Matrix metalloproteinase content and activity in low-platelet, low-leukocyte and high-platelet, high-leukocyte platelet rich plasma (PRP) and the biologic response to PRP by human ligament fibroblasts.

BACKGROUND: Recent work has shown the presence of catabolic cytokines in platelet-rich plasma (PRP), but little is known about endogenous catabolic proteases such as matrix metalloproteinases (MMPs). Hypothesis/ PURPOSE: To quantify MMP content in 2 commercially available PRP preparation systems: Arthrex Double Syringe System autologous conditioned plasma (ACP) and Biomet GPS (GPS). The hypothesis was that MMPs are actively secreted from PRP immediately after preparation. STUDY DESIGN: Controlled laboratory study. METHODS: PRP was prepared using either ACP (low platelet, low leukocyte) or GPS (high platelet, high leukocyte). MMP-2, MMP-3, and MMP-9 concentrations were measured using multiplex enzyme-linked immunosorbent assays for up to 6 days in 2 donors, and MMP activity was measured in 3 donors using kinetic activity kits able to detect the enzymatic cleavage of a fluorogenic peptide. Human ligament fibroblasts were cultured and exposed to both ACP and GPS from 1 donor each. MMP-2, -3, and -9 concentrations were assayed in culture media at 24 and 48 hours after exposure. RESULTS: GPS exhibited higher total MMP-2, -3, and -9 concentrations for up to 144 hours of release, while ACP had higher platelet-normalized MMP-2 and MMP-3 concentrations. GPS had significantly higher total and endogenous MMP-2 activity (P = .004 and .014, respectively), MMP-3 activity (P = .020 and .015, respectively), and MMP-9 activity (P = .004 and .002, respectively) compared with ACP. Once normalized to platelet count, differences in MMP activity were not significant between ACP and GPS. Compared with controls, cells stimulated with interleukin-1 beta (IL-1ß) and treated with ACP showed significantly higher fold changes of MMP-2 (P = .001) and MMP-3 (P = .003) concentrations at 24 hours than did cells treated with GPS. Total MMP-9 content was higher in the media of GPS-treated, IL-1ß-stimulated cells compared with ACP-treated cells (P = .001). At 48 hours, IL-1ß-stimulated cells treated with GPS exhibited higher fold changes of MMP-2 concentration (P = .002) compared with controls, but no difference in MMP-3 concentration was found. At 48 hours, there was a significantly higher concentration of MMP-9 in the cell culture media of ACP-treated cells compared with GPS-treated cells (P = .003). CONCLUSION: PRP prepared as both ACP and GPS contains MMP-2, -3, and -9, which is released over a period of at least 6 days. Furthermore, a large proportion of these MMPs are in their active form, and MMP activity is dependent on platelet count within the PRP preparation. Once exposed to ligament fibroblasts, both ACP and GPS cause the fibroblasts to release MMPs, most notably 24 hours after PRP exposure, and this release is dependent on prior IL-1ß stimulation. CLINICAL RELEVANCE: The results of this study demonstrate that PRP therapy delivers ng/mL-range concentrations of catabolic proteases, which could perpetuate inflammation and inhibit tissue healing.

The GraftLink ulnar collateral ligament reconstruction: biomechanical comparison with the docking technique in both kinematics and failure tests.

BACKGROUND: Ulnar collateral ligament (UCL) reconstruction aims to restore valgus stability, and numerous techniques have been described in the literature. HYPOTHESIS/ PURPOSE: To biomechanically compare the GraftLink (GL) technique with traditional bone tunnels used in the docking (DO) technique. It is hypothesized that the GL method will offer a stiffer, less lax construct compared with the DO. STUDY DESIGN: Controlled laboratory study. METHODS: Native and reconstructed states were tested in 7 matched pairs of cadaveric arms. To test kinematics, a 1.5-N·m valgus torque was applied and the resultant displacement at 15° to 90° of flexion was measured. Dissipated energy and the torque at the peak of the 10th cycle of preconditioning were analyzed during kinematic tests. Failure testing was performed by internal rotation of the humerus at 4.5 deg/s in 70° of flexion. Ulnotrochlear joint (UTJ) gapping was quantified during failure tests by use of video tracking. RESULTS: Kinematics testing revealed no differences between the native state and the reconstructed state in either the DO or the GL group at any flexion angle. Stiffness was lower in the reconstructed specimens in both the DO (39.92 N·m/rad) and GL (50.74 N·m/rad) groups compared with their matched native states (DO Native, 71.41 N·m/rad, P = .005; GL Native, 86.36 N·m/rad, P = .002). There was no difference in stiffness between DO and GL. Reconstructed specimens in the GL group had lower torque at failure compared with native specimens (17.404 N·m vs 24.63 N·m, P = .038), but there was no difference in the DO group at failure. There was no difference in torque at failure between DO and GL. The DO exhibited higher angular displacement at failure compared with the native state (34.21° vs 21.79°, P = .010) and compared with the GL when normalized (1.58-fold vs 1.19-fold, P = .039). Compared with their native states, both DO and GL had significantly higher UTJ gapping at 3 N·m and at failure. The DO had significantly higher normalized UTJ gapping than the GL at 3 N·m (P = .037) and at failure (P = .043). CONCLUSION: The DO and GL both restored joint kinematics under low loading conditions. Although less stiff, the GL exhibited lower joint gapping and laxity than did the DO. CLINICAL RELEVANCE: Understanding the biomechanics of UCL reconstruction has significant implications for postoperative management as it relates to early rehabilitation. Biomechanically inferior constructs could risk graft failure or early loosening during rehabilitation, and comparing the biomechanics of new techniques to established, widely used procedures such as the docking technique can provide important information about the immediate postoperative performance.

In vitro response of human chondrocytes to a combination of growth factors and a proteinase inhibitor.

Degenerative disk disease is an accelerating cascade of tissue degeneration in the intervertebral disk. A harsh catabolic environment perpetuates the degeneration of the intervertebral disk. Tissue engineering-based techniques offer effective treatment to slow the progression of degenerative disk disease and regenerate intervertebral disk tissue. The purpose of this study was to assess the efficacy of a regenerative therapy for degenerative disk disease by treating human chondrocytes with anabolic growth factors and a proteinase inhibitor. The use of both proved effective in upregulating important extracellular matrix markers of human chondrocytes. These successful in vitro results have implications for the regeneration of the intervertebral disk.

Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation.

1 Transient accumulation of extracellular ATP reflects both release of ATP from intracellular stores and altered rates of ATP metabolism by ecto-enzymes. Ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) and ecto-nucleotide pyrophosphatases (eNPPs) degrade ATP, while ecto-nucleotide diphosphokinases (eNDPKs) synthesize ATP from ambient ADP. 2 Although the methylene ATP analogs betagamma-meATP and alphabeta-meATP are widely used as metabolically stable tools for the analysis of purinergic signaling, their specific effects on eNTPDase, eNPP, and eNDPK activities have not been defined. This study compared the actions of these analogs on extracellular ATP metabolism by human 1321N1 astrocytes, rat PC12 pheochomocytoma cells, and rat C6 glioma cells. 3 Both analogs significantly reduced clearance of extracellular ATP by 1321N1 cells that express both eNTPDases and eNPPs, as well as by C6 cells that exclusively express eNPPs. In contrast, both analogs were much less efficacious in inhibiting ATP clearance by PC12 cells that predominantly express eNTPDases. Betagamma-meATP, but not alphabeta-meATP, was effectively hydrolyzed by the 1321N1 and C6 cells; PC12 cells did not significantly degrade this analog. 4 Alphabeta-meATP, but not betagamma-meATP, acted as a substrate for purified yeast NDPK to generate ATP via trans-phosphorylation of ADP. alphabeta-meATP also acted as substrate for the eNDPK activities expressed by 1321N1, PC12, and C6 cells and thereby induced extracellular ATP accumulation in the presence of ambient or exogenously added ADP. 5 These results indicate that methylene ATP analogs exert complex and cell-specific effects on extracellular ATP metabolism that can significantly modify interpretation of studies that use these reagents as probes of purinergic signal transduction in intact tissues.