Effect of Autologous Platelet-rich Plasma on Cell Proliferation and Collagen Synthesis of Human Anterior Cruciate Ligament Cells

INTRODUCTION: Activated platelets are known to release many kinds of growth factors such as Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor-β (TGF-β). Platelet-rich plasma (PRP) is a fraction of plasma in which platelets are concentrated and is reported to be utilized as a source of multiple growth factors that promote tissue repair. Recent studies has shown that platelet rich plasma (PRP) have an enhancing effect on cell proliferation and collagen synthesis in tendon and ligament fibroblasts [1],[2]. It was also reported that collagen-PRP gel treated with thrombin enhanced bovine anterior cruciate ligament (ACL) cell proliferation and migration in the gel [3], and the use of collagen-PRP scaffold stimulated healing of a central defect in the canine ACL [4]. For the clinical application of PRP to ACL deficient patients, it is essential to examine if PRP has a promotive effect on ACL cell metabolism using samples from adult human. However, no studies have clarified the effect of PRP on human ACL. We have hypothesized that human PRP also enhance the ACL cell functions, and our focuses were on testing the effect of autologuous prepared PRP on adult human ACL cells. MATERIALS AND METHODS: Plasma and cell preparation: Fresh blood (50ml) and ACL tissue were obtained from three patients underwent ACL reconstruction surgery and three patients underwent total knee arthroplasty, with their consent to our study. The blood was centrifuged using a platelet concentration system, Symphony (DePuy), and platelet poor plasma (PPP) and PRP were isolated separately. Isolated PPP and PRP were clotted with thrombin, followed by centrifugation and separation into fibrin gel and soluble supernatant. The soluble fraction was used for this experiment. ACL cells were isolate with enzymatic digestion of the ACL tissue. Cell culture: Isolated cells were cultured in DMEM/F12 containing 10%FBS at 37ûC and 5%CO2 air atmosphere. The cells whose number reached about 4.5x10 after cell passage (0-3 times) were used for the following experiments. After 2 day pre-culture, the cells were cultured in serum-free medium for 6 hours and then cultured for another 48 hours under the following conditions; in the absence of serum (serum free group), in the presence of 5% Fetal Bovine Serum, 5% platelet poor plasma and 5% platelet poor plasma releasates (FBS, PPP and PRP groups respectively). Cell proliferation and gene expression assay: For cell proliferation we performed a WST-8 assay (cell counting kit-8) using 96well plates. To examine type I collagen synthesis, a relative quantitative real time RT-PCR assay was performed. Total RNA was isolated using TRIzol reagent (Invitrogen) and then cDNA was made by reverse transcription of the isolated RNA. Amplification of the target gene was carried out by real time PCR system (Applied Biosystems 7500), with a pair of primers for type I collagen α1 chain (TaqMan Gene Expression Assays, Applied Biosystems). RESULTS: The concentrations of PDGF and TGF-β1 in the PRP releasate were much higher than those in the PPP releasate (measured with sandwich ELISA, data not shown). A direct sequential observation under microscope revealed that the PRP group have shown higher cell density than the other groups (Fig. 1). Similarly, the WST-8 assay on day 2 showed a higher absorbance in the PRP group compared with other groups (p=0.05) (Fig. 2), which reflect an enhancement of cell proliferation. The type I collagen expression of the PRP group was significantly higher than those of other groups as relative quantitative RT-PCR revealed (Fig. 3). Figure 1: Cell morphology of human ACL cells cultured in monolayer after 4 day treatment with different media. (A) serum free, (B) 5%FBS, (C) 5%PPP, (D) 5%PRP x10.