Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells
Joseph G. Bartolaccia,b, Arthi Shridharb, Stephen F. Badylaka-c
- McGowan Institute for Regenerative Medicine; b) Bioengineering Department of the University of Pittsbugh; c) Department of Surgery, University of Pittsburgh Medical Center.
Category: Basic Science/Biologics
Keyword: Immunomodulation, Polymer, Macrophage
FDA Device/Drug Status: Approved
Prior/Future Presentation: Yes, 2019, TERMIS Orlando
Poly-ether-ether-ketone (PEEK) has been the most commonly used biomaterial for spinal fusion for many years, primarily because it meets the necessary mechanical requirements to support the pressures to which the spinal column is subjected. However, the biologic response to PEEK has serious limitations including a persistent foreign body response, very modest osseointegration which can result in micromotion, pain, or worse, delamination requiring surgical reintervention. Recent advances in our understanding of immunobiology show that PEEK elicits a proinflammatory phenotype among cells of the innate immune system, including macrophages. Numerous studies have shown that macrophage phenotype can be predictive of clinical outcome. The host immune response to biomaterials used in spine surgery has been largely ignored in deference to mechanical and biomaterial properties such as peak load bearing, porosity, and surface characteristics. Pro-inflammatory (M1-like) macrophages have been shown to release several mediators of cell behavior such as IL-6, IL-1b, and TNF-a, that are osteoclastogenic, while pro-healing (M2-like) macrophages are associated with the release of osteoblastogenic signaling molecules. The emergence of “immunomodulatory biomaterials” is directed toward the biologic consequences associated with permanent implants such as spinal fusion materials. Surface modification of such materials represents one approach for developing more host friendly, biocompatible materials while retaining the required mechanical properties.
The objective of the present study was to characterize and compare the macrophage phenotype elicited by the ZFuze polymer, PEEK, Titanium-based alloys, or iterations of modified ZFuze polymer. The ability of these materials to promote differentiation of osteoblast-like cells in vitro was also determined.
Macrophage Activation Study: Primary bone marrow-derived derived macrophages were isolated from C57bl6/j mice and plated on one of the following test articles: ZFuzeTM, PEEK, rough surface Titanium alloy, or iterations of modified ZFuze polymer. Gene and protein expression were determined through qPCR and immunolabeling, respectively.
Osteoblast Differentiation Assay: Saos-2 cells were purchased from ATCC. 1*104 cells/cm2 were plated into 6 well tissue culture plates containing test articles composed of: ZFuzeTM, PEEK, rough surface Titanium alloy, or iterations of modified ZFuze polymer. Gene and protein expression were determined through qPCR and ELISA, respectively.
qPCR, Immunolabeling, and ELISA.
Culture of Bone Marrow Derived Macrophages: Bone marrow was isolated from the femurs and tibias of C57bl/6 mice and subsequently cultured in complete growth media, including Dulbecco’s modified Eagles medium, 10% fetal bovine serum (FBS), 10% L929 supernatant, 0.1% beta-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM nonessential amino acids, and 10 mM HEPES buffer, for 7 days with complete media changes every 48 h until mature bone marrow-derived macrophages were obtained.
Macrophage Activation: Mature macrophages were exposed to the following treatments for 24h: 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), PEEK, ZFuze, rough-surface Titanium alloy, or ZFuze surface modification iterations. For the cytokine challenge study, cells were exposed for 6h to 20 ng/ml IFN-γ and 100 ng/mL LPS, washed and then placed in 10% FBS 1% P/S DMEM for 24h.
Macrophage Immunolabeling: Fixed cells were washed with 1X PBS followed by incubation in a blocking solution composed of PBS, 0.1% Triton-X, 0.1% Tween-20, 4% goat serum, and 2% bovine serum albumin for 1 h at room temperature. Cells were then incubated in a solution of one of the following primary antibodies: anti-F4/80 at 1:100 dilution as a pan-macrophage marker, anti-inducible nitric oxide synthase (iNOS) at 1:100 dilution as an M1-like marker, and anti-Fizz1 and anti-Arginase1 at 1:200 dilution, each as M2-like markers. Nuclei were counterstained with DAPI.
Saos-2 Culture: Saos-2 cells were purchased from ATCC. Cells were grown in culture medium comprised of EMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin. Medium was changed every 48h. 1*104 cells/cm2 were plated into 6 well tissue culture plates containing test articles composed of either ZFuze, PEEK, rough surface Titanium, or iterations of modified ZFuze polymer. Upon reaching confluence, fresh medium was added for 24h, and subsequently collected for protein expression determination by ELISA, and then the cells were trypsinized for cell counting and subsequently lysed with TRIzol reagent for RNA isolation. Isolated RNA was then converted to cDNA and gene expression was assessed by qPCR: GAPDH, BMP2, and BMP4. Collected medium was assessed for relative levels of: osteocalcin, BMP2, BMP4, and BMP7. Relative gene and protein expression levels was normalized to cell number and quantified to make relative assessments of differentiation status as a function of test article.
Macrophage Gene and Protein Expression: Across three biological replicates, results of qPCR analyses show that ZFuze promoted an M2-like phenotype with expression of Fizz1, and decreased expression of IL-1b and TNF-a compared to PEEK and Titanium, which elicited a more “M1-like” macrophage phenotype. Further, zeolite-loaded ZFuze promoted significantly greater expression of Fizz1 by both gene and protein expression than unloaded ZFuze or other test article comparators.
Saos-2 Osteoblast-like Cell Gene and Protein Expression: Aggregated biological replicates show that BMP4 gene expression was upregulated in a statistically significant manner in Saos-2 cells exposed to ZFuze, but not PEEK or rough surface Titanium alloy. The expression of BMP2 was also significantly elevated at the translational level in ZFuze, but not PEEK or rough surface Titanium, exposed Saos-2 cells.
ZFuze and its iterations elicit a favorable and more biocompatible in-vitro immune profile than PEEK or Titanium. ZFuze also supports increased expression of osteoblastic differentiation markers differentiation of progenitor cells in vitro.