Sodium chloride (nacl)

Spiders were anesthetized using CO₂ gas and severed at the pedicel. One group of spiders (n = 7) was silked for about 15 min. To make the spiders spin major ampullate silk, they were gently made to fall several times from a wooden block on which the silk was collected, before they were sacrificed. The dissection was performed on a wax plate placed on ice using 9 mg/mL (154 mM) sodium chloride (Fresenius Kabi AG, Germany) with the help of a Leica M60 stereomicroscope equipped with a Leica IC80 HD camera. Individual glands and whole opisthosomas were fixed in 2.5% glutaraldehyde in 67 mM phosphate buffer, pH 7.2 for 24 h at 4 °C, and later rinsed in 67 mM phosphate buffer. The tissues were dehydrated in increasing concentrations of ethanol (50, 70, 90, and 100%, for 30 min each), infiltrated, and embedded in a water-soluble glycol methacrylate (Leica Historesin). Sections (1 and 2 μm) were cut with glass knives using a Leica RM 2165 microtome, stained with hematoxylin–eosin (HE), and mounted using Agar 100 resin. HE staining is one of the most common techniques used in histological analysis that allows for visualization of cells and tissues, with hematoxylin staining cell nuclei blue-purple and eosin staining cytoplasm and extracellular matrix pink.

To test gas transfer rates, we used fresh, pooled and heparinized porcine blood from an abattoir. Initially, 15,000 IU L⁻¹ heparin-sodium (B. Braun Melsungen), 1.8 mL L⁻¹ 50% glucose solution (B. Braun Melsungen), 1.2 mL L⁻¹ antibiotics solution (10 mg mL⁻¹ gentamycin, Biochrom) and 6 mL L⁻¹ 0.9% sodium chloride (B. Braun Melsungen) were added to the porcine whole blood.
All tests were performed with blood inlet conditions according to DIN EN ISO 7199.6 To meet the specifications, the heparinized blood was transferred to a simplified heart–lung machine setup comprising an open reservoir, a roller pump and an oxygenator with a heat exchanger to adjust the blood parameters according to Table 1. The hemoglobin concentration and the base excess were adjusted by adding 0.9% sodium chloride and 8.4% sodium bicarbonate (Fresenius Kabi), respectively. The oxygenator was supplied with an adjustable gas mixture of oxygen, nitrogen and carbon dioxide. Throughout the experiments, the blood parameters were checked regularly by blood gas analysis (ABL 800 Flex, Radiometer) and the settings were adjusted accordingly.Table 1

Blood conditions for in vitro testing of oxygen and carbon dioxide transfer rates specified by DIN EN ISO 7199:2017⁶.

Parameter	Range
Hemoglobin	12 ± 1 g dL−1
Oxyhemoglobin	65 ± 5%
Base excess	0 ± 5 mmol L−1
pCO2	6.0 ± 0.7 kPa (equivalent to 45 ± 5 mmHg)
Temperature	37 ± 1 °C

100 μL EDTA-anticoagulated blood samples were washed with sodium chloride (0.9%, Fresenius Kabi Austria, Linz, Austria) at 79 g for 3 minutes. Erythrocytes were incubated for 30 minutes at 37 °C with antibodies in ID-CellStab (buffer specially formulated for erythrocytes; Bio-Rad Laboratories, Cressier, Switzerland). Unbound antibodies were removed by washing three times with sodium chloride. Subsequently, cells were resuspended in ID-CellStab.

Homologous PRP was collected at Casa del Dono di Reggio Emilia by apheresis procedure from 1 single donor. The PRP was used to reconstitute platelet gel: briefly, 8 mL syringe of platelet-rich plasma was mixed with 1mL of calcium gluconate 1000 mg/10 mL (Bioindustria L.I.M.m Novi Ligure, Italy) and 2 mL of thrombin in order to achieve complete platelets activation and coagulum formation (Fig. 1). PRP and Thrombin are byproduct of blood routinely obtained at Transfusion Medicine Unit of the AUSL-IRCCS di Reggio Emilia.^{[18 ]} Medication was performed at home by expert nurse: the skin was cleansed with Amukine Med 0.05 % (Angelini, Rome, Italy), NaCl 0.9% (Fresenius, Modena, Italy) and dried with sterile gauze. Thereafter, platelet gel was applied and the lesions covered with a transparent dressing (Tegaderm^3M) for 24 hours.
The radiation oncology/toxicity grading scale^{[19 (link)]} and the common terminology criteria for adverse events score v4.03 (CTCAE v 4.03) score,^{[20 ]} were used to assess the grade of dermatitis. The numeric rating scale (NRS) from 0 (no pain) to 10 (worst pain) was used to assess pain.
Specific ethical approval was not required for the conduction of this case study. Data are presented without revealing the patient’s identity and we acquired both the patient’s and the legal guardian’s informed consent for the publication of the anonymised data.

The animals were randomized in two groups. The first group was treated with pamidronate (Pamifos® - Medac GmbH, Wedel) 0.6 mg/kg, the other group was treated with placebo (NaCl 0.9 % Fresenius Kabi AG, Bad Homburg). Anaesthesia was applied (Fentanyl 0.005 mg/kg, Midazolam 2.0 mg/kg und Medetomidin 0.15 mg/kg) intraperitoneally. First the intramedullary fixation through a median parapatellar insertion at the knee was performed as described by Gabet et al. [15 (link)]. A cannula was drilled into the fossa intercondylaris and advanced into the femur. Then the wound was closed and a standard trauma was applied to the right femur as described by Bonnarens [16 (link)]. Afterwards through a second surgical approach on the lateral femur, a sub vastus approach, the collagen matrix (soaked with pamidronate or placebo) was laid around the fracture region and fixed with a suture. The wound was closed again and anaesthesia was antagonized by Naloxon 0.12 mg/kg, Flumazenil 0.2 mg/kg and Atipamezol 0.75 mg/kg.