Joris van Dongen

290 Chapter 12 No difference in sulphated glycosaminoglycans There was no difference in amount of sGAGs in NAM and DAM samples with 0.93 +/- 0.31 and 0.69 +/-0.31 µg sGAG per mg dry weight ECM respectively (p>0.05) (Fig. 3A, supplemental content). This was confirmed by Alcian blue staining of control adipose tissue, tSVF as well as ECM samples: here similar levels of staining were detected (Fig. 3B, supplemental content). Viscoelastic relaxation properties of NAM hydrogels Proteolytic treatment at room temperature of NAM and DAM liquified these matrices, which formed stable hydrogels upon warming to 37°C, albeit of relatively low mechanical strength. Viscoelastic relaxation properties were determined for all NAM and DAM hydrogels, however, all DAM hydrogels and one NAM gel collapsed during the measurements and yielded no data. This indicates that structural differences are present in ECM derived from diabetic donors in comparison with ECM derived from non-diabetic donors. Average stiffness for all measured NAM hydrogels was relatively low with 1.81 +/- 0.02 kPa (Fig. 2A). For each donor, three independent pre-gel solutions derived from the same donor and ECM isolation were produced by pepsin digestion (Table 1). Different pre-gel solutions showed a large intra-donor variation of stiffness (Fig. 2A). The large intra-donor variation of stiffness warrants standardization of the gelation procedure. The intra-donor variation was also present in the relaxation properties of the measured NAM hydrogels. Relaxation of stiffness showed a fast decrease with the stiffness reaching zero within about 20 seconds (Fig. 2B, C, D). This was typical for most of the replicates i.e. NAM1, -2 and -3 with each requiring 2 to 3 (Table 1, Fig. 2E). The fast decrease shows that adipose tissue-derived ECM hydrogels are much more viscous than elastic. A more viscous gel is less resistant to mechanical stress i.e. more prone to collapse and therefore less suitable for clinical applications which relate to movement and friction such as wound healing. Stress relaxation as a function of time was measured with the use of a Maxwell model with each element in this model having a spring constant related to the elastic part of the gel and a relaxation time constant representing a viscous part of the gel. Two or three elements were sufficient and the addition of more elements did not result in an improved quality of the fit. The first element having a high relative importance and a relaxation time constant of less than 1 sec. was most likely liquid that was pushed out of the gel (Fig. 2). The second element with a relaxation time constant between 1 and 10 sec, which was most likely ECM. Interestingly, in some hydrogels a third element was shown with a relaxation time constant between 10 and 100 sec. Hydrogels that present a third element were also the hydrogels with the highest stiffness and lowest relaxation.