Acros organicstm

For preparation of thermosensitive hydrogels, the raw commercial hydroxyapatite (HA) (CAPTAL^® ‘R’ grade, Batch No: P218R) was supplied from Plazma Biotal (Buxton, UK). Chitosan (medium molecular weight, 100–300 kDa) and glacial acetic acid (Aca) (ACROS Organics^TM, Thermo Fisher Scientific, Geel, Belgium) and sodium bi-carbonate (NaHCO₃) (Fluka^®, Sigma Aldrich, USA) were purchased, and injectable grade sodium bovine heparin was kindly gifted by Extrasul Ext. An. Veg. LTDA (Sao Paulo, Brazil).

The ultracentrifuge-purified virus was inactivated in a 6-well tissue culture plate with 0.1% beta-Propiolactone (ACROS Organics^TM, Thermo Fisher, US). This was done overnight at 4 °C and further incubated at 37 °C in a cell culture incubator to hydrolyze the beta-Propiolactone. The virus was then grown on LLC-MK2 cells and checked by quantitative real-time PCR for virus growth.

Fucoidan extracted from Fucus vesiculosus (92.9% fucoidan phytonutrient content, high molecular weight, code 5619002900, batch FVF2016507) was a kind gift from Kraeber & Co. GMBH (Ellerbek, Germany). Sodium alginate (from Lessonia nigrescens, code 177775000, lot A0376873, sulfated ash 34.16% (on dried substance)) was purchased from ACROS OrganicsTM (Thermo Fisher Scientific, Waltham, MA, USA). (−)-Menthol was acquired from Fluka (Buchs, Switzerland). Bergamot oil was purchased from Pranarôm (Ghislenghien, Belgium). L-Linalool, (−)-β-pinene, calcein, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) hemisodium salt, and trypsin (from the porcine pancreas) were acquired from Sigma-Aldrich (St. Louis, MO, USA). The porcine ears were purchased at a local slaughter (Porto, Portugal). Double-deionized water was provided by an ultra-pure water system (Arium Pro, Sartorius AG, Gottingen, Germany). The reagents were weighted in a digital analytical balance Kern ACJ/ACS 80-4 (Kern & Sohn; Balingen, Germany). All other reagents were analytical grade and were used without any further purification.