Th homogenizer

The other halves of the explanted constructs at the 2 week time point (N=6 per group) were placed in 1ml TRI reagent (Sigma) and homogenized at 35,000 rpm for 60 s using a TH homogenizer (Omni International). The homogenized solution was then centrifuged at 12,000 g for 15 min using a microcentrifuge (accuSpin Micro 17R, Fisher Scientific), and then RNA was isolated according to the manufacturer’s instructions. Isolated RNA was used for cDNA synthesis using a cDNA synthesis kit (PrimeScript™ RT Reagent Kit with gDNA Eraser, Takara Bio, Mountain View, CA). SYBR^® Premix Ex Taq™ II (Tli RNase H Plus) kit (Takara Bio) and primer sequences (Table 2) were then added to cDNA, and qRT-PCR was performed on an ABI 7500 Real-Time PCR instrument (Applied Biosystems, Thermo Fisher Scientific) with each sample run in duplicate. The threshold cycle (Ct) for endogenous control GAPDH (Ct_GAPDH) was subtracted from that of the gene of interest (Ct_GOI) obtain a ΔCt_GOI. Then, the ΔΔCt_GOI was calculated by subtracting the ΔCt_GOI of the group 1 (control) form the ΔCt_GOI of the other groups (2, 3, 4, 5 and 6). The relative target gene expression levels (i.e., Noggin and Runx2) were calculated using the 2^−ΔΔCt equation [41 (link)].

Tissue samples (including kidneys, heart, spleen, liver and small intestine) were collected and immediately frozen in dry ice before being stored at −80 °C. During preparation for the catecholamine assay, the tissues were kept on ice and in 0.1 N perchloric acid. All tissues were homogenized by an Omni International TH homogenizer with disposable probes. The samples were sonicated for a minimum of 20 s then centrifuged twice at 3000 rpm for 20 min. Supernatant aliquots were centrifuged at 9500 rpm for 5 min and a clear liquid was collected for catechol analysis using HPLC (Keller et al., 2004 (link)). Plasma was collected by cardiac puncture and catechols were assayed using the method previously described (Eisenhofer, 1986 (link)). Briefly, samples were extracted with alumina, and 3,4-dihydroxybenzylamine was added as an internal standard. Eluate was run on an ESA HPLC system (Thompson Instruments, Clearbrook, Va.). Plasma controls from Biorad Inc. were included.

To determine the effect of the hydrogel stiffness changes on stem cell fate, hydrogel-hMSC constructs were cultured in chondrogenic differentiation media [1 % ITS+ Premix, 100 nM dexamethasone, 37.5 μg/ml l-ascorbic acid-2-phosphate, 1 mM sodium pyruvate, 100 μM nonessential amino acids, and 10 ng/ml TGF-β1 in HG-DMEM] for 4 weeks. After 2 weeks of culture, the hydrogel constructs were softened or stiffened as described above. After 4 weeks of culture in chondrogenic differentiation media, samples were fixed in 10 % neutral buffered formalin over night at 4 °C, embedded in paraffin, sectioned at a thickness of 10 μm, stained with Toluidine blue O and then imaged using a microscope (Leitz Laborlus S, Leica) equipped with a digital camera (Coopix 995, Nikon). To measure GAG production, chondrogenically differentiated hydrogel constructs were homogenized at 35000 rpm for 60 sec using a TH homogenizer (Omni International) in papain buffer (1 mL, pH 6.5) containing papain (25 μg m/l, Sigma), l-cysteine (2 × 10⁻³M, Sigma), sodium phosphate (50 × 10⁻³M, Thermo Fisher Scientific) and EDTA (2 × 10⁻³M, Thermo Fisher Scientific) and then digested at 65 °C overnight. GAG content was quantified by a dimethylmethylene blue assay [31 (link), 32 ] and DNA content was measured using the PicoGreen® assay.