Biphasic calcium phosphate (BCP) biomaterials, comprised of hydroxyapatite and beta-tricalcium phosphate in a ratio of 20:80 by weight, ranging in size from 0.5 to 1 mm, were supplied by Biomatlante (Vigneux de Bretagne, France). These materials were chosen as they have previously been successfully employed in preclinical5,36 and clinical trials8,43 as delivery vehicles for stromal cell therapy. The overall porosity (% volume) was 75% ± 5%, with a pore size distribution of 0–10 µm (70%), 10–100 µm (20%), and 100–300 µm (10%). Biomaterials were steam sterilized by autoclaving.
Isolation and characterization of MSCs
Bone marrow (BM) aspirates were acquired from the iliac crest of healthy donors (22.66 ± 4.51 years old; mean ± standard deviation), by the standard puncture and aspiration method in heparinized syringes. BMSCs were isolated from mononuclear cells cultured at 1×104 cells/cm2 by plastic adherence as described previously6. CBMSCs were isolated from mononuclear cells cultured at 1×106 cells/cm2 by plastic adherence as previously described44,45. Written informed consent was obtained from all the donors involved in the study. All research was performed in accordance with relevant guidelines/regulations. Experiments involving human cells were conducted according to the amended Declaration of Helsinki. Ethics evaluation was attained by the Ethical Committee of Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico n° 1982, 14th January 2020. Passage 4-5 MSCs from 3 donors of BM and 3 donors of CB were used. Cells were expanded ex vivo by the established plastic adhesion 2D culture method. Basal culture medium consisted of αMEM GlutaMax, supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin, and 20% FBS.
The morphology of MSCs was observed by fluorescent microscopy. Cells were fixed in 4% paraformaldehyde (PFA) and cell membranes were permeabilized with 0.1% Triton X-100 and 0.2% Tween in PBS followed by incubation with 1% BSA and 5% goat serum in PBS to reduce non-specific background staining. The actin cytoskeleton of cells was stained with rhodamine phalloidin (Alexa Fluor 488 Phalloidin, Invitrogen by Life Technologies, Saint Aubin, France) at a dilution of 1/40 in 1% BSA. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) at a concentration of 1/40 000 (Molecular Probes by Life Technologies). For phenotypic analysis of MSCs, flow cytometry was performed as previously described6. Briefly, cells were characterized by using the following antibodies: CD73-PE, CD90-FITC, CD105-FITC, CD3-FITC, CD34-PE, and CD45-FITC; CD3 was sourced from Beckman Coulter (Paris, France), CD73 was purchased from BD Biosciences (Le Pont de Claix, France), while all others were sourced from BioLegend through Ozyme (Paris, France).
Tri-lineage differentiation capacity of BMSCs and CBMSCs was also investigated. To assess osteogenic potential, MSCs were plated at a density of 5 × 103/cm2 in 24 well plates in basal media. After 1 day, differentiation was induced towards an osteogenic lineage by using standard osteogenic supplements (250 μM ascorbic acid, 10 mM β-glycerolphosphate, and 100 nM dexamethasone). Mineralization was detected after 14 and 21 days with a 40 mM alizarin red solution (pH 4.1–4.3), while extracellular alkaline phosphatase (ALP) activity was identified by staining with a mixture of naphthol AS-MX phosphate alkaline solution with fast blue RR salt (85L2-1KT; Sigma-Aldrich, Saint Louis, MI, USA). For adipogenic differentiation, MSCs were plated at a density of 2 × 104/cm2 in 24 well plates. Cells were cultured until 80% confluent in basal media and then induced towards adipogenic lineage by using StemPro Adipogenesis Differentiation Kit (Gibco, Life Technologies, France) according to the manufacturers’ instructions. After 21 days of differentiation, cells were stained with Oil Red O solution in 2-propanol, diluted to 60% using deionized water. Chondrogenic differentiation was induced in pellet culture (0.5 × 106 cells/pellet) by chondrogenic supplements (10 ng/mL TGF-b3, 50 mg/mL ascorbic acid, 4.7 mg/mL linoleic acid, 100 nM dexamethasone and 1× insulin-transferrin-selenium) for 21 days after which sections through pellets were stained with Alcian blue to identify glycosaminoglycans. Population doubling time and cumulative population doublings were assessed and calculated as described in previous studies45,46.
In vitro osteogenic or chondrogenic priming of BMSCs and CBMSCs
Prior to in vivo implantations, BMSCs and CBMSCs were cultured for 6 days in either basal media or media supplemented with; (a) standard osteogenic supplements: 10 mM β-glycerolphosphate, 250 μM ascorbic acid and 10nM dexamethasone, (b) BMP-4 (50 ng/mL), or (c) chondrogenic supplements (10 ng/mL TGF-b3, 50 mg/mL ascorbic acid, 4.7 mg/mL linoleic acid, 100 nM dexamethasone and 1× insulin-transferrin-selenium). Priming with chondrogenic induction factors prior to transplantation was performed in 2D cultures, unlike in the tri-linage differentiation assay. This was performed in order to have the same culture conditions and seeding densities as the other priming conditions. 2D monolayers, while not as efficient as 3D pellet cultures at in vitro chondrogenesis, have been shown to permit chondrogenic differentiation of MSCs47.
In vitro proliferation and intracellular ALP activity of BMSCs and CBMSCs
Cell number was measured by using a fluorescent Quant-iT PicoGreen dsDNA reagent assay kit (Invitrogen) according to the manufacturer’s instructions. Briefly, cells were lysed in a buffer consisting of 0.1% Triton x-100, 5 mM Tris-HCl, pH 8, followed by three freeze/thaw cycles, and the quantity of double stranded DNA was measured in the supernatant of the solutions. Fluorescent intensity was quantified at 485 nm Excitation and 535 nm Emission on a microplate reader (Tristar LB 941; Berthold Technologies, Thoiry, France) and converted to ng of DNA by using a standard lambda DNA solution. Osteogenic differentiation was investigated by intracellular alkaline phosphatase (ALP) expression in cell lysates by using an ALP Colorimetric Assay Kit (Abcam). The ALP enzyme converts the p-nitrophenyl phosphate (pNPP) substrate to an equivalent quantity of coloured p-Nitrophenol (pNP). Colorimetric absorbance was measured at 405 nm on a micro-plate reader.
Cytokine secretion by multiplex array analysis
Soluble secretions in MSC conditioned media was assessed by using Multiplex Arrays as per the manufactures guidelines (Millipore). Briefly, a Human Cytokine/Chemokine and a Human Bone Magnetic Bead Panels were employed, samples and standards were incubated with anti-immobilized beads, then with detection antibodies and streptavidin-phycoerythrin. All wash steps were performed on an automatic magnetic washer.
Gene expression by qRT-PCR
Total RNA was isolated using RNeasy Plus Mini-kit (Qiagen, Hilden, Germany), following manufacturer’s instructions. First strand cDNA was synthesized from 800 ng of total RNA in a 20 μL final volume reaction, using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. Real-time qRT-PCR was carried out using SsoFast EvaGreen Supermix (Bio-Rad Laboratories) in the CFX96 real-time PCR Detection System instrument (Bio-Rad Laboratories). To confirm product specificity, a melting curve analysis was performed after each amplification. Gene expression was calculated by the ΔΔCt method, using TATA-binding protein (TBP) as house-keeping gene. Bio-Rad CFX Manager software was used for expression data generation. Primer sequences will be provided upon request.
Attachment of MSCs to biomaterial prior to implantation
Scanning electron microscopy (SEM) was used to visualize the attachment of hMSCs onto the surface of the BCP particles after 1 h, the time allowed for cell attachment prior to in vivo implantation in this study. Cells were fixed with 4% paraformaldehyde (PFA), washed with PBS and dehydrated in graded series of ethanol: 50%, 70%, 95%, 100%. Samples were then mounted on aluminium stubs, sputter coated with gold, and viewed with a SEM (Hitachi 3000, Tokyo, Japan) operating at an acceleration voltage of 5 kV. Cell attachment was also verified by methylene blue staining and a stereomicroscope (Zeiss, Stemi 2000-C, Germany). Briefly, cells were fixed in 4% PFA, rinsed in PBS and stained with 1% (w/v) methylene blue (in dH20).
Culture of MSCs on BCP biomaterial
MSCs were resuspended at a concentration of 3.75 × 106 cells/mL in MSC basal medium. A volume of 80 μL of cell suspension was then seeded onto 50 mg of BCP biomaterial deposited at the bottom of 15 mL tubes (n = 4 experimental replicates each). MSCs were allowed to adhere for 4 h at 37 °C, 5% CO2, after which additional MSC basal medium was added. Cultures were maintained for 6 days, changing medium on day 4 to collect 48 h conditioned medium at day 6. Conditioned media was centrifuged at 300×g for 10 min and analysed. To take into account background from cell culture medium, the same experimental conditions were applied to BCP incubated with MSC medium only (n = 3). One out of four MSCs-BCP construct replicates was dedicated to microculture tetrazolium (MTT) assay to normalize the levels of secreted proteins to cell number. Briefly, MSCs-BCP constructs were washed with PBS, incubated with 1 mL of 0.5 mg/mL of Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich) for 2 h at 37 °C, 5% CO2. Next, after another PBS washing step, MSCs-BCP constructs were incubated with 100 μL of 96% ethanol to extract the formazan crystal. The absorbance of dissolved crystals was read at 570 nm on the GENios microplate reader (TECAN, Männedorf, Switzerland) running Magellan software (TECAN).
RNA extraction was performed from three replicates for each MSC population with Trizol (Fisher Molecular Biology, Rome, Italy), following standard procedures with modifications48. RNA concentration and quality were verified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Retro-transcription was performed with SuperScript IV VILO Master Mix (Thermo Fisher Scientific), while qRT-PCR was carried out as described above, using Power-Up SYBR Green Master Mix (Thermo Fisher Scientific), following manufacturers’ indications.
To quantify extracellular ALP, 100 μL of conditioned medium was incubated with 1 μL of AP Live Stain (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C. Background control medium alone or supplemented with 1–0.5–0.25 μL of 1 U/μL of Anza Alkaline Phosphatase (Thermo Fisher Scientific) was used as negative and positive controls, respectively. All samples were analysed in duplicate. Fluorescence was read on the GENios microplate reader, using 485/25 nm excitation and 535/35 nm emission filter set. To quantify IL-8 and IL-6 cytokines, pre-coated ELISA kits (Peprotech, Rocky Hill, USA) were used, following manufacturer’s instructions.
MSC transplantation into the subcutis of nude mice
All animal experiments were conducted in keeping with the Directive 2010/63/UE and ARRIVE guidelines and after approval (CEEA.2012.199) of protocols from the local and national ethical committee (CEEA, Pays-de-la-Loire, France). 4–10 implants were prepared per group, using cells from six different human donors: three for BM and three for CB. These were the same donors as those used for in vitro experiments. BMSCs and CBMSCs in passage 4–5 with/without priming were suspended in basal media and mixed with BCP granules (2 × 106 cells/50 mg BCP for each implant) for 1 h prior to implantation in order to allow cells to attach to the biomaterial. This cell/biomaterial ratio was previously determined to be optimal4. BCP biomaterials alone served as negative controls. Immunocompromised nude female mice (RjOrl: NMRI-Foxn1nu/Foxn1nu) at 4 weeks of age were purchased from Janvier Labs, Saint-Berthevin, France, were placed under general anaesthesia by inhalation of isoflurane. Two subcutaneous implants were performed on the dorsal side of each mouse. After 8 weeks, animals were sacrificed and implants were excised and fixed in 4% formal buffered solution.
Histology and histomorphometry
Explants were decalcified in a PBS solution containing 4.13% EDTA/0.2% PFA, pH 7.4 at 50 °C using a decalcifying microwave apparatus (KOS Histostation, Milestone Med. Corp. USA). Samples were dehydrated in ascending series of ethanol and butanol in a dehydration station (MicromMicrotech, Lyon, France). Samples were embedded in paraffin (Histowax; Histolab, Gottenburg, Sweden) and thin sections (3 µm thick) were stained by the Masson’s trichrome technique, staining cell nuclei blue/black with hematoxylin, cytoplasm, muscle and erythrocytes red using fuchsine, and collagen green using light green solution. Sections were scanned (NanoZoomer; Hamamatsu, Photonics, Hamamatsu City, Shizuoka, Japan) and observed using a virtual microscope (NDP view; Hamamatsu). Histomorphometry of images was completed using ImageJ with the color deconvolution plugin, and percentage of bone and bone marrow were calculated per area of explants.
IHC of CD146 to identify blood vessels and Vimentin to identify engrafted human cells was performed. For both protocols paraffin embedded tissue samples were cut with a microtome and adhered to poly-lysine slides, dewaxed, and rehydrated. Heat-mediated antigen retrieval was performed over night with Tris-EDTA (pH 9) at 60 °C, followed by quenching of endogenous peroxidase activity by 3% hydrogen peroxide. Nonspecific binding sites were blocked with 2% goat serum, 1% bovine serum albumin (BSA). All washing steps were conducted using T.B.S. 1× pH = 7.4 Tween 0.05%. Neovascularization was identified with the CD146 rabbit monoclonal antibody which identifies pericyte cells (ab75769; Abcam) at a dilution of 1:400 was used, followed by incubation with the goat anti-rabbit secondary antibody at a dilution of 1:400 (E0432; Dako). The engraftment of human cells at the implant site was assessed by using an antibody against human Vimentin, a protein expressed in mesenchymal cells and their derivatives. Samples were incubated with the primary antibody (Monoclonal Mouse anti-Vimentin clone V9, Dako, M0725) at a dilution of 1:800, followed by incubation with the secondary antibody (goat anti-mouse biotinylated, Dako, E0433) at a dilution of 1:500. For both protocols the target antigen signal was amplified using streptavidin peroxidase (DAKO, P0397), while diaminobenzidine (DAB) was used as the chromogen. All sections were dehydrated and counterstained using Gill’s hematoxylin and mounted using Pertex mounting medium. Sections were scanned (NanoZoomer; Hamamatsu, Photonics, Hamamatsu City, Shizuoka, Japan) and observed using a virtual microscope (NDP view; Hamamatsu). Images were processed using ImageJ and area of staining, number of blood vessel/mm2, and size of vessels were measured in the case of CD146 staining, while for Vimentin the number of implants with human cells present over the total number of implants was identified.
Experiments were performed using three human donors of CBMSCs and three human donors of BMSCs. Data were expressed as mean ± standard error of the mean (SEM), where not specified otherwise. When considering differences between MSCs of each origin, where multiple time points or priming conditions were considered, statistical differences between group means were tested by using either a one-way, or two-way ANOVA, followed by Holm–Sidak’s multiple comparison test. Statistical differences between CBMSC and BMSC groups where experiments were conducted at a single time point or single experimental condition were assessed by an unpaired Student’s t-tests. Statistical analyses were conducted with software package GraphPad Prism. A p-value ≤ 0.05 was considered statistically significant and indicated in the figures following figure legend indications. Statistically non-significant results were specified in the text only.