Slides were prepared from these spinal tissue samples as well as positive and negative control tissues. Muscle tissue from the spinal column at the operative level was collected and used for a quantitative analysis of various inflammatory markers using immunohistochemistry techniques. At the time of sacrifice index level functional spinal units (FSU), major organs, lymph nodes, and muscle tissues were collected.
Skeletally mature Montadale sheep were implanted with PEEK and PEEK zeolite interbody cages at the C2-C3 and C4-C5 index levels and survived to 12 or 26 weeks, as approved by the IACUC Committee. SYBR Green gene expression assays were used to determine the relative expression levels of: gapdh, inos, tnf-α, arg1, fizz1, il1β, il6, and gapdh. Reverse transcription of 500 ng of RNA to cDNA were performed via a high-capacity reverse transcriptase kit according to the manufacturer's instructions. Isolated RNA concentration was subsequently determined using a NanoDrop spectrophotometer. RNA were isolated from 8 × 106 cells using the RNeasy Mini Kit according to the manufacturer's instructions. After the incubation period at 37☌, cells were washed with sterile PBS and fixed for 30 minutes with 2% paraformaldehyde (PFA) for immunolabeling, or harvested with TRIzol lysis reagent for RNA assessment, respectively. For the cytokine challenge study, cells were exposed for 6 hours to 20 ng/ml IFN-γ and 100 ng/mL LPS, washed and then placed in 10% FBS 1% P/S DMEM for 24 hours. Mature macrophages were exposed to the following treatments for 24 hours: complete media (M0 control), 20 ng/ml IFN-γ and 100 ng/mL lipopolysaccharide LPS (M1 control), 20 ng/mL interleukin IL-4 (M2 control). For cell-based study, the presence of cytokines was noted as comparable, increased, or slightly increased compared to macrophages of the M0 phenotype. The levels of inflammatory cytokines interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) were quantified in muscle tissue directly anterior to the index level based on the intensity of fluorescence in each slide. The large animal model included two instrumented levels with one level implanted with a PEEK interbody cervical cage and the alternate level implanted with a PEEK zeolite composite interbody device.
In vitro cell culture study in combination with a large animal functional cervical spine model. Similarly, the immune response measured at the local sites in a large animal study was also studied in order to determine the inflammatory response at the local implanted site. The objective of this study was to compare the activation state of macrophages, as determined by selected gene and protein expression profiles, following in vitro exposure to PEEK zeolite and PEEK based spinal interbody devices. It was hypothesized that IL-1β, IL-6, and TNF-α cytokine expression levels results would be different for PEEK and a novel zeolite filled PEEK composite (ZFuze, DiFusion Technologies). The combination of results from three separate kinds of studies would potentially weave a better understanding of PEEK fibrous encapsulation around retrieved implanted PEEK devices. The cell studies in combination with larger animal studies help understand the importance of appropriate choice in materials. Studies involving macrophages facilitate the understanding behind the mechanism of fibrous capsule formation anecdotally reported with PEEK implants.
Based on the current understanding of the immune system, both the innate and adaptive components, a combination of tests on current design materials in spinal implant devices may help understand potential clinical complications and thus reduce the potential adverse events associated with specific design materials. Osteoimmunology, the study of the relationship between the musculoskeletal system and immune system, has emerged as an important consideration in biomaterial research.
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