RESUMO
Platelet-rich plasma (PRP) associated with high molecular weight hyaluronic acid (HA) has been clinically used for tissue regeneration in orthopedics. Despite the recognized beneficial clinical outcomes (e.g., early pain control, improvement of patients' functional limitation and longer-term effectiveness compared to PRP and HA alone in mild and moderate osteoarthritis treatments), its use is still challenging and controversial due to lack of standardization of association practical protocols. Moreover, most studies neglect the matrix structure, that generates the ultimate properties of the association among platelets, fibrin network and the microparticles. In the present work, we aimed to analyze the influence of the PRP/HA association with a controlled matrix structure on the stability, rheological behavior, release of growth factors and in vitro proliferation of human adipose-derived mesenchymal cells (h-AdMSCs). The attenuation of the negative charge of HA was also evaluated. Pure PRP (P-PRP) (i.e., plasma enriched with platelets and poor in leukocytes) was prepared by centrifugation and activated with serum and calcium chloride (AP-PRP). Autocrosslinked hyaluronic acid (AHA) was prepared by organocatalyzed auto-esterification and structured in microparticles (MPAHA) by shearing. The attenuation of the negative charge of MPAHA was performed with chitosan (CHT) by polyelectrolyte complexation yielding MPAHA-CHT. The results showed that microparticles (MPs) have viscoelastic properties, extrusion force and swelling ratio appropriate for injectable applications. The association of AP-PRP with the controlled structure of MPAHA and MPAHA-CHT formed a matrix composed of platelets and of a fibrin network with fibers around 160 nm located preferably on the surface of the MPs with an average diameter of 250 µm. Moreover, AP-PRP/MPAHA and AP-PRP/MPAHA-CHT associations were non-toxic and supported controlled growth factor (PDGF-AB and TGF-ß1) release and in vitro proliferation of h-AdMSC with a similar pattern to that of AP-PRP alone. The best h-AdMSC proliferation was obtained with the AP-PRP/MPAHA-CHT75:25 indicating that the charge attenuation improved the cell proliferation. Thus, the association of AP-PRP with the controlled structure of HA can be a valuable approach for orthopedic applications.
RESUMO
Platelet-rich plasma (PRP) is an autologous product prepared from whole blood (WB) that is widely used in regenerative medicine. In clinical practice, discontinuous centrifugation is used for both hand- and machine-prepared PRP. However, separation of WB fractions via centrifugation is a complex process, and the lack of clear mechanisms limits the understanding and evaluation of PRP preparation methods This paper focuses on the distribution, recovery and concentration factor of platelets and leukocytes in L-PRP (leukocyte and platelet-rich plasma) to define a concentration pattern for these blood components due to centrifugation conditions. WB collected from three healthy donors was centrifuged for 10min at 50-800 xg in a first step and then at 400 xg in a second step. The results from the first centrifugation step showed most platelets to be distributed in the upper layer (UL) and the buffy coat (BC), with approximately 14.5±5.2% retained in the bottom layer (BL). Most leukocytes were present in the BL. The greatest platelet recoveries from L-PRP were obtained at up to 150 xg (88.5±16.9%). The cumulative concentration factors with respect to the WB from the second centrifugation step were 6 and 1.2 for platelets and leukocytes, respectively. Thus, the concentration patterns delineated three centrifugation ranges with platelet/leukocyte ratios of 205±18, 325±15 and 107±4 and lymphocyte/granulocyte ratios of 1.54±0.74, 0.90±0.08 and 0.42±0.07. These findings contribute to a scientifically based standardization of L-PRP preparations.