Activation and platelets degranulation in the PRP (platelet-rich plasma): cytofluorimetric evaluation and kinetic analysis of TGF-β1 release through immunotest (ELISA)

Aim: The Platelet-Rich Plasma is a blood compound with high platelets density. By using immunotest ELISA. this study evaluates the activation and degranulation of the platelets during stocking procedure of the PRP, considering the role of the TGF-β1, a growth factor of the tissues, that is found in the α-platelet granules. Finally, to gain a better understanding of the quality and the quantity activationes state these datas are compared to the ones obtained with the cytofluorimetric analysis of the activation state of the platelets in the PRP. Methods: This study focuses on a sample of 19 patients (21-71 years-old), with no major deseases and waiting for an invasive oral-surgery. 24h before surgery each patient was sampled for 400ml of venous blood (+sodium citrate). Subsequently, the obtained blood sack was centrifugated to divided the erythrocytes from the plasma (900RPM, 13min, at 20°C). The plasma was then centrifugated (3300RPM, 15min) to respectively obtain the P.R.P. and the P.P.P. (Platelet- Poor Plasma). Thus, P.R.P. was then shaked to prevent platelet aggregation. Instead, from the P.P.P. were took the autologous-thrombin and cryoprecipitate. Finally, P.R.P. (5ml), cryoprecipitate (2ml), calcium-gluconate (1ml) and autologous-thrombin (2ml) were mixed in 10ml syringe; after gelification the compound was placed in the chirurgic site. Through test-ELISA were calculated the pg of TGF-β1 into P.R.P. and into compound after the gelification. Results: The results for the TGF-β1 obtained through test-ELISA are the following. 1) For the diluited solution SN (1:300) is has been extimated a release after 1 hour (h) of 22366.50 pgTGFβ/ml, after 6h of 30527.50 pgTGFβ/ml, after 24h of 19138.88 pgTGFβ/ ml and in post gel of 11791.63 pgTGFβ/ml. 2) For the diluited solution (1:150) is has been extimated a release after 1h of 15712.33 pgTGFβ/ml, after 6h of 26850.00 pgTGFβ/ml, after 24h of 29450.00 pgTGFβ/ ml and in post gel of 15321,43 pgTGFβ/ml. The TGF-β1 percentage released has been calculated using two formulas: (SN/SN+TOT) e (SN/TOT). The results using first formula were respectively after 1h of 7.6%, after 6h of 8.6% and after 24h of 20.5%. The results using second formula were after 1h of 8.4%, after 6h of 14.8% and after 24h of 19.9%. These results have been matched with the ones obtained through cytofluorimetric analysis of the samples marked with monoclonal antibodies. They reveal the following datas: for CD 42 the percentage of exposure are after 1h of 79.1%, after 6h of 90.2% after 24h of 87.6% and in post gel of 11.9%; for CD 62p is after 1h of 29.1%, after 6h of 32.4%, after 24h of 52.7%; for CD 63 is after 1h of 64.5%, after 6h of 67.1%, after 24h of 80.3%. Conclusion: From the cytofluorimetric analysis of the datas we state the procedure to produce P.R.P. it is satisfying since the gel has demostrated a high platelets density. When the activation and degranulation of the platelets rise (cytofluorimetric), the release of the TGF-β1 increases (ELISA), but only 20% of the total present in the platelets has been effectivly released in the first 24h. This implies that a great quantity of growth factors remains in the paltelets and is available in the chirurgic-site. Untill the 24h it has been observed an increasing of release of the TGF-β1, with a drop in the quantity once the gel was formed, bringing the levels close to the first hour.