Cell-Seeded Contraction Gel Model To Assess Fibroblast Function In Carpal
Tunnel Syndrome
Tai-Hua Yang, M.D., Anne Gingery, Ph.D., Andrew R Thoreson, M.S., Kai-Nan An, Ph.D., Chunfeng Zhao, MD, Peter C Amadio,
M.D..
Mayo Clinic, Rochester, MN, USA.
Disclosures:
T. Yang: None. A. Gingery: None. A. Thoreson: None. K. An: None. C. Zhao: None. P. Amadio: None.
Introduction: Non-inflammatory subsynovial connective tissue (SSCT) fibrosis is a characteristic finding of carpal tunnel
syndrome (CTS). This SSCT fibrosis is mediated by transforming growth factor β (TGF-β) signaling, which regulates the expression
of genes associated both with fibrosis and CTS, changing fibroblast behavior and increasing extracellular matrix (ECM) synthesis
in the SSCT of CTS patients. This study’s aim was to develop a bioassay of CTS fibroblast activity, by comparing differences in
contraction rate and material properties between normal SSCT and CTS SSCT derived cells, in cell-seeded collagen gels.
Methods: Harvesting and culturing of SSCT cells: SSCT tissue was harvested from CTS patients undergoing open carpal tunnel
release and from cadavers with no antemortem history of CTS to obtain and culture SSCT cells. Cells from passages 3-4 were
used. This study was approved by our institutional review board (IRB).
Preparation of SSCT cell-populated collagen gel: 1.0 x 106 cells/ml were suspended in 0.5 mg/ml collagen/MEM solution and
added to 3.5-cm diameter Petri dishes that included an inner cylinder, so that the gel and cells would form a ring around the
cylinder. After gelation, TGF-β media (5.0 ng/ml TGF-β1) or MEM vehicle was added. Gels were incubated at 37°C and 5% CO 2,
with media changed every other day.
Gel contractions quantification: The contracting gel ring was photographed every 4 hours for 3 days. All contracted area data
was plotted and fitted using linear regression and optimization to an exponential decay function of the form, N(t) = Ae -Bt + C, (A,
initial area at t = 0; B, the decay constant, and C, the offset of the asymptote as t→∞). Of these constants, the decay constant (B)
was considered to be the parameter that best describes contraction behavior of the gel. The decay constant is directly
proportional to the contraction rate of the gel.
Mechanical testing of gel ring: At the end of the contraction period, each group of contracted gel rings was subjected to uniaxial
failure testing using a custom-built micro test system to determine stiffness and tensile strength.
Statistical consideration: All measurements were expressed as the mean ± SD. The outcomes were analyzed with two-way
ANOVA with a Bonferroni post hoc test. A p-value of 0.05 or less was chosen to indicate significant differences between groups.
Results: Gel contraction rate: The interaction between the factors of cell type and treatment type was found to be significant for
the decay constant response (p = 0.0401). Gel type (p < 0.0001) and treatment type (p = 0.0015) both, individually, had a
significant effect on the decay constant. The decay constant was higher in gels seeded with patient cells than for gels seeded
with normal cells. The decay constant was also higher when cells received TGF-β1 treatment over treatment with medium
lacking TGF-β1 (Fig. 1).
Material properties of gel ring: The interaction between the factors of gel type and treatment type was found to be significant
for the stiffness response (p = 0.0382) (Fig. 2). Gel type (p = 0.0002) and treatment type (p < 0.0001) both, individually, had a
significant effect on the stiffness. No significant interaction effect was identified for the tensile strength (p = 0.9692) (Fig. 3).
However, a significant effect was identified for the individual factors of gel type (p = 0.0005) and treatment type (p < 0.0001).
Discussion: A cell-seeded gel contraction model has been used as a tool for observing mechanisms of ECM building since Bell et
al. [1] first described the contraction phenomenon as “tissue-like”. In our study, the decay constant and gel ring stiffness were
higher in the patient group than in the control group, and these effects were further intensified by treatment with TGF-β1.
These results are consistent with the results observed by Osamura et al. and Ettema et al., who noted increased stiffness in the
SSCT of CTS patients [2, 3].These results are consistent with the known role of TGF-β1 on matrix synthesis, cell division and
remodeling. They are also consistent with the over-expression of pro-fibrotic genes (CTGF, collagen types I & III), and increased
expression and activation of TGF-β second messengers (e.g. Smad 3) in the SSCT of CTS patients.
Significance: This cell-seeded gel contraction model is a potentially useful method to observe differences between CTS patient
SSCT cells and control cells in their ability to contract a collagen gel, and to screen compounds which might interfere with the
process of fibrosis in vitro.
Figure Legends:
Figure 1: The rate of cell-seeded collagen gel contraction in different groups. (C, control group; P, patient group; C+TGF, control
group with TGF-β1 treatment; P+TGF, patient group with TGF-β1 treatment)
Figure 2: Interaction diagram of stiffness.
Figure 3: Interaction diagram of tensile strength.
Acknowledgments: This study was supported by NIH/NIAMS (AR49823).
References: 1. Bell, E., B. Ivarsson, and C. Merrill, Production of a tissue-like structure by contraction of collagen lattices by
human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci U S A, 1979. 76(3): p. 1274-8.
2. Osamura, N., et al., Evaluation of the material properties of the subsynovial connective tissue in carpal tunnel syndrome.
Clinical Biomechanics, 2007. 22(9): p. 999-1003.
3. Ettema, A.M., et al., Flexor tendon and synovial gliding during simultaneous and single digit flexion in idiopathic carpal tunnel
syndrome. J Biomech, 2008. 41(2): p. 292-
8.
ORS 2014 Annual Meeting
Poster No: 1919