The current study included 8 volunteers (6 men and 2 women; mean±SD age, 31.6±10.9 years). Formal institutional review board approval to carry out the study was obtained (institutional review board #10-204-2). The study included healthy adult subjects who agreed to participate. Those with a history of blood-derived illness or use of medications known to affect platelet function or concentration for a minimum of 2 weeks before testing were excluded.
Venous blood (approximately 125 mL venous blood+5 mL acid citrate dextrose) was collected from new blood draws. Approximately 125 mL peripheral blood was drawn from each subject at 3 different times (at 2-day and 4-day intervals, as explained later) to allow sufficient platelet recovery. A 60-mL syringe prefilled with 5 mL acid citrate dextrose was used for the standardized blood draw. Acid citrate dextrose binds calcium and prevents blood clotting without affecting platelet function.
Platelet-rich plasma was isolated using 2 methods, a single-spin process (PRPSS) and a double-spin process (PRPDS). Both PRPSS and PRPDS were performed on each day of treatment. Two systems were examined: 1 that produced a high concentration of platelets and another that had lower platelet values. The single-spin process is quicker, but typically yields a lower platelet concentration.15 Marx16 recommended that the goal of a platelet-rich plasma product should be a platelet concentration 3 to 5 times greater than the baseline platelet count.17 Under a laminar flow hood and using sterile technique, 10 mL blood was transferred into the platelet-rich plasma preparation system or Arthrex Double Syringe (Arthrex Inc, Naples, Florida) to produce 3 mL platelet-rich plasma. The double syringe is a device used to isolate platelet-rich plasma from peripheral blood. According to the manufacturer’s recommendations, blood is drawn or loaded into the outside area of the double syringe, then centrifuged at 200×g for 5 minutes. The resulting top plasma layer is drawn into the inner syringe to retrieve the platelet-rich plasma product. Syringes were centrifuged at 1500 rpm for 5 minutes. This separated the blood into 3 layers: plasma, erythrocytes, and the buffy coat containing the platelets. The plasma and buffy coat were then isolated with the inner syringe and used as the platelet-rich plasma separation.
In preparing the PRPDS, after a first centrifugation at 1500 rpm for 5 minutes, the buffy coat was aspirated and centrifuged a second time (20 minutes at 6300 rpm). Finally, half of the superficial plasma layer was removed and the platelet pellet was suspended in the remaining half of the plasma volume.9
The composition of the treatment preparations may ultimately affect the outcome. The authors analyzed 3 specific components of these different treatments: platelet concentration, white blood cell concentration, and red blood cell concentration.
Human tenocytes were isolated from specimens of proximal biceps tendon extracted in the process of tenodesis during shoulder surgery. This portion of the study was carried out under a second institutional review board approval (institutional review board #07-22-4). Specimens were included if they appeared to have no gross degenerative changes. These changes were proven in previous studies to correlate with the histologic appearance.18 Tendons were cut and placed into a 2% collagenase solution. Tendon cells were successfully obtained via a primary digest at a concentration of 8 million cells per sample. The resulting tenocyte cell suspension was filtered, collagenase was removed, and the tenocytes were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, New York) with 10% fetal bovine serum and penicillin-streptomycin. The tenocyte genotype was confirmed by quantitative real-time polymerase chain reaction for the tenocyte markers tenascin-C, decorin, and collagen types I and III. Normal tenocyte morphologic findings were confirmed by microscopy. For all experiments, only cells from passage 1 were used.
For this study, the authors referred to tendon cells as tenocytes, but previous work based on phenotypic criteria showed the presence of collagen type I.19 Additional proteins were assessed as well, such as collagen type III, decorin, biglycan, and tenascin-C, which have been found to be expressed in tenocytes.19
The study had 2 limbs (Figure 1). The first limb included tenocytes exposed to experimental treatments at 2-day intervals. Three separate treatments of PRPSS, PRPDS, and media were given at 0, 2, and 4 days. The second limb included tenocytes exposed to experimental treatments at 4-day intervals. Three separate treatments of PRPSS, PRPDS, and media were given at 0, 4, and 8 days. On the day of each treatment, fresh blood draws were obtained from the 8 subjects. The PRPSS and PRPDS products were then prepared accordingly, as described earlier, for a total of 24 samples (8 blood draws each for PRPDS, PRPSS, and media) in both the 2-day and 4-day interval groups. Cultured human tenocytes underwent interval PRPSS, PRPDS (10% v/v), or control media (10% fetal bovine serum) treatments. Before experimentation, fetal bovine serum was reduced to 2% in all platelet-rich plasma groups to maintain cell viability but not promote growth. Tenocyte cultures underwent 3 separate and consecutive treatments with fresh PRPSS, PRPDS, or media products at each application, prepared from the separate same-day blood draws. In the 2-day-interval and 4-day-interval groups, these products were given on days 0, 2, and 4 or 0, 4, and 8, respectively.
Experimental setup. Abbreviations: Dpm, disintegrations per minute; PRPDS, platelet-rich plasma derived from a double-spin process; PRPSS, platelet-rich plasma derived from a single-spin process.
Cell proliferation, measured as disintegrations per minute, was examined with a radioactive thymidine assay.20 The use of disintegrations per minute provides a measure of actively replicating DNA within each individual cell; in this way, a higher number of disintegrations per minute correlates to increased cell number.21 Radioactive thymidine is incorporated into the DNA of dividing cells. Therefore, cellular proliferation correlates with the amount of radioactivity. After 4 days of culture, cells were labeled with radioactive thymidine by adding 5 mCi tritiated thymidine/mL to the wells during the last 24 hours of culture. At the end of the 24-hour period, the thin floating layer of fibrin, formed on addition of platelet-rich plasma, was removed from each well, and the remaining attached cells were washed twice with 10% trichloroacetic acid to remove unbound thymidine. Adding 0.5 M sodium hydroxide to each well lysed cells, and the number of proliferating cells was assayed by measuring the number of disintegrations per minute with a scintillation counter. Each well was measured in triplicate to assure consistency of results.