Animals
Ethical approval for all animal experimentation was obtained from the local ethics committee (ComEth Afssa/ENVA/UPEC, Maisons-Alfort, France) (#12-036) in accordance with the European Guidelines for Animal Care (Directive 2010/63/EU).
Pigs
Five female pigs (hybrid of Landrace and large white pigs) with a weight of 35 to 50 kg and age of 3 to 6 months were used (Lebeau Christian, Gambais, France). Pigs were managed in accordance with the instructions of the ethics committee.
Mice
Two severe combined immunodeficiency (SCID) mice (males, 7 weeks old) purchased from Charles River Laboratories (Chatillon, France) were used for the ectopic implantation procedure. The mice were anesthetized with isoflurane (Abbott, Rungis, France) and were euthanized with an overdose of pentobarbital (Centravet, Maisons-Alfort, France).
Biomaterials
Scaffolds of Tutoplast Process Bone (Tutogen Medical, Metz, France) were derived from human cancellous bone. The Tutoplast process consisted of a delipidization, an osmotic cell destruction treatment, hydrogene peroxide treatment, and washing cycles for removal of the non-collagen proteins followed by a solvant dehydrated step and finally a γ-irradiation procedure. Fragments of 2 to 4 mm were cut manually and were stored at room temperature (RT) under sterile condition. Bone scaffolds of equivalent size, volume, and weight (8.0 ± 1.0 mg) were used in this study to ensure a comparable surface area for in vivo analyses.
Bone marrow mesenchymal stromal cell cultures
Pig BMSCs were isolated from BM (5 to 10 mL) of pig humerus (pBMSCs). Human MSCs were isolated from BM (3 to 5 mL) collected from the iliac crest (hBMSCs) of patients undergoing standard BM transplantation procedures (AP-HP Hôpital Henri Mondor, Créteil, France), after having received their informed consent in accordance with the Declaration of Helsinki. The project was approved by the Ethical Committee of Ile de France (section 4 #DC-2009-1049). pBMSCs and hBMSCs were cultured in alpha-modified Eagle’s medium (αMEM) (PAA, Les Mureaux, France) supplemented with 10% of foetal calf serum (FCS) (Stem Cell Technologies, Grenoble, France) and 0.5% ciprofloxacine (Bayer Pharma, Puteaux, France). The hBMSCs used in this study were positive for CD90, CD105, and CD73 and negative for CD34 and CD45 and were able to differentiate into osteogenic, adipogenic, and chondrogenic lineages (data not shown) as previously described [7,24,25].
Functional characterization
To characterize pBMSCs, their capacity to differentiate into mesenchymal lineages was assessed. For osteogenic differentiation, at 50% confluence the growth medium was replaced by αMEM-10% FCS supplemented with 50 μM L-ascorbic acid-2-phosphate (AA), 10 mM βGlycerophosphate (βGly), 0.1 μM dexamethasone (Dex) (Sigma, Saint Quentin Fallavier, France), and 100 ng/mL rhBMP2 (recombinant human bone morphogenetic protein 2, Inductos; Laboratoire Wyeth Pharmaceuticals, Philadelphia, PA, USA). On day 10, the monolayers were fixed in 70% ethanol (Cooper, Melun, France) for 1 hour at 4°C and stained for 15 minutes with Alizarin Red S (Sigma) at RT.
For adipogenic differentiation, at 80% confluence the medium was replaced by a high-glucose medium (Invitrogen, which is part of Life Technologies, Villebon sur Yvette, France) supplemented with 10% FCS, 0.1 mM Dex, 0.2 mM indomethacin, 0.01 mg/mL insulin, and 0.5 mM IBMX (Sigma). On day 10, the monolayers were fixed by using 4% paraformaldehyde (VWR, Fontenay Sous Bois, France) for 5 minutes at RT and stained for 15 minutes with 0.3% Oil Red O (Sigma)/60% isopropanol (VWR). Chondrogenic differentiation was performed in pellet culture by using Stempro, a Chondrogenesis Differentiation Kit (Life Technologies), as described by the manufacturer. On day 21, pellets were fixed in 4% formaldehyde (Sigma) and embedded in paraffin. Sections (3 μm) were stained with Alcian Blue 8GX (Sigma) as described by the manufacturer and counterstained with hematoxylin (Sigma).
Surgical procedure in pigs
Pigs were managed in accordance with the instructions of the ethics committee. Access to FH was done in accordance with the previously described protocol by a percutaneous approach of the hip [26]. Pigs received an injection of 140 × 106 autologous pBMSCs (n = 1) or hBMSCs (n = 2) in 7 mL of 5% human serum albumin (Albunorm; Octapharma, Boulogne-Billancourt, France), and one pig served as a negative control. To push all the cells inside the FH and to allow the cells to migrate correctly to the necrotic site, a volume of air (2 to 5 mL) was injected and then the trocar was let 5 minutes before being removed.
Blood was collected before (T0) and after injection with a kinetic from 1 minute to 24 hours. Liver, kidneys, spleen, and lungs were collected at either 30 minutes or 24 hours after injection. BM was collected before (T0) and 24 hours after injection. Injected FH and adjacent tissues (that is, capsule, periarticular muscles, gluteus maximus muscle, and round ligament) were analysed 30 minutes after injection. Non-injected pig served as a negative control. Organs were dissected into several pieces and ground on a cell strainer with a suitable piston and used for cytometry and molecular biology.
Cell labeling with DiOC18
Before injection, pBMSCs (20 × 106 cells/mL) were incubated in 10 μg/mL of DiOC18 (3, 3′-dioctadecyloxacarbocyanine perchlorate) solution (Molecular Probes, part of Life Technologies) containing 3% FCS for 20 minutes at 37°C [27]. Finally, pBMSCs were washed with 1X Hanks’ balanced salt solution (PAA) three times to get rid of dye remnant and re-suspended in 5% human serum albumin (Albunorm; Octapharma).
Flow cytometry
Ground organs and body fluids of pigs that received an hBMSC injection were stained for BMSC marker CD73-APC (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for 15 minutes. Then the different samples of pigs that received an injection of DiOC18
+ pBMSCs (n = 1) or hBMSCs (n = 2) were examined by using FACSCanto™ II (Becton, Dickinson and Company). The data were analysed by using BD FACS DIVA™ software (Becton, Dickinson and Company). The efficacy of labeling was checked before injection with positive expression of stained cells defined as fluorescence greater than 95% of that of the corresponding control pBMSCs not labeled DiOC18 or hBMSCs not labeled CD73.
DNA purification and quantitative real-time polymerase chain reaction
Two specimens were sacrificed 30 minutes and 24 hours after hBMSC injection. Samples were immediately placed in DNA lysis buffer (Qiagen, Courtaboeuf, France) after collection. Total DNA was isolated by using a QIAmp DNA Mini Kit for blood, BM, organs, and tissue samples and using a QIAmp DNA Investigator Kit for FH samples as described by the manufacturer (Qiagen). FH samples were previously pulverized to a fine powder by using a ceramic ball of 6.35 mm and a Fast Prep System (MP Biomedical, Santa Ana, CA, USA). The human genomic DNA (gDNA) obtained was quantified by using human TaqMan Copy Number Reference Assay, RNase P (Applied Biosystems, part of Life Technologies, Courtaboeuf, France) with a 7500HT Fast Real-Time PCR System (Applied Biosystems). hBMSC standard range was realized with decreasing concentrations of hBMSC gDNA diluted in pig gDNA (5 ng/μL). The straight equation (y = −3.526 × 38.163; R2 = 0.9983) of standard curve had permitted us to obtain the number of cells corresponding to detected cycle threshold (Ct).
In situ hybridization of human Alu sequences
After euthanasia, half of FH was removed 30 minutes after hBMSC injection, fixed for 48 hours, and decalcified in 4.13% EDTA solution (pH 7.4) (Sigma). After dehydration, clearing, paraffin-embedding, and cutting steps, ISH was performed on the FH sections as previously described by Redwine and Armstrong [28]. Locked nucleic acid-based probes were ordered from Exiqon, Inc. (Woburn, MA, USA), and the sequence used was /5DigN/TCTCGATCTTCCTGACCTCATGA/3Dig_N/. Sections were deparaffinized, rehydrated, washed, and treated with 3% hydrogen peroxide for 15 minutes. After washing, sections were treated in 0.1 M triethanolamine pH 8.0 and 0.25% acetic acid for 20 minutes at RT and pre-hybridized for 1 hour at 56°C in buffer containing 4X SSC (sodium saline citrate) (VWR), 50% deionized formamide, 1X Denhardt’s solution, 5% Dextrane Sulfate, and 100 μg/mL Salmon Sperm DNA. Hybridization buffer was replaced by fresh buffer containing 70 nM of Alu probe and was denatured for 5 minutes at 95°C. Hybridization was carried out for 2 hours at 56°C in a wet chamber. Slides were washed twice for 5 minutes in 2X SSC and twice for 5 minutes in 0.5X SSC at 56°C each. Signals were detected by using anti-DIG horseradish peroxidase-conjugated Fab fragments (Roche, Boulogne Billancourt, France) and diaminobenzidine (Dako, Carpinteria, CA, USA) as substrate. Sections were counterstained with Gill-2 hematoxylin (Thermo Shandon Ltd., Runcorn, UK). Two negative controls were produced to compare and ensure consistent interpretation: negative controls of pig FH injected with human cells in omitting Alu probe and sections of FH injected with physiological saline only exposed to Alu probe. Slides were observed by using a DMRXA microscope (Leica, Nussloch, Germany).
Magnetic resonance imaging
Magnetic resonance imaging (MRI) was performed on a 1.5-T MRI device (Siemens Avanto, Erangen, Germany). Pigs were in the supine position, and their hind legs were tied in extension. T1-weighted and T2 with fat saturation (T2 FS)-weighted sequences were obtained in the coronal planes.
Ectopic implantation procedure in immunodeficient mice
Six subcutaneous dorsal pockets (0.5-cm incisions) were prepared in each of the SCID mice. In each pocket, one scaffold was implanted and 300,000 pBMSCs (P2) were injected onto the scaffold in the pocket. The skin was closed with 5-0 sutures (Ethicon, San Lorenzo, Puerto Rico, USA). Cell-free scaffolds were implanted under similar conditions and served as controls. pBMSCs from porcine BM of three independent pigs were tested in duplicate (n = 6 scaffolds).
Histology
Pig femoral head
Pig FHs were fixed with 4% formaldehyde solution (VWR), decalcified in 6.8% nitric acid (VWR) for 2 weeks, and rinsed abundantly in tap water before embedding in paraffin. Sections (3 μm) were stained with Masson’s Tri-chrome (hematoxylin: nuclear staining; acid fuchsin/xylidine ponceau: cytoplasmic staining; light green SF yellowish: collagen staining; all from VWR). Images were visualized by standard light microscopy and captured by using a UC30 Digital Color Camera and CellSens Entry software (Olympus, Rungis, France).
Mice scaffolds
After 7 weeks, scaffolds were excised from mice and immediately fixed in 70% ethanol, decalcified for 3 hours in 6.8% nitric acid (VWR), and rinsed in tap water before embedding in paraffin. Sections (3 to 5 μm) were stained with Masson’s Tri-chrome. Fifteen sections of each sample were analyzed (five at the beginning, five in the middle, and five at the end).