An Analysis of a Rapid, Simple, and Inexpensive Technique Used to Obtain Platelet-Rich Plasma for Use in Clinical Practice
The use of platelet-rich plasma (PRP) has become more generally accepted, and implant dentists are using PRP more frequently to promote the healing of oral surgical and/or periodontal wounds. Critical elements of PRP are thought to be growth factors contained within the concentrated platelets. These growth factors are known to promote soft-tissue healing, angiogenesis and osteogenesis. We present a rapid, simple, and inexpensive methodology for preparing PRP using the Cliniseal centrifuge method. This study demonstrates that platelets are concentrated approximately 6-fold without altering platelet morphology. Further we demonstrate that key growth factors, platelet-derived growth factor BB (PDGF-BB), transforming growth factor B (TGF-B1), vasculature endothelial growth factor (VEGF), and epidermal growth factor (EGF) are present in comparable or higher concentrations than those reported with the use of other techniques. Prolonged bench set time (>3 hours) after centrifugation resulted in decreased concentration of TGF-B1 but not decreased concentration of PDGF-BB, VEGF, or EGF. This study confirms the molecular aspects of PRP obtained using this inexpensive and efficient methodology.Abstract
Anticoagulated whole blood after centrifugation. Close-up of pelleted anticoagulated whole blood separated into the 3 layers. Note the placement of the label on the outside of the tube is dictated by matching the dotted line to the plasma/cell pellet interface.
and 3. Figure 2. Platelet enrichment in platelet-rich plasma (PRP) as a function of centrifugation time. Enrichment is expressed as the fold increase in platelet concentration over the whole blood sample from which the PRP was prepared. Note the great increase in concentration after 6 minutes. Although 6 minutes was found to have the highest fold increase in concentration, 10 minutes produced the most consistent results (SD = 1.49) out of all the times considered. WB = whole Blood; n = 7. Figure 3. Platelet enrichment in PRP as a function of bench set time. Centrifuged anticoagulated whole blood was allowed to set on the bench for up to 6 hours. The greatest variability occurred after 15 minutes, and it became more pronounced after 1 hour. The most effective time point was 0 minutes; it was noted as having the greatest concentration increase (7 × whole blood) with regard to consistency (SD = 370.99; n = 7).
The morphology of platelets in platelet-rich plasma (PRP) change as a function of bench set time. Centrifuged blood was allowed to set on the bench for up to 6 hours, and then PRP was harvested at various times. Samples were smeared, stained with Wright-Giemsa, and viewed/photographed using brightfield microscopy. All fields are ×1000 magnification. (A) Whole blood; note that very few platelets are present relative to the number of red blood cells (RBCs). (B) A 0 bench set time; note that the platelets are higher in number relative to the RBCs, indicating that platelets have been enriched using the procedures described previously. Also note that the platelets possess a normal morphology (discoid, light blue staining, with little to no adherence to one another [aggregation]). (C) A 30-minute bench set time; concentrated platelets continue to appear intact and healthy. (D) A 1-hour bench set time; platelets begin to aggregate but they are still ovoid and lightly stained. (E) A 2-hour bench set time; platelets form larger aggregates and have begun to lose ovoid shape developing bleb-like structures on the plasma membranes. (F) A 6-hour bench set time; note the aggregation occurring with prolonged bench set time with more than 100 platelets per aggregate. Platelets appear shrunken and misshapen, suggesting a loss of functional capacity.
Platelets in platelet-rich plasma (PRP) prepared using the Clinaseal Centrifuge method contain intact growth factors. Western blotting was used to qualitatively assess whether platelets in PRP prepared using the methods described contain platelet-derived growth factor (PDGF-BB) or transforming growth factor-β1 (TGFβ1). Lane 1: molecular weight markers that flank the growth factor protein bands of interest. Lane 2: purified recombinant human growth factors that serve as positive controls. Lane 3: lysate from 5 to 7 × 106 partially purified platelets contained within the PRP. Note the presence of bands in Lane 3 that correspond in migration and immunoreactivity to the standard growth factors in Lane 2. Also note that the bands in Lanes 2 and 3 migrate to the known molecular weight range for each growth factor monomer (PDGF-BB = 15 kD, TGFβ1 = 12.5 kD, nerve growth factor = 14 kD).
Contributor Notes
James L. Rutkowski, DMD, is in private practice in Clarion, Pa, and is currently pursuing his doctoral degree at Duquesne University, Pittsburgh, Pa. Address correspondence to Dr Rutkowski at 35 South 2nd Avenue, Clarion, PA 16214. (james.rutkowski@verizon.net)
Joseph M. Thomas, BS, is completing his master of science degree in biology, C. Larry Bering, PhD, is a professor of biochemistry in the Department of Chemistry, Julie L. Speicher, is completing her bachelor of science degree in molecular biology and biotechnology, and Douglas M. Smith, PhD, is a professor of cell biology and immunology in the Department of Biology at Clarion University of Pennsylvania, Clarion, Pa.
Nicholas M. Radio, PhD, is completing a postdoctoral fellowship at the National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Chapel Hill, NC.
David A. Johnson, PhD, is an associate professor of pharmacology and toxicology and director of graduate studies of pharmaceutical sciences at Duquesne University, Pittsburgh, Pa.