Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (Rh) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In Rh, ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by Rh of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger Rh obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and Rh of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.
Purpose: Unfavourable distribution of contact stress over the load bearing area is considered a risk factor for early coxarthritis and it is of interest to outline respective biomechanical parameters for its prediction. The purpose of the work was to develop a transparent mathematical model which can be used to assess contact stress in the hip from imaged structures of pelvis and proximal femora, in large population studies and in clinical practice. Methods: We upgraded a previously validated three-dimensional mathematical model of the human hip in the one-legged stance HIPSTRESS by introducing parameters independent from the size of the structures in the images. We validated a new parameter – dimensionless peak stress normalized by the body weight and by the radius of the femoral head (pmaxr2/WB) on the population of 172 hips that were in the childhood subjected to the Perthes disease and exhibited increased proportion of dysplastic hips. Results: The dimensionless parameter pmaxr2/WB exhibited smaller number of indecisive cases of hip dysplasia predicted by the model than the previously used parameter pmax/WB (6% vs. 81%, respectively). A threshold for an increased risk of early coxarthritis development by the HIPSTRESS parameter H = pmaxr2/WB was found to be 2. Conclusions: We proposed a dimensionless peak stress on the load bearing area with the border value of 2 as a decisive parameter over which hips are at risk for early development of degenerative processes and presented a method for determination of biomechanical parameters with the use of nomogram.
The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP.
Background: Cells in general secrete nanoparticles (NPs) which are believed to mediate intercellular communication. Recently, great efforts have been made to utilize them as delivery vectors. We aimed to harvest and identify NPs from liquid cultures of two marine microalgae Dunaliella tertiolecta and Phaeodactyum tricornutum. Methods: NPs were isolated from the culture conditioned media by differential ultracentrifugation by the protocol used for the isolation of extracellular vesicles. Microalgae and isolated NPs were examined by scanning electron microscopy (SEM) while isolated NPs were examined also by cryogenic transmission electron microscopy (cryo-TEM). The Triton X-100 detergent and temperature sensitivity of NPs was assessed by dynamic light scattering (DLS) through monitoring the intensity of the scattered light (I) and the distribution of hydrodynamic radii of NPs (Rh). Results: Two mechanisms of formation of NPs with average Rh 200 nm were observed in the D. tertiolecta culture: a disintegration of tubular protrusions, and cell decay. A part of the imaged D. tertiolecta NPs were membrane-enclosed vesicles, but the isolates also contained electron-dense NPs and nanofilaments. P. tricornutum NPs in the culture and in the isolate were homogeneous in size and shape. Their average Rh was 104 nm. The addition of surfactant to isolates resulted in a change in Rh distribution and a decrease of I in samples from both species, indicating decay of a part of NPs. Changes in the width of the I(Rh) peaks were observed at temperatures above 45 °C. Conclusions: A part of NPs found in isolates from microalgae D. tertiolecta and P. tricornutum were membrane-enclosed vesicles. However, the isolates obtained by a standard protocol for extracellular vesicle isolation by ultracentrifugation contained also a significant amount of other similar-sized nanoparticles. The isolates were partly susceptible to the addition of detergent and to temperature up to 80 degrees.
Uses of platelet and extracellular vesicles rich plasma (PVRP) are in many fields of medicine as it was found that PVRP has regenareative properties. Preparation of PVRP is performed by sep-aration of erythrocytes from the liquid they are immersed in. Erythrocytes are the most numerous blood cells; they are also relatively large and dense as they are filled with haemoglobin. Therefore they sediment due to gravitation or systemic centrifugation force thereby pushing plasma that carries smaller particles in the opposite direction. This mechanism that takes place during pro-cessing determines the composition of plasma. Its tuning is therefore key in acquiring a prepara-tion with desired properties. In particular, it is of interest to study the effect of erythrocyte sedi-mentation rate (ESR) on composition and volume of acquired plasma. Different animals have different ranges of ESR values which may enable insight into mechanisms of plasma preparation. In this contribution we present the basic mechanisms of plasma preparation and properties of blood of different animals.
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