Titriplex 3

Three researchers who were blinded to the experimental groups independently evaluated the cartilage macroscopically of the femur and tibia according to the criteria of the ICRS cartilage injury classification^{28 (link)}. The cartilage of the medial femoral condyle and the tibial plateau were analyzed and the medial tibial plateau was divided into the WB and MC regions (Fig. 10C). Each region was evaluated once by one researcher. The mean score of the researchers was then taken for the final evaluation.
The osteochondral specimens were decalcified in 10% (w/v) EDTA (Titriplex III, Merck, Darmstadt, Germany) for 3 weeks. When the decalcification was completed, the osteochondral specimens were sectioned in the coronal plane at the midpoint of the tibial plateau in the axial plane at the midpoint of the medial femoral condyle (Fig. 10B,C). Histological sections of medial FC were divided evenly into anterior (FC-A1/3), middle (FC-M1/3) and posterior (FC-P1/3) third regions. The sections of the medial tibial plateaus were divided into WB and MC regions. Each region of the histologic sections was graded with the Mankin grading system for hyaline cartilage degeneration^{29 (link)}. All histological sections were scored by three attending pathologists blinded to the procedures performed. The mean score of both investigators was used for the final evaluation.

Blood was drawn
from healthy donors
and collected in EDTA-coated tubes. Blood was diluted in PBS–2
mM EDTA (Titriplex III, Merck, Darmstadt, Germany) 1:2 and carefully
layered on top of Ficoll-Paque solution (Merck, Darmstadt, Germany)
in a 50 mL falcon tube. Subsequently, blood was centrifuged for 40
min, at 400g, at 20 °C without active deceleration
to obtain a buffy coat layer containing PBMC. Upper layer, containing
serum, was aspirated, and the buffy coat layer was collected and washed
in PBS containing 2 mM EDTA. PBMCs were diluted in RPMI-1640 supplemented
with 10% FBS. For later analysis, PBMCs were frozen in RPMI-1640–40%
FBS–15% DMSO and stored in liquid nitrogen.

Gas-phase H₂O₂ is measured using a commercial H₂O₂ analyzer with a detection limit of 100 ppt, corresponding
to 2.5 × 10⁹ molecules per cm³ at 298 K
and 1 atm (Aero-Laser, Germany, AL2021). The instrument relies on
a fluorometric method, by which H₂O₂ is stripped
from the gas phase in a glass coil and subsequently reacts with p-hydroxyphenyl acetic acid and peroxidase forming a fluorescence
dimer. This method detects all peroxides in the solution. The reagents
used for the analyzer were: Potassium phthalate (monobasic, 96148
Fluka), EDTA (Titriplex III p.a., 1.08418 Merck), NaOH (1 N, 71463
Fluka), formaldehyde (37 wt % in water, 252549 Sigma-Aldrich), p-hydroxyphenyl acetic acid (98%, H5,000-4 Sigma-Aldrich),
and peroxidase (from horseradish, P8250 Sigma-Aldrich). The reagents
are dissolved in double distilled water (3478.2 Roth). The instrument
is calibrated with diluted liquid solutions of H₂O₂ (Perhydrol 30% p.a., 7209 Merck), which are titrated against
a permanganate solution (permanganate 1/500 mol 38136 Fixanal) of
certified concentration.

Tissue samples from patients with metastatic CRC undergoing cytoreductive surgery (CRS) and Hyperthermic Intraperitoneal Chemotherapy (HIPEC) were collected from patients during surgery before HIPEC under written informed consent at the Amsterdam UMC (location VU University Medical Center, Amsterdam). Both cancer and normal (non-malignant) tissue were dissociated within 2 h after collection from the patient into a single-cell suspension using a dissociation medium consisting of RPMI, supplemented with 0.1% DNase I (Roche, Basel, Switzerland), 0.14% Collagenase A (Roche, Basel, Switzerland), 5% FCS, and PSG. The specimens were incubated at 37 °C for one to three 45-min dissociation rounds. Thereafter, the cell suspension was run through a 100-μm cell strainer (Corning, NY, USA), erythrocytes were lysed using a shock buffer containing NH₄Cl (Merck, Kenilworth, NJ, USA), KHCO₃ (Merck, Kenilworth, NJ, USA), and EDTA (Titriplex III, Merck, Kenilworth, NJ, USA), and the cells were counted. Cells were either used immediately or cryopreserved in liquid nitrogen until further use. Patient characteristics are shown in Supplementary Table S1.

All chemicals were of analytical grade reagent. Atomic spectroscopy standard (Perkin Elmer, USA) stock Cu(II) solution (1000 μg/mL) was used and diluted to the desired value. All required metal solutions were obtained from their respective nitrate salts and ICP standards as Ni, Mn, Zn, Cd, Pb, Co, Cr, and Cu(NO₃)₂ (Merck, Germany). Activated carbon (Sigma-Aldrich, USA), hydrazine hydrate (Merck, Germany), HNO₃ (65%, Merck, Germany), NaOH (Merck, Germany), HCl (37%, Merck, Germany), ethanol (Merck, Germany), and N-methoxymethyl melamine chemical basis a crosslinker (RUCO-COAT FX 8000, Rudolf Duraner, Bursa, Turkey), ethylendinitrilo tetraacetic acid, disodium salt dihydrate (Titriplex III) (Merck, Germany) were used in the experiment. NIST 1643e and soft drinking water (certified reference material) obtained from National Institute of Standard and Technology, (Gaithersburg, USA).

The specimens were washed in tap water for 24 h and immersed in 10% ethylenediaminetetraacetic acid (EDTA—a solution containing 4.13% Titriplex™ III Merck KGaA, Darmstadt, Germany and 0.44% sodium hydroxide Labsynth, São Paulo, Brazil), for a period of approximately 60 days [52 (link)]. Then, the collected bone fragments underwent successive standard histological staging and were finally included in Histosec^TM (Merck KGaA, Darmstadt, Germany). Semi-serial coronal cuts of 5 µm thickness were performed, prioritising the centre of the circular defect and stained with haematoxylin-eosin.