Semi-permeable membrane retention of synovial fluid lubricants hyaluronan and proteoglycan 4 for a biomimetic bioreactor

Abstract

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 microm 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-1beta + TGF-beta1 + TNF-alpha, 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 microg/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 microm 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 approximately 20 x 10(-8) cm/s (- cells) and approximately 5 x 10(-8) cm/s (+ cells), for 90 nm membranes was approximately 35 x 10(-8) cm/s (- cells) and approximately 19 x 10(-8) cm/s (+ cells), for 170 nm membranes was approximately 74 x 10(-8) cm/s (+/- cells), and for 3 microm membranes was approximately 139 x 10(-8) cm/s (+/- cells). The permeability of 450 kDa HA was approximately 40x lower than that of 30 kDa HA for 50 nm membranes, but only approximately 2.5x lower for 3 microm membranes. The permeability of 4,000 kDa HA was approximately 250x lower than that of 30 kDa HA for 50 nm membranes, but only approximately 4x lower for 3 microm membranes. The permeability for PRG4 was approximately 4 x 10(-8) cm/s for 50 nm membranes, approximately 48 x 10(-8) cm/s for 90 nm membranes, approximately 144 x 10(-8) cm/s for 170 nm membranes, and approximately 336 x 10(-8) cm/s for 3 microm 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 lubricant transport.

Figure 1

(A) SF lubricant retention by the synovium in the in vivo synovial joint can be (B) biomimetically modeled in a bioreactor where a bioengineered (synovial) fluid compartment containing lubricants (HA and PRG4) is separated from a nutrient fluid compartment by a semi-permeable membrane ± adhered synoviocytes. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

Figure 2

(A) Effects of substrate on synoviocyte proliferation over a 6-day culture period. Effects of substrate and cytokines (IL-1β + TGF-β1 + TNF-α) on (B) HA and (C) PRG4 secretion rates, n =4. D: Correlation of secretion rates of HA and PRG4.

Figure 3

(A) HA loss (with the mixture of MWs) and (B) PRG4 loss from the BF compartment into NF due to transport across indicated membranes ± adhered cells as a % of total, n =3–4.

Figure 4

HA loss, as a function of HA MW, from the BF compartment into NF due to transport across indicated membranes ± adherent cells, (A) 4,000, (B) 2,400, (C) 1,156, (D) 450, (E) 262, (F) 160, and (G) 30 kDa, n =3–4.

Figure 5

Permeability of ePTFE membranes ± adhered cells to (A) HA (with mixture of MWs) and (B) PRG4, n =3–4.

Figure 6

Permeability of ePTFE membranes ± adherent cells to HA, as a function of HA MW, (A) 4,000, (B) 2,400, (C) 1,156, (D) 450, (E) 262, (F) 160, and (G) 30 kDa, n =3–4.

Figure 7

Representative data of the mass of lubricant in BF vs. time (0, 2, 8, and 24 h time points) fit with exponential curves predicted by engineering analysis utilized in permeability calculations: (A) 4,000 kDa HA with 50 nm membranes, (B) 4,000 kDa HA with 3 μm membranes, (C) 30 kDa HA with 50 nm membranes, (D) 30 kDa HA with 3 μm membranes, (E) PRG4 with 50 nm membranes, (F) PRG4 3 μm membranes.