Speaker
Description
Osteogenesis Imperfecta (OI) is an inherited connective disorder mostly caused by type I collagen
(Col I) mutations resulting in low bone mass and increased fracture incidence [1]. Type I collagen is the structural protein that maintains the mechanical properties of bone. To date, analysis of collagen structure has been conducted using either patient bone biopsies or animal (mice) models.
This project aims to visualize an in vitro, human cell-based model of ECM deposition using electrospun Polycaprolactone (PCL) to understand how Col I mutations alter collagen structure in 3D. Second Harmonic Generation (SHG) has recently been used to visualize the extracellular matrix (ECM) of OI tissues [2]. PCL mats were fabricated by electrospinning into non-aligned and aligned fibers. Primary Human Dermal Fibroblasts (HDF) were obtained from PromoCell and OI HDF under informed consent from Sheffield’s Children Hospital. Three different donors’ cells denoted here 1, 2 and 3 were studied. All donors have Type I collagen mutation (COL1A1 gene). Donor 1 has type IV OI (qualitative defect), Donor 2 has type I OI (mild defect) and Donor 3 quantitative defect. Overall, in vitro collagen secretion by healthy and OI HDFs was determined by Sirius Red Assay. Cell-secreted collagen was structurally analysed using a Second Harmonic Generation (SHG) imaging on a laser scanning confocal microscope fitted with a Ti:sapphire laser. The results indicated that collagen secreted by healthy HDFs aligned in the direction of the electrospun PCL fibres [3]. Collagen secreted by OI fibroblasts cultured on aligned and nonaligned fibres also produced SHG signals but less strongly than healthy fibroblasts. The SHG signal produced by OI fibroblasts on aligned scaffolds are stronger and more elongated than nonaligned scaffolds and the collagen produced by OI HDF is less ordered. Sirius Red Assay supported the indication that OI HDF can secrete more collagen on aligned substrates.
Our results demonstrate that fibrous scaffolds can be used to create in vitro human cell based
models in 3D. This will be a tool to better understand the mechanisms behind
diseases of collagen such as OI. Reference: [1] Balasubramanian, Meena, et al. Clinical dysmorphology 24, no. 2 (2015): 45-54. [2] Nadiarnykh, O, et al. J. Biom. Opt 12.5 (2007): 051805 [3] Delaine-Smith, R et al. PloS one 9.2 (2014): e89761
Acknowledgement: Tugba Cebe is funded by Republic of Turkey Ministry of National Education.
Marie Skłodowska - Curie Actions: H2020 – MSCA – RISE - 2017 (777926). The project is funded by the European Union Nanosurf: R/153598
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