Low glucose dulbecco s modified eagle s medium lg dmem

Ti–6Al–4V powder (grade 23) was provided by AK Medical Co., Ltd. (Beijing, P. R. China). Kroll's reagent was bought from Aladdin Reagent Co., Ltd. (Shanghai, P. R. China). Van Gieson's picrofuchsin and calcein AM staining solution were purchased from Sigma-Aldrich (Shanghai, P. R. China). Low glucose Dulbecco's modified Eagle's medium (LG-DMEM) and fetal bovine serum (FBS) were bought from Gibco (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) for mammalian cells was obtained from Beyotime Co., Ltd. (Shanghai, P. R. China).

Cell culture substrates including low-glucose Dulbecco’s Modified Eagle's Medium (LG-DMEM) and fetal bovine serum (FBS) were bought from Gibco (Grand Island, NY, USA). Penicillin and streptomycin were purchased from Huabei Pharmaceutical Co., Ltd. (Shijiazhuang, China). Percoll density gradient, thrombin, OVA and interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and anti-ovalbumin antibody (anti-OVA Ab) ELISA assay kits and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (Shanghai, China). Complete Freund’s adjuvant (CFA) was acquired from Chondrex, (Washington, DC, USA). Rabbit anti-collagen type I and anti-collagen type II antibodies were purchased from Abcam, (Cambridge, MA, USA).

Chitosan (MW ∼120 kDa, degree of deacetylation 76%), 2-(N-morpholino) ethanesulfonic acid hydrate (MES), hydrocaffeic acid (HCA, 3,4-dihydroxyhydrocinnamic acid), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and NaOH were purchased from Sigma. Hydrochloric acid (35%) was purchased from Showa. Low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM) was purchased from Gibco. All materials were applied in commercial form.

Ti6Al4V powder was obtained from AK Medical (Beijing, China). Poloxamer 407 was supplied by Bayee Chemical Co., Ltd. (Hangzhou, China). Rabbit endothelial progenitor cells (BFN60810500) was purchased from ATCC (Manassas, VA, USA) and Rabbit BMSCs (RBXMX-01001) was supplied by Cyagen Biosciences (Guangzhou, China). Low Glucose Dulbecco’s Modified Eagle’s Medium (LG-DMEM), fetal bovine serum (FBS), and streptomycin double antibody were supplied by Gibco (Grand Island, NY, USA). Cell Counting Kit‑8 (CCK‑8) assay was supplied by (Dojindo, Shanghai, China) and Calcein-AM/PI Kits was purchased from Beyotime Co., Ltd. (Shanghai, China). Microfil (MV122, Flow Tech, Carver, MA, USA) was used for microangiography. RNA and the Revert Aid First Strand cDNA Synthesis Kit, and SYBR Premix Ex TaqTM kit were supplied by TaKaRa (Dalian, China). Trizol Reagent was obtained from Invitrogen (CA, USA).

Fibroblasts were isolated from human skin, obtained by donation with written informed consent. The skin was taken from healthy voluntary donors, using a cylindrical scalpel for 5 mm biopsies, in septic and antiseptic conditions; the skin thus obtained was immediately deposited in Hank solution with an antibiotic. In a laminar flow hood, the skin samples were cut into smaller fragments, and each fragment was grown in Dulbecco’s modified Eagle’s low-glucose medium (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, and 100 mg/mL gentamicin), all from Gibco BRL (Rockville, MD, USA), and incubated at 37 °C and in 5% CO₂. The culture medium was replaced every two days; after two weeks of culture, the explants (skin fragments) were removed. The fibroblasts were cultured to approximately 80% confluence, and the cells were separated with 0.05%/0.02% trypsin/EDTA and reseeded to generate sufficient cells for the subsequent tests [17 (link)].

Fibroblasts were isolated from human skin donated with written informed consent. The skin was taken from healthy voluntary donors using a cylindrical scalpel in 5 mm biopsies under septic and antiseptic conditions. The skin obtained was immediately deposited in Hank’s solution with an antibiotic. In a laminar flow hood, the skin samples were cut into smaller fragments, and each fragment was grown in Dulbecco’s modified Eagle’s low-glucose medium (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, and 100 mg/mL gentamicin), all from Gibco BRL (Rockville, MD, USA), and incubated at 37 °C with 5% CO₂. The culture medium was replaced every two days. After two weeks of culture, the explants (skin fragments) were removed. The fibroblasts were cultured to approximately 80% confluence, and the cells were separated with 0.05%/0.02% trypsin/EDTA and reseeded to generate a sufficient number of cells for the subsequent tests [8 (link)].