*4.7. Gene Expression Analysis*

Biphasic scaffolds were divided with a scalpel into the two phases prior to RNA extraction, for which three samples of each experimental group were used. During the RNA isolation procedure, cell lysates of the three samples of each group were pooled.

To dissolve the alginate in alginate-containing jellyfish collagen constructs, samples were mixed thoroughly with 55 mM sodium citrate (Fluka, distributed by Sigma-Aldrich, Taufenkirchen, Germany) with 0.9% sodium chloride (Sigma), in DEPC-water (Gibco), incubated at 37 ◦C for 45 min and afterwards mixed thoroughly again. Cells in the supernatant were centrifuged at 3600 rpm for 10 min, resuspended in PBS, and centrifuged again. Both the pellet and the collagen scaffold were treated with lysis buffer from peqGOLD MicroSpin total RNA Kit (Peqlab, Erlangen, Germany). Lysates from the pellets and scaffolds were pooled and RNA was extracted according to the manufacturer s instructions of the kit. To isolate RNA from the mineralized salmon collagen phase, cell seeded scaffolds were treated with lysis buffer from the RNA kit directly.

For polymerase chain reaction (PCR) 200 ng per experimental condition were transcribed into cDNA in a 20 μL reaction mixture containing 200 U of superscript II reverse transcriptase, 0.5 mM dNTPs (both Invitrogen), 12.5 ng/μL random hexamers (Eurofins MWG Operon, Ebersberg, Germany) and 40 U of RNase inhibitor RNase OUT (Invitrogen, Carlsbad, CA, USA). 1 μL cDNA in 20 μL reaction mixtures containing specific primer pairs were used for amplification in PCR analysis to detect transcripts of collagen I, collagen IIa, collagen X, ALPL and β-actin, respectively. Primer sequences (Eurofins MWG Operon), annealing temperatures and amplicon sizes for each gene are summarized in Table 2.

For analysis, PCR products were visualized in 2% agarose gels (Ultra PureTMAgarose, Invitrogen).


**Table 2.** Primer and conditions for reverse transcriptase PCR.

*4.8. Analysis of DNA Content, ALP Activity in Monophasic Scaffolds from Mineralized Salmon Collagen sGAG Content and Collagen II Content*

Frozen cell-seeded scaffolds were homogenized in ice-cold PBS (2 × 10 s at 5900 rpm) using a Precellys24 apparatus (Peqlab, Erlangen, Germany). After homogenization, 10% Triton X-100 in PBS were added to a final concentration of 1% Triton-X-100 and the samples were incubated on ice for 50 min. ALP activity and DNA content were analyzed from the same lysate. ALP activity was analyzed by conversion of p-nitrophenyl phosphate (Sigma, 1 mg/mL), in 0.1 M diethanolamine pH 9.8, 1% Triton X-100 and 1 mM MgCl2, to p-nitrophenol after 30 min of incubation at 37 ◦C and absorption measurement at 405 nm (Infinite® M200 Pro, Tecan, Männedorf, Switzerland). DNA content was quantified from the same lysate with Quantifluor dye (Promega, Madison, WI, USA) at an excitation/emission wavelength of 485/535 nm. Cell number was calculated from DNA content of defined cell numbers. ALP activity was related to the cell number of the respective sample.

#### *4.9. sGAG Content and Collagen II Content in Biphasic Scaffolds*

Biphasic scaffolds were divided with a scalpel into the two phases prior to freezing for subsequent biochemical analyzes which included always three biphasic scaffolds per group. After thawing of alginate-containing jellyfish collagen constructs, samples were covered with 500 μL of 55 mM sodium citrate (Fluka) with 0.9% sodium chloride (Sigma), in deionized water, homogenized (1 × 10 s at 5900 rpm, Precellys24, Peqlab) incubated at 37 ◦C for 30 min and afterwards mixed thoroughly again. After thawing mineralized salmon collagen constructs, samples were covered with 450 μL PBS, homogenized (2 × 10 s at 5900 rpm), 50 μL of 10% Triton X100 were added, and the mixture was incubated for 30 min at 37 ◦C. All samples were centrifuged at 3600 rpm for 10 min. The supernatants were used for collagen II ELISA, while the pellet was further processed for sGAG determination. Collagen II ELISA was performed as already published [25]. Briefly, each 50 μL of the supernatants was added to wells which were precoated with primary antibody (Mouse Anti-Chick Collagen II Capture Antibody (clone 35; Chondrex, Redmont, WA, USA) diluted 1:500 in PBS). A calibration line was established using human collagen II (Millipore) diluted in PBS with 3% normal goat serum (NGS; Life Technologies, Carlsbad, CA, USA). Afterwards 50 μL of secondary antibody (Biotin-labeled Detection Antibody (Mouse monoclonal anti-Type 2 Collagen (Chondrex)) diluted 1:100 in PBS with 3% NGS) were added. Detection was performed with streptavidin-horseradish-peroxidase (R&D Systems, Minneapolis, MN, USA) and 3,3 ,5,5 -tetramethylbenzidin substrate-solution (Sigma). Absorbance was assessed at 450 nm (reference 570 nm) in a microplate reader (Infinite® M200 Pro, Tecan). Absorbance values from scaffolds without cells carried along during the experiments were subtracted as correction factors.

sGAG content from the pellets was quantified as previously described [25]. Briefly, 1 mL of papain digestion solution (containing 125 μg/mL papain, 5 mM EDTA (both from Sigma), 100 mM Na2HPO4, and 5 mM cystein (both from Carl Roth, Karlsruhe, Germany) in deionized water) were added to each pellet. After incubation at 60 ◦C for 24 h. 50 μL of the digested solution were subjected to a commercially available sGAG assay (Kamiya, Seattle, WA, USA) according to manufacturer s instructions. Absorbance was assessed at 610 nm (Infinite® M200 Pro, Tecan).
