Angiogenesis in newly regenerated bone by secretomes of human mesenchymal stem cells

All animal experiments undertaken in this study were performed in strict accordance with the protocols approved by the Guidelines for Animal Experimentation of the Nagoya University School of Medicine (approval nos. 25,374 and 26,063). Human mesenchymal stem cells (hMSCs) were purchased from Lonza, Inc. (Walkersville, MD, USA) and cultured in MSC basal medium (Lonza, Inc.) containing MSCGM SingleQuots (Lonza, Inc.) at 37 °C in 5% CO₂ air. After primary culture, the cells were subcultured at a density of approximately 1 × 10⁴ cells/cm². hMSCs at the third to sixth passages were used for experiments.

hMSCs that were 80% confluent were re-fed with serum-free Dulbecco’s modified Eagle medium (DMEM(−)) containing antibiotic-antimycotic solution. The cell-cultured conditioned medium was collected after 48 h of incubation. The collected cultured conditioned medium was defined as MSC-CM and was stored at −80 °C before being used for the following experiments.

For the tube formation assay, MSC-CM was then concentrated and stored as described. Briefly, MSC-CM was centrifuged for 5 min at 407 g and then for another 3 min at 1630 g to remove any cells. Five milliliters of MSC-CM was mixed with 45 mL of 100% ethanol and incubated at −20 °C for 1 h. The mixture was centrifuged for 15 min at 24,229 g at 4 °C, and the supernatant was discarded. The precipitate was suspended again in cold 90% ethanol and centrifuged for 15 min at 24,229 g at 4 °C. The final precipitate was frozen at −80 °C, lyophilized, and stored at −80 °C until use.

When the tube formation assay was performed, this frozen precipitate was dissolved with optimized angiogenesis medium (AM) at the same concentration of MSC-CM. The concentrations of each growth factor after freeze-thawing did not show significant differences by an enzyme-linked immunosorbent assay (ELISA) (data not shown).

The concentrations of IGF-1, VEGF, and TGF-β1 in MSC-CM were investigated using a Human Quantikine ELISA kit (R&D Systems, Hornby, Ontario, Canada) according to the manufacturer’s instructions. Briefly, 200 mL of MSC-CM, DMEM-0%FBS, or DMEM-30%FBS was added to 96-well microplates that were coated with a monoclonal antibody to the factor of interest and incubated for 2 h. After washing with phosphate-buffered saline (PBS), a horseradish peroxidase-conjugated cytokine or growth factor-specific antibody was added to each well, incubated for 2 h, and washed. Substrate solution was added and incubated for 30 min, and the reaction was terminated by addition of stop solution. Cytokine/growth factor levels were determined by measurement of the optical density at 450 nm, using a microplate spectrophotometer (PowerScan4; DS Pharma Biomedical, Osaka, Japan).

MSC-CM was combined with neutralizing antibodies from a commercial source (R&D Systems) against VEGF (MAB293). The final concentration of the antibodies was 10 μg/mL and, therefore, was 100-fold greater than the concentration for half-maximal inhibition of 10 ng/mL of the recombinant proteins (R&D Systems).

An angiogenesis assay kit (KZ-1000; Kurabo, Osaka, Japan) was used according to the manufacturer’s instructions [11]. This kit comprised a 24-well culture dish in which HUVECs and human diploid fibroblasts were seeded in the optimal conditions for capillary tube formation. The optimized AM in each well was changed on days 1, 4, 7, and 9 with fresh medium containing VEGF (10 ng/mL), MSC-CM, MSC-CM + anti-VEGF antibody, or none. After 11 days, cells were fixed in 70% ethanol and incubated with diluted primary antibody (mouse anti-human CD31, 1:4000) for 1 h at 37 °C and with secondary antibody (goat anti-mouse immunoglobulin (Ig) G alkaline phosphatase-conjugated antibody, 1:500) for 1 h at 37 °C, and visualization was achieved using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. Images were obtained from five different fields (5.5 mm²/field) for each well, and tube length (the total lengths of the tubes) and joints (the number of capillary connections) were quantified using the Angiogenesis Image Analyzer Ver.2 (Kurabo).

Atelocollagen (Terudermis®; Olympus Terumo Biomaterials, Tokyo, Japan), which was cut into the desired form, was suspended in MSC-CM, MSC-CM + anti-VEGF antibody, or PBS, respectively; the following groups were defined: (1) MSC-CM: MSC-CM/Terudermis®; (2) MSC-CM + antiVEGF: MSC-CM + anti-VEGF antibody/Terudermis®; (3) PBS: PBS/Terudermis®; (4) Defect: unfilled defect.

Then, the periosteum and scalp were closed with interrupted 4–0 nylon sutures.

All 8-mm defect samples were perfused with Microfil (Flowtech, Carver, MA, USA) after euthanasia to evaluate blood vessel formation as previously described [12]. Briefly, the chest was shaved and an incision was made from the front limbs down to the xiphoid process. Scissors were used to cut along one side of the sternum, and the rib cage was retracted laterally. The descending aorta was clamped, and an angiocatheter was used to penetrate the left ventricle. The inferior vena cava was incised, and immediately after, perfusion of 20 mL of heparinized saline was started (100 U/mL at 2 mL/min using a syringe pump). A solution of Microfil was prepared in a volume ratio of 4:5 of Microfil:diluent with 5% curing agent. Following perfusion with saline, 20 mL of the Microfil solution was perfused at a rate of 2 mL/min. Finally, the Microfil was allowed to set overnight at 4 °C.

Surgical sites were dissected, fixed in 4% PFA, and subjected to micro-CT analysis using a laboratory X-ray CT device (Rigaku, Tokyo, Japan). Using the software supplied with the instrument, three-dimensional (3D) images were reconstituted. After radiographical assessment, explants were decalcified using K-CX solution (Falma Co., Tokyo, Japan) and were then dehydrated using graded ethanol, cleared with xylene, and embedded in paraffin. The specimens were cut in a sagittal direction to make 5-μm-thick histological sections in the buccal-palatal plane and were stained with hematoxylin and eosin. Histological analysis was performed by light microscopy (CK40; Olympus).

All 4-mm defect samples were collected after 2 weeks. Fresh-frozen sections of these samples were made according to the Kawamoto’s method, using a Multi-Purpose Cryosection Preparation Kit [13]. Cryofilm type 2C was applied to the cutting surface of the completely frozen block, which was cut with a tungsten carbide knife at −25 °C in a cryostat chamber (Leica CM3050S; Leica Microsystems). The section was fixed with 100% ethanol for 10 min and then washed with PBS for 3 min. CD31, a monoclonal mouse antibody (BD Biosciences, San Jose, CA, USA), was used as a marker for rat endothelial cells. CD105, a polyclonal rabbit antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), was used as a marker for rat stem cells. FLK-1, a monoclonal mouse antibody (Santa Cruz Biotechnology), was used as a marker for VEGF-R2. Alexa Fluor 633 conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor 488 conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific) were used as secondary antibodies. After 4′,6-diamidino-2-phenylindole staining, the section was washed with PBS and mounted between a glass slide and the adhesive film. The section was enclosed by mounting the resin SCMM R2 on the glass slide, and the resin was hardened with ultraviolet (UV) irradiation for 1 min by means of the UV Quick Cryosection Mounter (ATTO Bio-Instrument). After fixation, the specimen was observed by fluorescence microscopy (BZ9000; Keyence Co., Osaka, Japan).

All experiments were conducted in triplicate and repeated at least twice. Group means and standard deviations were calculated for each measured parameter. Statistical differences were evaluated with Tukey’s honestly significant difference (HSD) test. Statistically, a value of p < 0.05 was considered significant; p < 0.01, very significant.