"DEVELOPMENT OF AN IN VITRO SYNTHETIC POLYMER-BASED 3D CONTRACTION MODEL FOR FIBROSIS
J. Kumari1,2 M.Sc, F.A.D.T.G Wagener2 PhD, and P.Kouwer1 PhD
1 Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands, 2 Department of Dentistry, Section of Orthodontics and Craniofacial Biology, Radboud University Medical Centre, Nijmegen, The Netherlands
Jyoti.email@example.com; Frank.Wagener@radboudumc.nl; firstname.lastname@example.org
Fibrosis contributes to 35-40 percent of deaths worldwide1. The key factors involved in fibrosis at the cellular level are increased differentiation of fibroblasts into myofibroblasts and hampered myofibroblast apoptosis, leading to persistent collagen deposition and tissue contraction2. Currently only very few drugs are clinically available for fibrosis treatment therefore there is an urgent demand for developing novel therapeutic drugs and in vitro fibrosis models to check the efficacy of these putative drugs. In this study, we developed a novel fibrosis model based on synthetic polyisocyanides (PIC-RGD) hydrogels. The model not only measures contraction but, additionally, allows for molecular and cellular analysis.
Fibroblasts were seeded in PIC-RGD gels in the absence or the presence of 10 ng/ml TGFβ1 to facilitate differentiation into myofibroblasts up to day 6. Antifibrotic drugs were added at day 6 and inhibitory effects of drug were checked at day 7.
The presence of myofibroblasts was confirmed by gene expression and immunostaining of, respectively, alpha smooth muscle actin (????SMA) and collagen 1⍺1 (Col1⍺1) in the hydrogels. Tailoring gel concentration allows for excellent discrimination between fibroblasts and myofibroblasts in terms of concentration. As proof of principle, nintedanib and pirfenidone (both are FDA-approved drugs for pulmonary fibrosis) were probed on our developed fibrosis model. The inhibiting effect of nintedanib and pirfenidone is clearly observed in reduced myofibroblast contraction. The results were confirmed by bright field imaging, immunostaining of ????SMA and live-dead staining processes.
In summary, the PIC-RGD hydrogel is highly suitable as an in vitro contraction and fibrosis platform to monitor the efficacy of various drugs and chemicals on fibrosis, scarring and molecular and cellular analyses. Our newly developed contraction and fibrosis platform offers several advantages: 1) it is synthetic and easy to modify with any desirable peptide and growth factor, 2) it has no batch to batch variations, 3) it is temperature sensitive, which allows easy isolation of cells for downstream assays and analysis such as PCR 4) no excess proteins interfere during the assays 5) the platform has virtually no autofluorescence but can easily be equipped with any fluorophore. The combination of these properties make our model an attractive candidate for high throughput screening of putative drugs against fibrosis and scarring.
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2. Nanchahal, J., Proc Natl Acad Sci U S A. 113, 7291-7293 (2016).
3. Gurtner GC., Nature. 453,314-321 (2008)"