Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet-rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF-b1, TGF-b2, EGF, IGF-1, PDGF-AB, PDGF-BB, and VEGF) and antiinflammatory (IL-1ra, sTNF-RI, sTNF-RII, IL-4, IL-10, IL-13, and IFNg) cytokines but low concentrations of catabolic cytokines (IL-1a, IL-1b, TNFa, IL-6, IL-8, IL-17, and IL-18). Human articular chondrocytes were pre-incubated with the antagonists IL-1ra, sTNF-RI, or APS prior to the addition of recombinant human IL-1b or TNFa. Following exposure to inflammatory cytokines, the levels of MMP-13 in the culture medium were evaluated by ELISA. MMP-13 production stimulated in chondrocytes by IL-1b or TNFa was reduced by rhIL-1ra and sTNF-RI to near basal levels. APS was also capable of inhibiting the production of MMP-13 induced by both IL-1b and TNFa. The combination of anabolic and anti-inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. ß

The objective of this clinical study was to test if blood from osteoarthritis (OA) patients (n = 105) could be processed by a device system to form an autologous protein solution (APS) with preferentially increased concentrations of anti-inflammatory cytokines compared to inflammatory cytokines. To address this objective, APS was prepared from patients exhibiting radiographic evidence of knee OA. Patient metrics were collected including: demographic information, medical history, medication records, and Knee Injury and Osteoarthritis Outcome Score (KOOS) surveys. Cytokine and growth factor concentrations in whole blood and APS were measured using enzyme-linked immunosorbent assays. Statistical analyses were used to identify relationships between OA patient metrics and cytokines. The results of this study indicated that anti-inflammatory cytokines were preferentially increased compared to inflammatory cytokines in APS from 98% of OA patients. APS contained high concentrations of anti-inflammatory proteins including 39,000 ± 20,000 pg/ml IL-1ra, 21,000 ± 5,000 pg/ml sIL-1RII, 2,100 ± 570 pg/ml sTNF-RI, and 4,200 ± 1,500 pg/ml sTNF-RII. Analysis of the 82 patient metrics indicated that no single patient metric was strongly correlated (R2 > .7) with the key cytokine concentrations in APS. Therefore, APS can be prepared from a broad range of OA patients.

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro-and antiinflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL-1a-or TNFa-challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL-1a and TNFa. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL-1ra, sTNF-RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin-O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL-1a-induced collagen release over a 21-day culture period. APS treatment reduced the degree of Safranin-O staining loss when cartilage explants were cultured with IL-1a or TNFa. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA. ß

Objective The purpose of this research was to determine if an autologous protein solution (APS), prepared from platelet-rich plasma (PRP), could reduce the deleterious effects of inflammatory cytokines in vitro. Methods APS was prepared by processing human blood in a tuned density buoy separation device (Platelet Separation System, Biomet Biologics, LLC) to produce platelet-rich plasma (PRP) and processing the PRP in a concentration device containing polyacrylimide beads to produce a highly concentrated anti-inflammatory solution. A functional assay was designed using recombinant interleukin (IL)-1b to upregulate IL-8 production by human monocytes. Either recombinant human interleukin-1 receptor antagonist (rhIL-1ra) or APS was added to some samples to determine if a reduced inflammatory response could be identified in vitro. The enzyme-linked immunosorbent assay (ELISA) method was employed to perform cytokine analyses, and Student's t test (a = 0.05) was used for all statistical analyses. Results Both the rhIL-1ra and the APS reduced the effect of IL-1b on human macrophages in vitro. This was measured by the reduced production of IL-8 and tumor necrosis factor (TNF)-a. Further analysis of the supernatants confirmed the presence of high concentrations of IL-1ra and soluble TNF receptor I (sTNF-RI) with the APS treatment. Conclusion The ability of the APS to reduce the effect of IL-1b and limit the expression of other inflammatory cytokines in vitro validates its potential use as an autologous treatment for osteoarthritis.