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Effects of Platelet-Rich Plasma Composition on Anabolic and Catabolic Activities in Equine Cartilage and Meniscal Explants

Take Home Message

Clinical preparations of platelet-rich plasma (PRP) from autologous blood are created using commercial kits, of which there are numerous options that produce different platelet and growth factor doses. It is not known which PRP preparation is most appropriate for applications to cartilage and the meniscus. Therefore, we evaluated the response of equine cartilage and meniscal explants to single or double spin PRP preparations, the latter of which is necessary to obtain a high density of platelets. In general, protein and proteoglycan synthesis for single spin PRP was higher than double spin PRP. Gene expression of the proteoglycan catabolic enzyme ADAMTS-4 was lowest for single spin PRP. This study suggests that single-spin PRP preparations may be the most advantageous for intra-articular applications, and that double-spin systems should be considered with caution.

Introduction

Platelet-rich plasma represents an accessible and inexpensive source of growth factors that has received considerable attention for the treatment of musculoskeletal tissues, although few studies have evaluated the effect of PRP on articular cartilage and the meniscus (1). An important factor in determining the potential of PRP to heal joint tissues is the dose of platelets that best stimulates cartilage or meniscal growth. PRP containing less than a two-fold increase in platelet concentration relative to blood may be obtained by a single spin protocol (2). To further concentrate the platelets, a second higher force spin may be used (3). Given that higher platelet concentrations translate into a higher growth factor dose, we hypothesized that double spin PRP preparations will stimulate higher extracellular matrix synthesis without a concomitant increase in catabolic activities in cartilage and meniscal explants than single spin protocols. This project was a collaborative study by Drs. Kisiday, Frisbie, and McIlwraith of the ORC with Drs. Rodkey and Steadman of the Steadman Philippon Research Institute.

Methods

Samples were obtained from five healthy two- to five-year-old horses. Blood and tissues. Blood was drawn into anticoagulant citrate dextrose at a ratio of 10:1. Articular cartilage and menisci were retrieved from the femorotibial joint. Full-thickness (1-2 mm) sections of articular cartilage were removed from the femoral condyle and divided into 5 mm by 5 mm explants. Three mm thick meniscal explants were harvested from the femoral articulating surface of the avascular portion of medial menisci. PRP. PRP was prepared using Arthrex ACP™ according to the manufacturer's single protocol (referred to as Single Spin Kit). PRP from Harvest Smart Prep 2™ was prepared according to the manufacturer's double spin protocol (referred to as Double Spin Kit). Laboratory PRP was created by centrifuging blood at 200 g for 18 minutes and harvesting the plasma. A portion of this PRP (referred to as Single Spin Lab), was set aside for experimentation. The remaining PRP was centrifuged at 1000 g for 15 minutes. The platelet-poor plasma (PPP) supernatant was removed, and PPP was added back to the centrifuged platelets in reduced volumes that resulted in 3x, 6x, and 9x concentrations of platelets relative to Single Spin Lab PRP. Hereafter, these PRP preparations are referred to as Double Spin 3x, 6x, and 9x, respectively. Culture medium: PRP was mixed with equal volumes of low glucose DMEM plus ascorbate and antibiotics. Half of the cultures received 10 ng/ml of recombinant human IL-1β resuspended to produce a proinflammatory environment (4). Evaluation of extracellular matrix synthesis: Explants were evaluated for 35S-sulfate and 3H-proline incorporation as measures of proteoglycan and protein synthesis, respectively. These data were normalized to total DNA (5). Gene expression of catabolic enzymes: RNA was extracted from explants and reverse transcribed to cDNA using random hexamers. cDNA and TaqMan Gene Expression Master Mix were mixed with primer/probes for the catabolic enzymes ADAMTS-4, ADAMTS-5, MMP1, MMP13, and the housekeeping gene GAPDH, and expression levels were determined using semi-quantitative real-time PCR. Gene expression was normalized to GAPDH. (6). Data analysis: The analysis of cartilage and meniscal explants was repeated for blood and tissue samples from five donor horses, with blood and tissues paired in an autologous fashion. All data were analyzed using a mixed model analysis of variance, with the donor animal used as a random effect. Individual comparisons were made using least square means procedure. Individual comparisons of main effects or interactions were indicated based on a protected f-test. p-values less than 0.05 were considered significant. Data are reported as mean +/- sem.

Results

Cellular content of PRP preparations. The concentration of platelets in Single Spin Lab PRP (317 +/- 22 thousand/βl, was not significantly different from Single Spin Kit PRP (276 +/- 8 thousand/βl) (p=0.20). Double Spin Kit PRP contained 2.3 and 2.6-fold higher platelet concentrations (725 +/- 95 thousand/βl) relative to Single Spin Lab and Single Spin Kit PRP, respectively. White blood cell counts in Single Spin Lab PRP (0.04 +/- 0.001 thousand/βl) were not significantly different from Single Spin Kit PRP (0.03 +/- 0.01 thousand/βl) (p=0.98). Double Spin Kit PRP contained at least a 400-fold higher concentration of white blood cells (14.8 +/- 3.0 thousand/βl) relative to Single Spin Lab and Single Spin Kit PRP.
 
Extracellular matrix synthesis. Cartilage (Fig. 1): 3H-proline – In the absence of IL-1β, 3H-proline incorporation in Single Spin Lab cultures was not significantly different from Single Spin Kit cultures (p=0.22). 3H-proline incorporation in Single Spin Lab PRP was 31-64% higher than Double Spin 3x, 6x, and 9x, and Double Spin Kit cultures. In Single Spin Kit cultures, 3H-proline incorporation was 31% and 20% higher than Double Spin 3x and Double Spin Kit cultures, respectively. In Double Spin Kit cultures, 3H-proline incorporation was not significantly different from Double Spin 3x PRP (p=0.25). No significant differences were found among Double Spin 3x, 6x, and 9x cultures (p=0.07-0.58). 3H-proline incorporation decreased with the addition of IL-1β for Single Spin Lab cultures only. In IL-1β medium, 3H-proline incorporation was not significantly different among Single Spin Lab, Double Spin 3x, and Single and Double Spin Kit cultures. (p=0.22-0.98). 3H-proline incorporation in Double Spin 6x and 9x cultures was approximately 15% lower than Single and Double Spin Kit cultures. 35S-sulfate – In the absence of IL-1β, 35S-sulfate incorporation in Single Spin Lab cultures was not significantly different from Single Spin Kit cultures (p=0.28). 35S-sulfate incorporation in Single Spin Lab cultures was 32-72% higher than Double Spin 3x, 6x, and 9x, and Double Spin Kit cultures. 35S-sulfate incorporation in Single Spin Kit cultures was 38-50% higher than higher than that in Double Spin 3x, 6x, and 9x cultures. No significant differences were detected among Double Spin 3x, 6x, and 9x PRP (p=0.47-0.97). 35S-sulfate incorporation in Double Spin Kit cultures was not significantly different from Single Spin Kit and Double Spin, 3x, 6x, and 9x PRP cultures (p=0.07-0.27). 35S-sulfate incorporation decreased with the addition of IL-1β for Single Spin Lab, Single Spin Kit, and Double Spin Kit cultures. In IL-1β cultures, 35S-sulfate incorporation among all media was not significantly different (p=0.47-0.99). Meniscus (Fig. 2): 3H-proline – Interactions between PRP preparations and IL-1β were not significant (p=0.78). When considering the effect of PRP independent of IL-1β, 3H-proline incorporation in Single Spin Lab and Double Spin 3x cultures was not significantly different (p=0.46). 3H-proline incorporation in Double Spin 6x and 9x cultures were not significantly different (p=0.15), and both were significantly lower than Single Spin Lab and Double Spin 3x cultures. Single Spin Kit cultures were not significantly different from all other cultures (p=0.06-0.35) except for Double Spin 9x PRP. 3H-proline incorporation in Double Spin Kit cultures was less than Single Spin Lab and Double Spin 3x cultures only. 35S-sulfate – In the absence of IL-1β, 35S-sulfate incorporation was not significantly different among Single Spin Lab, Double Spin 3x, and Single Spin Kit cultures (p=0.28-0.67). 35S-sulfate incorporation in Double Spin 6x and 9x, and Double Spin Kit cultures was not significantly different (p=0.40-0.74). 35S-sulfate incorporation in Double Spin 6x and 9x, and Double Spin Kit cultures was approximately 40% less than Single Spin Lab, Double Spin 3x, and Single Spin Kit cultures. 35S-sulfate incorporation decreased with the addition of IL-1β for Single Spin Lab, Double Spin 3x, and Single Spin Kit PRP. IL-1β cultures, 35S-sulfate incorporation among all media was not significantly different (p=0.30-0.89).
 
Gene expression of catabolic enzymes. Cartilage: Interactions between PRP formulation and IL-1β were not significant for MMP1 (p=0.47), MMP13 (p=0.12), and ADAMTS-5 (p=0.19). In the absence of IL-1β, ADAMTS4 expression in Double Spin 6x cultures was 3.4- to 8.9-fold higher than Single Spin Lab, Single Spin Kit, and Double Spin 3x cultures. ADAMTS-4 expression in Double Spin Kit cultures was not significantly different from Double Spin 6x cultures (p=0.78), and significantly higher than all other IL-1β-free cultures. The addition of IL-1β to the culture medium increased ADAMTS-4 expression for Double Spin 9x cultures (11-fold) only. In IL-1β medium, ADAMTS expression was not significantly different between Single Spin Lab and Double Spin 3x cultures (p=0.97). ADAMTS-4 expression increased approximately 5-fold for Double Spin 6x and 22-fold for Double Spin 9x cultures. ADAMTS-4 expression in Single Spin Kit cultures was not significantly different from all laboratory preparations (p=0.16-0.18) except for Double Spin 9x. ADAMTS-4 expression in Double Spin Kit cultures was 2.1-fold higher than Single Spin Kit, approximately 5-fold higher than Single Spin Lab and 3x, and not significantly different from Single Spin 6x (p=0.68) cultures. Meniscus: Expression levels of MMP1 and 13 were not analyzed as many samples resulted in expression levels near the PCR detection limit. Interactions between PRP formulations and IL-1β were not significant for ADAMTS-4 (p=0.48) and ADAMTS-5 (p=0.33) (data not shown).

Conclusions

This study evaluated PRP formulations across a range of platelet concentrations as an indicator of whether differences exist among the various PRP kits that are commercially available. These data reject the hypothesis that increases the concentration of platelets in PRP stimulates ECM synthesis in cartilage and the meniscus; furthermore, these findings suggest that high platelet concentrations for intra-articular injection should be considered with caution. When projecting the results of in vitro studies to clinical applications, certain limitations of laboratory models must be considered. While the dilution of PRP by 50% was intended to mimic the dilution of an intra-articular injection in synovial fluid, tissue culture does not allow for clearance of the growth factors (7) as may occur in joints. Therefore, the laboratory model may overestimate the concentration of growth factors that is present in vivo. Importantly, the acute response of cartilage and meniscal explants does not necessarily recapitulate the response that occurs over weeks of clinical healing. In vivo testing will be necessary to address these limitations.

Acknowledgments

Funded by the Steadman Philippon Research Institute, Vail, Colo., and discretionary funds from the Orthopaedic Research Center at Colorado State University. We thank Dr. David Karli of the Steadman Clinic for his technical assistance.

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