Leukocyte- and Platelet-Rich Fibrin (L-PRF) Obtained from Smokers and Nonsmokers Shows a Similar Uniaxial Tensile Response In Vitro.

We evaluated and compared the biomechanical properties of Leukocyte-and Platelet Rich Fibrin L-PRF clots and membranes derived from smoker and nonsmoker donors. Twenty venous-blood donors (aged 18 to 50 years) were included after signing informed consent forms. L-PRF clots were analyzed and then compressed to obtain L-PRF membranes. L-PRF clot and membrane samples were tested in quasi-static uniaxial tension and the stress-stretch response was registered and characterized. Furthermore, scanning electron microscope representative images were taken to see the fibrin structure from both groups. The analysis of stress-stretch curves allowed us to evaluate the statistical significance in differences between smoker and nonsmoker groups. L-PRF membranes showed a stiffer response and higher tensile strength when compared to L-PRF clots. However, no statistically significant differences were found between samples from smokers and nonsmokers. With the limitations of our in vitro study, we can suggest that the tensile properties of L-PRF clots and membranes from the blood of smokers and nonsmokers are similar. More studies are necessary to fully characterize the effect of smoking on the biomechanical behavior of this platelet concentrate, to further encourage its use as an alternative to promote wound healing in smokers.

Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. METHODS: Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. RESULTS: A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. CONCLUSION: Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.

Uncoupled inflammatory, proliferative, and cytoskeletal responses in senescent human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration. METHODS: Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-κB. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test. RESULTS: Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-κB activation. CONCLUSION: Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium.

Comparative effect of platelet-rich plasma, platelet-poor plasma, and fetal bovine serum on the proliferative response of periodontal ligament cell subpopulations.

OBJECTIVES: Cell-based therapies involve the need to expand cell cultures ex vivo for their subsequent implantation in an autologous manner. An important limitation regarding this technology is the use of fetal bovine serum (FBS) that has critical safety limitations. Platelet-derived fractions represent an autologous source of growth factors that may be used for the expansion of these cell cultures. Periodontal ligament (PDL) cells comprise a heterogeneous cell population that may not necessarily respond in a uniform manner to proliferative stimuli. The aim of this study was to evaluate the ability of two platelet-derived fractions, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) and FBS on the proliferative response of different subpopulations of PDL cell cultures. MATERIALS AND METHODS: PDL cells were characterized and then exposed to PRP, PPP, or FBS during 2, 5, or 14 days to analyze cell proliferation and clonogenic capability. Cell proliferation was evaluated through immunofluorescence for Ki67 and by tracing carboxyfluorescein diacetate succinimidyl ester (CFSE) dye in combination with mesenchymal stem cell markers using flow cytometry. RESULTS: Both PRP and PPP stimulated PDL cell proliferation and their clonogenic ability. We found a significant increase of CD73- and CD90-positive cells after PRP or PPP treatment, compared to FBS. Otherwise, no differences were found regarding the response of CD146-or CD105-positive cells when stimulated with PRP, PPP, or FBS. CONCLUSION: PRP and PPP can stimulate the proliferation and clonogenicity of PDL cell populations including cells positive for CD90 and CD73 markers. CLINICAL RELEVANCE: These findings may have implications for future therapies aiming to stimulate periodontal regeneration using autologous growth factors.

Platelet-Poor and Platelet-Rich Plasma Stimulate Bone Lineage Differentiation in Periodontal Ligament Stem Cells.

BACKGROUND: Plasma-derived fractions have been used as an autologous source of growth factors; however, limited knowledge concerning their biologic effects has hampered their clinical application. In this study, the authors analyze the content and specific effect of both platelet-rich plasma (PRP) and platelet-poor plasma (PPP) on osteoblastic differentiation using primary cultures of human periodontal ligament stem cells (HPLSCs). METHODS: The authors evaluated the growth factor content of PRP and PPP using a proteome profiler array and enzyme-linked immunosorbent assay. HPLSCs were characterized by flow cytometry and differentiation assays. The effect of PRP and PPP on HPLSC bone differentiation was analyzed by quantifying calcium deposition after 14 and 21 days of treatment. RESULTS: Albeit at different concentrations, the two fractions had similar profiles of growth factors, the most representative being platelet-derived growth factor (PDGF) isoforms (PDGF-AA, -BB, and -AB), insulin-like growth factor binding protein (IGFBP)-2, and IGFBP-6. Both formulations exerted a comparable stimulus on osteoblastic differentiation even at low doses (2.5%), increasing calcium deposits in HPLSCs. CONCLUSIONS: PRP and PPP showed a similar protein profile and exerted comparable effects on bone differentiation. Further studies are needed to characterize and compare the effects of PPP and PRP on bone healing in vivo.

The influence of platelet-derived products on angiogenesis and tissue repair: a concise update.

Platelet degranulation allows the release of a large amount of soluble mediators, is an essential step for wound healing initiation, and stimulates clotting, and angiogenesis. The latter process is one of the most critical biological events observed during tissue repair, increasing the growth of blood vessels in the maturing wound. Angiogenesis requires the action of a variety of growth factors that act in an appropriate physiological ratio to assure functional blood vessel restoration. Platelets release main regulators of angiogenesis: Vascular Endothelial Growth Factors (VEGFs), basic fibroblast growth factor (FGF-2), and Platelet derived growth factors (PDGFs), among others. In order to stimulate tissue repair, platelet derived fractions have been used as an autologous source of growth factors and biomolecules, namely Platelet Rich Plasma (PRP), Platelet Poor Plasma (PPP), and Platelet Rich Fibrin (PRF). The continuous release of these growth factors has been proposed to promote angiogenesis both in vitro and in vivo. Considering the existence of clinical trials currently evaluating the efficacy of autologous PRP, the present review analyses fundamental questions regarding the putative role of platelet derived fractions as regulators of angiogenesis and evaluates the possible clinical implications of these formulations.