Fibrin-genipin annulus fibrosus sealant as a delivery system for anti-TNFα drug.

BACKGROUND CONTEXT: Intervertebral discs (IVDs) are attractive targets for local drug delivery because they are avascular structures with limited transport. Painful IVDs are in a chronic inflammatory state. Although anti-inflammatories show poor performance in clinical trials, their efficacy treating IVD cells suggests that sustained, local drug delivery directly to painful IVDs may be beneficial. PURPOSE: The purpose of this study was to determine if genipin cross-linked fibrin (FibGen) with collagen Type I hollow spheres (CHS) can serve as a drug-delivery carrier for infliximab, the anti-tumor necrosis factor α (TNFα) drug. Infliximab was chosen as a model drug because of the known role of TNFα in increasing downstream production of several pro-inflammatory cytokines and pain mediators. Genipin cross-linked fibrin was used as drug carrier because it is adhesive, injectable, and slowly degrading hydrogel with the potential to seal annulus fibrosus (AF) defects. CHS allow simple and nondamaging drug loading and could act as a drug reservoir to improve sustained delivery. STUDY DESIGN/SETTING: This is a study of biomaterials and human AF cell culture to determine drug release kinetics and efficacy. METHODS: Infliximab was delivered at low and high concentrations using FibGen with and without CHS. Gels were analyzed for structure, drug release kinetics, and efficacy treating human AF cells after release. RESULTS: Fibrin showed rapid infliximab drug release but degraded quickly. CHS alone showed a sustained release profile, but the small spheres may not remain in a degenerated IVD with fissures. Genipin cross-linked fibrin showed steady and low levels of infliximab release that was increased when loaded with higher drug concentrations. Infliximab was bound in CHS when delivered within FibGen and was only released after enzymatic degradation. The infliximab released over 20 days retained its bioactivity as confirmed by the sustained reduction of interleukin (IL)-1ß, IL-6, IL-8, and TNFα concentrations produced by AF cells. CONCLUSIONS: Direct mixing of infliximab into FibGen was the simplest drug-loading protocol capable of sustained release. Results show feasibility of using drug-loaded FibGen for delivery of infliximab and, in the context with the literature, show potential to seal AF defects and partially restore IVD biomechanics. Future investigations are required to determine if drug-loaded FibGen can effectively deliver drugs, seal AF defects, and promote IVD repair or prevent further IVD degeneration in vivo.

Inflammatory Kinetics and Efficacy of Anti-inflammatory Treatments on Human Nucleus Pulposus Cells.

STUDY DESIGN: Human nucleus pulposus (NP) cell culture study investigating response to tumor necrosis factor-α (TNFα), effectiveness of clinically available anti-inflammatory drugs, and interactions between proinflammatory cytokines. OBJECTIVE: To characterize the kinetic response of proinflammatory cytokines released by human NP cells to TNFα stimulation and the effectiveness of multiple anti-inflammatories with 3 substudies: Timecourse, Same-time blocking, Delayed blocking. SUMMARY OF BACKGROUND DATA: Chronic inflammation is a key component of painful intervertebral disc degeneration. Improved efficacy of anti-inflammatories requires better understanding of how quickly NP cells produce proinflammatory cytokines and which proinflammatory mediators are most therapeutically advantageous to target. METHODS: Degenerated human NP cells (n = 10) were cultured in alginate with or without TNFα (10 ng/mL). Cells were incubated with 1 of 4 anti-inflammatories (anti-IL-6 receptor/atlizumab, IL-1 receptor anatagonist, anti-TNFα/infliximab and sodium pentosan polysulfate/PPS) in 2 blocking-studies designed to determine how intervention timing influences drug efficacy. Cell viability, protein, and gene expression for IL-1ß, IL-6, and IL-8 were assessed. RESULTS: Timecourse: TNFα substantially increased the amount of IL-6, IL-8, and IL-1ß, with IL-1ß and IL-8 reaching equilibrium within ∼72 hours (IL-1ß: 111 ± 40 pg/mL, IL-8: 8478 ± 957 pg/mL), and IL-6 not reaching steady state after 144 hours (1570 ± 435 pg/mL). Anti-TNFα treatment was most effective at reducing the expression of all cytokines measured when added at the same time as TNFα stimulation. Similar trends were observed when drugs were added 72 hours after TNFα stimulation, however, no anti-inflammatories significantly reduced cytokine levels compared with TNF control. CONCLUSION: IL-1ß, IL-6, and IL-8 were expressed at different rates and magnitudes suggesting different roles for these cytokines in disease. Autocrine signaling of IL-6 or IL-1ß did not contribute to the expression of any proinflammatory cytokines measured in this study. Anti-inflammatory treatments were most effective when applied early in the inflammatory process, when targeting the source of the inflammation. LEVEL OF EVIDENCE: N/A.

TNFα transport induced by dynamic loading alters biomechanics of intact intervertebral discs.

OBJECTIVE: Intervertebral disc (IVD) degeneration is an important contributor to the development of back pain, and a key factor relating pain and degeneration are the presence of pro-inflammatory cytokines and IVD motion. There is surprisingly limited understanding of how mechanics and inflammation interact in the IVD. This study investigated interactions between mechanical loading and pro-inflammatory cytokines in a large animal organ culture model to address fundamental questions regarding (i.) how inflammatory mediators arise within the IVD, (ii.) how long inflammatory mediators persist, and (iii.) how inflammatory mediators influence IVD biomechanics. METHODS: Bovine caudal IVDs were cultured for 6 or 20-days under static & dynamic loading with or without exogenous TNFα in the culture medium, simulating a consequence of inflammation of the surrounding spinal tissues. TNFα transport within the IVD was assessed via immunohistochemistry. Changes in IVD structural integrity (dimensions, histology & aggrecan degradation), biomechanical behavior (Creep, Recovery & Dynamic stiffness) and pro-inflammatory cytokines in the culture medium (ELISA) were assessed. RESULTS: TNFα was able to penetrate intact IVDs when subjected to dynamic loading but not static loading. Once transported within the IVD, pro-inflammatory mediators persisted for 4-8 days after TNFα removal. TNFα exposure induced changes in IVD biomechanics (reduced diurnal displacements & increased dynamic stiffness). DISCUSSION: This study demonstrated that exposure to TNFα, as might occur from injured surrounding tissues, can penetrate healthy intact IVDs, induce expression of additional pro-inflammatory cytokines and alter IVD mechanical behavior. We conclude that exposure to pro-inflammatory cytokine may be an initiating event in the progression of IVD degeneration in addition to being a consequence of disease.

Integration of statistical modeling and high-content microscopy to systematically investigate cell-substrate interactions.

Cell-substrate interactions are multifaceted, involving the integration of various physical and biochemical signals. The interactions among these microenvironmental factors cannot be facilely elucidated and quantified by conventional experimentation, and necessitate multifactorial strategies. Here we describe an approach that integrates statistical design and analysis of experiments with automated microscopy to systematically investigate the combinatorial effects of substrate-derived stimuli (substrate stiffness and matrix protein concentration) on mesenchymal stem cell (MSC) spreading, proliferation and osteogenic differentiation. C3H10T1/2 cells were grown on type I collagen- or fibronectin-coated polyacrylamide hydrogels with tunable mechanical properties. Experimental conditions, which were defined according to central composite design, consisted of specific permutations of substrate stiffness (3-144 kPa) and adhesion protein concentration (7-520 microg/mL). Spreading area, BrdU incorporation and Runx2 nuclear translocation were quantified using high-content microscopy and modeled as mathematical functions of substrate stiffness and protein concentration. The resulting response surfaces revealed distinct patterns of protein-specific, substrate stiffness-dependent modulation of MSC proliferation and differentiation, demonstrating the advantage of statistical modeling in the detection and description of higher-order cellular responses. In a broader context, this approach can be adapted to study other types of cell-material interactions and can facilitate the efficient screening and optimization of substrate properties for applications involving cell-material interfaces.