Bone marrow stromal cell isolation and culture
For the use of animals the NIH guidelines for the care and use of laboratory animals have been followed. Rat bone marrow stromal cells (rBMSCs) were harvested from both femora of four 8 weeks old male Sprague-Dawley rats. Briefly, rats were euthanized using 4% isofluorane in CO2. Both femora were aseptically excised and surrounding soft tissue was removed. Both ends of the femora were clipped, and the bone marrow was flushed from the shaft into DMEM/F-12 cell culture medium supplemented with 10% fetal bovine serum (FBS), fungizone 20 μg/ml and gentamicin sulfate 20 μg/ml (all Life Technologies Inc., Grand Island, NY) using a 5 ml syringe and a 18-gauge needle (both Becton Dickinson & Co, Franklin Lakes, NJ). The antibiotic composition differed from that used in other cell culture procedures described here and was chosen to avoid contamination during the cell harvest. Subsequently, rBMSCs were centrifuged and the cell pellets were re-suspended in DMEM/F-12 medium containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (all Life Technologies Inc., Grand Island, NY). Cells derived from different animals were combined and grown in 75 cm2 cell culture flasks (Becton Dickinson & Co, Franklin Lakes, NJ) at 37°C and 5% CO2 in a humidified atmosphere. Before the cell layer became confluent, cells were detached with trypsin (Life Technologies, Grand Island, NY) and cultured for 2 additional passages. The medium was changed every second day. To induce osteogenic differentiation, 100 μg/ml L-ascorbic acid phosphate (Wako Pure Chemical Industries LTD, RI), 10 mM β-glycerophosphate, and 2.55 mM dexamethasone (both Sigma-Aldrich Corp., St. Louis, MO) were supplemented to the medium.
Collagen type I hydrogel preparation
Purified bovine dermal collagen type I (Vitrogen®, Cohesion, Palo Alto, CA) was used as scaffold material. The hydrogel was formed by collagen fibril self-assembly inside of a trans-well insert (12 mm diameter, 4 μm membrane pore size) (Corning Inc, Corning, NY). Briefly, the pH of the collagen type I solution was adjusted to 7.4 according to the manufacturer’s protocol. Next, liquid collagen type I was transferred to a trans-well insert and filled up to 2/3 of the insert. Collagen type I fibril self-assembly was achieved by subsequent incubation at 37°C in normal atmosphere for 60 minutes. Collagen type I gels were sterilized over night using anprolene sterilizing gas ampoules (Anderson Products, Inc., Haw River, NC). After rinsing the collagen type I hydrogels two times with 1x phosphate-buffered saline (PBS), the hydrogels were conditioned for 12–24 h using cell culture medium.
Preparation of cell-seeded collagen type I hydrogels and sample processing
Conditioned collagen type I hydrogels and control tissue culture plates were seeded with 5 × 104 rBMSCs/cm2. Collagen type I constructs were harvested after 7, 14, and 21 days. Cells from control cultures were harvested and count by day 7 and by day 14. For each time point, 9 hydrogels were seeded with rBMSCs and all assays were performed in triplicate. For subsequent DNA quantification and measurement of alkaline phosphatase activity, hydrogels were immediately frozen at −80°C. For cryosectioning and subsequent histomorphometric analysis, hydrogels were embedded in Tissue-Tek® O.C.T. Compound 4583 (Sakura Finetek, Torrance, CA) and stored at −80°C until analysis. Laser scanning confocal microscopy was performed using fresh and hydrated gels immediately after labeling with the Live/Dead® Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR) as recommended by the manufacturer.
Frozen hydrogels were embedded In Tissue-Tek® O.C.T. Compound 4583 and cut into 6 μm sagittal sections at −20°C using a cryostat (Leica Jung CM 3000, Wetzlar, Germany). Sections were taken from the central portion of three different hydrogels at each time point and air dried at room temperature on a microscope slides (Fisher Scientific, Pittsburgh, PA). Samples were labeled using the Live/Dead® Viability/Cytotoxicity Kit (Molecular Probes, Inc., Eugene, OR), air dried, overlaid with mounting medium, and covered with a cover slip. All samples were protected from light and kept at room temperature until fluorescent microscopy was performed.
For each analytic time point, 6 labeled cryo-sections derived from the center of three different hydrogels (18 sections in total) were visualized using a fluorescent microscope (Olympus BH2-RFCA, Olympus, Melville, NY). The number of cells that remained on the hydrogel surface and the number of cells that migrated into the hydrogel was determined as well as the distance between each migrated cell and the hydrogel surface. Each sample was analyzed in three rows with six fields per row using the Osteomeasure Bone Histomorphometry System 4.0 (Osteometrics Inc., Atlanta, GA).
Three calcein AM/Ethidium homodimer-1-stained hydrogel/cell- constructs per time point were placed on a glass slide (Fisher Scientific, Pittsburgh, PA), and analyzed using a LSM 510 confocal laser-scanning microscope (Carl Zeiss Inc., Thornwood, NY). Calcein AM was excited at 488 nm by an argon/krypton laser. Emission was selected with a 505–550 nm bandpass filter. Ethidium homodimer-1 was excited at 568 nm by the argon/krypton laser. Emission was selected with a 585 nm longpass filter. Images were collected at 512×512 pixel resolution using a C-Apochromat 10x/0.45 water immersion lens. The software used was part of the LSM 510 system.
Alkaline phosphatase assay
In order to determine the commitment of the mesenchymal precursor cells to the osteoblast lineage, alkaline phosphatase (ALP) activity was assayed (Sigma Chemical Co., St. Louis, MO) in 3 samples in triplicate. Briefly, cell culture medium was removed and cell-seeded collagen type I hydrogels were washed twice with 1 × PBS and kept at −80°C until analysis. Cell-seeded hydrogels were thawed and gently homogenized in the trans-well insert using 0.3 ml AP-substrate/AP-Buffer mix (1:1) followed by an incubation at 37°C for 30 minutes. The reaction was stopped after adding 0.3 ml NaOH (50 mM). Aliquots (200 μl) of the samples were transferred to a 96-well plate (Corning Inc, Corning, NY). A standard calibration curve was established using a serial dilution of p-nitrophenol according to the manufacturer’s protocol. Emission was detected at 562 nm using a plate reader (Spectra Max Plus, Molecular Devices, Sunnyvale, CA) followed by the analysis of the excitation at 645 nm. Quantifications were done at least in duplicate. All data were normalized to the total amount of DNA.
To normalize the ALP activity, dsDNA derived from the cell-containing collagen type I hydrogels was quantified using the cyanine dye PicoGreen® according to the manufacturer’s recommendations (Molecular Probes, Inc., Eugene, OR). After excitation at 480 nm fluorescence emission intensity was detected at 520 nm using the plate reader. Samples were analyzed in triplicate.
All results were expressed as the mean ± SEM. Statistical analysis was performed by ANOVA-test with Fisher’s PLSD and paired t-test. P-values < 0.05 were considered statistically significant.