Preclinical Evidence for Biologics in Cartilage Repair and Early Osteoarthritis

Introduction

Numerous biologics have been investigated in vitro and in preclinical animal models and show significant promise as methods of cartilage repair or as treatments for osteoarthritis (OA). Although there is mounting preclinical and clinical evidence for the use of certain biologics in treating articular disease, data for each biologic should be carefully weighed against where and how the drug has been tested. In the laboratory, it is not uncommon for biologics to be applied to chondrocytes isolated from their native matrix environment to test their effect. Similarly, in animal models of cartilage repair or OA, it is also not uncommon for outcome measures to focus on the articular cartilage surfaces alone. Although chondrocyte and cartilage homeostasis are undoubtedly important, it should be remembered that these biologics will affect the whole joint including the synovial membrane, meniscus, any exposed subchondral bone, and intra-articular ligaments in addition to their notable effects on chondrocytes. Furthermore, any chondrogenic or chondroprotective effect mediated by these biologic adjuncts are only of benefit if there is mechanical stability and alignment in the joint.

Biologics are often promoted as therapeutics based on the presence of growth factor and/or as stem cell therapies containing mesenchymal stromal cells (MSCs). Despite recent advances in our understanding of the role of MSCs in producing secretome with trophic effects on the local immune response, antiinflammatory, and regenerative effects, the precise mechanism of these actions is not yet fully elucidated. Similarly, it is nearly impossible to identify the precise mechanisms of action of growth factors contained within autologous biologic adjuncts because they all contain mixtures of growth factors, pro- and antiinflammatory cytokines, and chemokines that have direct and indirect effect on local cells to effectuate tissue restoration. This combination of bioactive factors will have pleiotropic effects on the complex disease processes associated with loss of joint homeostasis.

By understanding the integration of biologic agents into treatment paradigms that respect the biomechanics, and variability due to disease stage and patient physiology, we can provide better integrated patient care. Ultimately, continued research will guide patient protocols regarding the optimal dose and timing of biologic treatment for each specific disease stage.

Platelet-Rich Plasma

Platelet-rich plasma (PRP) is autologous plasma that contains more platelets than the starting blood sample. Growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-B (TGF-β), vascular endothelial growth factor (VEGF), and other growth factors within platelet alpha granules are commonly cited as responsible for the anabolic and reparative effects of PRP. White blood cells or leukocytes (lymphocytes, granulocytes, and monocytes) are also abundant in PRP and are typically associated with catabolic or pro-inflammatory effects of PRP. This “growth factor = good, leukocyte = bad” paradigm is an oversimplification given that there are also red blood cells and over 1500 proteins in PRP. Nonetheless, platelet and leukocyte concentrations still form the basis for PRP classification schemes.

There are numerous commercial devices marketed for generation of PRP and several classification systems for PRP products, resulting in lack of standardization in classification and treatment protocols. Very broadly, PRP preparations can be divided into leukocyte-rich (LR-PRP), leukocyte-poor (LP-PRP), and platelet-rich fibrin (PRF). To answer the important and prevailing clinical questions of dosing, timing, and type of PRP (LP or LR) that is indicated for each musculoskeletal injury, it is critical that investigators of basic science, preclinical, and clinical studies analyze and report the contents of PRP used in studies. This can be done with automated machine counting or equally as accurately with simple, inexpensive manual direct smear analysis.

Once administered, the cellular and soluble contents of PRP including growth factors, chemokines, and cytokines will have direct and indirect effects on local tissue cells. Although the intent might be to treat a focal cartilage defect or to enhance repair of an anterior cruciate ligament, it should be remembered that any injection will affect all the tissues of the joint including the synovial membrane, cartilage, meniscus, exposed subchondral bone, and peri- or intra-articular structures. Growth factors in PRP will directly affect local fibroblasts, endothelial, and articular cartilage cells resulting in cell proliferation, angiogenesis, synthesis of collagens types I and II, extracellular matrix formation, migration, and facilitated recruitment of cells including mesenchymal stromal cells and macrophages ( Fig. 5.1 ). This will often culminate in decreased inflammation and pain restoring joint homeostasis.

Growth factor signaling pathways have both positive and negative feedback mechanisms that are critical for regulating their activity. Ex vivo studies on several tissues of the musculoskeletal system suggest that a negative feedback loop also exists for PRP where there is a plateau effect of matrix synthesis occurring beyond which further increased platelet concentration is of no further benefit. The optimal log dose response curve is not yet known, but it is unlikely that repeated administration of PRP into a joint will have a cumulative beneficial effect on joint homeostasis.

Leukocyte subtypes are commonly grouped together when defining PRP and have been negatively correlated with in vitro and in vivo results. However, the preponderance of evidence indicates that it is the neutrophils that impart inflammatory and catabolic characteristics to a PRP preparation. Neutrophil concentrations have been shown to correlate with catabolic cytokines interleukin-1 and matrix metalloproteinase-9. In contrast, monocytes appear to be beneficial in a PRP preparation. Peripheral blood monocytes are divided into three subsets based on phenotype and function with the heterogeneity and phenotypic plasticity of these cells having implications in several human diseases. Although monocyte subsets have not been studied in relation to PRP, monocyte-enriched PRP can be generated by two commercial systems: Arthrex Angel System (Arthrex Inc., Naples, FL) and EmCyte PurePRP (EmCyte Corp., Fort Myers, FL). In both of these commercial systems, within the leukocyte population monocytes are enriched and neutrophils are relatively decreased in concentration. Further basic and clinical research will guide development of commercial systems with the ability to generate personalized PRP depending on donor and patient variabilities.

Identifying patient- and donor-related factors associated with successful PRP treatment are probably as important as defining and standardizing PRP formulations for achieving predictable outcomes. Differences in clinical response have been attributed to patient donor factors and disease state of the patient ( Table 5.1 ). More recently, a focus on the physiology of the donor has identified several variables that negatively affect the quality of resultant PRP independent of the disease state targeted for therapy. Growth factor concentrations in PRP have been reported to be negatively affected by daily low-dose aspirin, naproxen, antiplatelet drugs, diabetes mellitus, age, and female compared with male gender. Increased age has been associated with an increase in the pro-inflammatory cytokines interleukin-1β and tumor-necrosis-factor alpha, and in another study, age, but not gender, was negatively correlated with growth factors in PRP. In contrast, platelet and TGF-β concentrations in PRP were significantly increased after a burst of high-intensity exercise but neither RBC nor WBC concentrations were affected. There are other, not yet identified, donor-related factors that can alter the biological effect of PRP. One study reported that PRP generated from older patients with knee osteoarthritis suppressed chondrocyte matrix synthesis, and promoted macrophage inflammation in vitro independent of the concentration of inflammatory mediators interleukin-1β and tumor-necrosis-factor alpha, or the growth factors insulin-like growth factor 1 or transforming growth factor-β1. This might suggest that autologous PRP from patients with OA could accentuate the disease process, but it should be cautioned that the OA patients in this study had end-stage OA and the PRP was generated at the time of knee arthroplasty, which might not reflect the clinical scenario.

Although there is sufficient evidence that PRP is an effective treatment for OA, to improve the ability of PRP to restore joint homeostasis, basic science and preclinical studies need to continue to go beyond measuring bulk cellular contents and common growth factors in PRP. Further refinement of donor factors including monocyte subtypes and other donor factors reflecting the physiology of the patient need to be identified so that the quality of a PRP preparation can be defined prior to use in a patient.