AIM: To work out the technique of saturation of the preserved amniotic membrane (AM) with platelet rich plasma (PRP) lysate and to evaluate the growth-stimulating effect of a combination of AM and PRP lysate in vitro. MATERIALS AND METHODS: In the experiment, AM samples preserved in 3 ways were used: silicate drying, lyophilization, cryopreservation. PRP lysate was prepared on the basis of volunteers blood. During the exposure of AM with PRP lysate, the optimal saturation time of canned AM with lysate was determined, the volume of lysate that 1 cm2 of AM could adsorb was estimated. The growth-stimulating effect of AM transplants was evaluated in the culture of human buccal epithelium. The dynamics of cell growth was evaluated after 1, 2 and 3 days from the moment of sowing. RESULTS: In the presence of PRP lysate, the mass of silicatedried AM increased 4.2 times, lyophilized AM 4.8 times, cryopreserved AM 1.8 times. AM samples obtained by lyophilization adsorbed PRP lysate most effectively. Five minutes of exposure with PRP lysate are enough to fully saturate the AM. AM without PRP lysate did not give a growth-stimulating effect. CONCLUSIONS: When comparing experiments with PRP lysate without AM and AM with PRP lysate, it was found that the greatest stimulation of cell growth occurred when using lyophilized AM and PRP lysate. Saturation of cryopreserved AM with PRP lysate was ineffective, and when using silicate-dried AM impregnated with PRP lysate, the greatest growth-stimulating effect was observed on the 1st day.
Purpose: To study the biological effect of a combination of platelet lysate and amniotic membrane, preserved by various techniques, on human buccal epithelium culture. Materials and methods. Human amnion transplants were preserved using 3 methods: silicate drying, lyophilization, cryopreservation. The blood of healthy volunteers was used as a source of platelets. Platelet-rich plasma (PRP) with a platelet content over 1000 thousand/mcl and more was isolated from the donors blood, frozen at -80 °С and defrosted at 0–4 °С to prepare platelet lysate. Growth-stimulating effect of the amnion transplants was studied in different groups: control group 1 — without amnion and without PRP lysate; control group 2 — PRP lysate without amnion; experimental group 1 — amnion without PRP lysate; experimental group 2 — amnion samples combined with PRP lysate. The study was carried out on the example of human buccal epithelium culture of 3–5 passages. The dynamics of cell growth was evaluated after 1, 2 and 3 days from the moment of seeding. The number of cells and their viability were evaluated using original methods based on vital cell staining and their examination in a fluorescent microscope. Results. All samples of preserved amnions were non-toxic and did not damage the structural and functional characteristics of the buccal epithelium. On the other hand, the use of amnion without PRP lysate did not have a growth-stimulating effect on cells. Among the amnion samples combined with PRP lysate, the combination of lyophilized amnion and PRP lysate was the most effective during the entire study period. Conclusions. Silicate drying, lyophilization and cryopreservation of the amniotic membrane makes it possible to obtain biocompatible and non-toxic transplants, based on human amnion. Lyophilized amnions are the most optimal for saturating PRP lysate. The combination of lyophilized amnion and PRP lysate stimulates cell growth in vitro without violating their structural integrity.
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