Biomimetic Nanocomposite Scaffold For Bone Tissue Engineering Biology Essay

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In vitro evaluation of biomimetic nanocomposite scaffold using endometrial stem cell derived osteoblast-like cells

Jafar Ai1,2,3, Mahmoud Azami1*, Somayeh Ebrahimi Barough4, Mehdi Farokhi1, Sahar E.Fard5

1Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

2Research Center for Science and Technology in Medicine, Tehran University of Medical Sciences, Tehran, Iran

3Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran

4Department of Biology, Faculty of Biological science, University of Kharazmi (TMU), Tehran, Iran

5 Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ, USA

______________

*Correspondence Author:

Mahmoud Azami, m-azami@tums.ac.ir

Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran, Tel&Fax:+98-21-88991117

Abstract

Current study encourages the differentiation of human endometrial stem cells into osteoblast-like cells using osteogenic media for potential bone tissue engineering purposes. A biomimetic nanocomposite scaffolds based on GEL/Calcium phosphate were fabricated and behavior of differentiated osteoblast cells was evaluated after seeding on this scaffold. Prepared scaffolds were assessed in terms of attachment, alkaline phosphatase activity, gene expression and proliferation of osteoblast cells. The matrix mineralization was approved by Alizarin red and the treated cultures with osteogenic media and BMP2 were positive for osteopontine and osteocalcin antibodies. RT-PCR confirmed presence of osteopontin, osteonectin and alkaline phosphatase mRNA after differentiation in EnSCs-derived osteoblast-like cells. Also, it has been shown that the biomimetic nanocomposites possess appropriate chemical and physical properties to support the attachment and proliferation of differentiated osteoblast cells.

Key Words: Endometrial stem cells, Osteoblast cells, Biomimetic nanocomposite, Bone tissue engineering

Introduction

A healthy bone has the ability to regenerate if the volume of the defect does not exceed a certain size [1]. In cases involving large defects, bone graft biomaterials can be used to both bridge the defects and to facilitate bone formation in the defective areas [2]. However, all of the mentioned techniques have some limitations such as disease transfer, histo-incompatibilities, limitation of supply of autograft tissue and insufficient mechanical support of synthetic grafts. It has shown that engineered biomaterials can use for bone regeneration as a new treatment [3-8].

A well-designed three-dimensional scaffold is one of the fundamental tools to guide tissue formation in vitro and in vivo. Scaffolds for bone tissue engineering purposes should mimic in part the structure and biological function of the extracellular matrix (ECM) [9]. The scaffolds should provide mechanical support, deliver inductive molecules or cells to the repair site and provide cues to control the structure and function of newly formed tissue [10].

Many techniques have been developed to fabricate three-dimensional porous architectures to fulfill this role, such as by progen leaching [11], phase separation [12], solvent casting/freeze drying [13, 14, 15].

However, many of these scaffolds do not adequately mimic the structure of the natural ECM in terms of architecture, which may be one of the reasons for suboptimal outcomes in generating functional bone tissues. Therefore, in this study we have tried to use biomimetic methods for fabrication of the scaffold. Biomimetic techniques have been used to fabricate bone scaffolds with the ability to mimic the structure and composition of natural bone. Natural polymers such as collagen, gelatin, and mineral compounds similar to those in natural bone (e.g., HA) have been used as biomimetics. In addition to the resemblance of chemical compounds to bone material, the formation and structure of these compounds is of essential importance in designing bone biomimetics. Most biomimetic synthetic processes occur at a temperature (37°C) and pH (7.4) [16-19] that are most similar to those of the human body. However, other studies have tried to more closely imitate the body's biological environment. Diffusion based methods [20-22] and electrophoresis [23] are processes that can be carried out under physiological conditions. Diffusion of calcium and phosphate ions into a gel like matrix closely mimics the process of bone formation in the body, because the gel simulates the cartilaginous precursor template. In the body, ions move to the bone formation area through the vessels' wall and the extracellular matrix to enter the preliminary cartilage through diffusion. The sedimentation phase, shaped inside the gel on the polymer chains nucleates, grows and has an ideal combination with the matrix. Thus, composite formed under these conditions are very similar to natural bone. From an engineering point of view, the mineralized phase in this nanocomposite works as a particulate reinforcing phase. As the particles get smaller and their links to the matrix stronger, they show better mechanical properties.

It is believed that amorphous calcium phosphate (ACP) exists in the bone of vertebrate animals during the biomineralization process [24-28]. ACP plays an important role in the tissue mineralization process. The amount of biogenic ACP produced due to biomineralization in the early stages of bone formation is higher than that in mature adult tissue [27,29]. ACP has excellent bioactivity and good biodegradation rate [30- 32]. Therefore, it can be used as a precursor to prepare the crystalline phases, including HA, tricalciumphosphates (TCP), and calcium pyrophosphates in various forms. ACP remains stable in the dried state, but it transforms into apatite by dissolution and recrystallization in solution and in the presence of moisture [28].

Posner et al. first observed the existence of synthetic ACP during HA synthesis and considered it as an intermediate phase [33]. Many others have tried to synthesize ACP in vitro via addition of inorganic substance such as Mg2+, P2O74-, CO32-, F-, ZrOCl2, and Si [35-37]and biomolecules such as adenosine triphosphate, casein phosphopeptide, and poly-l-lysine [28, 34, 37, 38].

The source of osteoblast cells is the most important cues that should be considered in bone tissue engineering [5]. Recently, several attempts have shown that mesenchymal stem cells (MSCs), hematopoietic stem cells (HSC) and embryonic stem cells (ES) can differentiate into osteoblast cells[8,39,40]. Human endometrial stem cells (EnSCs) are another choice for osteogenic differentiation due to its dynamic nature [41-43].

This study aims to fabricate GEL-ACP nanocomposite scaffold and comprehensively investigate the porosity, physical and chemical properties of the scaffold. Moreover, the differentiation of endometrial stem cells to osteoblast cells was evaluated. Cell viability and activity was also inspected in order to evaluate the ability of these nanocomposites to support cell attachment and proliferation.

2. Materials and methods

2.1. Differentiation of endometrial stem cells into osteoblast cells

2.1.1. Sample collection and culture of Human EnSCs

Endometrial samples were collected from ten reproductive aged women referred to the hospital for infertility treatment. A written informed consent form describing the proce­dures and aims of the study was obtained from each donor in compliance with regula­tions concerning the use of human tissues. Isolation and culture of Human EnSCs were performed based on our previous report [43]. Briefly, the biopsy tissue was washed in Dulbecco's phosphate buffered saline (DPBS), minced and treated with collagenase I type A (2mg/ml, Gibco, USA). Following tissue digestion, epithelial and stromal cells were separated using filtration with a 70µM seive. The cells were then centrifuged and were undergo Ficoll purifi­cation. The isolated cells were cultured in DMEM/F12 medium containing 10% FBS, 1% antibiotic penicillin/streptomycin and 1% Glutamine and then incubated at 37°C with 5% CO2.

To confirm stem cell identification of isolated cells, flow cytometry analysis was conducted.

The list of antibodies used for flow cytometry are as below: FITC- conjugated anti-human CD90, PE-conjugated anti-human CD105, FITC-conjugated anti-human CD44, FITC- conjugated anti-human CD34, and PE-conjugated anti-human CD31, PE-conjugated anti-human CD133. Moreover, FITC-conjugated mouse IgG1 and PE-conjugated mouse IgG1 were used as negative controls (all from Santa Cruz Biotechnology, USA). The intracellular antibody used was OCT4 (Abcam, UK)[43].

2.1.2. Osteogenic differentiation

EnSCs were seeded at a concentration of 2 Ã- 104 cells/ml with 0.5 ml completed DMEM media per well. The follow­ing study groups were examined: Treatment Group, BM2 (10ng/ml) + 10-7 M dexamethasone +50 μg/ml L-ascorbic acid-2-phosphate + 10 mM β-glycerophosphate (10ng/ml) and a control group without osteogenic media and BMP-2. The cells were cultured at 37 °C in 5.5% CO2 up to 28 days. The medium was changed every 3 days and cultures were observed twice weekly with an inverted microscope to evaluate their appearance. After 28 days the cells were stained with Alizarin Red (Sigma, USA) and visualized under microscope.

2.1.3. Alizarin red staining

Alizarin Red stain (ARS) was used to verify the extracellular matrix mineralization and calcification nodules [20]. On day 28, the cultures were fixed in 4% paraformaldehyde for 30 min and then stained with 2% alizarin red (Sigma, USA) at room temperature. Alizarin red stained areas were evaluated via phase-contrast microscope. The results were analyzed qualitatively based on the intensity of the staining and the extension of the AR-stained positively areas (i.e. red spots).

2.1.4. Immunocytochemical analysis

The 28 cultured cells were fixed with 4% paraformaldehyde (Sigma, USA) for 30 min at room temperature. After permeabilization with 0.2% Triton-X 100 (Sigma-Aldrich) for 15 min, the cells were blocked with goat serum5% and incubated with primary antibody anti-osteopontin (mouse anti-human, Santa Cruz, USA) and anti-osteocalcin (mouse anti-human, Santa Cruz, USA) at a 1:200 dilution; at 4°C and 2h with secondary antibody (rabbit anti mouse IgG-FITC, at a 1:200 dilution; Santa Cruz, USA) at 37°C. Between each step, slides were washed with PBS and nuclei staining were performed using 4´, 6-diamidino-2-phenylindole (DAPI, Sigma, USA). Cells were examined by fluorescence microscope (Olympus BX51, Japan).

2.1.5. Reverse transcription-polymerase chain reaction (RT-PCR)

RT-PCR was used to detect the gene expression of the β-actin, alkaline phosphatase (ALP), osteopontin (OP) and osteonectin (ON). Primer sequences for RT-PCR were shown in Table 1. The cells were harvested in extraction solutions for 10 days and then RNA was isolated from the cells. The cells were first lysed with 1ml of TRIzol Reagent (Gibco, USA) and then 0.5 mL chloroform (Sigma, USA) was added to the lysis cells. The samples were supplemented in 0.5 mL absolute ethanol for several minutes and then the amount of RNA pellet was measured by spectrophotometrically (Eppendorf, Germany) at the wavelength of 260 nm. Subsequently, RNA was converted to cDNA according to the manufacturer's commercial protocol (Fermentas, Lithuania). Finally, samples were amplified by incubation at 94°C for 2 minutes followed by cycles of denaturation at 96°C for 30s, annealing at an appropriate temperature (usually between 50 and 60°C) for 45s and extension temperature of 72°C for 45s.

2.2. Fabrication and characterization of nanocomposite scaffold

2.2.1. Scaffold fabrication

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. Hydroxymethylaminomethane (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 gelation occurred. Afterward, gel was extracted and soaked into the phosphate containing solution and incubated at 4° 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. To achieve a fine powder from fabricated scaffolds for some of the characterization methods, the samples were chopped and milled.

2.2.2. Scaffold characterization

2.2.2.1. Porosity measurement

The porosity was measured by liquid substitution method using isopropanol and the following formula:

ε = (V1−V3)/(V2−V1)

Where ε is the porosity of the scaffold; V1 is the volume of isopropanol before submersion of the scaffold; V2, the volume reading of the liquid after submersion of the scaffold; V3, the volume of isopropanol after the liquid was pressed into the pores of the sample and the sample was taken out of the liquid.

2.2.2.2. Scanning electron microscopy (SEM) and elemental map analysis

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 used to evaluate the pore size of the nanocomposites. Elemental map analysis was also used to determine the distribution of minerals formed within hydrogel.

2.2.2.3. X-ray diffraction (XRD)

The prepared powders from the scaffolds before and after incubation at 37°C were analyzed by XRD with a Siemens-Brucker D5000 diffractometer. This instrument uses Cu-Kα radiation (1.540600 Å), with a slit width of 1 mm and was set at 40 kV and 40 mA. For qualitative analysis, XRD diagrams were recorded in the interval 20° ≤ 2θ ≤ 70° at a scan speed of 2°/min; step size, 0.02°; and step time, 1 s.

Crystallographic identification of the phases of synthesized powder was accomplished by comparing experimental XRD patterns to standards compiled by the International Center for Diffraction Data (ICDD); card #09-0432 for HA; card # 09-0077 for dicalcium phosphate dehydrate (brushite, DCPD). The average size of individual crystallites was calculated from XRD data using the Scherrer approximation (Eq. 1):

(1)

Where t is the crystallite size, λ is the wavelength of Cu-Kα radiation (1.540560 Å), and β1/2 is the full width at half-maximum intensity.

2.2.2.4. Transmission electron microscopy

Transmission electron microscopy (TEM; EM208S; Philips, Netherland) was used to characterize the precipitate particles within GEL polymeric matrix. A dilute suspension of scaffold powder was prepared in ethanol and deposited onto a Cu grid, which supports a carbon film. The particle shape and size were characterized by diffraction (amplitude) contrast and by high-resolution (phase contrast) imaging for crystalline materials.

2.2.2.5. Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy was used to evaluate the formation of calcium phosphate within the scaffold using a Bomem MB 100 spectrometer. For IR analysis, 1 mg of powdered sample was carefully mixed with 300 mg of KBr (infrared grade) and pelletized under vacuum. Resulting pellets were analyzed in the range of 500 to 4000 cm-1 at a scan speed of 23 scan/min with 4 cm-1 resolution.

2.2.3. Cell attachment study

Differentiated osteoblast cells were used to evaluate the in vitro cytocompatibility of the scaffolds. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and streptomycin/penicillin 100 U/mL (1%). For seeding, the cells were trypsinized (0.05% trypsin/0.53 mM EDTA in 0.1 M PBS without calcium or magnesium), centrifuged and resuspended in complete culture medium. Aliquots of 200 μl containing 3  105 cells were then seeded on the top of each scaffold sample pre-soaked in the medium. Cells/scaffold constructs were incubated for 4 days at 37°C in a humidified atmosphere containing 5% CO2. Then, the samples were washed twice with phosphate-buffered saline (PBS) prior to fixation. For fixation, samples were soaked in 2.5% glutaraldehyde for 1 h. Post-fixation was performed in 1% osmium tetroxide, and dehydration in a graded acetone series solution. Samples were then freeze-dried and kept dry using silica gel. Subsequently, they were kept in a hood for air drying and used for SEM observation.

2.2.4. MTTassay

The cell viability was analysed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay at days 1, 3 and 5. The test was performed with a 5mg/ml MTT solution prepared by dissolving MTT powder (Sigma, Germany) in warm PBS (37°C). About 1Ã-104 cells/well were incubated into 96-microtiterplates (NUNC, Denmark) and after 24 hours, 20 μl of MTT solution was added to each well and was incubated for 4 h in a 5% CO2 in incubator at 37°C. The solution was then removed and the construct was blotted with filter paper. Finally, 200 μl of dimethylsulfoxide 99.5% (DMSO, Sigma, Germany) was added to each well and the plate was shaken for 5 min with a shaker. For colorimetric reading, 100 μl of this solution was transferred to an ELISA plate and the absorbance was read by an ELISA reader at 570 nm, with the reference wavelength of 690 nm. In this study, five wells of culture plates in the absence of GEL/Calcium phosphate nanocomposite were used as negative controls [tissue culture polystyrene (TPS)].

2.2.5 Alkaline phosphatase production

In this study the ALP activity was measured by commercial kinetic kit (Pars azmun, Iran). Briefly, the cells were seeded into the 24-well culture plates at a density of 2Ã-103 cells/well and then the composite samples were placed into the each well. One blank well was used as negative control group (TPS). The cells were harvested next to each composite sample for 1, 3 and 5 days. At the end of these times, about 10 µl supernatant of each well was removed and mixed with 800 µl of diethanolamin (1.0 Mol/L)/magnesium chloride (0.5 mMol/L) solution accompanied by 200 µl of p-nitrophenolphosphate (10 mMol/L). The ALP activity was measured by the conversion of p-nitrophenylphosphate to p-nitrophenol and phosphate at 37°C and pH 9.8. The absorbance change was monitored by using spectrophotometrically at 405 nm at 37°C temperature. The study was repeated three times.

Statistical analysis

All quantitative data were analyzed using SPSS software. Data are reported as mean values ± standard deviation (SD) and value were considered significant at P<0.05. Statistical comparisons were performed using parametric analysis of variance [ANOVA (Tukey)].

3. Results

3.1. Characterization of isolated human EnSCs

3.1.1. EnSCs seeding

Human EnSCs could be isolated easily by their adherence to plastic flask. After plating up for 24 h, some heterogeneous adherent MSCs appeared in flask. About 10 days later, these cells developed as several clusters, which could be used subsequently for sub-culture. After 3-4 passages, human EnSCs became relatively homogeneous and with relatively elongated or spindle shapes (Fig. 1).

The immunophenotype was based on the flow cytometry analysis of a subset of embryonic stem cell marker (OCT4), mesenchymal stem cell markers (CD90, CD105 and CD44), hematopoietic marker (CD34) and endothelial marker (CD31 and CD133). The result obtained from flow cytometric which was published in our previous report [50], showed that they were positive for CD90, CD105, CD44 and OCT4, and were negative for CD133, CD31 and CD34.

3.1.2. Matrix mineralization and differentiation analysis

Fig.2 represents cultured cells stained with alizarin red after 28 days in differentiation media. As shown, the cells were demonstrated by dark red staining of calcium deposition after exposure to BMP-2/osteogenic media. Immunocytochemistry was performed using antibodies specific to osteoblast cells markers such as osteopointin and osteocalcin (Fig.3). The osteoblast cells obtained from endometrial stem cells cultured in osteogenic media in the presence of BMP2 for 28 days. The cells that weren't undergo osteogenic differentiation (as a control groups). Expression of the osteopointin and osteocalcin were positive in treatment group and no positive result observed in control group (Fig. 3). RT-PCR showed that osteoblast cells expressed phenotypic markers like ALP, osteopontin and osteonectin after incubation time as compared with control group (Fig. 4).

3.2. Scaffold characterization

3.2.1. Porosity measurement

Diffusion of PO43- into the gel resulted in formation of a ring like white precipitates. Porosity percentage of the synthesized scaffold was estimated about 82%. This amount of porosity fall in the range of ideal porosity percentage for a scaffold in tissue engineering.

3.2.2. Scanning electron microscopy (SEM)

SEM was used to evaluate the morphology of the nanocomposites. Fig. 5 shows images captured from the surfaces of prepared porous nanocomposite scaffolds with SEM. As shown in Fig. 5a, 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. Fig. 6 shows the digital image taken from a mineralized sample along with Ca atom map analysis obtained from EDS for the scaffold. Gradated formation of precipitate phase within the hydrogel from the digital image and also gradated distribution of Ca atoms can be obviously seen.

3.2.3. X-ray diffraction analysis

Crystallographic properties of precipitated phase within the GEL matrix were analyzed using XRD. Fig. 7 shows diffractograms for the samples mineralized at 4°C.The diffractogram has weak peaks representing a type of amorphous or semi-crystalline nature. These peaks can be ascribed to both HA and DCPD (marked in this figure), according to the ICCD database.

3.2.4. Transmission electron microscopy

TEM micrographs obtained from nanocomposite scaffolds at 4°C are shown in Fig. 8. For these samples, GEL/mineral nanocomposite particles of 50-200 nm can be seen as discrete regions. This observation supports the formation of these minerals within the GEL matrix and indicates adhesion of precipitated minerals to GEL polymeric chains, since samples underwent drying and milling processes. Precipitated nano-crystals were spherical in shape, and their sizes ranged from 2.5 to 10 nm.

3.2.5. FTIR analysis

In order to study the chemical characteristics of prepared nanocomposite scaffolds, FTIR analysis was performed. Fig. 9 shows the FTIR spectra for scaffolds obtained by double diffusion precipitation at 4°C. Table 2 shows detected chemical bonds. For this sample, the 2 series of obtained peaks, 1238, 1453, 1541, 1650, 2931 cm-1 and 560, 622, 868, 1040 cm-1 are due to GEL and calcium phosphate chemical structures, respectively. Interestingly, a peak related to chemical bonds formed between the GEL matrix and the precipitated phase appears at approximately 1340 cm-1. This peak indicates formation of a chemical bond between a carboxyl group from GEL and Ca+2 ion from HA. This peak is also consistent with data from previous studies [44,45].

3.2.6. MTT assay

As shown by MTT assay, scaffolds did not have any negative effects on proliferation rate of osteoblasts in comparison with cell culture plastic surfaces. This result demonstrates that the fabricated scaffold could preserve the cytocompatibility of the scaffold despite cross-linking with glutaraldehyde which have been done for achieving a good mechanical property. (Fig 10).

3.2.7. Cell adhesion and proliferation on the scaffold

A representative SEM picture of endometrial stem cells obtained after culture of these cells on the nanocomposite scaffold sample is shown in Fig. 11. These cells demonstrate adhesion and penetration to the pores of the scaffold. Cultured cells are attached to the scaffold surfaces. ECM gave an unsmooth appearance to the cells.

3.2.8. ALP activity

Results concerning ALP activity are shown in Fig. 12. There was an equal volume of ALP production in the all days compared to control group. However, no significant ALP production rate was observed.

4. Discussion

Bone tissue engineering aims to provide functional bone substitutes for clinical treatment of bone defects by integrating osteoblast cells into porous scaffolds in vitro. The first biological object of this study was to differentiate human endometrial stem cells to osteoblast cells by using osteogenic media containing BMP-2. Secondly, different behaviors of human osteoblast diffrerntiated cells on the surface of biomimetic nanocomposite scaffolds were evaluated in terms of proliferation, alkaline phosphatase secretion, cell attachment, calcium deposition, gene expression and morphology.

The differentiation of human endometrial stem cells into osteoblast-like cells was confirmed by morphological and by molecular criteria. The extracellular mineralized matrix secreted by the osteoblast-like cells was shown by alizarin red staining. Immunocytochemistry revealed that osteopontin and osteocalcin were expressed in the culture treated with osteogenic media. Mineralization was in accordance with previous reports for mesenchymal stem cells suggesting similar osteogenic potentials [46,47].

Therefore, we isolated hEnSCs and treated with osteogenic media and BMP-2 and differentiation markers studied constitute an interesting model for the evaluation of biological properties of tissue engineering scaffolds and different biomaterials or bioactive molecules [48]. This issue is highly supported with previous studies. According to Wozney, the osteoinductive activity of BMP's and their presence in bone tissue suggest that they are important regulators in bone repair process and may be involved in such tissues'maintenance [49]. Furthermore, Ripamonti and Reddi [50] stated that BMPs maybe play multiple roles in embryogenic and organogenic development, including skeletogenesis, craniofacial and dental tissues development.

Our data showed that BMP-2 have influence on mineralization of endometrial stem cells and this was shown by mineralization nodules in treated group studied by alizarin red staining. Moreover, increase in the expression levels of osteopontin and osteocalcin in treated cells with osteogenic media with BMP-2 suggests that BMP-2 plays a significant role in the differentiation of human endometrial stem cells into osteoblast-like cells. Furthermore, the data concerning osteoblast specific gene markers including osteopontin, osteonectin and alkaline phosphates shows that endometrial stem cells can differentiate to osteoblast-like cells.

In bone defects, providing the mechanical and functional integrity is the most important issue in the patient's rehabilitation. Hydroxyapatite (HA) due to its bone-like chemical constitution and mechanical properties is of important interest in the field of bone substitution. HA is known as the mineral phase of natural bones, but precipitation of Ca2+ and PO43- in the body environment does not directly lead to formation of HA. These data indicate that there are precursor phases such as DCPD, ACP, and octacalcium phosphate (OCP), which are formed before HA and then converted into HA in the body.

Li et al. [30] aimed to synthesize ACP through drop-wise addition of a phosphate solution into a polyethylene glycol (PEG, a stabilizer) containing calcium solution at 5°C and investigated the effect of ageing, pH, and Ca/P atomic ratios on the final product. Although they obtained ACP under different conditions, they observed some crystalline peaks at 2ө = 11.6, 20.9, 29.2, 30.4, and 34.0 in XRD diffractograms. The authors ascribed these peaks to DCPD that appeared in the sample, which was synthesized at an initial PH=6. Therefore, formation of DCPD along with ACP was expected in this study.

According to a previously published report [51], we conclude that if the GEL/ACP scaffold is implanted in the body, it will ultimately be converted to HA crystals through a process similar to natural bone formation in the body. Precipitation of calcium phosphate mineral via the methods described can give a more homogenous mixture and better adhesion of ceramic particles with GEL matrix in comparison with conventional mixing of previously prepared ceramic particles with GEL [13-15].

Furthermore, cytotoxicity tests using cell cultures have been applied as the first step in identifying active compounds and for bio-safety testing. From cell viability assays as are shown in Fig.10, the ability of biomimetic nanocomposites to support cell viability was verified. The whole set of evaluated nanocomposites exhibited comparable biocompatibility where the cellular proliferation rates of osteoblast cells were similar to control group. Beyond that, cell proliferation and growth were assessed via adhesion and spreading test, where general morphology was observed by SEM. It can be noted that the osteoblast differentiated cells seeded on nanocomposites with good adhesion and spreading morphology regular for these cells (Fig.11). Since cellular attachment, adhesion, and spreading belong to the first phase of cell/material interactions, the quality of this phase will influence the proliferation of cells on biomaterials surfaces. Based on the SEM results obtained in this study (Fig.11), one may attribute the osteoblast-like cells spreading and adhesion verified on the biomimetic nanocomposites to be reliable proof of biocompatibility and non-cytotoxicity of samples. In addition, Results concerning ALP activity showed that the amount of the enzyme next to nanocomposite was equal to the production of ALP in comparison to control group. These data demonstrated that the nano-sized composites didn't have harmful effect on ALP expression.

Finally, it is demonstrated that biomimetic nanocomposites have considerable biocompatibility without exerting any significant cytotoxic effects which could be used as potential biomaterials for bone tissue engineering applications.

5. Conclusion

In this study, human endometrial stem cells were successfully differentiated to osteoblast like-cells by osteogenic media.This may propose EnSCs as an attractive alternative candidate for repair of bone tissue defects, as they exhibit several important and potential advantages over other stem cells. Moreover, in situ formed nanocomposite scaffolds were designed and fabricated using a biomimetic approach. To obtain a better biological response, it was decided to produce a GEL/Calcium phosphate nanocomposite under conditions similar to those for bone formation in the human body. Ca2+ and PO43-, the main components of the bone mineral phase, were diffused into a GEL matrix from opposite sides. A mineral containing ACP and DCPD precipitate was formed within the GEL matrix at 4°C. Thus, our results highlight the potential use of engineered biomimetic bone tissue scaffolds in the bone tissue repair process.

6. Acknowledgment

We thank Tehran University of Medical Sciences Research assistant, Iranian Council of Stem Cell Technology and Iran National Science Foundation (INSF) for financial supported.

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