Impact of interstitial flow on cartilage matrix synthesis and NF-kB transcription factor mRNA expression in a novel perfusion bioreactor.

This work is focused on designing an easy-to-use novel perfusion system for articular cartilage (AC) tissue engineering and using it to elucidate the mechanism by which interstitial shear upregulates matrix synthesis by articular chondrocytes (AChs). Porous chitosan-agarose (CHAG) scaffolds were synthesized and compared to bulk agarose (AG) scaffolds. Both scaffolds were seeded with osteoarthritic human AChs and cultured in a novel perfusion system with a medium flow velocity of 0.33 mm/s corresponding to 0.4 mPa surfice shear and 40 mPa CHAG interstitial shear. While there were no statistical differences in cell viability for perfusion versus static cultures for either scaffold type, CHAG scaffolds exhibited a 3.3-fold higher (p < 0.005) cell viability compared to AG scaffold cultures. Effects of combined superficial and interstitial perfusion for CHAG showed 150- and 45-fold (p < 0.0001) increases in total collagen (COL) and 13- and 2.2-fold (p < 0.001) increases in glycosaminoglycans (GAGs) over AG non-perfusion and perfusion cultures, respectively, and a 1.5-fold and 3.6-fold (p < 0.005) increase over non-perfusion CHAG cultures. Contrasting CHAG perfusion and static cultures, chondrogenic gene comparisons showed a 3.5-fold increase in collagen type II/type I (COL2A1/COL1A1) mRNA ratio (p < 0.05), and a 1.3-fold increase in aggrecan mRNA. Observed effects are linked to NF-κB signal transduction pathway inhibition as confirmed by a 3.2-fold (p < 0.05) reduction of NF-κB mRNA expression upon exposure to perfusion. Our results demonstrate that pores play a critical role in improving cell viability and that interstitial flow caused by medium perfusion through the porous scaffolds enhances the expression of chondrogenic genes and extracellular matrix through downregulating NF-κB1.

Pd@Pt nanoparticle-linked immunosorbent assay for quantification of Collagen type II.

The evaluation of specific protein content in engineered tissues provides a gateway for developing regenerative medicine treatments. Since collagen type II, the major component of articular cartilage, is critical for the blossoming field of articular cartilage tissue engineering, the interest in this protein is growing rapidly. Accordingly, the need for quantification of collagen type II is increasing as well. In this study, we provide recent results for a new quantifying nanoparticle sandwich immunoassay technique for collagen type II. Since mesoporous palladium@platinum (Pd@Pt) nanoparticles have peroxidase-like catalytic activities, these nanoparticles were utilized in an enzyme-linked immunosorbent assay (ELISA)-like format to circumvent the need for traditional enzymes. These nanoparticles were easily conjugated with anti-collagen type II antibodies by the natural affinity interaction and used to develop a direct sandwich ELISA-like format for nanoparticle-linked immunosorbent assays. Using this method, we obtained a limit of detection of 1 ng mL-1, a limit of quantification of 9 ng mL-1. and a broad linear range between 1 ng mL-1 and 50 µg mL-1 for collagen type II with an average relative standard deviation of 5.5%, useable over a pH range of 7 - 9 at least. The assay was successfully applied to quantify collagen type II in cartilage tissues and compared with the results of commercial ELISAs and gene expression by reverse transcription-quantitative polymerase chain reaction. This method provides a thermally stable and cost-efficient alternative to traditional ELISAs. It also extends the application of nanoparticle-linked immunosorbent assays, thereby providing the potential to quantify other proteins and apply the technology in the medical, environmental, and biotechnology industry fields.

Combining stretching and gallic acid to decrease inflammation indices and promote extracellular matrix production in osteoarthritic human articular chondrocytes.

Osteoarthritis (OA) patients undergo cartilage degradation and experience painful joint swelling. OA symptoms are caused by inflammatory molecules and the upregulation of catabolic genes leading to the breakdown of cartilage extracellular matrix (ECM). Here, we investigate the effects of gallic acid (GA) and mechanical stretching on the expression of anabolic and catabolic genes and restoring ECM production by osteoarthritic human articular chondrocytes (hAChs) cultured in monolayers. hAChs were seeded onto conventional plates or silicone chambers with or without 100 µM GA. A 5% cyclic tensile strain (CTS) was applied to the silicone chambers and the deposition of collagen and glycosaminoglycan, and gene expressions of collagen types II (COL2A1), XI (COL11A2), I (COL1A1), and X (COL10A1), and matrix metalloproteinases (MMP-1 and MMP-13) as inflammation markers, were quantified. CTS and GA acted synergistically to promote the deposition of collagen and glycosaminoglycan in the ECM by 14- and 7-fold, respectively. Furthermore, the synergistic stimuli selectively upregulated the expression of cartilage-specific proteins, COL11A2 by 7-fold, and COL2A1 by 47-fold, and, in contrast, downregulated the expression of MMP-1 by 2.5-fold and MMP-13 by 125-fold. GA supplementation with CTS is a promising approach for restoring osteoarthritic hAChs ECM production ability making them suitable for complex tissue engineering applications.

Enhanced matrix production by cocultivated human stem cells and chondrocytes under concurrent mechanical strain.

Conventional treatments of osteoarthritis have failed to re-build functional articular cartilage. Tissue engineering clinical treatments for osteoarthritis, including autologous chondrocyte implantation, provides an alternative approach by injecting a cell suspension to fill lesions within the cartilage in osteoarthritic knees. The success of chondrocyte implantation relies on the availability of chondrogenic cell lines, and their resilience to high mechanical loading. We hypothesize we can reduce the numbers of human articular chondrocytes necessary for a treatment by supplementing cultures with human adipose-derived stem cells, in which stem cells will have protective and stimulatory effects on mixed cultures when exposed to high mechanical loads, and in which coculture will enhance production of requisite extracellular matrix proteins over those produced by stretched chondrocytes alone. In this work, adipose-derived stem cells and articular chondrocytes were cultured separately or cocultivated at ratios of 3:1, 1:1, and 1:3 in static plates or under excessive cyclic tensile strain of 10% and results were compared to culturing of both cell types alone with and without cyclic strain. Results indicate 75% of chondrocytes in engineered articular cartilage can be replaced with stem cells with enhanced collagen over all culture conditions and glycosaminoglycan content over stretched cultures of chondrocytes. This can be done without observing adverse effects on cell viability. Collagen and glycosaminoglycan secretion, when compared to chondrocyte alone under 10% strain, was enhanced 6.1- and 2-fold, respectively, by chondrocytes cocultivated with stem cells at a ratio of 1:3.