Unraveling the Complexities of Leukocytes in PRP: Macrophage Plasticity and its Implications for Tissue Repair and Regeneration

This blog post aims to better understand the different white cell populations that are often injected with platelets (PLTs) in everyday cell therapies and questions "old school" PRP nomenclature while diving deeper on macrophage plasticity and its effect on tissue repair and regeneration in Orthobiologics. 

The human body is a remarkable symphony of cooperating cells, each playing a vital role in maintaining homeostasis and orchestrating the intricate processes of tissue repair and regeneration. In a recent Editorial piece “Nothing Heals Without Cells” in The American Journal of Sports Medicine, Plymouth Medical’s esteemed client Dr Scott Rodeo from the Hospital of Special Surgery reminds us that “simply studying the physiology and function of individual cells provides an incomplete view of how cells might function when used in cell therapy approaches”. One must therefore look to understand how cells behave in concert with each other and not “simply continue to study cell populations in isolation”[1]. A good reminder indeed and frankly, a great opportunity to better understand the different white cell populations that are often injected with platelets (PLT) in everyday cell therapies.  It may also be time to question some older PRP definitions that were marketed and promoted primarily by PRP kit manufacturers. 

Leukocyte-Poor vs Leukocyte-Rich PRP: Can we all agree this is an outdated way to define PRP?

The definition and characterization of Platelet Rich Plasma has always been a hot topic for our PLYMOUTH MEDICAL team. Leukocyte-poor platelet-rich plasma (LP-PRP) and leukocyte-rich platelet-rich plasma (LR-PRP) have been marketed as two different formulations of platelet-rich plasma (PRP). The basic difference between these two formulations lies in the presence or absence of ALL leukocytes (white blood cells). As with everything, it’s a lot more complex than this since it is argued that different leukocytes (neutrophils, lymphocytes and monocytes) will have very different effects on the tissues and joints being injected with PRP. 

The absence of leukocytes is believed to reduce the inflammatory response and potential catabolic effects associated with the release of pro-inflammatory cytokines from leukocytes [2, 3]. A group of French-speaking PRP experts, which include some PLYMOUTH MEDICAL clients, have also stated that LP-PRP has also been widely used in various clinical applications, such as orthopedics and sports medicine due to its potential for promoting tissue repair and regeneration [4]. LP-PRP is prepared by centrifugation techniques that selectively “concentrate” platelets while minimizing the presence of all leukocytes (granulocytes and agranulocytes) in the final product as seen in Arthrex ACP and Regenlab test tube PRP kits. Trouble is, the latter kits also minimize the presence of platelets and these are now understood to be subtherapeutic for MSK applications such as knee osteoarthritis [5,6] at their low doses of 1.4-2.0x PLT concentration. 

Conversely, the presence of neutrophils in PRP is believed to contribute to the antimicrobial properties of LR-PRP and enhance the immune response [5]. LR-PRP which includes neutrophils has been studied for applications in oral and maxillofacial surgery, periodontal regeneration, as well as tendon and bone regeneration, where the antimicrobial and angiogenic properties may be beneficial [7, 8, 9, 10]. The late great Dr Greg Lutz also postulated that NR-PRP was his root cause treatment of choice for Chronic Low Back Pain patients with Degenerative Disc Disease; his team characterized the various PRP preparations studied in each co-culture of Cutibacterium acnes with our 3 part-diff Horiba Hemocytometer to evaluate optimal leukocytic PRP profiles and their bactericidal properties [11].  

While both LP-PRP and LR-PRP have been studied for various clinical applications, PLYMOUTH MEDICAL have always believed the terms Leukocyte-Rich PRP or Leukocyte-Poor PRP to be misnomers when it comes to our high dose PRP protocols.PLYMOUTH MEDICAL have been using the terms Granulocyte or Neutrophil-Poor PRP (NP-PRP Protocol A) or Granulocyte or Neutrophil-Rich (NR-PRP Protocol B) which are covered in our trainings and outlined in our instructions for use. In other words, calling our high dose PRP protocol A “leukocyte-poor” is incorrect since it concentrates monocytes and lymphocytes (aka agranulocytes); our NP-PRP demonstrates a 61% mononuclear cell recovery while depleting the neutrophil content by over 98% compared to baseline whole blood. Eosinophils and basophils typically do not survive the centrifugation process and are therefore moot in PRP cell counting. Proper characterization of all types of leukocytes and proper nomenclature ensures our clients are leveraging their preferred PRP formulations based on clinical evidence and the indications treated (i.e. fractures with NR-PRP vs knee OA with NP-PRP).

What about monocytes? Macrophages may be the unsung heroes of Orthobiologics

At the heart of tissue repair lie the versatile macrophages - immune cells that possess the remarkable ability to adapt and respond to the dynamic microenvironmental cues they encounter as part of this cellular orchestra. Understanding the nuances of macrophage plasticity has emerged as a crucial frontier in the field of Orthobiologics, as these cells hold the potential to either hinder or facilitate the body's natural healing mechanisms.

Monocytes that migrate from the circulation into tissue mature into macrophages. In the circulation, the normal range of monocytes is between 2% and 8% of the white blood cell count, corresponding to 200–800 monocytes per microliter of blood (uL). Monocytes are round cells with a kidney-shaped nucleus and are 25–30 µm in size [9]. Macrophages exist along a spectrum of activation states, each with its own unique functional profile. The two most well-studied subtypes are M1 macrophages, driven by pro-inflammatory signals such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), which are characterized by their potent microbicidal and cytotoxic activities. In contrast, M2 macrophages, shaped by anti-inflammatory cytokines like interleukin-4 (IL-4) and interleukin-13 (IL-13), exhibit enhanced phagocytic capacity and promote tissue repair and remodeling [13].

The delicate balance between M1 and M2 macrophages is crucial for the successful orchestration of tissue repair following injury. In the acute phase of the inflammatory response, the pro-inflammatory M1 phenotype predominates, clearing cellular debris and pathogens. However, a timely transition to the anti-inflammatory, pro-regenerative M2 phenotype is essential for the resolution of inflammation and the initiation of the tissue healing process [13]. 

The M1-to-M2 Shift: A Hallmark of Successful Tissue Repair

The shift from the M1 to the M2 macrophage phenotype is a hallmark of successful tissue repair. M2 macrophages secrete a plethora of growth factors, cytokines, and chemokines that promote angiogenesis, extracellular matrix (ECM) deposition, and the recruitment of other reparative cell types, such as fibroblasts and stem/progenitor cells [13]. 

Moreover, a failure to transition from the M1 to the M2 phenotype, or a persistent dominance of the M1 phenotype, can have detrimental consequences for tissue repair. Sustained M1 macrophage activity leads to excessive inflammation, tissue damage, and the formation of inhibitory scar tissue, ultimately impairing the regenerative capacity of the affected tissue [14]. 

Given the pivotal role of macrophage plasticity in tissue repair and regeneration, the strategic modulation of this dynamic process has emerged as a promising therapeutic approach. Researchers have explored various strategies to tip the balance in favor of the pro-regenerative M2 phenotype, including the use of stem cell therapies, biomaterial scaffolds, and targeted pharmacological interventions [13].

Mesenchymal Stem Cell Therapies and Macrophage Polarization

Mesenchymal stem cells (MSCs) have been shown to possess the remarkable ability to shift the macrophage phenotype from M1 to M2 [15]. Lymphocytes and T lymphocyte-derived cytokines have also been shown to strengthen macrophage polarization, which is fortuitous given lymphocytes and monocytes often get concentrated together in our NP-PRP protocol given their similar cell densities. Through the secretion of anti-inflammatory cytokines and the modulation of the local immune microenvironment, MSCs can create a milieu that favors the M2 macrophage phenotype, thereby enhancing tissue repair and regeneration [15].

In summary, taking macrophage plasticity into consideration is crucial to understanding cellular-based tissue repair and regeneration. By evaluating the delicate balance between M1 and M2 macrophages, and their dynamic interplay with other cellular players within the microenvironment or even, as some studies suggest, with concomitant MSC-rich injectates, researchers and clinicians can optimize effective Orthobiologic therapies in the treatment of various musculoskeletal conditions. In light of the fact data on neutrophils, lymphocytes and monocytes are often missing from Orthobiologic clinical studies, it will be very interesting for our field to evaluate real world data such as the correlation between PLT dose and neutrophil/monocyte/lymphocyte yields to the complex outcomes patients experience across a variety of musculoskeletal and tissue-related conditions. 

Key Takeaways

  1. LR-PRP and LP-PRP may be outdated definitions for the orthobiologics PRP preparations our clients are leveraging in everyday cell therapy applications

  2. Plymouth Medical differentiate between Granulocyte or Neutrophil-Poor (NP-PRP protocol A) and Granulocyte or Neutrophil-rich (NR-PRP protocol B) PRP preparations as all our protocols provide high doses of Platelets in addition to higher recovery rates of mononuclear leukocytes such as monocytes and lymphocytes. Protocol B is Neutrophil-Rich PRP (NR-PRP). 

  3. Circulatory Monocytes transform into macrophages when they migrate into tissue and exist along a spectrum of activation states, with the classically activated M1 and alternatively activated M2 phenotypes playing distinct roles in the tissue repair and regeneration process.

  4. The timely transition from the pro-inflammatory M1 to the anti-inflammatory, pro-regenerative M2 macrophage phenotype is a hallmark of successful tissue repair.

  5. Persistent M1 macrophage dominance can lead to excessive inflammation, tissue damage, and the formation of inhibitory scar tissue, impairing the regenerative capacity of the affected tissue.

  6. Strategies to modulate macrophage plasticity, such as Mesenchymal stem cell therapies, biomaterial scaffolds, and targeted pharmacological interventions, hold promise for enhancing tissue repair and regeneration.

What type of PRP are you leveraging for the different MSK conditions treated? 

Citations

[1] Rodeo SA. Nothing Heals Without Cells. The American Journal of Sports Medicine. 2024;52(7):1669-1670. doi:10.1177/03635465241253491

[2] Dohan Ehrenfest DM, Andia I, Zumstein MA, Zhang CQ, Pinto NR, Bielecki T. Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich Fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives. Muscles Ligaments Tendons J. 2014 May 8;4(1):3-9. PMID: 24932440; PMCID: PMC4049647.

[3] Mishra, Allan & Woodall, James & Vieira, Amy. (2009). Treatment of Tendon and Muscle Using Platelet-Rich Plasma. Clinics in sports medicine. 28. 113-25. 10.1016/j.csm.2008.08.007.

[4] Eymard F, Ornetti P, Maillet J, Noel É, Adam P, Legré-Boyer V, Boyer T, Allali F, Gremeaux V, Kaux JF, Louati K, Lamontagne M, Michel F, Richette P, Bard H; GRIP (Groupe de Recherche sur les Injections de PRP, PRP Injection Research Group). Intra-articular injections of platelet-rich plasma in symptomatic knee osteoarthritis: a consensus statement from French-speaking experts. Knee Surg Sports Traumatol Arthrosc. 2021 Oct;29(10):3195-3210. doi: 10.1007/s00167-020-06102-5. Epub 2020 Jun 24. Erratum in: Knee Surg Sports Traumatol Arthrosc. 2021 Oct;29(10):3211-3212. doi: 10.1007/s00167-020-06331-8. PMID: 32583023; PMCID: PMC8458198.

[5] Andia I, Maffulli N. Platelet-rich plasma for managing pain and inflammation in osteoarthritis. Nat Rev Rheumatol. 2013 Dec;9(12):721-30. doi: 10.1038/nrrheum.2013.141. Epub 2013 Oct 1. PMID: 24080861.

[6] Bennell KL, Paterson KL, Metcalf BR, et al. Effect of Intra-articular Platelet-Rich Plasma vs Placebo Injection on Pain and Medial Tibial Cartilage Volume in Patients With Knee Osteoarthritis: The RESTORE Randomized Clinical Trial. JAMA. 2021;326(20):2021–2030. doi:10.1001/jama.2021.19415

[7] Sundman EA, Cole BJ, Fortier LA. Growth factor and catabolic cytokine concentrations are influenced by the cellular composition of platelet-rich plasma. Am J Sports Med. 2011 Oct;39(10):2135-40. doi: 10.1177/0363546511417792. Epub 2011 Aug 16. PMID: 21846925.

[8] Amable PR, Carias RB, Teixeira MV, da Cruz Pacheco I, Corrêa do Amaral RJ, Granjeiro JM, Borojevic R. Platelet-rich plasma preparation for regenerative medicine: optimization and quantification of cytokines and growth factors. Stem Cell Res Ther. 2013 Jun 7;4(3):67. doi: 10.1186/scrt218. PMID: 23759113; PMCID: PMC3706762.

[9] Kovtun A, Bergdolt S, Wiegner R, Radermacher P, Huber-Lang M, Ignatius A. The crucial role of neutrophil granulocytes in bone fracture healing. Eur Cell Mater. 2016 Jul 25;32:152-62. doi: 10.22203/ecm.v032a10. PMID: 27452963.

[10] Miron RJ, Fujioka-Kobayashi M, Bishara M, Zhang Y, Hernandez M, Choukroun J. Platelet-Rich Fibrin and Soft Tissue Wound Healing: A Systematic Review. Tissue Eng Part B Rev. 2017 Feb;23(1):83-99. doi: 10.1089/ten.TEB.2016.0233. Epub 2016 Oct 10. PMID: 27672729.

[11] Prysak, M. H., Lutz, C. G., Zukofsky, T. A., Katz, J. M., Everts, P. A., & Lutz, G. E. (2019). Optimizing the Safety of Intradiscal Platelet-Rich Plasma: An in Vitro Study with Cutibacterium Acnes. Regenerative Medicine, 14(10), 955–967. https://doi.org/10.2217/rme-2019-0098

[12] Lendeckel U, Venz S, Wolke C. Macrophages: shapes and functions. ChemTexts. 2022;8(2):12. doi: 10.1007/s40828-022-00163-4. Epub 2022 Mar 10. PMID: 35287314; PMCID: PMC8907910.

[13] Mantovani A, Biswas SK, Galdiero MR, Sica A, Locati M. Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol. 2013 Jan;229(2):176-85. doi: 10.1002/path.4133. Epub 2012 Nov 29. PMID: 23096265.

[14] Kawamura S, Ying L, Kim HJ, Dynybil C, Rodeo SA. Macrophages accumulate in the early phase of tendon-bone healing. J Orthop Res. 2005 Nov;23(6):1425-32. doi: 10.1016/j.orthres.2005.01.014.1100230627. Epub 2005 Aug 19. PMID: 16111854.

[15] Nakajima H, Uchida K, Guerrero AR, Watanabe S, Sugita D, Takeura N, Yoshida A, Long G, Wright KT, Johnson WE, Baba H. Transplantation of mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury. J Neurotrauma. 2012 May 20;29(8):1614-25. doi: 10.1089/neu.2011.2109. Epub 2012 Apr 18. PMID: 22233298; PMCID: PMC3353766.

06/01/24