The acellular matrix for bladder tissue-engineering: a quantitative MRI study

squares, dehydrated in ethanol and lyophilized for 24 hours, and rehydrated in increasing concentrations of HA (0.05, 0.1, 0.2, 0.5 mg/100mL) (Sigma). Alcian blue staining was performed to confirm HA uptake. Both ACM with HA (HA-ACM) and without HA (non-HA-ACM) were prepared (N=17 total) for MRI. A separate group of ACMs (N=17) was prepared for hydration and biochemical assays. MRI was performed on a 1.5T scanner (Signa EXCITE Twinspeed, GE Healthcare) using a 3-inch surface coil on ACM samples placed in 5mL round Falcon tubes. Quantitative T1, T2, and diffusion coefficients were measured. T1 was measured with a spoiled gradient echo sequence (FA=2,3,10,20°, NEX=4) [7]. Both single T2 (spin echo: TE=9-300ms, TR=3s, NEX=1) and multicomponent T2 (96-echo CPMG: TE=11.41094ms, TR=2.5s, NEX=2 [8]) were measured. Diffusion coefficients (D) were measured using a spin-echo diffusion-weighted sequence (b-value=03000s/mm 2 in steps of 200s/mm 2 , TR=4s, NEX=16). In-plane resolutions were 0.4mm (T1,T2) and 0.8mm (D), and slice thickness was 5mm for CPMG and 3mm for all other sequences. Data analysis was performed using in-house developed software in Matlab (v.7.0). Water content in ACMs was determined by measuring its wet weight and dry weight prior to and after, respectively, dehydration with ethanol and lyophilization. The total water uptake was calculated as the difference between wet and dry weights. Biochemical assays were performed to measure the glycosaminoglycan (GAG) content. Since HA is a non-sulfated GAG and is distinct from sulfated GAG (sGAG), two assays were performed. The Dimethyl-Methylene Blue (DMMB) method was used to determine total GAG content [9]. For this, dried ACM pieces were digested using 250μg/mL papain from papaya latex, mixed with 1,9-DMMB, and measured for absorbance at 530 nm, using chondroitin sulfate sodium salt as the standard. The second assay used Stains-all® at 480 nm to determine only sGAG content, according to a previous protocol [10]. All products were from Sigma. RESULTS Biochemical and physical changes were observed from incorporating HA in the ACM. The hydration assay (Table 1) showed significantly higher absolute water uptake and retention, by nearly two-folds (P<0.05). Biochemical assays (Table 2) showed a nearly three-fold increase in total GAG (P<0.01) attributed to HA only, since sGAG content was unchanged. On MRI, single T2 and diffusion measurements (Fig.1) were significantly higher in HA-ACM (P<0.01), consistent with the overall effect of greater hydration, increased space in the extracellular matrix, and higher GAG content. Multicomponent T2 appeared more specific and able to separate effects of increased GAG and hydration (Table 3, *P<0.05). The fast T2 increased likely due to improved mobility of water now associated with HA. The slow T2 increase reflects greater hydration. CONCLUSIONS This study has provided baseline quantitative MRI measurements of the bladder ACM, which is necessary for understanding MRI changes with further manipulation such as cell-seeding. The effect of HA incorporation was also assessed. Both increased GAG content and two-fold water uptake in HA-ACM were detected on T1, T2, and diffusion measurements, with multicomponent T2 being most specific. These results are valuable in guiding further regeneration development using ACMs and tissue-engineering strategies involving HA.