Papain solution

The compressive loading-displacement curve and Young’s modulus of PGA-PLA scaffolds and BEC-vitro at 2, 4, and 8 weeks (conducted by static compression, biomechanical analyser, Instron-5542, Canton, MA, USA, n = 5 for each time point) were recorded and analysed according to a previously establish method44 (link). After mechanical testing, samples were collected and minced for quantitative analysis of total GAG49 (link), total collagen49 (link), COL I, and COL II44 (link)50 (link). To determine the degradation rate of PGA scaffolds, BMSCs were digested from BMSC-scaffold constructs at various time points (1, 2, 4, 6, 8, 10, and 12 weeks) with papain solution (1:50, Sigma-Aldrich). Next, remnant PGA fibres were collected, dried and weighed (PGA fibres were not digested by papain). Dry weight of remnant PGA fibres were recorded at each time point and plotted to form a PGA degradation curve (n = 3).

The sGAG content in the DEWJ was measured using a Blyscan sulfated GAG assay kit (Biocolor, UK) according to the manufacturer’s instructions. Briefly, to extract sGAG, the samples were digested with a papain solution (with a concentration of 125 mg mL⁻¹ papain in GAG buffer) (Sigma-Aldrich, USA) and placed in a water bath at 60 °C overnight. The suspension was centrifuged at 10,000g for 10 min. About 100 µL of the supernatant containing sGAG was mixed with 1 mL Blyscan dye and shaken for 30 min. The precipitate was collected by centrifugation at 12,000 rpm for 10 min and then dissolved in 1 mL of dissociation reagent. The absorbance was measured in a 96-well plate at 656 nm using a multiplate reader (H4, BIO-TEK Instruments Inc., USA).

Primary mouse neurons were isolated from cortices of early postnatal (P0) Balb/c mice as described previously [43 (link)], except that neurons were plated and maintained in NeuroCult™ SM1 media (Stemcell™ Technologies, Vancouver, Canada). In brief, pups were decapitated and the brain was collected, washed, and placed into the dissection media. Meninges and non-cortical forebrain tissues were removed with fine-point forceps. The cortex was collected and separated into a single-cell suspension by incubating in 20 U/mL papain solution (Sigma-Aldrich) for 10 minutes, followed by the addition of 100 U DNase I (Sigma-Aldrich) and incubation for 5 more minutes, gentle trituration with a fire-polished glass Pasteur pipette, and filtration through a 0.45-μm cell strainer. Cells were then resuspended in NeuroCult™ SM1 plating medium and 1 × 10⁵ cells were plated into wells of a 24-well plate coated with poly-D-lysine (molecular weight, 30 to 70 K; Sigma-Aldrich). A half volume of the culture medium was replaced with fresh NeuroCult™ SM1 maintenance medium every 3 days. Neurons were maintained at 37°C in 5% CO₂ for 6 days before treatments.

After 8 weeks, the subcutaneous implanted samples were harvested for gross view, measured weight and digested in a papain solution (Sigma, USA) at 60 °C overnight (n=3 in each group). The content of DNA measured using the Hoechst 33258 fluorometric assay (Polysciences Inc, USA). The fluorescence intensities were then measured at 360 and 460 nm for excitation and emission, respectively. The DNA content was obtained according to a standard curve of calf thymus DNA (Sigma). The DNA contents were normalized to the disk wet weight [34 (link)]. The glycosaminoglycan (GAGs) determined using 1,9-Dimethylmethylene blue (DMMB; Sigma St. Louis, MO, USA) dye-binding assay to quantify the sulfated GAGs. The absorbance was measured on a Varioskan Flash instrument at 525 and 460 nm. The GAGs content was determined according to a standard curve based on chondroitin 6-sulfate from shark (Sigma) [35 (link)].

For primary rat neuronal cultures, cerebral cortices were collected from embryonic day 18 rats and cells were dissociated using papain solution (10 U/mL, Sigma-Aldrich). Mechanical trituration was performed to attain single cell suspension that was then plated. Four hours after incubation, the culture medium was replaced with Neurobasal medium (Life Technologies) supplemented with 2% B27 and 0.5 mM GlutaMax (Life Technologies). For primary rat astrocytic cultures, cells from cerebral cortices of 2-day-old rat pups were prepared as previously reported. Cells were dissociated, plated in flasks, and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. After 10 days, flasks were shaken to remove microglia and cells were passaged onto plates. Astrocyte cultures were serum-deprived for 18 hour prior to albumin treatment by replacing the medium with serum-free high-glucose DMEM supplemented with 1% penicillin/streptomycin. Bovine serum albumin (BSA with a minimum of 98%, fraction V; Sigma-Aldrich) in serum-free media was then given for 24 hours with the final concentration of 0.2 mM. SJN2511 (30 µM, final concentration; obtained from Tocris Biosciences, Minneapolis, MN), a TGFβ receptor I blocker, was added 1 hour prior to BSA treatment. All cells were maintained in 5% CO₂ at 37 °C.

Papain solution (0.1 mg/mL) (Sigma-Aldrich) was prepared in PBS supplemented with 20 mM L-cysteine and 20 mM EDTA (Sigma-Aldrich) and the pH was then adjusted to 7.2. An equal volume of purified 3.3 or 2B5 anti-PEG antibody (2 mg/mL) was added to the Papain solution and incubated at 37 °C for 2.5 h. One-tenth volume of 0.3 M iodoacetamide solution (Sigma-Aldrich) was added to stop the reaction. The 3.3 and 2B5 anti-PEG Fab fragments were purified by affinity chromatography on a PEG affinity column, generated by swelling 1 g of CNBr-activated Sepharose 4B (GE Healthcare) in 1 mM HCl (pH 3) for 30 min, washing with coupling buffer (0.1 M NaHCO₃, pH 8.3) and adding 5 moles of methoxy-PEG_30K-amine (Laysan Bio) per mL gel in coupling buffer for 4 h at 25 °C. Remaining active groups on the CNBr-activated Sepharose were blocked by adding 1/10 volume of 1 M Tris (pH 8) to the gel at 25 °C for 2 h. The PEG coupled Sepharose was washed with 0.1 M acetate buffer (pH 4) containing 0.5 M NaCl followed by 0.1 M Tris (pH 8) containing 0.5 M NaCl. Papain digested antibodies were loaded to the PEG-resin column at 4 °C for 45 min and washed with cold PBS to remove papain and Fc fragments. The PEG-resin bound anti-PEG Fab fragments were eluted with 100 mM glycine buffer (pH 3) and dialyzed against 20 mM Tris buffer (pH 7.5).