeer, Mengzhen Wang, Gulzar Alam, Arshad Ahmed Padhiar, Dexiu Zheng, Zhiming Luo, Irene Shuping Zhao, Guangqian Zhou, Jeroen van den Beucken, Huanan Wang, Yang Zhang Journal Biomaterials Abstract Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbi (show more...)Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor β1 (TGFβ1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: HSPA8/ CD9/ CD81/ TSG101/ beta actin non-EV: calnexin Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Osteoclasts EV-harvesting Medium Serum free medium Cell count 3000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70.1Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 8 Wash: time (min) 60 Wash: Rotor Type Type 70.1Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins HSPA8/ CD9/ CD81/ TSG101/ beta actin Not detected contaminants calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 180 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 155

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EV-TRACK ID	EV220429
species	Mus musculus
sample type	Cell culture
cell type	Osteoclasts
condition	Control condition
separation protocol	dUC
Exp. nr.	1
EV-METRIC %	78