Lewis S. Hanna

Platelet-rich plasma increases matrix metalloproteinases in cultures of human synovial fibroblasts.

BACKGROUND The effect of platelet-rich plasma on chondrocytes has been studied in cell and tissue culture. Less attention has been given to the effect of platelet-rich plasma on nonchondrocytic cell lineages within synovial joints, such as fibroblast-like synoviocytes, which produce cytokines and matrix metalloproteinases (MMPs) that mediate cartilage catabolism. The purpose of the present study was to determine the effect of platelet-rich plasma on cytokines and proteases produced by fibroblast-like synoviocytes. METHODS Platelet-rich plasma and platelet-poor plasma from harvested autologous blood were prepared with a commercially available system. Fibroblast-like synoviocytes were treated with platelet-rich plasma, platelet-poor plasma, recombinant PDGFββ (platelet-derived growth factor ββ), or phosphate-buffered saline solution and incubated at 37°C for forty-eight hours. The concentrations of IL-1β (interleukin-1β), IL-1RA (IL-1 receptor antagonist), IL-6, IFN-γ (interferon-γ), IP-10 (interferon gamma-induced protein 10), MCP-1 (monocyte chemotactic protein-1), MIP-1β (macrophage inflammatory protein-1β), PDGFββ, RANTES, TNF-α (tumor necrosis factor-α), VEGF (vascular endothelial growth factor), MMP-1, MMP-3, and MMP-9 in the culture medium were determined by multiplex immunoassay. RESULTS Platelet-rich plasma cultured in medium contained multiple catabolic mediators in substantial concentrations, including MMP-9 (15.8 ± 2.3 ng/mL) and MMP-1 (2.5 ± 0.8 ng/mL), as well as proinflammatory mediators IL-1β, IL-6, IFN-γ, IP-10, MCP-1, MIP-1β, RANTES, and TNF-α in concentrations between 20 pg/mL and 20 ng/mL. Platelet-poor plasma contained significantly lower concentrations of these compounds. Platelet-rich plasma was used to treat human fibroblast-like synoviocytes, and the resulting concentrations of mediators were corrected for the concentrations in the platelet-rich plasma alone. Compared with untreated fibroblast-like synoviocytes, synoviocytes treated with platelet-rich plasma exhibited significantly greater levels of MMP-1 (363 ± 94.0 ng/mL, p = 0.018) and MMP-3 (278 ± 90.0 ng/mL, p = 0.018). In contrast, platelet-poor plasma had little effect on mediators secreted by the synoviocytes. PDGFββ-treated fibroblast-like synoviocytes exhibited a broad proinflammatory cytokine response at four and forty-eight hours. CONCLUSIONS Platelet-rich plasma was shown to contain a mixture of anabolic and catabolic mediators. Synoviocytes treated with platelet-rich plasma responded with substantial MMP secretion, which may increase cartilage catabolism. Synoviocytes responded to PDGF with a substantial proinflammatory response.

Outcome of Lumbar Epidural Steroid Injection Is Predicted By Assay of a Complex of Fibronectin and Aggrecan From Epidural Lavage

Study Design. A single-center, prospective, consecutive case series of patients undergoing epidural lavage before the treatment of radiculopathy due to lumbar disc herniation. Objective. To determine whether a novel complex of fibronectin and aggrecan predicts clinical response to epidural steroid injection (ESI) for the indication of radiculopathy from lumbar herniated nucleus pulposus (HNP). Summary of Background Data. ESI for lumbar radiculopathy due to HNP is widely used despite variable effectiveness for this indication. With increased attention aimed at cost containment, it would be beneficial to identify those in whom ESI may be helpful. There are currently no accurate diagnostic tests to predict response to ESI in back pain and sciatica syndromes. We have previously investigated biomarkers of disc degeneration associated with radiculopathy. Methods. We embarked to determine whether a molecular complex of fibronectin and aggrecan predicts clinical response to ESI for the indication of radiculopathy from HNP. This prospective study was conducted at a single center and included 26 patients with radiculopathic pain and magnetic resonance imaging positive for HNP, who elected ESI. Epidural lavage with physiologic saline was performed immediately before ESI. The lavage fluid was assayed for the fibronectin–aggrecan complex (FAC) by using a heterogeneous sandwich enzyme-linked immunosorbent assay. The results were compared with the interval improvement in the physical component summary (PCS) score of the Medical Outcomes Study Short Form-36 instrument (SF-36) after injection compared with baseline. Results. The mean improvement from baseline PCS in patients with the FAC was 22.9 (SD, 12.4) and without the complex was 0.64 (SD, 3.97; P < 0.001). Differences in total SF-36 improvement were also highly significant (P < 0.001). The presence of the FAC predicts a clinically significant increase in PCS after lumbar ESI by receiver-operating-characteristic analysis (area under the curve = 0.97; P < 0.001). There was no significant difference in age (P = 0.25), sex (P = 0.84), laterality (P = 0.06), lumbar spinal level (P = 0.75), or payer type (workers compensation vs. private insurance; P = 0.90) between groups with and without the marker. Conclusion. A molecular complex of fibronectin and aggrecan predicts response to lumbar ESI for radiculopathy with HNP. The biomarker is accurate, objective, and not affected by demographic or psychosocial variables in this series.

Identification of a complex between fibronectin and aggrecan G3 domain in synovial fluid of patients with painful meniscal pathology

OBJECTIVES We previously described a panel of four cytokines biomarkers in knee synovial fluid for acute knee pain associated with meniscal pathology. The cytokine biomarkers included interferon gamma (IFN-gamma), interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1beta). Validation studies using other immunologic techniques confirmed the presence of IL-6, MCP-1 and MIP-1beta, but not IFN-gamma. Therefore we sought the identity of the IFN-gamma signal in synovial fluid. METHODS Knee synovial fluid was collected from patients with an acute, painful meniscal injury, as well as asymptomatic volunteers. A combination of high-pressure chromatography, mass spectrometry and immunological techniques were used to enrich and identify the protein components representing the IFN-gamma signal. RESULTS A protein complex of fibronectin and the aggrecan G3 domain was identified in the synovial fluid of patients with a meniscal tear and pain that was absent in asymptomatic controls. This protein complex correlated to the IFN-gamma signal. A novel enzyme-linked immunosorbent assay (ELISA) was developed to specifically identify the complex in synovial fluid. CONCLUSIONS We have identified a protein complex of fibronectin and aggrecan G3 domain that is a candidate biomarker for pain associated with meniscal injury.

Identification of a novel fibronectin-aggrecan complex in the synovial fluid of knees with painful meniscal injury.

BACKGROUND Molecular biomarkers associated with knee pain may be useful as diagnostic modalities, prognostic indicators, and surrogate end points for therapeutic trials. The present study describes a novel complex of fibronectin and aggrecan that is present in the affected knee of patients with pain and meniscal abnormality. METHODS The present prospective study included thirty patients with knee pain, mechanical symptoms, and magnetic resonance imaging findings that were positive for a meniscal tear who chose arthroscopic partial meniscectomy after unsuccessful nonoperative management. Synovial fluid was aspirated at the time of surgery and was assayed for the fibronectin-aggrecan complex with use of a heterogeneous enzyme-linked immunosorbent assay (ELISA). The results were compared with knee aspirates from ten asymptomatic volunteers with no pain who underwent magnetic resonance imaging of the knee. RESULTS The mean optical density (and standard deviation) of the fibronectin-aggrecan complex was significantly greater in synovial fluid from knees undergoing arthroscopic surgery as compared with fluid from asymptomatic controls (13.29 ± 8.48 compared with 0.03 ± 0.09; p < 0.001). The mean age in the study group was significantly greater than in control group (46.0 ± 12.6 compared with 38.5 ± 6.0 years; p = 0.02), but controlling for age did not affect the results. Post hoc, an optical density cutoff value of 0.3 distinguished the study group from the control group with 100% accuracy. CONCLUSIONS A novel fibronectin-aggrecan complex is present in the synovial fluid of painful knees with meniscal abnormality. The fibronectin-aggrecan complex may prove to be useful as a clinical biomarker or therapeutic target. Further research is warranted to correlate functional outcome after surgery with the fibronectin-aggrecan complex and other cartilage biomarkers.

Correlation of intra-articular ankle pathology with cytokine biomarkers and matrix degradation products.

Background: Articular cartilage degeneration is mediated by inflammatory cytokines and fragments of structural matrix proteins. Few studies have examined the role of these biomarkers in intra-articular pathology of the ankle. Methods: Four groups of patients with increasing ankle pathology were enrolled. Group 1 included controls with no pain who underwent unrelated forefoot surgery. Group 2 included patients undergoing arthroscopy with intraoperative mild chondrosis. Group 3 included patients undergoing arthroscopy with moderate/severe chondrosis, osteochondral lesions, impingement, or loose bodies. Group 4 included positive controls with severe arthrosis undergoing ankle arthrodesis/arthroplasty. Ankle fluid was obtained by intra-articular aspiration and was assayed for IL-6, IFN-γ, MCP, MIP-1β, and fibronectin-aggrecan complex (FAC), a matrix-degradation marker. There were 36 patients total, 21 males and 15 females with a mean age 45 (±16; range 18 to 76) years and a mean VAS for pain of 4.7 (±3.5; range 0 to 9). In groups 1 through 4, there were 11, 6, 15 and 4 patients respectively. Results: The mean values of MCP-1 were 49.8 (±8.0) for minimal pathology and 133.9 (±33.0) for substantial pathology (pg/ml). The mean values of the FAC were 2.83 (±1.16) for minimal pathology and 9.62 (±2.23) for substantial pathology (optical density at 450 nm). The groups differed significantly in age, preoperative VAS, FAC, IL-6, and MCP-1 (p < 0.05). Conclusion: There are differences in FAC and MCP-1 with increasing grades of severity of intra-articular pathology. Clinical Relevance: These tests may play a role in determining the necessity for arthroscopy or intra-articular procedures in equivocal candidates. Level of Evidence: II

Does a fibronectin and aggrecan complex play a role in painful vertebral disks

To determine the presence of a fibronectin‐aggrecan complex (FAC) in the disk space of persons with chronic low back pain as relates to provocative diskography.

Poster 10 Is There a Chondroprotective Effect of Autologous Platelet Integrated Concentrate (APIC) on an Osteoarthritis (OA) Rabbit Model? A Pilot Study

exam, was otherwise normal. He was subsequently sent for radiographs of the pelvis and bilateral (B/L) hips which revealed advanced degenerative joint disease (DJD). On AP view, radiographic measurements of the neck shaft angles were found to be greater than 142°, while lateral radiographs showed an anteverted neck in relation to the femoral shaft. Setting: Outpatient physiatry office. Results or Clinical Course: The radiographic findings, in combination with clinical presentation, helped confirm the diagnosis of B/L pistol grip hip deformity (PGD). Such deformity led to severe progressive DJD, which limited both ROM and the performance of activities of daily living. As a result, the patient underwent B/L total hip arthroplasty (THA) without complication. Discussion: PGD is a pre-arthritic condition with a greater prevalence in adults who are younger than 50 years old and are very physically active. Such patients presenting with groin pain and decreased hip ROM should be evaluated for PGD. The triad of Cam type anterior femoral acetabular impingement, DJD, and a femoral neck-shaft angle 140° or 115° with radiographic evidence of an abnormally shaped femoral head is suggestive of PGD. Significant benefit may arise from early recognition and orthopedic evaluation in terms of diagnosis and treatment. Conclusions: PGD left untreated is progressive and ultimately leads to THA. With early recognition, there is potential for successful treatment via arthroscopy. It is therefore imperative that the rehabilitation physician be familiar with this condition so that such a condition does not go unnoticed.

Are Persistently Symptomatic Vertebral Compression Fractures Associated With Abnormal Inflammatory Profiles? A Prospective Study

Study Design A case-control study with prospectively collected samples for laboratory analysis in a series of patients with spinal fragility fractures and a series of patients without fracture who underwent fusion for LBP. Objective Was an exploratory data analysis for candidate cytokine biomarkers present in the fracture milieu of patients with persistent back pain associated with vertebral compression fracture. Summary of Background Data Lumbar and thoracic compression fractures are common. Little is known about the presence of inflammatory mediators within fractured vertebra in the clinical setting. Methods Thirty patients diagnosed with a single thoracic or lumbar compression fracture were treated with single level vertebroplasty. At the time of intervention, needle aspiration was carried out at the fractured level. A multiplexed bead assay was used to assess the presence of 27 different cytokines and inflammatory mediators. A control group consisted of needle aspiration samples of 30 lumbar vertebra from 13 patients with chronic pain but no fracture undergoing open instrumented fusion. Results Thirty patients with 30 fractures consisted of 23 female and 7 male with a mean age of 77.5 years (SD 13.6; range 42 to 97) and a mean of 3.9 weeks of pain (SD 3.1; range 1 to 12). The highest levels of inflammatory mediators were (in order): IL-1 receptor antagonist, PDGF, RANTES, IP-10, IL-8, and eotaxin. These mediators were present at concentrations>200 pg/mL. Compared with controls with chronic pain, significant differences were present for 4 mediators: TNF, MIP-1b, IL-9, and IL-12. The panel of these 4 markers was 93.3% specific and 66.7% sensitive for fracture compared with the control group. Conclusions Inflammatory mediators are present in needle aspirates of symptomatic vertebral compression fractures. Some of these mediators show different levels than in patients with chronic pain but no fracture. Level of Evidence Diagnostic level of evidence II.

Alpha-2-Macroglobulin: Protease Inhibitor Treatment (PRP Variant)

Alpha-2-macroglobulin (A2M) is a major plasma glycoprotein best known for its ability to inhibit a broad spectrum of inflammatory mediators, such as metalloproteases and inflammatory cytokines by a unique “bait-and-trap” method. A2M has emerged as a unique potential treatment for cartilage-based pathology and inflammatory arthritides. A2M can be concentrated from an autologous source and injected into diseased tissue to enhance healing, prevent further degradation, and protect normal tissue. A2M not only inhibits the associated inflammatory cascade, but also disrupts the catabolic process of cartilage degeneration. Any pathology that is protease-mediated may benefit from A2M therapy. The half-life is quite long, so successful treatment can result in months of pain relief. Autologous concentrated A2M from plasma is currently used successfully to treat various painful arthritides, including mild to moderate osteoarthritis, post-traumatic osteoarthritis, enthesopathies, and spinal discogenic pain. Recent work has shown that recombinant A2M may be able to enhance cartilage regeneration. The discovery of A2M as the body’s own healing mechanism, with anti-inflammatory and disease-modifying potential, offers great promise.

Poster 13 Can Cartilage Degradation be Prevented by Platelet Rich Plasma (PRP) Preparations on Bovine Cartilage Explants

nitisinone. Design: Prospective randomized treatment study. Setting: Biomedical research facility. Participants: 40 subjects with alkaptonuria (27 male and 13 female). Baseline 38 years old to 65 years old (mean age 51). Interventions: Nitisinone (IND 71780). Main Outcome Measures: Human activity profile (HAP), a measure of physical activity, SF-36, Health Assessment Questionnaire (HAQ), a measure of ADL and health status, Pain Disability Index (PDI), Wisconsin Brief Pain questionnaire (BP), the fatigue assessment instrument (FAI). Results: On the HAP adjusted activity score (AAS) over 3 years, the treatment group (TG) had 3 fewer subjects in the impaired category, 1 more subject in the moderately active group, and gained 2 subjects in the active group. The control group (CG) had 1 more subject in the impaired category, 1 fewer subject in the moderately active group, and no change in the active group. In controlling for the subjects’ homogentisic acid, the TG significantly increased its AAS and HAP maximum activity score (MAS) in the 3rd year (P .040 and P .032, respectively). In the TG, the AAS increased from 56.50-61.61 and the CG decreased from 64.06-63.87. In the TG, the average MAS increased from 71.22 to 73.11 and the CG average decreased from 78.44 to 76.19. On the HAQ, the TG had 2 more subjects with no functional limitations and 2 fewer with moderate limitations; the CG had 3 more with mild limitations and 3 fewer with moderated limitations. The TG and CG are associated with increased SF-36 physical composite score (PCS) (P .045 and P .043, respectively). The CG is associated with increased SF-36 mental composite score (MCS) (P .009). In the 1st year, the TG is associated with lower BP right now pain level (BPRN) (P .015) then at 3 years, both groups have lower BPRN (P .015 (TG) and P .026 (CG)). Conclusions: In a 3-year follow up of this alkaptonuria cohort, the TG is associated with an increase in maximum and average activity level. However, the TG and the CG have improvement in physical disability. Both groups have a lower pain level at 3 years but the TG had a significant earlier reduction in pain. Further long-term treatment studies are warranted to determine if nitisinone treatment is associated with improvement in activity, pain, and disability over time.