Mscgm cd

Human PDLSCs were seeded on titanium disks, CTRL and TEST, with MSCGM-CD (Lonza). The culture medium was changed twice a week. Cultures were maintained for 8 weeks, then were processed as described below for further examination. The inverted light microscopy DMIL (Leica microsystem, Milan, Italy) was used to carried out the microphotographs in order to evaluate the morphological arrangement of hPDLSCs and titanium disks.

hGMSCs were seeded at 8 × 10³ cells/cm² in chemically defined MSC growth medium (MSCGM-CD) (control medium) (Lonza, Basel, Switzerland) and in osteogenic differentiation medium (Lonza) in the presence of all scaffold designs (Fig. 1a–e). To evaluate the performance of different scaffold designs in terms of osteogenic differentiation, the expression of RUNX2 after 1 week of culture was performed by Western blotting analysis (Fig. 1g).

To collect hPDLSCs, we have enrolled six patients in good general health and without oral cavity diseases. They were to undergo surgical procedures to start the orthodontic treatment. After the collection, the periodontal ligament fragments were washed five times with phosphate-buffered saline solution (PBS, Lonza, Basel, Switzerland) [28 (link)]. The washed tissue fragments were placed in a culture dish in an incubator at 37°C in a humidified atmosphere of 5% CO₂ in air. The mesenchymal stem cell growth medium-chemically defined (MSCGM-CD, Lonza) was used as a medium and changed every two days. Isolated cells were migrated spontaneously from the tissue fragments after two weeks of culture [29 (link), 30 (link)].

hUC-MSCs derived from five different donors were isolated from Wharton’s jelly by enzymatic digestion [22 (link)] and frozen in a master cell bank after short-term expansion in SCM. This study is approved by the Institutional Review Board of the Chinese Academy of Medical Science and Peking Union Medical College. Umbilical cords were obtained following the ethical guidelines with written informed consent from donors. All experimental research of this study was in compliance with the Helsinki Declaration. After recovery from the master cell bank, hUC-MSCs were cultured on a tissue culture surface with SCM that contained 10% fetal bovine serum (ExCell Bio, Shanghai, China) or on a chemically treated cell culture surface (CellBIND; Corning Incorporated, Corning, NY, USA) with a chemically defined SFM (MSCGM-CD; Lonza, Walkersville, MD, USA), at 37°C and 5% carbon dioxide. After reaching 90% confluence, hUC-MSCs were detached and subcultured at a ratio of 1:3 until reaching senescence. The time needed to obtain confluence for every passage was recorded to calculate the population-doubling time. β-galactosidase were analyzed at late passage by a cellular senescence assay kit (Millipore, Billerica, MA, USA) following the manufacturer’s protocol. In this assay, senescent cells were stained as a distinctive blue color.

Human gingival mesenchymal stem cells were treated with 5-Aza (5 μm) for 48 h in a humidified atmosphere 5% CO₂ at 37°C. Subsequently, 5-Aza treatment was removed and then cells were cultured in MSCGM-CD (Lonza) for 10 days. Untreated cells were used as control.