Speaker
Description
"It is well known that cartilage possesses a low intrinsic regenerative potential, causing tissue damage that remains unhealed and contributes to a high socioeconomical burden for affected patients. New strategies to restore the properties of load bearing, friction-reducing hyaline cartilage are thus timely. Tissue engineering and regenerative medicine approaches must deal with the high donor-to-donor variability typical of primary human cells, including bone marrow derived-mesenchymal stromal cells (BMSCs). Recently, we have demonstrated that the chondrogenic potential of BMSCs can be predicted on a donor-specific basis by the ratio between the gene expression levels of two main TGF-β receptors, namely TGFBR1 and TGFBR2 [1]. Also, a transient downregulation of the TGF-β receptors TGFBR2 and ACVRL1 was sufficient to reverse the phenotype of cells that poorly responded to TGF-β1 stimulation and increased their chondrogenic commitment in pellet culture. In the field of regenerative medicine, major efforts focus on engineering hyaline cartilage of clinically relevant sizes which, in addition to the cell type, requires an adequate 3-dimensional cell culture substrate or scaffold. Therefore, the aim of this study is to validate the translational potential of those findings and explore the feasibility of siRNA delivery in hydrogels to improve chondrogenic differentiation of human BMSCs.
For this purpose, human BMSCs were isolated and expanded from the bone marrow of patients undergoing spinal surgery, with full ethical approval (Bern Req-2016-00141) and written informed consent. Gelatin methacryloyl (GelMA) was synthesized using Gelatin type A from porcine skin and methacrylic anhydride to reach a 50% degree of substitution. Gels were prepared as 8% w/v GelMA in PBS containing 0.3% Irgacure and cured at 365 nm. Fibrin was prepared using Fibrinogen and Thrombin from human plasma (Sigma-Aldrich) to a final concentration of 25 mg/ml fibrinogen and 1 U/ml thrombin, respectively. Cells were encapsulated in either GelMA or fibrin at a density of ~20x106 cells/ml. For siRNA delivery, gels were supplemented with either a negative control or a TGFBR2-targeting siRNA (10 pmol, Thermo Fisher) complexed with Fuse-It-siRNA reagent (Ibidi). Constructs were cultured for up to 3 days for RNA isolation and RT-qPCR analysis. Samples were stained with calcein green and ethidium homodimer for analysis of cell viability at 7 days.
Confocal microscopy was used to evaluate the Live/Dead staining of constructs after 7 days in culture, showing good viability and even distribution of embedded cells. Up to 3 days, gene expression analysis from fibrin constructs showed a consistent downregulation of TGFBR2, resulting in an increase of the TGFBR1/TGFBR2 ratio. This phenomenon was much less pronounced in GelMA, potentially because of the lower migration ability of cells within this hydrogel. Future evaluations will explore the chondrogenic differentiation potential of distinct BMSC donors within different hydrogels and in response to siRNA delivery. We suggest that this method will increase the therapeutic efficacy of patient-specific cell-hydrogel based constructs for cartilage regeneration.
The work is supported by AO Foundation and AO Research Institute Davos. GC was funded by the Chinese-Swiss Young Researchers' Exchange Programme (EG-CN_04-032019).
[1] Rothweiler et al. (2020) Front Bioeng Biotechnol 8:618."
52354515324
