Multipotent Stromal Cell Extracellular Vesicle Distribution in Distant Organs after Introduction into a Bone Tissue Defect of a Limb

Abstract

When administered intravenously, extracellular vesicles derived from multipotent stromal cells (MSC EVs) immediately pass through the lungs along with the blood and regularly spread to all organs. When administered intraperitoneally, they are absorbed either into the blood or into the lymph and are quickly disseminated throughout the body. The possibility of generalized spread of MSC EVs to distant organs in case of local intratissular administration remains unexplored. However, it is impossible to exclude MSC EV influence on tissues distant from the injection site due to the active or passive migration of these injected nanoparticles through the vessels. The research is based on findings obtained when studying the samples of lungs, heart, spleen, and liver of outbred rabbits of both sexes weighing 3–4 kg at various times after the injection of EVs derived from MSCs of bone marrow origin and labeled by PKH26 into an artificially created defect of the proximal condyle of the tibia. MSC EVs were isolated by serial ultracentrifugation and characterized by transmission electron microscopy and flow cytometry. After the introduction of MSC EVs into the damaged proximal condyle of the tibia of rabbits, these MSC EVs can be found most frequently in the lungs, myocardium, liver, and spleen. MSC EVs enter all of these organs with the blood flow. The lungs contained the maximum number of labeled MSC EVs; moreover, they were often associated with detritus and were located in the lumen of the alveoli. In the capillary network of various organs except the myocardium, MSC EVs are adsorbed by paravasal phagocytes; in some cases, specifically labeled small dust-like objects can be detected throughout the entire experiment—up to ten days of observation. Therefore, we can conclude that the entire body, including distant organs, is effected both by antigenic detritus, which appeared in the bloodstream after extensive surgery, and MSC EVs introduced from the outside.

First Experimental Study of the Influence of Extracellular Vesicles Derived from Multipotent Stromal Cells on Osseointegration of Dental Implants

“…The culture nutrient medium containing fetal bovine serum and used for MSC growth was preliminarily subjected to complete gradient centrifugation and purified from its own EVs. At the stage of stationary growth of a stable culture of the 3rd MSC passage, when the confluence of the cell monolayer reached 80-90%, a conditioned medium was collected from which MSC EVs were isolated as recommended in the literature [14,15,[25][26][27]. To remove cells, cell debris, apoptotic bodies, and large vesicles, the conditioned medium was centrifuged sequentially: 10 min in the case of 300× g, 10 min in the case of 2000× g, and 30 min in the case of 12,000× g. EVs were precipitated by centrifuging the supernatant for 2 h in the case of 100,000× g and resuspended in saline with phosphate buffer.…”

“…Surgical intervention was performed in compliance with all the rules of asepsis and antiseptics under general intravenous anesthesia with propofol. In both proximal tibial condyles of rabbits, standardized 4 mm holes were created with a 2 mm dental bur and cooled by sterile saline solution [14,27,31].…”

“…Next, an insulin syringe was used to fill the bone defect with physiological saline prepared in phosphate buffer (pH = 7.3) (control, 9 rabbits, 3 pieces for each experiment date) [32] or 19.2 µg of MSC EVs in saline solution injected for each limb (experiment, 10 animals, 4 rabbits for a period of 3 days, and 3 animals for each observation period). The MSC EVs dose was selected based on the average dose recommended by other researchers: 10-20 µg/mL [11]; 0.6 µg, 5 µg or 50 µg [33]; 50 µg for the same bone tissue defect of the proximal tibial condyle [14,27]; 100 µg immediately after surgery and weekly for 12 weeks [32]. After 10-20 s, titanium screw implants (catalog number IS 358; 3.5 × 8 mm with a rough surface; 3S, Israel) were inserted with a stable primary fixation up to 30 Ncm, and the surgical wound was sutured layer by layer without tension [14,27,31].…”

“…The MSC EVs dose was selected based on the average dose recommended by other researchers: 10-20 µg/mL [11]; 0.6 µg, 5 µg or 50 µg [33]; 50 µg for the same bone tissue defect of the proximal tibial condyle [14,27]; 100 µg immediately after surgery and weekly for 12 weeks [32]. After 10-20 s, titanium screw implants (catalog number IS 358; 3.5 × 8 mm with a rough surface; 3S, Israel) were inserted with a stable primary fixation up to 30 Ncm, and the surgical wound was sutured layer by layer without tension [14,27,31].…”

Morphology of the Myocardium after Experimental Bone Tissue Trauma and the Use of Extracellular Vesicles Derived from Mesenchymal Multipotent Stromal Cells

“…The literature contains a large number of publications devoted to the influence of exosomes of various origins on the heart structures in the time of health and disease. At the same time, data on the distribution of EVs throughout the body after different routes of administration are practically absent: found only the post of Z. Wan et al [9] and the opinion of S. Femminò et al [8] that circulating EVs can target cardiovascular structures, and EVs derived from MSC of bone marrow origin can be transmitted to distant tissues, including the myocardium, via blood flow after local injection into distant organs and tissues [10]. It is also necessary to note a certain inconsistency in the results of various researchers: from articles on the direct absorption of MSC EVs by cardiomyocytes [11] to the data that the heart is inherently immune to drug delivery mediated by nanoparticles [12].…”