Abstract
8. The immune response of two separate polymer spinal interbody device materials
The spine journal, v 19(9), pp S4-S5
Sep 2019
Abstract
Osteoimmunology, the study of the relationship between the musculoskeletal system and immune system, has emerged as an important consideration in biomaterial research. 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. Studies involving macrophages facilitate the understanding behind the mechanism of fibrous capsule formation anecdotally reported with PEEK implants. The cell studies in combination with larger animal studies help understand the importance of appropriate choice in materials. 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. 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 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. 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.
In vitro cell culture study in combination with a large animal functional cervical spine model. 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.
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. For cell-based study, the presence of cytokines was noted as comparable, increased, or slightly increased compared to macrophages of the M0 phenotype.
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 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. After the incubation period at 37°C, 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. RNA were isolated from 8 × 106 cells using the RNeasy Mini Kit according to the manufacturer's instructions. Isolated RNA concentration was subsequently determined using a NanoDrop spectrophotometer. Reverse transcription of 500 ng of RNA to cDNA were performed via a high-capacity reverse transcriptase kit according to the manufacturer's instructions. SYBR Green gene expression assays were used to determine the relative expression levels of: gapdh, inos, tnf-α, arg1, fizz1, il1β, il6, and gapdh. 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. At the time of sacrifice index level functional spinal units (FSU), major organs, lymph nodes, and muscle tissues were collected. 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. Slides were prepared from these spinal tissue samples as well as positive and negative control tissues. Lymph nodes and muscle tissues distant from the surgical site were collected during necropsy and served as the positive and negative controls respectively.
In vivo expression was normalized to represent a percentage of intensity measured in the negative control. Independent T-test was performed to compare cytokine expression at both 12 and 26 week time points. Expression of IL1-β was found to be significantly lower in PEEK zeolite samples at 12 weeks (M=0.23, SD=0.28) than standard PEEK samples at 12 weeks (M=1.37, SD=0.49) t(8)=-4.582, p=0.002. PEEK zeolite composite samples were also found to have significantly lower IL1-β expression at 26 weeks (M=0.02, SD=0.009) when compared to standard PEEK samples (M=0.60, SD=0.23) t(11)=-6.667, p<0.001. While there was no significant difference between expression of IL-6 or TNF-α, it should be noted that PEEK zeolite composite samples trended lower in all cases. These results were corroborated by the in vitro cell studies. IL-1 expression was greater in macrophages exposed to PEEK test articles compared to ZFuse. Similarly, expression of TNF-α was twofold greater with macrophages exposed to PEEK samples rather than ZFuse. Results of qPCR show that ZFuse samples promote greater Fizz1 expression relative to PEEK samples, indicating that ZFuse would direct the differentiation of macrophages toward the favorable M1 phenotype.
The macrophage study in combination with larger animal studies help understand the importance of appropriate choice in design materials. With the above strategies in mind, it was possible to conduct in vitro and/or in vivo studies that comprehensively evaluate the macrophage response to a biomaterial. Results of the macrophage study suggest that PEEK-zeolite, but not PEEK, increases the expression at the translational level of an M2-associated marker, Fizz1. Results of protein expression data support the hypothesis that PEEK zeolite promotes a pro-repair M2-like macrophage phenotype. Similarly, the ovine study showed a novel PEEK zeolite composite elicits reduced pro-inflammatory cytokine excretion when compared to standard PEEK implants. Specifically, the tissues surrounding PEEK-zeolite composite implants had significantly lower levels of IL1-β and IL-6. These results may explain the increased bone growth observed in the pilot rabbit study and suggest the new material is well suited for clinical application including spinal implants that benefit from host bone implant apposition.
Unavailable from authors at time of publication.
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Details
- Title
- 8. The immune response of two separate polymer spinal interbody device materials
- Creators
- Boyle C. Cheng - Allegheny General HospitalIsaac Swink - Pittsburgh, PA, USDaniel T. Altman - Allegheny Health Network
- Publication Details
- The spine journal, v 19(9), pp S4-S5
- Publisher
- Elsevier
- Resource Type
- Abstract
- Language
- English
- Academic Unit
- Orthopedic/Orthopaedic Surgery; Surgery
- Other Identifier
- 991021897301304721