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.

Keywords: bone tissue defect of a limb; exosomes; extracellular vesicles distribution; heart; liver; lungs; multipotent stromal cell extracellular vesicles; spleen.

Figure 1

Lungs of rabbits at various times after injecting extracellular vesicles derived from multipotent stromal cells (MSC EVs) into the tibial defect. Alignment of images obtained using Alexa 488 and rhodamine filters in the luminescent mode of the microscope. (a) After 3 days, numerous objects of very small size, dust-like, with bright fluorescence when using the rhodamine filter (arrows) were found in the alveoli. (b) In the alveolar lumen, objects with luminescence when applying the rhodamine filter were associated with a structureless substance (arrows) on day 3. (c) After 3 days, numerous objects with very bright fluorescence when using the rhodamine filter were enclosed in a homogeneous substance in the alveolus of the lung (arrows). (d) Cytoplasmic inclusions in large cells of various shapes have a red glow when applying the rhodamine filter in the lung parenchyma near a large vessel (the lumen is indicated by an arrow) on day 3. (e) By the 7th day, an object of about 5 μm in size (arrow) with intense fluorescence when using the rhodamine filter was found in the alveolus. (f) After 10 days, the alveolus contained an object with a diameter of 5–7 μm (arrow) with a very bright glow when a rhodamine filter was installed.

Figure 1

Lungs of rabbits at various times after injecting extracellular vesicles derived from multipotent stromal cells (MSC EVs) into the tibial defect. Alignment of images obtained using Alexa 488 and rhodamine filters in the luminescent mode of the microscope. (a) After 3 days, numerous objects of very small size, dust-like, with bright fluorescence when using the rhodamine filter (arrows) were found in the alveoli. (b) In the alveolar lumen, objects with luminescence when applying the rhodamine filter were associated with a structureless substance (arrows) on day 3. (c) After 3 days, numerous objects with very bright fluorescence when using the rhodamine filter were enclosed in a homogeneous substance in the alveolus of the lung (arrows). (d) Cytoplasmic inclusions in large cells of various shapes have a red glow when applying the rhodamine filter in the lung parenchyma near a large vessel (the lumen is indicated by an arrow) on day 3. (e) By the 7th day, an object of about 5 μm in size (arrow) with intense fluorescence when using the rhodamine filter was found in the alveolus. (f) After 10 days, the alveolus contained an object with a diameter of 5–7 μm (arrow) with a very bright glow when a rhodamine filter was installed.

Figure 2

Myocardium (a,b), spleen (c,d), and liver (e,f) of rabbits at different dates after damage to the tibia with the introduction of MSC EVs. Alignment of images obtained using Alexa 488 and rhodamine filters in the luminescent mode of the microscope. (a) On day 3, a very fine, dust-like object with strong fluorescence was located on the capillary endothelium (arrow) when a rhodamine filter was applied. (b) After 7 days, the capillary contained several very small, dust-like objects with a bright glow (arrow) when a rhodamine filter was installed. (c) After 3 days, a very small, dust-like object with a strong glow was noticeable close to a macrophage with intense autofluorescence located in the red pulp (arrow) when a rhodamine filter was used. (d) Individual inclusions in the cytoplasm of red pulp macrophages fluoresce with a noticeable red tint (arrows) by day 7 when a rhodamine filter was installed. (e) After 3 days, a macrophage in a sinusoid had a red tint in the glow of cytoplasmic inclusions (arrows) when a rhodamine filter was applied. (f) After 7 days, numerous macrophages were located along the sinusoids fluoresce with a clear red tint (arrows) when a rhodamine filter was installed.