The Effect of a Single Freeze-Thaw Cycle on Matrix Metalloproteases in Different Human Platelet-Rich Plasma Formulations: A Prospective Cohort Study
Authors: Whitney Kaitlyn E. , Kennedy Mitchell , Dornan Grant , Chahla Jorge , Evans Thos A. , Philippon Marc J. , LaPrade Robert F. , Huard Johnny 
Published: July 2019

Objectives: The possibility of preserving platelet-rich plasma (PRP) from young, healthy individuals for future use is a compelling approach to reduce or delay degenerative processes, presuming that the retention of the biological properties are maintained. The purpose of this study was to measure and compare matrix metalloproteinases (MMP) isoform concentrations between whole blood (WB), leukocyte-rich PRP (LR-PRP) inactivated (LR-I) and activated (LR-A), leukocyte-poor PRP (LP-PRP) inactivated (LP-I) and activated (LP-A). Methods: Following institutional review board approval (2017-36), 24 donors that were physically and mentally healthy were prospectively enrolled in the study. Approximately 60 mL of WB was drawn from each donor to produce inactivated and activated LP-PRP and LR-PRP using manual processing methodology, as previously described. A complete blood count for WB and inactivated PRP products was obtained to verify that concentration of platelets was achieved. WB, LP-I, and LR-I samples were set aside for immunoassay and analysis. The LP-I and LR-I products were activated with 10% calcium chloride and recombinant thrombin in a red-top 10 mL vacutainer tube. Blood fractions were either immediately assayed and analyzed (fresh) or stored at -80℃ for 24 hours, 72 hours, and 160 hours. Commercial kits (EMD Millipore) were used according to manufacturer’s instructions for protein content: MMP-1, MMP-3, MMP-9, MMP-10, and MMP-12. A standard methodology for the Luminex 200® system was used as previously published. A pairwise Wilcoxin rank test was performed for statistical calculation. Results: Twenty-two healthy donors (n = 12 females, n = 10 males) with a mean age of 37.7 (range: 21 to 60), and average BMI of 23.7 kg/m2, were used in the final analysis. MMP-1 significantly increased between fresh and 160 hours in WB (p<0.05) (Figure 1), and significantly increased between fresh and 24 hours and 160 hours in LR-A (p<0.05) (Figure 4). MMP-3 significantly decreased between fresh and 24 hours, 72 hours, and 160 hours in LR-A (p<0.05) (Figure 4). MMP-9 significantly increased between fresh and 160 hours in WB, LR-A, and LR-I (p<0.05) (Figures 1, 2 & 4). MMP-12 significantly decreased between fresh and 24 hours in LR-A (Figure 4), while MMP-12 significantly decreased between fresh and 24 hours, 72 hours, and 160 hours in WB, LR-I, and LP-I (p<0.05) (Figures 1, 2 & 3). MMP-10 was not statistically different amongst fresh and freezing time points in all WB and PRP preparations. Interestingly, there was no statistical difference between MMP concentrations and freezing timepoints in LP-A. There were no significant correlations between MMPS and age, BMI or sex. Conclusion: In this study, we evaluated the influence of short-term freezing (-80℃) on MMP concentrations in WB, inactivated PRP, and activated PRP formulations. Our results suggest that certain MMP isoforms, can either increase or decrease in response to freezing in WB, inactivated PRP, and activated PRP formulations. The development of PRP preservation approaches through minimal manipulation, without attenuating its biological properties, represents an important step in PRP mediated tissue regeneration and repair. [Figure: see text][Figure: see text][Figure: see text][Figure: see text]