Original Research

The In Vivo Impact of Leukocyte Injections on Normal Rat Achilles Tendons: Potential Detriment to Tendon Morphology, Cellularity, and Vascularity

Publish date: October 1, 2018

References

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Tendinopathies are debilitating conditions affecting patients worldwide every day. They arise most frequently from tendon overuse resulting in pathology.¹ There are 2 major subtypes of tendinopathy: tendinosis and tendinitis. Tendinosis, the more common condition, is characterized by long-term, chronic degradation of tendon tissue resulting in fibrosis from infiltrating fibroblasts.² Tendinitis, the less common condition, is characterized by an acute inflammatory response and inflammatory cell infiltrate.² Both conditions are common, with Achilles tendinopathy affecting 11% of runners and lateral epicondylitis affecting 1% to 3% of the general population.^3,4 Many sports-related overuse injuries, such as tendinopathies, go undiagnosed for extended periods of time because medical attention is avoided in order to prevent time loss from training or competing.⁵ These delays could be eliminated if a non-surgical option for treating tendon pathology was available.

Tendinopathies are believed to result from tendon overuse that causes micro-damage to collagen, as well as from significant changes in protein and enzyme composition within the tendon.⁶ The damage accumulates over time and eventually leads to chronic inflammation or fibrotic change within tendons, in both cases weakening the tendon and causing pain. Currently, accepted treatments for tendinopathies include: nonsteroidal anti-inflammatory drugs, physical therapy, ultrasound, laser-therapy, corticosteroids, glyceryl trinitrate patches, extracorporeal shock wave therapy, sclerotherapy, and surgery.⁷ Recently, platelet-rich plasma (PRP) therapy has emerged as a promising treatment for tendinopathies, as well as a variety of other orthopedic indications.

PRP consists of autologous blood from the patient, centrifuged to increase the amount of platelets in the sample above baseline, and subsequently injected around an affected tendon or joint.⁸ PRP is used to treat tendinopathy because it can supply injured tendons with blood components that aid in healing, which tendons do not receive due to poor vascularity.⁹ These components include growth factors, such as platelet derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), endothelial growth factor, and leukocytes that can stimulate an inflammatory response within the injured tissue.¹⁰ The inflammatory response from the PRP induces a more robust reconstruction and revascularization of the injured tissue, stimulating proliferation, and remodeling.^11,12However, significant variability exists within the platelets, leukocytes, and growth factors that comprise PRP. This is attributed to 3 major causes. First, current commercial preparations of PRP result in differing platelet concentrations, as well as leukocyte-rich and leukocyte-poor compositions.^13,14 Variability in platelet concentrations results in unreliable amounts of growth factors, including cytokines, TGF-β, PDGF, VEGF and basic fibroblast growth factor in each preparation, while leukocyte levels affect inflammation, all leading to variable effects for each preparation.^15,16Second, despite sex and age of the PRP donor not being significant factors influencing variation in growth factor concentrations, the existence of an unexplained variation in concentrations of growth factors between different donors has been observed.¹⁷ Third, the selection of activating agents, bovine thrombin or calcium chloride, and their application, whether to the elbow, shoulder, or knee, produces variability.¹⁸

While the effects of platelets and growth factors in PRP have been well studied, less is known about the effects of differing cell types. Recently it was reported that the concentrations of leukocytes directly affect the outcomes of PRP injections. McCarrel and colleagues^19,20 found that as the number of leukocytes increased, there was a concomitant increase in the expression of inflammatory cytokines and catabolic activity. This effect may result in inferior healing of injured tissues and is attributed to the release of pro-inflammatory cytokines such as interleukin-1β from the leukocytes.²¹ There is also evidence that minimizing the catabolic effect of leukocytes may be just as important to tissue healing as the maximizing anabolic effect of platelets and growth factors.²²

The use of PRP has been highly disputed in recent years due to conflicting reports of its success in treating orthopedic conditions. Numerous favorable studies have shown benefit for treating chronic and acute orthopedic injuries including; rotator cuff tear repair, chronic refractory patellar tendinopathy, and chronic lateral tendinosis/epicondylitis.^23-26 Concurrently, articles demonstrating no significant effects from PRP have also been published. One study claiming that PRP injections did not improve outcomes of chronic Achilles tendinopathy did not differentiate whether patients had tendinosis or tendinitis, and did not consider leukocyte concentration in their PRP preparations²⁷ Another study that determined PRP is not beneficial to the healing of ruptured Achilles tendons after surgical repair also failed to consider the concentration of leukocytes in their PRP preparations.²⁸ One of the difficulties in comparing these studies is their heterogeneous nature. This arises from the use of different conditions in each study that makes the studies incomparable. Variations in PRP preparations lead to different concentrations of growth factors, platelets, and leukocyte concentrations. Additionally, tendinopathy models were not specified as tendinosis and tendonitis, and models or patients were not controlled for age, sex, or comorbidities. Given that leukocyte-rich and leukocyte-poor PRP preparations are currently widely used in clinical practice, the discovery of which type of preparation is indicated in which setting is paramount to evidence-based use of this treatment modality. Due to reports suggesting that leukocytes may be detrimental to tendon healing, determining which types of leukocytes are responsible for these effects is vital. As such, the purpose of this study is to determine the in vivo effects of sub-populations of leukocytes on normal rat tendons. This study design allowed us to isolate the effects of the injections to induce a response and remove confounding effects of normal healing response to a damaged tendon and effects from the injection itself. Our hypothesis was that the injection of leukocytes would cause an inflammatory response in rat tendons, leading to catabolic outcomes.

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