Introduction: The increasing applications of genetically engineered materials, such as elastin-like recombinamers, in the field of biomedicine have led to the developing of different devices for regenerative medicine, e.g. scaffolds that simulate the extracellular matrix (ECM)[1].
On this basis, a novel biocompatible ELR has been designed, including silk-like domains able to form β-sheet secondary structures that can lead to the formation of stable physically cross-linked hydrogels at physiologic conditions, being soluble and injectable as a cold solution[2]. Furthermore, RGD cell-adhesion motifs have been fused to the Silk-ELR (SELR) to achieve specific cell-matrix interactions. 2D and 3D in vitro experiments were performed with human mesenchymal stem cells (hMSCs), which have been widely used in cell therapy[3], showing good results regarding spreading and proliferation.
Materials and Methods: Recombinant DNA techniques were used for the synthesis of the SELR-RGD, which included plasmid DNA evaluation by agarose gel electrophoresis. The recombinamer was expressed and bioproduced in E. coli in a bioreactor. Physicochemical characterization of the material included SDS-PAGE, MALDI-TOF mass spectrometry and differential scanning calorimetry (DSC) to determine transition temperature (Tt). 2D and 3D in vitro cell cultures were performed with hMSCs. Proliferation was assessed by resazurin (Alamar Blue) reduction and fluorescence measurement. Further nuclei (DAPI) and actin (Alexa Fluor 488 labelled Phalloidin) staining were achieved and fluorescence microscopy pictures were taken.
Results and Discussion: SELR-RGD molecular weight and pureness was confirmed as satisfactory by both SDS-PAGE and MALDI-TOF, while transition temperature was found to be 27ºC, assuring the suitability of the recombinamer in biomedical applications, at physiologic temperature (assumed as 37ºC).
Regarding in vitro cell cultures, a first experiment to evaluate the adhesion was performed by adsorbing SELR molecules on tissue culture plates (TCPs) and adding cells on the modified wells. Hydrogel were formed and cells were seeded on its surface for further 2D studies. In addition, 3D scaffolds were prepared by mixing a suspension of cells with a SELR cold solution. After homogeneization, hydrogels were deposited on a TCP and allowed to stabilize. Then, culture medium was added and cells were cultured for several days, like in 2D cultures. Pores could be directly observed by phase contrast microscopy, found to be from 50 to 200 µm diameter.
The evaluation of the proliferation showed an increase of the cell metabolic activity in every case, by means of Alamar Blue reduction, which is closely related to cell number. Stained spread cells could be clearly observed in all the conditions tested (Fig. 1), being, in the case of 3D cultures, mostly adhered to the pore surface (Fig. 2), suggesting that a cell-friendly artificial ECM has been developed.
Conclusion: In conclusion, a novel SELR with the cell-adhesion property via the fusion of RGD domains has been designed and bioproduced. Its use as an artificial ECM has been proven, since proliferation and spreading results showed a cell behaviour similar to what is expected to be found in living tissues, having a lot of potential as a scaffold suitable for cell therapy.
EU FP7 THE GRAIL Project (Grant no. 278557); PRI-PIBAR-2011-1403; MAT2012-38043; MAT2013-42473-R; MAT2013-41723-R; VA152A12; VA155A12; VA313U14; BIO/VA43/14; CIBER-BBN; Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León; European social Fund (ESF); European Regional Development Fund (ERDF)
References:
[1] Rodriguez-Cabello JC, Martin L, Girotti A, Garcia-Arevalo C, Arias FJ, Alonso M. Emerging applications of multifunctional elastin-like recombinamers. Nanomedicine 2011; 6:111-22.
[2] Fernández-Colino A, Arias FJ, Alonso M, Rodríguez-Cabello JC. Self-organized ECM-mimetic model based on an amphiphilic multiblock silk-elastin-like corecombinamer with a concomitant dual physical gelation process. Biomacromolecules. 2014; 15(10):3781-93
[3] Wei X, Yang X, Han ZP, Qu FF, Shao L, Shi YF. Mesenchymal stem cells: a new trend for cell therapy. Acta pharmacologica Sinica. 2013; 34(6):747-54.