Porous GEL/AP nanocomposites were fabricated as follow: at first, gelatin (Merck, microbiology grade, catalogue number 104070) was added to deionized water, making a 10% (w/v) solution. Hydroxymethy-laminomethane (Tris) (Merck, catalogue number 8382) and hydrochloric acid (HCl) (Merck, catalogue number 280211) were added to solution to prepare a buffer gel solution adjusted to pH 7.4. Disodium hydroxide phosphate (1 M) (Merck, catalogue number 106573) and calcium chloride (0.6 M) (Merck, catalogue number 2380) were prepared and the pH of both solutions were brought to about 7.4 using Tris and HCL. In the next step, the GEL-Calcium containing solution was poured into a cylindrical mold and kept at 4˚C for 2 hours until physical gelatin occurred. Afterward, gel was extracted and soaked into the phosphate containing solution and incubated at 4˚C for about 48 hours. Following diffusion of ions into the gel, a white precipitate formed within the gel in a gradient manner from the border to the center of gel. The resulting nanocomposite was extracted and freeze dried to create a porous structure through solvent sublimation. Scaffolds were incubated in a 1% glutaraldehyde (GA) (Merck, Catalogue number 820603) solution for 24 hours. Samples were washed with deionized water to remove remnants of the GA.

The morphology of the nanocomposites was assessed by scanning electron microscopy (SEM; Philips XL30 microscope). The images were obtained using an accelerating voltage of 15 kV. All samples were coated with a gold sputtering device (EMITECH K450X, England) before being investigated under the scanning electron microscope. The obtained images were also used to evaluate the pore size of the nanocomposites.

The endometrium derived from stem cells isolation and culture were obtained from a biopsy sample of the endometrium collected during the phase of proliferation of a fertile of 26 years woman who had not undergone a hormonal exogenous treatment during at least 3 months before surgery. Informed consent was obtained and approval was granted by the ethics committee of the University of Tehran of Medical Sciences. The sample was taken in buffered HEPES Dulbecco Modified Eagle Medium/Hams F-12 (DMEM/F-12; Invitrogen^®) or DMEM glucose high (DMEM/High; Invitrogen^®) supplemented with 10% fetal bovine serum (FBS; Invitrogen^®), stored at 4˚C. and treated within 24 h.The sample was washed twice in buffer saline phosphate (PBS), finely chopped with a (scalpel and placed in a Falcon tube 15 ml and incubated in 10 ml of collagenase Worthington^®) for 45 min at 37˚C in a water bath. The supernatant was eliminated with a pipette Pasteur sterile, washed once with 7 ml of DMEM/F-12 supplemented with FBS to 10% in 15 ml of a hawk, and sedimented by centrifugation at 400 g for 5 min at temperature ambient. Cells were then plated in DMEM/F-12 (5 ml) supplemented with 10% FBS, 100 IU/ml penicillin (Invitrogen^®) and 100 IU/ml streptomycin (Invitrogen^®) in vials plastic (25 cm²), and maintained in a humidified atmosphere of 5% CO₂ in air at 37˚C. The culture medium used for the development was first changed every 72 h and regularly replaced twice by week thereafter.

The hESCs were first trypsinized and counted. Tubes containing about 1 × 10⁶ cells were incubated on a rocking and centrifuged at 300 g for 6 minutes, then 2% human serum added to the repository. Mixtures resulted remained at room temperature for 30 minutes and centrifuged again as above. In addition to the PBS was added to the repository. The cell mixture was passed through a nylon mesh, 100 pl of the mixture was added to each tube with the following antibodies: anti-CD105, anti-CD73, anti-CD45, anti-CD166, anti-CD90 and anti-CD34 (all products Abcam). In addition, these tubes were incubated at 4˚C in a dark room for 45 minutes. After the washing process, the cells were fixed in 100 ul of 1% paraformaldehyde. Finally, flow cytometric analysis was performed. The ratios of fluorescence signals from scattered signals were calculated by the flow cytometer (Partec). Histograms were generated using the software WinDmi 2.9.

After two passages, when the cells (hESCs) reached almost confluence completely, then they were digested with 0.25% trypsin/EDTA (Gibco), counted and seeded on scaffolds at a density of 1 × 10⁵ cells/ml in plate wells 6-flat bottom. The samples were sterilized by exposure to UV light for 2 hours on both sides facing cell seeding. Cultures were maintained in an incubator at 37˚C in an atmosphere of 5% CO₂/95% air, and the cell culture medium was changed every 3 days. The MTT assay is a rapid color to determine the number of viable cells, which is based on the mitochondrial conversion of MTT to formazan. Briefly, the cell containing the sample was rinsed with serum-free medium to remove cells only 200 µl and serum-free medium and 20 µl of MTT stock solution (5 mg/ml in RPMI 1640) were added to each sample and incubated for 4 h (37 c) MTT fomarazan training. After adding 200 µl of DMSO solution was used for the density (OD) measurement with optical absorbance detection microplate reader at 570 nm (Rayeto, USA).

Adipogenic differentiation: 1 × 10⁵ hESCs of the 5th to 8th passages were treated with adipogenic medium for 14 days. Adipogenic medium consisted of IMDM supplemented with 0.5 mmol/L 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1 mol/L hydrocortisone (Sigma-Aldrich), 0.1 mmol/L indomethacin (Sigma-Aldrich), and rabbit serum 10% (Sigma-Aldrich). For the evaluation of adipocytes, cells were fixed with 4% formaldehyde and stained with oil red O (Sigma).

Osteogenic differentiation: 1 × 10⁵ hESCs of the 5th to 8th passages were treated with osteogenic medium for 3 weeks. Osteogenic medium consists of DMEM-LG (Gibco) supplemented with 50 lg/ml ascorbate-2 phosphate, 10-8 dexamethasone, and 10 mM b-glycerophosphate (all from Sigma, St. Louis, MO). Osteogenesis was assessed by staining with alizarin red. For the evaluation of the mineralized matrix, and induced hESCs were treated with silver nitrate 2% (Sigma) under UV radiation for 1 h. The cells were fixed with sodium thiosulfate 2.5% (Sigma) for 5 min and washed in dH₂O. The cells were then stained with cons-1% neutral red (Sigma) for 1 min (Chen et al.).

Neurogenic differentiation: 1 × 10⁵ hESCs of the 5th to 8th passages were treated with serum-free medium modified neurogenic selection for two weeks. Serum-free medium for the selection of neural precursor cells contained DMEM/F12 1:1 (Gibco) and was supplemented with 0.6% glucose, 25 pg/ml insulin, 100 µg/ml transferrin, 20 nM.

Twenty male Wistar rats with starting weight 200 - 250 gr were allowed to acclimatise for 7 days. They were maintained in individually ventilated cages at 23˚C ± 2˚C with a 12:12 h light-dark cycle and food and water were provided ad libitum. The rats were anesthetized i.p. with a mixture of ketamine (85 mg/kg) and xylazine (15 mg/ kg) were fixed on the stereotax. Scalps were then shaved, shaved and prepared for surgery. The skull was exposed through an incision in the scalp midline, and the skin and periosteum are reflected laterally border ridges (Figure 1). Default monolateral (n = 10) were created in the parietal bones, using a 5 mm drill burr. Great care has been taken to ensure that the time was left intact because of its osteoinductive properties. The defects were randomly filled with one of two treatments: 1) Gel/Ap scaffold alone (n = 10), 2) the absence of treatment (default empty) (n = 6), 3) the endometrium Stem cells grown in Gel/Ap scaffolds (n = 10). The implants were held in place with components of the periosteum and the scalp incision was closed with sutures.

8 weeks after surgery, all rats were euthanized by IV injection (40 mg/kg) of pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL) and the calvaria were harvested for radiographic and histologically. Radiographs were obtained by Faxitron (MX-20 X-Ray Faxitron Corporation, Chicago, IL). Radiographs were seen under a microscope Leica MZ12 Stereo Zoom and Northern Eclipse (v 5.0) software image analysis (Empix Imaging, Inc., Cheektowaga) was used to draw the perimeter of each defect and evaluate the total area. Means and standard deviations for fault zone were calculated and compared between groups using, One-way analysis of variance (ANOVA). All data were analyzed using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL). Differences were considered significant if P value was less than 0.05. The samples were then fixed in 10% buffered formalin neutral (Sigma), demineralized in EDTA (Sigma) solution, dehydrated in a series of washes alcohol and embedded in paraffin.

The samples were cut in the coronal plane at a thickness of 5 - 7 m and stained with hematoxylin and eosin for conventional qualitative analysis Light field optical microscopy.

SEM was used to evaluate the morphology of the nanocomposites. Figure 1 shows images captured from the surfaces of prepared porous nanocomposite scaffolds with SEM. As shown in Figure 2, a network of interconnected pores with a fairly uniform honeycomb-like shape can be seen. These scaffolds have pore diameters ranging from 150 to 350 µm, which are optimal for bone cell growth. The formation of distributed clusters of precipitates, including spherical agglomerated nanoparticles within the GEL matrix could be observed from the taken micrographs.

The MTT results after 48 hours showed that 96% of hESCs are alive in the presence of Gel/Ap nanocomposite scaffold which confirms the biocompatibility of the prepared scaffold.

The rats were anesthetized with a mixture of ketamine and xylazine and CT-Scan 3 tesla using Bone gread software was performed after 8 weeks of surgery to evaluate the rate of bone repair. The amount of healing, bone repair and their position for the group with scaffold only and the group with hESCs are shown in Figures 3(a) and (b). The whole defect was repaired in scaffold with hESCs (Figure 4).

Mild inflation was found in scaffold with hESCs (group 2). There was a moderate inflation in control group. The foreign body reaction did not report.

The woven and lamellar bones were observed in group 1 and 2, as lamellar bone was greater (Figures 5(a)-(c)).

The amount of new bone was significantly higher in group 2 compared with both group 1 and control (p < 0.05) (Table 1).