A total of 27 male Sprague Dawley (SD) rats (220–240 g; 6–8 weeks) and 27 male Balb/c nude mice (18–22 g; 6–8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The SD rats received free access to food and water and were housed at a temperature of 20–26°C, a humidity of 50–60% and a 12 h light/dark cycle. The Balb/c nude mice were put in individual ventilated cages for mating in the barrier system with ad libitum access to standard murine food and water. The temperature, humidity and light/dark cycle were the same as the rats as mentioned above.

The Institute of Radiation Medicine of the Chinese Academy of Medical Sciences (Tianjin, China) provided an animal experiment platform in which the animal experiments were performed. The protocol was approved by the medical ethics committee of the Institute of Radiation Medicine Chinese Academy of Medical Sciences (Tianjin, China).

BMSCs and UCMSCs were purchased from Cyagen Biosciences, Inc. (Guangzhou, China). Cells were revived and seeded in culture flasks, and maintained at 37°C in humidified atmosphere containing 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM)/F-12 (HyClone^™; GE Healthcare, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Lanzhou Bailing Biotechnology Co., Ltd., Lanzhou, China) and 1% penicillin and streptomycin (P/S; North China Pharmaceutical Group Co., Ltd., Hebei, China). Cells were allowed to expand until they reached 80% confluence. Passage-3 cells were used in the present study.

Routine characterization of the BMSCs and UCMSCs was performed according to the manufacturer's protocol, as described previously (17,18). After reaching 80% confluence, the cells were treated with 0.25% trypsin-EDTA. Cells were incubated with fluorochrome-conjugated primary antibodies against CD34 (cat. no. 555822), CD44 (cat. no. 555479), CD45 (cat. no. 555483), CD73 (cat. no. 550257), CD90 (cat. no. 555596) and CD105 (cat. no. 560539; all from BD Biosciences, San Jose, CA, USA) at 4°C for 30 min. The samples were subsequently measured on a flow cytometer (BD FACSCanto II; BD Biosciences) and data was analyzed with FlowJo Version 7.6 software (Tree Star, Inc., Ashland, OR, USA).

A Cell Counting Kit-8 (CCK-8; Tongren Shanghai Co., Shanghai, China) was used to evaluate the proliferation of BMSCs and UCMSCs. In brief, 2×10³ cells were seeded onto 96-well plates and cultured for 8 days. Every day, designated wells were stained with CCK-8 reagent according to the manufacturer's protocols, the absorbance at 450 nm was determined using a microplate reader.

BMSCs and UCMSCs at passage 3 were cultured in 6-well plates with DMEM/F-12 medium. The next day, the media in the experimental groups were replaced with osteo-inductive medium (OIM) consisting of 50 µM ascorbate-2-phosphate, 10 mM β-glycero-phosphate and 10 nM dexamethasone (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and control groups were replaced with DMEM/high glucose medium (HyClone™; GE Healthcare) with 10% FBS (6). Alkaline phosphatase (ALP) staining was performed using the Alkaline Phosphatase kit (cat. no. 86R-1KT; Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions at day 9. Staining with Alizarin red S (cat. no. A5533, Sigma-Aldrich; Merck KGaA) was performed at day 18. Cells were washed with PBS, fixed with 95% ethanol at room temperature for 15 min and stained with 0.1% Alizarin Red S (pH 4.2; Sigma-Aldrich; Merck KGaA) at 37 °C for 30 min. The medium was replaced every 3 days during culture.

BMSCs and UCMSCs at passage 3 were used in the subsequent experiments. First, the CPC scaffolds (Shanghai Rebone Biomaterials Co., Ltd., Shanghai, China) were cut into an 8 mm diameter round sheet with an average thickness of 2 mm, or to the size of 2×2×6 mm. The CPCs were placed at the bottom of 24-well plates in a 100-µl suspension (1×10⁶ cells/ml) of BMSCs or UCMSCs. Subsequently, the scaffold and cell complexes were cultivated at 37°C with DMEM/F-12 for 24 h. As a control, certain CPC pieces were immersed in medium lacking cells prior to transplantation into the animal model.

A total of 27 mature SD rats were used for the generation of the tibia bone defect model as described previously (19,20). In brief, general anesthesia was performed by administering sodium pentobarbital (40 mg/kg body weight; intraperitoneal injection; Merck KGaA), and a 1–2 cm skin incision was made to separate and expose the tibia following disinfection. A unilateral 2×2×6-mm defect was created in the middle part of the tibia. Rats were then randomly divided into three groups (n=3 per group): The BMSCs+CPC scaffold, the UCMSCs+CPC scaffold and the control group. For the control group, the right side of the rats were implanted with the cell-free CPC scaffold, whereas the left side remained untreated. Finally, the surgical site was sutured. The grafts from each group were harvested at 4, 6 and 8 weeks after the surgery, and these were then evaluated using X-ray analysis, as well as histological and immunohistochemical techniques.

A total of 27 BALB/c-nude mice aged 6–8 weeks were used for the ectopic bone formation experiment (21). Mice were randomly divided into three groups (n=3 per group): BMSCs+CPC scaffold, UCMSCs+CPC scaffold and CPC scaffold (cell-free). Following anesthesia, an incision was made in the dorsal side of the nude mouse and the scaffold construct (8 mm in diameter) was implanted subcutaneously. The wound was then closed in layers. The grafts were harvested at 4, 6 and 8 weeks following surgery and fixed in 4% paraformaldehyde.

Grafts were embedded in paraffin and cut into 5-µm sections. These specimens were divided into two parts. One part was stained with H&E, as described previously (22), whereas the other part was used for immunofluorescence experiments. Osteopontin (OPN; cat. no. sc-21742; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), type I collagen (COL-1; cat. no. ab34710; 1:200; Abcam, Cambridge, UK), Runt-related transcription factor 2 (Runx2; cat. no. sc-10758; 1:50; Santa Cruz Biotechnology, Inc.) and vascular endothelial growth factor (VEGF; cat. no. ab46154; 1:200; Abcam) were assessed according to protocols of previous studies (20,23). Immunohistochemistry was performed using the streptavidin-peroxidase method (SPlink Detection kit; cat. no. SP-9001; OriGene Technologies, Inc., Rockville, MD, USA) according to the manufacturer's protocol. The kit comprised Reagent A (goat serum), B (goat anti-rabbit immunoglobulin G) and C (horseradish peroxidase). Endogenous peroxidase activity was quenched using 1% H₂O₂/methanol for 10 min and antigen retrieval was performed with citrate buffer in the microwave for 10 min. Slides were blocked with reagent A at room temperature for 20 min and stained with the aforementioned primary antibodies overnight at 4°C. Reagent B was then added and incubated at 37°C for 20 min. Reagent C was added and incubated for 15 min at 37°C. DAB (cat. no. ZLI-9019; OriGene Technologies, Inc.) was used for further staining at room temperature for 6 min. Finally, sections were counterstained with hematoxylin for 1 min at room temperature and observed under an optical microscope (magnification, ×100; Nikon Ti-S; Nikon Corporation, Tokyo, Japan) (20). In order to semi-quantitatively analyze the immunoreactivity of OPN, COL-1 and VEGF, the H-score was determined. The H-score was calculated by combining the percentage of immunoreactive cells (%) with an estimate of the staining intensity (0, 1, 2 or 3), as described previously (24). An H-score of 9–12 was considered to represent strong immunoreactivity (‘+++’), 5–8 was considered moderate (‘++’), 1–4 was considered weak (‘+’) and 0 was scored as negative (‘−’) (25,26).

Data were analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Values are expressed as the mean ± standard deviation. One-way analysis of variance and Tukey's post hoc test were used to determine significant differences between the groups. P<0.05 was considered to indicate a statistically significant difference.

Assessment of the growth activities of BMSCs and UCMSCs indicated that they exhibited a similar proliferation rate within all phases of the growth period. In each of the two cases, cells proliferated slowly at first, but then entered the exponential growth phase, which continued for 3–5 days. Finally, the cells became confluent and reached the period of stagnation (Fig. 1).

Flow cytometry was subsequently performed to identify the typical cell-surface markers for MSCs. The results indicated that BMSCs and UCMSCs were positive for the MSC markers CD44, CD90 and CD105, but negative for the markers CD14, CD34 and CD45 (Fig. 2). These results suggested that the expression of cell markers was similar for BMSCs and UCMSCs, and that the expression patterns conformed with the typical surface markers for MSCs.

After having been cultured in OIM for 9 days, the expression levels of ALP were high in the BMSCs and the UCMSCs. After 18 days of culture in OIM, mineralized nodules were observed in the BMSCs and UCMSCs, as demonstrated by Alizarin Red staining (Fig. 3).

To compare the effects of BMSCs and UCMSCs on bone repair, the SD rat tibia defect model was constructed. The bone structure in the defective area was observed by X-ray analysis at 4, 6 and 8 weeks after surgery. Fewer, or no new osseous were observed in the defect area in control group (Fig. 4).

In addition, in the area of the defect, flaky cartilage, bone-like matrixes and lacunae were observed in numerous instances. A certain amount of fibrous tissue and vascular hyperplasia were also observed. Lamellar bone was identified on each side of the bone, which was densely arranged and close to having reached maturity. The surface of the lamellar bone was lined with osteoblasts, and a normal bone marrow cavity was observed at 8 weeks following the surgery in the BMSCs+CPC and UCMSCs+CPC groups. However, in the CPC group, only a small amount of vascular hyperplasia was observed, a large number of neutrophlis had infiltrated into the area of the defect, and comparatively less mature cancellous bone and normal bone tissue was observed in the surrounding area on H&E staining (Fig. 5A).

At 4 weeks after the surgery, the expression of OPN and COL-1 was detected in the BMSCs+CPC and UCMSCs+CPC groups, although the staining lacked structure and definition (Fig. 5B and C). At 8 weeks after the surgery, a markedly increased expression of OPN and COL-1 was identified at the defect site of the experimental cell treatment groups compared with that in the CPC group (Fig. 5B and C). The H-scores for OPN in the BMSCs+CPC and the UCMSCs+CPC groups were greater compared with that in the CPC group (P<0.05), while no significant differences were identified between the BMSCs+CPC and UCMSCs+CPC groups (Fig. 6A). Furthermore, the H-scores for COL-1 in the BMSCs+CPC and the UCMSCs+CPC groups were larger than that in the CPC group (P<0.05), while no significant difference was identified between the BMSCs+CPC and UCMSCs+CPC groups (Fig. 6B). These results indicated that BMSCs and UCMSCs did indeed accelerate the healing of the bone defect, although no significant differences were observed between the two cell types in terms of these effects.

To further evaluate the osteogenic capacity of BMSCs and UCMSCs in vivo, engineering of cell-scaffolds or cell-free scaffolds were implanted under the skin of nude mice. The expression of Runx2 and VEGF on the CPC was detected by immunofluorescence and immunohistochemical analyses. Runx2 is the transcription factor that regulates the differentiation and maturation of osteogenic differentiation. For this reason, the expression of Runx2 provides the hallmark of osteoblast differentiation; it is the earliest one that may be observed and the most specific marker gene in bone formation. In the present study, a stronger intensity of fluorescence staining of Runx2 was observed in the defect areas of the BMSCs+CPC and UCMSCs+CPC groups compared with that in the CPC group (Fig. 7A). An increase in the levels of VEGF was observed in all groups from 4–8 weeks post-surgery (Fig. 7B). At week 8 post-surgery, the H-score of VEGF in the BMSCs+CPC and the UCMSCs+CPC groups were greater compared with that in the CPC group (P<0.05), while no significant differences were observed between the BMSCs+CPC group and the UCMSCs+CPC group (Fig. 6C). Therefore, these results suggested that the presence of BMSCs and UCMSCs led to an elevation in the rate of angiogenesis, and that this effect was not significantly different between the two cell types. The results of the ectopic bone formation experiments therefore exhibited the same trend as those of the experiments using the tibia defect model of bone regeneration.