Cytokine regulation of synovial fluid (SF) lubricants, hyaluronan (HA), and proteoglycan 4 (PRG4) is important in health, injury, and disease of synovial joints, and may also provide powerful regulation of lubricant secretion in bioreactors for articulating tissues. This study assessed lubricant secretion rates by human synoviocytes and the molecular weight (MW) of secreted lubricants in response to interleukin (IL)-1b, IL-17, IL-32, transforming growth factor-beta 1 (TGF-b1), and tumor necrosis factor-alpha (TNF-a), applied individually and in all combinations. Lubricant secretion rates were assessed using ELISA and binding assays, and lubricant MW was assessed using gel electrophoresis and Western blotting. HA secretion rates were increased *40-fold by IL-1b, and increased synergistically to *80-fold by the combination of IL-1b þ TGF-b1 or TNF-a þ IL-17. PRG4 secretion rates were increased *80-fold by TGF-b1, and this effect was counterbalanced by IL-1b and TNF-a. HA MW was predominantly <1 MDa for controls and individual cytokine stimulation, but was concentrated at >3 MDa after stimulation by IL-1b þ TGF-b1 þ TNF-a to resemble the distribution in human SF. PRG4 MW was unaffected by cytokines and similar to that in human SF. These results contribute to an understanding of the relationship between SF cytokine and lubricant content in health, injury, and disease, and provide approaches for using cytokines to modulate lubricant secretion rates and MW to help achieve desired lubricant composition of fluid in bioreactors.

Synovial fluid (SF) contains lubricant macromolecules, hyaluronan (HA), and proteoglycan 4 (PRG4). The synovium not only contributes lubricants to SF through secretion by synoviocyte lining cells, but also concentrates lubricants in SF due to its semi-permeable nature. A membrane that recapitulates these synovium functions may be useful in a bioreactor system for generating a bioengineered fluid (BF) similar to native SF. The objectives were to analyze expanded polytetrafluoroethylene membranes with pore sizes of 50 nm, 90 nm, 170 nm, and 3 μm in terms of (1) HA and PRG4 secretion rates by adherent synoviocytes, and (2) the extent of HA and PRG4 retention with or without synoviocytes adherent on the membrane. Experiment 1: Synoviocytes were cultured on tissue culture (TC) plastic or membranes ± IL-1β + TGF-β1 + TNF-α, a cytokine combination that stimulates lubricant synthesis. HA and PRG4 secretion rates were assessed by analysis of medium. Experiment 2: Bioreactors were fabricated to provide a BF compartment enclosed by membranes ± adherent synoviocytes, and an external compartment of nutrient fluid (NF). A solution with HA (1 mg/mL, MW ranging from 30 to 4,000 kDa) or PRG4 (50 μg/mL) was added to the BF compartment, and HA and PRG4 loss into the NF compartment after 2, 8, and 24 h was determined. Lubricant loss kinetics were analyzed to estimate membrane permeability. Experiment 1: Cytokine-regulated HA and PRG4 secretion rates on membranes were comparable to those on TC plastic. Experiment 2: Transport of HA and PRG4 across membranes was lowest with 50 nm membranes and highest with 3 μm membranes, and transport of high MW HA was decreased by adherent synoviocytes (for 50 and 90 nm membranes). The permeability to HA mixtures for 50 nm membranes was ~20 × 10 −8 cm/s (− cells) and ~5 × 10 −8 cm/s (+ cells), for 90 nm membranes was ~35 × 10 −8 cm/s (− cells) and ~ 19 × 10 −8 cm/s (+ cells), for 170 nm membranes was ~74 × 10 −8 cm/s (± cells), and for 3 μm membranes was ~139 × 10 −8 cm/s (± cells). The permeability of 450 kDa HA was ~40× lower than that of 30 kDa HA for 50 nm Correspondence to: Robert L. Sah, rsah@ucsd.edu. NIH Public AccessAuthor Manuscript Biotechnol Bioeng. Author manuscript; available in PMC 2011 May 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript membranes, but only ~2.5× lower for 3 μm membranes. The permeability of 4,000 kDa HA was 250× lower than that of 30 kDa HA for 50 nm membranes, but only ~4× lower for 3 μm membranes. The permeability for PRG4 was ~4 × 10 −8 cm/s for 50 nm membranes, ~48 × 10 −8 cm/s for 90 nm membranes, ~144 × 10 −8 cm/s for 170 nm membranes, and ~336 × 10 −8 cm/s for 3 μm membranes. The associated loss across membranes after 24 h ranged from 3% to 92% for HA, and from 3% to 93% for PRG4. These results suggest that semi-permeable membranes may be used in a bioreactor system to modulate lubricant retention in a bioengineered SF, and that synoviocytes adherent on the membranes may serve as both a lubricant source and a barrier for ...