Epithelial cell and fibroblast culture
Rat oral epithelial cells (OECs) and fibroblasts (FBs) cultures were established as previously described [11] (Fig. 1A). Briefly, these cells were individually isolated from oral mucosa from 4-day-old Wistar rats. Mucosal OECs were cultured in defined keratinocyte serum-free medium (DK-SFM; Invitrogen, Grand Island, NY, USA), and FBs from the connective tissue were grown in minimal essential medium (MEM; Invitrogen) containing 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA). This experiment followed the guidelines established by Kyushu University (approval number: A29-227-0).
Scanning electron microscopy observation
The OECs and FBs morphology on CO3Ap or HAp plates (diameter 10.0 mm, thickness 0.8 mm), which was kindly provided by GC (Tokyo, Japan), was evaluated by scanning electron microscopy (SEM) [12] (Fig. 2A). All samples were fixed with 2.5% glutaraldehyde, dehydrated by graded ethanol solutions, and then freeze-dried. Samples were mounted on stubs, coated with an Au/Pd alloy, and evaluated microscopically.
Evaluation of OEC and FB adhesion
Adhesion assays were performed following methods described in our previous report. Briefly, non-adherent cells were removed by shaking the specimens using a rotary shaker (NX-20; Nissin, Tokyo, Japan), and adherent cells were counted.
Collagen production from FB
To quantify collagen production, a colorimetric assay based on Sirius red staining was performed after culture with CO3Ap and HAp plates or on culture dishes. Samples were washed with PBS and treated with 0.2% aqueous phosphomolybdic acid, then stained with Sirius red dye (Wako Pure Chemical Industries, Osaka, Japan) dissolved in saturated aqueous picric acid (pH 2.0). The samples were then treated for 30 min with 0.1 N sodium hydroxide to de-stain and release bound dye into the solution. The optimal density (OD) of this solution was then identified using a spectrophotometer at 550 nm, with 0.1 N sodium hydroxide as the blank.
Transwell analyses
Cells were co-cultured indirectly with materials using a Transwell® insert as a separator. Briefly, OECs and FBs were cultured on a culture well and then Transwell inserts with or without materials (CO3Ap and HAp) in the upper chamber served as experimental or control groups (Fig. 3) for the various assays described below.
Proliferation analyses
OECs or FBs were cultured in the bottom chamber on a Transwell system. As shown in Fig. 1A, the upper chamber contained CO3Ap or HAp. Cell proliferation was detected using a Cell Proliferation Kit (GE Healthcare). Briefly, OECs or FBs were exposed to 5-bromo-2′-deoxyuridine (BrdU) in culture medium for 24 h, fixed in 70% methanol for 30 min, and incubated with an anti-BrdU antibody for 1 h. The cells were then incubated with FITC-conjugated anti-mouse IgG (Thermo Fisher Scientific; 1:100) and counted.
Fluorescence-activated cell sorting (FACS) analyses
For apoptosis analyses, OECs or FBs on CO3Ap, HAp plates, or cultures dishes were incubated with Annexin-V-FITC and 7AAD-peridinin chlorophyll protein (Apoptosis Detection Kit; BD Biosciences Ltd., Franklin Lakes, NJ, USA). The analysis was carried out using a FACSCalibur system (BD Biosciences Ltd.) [13].
Migration assays
As previously described [14], confluent monolayers of OECs or FBs were wounded with a cell scraper and cultured for 48 h. OECs or FBs at the edge of the wound were observed by immunofluorescence using antibodies against actin for cell visualization.
Animals
Rats received care following the guidelines established by Kyushu University (approval number: A29-227-0). Extraction and transplant was performed as previously reported [12]. Briefly, 6-week-old Wistar rats (72 males; 120–150 g) had extraction of the maxillary right first and second molars under systemic anesthesia. The extraction socket was enlarged with a dental round bar, and the CO3Ap (Cytrans Granule, GC; particle size 0.3–0.6 mm) or HAp bone substitute (Bonetite granule perio, HOYA Technosurgical, Tokyo Japan; particle size 0.3–1.0 mm) was implanted [12].
Micro-CT
After the rats were killed, their maxillae were corrected, and fixed in 10% paraformaldehyde (Merck, Darmstadt, Germany) for 24 h. Micro-CT imaging was performed (SkyScan 1076; Bruker micro-CT; tube current: 201 μA; voltage: 49 kV; pixel size: 18 μm) and three-dimensional analysis software (CTAn, Bruker micro-CT) was used for analysis (Fig. 4A).
Wound healing
The length of distance between the edges of the epithelial surfaces was calculated for three sections of the maxillae, namely the midsection of the extraction socket and sections 100 μm mesial and distal to the midsection. All measurements were done three times and the average calculated.
Histochemistry with light microscopy
The oral mucosa from the rat was cut into sections on the coronal plane using a cryostat. For immuno-histochemical staining, these sections were stained with rabbit anti-Ln-332 (Chemicon International Inc., Temecula, CA, USA), biotinylated anti-rabbit IgG (Sigma, St. Louis, MO, USA), and visualized by a diaminobenzidine (DAB) staining kit (Vector Laboratories, Burlingame, CA, USA) as previously reported [13]. Some sections were stained with Ladewig’s fibrin stain to observe the connective tissue (Fig. 5A).
Statistical analysis
Our experiment used six samples in each group, and an a priori Shapiro–Wilk test was performed to test for normality. One-way analysis of variance (ANOVA) with Scheffe’s post hoc was performed. Values of p < 0.05 were considered statistically significant. Data are indicated as the mean ± standard deviation (SD).