Approximately 1 cm² of periosteum was removed during surgical procedures, including tooth extraction and periodontal surgery, from otherwise healthy patients visiting the dental clinic of Uijeongbu St. Mary's Hospital. The participants did not have any systemic diseases. We obtained the approval of the Institutional Review Board at Uijeongbu St. Mary's Hospital's College of Medicine and the Catholic University of Korea (UC12TISI0046).

Isolation and culture of stem cells derived from the periosteum were performed as follows. The periosteum was placed in sterile phosphate-buffered saline (PBS; Gibco BRL, Grand Island, NY, USA) containing 1% antibiotic and antimycotic (Gibco BRL). The periosteum was then washed with PBS (Gibco BRL) and cut into 1–2 mm² fragments in sterile conditions. The tissues were digested with 0.06% collagenase type II (Invitrogen Corporation, Carlsbad, CA, USA) and incubated at 37°C in a humidified incubator (Forma Scientific Inc, Marietta, OH, USA) with 5% CO₂ and 95% air for 4 hours. The periosteum-derived cells were filtered through a 70-μm cell strainer (BD Bioscience, Two Oak Park, Bedford, MA, USA), and centrifuged at 1,500 rpm for 5 minutes. The precipitates were suspended in Dulbecco's modified Eagle medium (Gibco BRL) containing 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco BRL) and cultured in an incubator. Each day, the morphological characteristics of cells were observed under a microscope. When necessary, the culture medium was exchanged. Cells not attached to the culture dish were removed, and only the attached cells were cultured. Upon reaching 90% confluence, cells were subcultured with a coenzyme solution composed of trypsin (Gibco BRL) and ethylenediaminetetraacetic acid (EDTA; Gibco BRL). Stem cells were identified by the surface markers STRO-1 (R&D Systems, Minneapolis, MN, USA) and CD146 (R&D Systems) using a fluorescence-activated cell sorter (FACS, BD Biosciences, Bedford, MA, USA). Stem cells were subcultured several times to obtain a sufficient number of cells (Figure 1A). Fluorescent staining was performed to confirm the survival of transplanted stem cells (Figure 1B). In short, the viability of cell spheroids was qualitatively analyzed using a Live/Dead Viability/Cytotoxicity Kit for Mammalian Cells (Thermo Fisher Scientific, Waltham, MA, USA). The cells were washed and suspended in 10 mL of phosphate-buffered saline, followed by addition of 5 μL of 50 mM calcein acetoxymethyl ester solution and 20 μL of ethidium homodimer-1 for 20 minutes at room temperature.

New Zealand white rabbits weighing between 2.5 and 3.0 kg were used as experimental animals. All animal experiments were strictly carried out following the approval of the Ethics Committee for Animal Experiment of Uijeongbu St. Mary's Hospital, the Catholic University of Korea (UJA2017-05A).

General anesthesia was induced with tiletamine and zolazepam (15 mg/kg, Zoletil 50; Virbac Laboratories, Carros, France) and xylazine (5 mg/kg, Kepro BV, Deventer, Netherlands) intramuscularly and hair removal was performed. After disinfection with povidone-iodine (Sung Kwang Pharm, Seoul, Korea), local anesthesia was achieved with a 2% lidocaine solution containing epinephrine (Yuhan Co., Seoul, Korea).

A skin incision was made from the nasofrontal area to the external occipital area, and a periosteal flap was raised to expose the cranium. Four symmetrical circular defects 6 mm in diameter were made using a trephine drill (Figure 2A). The defects were grafted with 1) synthetic bone (β-tricalcium phosphate/hydroxyapatite [β-TCP/HA] with a particle size of 410–1,000 μm and a β-TCP-to-HA ratio of 7:3; Cowellmedi Co., Ltd, Seoul, Korea) and 1×10⁵ MSCs, 2) a collagen membrane (type I collagen membrane, CollaTape^®, Zimmer Dental Co., Carlsbad, CA, USA) and 1×10⁵ MSCs, 3) β-TCP/HA+a collagen membrane and 1×10⁵ MSCs, or 4) β-TCP/HA, a chipped collagen membrane, and 1×10⁵ MSCs (Figure 2B). Efforts were made to apply the same amount of synthetic bone and/or collagen membrane in each group. The stem cells were incubated with the graft material for 1 hour before transplantation. The defects were then covered with a Teflon sheet (W.L. Gore & Associates Inc., Flagstaff, AZ, USA) and titanium screws (Figure 2C). The periosteum was released at its base, and the wound margins were reapproximated and closed with a non-absorbable suture (3-0 black silk; Ailee Co. Ltd, Busan, Korea).

After surgery, all rabbits received subcutaneous injections of antibiotics (4 mg/kg gentamicin) and anti-inflammatory analgesics (1 mg/kg ketoprofen) once per day. An E-collar (Saf-T-Shield, EJAY International Inc., Glendora, CA, USA), was placed to prevent injuries to the surgical field and fresh vegetables, including cabbage, were supplied to promote the animals' recovery. The transplanted cells and scaffold were retrieved 24 hours later using various instruments, including curettes, and the remaining portion was retrieved by pipetting (Figure 2D).

The cells were retrieved through the following procedure. Tissues from the rabbit skulls were placed in a 1.5 mL tube and centrifuged. After removing the supernatant, the cells were washed with phosphate-buffered saline and then treated with collagenase type II (Gibco BRL) at 1 mg/mL for groups 2, 3, and 4, which contained collagen membranes. The cells were incubated for 30 minutes to 1 hour to dissolve the collagen. All tissues were centrifuged twice at 1,200 rpm for 5 minutes. The cells were then mixed with 9 mL of distilled water and 1 mL of ×10 PBS (Gibco BRL) for 15 seconds and then centrifuged at 1,700 rpm for 6 minutes to hemolyze the red blood cells. After removing the supernatant, 1 mL of PBS and 1 mL of trypan blue solution (T-8154, Merck KGaA, Darmstadt, Germany) were mixed. Next, the number of total cells, the number of live cells, and the ratio of live cells were analyzed using an automated cell counter (TC 20TM, Bio-Rad Laboratories, Berkeley, CA, USA). Before transplantation, the fluorescent-stained stem cells were observed with an inverted microscope (IX71; Olympus Co., Tokyo, Japan) and were photographed with a digital camera (DP70; Olympus Co.). A quantitative evaluation was performed by comparing and analyzing the measured values for each defect.

Data were presented as mean ± standard deviation. Statistical analysis was performed using SPSS version 12 for Windows (SPSS Inc., Chicago, IL, USA). The normality of the distribution of the data was tested, and one-way analysis of variance with the Tukey B post hoc test was used to evaluate between-group differences. The significance level of this study was set to P<0.05.

The percentage of viable cells in the tissues was calculated as follows: cellular viability=number of total viable cells/number of total cells collected [18]. In group 2, this percentage was significantly higher than in the other groups (Figure 4).

The rate of cell migration was calculated by dividing the number of total cells collected by the number of total cells transplanted [19]. The number of total cells reflected the sum of the number of transplanted cells that were not lost and the number of cells that migrated from neighboring areas. The cell migration rate was highest in group 2, but a statistically significant difference was not found (P>0.05) (Figure 5).

The cell survival rate was calculated by dividing the total number of viable cells collected by the number of surviving transplanted cells [20]. The values were highest in group 2 (P<0.05). However, there was no statistically significant difference among groups 1, 3, and 4 (P>0.05) (Figure 6).