The decellularization strategy will allow obtaining skECM from i) healthy, ii) regenerating at day 4 and day 8 and iii) dystrophic muscle (fibrotic mdx and Scgβ null), for a total of 5 conditions, in which healthy (i) and regenerating (ii) muscles will serve as steady-state and remodelling controls, respectively. Animal models: 8-week-old mdx fibrotic (DMD model) and Scg-β null (LGMD-2E model) mice will be used. We decided to use D2.mdx mice1 10 weeks old as model for DMD, instead of 8-week-old mdx fibrotic mdx mice, since the D2.mdx mouse model show fibrotic tissue formation during the development of the disease more similar to the human DMD. DBA/2J mice served as healthy controls.
First round of experiments started from the WP2, because this is a key WP that will allow the outcome of all the other experimental WPs of the project. Moreover, WP2 is required to choose the best decellularization protocol in order to reduce animal and chemical usage within the project.
WP2. Obtainment of ECMs from healthy, regenerating and dystrophic muscles (1-6 mo). TA muscles from healthy, regenerating and dystrophic mice will be decellularized under sterile conditions according to 3 protocols (#1 #2 #3) chosen for effective decellularization, short time (2-3 days) and researcher's personal experience. ECMs will be checked for quality control and for an effective decellularization using: i) lack of visible nuclear material in frozen tissue sections stained with DAPI and hematoxylin and eosin (H&E) staining; ii) <50ng dsDNA per mg of ECM dry weight; iii) <200bp DNA fragment length by electrophoresis and will be compared for the first time in respect to effectiveness of the protocol using the same parameters (D2.1). This WP will allow for the achievement of decellularized ECMs from healthy, regenerating and dystrophic muscle (M2.1).
Obtainment of ECMs from healthy, regenerating and dystrophic muscles.
Results: both the decellularization protocols (#1 and #3) failed an effective decellularization. As shown in the panel of images, named “Data Report Panel”, the murine TA muscles decellularized through both protocols still show muscle fibers. For protocol #1, 1% SDS solution often precipitated at room temperature. At +24 °C the movement of the samples required for the decellularization process cannot be maintained the same described in the reference protocol, for technical reasons. We suggest to use a fresh made 1% SD solution directly from the SDS powder, instead of from the solution at 20% SDS available in the laboratory. For the protocol #3, we suggest to keep the samples in agitation in the decellularization solutions for longer time. Moreover, we think to change the instrument used for the agitation and to buy a rotator acting along the longitudinal axis of a tube. The protein extraction failed in obtaining a detectable amount of protein to be analysed. We think to reduce the volume of the solutions used for the extraction and to use smaller dialysis cassettes. The DNA extraction failed in obtaining a detectable amount of DNA even in the no decellularized control. We think to reduce the volume of the solutions used for the DNA extraction.
