Study participants
This study involved five consecutive patients with long bone atrophic nonunion (Table 1, Fig. 1). The participants’ average age was 25.4 years (range, 18 to 34 years). There were four males and one female, and the sites of nonunion were tibias in three and the femurs in two. One patient underwent initial surgery using intramedullary locking nail, and the four other patients underwent initial treatment with plate-and screw fixation. There is no evidence that patients have become infected after surgeries. The average interval time between the first and the current surgery for nonunion treatment was 21.0 months (range, 16–27 months). We have excluded the patients with infections, tumors, or some systemic bone-related diseases, or patients who underwent treatment with hormones, steroids, and someone with alcohol abuse, but treatment with nonsteroidal anti-inflammatory drugs is allowed for patients [4]. We obtained some nonunion tissues from the enrolled patients during surgery. All atrophic nonunion were healed at last follow-up through our surgical treatment method [11, 24]. This study was ethically approved by our hospital committee (No.2010-S02) and written informed consent was obtained from the patients or their legal guardians.
Table 1
Case | Gender | Age (years) | Nonunion site | Duration from fracture (months) | Results |
1 | male | 18 | femur | 18 | Union |
2 | female | 25 | tibia | 16 | Union |
3 | male | 21 | tibia | 23 | Union |
4 | male | 29 | femur | 21 | Union |
5 | male | 34 | tibia | 27 | Union |
Isolate and culture human NCs
We got a small amount of nonunion tissues at the nonunion sites after exposing the nonunion sites at the current operation. The nonunion tissues were obtained with attention paid to avoiding contaminating the bone, periosteum, and muscle after exposing the nonunion sites adequately. The NCs were isolated by the method as the previous study carried out [4, 25]. Then we cut the nonunion tissues into small pieces after washing them with phosphate-buffer saline (PBS). The cells were cultured on 100 mm diameter culture dish with complex medium, including α-Modified Minimum Essential Medium (Sigma, St. Louis, MO, USA), 10% heat-inactivated fetal bovine serum (FBS; Sigma), 2 mM L-glutamine (Gibco, Carlsbad, CA, USA), and antibiotics. Finally, the cells were incubated at 37℃ with 5% humidified CO2. The nonviable cells and debris were removed at the seven days after initial incubation. Thereafter, we changed the culture medium twice a week. About two or three weeks later, we obtained the adherent cells with 0.05% trypsin-0.02% Ethylene Diamine Tetraacetic Acid (EDTA) (Sigma, St. Louis, MO, USA), and these adherent cells were preserved to passage for further expansion. The cells from passage three or four were widely applied in the following differentiation assay. All experiments were performed with all the five samples.
Flow cytometry analysis
After the first passage, we had harvested the adherent cells from nonunion tissues. The cell-surface protein expressions of NCs were evaluated using the method of flow cytometry. In order to make a comparison with BMSCs, BMSCs were kindly provided by our hospital laboratory and were cultured in the same medium like NCs’ conditions. The cells were washed with PBS-3% FBS for several times, and then were incubated with phycoerythrin (PE)-conjugated anti-mouse antibodies for 30 minutes at 4℃ in the dark, which includes CD29, CD34, CD44, and CD45 (BD Biosciences, San Jose, CA, USA). We applied the nonspecific mouse PE-conjugated IgG (BD Biosciences) as an isotype control. After the incubation, we have washed the cells with PBS-3% FBS twice, and applied the facsaria flow cytometry system (BD Biosciences) and CellQuest Pro (BD Biosciences) for data analyses. The positive staining of cells was scored as literature reported [19]. At least 10000 list mode events were collected for each sample.
Differentiation studies
Osteogenic induction
Aiming to induce osteogenic differentiation, we cultured the NCs under an osteogenic medium for three weeks, which have added 10 nM dexamethasone (Sigma), 10 mM β-glycerophosphate (Sigma), and 50 mg/mL ascorbic acid (Sigma) into the original medium [4]. However, we cultured the control NCs under the original medium without the osteogenic medium. Three weeks later, we evaluated the calcium deposition which is one common characteristic of osteogenic differentiation, and made a photo record of the cells staining after staining with 1% Alizarin Red S (Sigma).
Chondrogenic induction
Aiming to induce chondrogenic differentiation, we applied the three-dimensional culture structure, and constructed a pellet after centrifuging cells in the polypropylene tube at 2000 rpm for 4 minutes [4]. We cultured the NCs under an chondrogenic medium, which have added 100 nM dexamethasone, 50 ug/mL L-ascorbic acid-2-phosphate (Sigma), 0.4 mM proline (Sigma), 1% ITS+1(Sigma), 10 ng/mL recombinant human transforming growth factor (TGF)-ß3 (R&D Systems, Minneapolis, MN), and 500 ng/mL recombinant human bone morphogenetic protein (BMP)-6 (Sigma) into the original medium [4]. However, we cultured the control NCs under the original medium without chondrogenic medium. Three weeks later, chondrogenic differentiation was evaluated by staining with toluidine blue (Sigma) and the cells staining were photographed.
Adipogenic induction
Aiming to induce adipogenic differentiation, we cultured the NCs under an adipogenic medium for three weeks, which have added 1 uM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 10 mg/mL insulin (Sigma), 0.2 mM indomethacin (Sigma) into the original medium [4]. However, we cultured the control NCs under the original medium without adipogenic medium. Three weeks later, we evaluated the cellular accumulation of neutral lipid vacuoles which is one common characteristic of adipogenic differentiation, and made a photo record of the cells staining after staining with Oil-red O (Sigma).
Western blots analysis
Aiming to evaluate protein expressions which were related to each differentiation events, we obtained the differentiated and undifferentiated NCs. The NCs were collected and washed with PBS three times. The Western blots were implemented as previously described [26]. The cells were lysed with RIPA containing 1 mM protease inhibitor cocktail and 1 mM phosphatase inhibitor cocktail (Boster). We transferred the 30 ug protein samples, which were separated by SDS-polyacrylamide gels, into PVDF membranes. Then we blocked the membranes with 5% bone serum albumin for 1 hour, and incubated with appropriate antibodies at 4℃ overnight. Subsequently, we had incubated blots with horse-radish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Then we used the Western ECL Substrate Kit (Thermo Pierece, USA) to detect the bands. Protein expressions about Alkaline Phosphatase (ALP), osteocalcin (OC), Col Ⅱ, SOX9, lipoprotein lipase (LPL) and PPAR-γ2 were determined by mormalizing to GAPDH, and representative bands were shown.
Statistical analysis
We used the SPSS 21.0 software (SPSS Inc., Chicago, IL, USA) to analyze the data. Measurement data was described as the mean ± standard deviation, and difference was assessed by student’s t test. We considered it statistically significant when the p value was less than 0.05 for all tests.