透過細胞模型評估血漿血小板於再生醫學之抗發炎能力

Platelet materials, known as platelet-rich-plasma (PRP), platelet gel (PG) or platelet lysate (PL), are increasingly used in regenerative medicine, e.g. in oral and maxillo-facial surgeries, cartilage regeneration, and soft tissue healing. In addition to promoting tissue repair and bone regeneration, these platelet materials have been claimed to limit post-surgical inflammation and reduce pain in patients, possibly thanks to their content in anti-inflammatory growth factors, such as transforming growth factor (TGF)-β1 and hepatocyte growth factor (HGF). However, by lack of standardization in preparation methods, there is a large diversity in the type of platelet fractions used in clinics which makes it difficult to objectively appreciate their therapeutic benefits. In particular, these products may differ substantially in protein composition and content of possible inflammatory mediators. Only a few studies have examined the variability in anti-inflammatory factors present in platelet biomaterials and their functional role in regulating inflammation. Therefore, there is a need to better elucidate the key factors responsible for the possible anti-inflammatory properties of platelet materials as a means to standardize and improve the quality and reproducibility of these products and the clinical outcomes.
The aim of this study was (a) to compare the anti-inflammatory effects of various plasma/platelet fractions in an in vitro lipopolysaccharide (LPS)–stimulated RAW 264.7 mice macrophages inflammatory model, and (b) to understand the anti-inflammatory role of platelet growth factors/cytokines and the molecular mechanisms involved in the control of inflammation by platelet factors.
Our experimental design was divided into two main sections. First, 5 platelet fractions, representative of products used in clinics, were prepared from apheresis platelet concentrates (PC) collected from 4 volunteer donors : platelet poor plasma (PPP), platelet gel releasate (PGR), frozen-thawed platelet lysate with (PL) or without (CFPL) blood cell debris, and solvent-detergent (S/D)-treated platelet lysate (S/D-PL). The proteins in platelet fractions were determined, SDS-PAGE patterns compared, and the presence of growth factors and cytokines was identified and quantified by Enzyme-linked immunosorbent assay (ELISA), multiplex bead immunoassays (Luminex), and cytokine antibody array. Second, the anti-inflammatory effects of plasma/platelet fractions were evaluated in a macrophage in vitro model by measuring inflammatory markers. RAW 264.7 mice cells were grown in medium supplemented with 10% of fetal bovine serum (FBS; control) or plasma/platelet fractions, in the presence or not of LPS (500 ng/mL). Cell growth was determined by cell count determination and cell viability by MTT and other assays. The level of nitric oxide (NO) production and tumor necrosis factor (TNF)-α in cell culture supernatant was measured by colorimetric assay and ELISA, respectively. Expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in cell extracts was detected by Western Blot. PRP from mice and quercetin, a known anti-inflammatory compound, were used as controls. Cell morphology was investigated by optical microscope (OM) and scanning electron microscope (SEM).
Results showed that the five plasma/platelet fractions differed in protein composition and had variable content in growth factors and cytokines, as expected. In particular, PPP contained significantly less of most platelet growth factors. RAW 264.7 cell morphology was not affected when cells were cultured in medium supplemented with the different plasma/platelet fractions. The viability of the cells cultured in medium supplemented with different plasma/platelet fractions followed by exposure to 500 ng/mL LPS treatment was equivalent. All platelet fractions reduced NO production and iNOS expression by LPS-stimulated RAW 264.7 cell compared to FBS control. Interestingly, S/D-PL inhibited cell morphologic changes induced by LPS and strongly decreased NO production, and iNOS, COX-2 and TNF-α expression.
In conclusion, this study supports that all fractions, including PPP, exert an anti-inflammatory effect on RAW 264.7 macrophages stimulated by LPS. S/D-PL exhibits a stronger overall anti-inflammatory activity revealed by inhibition of cell morphological changes and TNF-α and COX-2 expression. These data, which highlight differences in platelet anti-inflammatory role, may help selecting an optimal platelet fraction for clinical use when an anti-inflammatory effect is targeted, such as in implantology and osteoarthritis treatment.