Assessing clinical implications and perspectives of the pathophysiological effects of erythrocytes and plasma free hemoglobin in autologous biologics for use in musculoskeletal regenerative medicine therapies. A review

Abstract

Autologous biologics, defined as platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMC), are cell-based therapy treatment options in regenerative medicine practices, and have been increasingly used in orthopedics, sports medicine, and spinal disorders. These biological products are produced at point-of-care; thereby, avoiding expensive and cumbersome culturing and expansion techniques. Numerous commercial PRP and BMC systems are available but reports and knowledge of bio-cellular formulations produced by these systems are limited. This limited information hinders evaluating clinical and research outcomes and thus making conclusions about their biological effectiveness. Some of their important cellular and protein properties have not been characterized, which is critical for understanding the mechanisms of actions involved in tissue regenerative processes. The presence and role of red blood cells (RBCs) in any biologic has not been addressed extensively. Furthermore, some of the pathophysiological effects and phenomena related to RBCs have not been studied. A lack of a complete understanding of all of the biological components and their functional consequences hampers the development of clinical standards for any biological preparation. This paper aims to review the clinical implications and pathophysiological effects of RBCs in PRP and BMC; emphasizes hemolysis, eryptosis, and the release of macrophage inhibitory factor; and explains several effects on the microenvironment, such as inflammation, oxidative stress, vasoconstriction, and impaired cell metabolism.

Keywords: BM-MSCs, bone marrow-mesenchymal cells; BMA, bone marrow aspiration; BMC, bone marrow concentrate; Bone marrow mesenchymal cells; Eryptosis; HSCs, hematopoietic stem cells; Hb, hemoglobin; Hp, haptoglobin; Hx, hemopexin; Inflammation; MIF, Macrophage migration inhibitory factor; MNCs, mononucleated cells; Macrophage migration inhibitor factor; NO, nitric oxide; OA, osteoarthritis; Oxidative stress; PAF, platelet activating factor; PFH, plasma free hemoglobin; PRP, platelet-rich plasma; PS, phosphatidylserine; Plasma free hemoglobin; Platelet-rich plasma; RBC, red blood cell; ROS, reactive oxygen species.

Fig. 1

Cellular whole blood density separation following the first centrifugation procedure with the EmCyte PurePRP®SP. The whole blood cellular components (indicated by the red lines) are separated in the PurePRP®SP concentrating device as a result of the different cell densities in two basic layers . The top layer is the platelet plasma suspension, consisting of plasma and the multicomponent buffy coat layer, containing platelets, monocytes, lymphocytes, and neutrophils. The second basic layer consists of the erythrocyte cellular pack. The range of the specific cell densities varies between individuals. After a second centrifugation procedure approximately 7 mL of PurePRP is aspirated from the bottom chamber to be used for regenerative therapies. (PurePRP®SP: Pure Platelet-Rich Plasma Supra-Physiologic).

Fig. 2

Bone marrow concentrate density separation following EmCyte Aspire ™ BMA harvesting and PureBMC® second spin centrifugation. Anticoagulated aspirated bone marrow was initially injected in the concentration device for the first spin cycle. After the second centrifugation procedure the separation of bone marrow components, according to their different density gradients, follows in the concentrating accessory device. The HSC’s and MSC’s are located on top of the erythrocyte and white blood cell layer and are extracted via the aspirating pipe. (BMA: bone marrow aspirate; PureBMC®: Pure Bone Marrow Concentrate; HSC: hematopoietic stem cell; MSC: mesenchymal stem cell).

Fig. 3

Schematic summary illustration showing the pathophysiological effects and reactions of RBC hemolysis and eryptosis. The pathophysiological consequences of RBC hemolysis and PFH development in a biological treatment vial. Under normal circumstances PFH and its split products oxyHb (Fe²⁺), ferric Hb (Fe³⁺), and free hemin are released into plasma where they are cleared by natural occurring scavengers and compensatory mechanisms like Hp, Hx, and NO vascular reactions. However, in their absence and due to excessive PFH, a build-up of ferric and heme products continues, potentially leading to toxic consequences like direct pro-inflammation and pro-oxidant effects, endothelial cell dysfunction, and vasoconstriction. A biologic formulation which contains a high concentration of RBCs, combined with oxidative and hemolytic components, applied to tissue microenvironments, will lead to RBC cell membrane asymmetry and membrane disruption. This will lead to eryptosis, while displaying PS, leading to inflammation, and endothelial cell reactions with decreased microcirculatory activity. Another consequence of RBC disintegration and PFH is an abundant release of MIF cytokines, playing a profound role in pro-inflammatory processes (Adapted in part and modified from Schaer et al. [23]).