Signed informed consent from all participants in this study was obtained from each patient or their legal guardian(s). Study approval was granted by the ethics committee of the Biosciences Institute of the University of São Paulo. The laboratory experiments were carried out at Hospital Sírio-Libanês, at Human Genome Research Center both in São Paulo, Brazil, and at Regenerative Bioengineering and Repair Laboratory, Department of Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA).
Levator veli palatini muscle (LVPM) collection and processing were performed at Good Practices of Manipulation(GMP)
LVPM fragments (n = 5) were obtained during palatoplasty of CL/P patients using a surgical technique (modified von Langenbeck repair) (14) with radical intravelar veloplasty (Fig. 1A, B). Surgical procedures were performed at Hospital Municipal Infantil Menino Jesus, São Paulo, Brazil and at Sobrapar hospital, Campinas Brazil. Harvesting of LVPM was executed at surgical operating room after antisepsis and asepsis and transported to Sírio-Libanês Hospital Laboratory. All procedures were performed at GMP conditions with maximum degree of decontamination and sterility.
According to regulatory local committee and under Brazilian Laws and resolution to regulate advanced cell therapies (National Sanitary Vigilance Agency – ANVISA – RDC n214, February 8th 2018) the Sírio-Libanês Hospital Laboratory facilities have recommended clean rooms infrastructure including air particulate control (HEPA filter) and airflow. There is also antechamber for individual protection vestment. Only human cells can be processes at laboratory site and all reagents from cell isolation to cryopreservation are certified, prion-free and apyrogenic following the guidelines for stem cell research and the development of new clinical therapies 2016, ISSCR (internet).
Each sample was collected in HEPES-buffered Dulbecco Modified Eagle Medium/Hams F-12 1:1 (DMEM/F-12; Invitrogen, Carlsbad, CA) with 200 U/mL penicillin (Invitrogen, Carlsbad, CA) and 200 µg/mL streptomycin (Invitrogen, Carlsbad, CA), kept in 4 °C and processed within 24 hours. All LVPM samples were washed twice in phosphate-buffered saline (PBS, Gibco, Invitrogen, Carlsbad, CA), finely minced with a scalpel, put inside a 15 mL centrifuge tube, and incubated in 5 ml of TrypLE Express, (Invitrogen, Carlsbad, CA) for 30 minutes, at 37ºC. Subsequently, supernatant was removed with a sterile transfer pipette, washed once with 7 mL of DMEM/F-12 supplemented with 10% fetal bovine serum (FBS, HyClone, Hyclone Laboratories, Logan, UT), and pelleted by centrifugation at 400xg for 5 minutes at room temperature. The fragments were resuspended and transferred to 35-mm Petri dishes (Corning, NY) containing D-MEM/F12 culture medium with 15% FBS, 2 mM L-glutamine, 2 mM non-essential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA). After 2 weeks, cells were washed with PBS, then dissociated in trypsin solution and seeded at 1.0 × 104 cells per 25 cm2 for the first passage. In order to prevent cell differentiation, cultures were maintained semi-confluent and they were subcultured every 4–5 days, with medium changes every 2–3 days.
To analyse the presence of aerobic and anaerobic bacteria and fungi in culture, it was used the automated microbial detection system Bact/Alert TM 3D (Bact/Alert- BioMérieux-Durham, NC) and for Micoplasm detection it was used MycoAlertTM (MycoAlert PLUS Mycoplasma detection Kit – Lonza). After these tests any positive results for them must be discarded and new harvesting is recommended according with the GMP laboratory rules.
Flow cytometry analysis was performed by flow cytometry in a FACSCalibur flow cytometer (BD, Becton Dickinson Franklin Lakes, NJ) and analyzed in the CellQuest program (BD, Becton Dickinson Franklin Lakes, NJ). Cells were pelleted, resuspended in PBS (Gibco-Invitrogen, Carlsbad, CA) at a concentration of 1.0 × 106 cells/mL and stained with saturating concentration of antibodies. After a 45-minute incubation in the dark at room temperature, cells were washed three times with PBS and resuspended in 0.25 mL of cold PBS. In order to analyse expression of typical cell surface markers, cells were treated with the following anti-human conjugated antibodies: CD29-PE; CD31-FITC; CD45-PE; CD73-FITC; CD90-FITC, (Becton Dickinson, Franklin Lakes, NJ). Unstained cells were gated on forward scatter to eliminate particulate debris and clumped cells. A minimum of 5,000 events were acquired for each sample.
Mesenchymal stem cell (MSC) differentiation
To evaluate the properties of mesenchymal stem cell differentiation, adherent cells (4th passage) underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation according to the following protocols:
Cells were seeded into 6-well plates (Corning, NY), at a density of 2.0 × 105 cells/well, in DMEM/High Glucose (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone), 1 µM dexamethasone, 100 µM indomethacin, 500 µM 3-isobutyl-1-methylxanthine, and 10 µg/mL insulin (all from Sigma-Aldrich, St. Louis, MO).
Fifteen days after induction, Oil Red-O (Sigma) staining was used to identify intracellular accumulation of lipid-rich vacuoles (12). Briefly, cells were fixed with 4% paraformaldehyde in PBS for 30 minutes, washed with PBS, and stained with a working solution of 0.16% Oil Red-O in PBS for 20 minutes (12).
Approximately 2.5 × 105 cells were centrifuged in a 15 mL polystyrene tube at 400xg for 5 minutes, and the pellet was resuspended in 10 mL of basal medium. The basal medium consisted of DMEM/High Glucose (Invitrogen, Carlsbad, CA) supplemented with 1% insulin, transferrin, selenite (ITS Premix, Becton Dickinson, Franklin Lakes, NJ), 1% 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO), 1 mM sodium pyruvate (Gibco - Invitrogen, Carlsbad, CA), and 50 µM ascorbic acid-2 phosphate (Sigma-Aldrich, St. Louis, MO).
Without disrupting the pellet, cells were resuspended in 0.5 mL of chondrogenic medium, consisting of basal medium supplemented with 10 ng/mL transforming growth factor (TGF) β1 (R&D Systems, Minneapolis, MN) and 10% FBS, and maintained in a humidified atmosphere with 5% CO2 at 37ºC.
On day one, tubes were gently turned over to acquire a single floating cell sphere. Medium was changed every four days. On day 21, samples were fixed in 10% formalin for 24 hours at 4ºC, and paraffin-embedded.
Cryosections (5 µm thick) were cut from the harvested micromasses and stained with toluidine blue to demonstrate extracellular matrix mucopolysaccharides (12).
Cells were cultured in DMEM/High Glucose supplemented with 10% FBS (Hyclone), 5% horse serum (Sigma-Aldrich, St. Louis, MO), 0.1 µM dexamethasone, 50 µM hydrocortisone, and 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA) for 60 days (12). Differentiated LVPM cells were stained using immunofluorescence.
Immunofluorescence localisation of myosin and dystrophin was performed on muscle-differentiated cells of LVPM to confirm myogenic differentiation. Cells were washed twice with cold PBS, fixed with 4% paraformaldehyde/PBS for 20 minutes at 4ºC, and permeabilised with 0.05% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS for five minutes. After blocking non-specific binding with 10% FBS/PBS for one hour at room temperature, incubations with the primary antibody (anti-dystrophin; Ab15277; Abcam, Cambridge, UK and monoclonal anti-myosin skeletal, Sigma) overnight at 4ºC and the secondary antibody (FITC IgG; Chemicon, Temecula, CA) for one hour at room temperature were performed. Nuclei were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO) for visualisation. As positive controls, we used normal human differentiated myotube cultures. As negative controls, we used non-differentiated LVPM-derived cells. The immunofluorescence slides were examined using an Axiovert 200 microscope (Axio Imager Z1, Carl Zeiss, Oberkochen, Germany).
LVPM cells were cultured in osteogenic medium containing DMEM/Low Glucose (Invitrogen, Carlsbad, CA) with 0.1 µM dexamethasone and 50 µM ascorbic acid 2-phosphate. On day 9, β-glycerolphosphate (10 mM) was added to induce mineralisation, and on day 21, Von Kossa staining was performed in order to identify accumulation of mineralised calcium (12).
Immunocompetent rat model used to test the in vivo osteogenic potential of LVPM cells
The Animal Research Ethics Committee at the University of São Paulo approved the use of Wistar immunocompetent 9-month-old male rats, body weight 320–420 g, in this experimental protocol (n = 5). The animals were kept in ventilated stands (Alesco, São Paulo, Brazil), in standardised air and light conditions, at a constant temperature of 22 °C with a 12-hour light/day cycle. They had free access to drinking water and standard laboratory food pellets.
The animals were anaesthetised with an intraperitoneal injection (0.3 mL/100 g of body weight) using a combination of ketamine hydrochloride (5%) and xylazine (2%). The heads of the rats were positioned in a cephalostat during the surgical procedure. A midline skin incision was performed from the nasofrontal area to the external occipital protuberance. The skin and underlying tissues, including the periosteum and the temporalis muscles were reflected laterally to expose the full extent of the calvaria.
Fabrication of scaffold carriers
CellCeram™ (Scaffdex, Finland) was designed in a cylindrical shape with 4-mm diameter of a bioabsorbable 60% hydroxyapatite and 40% ß-tricalciumphosphate composite with a foam-type structure of 83% average porosity, and 200–400 µm of average pore size, with an overall range of 100–800 µm. The dimensions of the scaffolds were designed to match the planned calvarial rat defects in these experiments
Cell preparation for transplantation procedure
We used CellCeram™ (Scaffdex, Finland) as a framework to seed 105 undifferentiated LVPM stem cells and placed on a 35-mm plate (6-well plate; Corning, NY). The cells were supplemented with 2.5 mL of medium used for undifferentiated LVPM stem cells and incubated at 37 °C and 5% CO2 for 24 h prior to transplantation in order to adhere to the scaffold.
CellCeram™ scaffolds with adherent LVPM stem cells were transferred to the right cranial bone defect, and the cell-bearing CellCeram™ surface was positioned in direct contact with the dura mater.
Creation and repair of calvarial defects
To evaluate the osteogenic potential of the LVPM cells, we performed two symmetric full-thickness cranial defects of 4 mm diameter in size on each parietal region of the animals. The cranial defect was created with a 4 mm diameter trephine drill, and constant irrigation with sterile physiological solution was used to prevent overheating of the bone.
The left sides (LS) of the skulls were arbitrarily selected as the control sides and were reconstructed with CellCeram™ scaffolds (Scaffdex, Finland). By comparison, the right-sided defects (RS) were reconstructed with CellCeram™ scaffolds that were seeded with 105 undifferentiated LVPM stem cells. Scalps were repaired with 4 − 0 nylon sutures (Ethicon, São Paulo, Brazil), and the animals euthanised 30 days after cell transplantation. Calvaria were harvested for analysis at the time of euthanasia.
The calvaria of the animals were obtained for histological assessment following euthanasia at day thirty following surgery. Tissue samples were fixed in 10% formalin solution for 24 hours, decalcified in 5% formic acid for 48 hours, and paraffin-embedded. For the morphological study, 5-µm sections were stained with hematoxylin and eosin, and examined under a conventional light microscope.