Matrix-Embedded Cytokines to Simulate Osteoarthritis-Like Cartilage Microenvironments

Abstract

In vivo, cytokines noncovalently bind to the extracellular matrix (ECM), to facilitate intimate interactions with cellular receptors and potentiate biological activity. Development of a biomaterial that simulates this type of physiological binding and function is an exciting proposition for designing controlled advanced delivery systems for simulating in vivo conditions in vitro. We have decorated silk protein with sulfonated moieties through diazonium coupling reactions to noncovalently immobilize pro-inflammatory cytokines interleukin-1 beta (IL1b) and tumor necrosis factor alpha (TNF-a) in such a biomimetic manner. After adsorption of the cytokines to the diazonium-modified silk matrix, constant release of cytokines up to at least 3 days was demonstrated, as an initial step to simulate an osteoarthritic (OA) microenvironment in vitro. Matrix-embedded cytokines induced the formation of multiple elongated processes in chondrocytes in vitro, akin to what is seen in OA cartilage in vivo. Gene expression profiles with this in vitro tissue model of OA showed significant similarities to profiles from explanted OA cartilage tissues collected from patients who underwent total knee replacement surgery. The common markers of OA, including COL, MMP, TIMP, ADAMTS, and metallothioneins, were upregulated at least 35-fold in the in vitro model when compared to the control-non-OA in vitro generated tissue-engineered cartilage. The microarray data were validated by reverse transcriptase-polymerase chain reaction. Mechanistically, protein interaction studies indicated that TNF-a and IL-1b synergistically controlled the equilibrium between MMPs and their inhibitors, TIMPs, resulting in ECM degradation through the MAPK pathway. This study offers a promising initial step toward establishing a relevant in vitro OA disease model, which can be further modified to assess signaling mechanisms, responses to cell or drug treatments and patient-specific features.

A Three-Dimensional Chondrocyte-Macrophage Coculture System to Probe Inflammation in Experimental Osteoarthritis

“…Our NC-AM ECM formation and degradation (i.e., Aggrecan MMPs) gene expression data are also consistent with previous in vitro results from several groups who reported increased expression of Aggrecan, MMP-1, and MMP-3 mRNA by 3D cartilage explants following either coculture with mouse macrophages or media conditioned by THP-1 cells. 28,29,33 Similarly, our inflammatory cytokine data showing significantly increased production of IL-1b, TNF-a, IL-8, IFN-g, and MCP-1 also matches well with other groups who focused on chondrocyte-produced inflammatory markers. These studies have demonstrated the enhanced production of key inflammatory mediators from chondrocytes exposed to OA synovial fluid or the synovium itself, which may act to further stimulate macrophage inflammation.…”

“…[24][25][26][27] Indeed, an increasing number of studies recently reporting in vitro OA models have cultured chondrocytes within 3D microenvironments. [28][29][30][31][32] However, important auxiliary cell types such as macrophages have continued to be cultured in 2D, 28,31,33 despite the primary location of the macrophages in the knee joint being within the synovial membrane. Moreover, phenotypic changes in macrophages cocultured with chondrocytes have largely been unexplored in previous in vitro studies.…”

Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

“…15−17 Since the in vivo OA conditions of chondrocytes can only be maintained for a short time under standardized in vitro cell culture conditions, long-term evaluations require the addition of OA-inducing agents such as synovial fluid from OA patients, 18 or at least OA mediators such as tumor necrosis factor alpha (TNF-α). 19,20 In order to find an in vitro OA model for preclinical tests suitable for screening in high-throughput approaches, we evaluated the potential of porcine chondrocyte micromass culture to mimic essential aspects of OA. Therefore, suspension of porcine primary chondrocytes was maintained for 14 days in medium containing 10% serum without any inducing agents to develop cartilage-like expression pattern and ECM rich in proteoglycans and collagens.…”

Elucidation of differential mineralisation on native and regenerated silk matrices

“…Silk solution was prepared directly from the fibres according to a protocol used routinely [23,24]. Briefly, 5 gm of B.mori fibres were weighed and cut into fine pieces, followed by boiling in 0.02 M Na 2 CO 3 for 30 min to remove sericin.…”