Five BRONJ patients, ages 54–81 years, underwent surgery at the Department of Oral Surgery, Ninth Peoples Hospital. Debridement of the affected bone and gingiva was extended to reach healthy-appearing tissues (12, 13). BRONJ patients were considered eligible for this study if they had a histologically confirmed advanced solid cancer and radiographic confirmation of bone metastases, receiving intravenous BPs and presented with exposed necrotic bone in the maxillofacial region at least eight. Patients were considered ineligible when they had received any radiotherapy, chemotherapy, immunotherapy, or hormonotherapy before the study. The control group included five patients, older than 50, without bone metabolism diseases. They underwent third molar extraction, meanwhile, gingival tissues surrounding the tooth were collected. All patients provided written informed consent to participate in this study. Detailed patient information is listed in Supplement 1.
Gene expression profiling was performed using the Affymetrix GeneChip (Affymetrix, Santa Clara, CA, US). The details of RNA sample extraction and quality control were in Supplement 2. Raw data were normalized by RMA algorithm, Affymetrix packages in R. Differentially expressed genes were selected at ≥2-fold and p < 0.005. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using the clusterProfler R/bioconductor package version 3.16.0. Only pathways with ≥2 genes were included in the analysis. P-values of hypergeometric tests were adjusted for multiple testing via the Benjamini-Hochberg method. For all pathways with adjusted P‑value ≤0.05.
Gingival samples fixed in 4% paraformaldehyde were embedded in paraffin and sliced for histological evaluation. Paraffin sections were stained with hematoxylin and eosin (H&E) as well as Masson staining. For IH staining, after deparaffinization, rehydration, antigen retrieval, permeabilization and blocking non-specific binding, sections were incubated in primary antibodies against collagen type I A1(COLIA1) (Abcam; 1:200), transforming growth factor beta 1 (TGF-β1) (Abcam; 1:300), caspase 3 (Abcam; 1:200) at 4℃ overnight and secondary antibodies (Servicebio; 1:500) for 1 hour at room temperature. DAPI (Abcam) at 1:500 was used as nuclear counterstain. Results were detected by fluorescence microscope (Olympus). BRONJ patients’ gingival samples were also assessed by TUNEL staining with Cell Death Fluorescein Detection Kit (Roche) following the manufacturer’s instructions. The experimental method was conducted as previously reported (3).
Isolation of GMSCs from BRONJ and healthy gingival tissues
GMSCs were isolated from gingiva as previously described (14), GMSCs under 3-5 passages were used. The cell morphology was analyzed with direct microscopic observation and immunofluorescence assay. First, microscopic images of the cells were acquired using an inverted contrast-phase microscope (Nikon, Tokyo, Japan). Then the cells were stained with a fluorescent dye for actin called (Tetramethyl Rhodamine Isothiocynate) TRITC phalloidin (YEASEN, USA). Fluorescence images were obtained using a fluorescence microscope (Olympus, Tokyo, Japan).
Surface antigens of GMSCs were analyzed by flow cytometry. Briefly, 2 × 105 cells were incubated with mouse anti-human CD45, CD31, CD146, CD90 and CD105 for 30 min at 37◦C. Labeled cells were analyzed using a flow cytometer (Beckman, USA).
Cell Proliferation, adhesion and scratch Assay
GMSCs were seeded at a density of 3×103 cells/mL into 96-well plate. The cell number was assessed on 1,3,5,7 days using Cell Counting Kit-8 (Beyotime) assay. The optical density was measured at 450nm using the SparkTM 10M Multimode Microlpate Reader (TECAN). The experiments of cell adhesion and scratch were done according to previously reported protocol(15). For cell adhesion, GMSCs were divided into 5.0 × 104 cells/ml and subsequently seeded on to type I collagen coated 6 well plates and incubated for 30 min at 37。C. Then the wells were rinsed vigorously three times with phosphate buffered saline (PBS), and the remaining cells were stained using 0.1% crystal violet dye. Data were expressed as adherent cells per field. For cell scratch, GMSCs were plated at 200,000 cells/well in 6-well plates. Once confluent, a scratch wound was performed using a sterile 10μl pipette tip. The size of the gap was measured microscopically immediately (0 h) and 24 h later.
Cell Cycle and Apoptosis
GMSCs were seeded at a density of 5×103 cells/ml into 6-well plate. After cell were detached, cell cycle was analyzed using CycleTESTTM PLUS DNA Reagent Kit (BD, Biosciences). After cells were fixed in 75% ice-cold ethanol, cell apoptosis was analyzed using FITC Annexin V Apoptosis Detection Kit I (BD, Biosciences). Finally, the samples were filtered through 22-µm nylon mesh and evaluated by flow cytometer (Beckman, USA).
Total mRNA was isolated using TRIzol reagent (Invitrogen Life Technologies), cDNA was prepared using GoScript Reverse Transcription System (Promega), and an ABI Prism 7500 (Bioscience) was used to perform quantitative RT-PCR. The relative mRNA expression levels was determined by normalizing to the β-Actin threshold cycle and calculated using the △△Ct method. Primers are shown in Supplement 3.
Proteins were extracted from GMSCs, and Western blot assays were performed as previously described (16). Primary antibodies against β-actin, TGF-β1, COLIA1 and p-Smad3 were used.
In vivo Wound Healing Assay
Lentiviral vector Transduction
Lentiviral vector PCHMWS-GFP-T2A-Fluc was purchased from Dr. A. Ibrahimi (Katholieke Universiteit Leuven). This vector contained a fused gene encoding for the firefly luciferase (Fluc) and GFP. Briefly, GMSCs were plated at 100,000 cells into 25-cm2 flask. After overnight culture, cells were transduced with 2 ml medium that contained lentiviral vector at 37°C for 3-4 hours by the multiplicity of infection (MOI) of 20 and then replaced with fresh medium. Three runs of cell transduction were carried out. Four days after the first transduction, the transduced GMSCs reached confluency and were subcultured at a density of 1000 cells/cm2 in 150-cm2 flasks. After 7 days, when these cultures were near confluency, the GMSCs were cryopreserved at 106/vial (passage 3) at -80°C. The cells were selected with puromycin (Genomeditech, China) at a low concentration (2 μg /mL) and cultured for 5 days. A GFP-positive signal was detected in 95% of the selected cells under an inverted fluorescence microscope (Nikon, Japan).
In Mice Skin wound healing model
Luciferase/GFP-labeled GMSCs were implanted in wound healing model as described previously (17-19). In brief, 5-week-old immunocompromised mice were individually anesthetized using an intraperitoneal injection of ketamine (75 mg/kg) and rinsed with an alcohol swab and sterilely prepped with betadine and draped. A sterile 8mm diameter full-thickness wound was created on the on the dorsum of the nude. A donut-shaped splint with a 10mm inner diameter and 10mm outer diameter was fashioned from a 0.5 mm-thick silicone sheet (Grace Bio-Laboratories, Bend, OR). An immediate-bonding adhesive (Tegaderm, 3M) was used to fix the splint to the skin followed by interrupted 5–0 nylon (Ethicon, Inc,Somerville, NJ) sutures to ensure position. Mastisol (Fernadale, MI) was applied to the perimeter of the wound to improve adherence of the occlusive dressing (Tegaderm, 3M) placed to cover the wounds. The animals were placed in individual cages under a warming lamp and allowed to recover fully from anesthesia. The wound dressings in each group were changed every 3 days according the above methods. 20 mice were randomly divided into four groups: Group A, hydrogel alone; Group B, hydrogel/control GMSCs; Group C, hydrogel/center-BRONJ GMSCs; Group D, hydrogel/peri-BRONJ GMSCs, n = 5. Figure 5 showed the experimental design and schematic representation of wound healing model in nude mice.
On days 7 and 14 post transplantation, in vivo cell viability was confirmed by measuring the luciferase activity with an IVIS bioluminescence imaging system (IVIS Lumina III, PerkinElmer, USA). Briefly, prior to anesthesia, D-luciferin (potassium salt, Yeasen, China) was injected into the mice at 150 mg/kg. The mice were imaged 20 min after injection. Photon flux was measured and quantified by the system software.
Wound closure measurements
Every day, nude mice were observed and digital images were taken. Wound area was measured by tracing the wound margin and calculating the pixel area using Image-J 1.52a software. The wound healing rates were calculated as follows: wound closure rate = (A0 − At)/A0 (20). A0 is the initial wound area, and At is the wound area at 5, 10 and 14 days post-surgery.
After 2 weeks, all mice were sacrificed and the wound tissues were harvested with a rim healthy normal skin tissue. Tissue samples were fixed in 10 % formalin. Frozen sectioning and fluorescence microscope detected cell viability as described previously (21). After imaging by a fluorescence microscope, five random fields were selected to calculate GFP signal areas. Then, the samples were stained with H&E and Masson staining. The lengths of neo-epithelium in H&E staining were calculated according to previously described methods (20). Masson’s staining was used to determine the content and maturity of collagen in the wound beds. The fraction of collagen was calculated by detecting the blue area in five random files under the 400× magnification fields of each group using Image-J 1.52 software. IH staining was the same as the previous experiment.
All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software). The outcome measurements are expressed as the mean±standard deviation. Differences between two groups were analyzed by t-test. P≤0.05 was considered as the statistically significant difference for all comparisons. All experiments were conducted in triplicate.