2017 ISAKOS Biennial Congress ePoster #311

 

The Impact Of Leukocyte Injections: Are They Detrimental To Tendon Morphology, Cellularity, And Vascularity?

David Komatsu, PhD, Stony Brook, NY UNITED STATES
Lucas King, East Setauket, NY UNITED STATES
James M. Paci, MD, East Setauket, NY UNITED STATES

Stony Brook University, Department of Orthopaedics, Stony Brook, New York, UNITED STATES

FDA Status Not Applicable

Summary

This study assessed the effects of differing leukocyte concentrations in PRP preparations by injecting sub-populations of leukocytes into rat Achilles tendons and found that the injection of granulocytes produced an inflammatory response, suggesting that leukocyte-rich PRP preparations are contraindicated for inflammatory conditions.

Abstract

Platelet- rich plasma (PRP) therapy is a potential treatment for multiple musculoskeletal injuries in orthopedics. Despite its increasing clinical utilization, consensus has not been achieved on the most effective PRP preparation. This is largely driven by the high variability of resultant components, particularly the concentrations of leukocytes. This study sought to determine the differential effects of leukocytes on tissue morphology, cellularity, and vascularity by injecting sub-populations of leukocytes into rat Achilles tendons. Twenty-four male Sprague-Dawley rats were randomized to three treatment groups (N=8/group): 1) Monocytes; 2) Granulocytes; and 3) Plasma. Allogenic blood was collected, treated with red cell lysis buffer, pelleted, and resuspended in saline. The suspension was then sorted into fractions containing monocytes and granulocytes using fluorescence activated cell sorting. A fraction containing only platelet poor plasma was also collected. The left Achilles tendons were then percutaneously injected 100ul of sorted cells containing 2x10^5 and 9x10^5 monocytes and granulocytes, respectively. Controls were similarly injected with 100ul of platelet poor plasma. On days 7 and 14, 4 rats from each group were euthanized and the Achilles tendons were harvested, fixed, and processed for paraffin microtomy. In order to assess cellularity and morphology, sections were stained with H&E and blindly rated by three independent observers. Similarly, vascularity was assessed by performing immunohistochemical staining for CD31. Automated image analyses were then performed using a global thresholding algorithm (Bioquant). For all outcomes, the results for the injected tendons were normalized to those of their contralateral untreated controls and reported as a percentage. Differences between groups at each time-point were assessed using Independent Samples Median Tests (SPSS, V.19) with significant differences determined for values of p<0.05. No differences in cellularity between groups were seen at day 7 (p=0.368). However, a significant difference between groups was seen at day 14 (P=0.050). Pairwise tests showed there to be a significant increase in cellularity in tendons treated with granulocytes, as compared to both monocytes and plasma. For morphology, no significant differences were seen between groups at either time-point (p= 0.091 for day 7 and p=1.000 for day 14). Similarly, no differences were seen for vascularity between treatment groups at either time-point (p= 0.368 for day 7 and p=0.535 for day 14). Both leukocyte-rich and leukocyte-poor PRP preparations are currently employed in clinical practice. We evaluated the effects of the leukocyte components on intact rat Achilles tendons and revealed that injecting granulocytes caused an increase in inflammation, as judged by the cellularity of the tendon, when compared to monocytes and plasma injections. Our findings of increased inflammation confirm previous reports linking leukocytes to inflammation and support the growing body of evidence indicating that leukocyte-rich PRP preparations are pro-inflammatory and that their use may be contraindicated for inflammatory conditions. In conclusion, further investigations of differing PRP preparations are clearly warranted. These studies should rigorously control for differences in PRP components, particularly platelets, leukocytes, and growth factors, in order to identify the optimal PRP preparation for each clinical situation.