Blyscan assay

Total sulphated GAG quantification in patient samples was performed using the Blyscan Assay (Biocolor Ltd., UK) as previously described [32 (link)]. GAG levels were corrected for total protein content and expressed as μg GAG mg^-1 total protein.

Collagen content was measured indirectly through hydroxyproline content measurements, as described previously23 (link), and the glycosaminoglycan content was determined using Blyscan Assay (Biocolor, Ltd., Newtownabbey, UK), according to the manufacturer’s instructions, in order to compare the amount of ECM components in native rat livers and decellularised liver scaffolds. The measured values were normalised to the weight of normal liver. DNA content was measured using Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA), to ensure the complete removal of native cells from the liver scaffolds. DNA content measurements were used also to assess the viability of engrafted cells in the liver scaffolds, based on the fact that the amount of DNA in each cell remains constant for a given cell type24 (link). The DNA levels of livers recellularised with 6 × 10⁶ adult hepatocytes were normalised to the DNA levels of 6 × 10⁶ pre-seeding freshly-isolated adult hepatocytes, while the levels of livers recellularised with 6 × 10⁶ foetal hepatocytes were normalised to those of 6 × 10⁶ pre-seeding foetal hepatocytes.

Frozen samples of both regenerative and reparative skin (N = 9) underwent freeze-dried for 24 h at −60 °C and 0.05 Pa (SJIA-10N, SJ Lab, Ningbo, China). Quantitative analysis of sGAG content was performed using Blyscan Assay (Biocolor, Belfast, UK) following the supplier’s instructions. All samples were analyzed in duplicate and read at 656 nm in a spectrophotometer (GENESYS 10 uv, Thermo, Waltham, MA, USA). The final concentration of sGAGs was determined from a calibration curve built from the provided standards, with an r² value of 0.99 or higher.

Samples of rat tails were harvested at 16 weeks after injection. The samples were fixed in 4% paraformaldehyde for 3 days before being immersed in decalcifying solution. The samples were dehydrated, embedded in paraffin, and sectioned at 3.5 μm thickness using a microtome. For histological analysis, haematoxylin and eosin (H&E) and safranine O–fast green staining were performed separately on consecutive tissue sections. Cellularity and morphology were assessed using a previously described grading scale and presented as a histological score (Han et al., 2008 (link)). For immunohistochemical analysis, tissue sections were treated with 3% H₂O₂ for 10 min and blocked with goat serum for 30 min at room temperature. The tissue sections were incubated with anti–collagen II antibody (Abcam) overnight at 4°C, then with a biotin–conjugated secondary antibody for 1 h at room temperature and detected by the SABC method. Images were obtained using a microscope (Leica). For biochemical analysis, NP samples of rats were frozen at −80°C and lyophilized for 24 h, and the dry weight was recorded. The glycosaminoglycan (GAG) content was determined by Blyscan assay (Biocolor, Beijing, China) according to the manufacturer’s protocols and normalized to the dry weight.

Total sulphated GAG in cell culture media were determined colorimetrically by DMMB method (described in [31 (link)] adapted for cell culture media). Total sulphated GAG in snap frozen liver and kidney samples and urine were measured using Blyscan assay from Biocolor Company (UK) following manufacturer’s instructions. Urine samples were processed directly according to the kit manual. For GAG in tissues quantification, individual pieces (approximately 50 mg) were removed from the frozen samples (liver and kidney), weighed and digested in 1 mL papain solution prepared according to the Blyscan kit protocol. After the extraction, 20 μL from each reaction were used for sulphated GAG determination following the Blyscan kit protocol. The obtained values for sulphated GAG were adjusted to the volume of papain reaction and were normalized to the weight of the tissue used or, in the case of urine samples, to creatinine levels.