Biomaterial
BCP, a ceramic composed of hydroxyapatite/beta-tricalcium phosphate in a ratio of 20/80 by weight, was used because this composition has previously demonstrated bone induction [19]. BCP granules, ranging in size from 1 to 2 mm, were supplied by Biomatlante (Vigneux de Bretagne, France) under the brand name MBCP+. MBCP + is CE and US Food and Drug Administration 510(k) approved as a synthetic bone substitute. BCP discs, 7 mm in diameter and 2 mm in thickness, were used for calvaria regeneration. The overall porosity (%vol) was 75 ± 5%, with a pore size distribution of 70% (0 to 10 μm), 20% (10 to 100 μm) and 10% (100 to 300 μm). The BCP biomaterials were supplied in double-sealed packaging and gamma-sterilized at 25 kGray.
Isolation, expansion and characterization of clinical batches of GMP-grade BMSCs
Bone marrow aspirates were obtained from the iliac crest, by standard puncture and aspiration, of healthy human donors (21 to 26 years old) after receiving informed consent according to the Declaration of Helsinki. The project was approved by the Ethical Committee of Ulm University. A maximum of 37 ml bone marrow was aspirated using up to four 20 ml Omnifix syringes (B. Braun, Melsungen, Germany) and transplant aspiration needles (Somatex, Teltow, Germany). Each syringe was prefilled with 1,000 IU heparin in 2 ml NaCl (B. Braun). BMSCs used in this study are of GMP grade and were expanded according to previously published protocols [6]. In brief, BMSCs were isolated from heparinized bone marrow aspirates by seeding 50,000 white blood cells/cm2 on two-chamber CellStacks (Corning/VWR, Ulm, Germany) in alpha minimum essential medium (Lonza, Basel, Switzerland) supplemented with 5% GMP-grade human platelet lysate (IKT, Ulm, Germany) in order to avoid animal products [5]. Cells were cultured for 10 or 14 days with medium exchange twice per week. Cells were detached and reseeded at a density of 4,000 BMSCs/cm2 on two-chamber CellStacks in alpha minimum essential medium supplemented with 8% PL for a further 5 or 7 days. A production license for this protocol has been granted from the regional government (Regierungspräsidium Tübingen, Germany; production and import license: DE_BW_01_MIA_2013_0040/DE_BW_01_IKT Ulm). To assess the quality of the aspirates, measures such as colony-forming units (CFUs-F), BM-MNC content and doubling times were measured. For phenotypic characterization, flow cytometry was performed as described previously [5, 6]. Fluorescent intensities of 50,000 to 100,000 BMSCs were acquired. Briefly, BMSCs were stained for 15 minutes in 100 μl phosphate-buffered saline using the following combinations of antibodies: IgG-FITC (clone X40), IgG-PE (clone X40), IgG-PerCP (clone X40); CD90-FITC (clone 5E10), CD34-PE (clone 8G12), CD45-PerCP (clone 2D1); CD105-FITC (clone SN6), CD73-PE (clone AD2), CD3-PerCP (clone SK7); and HLA-DR,DQ,DP-FITC (clone Tü39), HLA-A,B,C-PE (clone G46-2.6). All antibodies were sourced from Becton Dickinson (Heidelberg, Germany), except CD105 that was from AbDSerotec (Puchheim, Germany). After washing with phosphate-buffered saline, cells were analyzed using a BD FACScan (BD Biosciences, Heidelberg, Germany).
Differentiation capacity of expanded BMSCs was performed as described previously [5, 6]. Briefly, BMSCs differentiation was induced using adipogenic differentiation medium from Lonza or chondrogenic or osteogenic differentiation media from Miltenyi (Bergisch Gladbach, Germany) according to the manufacturers’ instructions. For detection of adipogenic differentiation, cells were stained with Oil Red O solution in 2-propanol, diluted to 60% using deionized water. Chondrogenic differentiation was detected by Alcian Blue staining, while mineralization was detected by Alizarin red staining.
Cell numbers required for bone formation
Prior to the transportation of large batches of fresh BMSCs for bone repair, it was necessary to quantify the optimal dose of cells for bone formation in proportionally lower numbers. Different quantities of BMSCs in passage 2 from three different human donors were mixed with 50 mg BCP particles and allowed to attach for 1 hour prior to subcutaneous implantation in nude mice. Number of cells per implant and number of implants per group were as follows: 0 cells, n =5; 0.1 × 106 cells, n =9; 2 × 106 cells, n =9; and 4 × 106 cells, n =9.
Transportation of fresh bone marrow stromal cells
Cells were harvested and washed, and 100 × 106 passage 1 BMSCs from each of the five donors were suspended in 7 ml saline solution supplemented with 4%, 5% or 20% HSA solution (CSL Behring, Hattersheim am Main,Germany) in a sterile luer lock 20 ml syringe (B. Braun). Cells were transported within 24 hours from Ulm (Baden-Wurttemberg, Germany) to Nantes (Pays de la Loire, France) at 20°C via TNT Express overnight courier. Upon arrival, cell viability was confirmed using the trypan blue exclusion method. Each syringe containing BMSCs was connected to a syringe containing 5 cm3 BCP particles (2.5 g) using a double luer lock. Cells were allowed to attach to the BCP particles for 1 hour, prior to implantation in nude mice. Cell attachment was confirmed by methylene blue staining.
Bone marrow stromal cell/biomaterial implantation in the subcutis and calvaria of nude mice
All animal experiments were performed according to Directive 2010/63/UE and after approval of protocols from the local ethical committee (CEEA, Pays-de-la-Loire, France). Immunocompromised female mice (RjOrl: NMRI-Foxn1nu/Foxn1nu) were purchased from a professional breeder (Janvier Labs, Saint-Berthevin, France) at 4 weeks of age. Mice were placed in cages as groups of five in HEPA-filtered closets with water and food ad libitum, and were quarantined for a minimum of 10 days prior to surgery. Once under general anesthesia by inhalation of isoflurane, the skin was disinfected with 1% iodine alcoholic solution. For subcutaneous implants, BCP granules alone (BCP) or in association with BMSCs (BCP + BMSCs) were implanted on the dorsal side in two subcutis pockets that were created by skin incisions and tissue dilacerations with sterile instruments. Then 20 × 106 BMSCs (passage 1) with 1 cm3 BCP were implanted per subcutaneous site in randomly assigned nude mice. BMSCs from five donors that were transported were implanted subcutaneously: Donor 1, n =6; Donor 2, n =6; Donor 3, n =4; Donor 4, n =8; and Donor 5, n =7.
For calvaria implants, the mouse was maintained on a stereostatic frame and a skin incision of 1 cm was made to expose the skull. A critical-sized defect 4 mm in diameter was created in the calvaria bone [26] using a trephine and a dental micromotor (Nouvag NM3000; NOUVAG, Goldach, Switzerland). Constant saline irrigation was used during drilling. Then 3 × 106 BMSCs in passage 2 or 3 were seeded onto one side of a BCP disc 1 hour prior to implantation. Cells from three different human donors were used. Discs were overlain cell-side down over the calvaria defect. Calvaria defects overlain with BCP discs alone or defects that were left empty served as controls. Skin incisions were closed with sutures (Filapeau; Peters Surgical, Bobigny, Ile-de-France, France). Analgesic (20 μg/kg; Buprenorphine, Axience, France) was injected intramuscularly before surgery and every 8 hours for 3 days after surgery. Animals were observed daily and body weights were determined weekly. After 4 or 8 weeks, the mice were euthanized by inhalation of an overdose of carbon dioxide gas. Sample sizes for calvaria implantations were as follows: 4 weeks empty, n =3; 4 weeks BCP, n =3; 4 weeks BCP + BMSCs, n =4; 8 weeks empty, n =3; 8 weeks BCP, n =6; and 8 weeks BCP + BMSCs, n =5.
Decalcified histology preparation, staining and histomorphometry
Explants were observed for signs of tissue necrosis, inflammation or infection, dissected and fixed in 10 volumes of buffered 4% formaldehyde for 72 hours. The skulls were further dissected using a diamond saw. Explants were decalcified in 4.13% ethylenediamine tetraacetic acid/0.2% paraformaldehyde in phosphate-buffered saline, pH 7.4 for 96 hours at 50°C using an automated microwave decalcifying apparatus (KOS Histostation; Milestone Medical, Kalamazoo, Michigan, USA). Samples were then dehydrated in ascending series of ethanol baths (80, 95 and 100%) and finally in butanol for 30 minutes in an automated dehydration station (Microm Microtech, Lyon, France). Samples were then impregnated in liquid paraffin at 56°C (Histowax; Histolab, Gottenburg, Sweden) and embedded at −16°C. Blocks were cut using a standard microtome (Leica RM2255; Leica Biosystems, Nanterre, Ile-de-France, France). Thin histology sections (3 to 5 μm thick) were made perpendicular to the plane of the skin for subcutis implants and in the middle of calvaria defects. Sections were stained by Masson trichrome technique using an automated coloration station (Microm Microtech). This staining combined hematoxylin for cell nuclei (blue/black), fuchsine for cytoplasm, muscle and erythrocytes (red), and light green solution for collagen (green). Stained slices were scanned (NanoZoomer; Hamamatsu, Photonics, Hamamatsu City, Shizuoka Prefecture, Japan) and observed with the virtual microscope (NDP view; Hamamatsu). Histomorphometry of images were processed on the whole implant sections using Image J software (National Institute of Health, Bethesda, Maryland, USA) and the percentage areas of biomaterial, bone tissue, and bone marrow per implant were calculated.
Nondecalcified histology and backscattered scanning electron imaging
Fixed explants were dehydrated in ascending graded ethanol series followed by one bath of pure acetone and then impregnated with methylmethacrylate for 4 days at 4°C. Each resulting block was cut in half with a circular diamond saw (Leica SP1600) and polished with 4,000-grit silicon carbide sandpaper. Samples were sputtered with a thin layer of gold–palladium (JEOL JFC - 1100E; JEOL, Akishima, Japan) and backscattered electron images were taken using a scanning electron microscope, operating at an accelerating voltage of 15 kV (HITACHI TM-3000; HITACHI, Tokyo, Japan).
In situ hybridization
In situ hybridization using the human-specific repetitive Alu sequence, which comprises approximately 5% of the total human genome, was performed for identification of human cells as described previously [27] with minor changes. Analysis was performed on ectopic explants of 50 mg BCP particles with or without 2 × 106 of passage 2 BMSCs. Briefly, sections were treated with 3% hydrogen peroxide for 15 minutes at room temperature, then with 10 μg/ml proteinase K (Sigma-Aldrich, Lyon, France) for 10 minutes at 37°C, followed by 0.25% acetic acid in 0.1 M triethanolamine, pH 8.0 for 20 minutes at room temperature. Prehybridization was performed for 3 hours at 56°C in a hybridization buffer containing 4× SSC (Sigma-Aldrich), 50% deionized formamide, 1× Denhardt’s solution, 5% dextran sulfate, 100 μg/ml salmon sperm DNA and molecular-grade water. Hybridization buffer was refreshed with the addition of 70 nM custom DIG-labeled human locked nucleic acid Alu probe 5DigN/5′-TCTCGATCTCCTGACCTCATGA-3′/3DigN (Exiqon, Vedbaek, Denmark) and then target DNA and the probe were denatured for 5 minutes at 95°C. Hybridization was carried out for 19 hours at 56°C. The hybridized probe was detected by immunohistochemistry using biotin-SP-conjugated IgG fraction monoclonal mouse anti-digoxin (Jackson Immunoresearch, West Grove, Pennsylvania, USA) diluted 1/200 in Tris-buffered saline with Tween, 2% bovine serum albumin for 35 minutes at 37°C. Stretoperoxidase was added (1/200 in Tris-buffered saline with Tween) for 45 minutes at 37°C before diaminobenzidine substrate addition (Dako, Les Ulis, Ile-de-France, France). Sections were counterstained with Gill-2 hematoxylin (Thermo Shandon Ltd, Runcorn, UK).
Bioluminescence imaging of luciferase-expressing BMSCs
To monitor the biological activity of BMSCs implanted subcutaneously, BMSCs were genetically modified using lentiviral units to co-express luciferase and green fluorescent protein (eGFP) reporter genes as described previously [28]. Briefly, cells were amplified in alpha minimum essential medium supplemented with 8% human PL, 100 U/ml penicillin and 100 μg/ml streptomycin, 1 U/ ml heparin and 1 ng/ml basic fibroblast growth factor. The percentage of luciferase and eGFP expressing cells (Luc/eGFP MSCs) was quantified by measuring the eGFP expression by flow cytometry (FC-500 Flow cytometer; Beckman Coulter, Nyon, Switzerland). Luciferase activity was measured for 1 × 105 cells in a 96-well plate with 100 μl lysis substrate buffer (Steady-Glo Luciferase Assay; Promega, Charbonnieres-les Bains, France) and 100 μl culture medium using a VICTOR plate reader (Perkin Elmer, Waltham, Massachusetts, USA). Cells in passage 8 were used for bioluminescence imaging (BLI) experiments. The experimental group consisted of 1 × 106 Luc/eGFP MSCs in unison with 25 mg BCP implanted subcutaneously in nude mice (n =6) as described above. A control group consisted of a subcutaneous injection of 1 × 106 Luc/eGFP MSCs on the right dorsal side of mice and 25 mg BCP implanted alone on the left dorsal side (n =6). Luciferase activity was monitored at days 0, 2, 4, 8, 11, 14, 18, 21, 28 and 37 using a photon imager (Biospace, Paris, France). Before each acquisition, 3 mg luciferin-D in 250 μl distilled, sterile water was injected intraperitoneally. The BLI results are expressed as counts per minute.
Statistical analysis
All experiments were blinded and data are expressed as mean ± standard error of the mean. Statistical comparison between groups was performed using a one-way analysis of variance. Minitab statistical software was used (Minitab 16; Minitab Ltd, Coventry, UK). Statistical significance was set as P <0.05.