Signed informed consent from all participants in this study was obtained from each patient or their legal parent or 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 and the Human Genome Research Center in São Paulo, Brazil, and at the Regenerative Bioengineering and Repair (REBAR) Laboratory, Department of Surgery, Division of Plastic and Reconstructive Surgery at the David Geffen School of Medicine at UCLA.
LVPM fragments (n=5) measuring 3-5 mm3 were obtained during palatoplasty in five individual CL/P patients undergoing modified von Langenbeck repair with intravelar veloplasty (Fig. 1A, B)(14). Surgical procedures were performed at Hospital Municipal Infantil Menino Jesus, São Paulo, Brazil and at Sobrapar hospital, Campinas, Brazil. LVPM fragments that were harvested at the two hospitals were transported to Sírio-Libanês Hospital Laboratory.
According to local regulatory committees and pursuant to the relevant Brazilian Laws regulating advanced cell therapies (National Sanitary Vigilance Agency – ANVISA – RDC n214, February 8 th 2018), all tissues were processed at the Sírio-Libanês Hospital Laboratory facilities using clean rooms infrastructure, air particulate control (HEPA filter) and airflow, and standard best practices for scientific investigation. These include the use of an antechamber for donning and doffing of personal protective equipment (PPE), exclusive processing of human cells and tissues at the laboratory site, and the use of certified prion-free and apyrogenic reagents for cell isolation and cryopreservation, based on guidelines for stem cell research and the development of new clinical therapies as set forth by the International Society for Stem Cell Research (ISSR, www.issr.org).
Each muscle 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 pellets were resuspended and cultured in 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 x 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. After 3-4 passages, cultures yielded between 4 X106 to 8X106 LVPMDSC.
The automated microbial detection system Bact/Alert TM 3D (Bact/Alert- BioMérieux-Durham, NC) was used to analyse the presence of aerobic and anaerobic bacteria and fungi in culture, and for MycoAlertTM (MycoAlert PLUS Mycoplasma detection Kit – Lonza, Basel, Switzerland) was use for mycoplasma surveillance. Any cultures with a positive test suggesting infection were discarded.
Flow cytometry
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 x 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-PE; CD34-FITC; CD44-PE; CD45-FITC; CD73-FITC; CD90-PE; CD105-FITC and 7-Amino-Actinomycin D (7-AAD) staining for viability analysis (Becton Dickinson, Franklin Lakes, NJ). 7-AAD was used for the exclusion of non-viable cells in combination with PE (phycoerythrin), and FITC conjugated antibodies in flow cytometry analysis. 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 stromal cell (MSC) differentiation
To evaluate the properties of mesenchymal stromal cell differentiation, adherent cells (4th passage) underwent in vitro adipogenic, chondrogenic, and osteogenic differentiation according to the following protocols:
Adipogenic differentiation
Cells were seeded into 6-well plates (Corning Inc., Corning, NY), at a density of 2.0 x 105 cells/well, in DMEM/High Glucose (Invitrogen, Carlsbad, CA), supplemented with 10% FBS (Hyclone, Hyclone Laboratories, Logan, UT), 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-Aldrich, St.Louis, MO) staining was used to identify intracellular accumulation of lipid-rich vacuoles (13). 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 (13).
Chondrogenic differentiation
Approximately 2.5 x 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 (13).
Osteogenic differentiation
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 mineralization. On day 11, calcium content was evaluated by a Calcium Detection Assay kit (abcam, Cambridge, United Kingdom) according to the company manual. On day 21, Alizarin Red staining was performed in order to identify accumulation of mineralised calcium. The wells were washed twice with PBS and Briefly, cells were fixed with 70% ethanol (Sigma-Aldrich, St. Louis, MO) for 30 minutes. After fixation, the wells were stained with 0.2% alizarin red S solution (pH 4.2; Sigma-Aldrich, St. Louis, MO) for 30 minutes. For the final wash, each well was washed with PBS (Gibco Invitrogen, Grand Island, NY) three times (15).
Immunocompetent rat calvarial defect model
The Animal Research Ethics Committee at the University of São Paulo approved the use of Wistar immunocompetent 9-month-old male rats, body weight 320–420g, in this experimental protocol (n=5). The animals were kept in ventilated stands (Alesco, São Paulo, Brazil), in standardized 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 anesthetised 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.
We next performed two symmetric full-thickness cranial defects of 4mm diameter in size on each parietal region of the animals. The cranial defect was created with a 4mm 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 CellCeramTM scaffolds (Scaffdex, Finland). By comparison, the right-sided defects (RS) were reconstructed with CellCeramTM scaffolds that were seeded with 1X 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.
Fabrication of scaffold carriers
CellCeramTM (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 CellCeramTM (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 24h prior to transplantation in order to adhere to the scaffold.
CellCeramTM scaffolds with adherent LVPM stem cells were transferred to the right cranial bone defect, and the cell-bearing CellCeramTM surface was positioned in direct contact with the dura mater.
Histological preparation and quantitative analysis
The calvaria of the animals were harvested 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 (HE) and examined under a conventional light microscope.
Quantification for regenerated bone was performed using ImageJ (NIH, Bethesda, MD) with reference to the methods established in published manuscripts (16,17). Briefly, the split channels function was used to split the original RGB image into red, blue and green channels. Then the blue channel image was subtracted from the red one and the threshold range was set to 53-255, comparing to original HE images to include all regenerated bone tissue into the positive region. Then, range of interest (ROI) for all the bone defect and regenerated bone tissue were selected with the polygon selection function. Finally, the percent area of positive region in ROI which was regenerated bone was determined.
Immunohistochemistry.
The sections were deparaffinized with two 5 minutes washes in Xylene, hydrated in graded ethanol series and then rinsed in distilled water. For antigen retrieval, slides were incubated for 40 min in citrate buffer (95-100°C) and then cooled for 20 minutes at room temperature, rinsed in PBS, and blocked for 1 hour in immunofluorescent blocking buffer (IBB-5% BSA, 10% FBS, 1xPBS, and 0.1% Triton x-100). Samples were then incubated for 1 hour at room temperature with 1:100 mouse anti-human nuclei monoclonal antibody (HuNu; Chemicon, Temecula, CA), washed with PBS, and incubated with secondary antibody (1:600 Alexa Fluor 594 anti mouse IgG; Thermo Fisher Scientific) for 1 hour at room temperature. Tissue was counter-stained with diamidino-2-phenylindole DAPI (Thermo Fisher Scientific) and mounted using ProLong anti-fade (Thermo Fisher Scientific).