Artificial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–8 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.909825
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Synthesis of calcium phosphate-zirconia scaffold and human endometrial adult stem cells for bone tissue engineering Aliakbar Alizadeh1,3, Fathollah Moztarzadeh2, Seyed Naser Ostad1, Mahmoud Azami1, Bita Geramizadeh3, Gholamreza Hatam4, Davood Bizari2, Seyed Mohammad Tavangar1, Mohammad Vasei1 & Jafar Ai1,5 1Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences,
Tehran, Iran, 2Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran, 3Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, 4Department of Parasitology, Shiraz University of Medical Sciences, Shiraz, Iran, and 5Brain and Spinal Injury Research Center, Imam Hospital, Tehran University of Medical Sciences, Tehran, Iran
a biocompatible inert scaffold to deliver the cells to the damaged region (Biazar et al. 2010, 2013a, Biazar and Heidari 2013a, 2013b, 2013c, 2014, Biazar 2013). Highly porous calcium phosphate-based bioceramic scaffolds have been widely investigated as three-dimensional (3D) templates for cell adhesion, proliferation, and differentiation promoting the bone regeneration. Their fragility, however, limits their clinical application especially for a large bone defect. Open pore structure of the scaffold enhances the new bone tissue formation, because the structure readily allows the nutrient supply, gas diffusion, and metabolic waste removal, which are important for cell survival and activity (Biazar et al. 2013b, 2014, Ai et al. 2012, Azami et al. 2012). Calcium phosphate is the first choice as a source material for bone reconstructive scaffolds because it is the main component of bone and is known for its excellent cellular and tissue affinity (Lee et al. 2007). Porous sintered hydroxyapatite (HAp), however, does not have sufficient mechanical properties (Jo et al. 2007). The compressive strength of pure HAp porous blocks synthesized in previous works is only 0.3 MPa, whereas that of trabecular bone is 12 MPa and that of cortical bone is 200 MPa (Mosekilde and Mosekilde 1986). Zirconia (ZrO2) would be a good additive material because it is a stable inorganic material with high biocompatibility and good mechanical properties. ZrO2 itself, however, does not have good cellular and tissue affinity. ZrO2 and calcium phosphate mixtures having porous structure were therefore prepared in efforts to obtain a nondegradable and bone reconstructive substrate that can enhance the bone regeneration as well (Masonis et al. 2004). The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. Human endometrium contains a low number of endometrial stem cells (ESCs) that seem to belong to the family of the mesenchymal stromal/stem cells (MSC). These cells are engaged in the monthly restructuring and
Abstract To address the hypothesis that using a zirconia (ZrO2)/ b-tricalcium phosphate (b-TCP) composite might improve both the mechanical properties and cellular compatibility of the porous material, we fabricated ZrO2/b-TCP composite scaffolds with different ZrO2/b-TCP ratios, and evaluated their physical and mechanical characteristics, also the effect of threedimensional (3D) culture (ZrO2/b-TCP scaffold) on the behavior of human endometrial stem cells. Results showed the porosity of a ZrO2/b-TCP scaffold can be adjusted from 65% to 84%, and the compressive strength of the scaffold increased from 4.95 to 6.25 MPa when the ZrO2 content increased from 30 to 50 wt%. The cell adhesion and proliferation in the ZrO2/b-TCP scaffold was greatly improved when ZrO2 decreased. Moreover, in vitro study showed that an osteoblasts-loaded ZrO2/b-TCP scaffold provided a suitable 3D environment for osteoblast survival and enhanced bone regeneration. We thus showed that a porous ZrO2/b-TCP composite scaffold has excellent mechanical properties, and cellular/tissue compatibility, and would be a promising substrate to achieve both bone reconstruction and regeneration needed during in vivo study for treatment of large bone defects. Keywords: β-tricalcium phosphate, bone tissue engineering, endometrial stem cells, porous scaffold, zirconia
Introduction Tissue engineering (TE) is defined as the combination of living cells and biocompatible scaffolds to generate a biologic substitute capable of sustaining itself and integrating with functional native tissue. In TE, there are many issues to consider in the creation of a functional, implantable replacement tissue. Most importantly, there must be an easily accessible, readily abundant cell source with the capacity to express the desired tissues’ phenotype, and
Correspondence: Jafar Ai, Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. Tel: ⫹ 982188991118. E-mail: [email protected] (Received 5 March 2014; accepted 26 March 2014)
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A. Alizadeh et al. (Sigma–Aldrich, USA) and β-TCP mixed at various wt% ratios (ZrO2.Y2O3/β-TCP: A1: 50/50, A2: 40/60 and A3: 30/70) in 20 ml of distilled water containing 0.02%wt of tripolyphosphate (TPP, Sigma–Aldrich, USA) and 0.04%wt carboxymethyl cellulose (CMC, Sigma–Aldrich, USA), and colloidal silica for 90 min. Porous scaffolds were fabricated by impregnating the struts of a polyurethane sponge (60 pores per inch) with the slurry. The sponge block was dipped into the slurry and compressed slightly to remove the excess slurry on the foam. After the sponge was dried at 60°C for 1 h, heated at thermal cycle 0–600°C to burn out the sponge block and binder, and then sintered at 1500°C for 4 h. Figure 1 shows designed scaffolds with different ratios.
Scaffold characterization Figure 1. Designed ZrO2/β-TCP scaffolds with different ratios.
remodeling of a human endometrium (Jabbour et al. 2006, Dimitrov et al. 2008, Gargett et al. 2007, Ai and Mehrabani 2010, Ai et al. 2013). Previous studies have shown the potential differentiation of the ESCs into mesoderm-derived cells, such as chondrogenic and osteoblastic lineages, when cultured in the appropriate induction media (Schwab et al. 2005, Kato et al. 2007). Since endometrial stromal cells (EnSC) are easy to isolate, expand rapidly without leading to major ethical and technical problems, and produce a higher overall clonogenicity, they have a unique potential as autologous therapeutic agents. Therefore, endometrium may be an alternative source of MSC-like cells for tissue engineering purposes, obtainable with no more morbidity than any other source of stem cells (Gargett 2006, Schwab and Gargett 2007, Patel et al. 2008). To evaluate the usefulness of the porous ZrO2/calcium phosphate composite material for bone tissue repair, in this study we investigated physical properties and ESCs compatibility of the material.
Materials and methods ZrO2/β-tricalcium phosphate (β-TCP) slurries were prepared by dispersing 80 g of powder consisting of (ZrO2)0.97(Y2O3)0.03
The morphology and pore size of the obtained scaffold were observed using a scanning electron microscope (SEM, Camscan MV2300, USA). X-ray diffraction analysis (XRD, PHILIPS PV 3710, the Netherlands) was carried out to identify the phase composition of sintered scaffolds with different ZrO2/HAp ratios. The porosity of the porous scaffolds was measured based on Archimedes’ principle (Jo et al. 2007, Mosekilde and Mosekilde 1986). The compressive strength of the porous scaffolds was measured by a mechanical tester (ZWICK/ROELL 2005, Germany) with a crosshead speed of 1 mm/min. Five samples were tested for each group, and the average and standard deviation were calculated.
Cell analyses Informed consent was obtained and approval was granted by the ethics committee of the University of Tehran of Medical Sciences (Iran). Stem cells isolation and culture were obtained from a biopsy samples of the endometrium collected during the proliferation phase of a fertile 20–26year-old woman who had not undergone a hormonal exogenous treatment during at least 3 months before surgery. ESC separation was done as follows: human endometrial tissue was digested into single-cell suspensions using Collagenase type 3 (300 mg/ml; Worthington Biochemical
Figure 2. XRD analyses for the scaffolds with different ratios.
Osteoconductivity of calcium phosphate-zirconia composite 3
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Figure 3. SEM image of the samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
Corporation, Freehold, NJ, USA) and mechanical digestion as previously described (Ai and Mehrabani 2010, Ai et al. 2013). Red blood cells were removed using Ficoll-Paque density gradient centrifugation (Pharmacia Biotechnology, Uppsala, Sweden). The cells were then plated in DMEM/F-12 supplemented with 10% FBS, 100 IU/ml penicillin (Invitrogenâ), and 100 IU/ml streptomycin (Invitrogenâ) in tissue culture flask (25 cm2), and incubated in 5% CO2 at 37°C. The culture medium used for the development was first changed every 3–4 days and regularly replaced twice a week thereafter. For evaluation of cell surface marker by flowcytometry, human endometrial stem cells (hESCs) were first trypsinized and counted. Tubes containing about 1 ⫻ 106 cells were incubated on rocking and centrifuged at 300 g for 6 min, and then 2% human serum added to the repository. The cell mixture was passed through a nylon mesh, 100 μl of the mixture was added to each tube with the following antibodies: anti-CD105, anti-CD73, anti-CD45, anti-CD146, anti-CD90, and anti-CD34 (all products Abcam). In addition, these tubes were incubated at 4°C in a dark room for 45 min. After the washing process, the cells were fixed in 100 μl of 1% paraformaldehyde. Finally, flowcytometric
analysis was performed. The ratios of fluorescence signals from scattered signals were calculated by the flowcytometer (Partec). Histograms were generated using the software WinDmi 2.9. The MTT assay is a rapid colorimetric method 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 serumfree 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 formazan training. After adding 200 μl of dimethylsulfoxide solution was used for the density (OD) measurement with optical absorbance detection microplate reader at 570 nm (Rayeto, USA). For osteoconductivity assay, 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 M dexamethasone, and 10 mM b-glycerophosphate (all from Sigma, St. Louis, MO). Osteogenesis was assessed by staining with alizarin red and 4,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich). Values are presented as mean ⫾ standard
Figure 4. SEM image of the cross section of the bridges for samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
Figure 5. EDAX image of distribution of zirconium and calcium from ZrO2.Y2O3/β-TCP in the samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
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A. Alizadeh et al. All data were analyzed by one-way ANOVA with Duncan’s multiple range tests (ANOVA; Duncan multiple range test, p ⬍ 0.05 and 0.01).
Results and discussion
Figure 6. The porosity and density diagram for samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
deviation unless otherwise noted. Statistical analysis was performed using Originâ 6.1 (OriginLab, Northampton, MA). Experimental results were expressed as means ⫾ SD. A1
A2
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y = 56.761x + 0.1339 R2 = 0.9592
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A3 y = 65.971x – 4.3687 R2 = 0.9698
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The XRD profile of the scaffold sintered at 1500°C had β-TCP, and CaZrO3 peaks addition to the original ZrO2 peaks for all the samples (Figure 2). The partial transition of the tetragonal phase of ZrO2 to the cubic phase of ZrO2 was also detected in XRD profile of the samples. The porous structure of the synthesized scaffold altered according to concentrations of slurry containing the ceramic powders (Figure 3). The obtained scaffold with the highest porosity had interconnected pores (A1), but the scaffolds with lower porosity had pores with only limited interconnection (A3). Differences in porosity and pore size
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Characterization of synthesized scaffolds
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Figure 7. The stress-strain curves for samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
Figure 8. Flow cytometric analysis of isolated EnSC for mesenchymal stem cell markers (CD90, CD105, and CD44), haemopoietic marker (CD34 and CD133), endothelial marker (CD31) and ES cell marker (OCT4). As shown the isolated cells are positive for CD90, CD105, CD44, and OCT4 and are negative for CD31, CD34.
among samples are due to removal of water vapor and gas from the surface. Figure 4 shows cross-section of bridges between pores in all samples that reaches enough strength. In the sample A3, it is clear that the materials show ingredients well-formed together and its bridges show more strength than the other samples. Results from EDAX elemental analysis, show more distribution of zirconium and calcium from ZrO2/β-TCP in the A1 sample than other samples (Figure 5). Figure 6 shows the porosity (A1: 68 ⫾ 2.5%, A2: 78 ⫾ 1.5%, and A3: 84.2 ⫾ 2.8%) and density amounts (A1: 2.67 ⫾ 0.14%, A2: 2.17 ⫾ 0.32%, and A3: 1.85 ⫾ 0.28%) of scaffolds with different ratios of ZrO2.Y2O3/β-TCP. The compressive strength of 6.25 ⫾ 0.01 (A1), 5.65 ⫾ 0.23 (A2), and 4.95 ⫾ 0.94 (A3) MPa and elasticity modulus of 63 ⫾ 0.01 (A1), 54 ⫾ 0.23 (A2), and 48 ⫾ 0.94 (A3) MPa were obtained for scaffolds. Results show decreasing strength and increasing porosity for the samples when ZrO2 content decreased from 50% to 30% in the scaffold (Figure 7).
(E)
Cell results After plating for 24–48 h, some adherent cells appeared in the flask, and the cells were heterogeneous in appearance. Approximately 10 days later, these cells developed into many clusters, and could be used for sub-culturing. After 3 passages, human ESCs became relatively homogeneous in appearance, being relatively elongated or spindle-shaped The immunophenotype was based on the flowcytometric analysis of a subset of stem cell marker (OCT4), MSC markers (CD90, CD105 and CD44), haemopoietic markers (CD34 and CD133), and endothelial marker (CD31). The analysis showed that they were positive for CD90, CD105, and OCT4 and negative for CD31 and CD34 (Figure 8). Given the phenotypic, morphological, proliferative characteristics of the ESCs, we determined whether these cells were capable of differentiating into various lineages as can other stem cell types. Differentiation into osteocytes was demonstrated by culturing of EnSC using standard commercially available culture reagents and methodologies.
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Osteoconductivity of calcium phosphate-zirconia composite 5
genes relative expression ratio plot [ mean ±S.E. ] Figure 9. Osteogenic capacity under in vitro condition (A, B), immunocytochemistry for osteocyte special marker osteopontin (C) and osteocalsin (D) [Scale bar: 100 μm], evaluation of gene expression of critical genes (SPP1, ALP, SPARC) for osteoconductivity by Real time PCR in 3D culture condition (E).
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Figure 10. MTT result after 3 and 6 days co-culture ESC and ZrO2/ β-TCP composite. Y2O3/β-TCP in the samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
EnSCs are easier to isolate and expand with less technical problems compared to bone marrow MSCs. Here we show that EnSCs can be differentiated into osteogenic cells as one of the most important issues in orthopedic surgery is associated with bone loss in traumas, infections, tumors, or congenital disorders (Figure 9B) and also show immunocytochemistry for osteocyte special marker osteopontin and osteocalcin (Figure 9C and D). Evaluation of gene expression of critical genes (SPP1, ALP, SPARC) for osteoconductivity has been performed by Real time PCR in 3D culture condition. The MTT results after 48 hours showed that 96% of hESCs are alive in the presence of ZrO2/β-TCP nanocomposite scaffold which confirms the biocompatibility of the prepared scaffold (Figure 10). At day 3 of culture, the cells on the surface of a scaffold composed of 30/70 ZrO2/β-TCP had a well-spread morphology but the cells on the surface of a scaffold composed of 50/50 ZrO2/β-TCP were less attached and remained spindle shaped. A plot of cell number against culture period showed that cell proliferation was significantly
higher in the scaffolds containing less than 50% ZrO2. The EnSC proliferation test and MTT assay results demonstrated that ZrO2 scaffolds have good biocompatibility and cell viability. Staining with osteoblasts specific markers showed that these cells expressed osteoblast cell markers (osteopontin and osteocalcin) 6 days after implantation of ESCs on the scaffold. These results indicate that the nanocomposite scaffolds is suitable for ESCs and their differentiation to osteoblast cells (Figure 11). SEM images of differentiated cells cultured in the scaffolds with different compositions are shown in Figure 12. The osteocyte cells adhesion and proliferation in the ZrO2/β-TCP scaffold was greatly improved in the samples with ratios of ZrO2.Y2O3/β-TCP: 30/70. The material chosen for a bone scaffold is very important because it must possess or be able to be modified to achieve adequate biocompatibility, porosity, pore size, surface texture, mechanical properties, and biodegradability. To date, ceramics and polymers from both natural and synthetic origins have been used to construct bone scaffolds for bone defects. However, most ceramics are not biodegradable, which presents a problem in bone restoration applications that require the scaffold material to degrade over time. This limits scaffold material choices to a small number of ceramics and biodegradable polymers. Ceramics, such as HAp, bioactive glasses, and calcium phosphates, have been widely used for bone regeneration and replacement (Oe et al. 2007). All three materials exhibit a bioactive and biocompatible behavior and have been traditionally used as coatings on metals for orthopedic/ dental implants but more recently have been proposed as filler material for bone defects and as bone scaffolds (Oe et al. 2007). These materials are unique in that they form a calcium-deficient (non-stoichiometric) apatite surface layer in the presence of biological fluids (Einhorn 1998a). This surface layer is thought to stimulate bioceramic-bone binding and, in some cases, promote new bone formation
Figure 11. Immunocytochemistry for the expression of osteoblast markers, 6 days after the implantation of differentiated cells on the scaffolds. Osteopontin (A: osteopontin specificity marker, B: 4,6-diamidino-2-phenylindole dihydrochloride, and C: negative control) and osteocalsin (D: osteocalcin specificity marker, E: 4,6-diamidino-2-phenylindole dihydrochloride, and F: negative control); Scale bar: 100 μm.
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Osteoconductivity of calcium phosphate-zirconia composite 7
Figure 12. SEM images of the culture ESC on ZrO2/β-TCP composite (Y2O3/β-TCP: 30/70) in different magnifications (A: 250X, B: 500X, C: 1000X, and D: 5000X).
(El-Ghannam 2005). Calcium phosphate based materials, in particular, have been widely investigated for use as bone replacement materials due to their biocompatibility and osteoconductivity (Chao and Inoue 2003, Khan et al. 2005, Fleming et al. 2000). Synthetic β-TCP is the two most common calcium phosphate materials used as hard-tissue replacements (Oe et al. 2007). Several studies have shown that using these two bioceramics as bone scaffolds, both seeded with and seeded without bone marrow cells, have yielded bone regeneration (Liao et al. 2004, Oest et al. 2007, Tanaka et al. 2010). Calcium phosphates are biocompatible and osteoconductive, although they have some drawbacks for use as bone scaffolds. These materials are brittle and have low mechanical stability, which limits their use in large defects that require load bearing (Einhorn 1998a). Furthermore, their dissolution and degradation rates are difficult to control in vivo (Einhorn 1998a). These factors could present a problem because if the scaffold degrades too fast, the mechanical stability would be compromised and could fail. In this study cell adherence ability was used to assess cell biocompatibility of the material. The results showed that ZrO2/β-TCP composites, especially the graded one, were more conducive to cell adhesion and proliferation. Significantly increased adhesion rate of graded composite may be due to many reasons. SEM images showed that the surface of the graded composite was porous with uniform pore diameter of 100–200 μm. XRD analysis showed that after ZrO2/β-TCP composite was sintered at 1500°C, new phases of β-Ca3(PO4)2, α-Ca3(PO4)2, and CaZrO3 were generated. The biocompatibility of composite materials was achieved, because after the high-temperature phosphate contacted with water or body fluid at 37°C, β-phase was formed as
well as Ca2⫹, HPO42⫺ were decomposed and deposited on the surface of bioceramic substrate. Over a period of time of contact, composition in bioceramic-based body changed slightly. ZrO2/β-TCP phase decreased and HAp increased. It is helpful for adsorbing serum protein and forming cell adhesion. We speculate that the adhesion of all the attachment-dependent cells on the biological scaffold material is enhanced. The increased proliferation rate of MSCs on the surface of ZrO2/β-TCP composites shows some characteristics of the material or components. Moreover, it is also due to the rough surface of substrates. Many studies have shown that the substrate surface roughness relates to the rate of cell proliferation (Einhorn 1998b, El-Ghannam 2005). Adherence enhancement may be another reason leading to increased proliferation rate. The adhesion enhancement of initially inoculated cell can result in a higher increase in cell density, thus affecting the biological behavior, such as proliferation rate.
Conclusions In this study, we have designed a new bioceramic composite consisting of porous calcium phosphate and strong load-bearing ZrO2 ceramic. Nanocomposite scaffold were fabricated via foam casting with different compositions. These scaffolds were sintered at 1500°C to obtain porous scaffolds. Porous ZrO2/β-TCP composite scaffold has excellent mechanical properties, supports cellular attachment and differentiation. We showed that ESCs differentiate into osteogenic cells using this scaffolds. This porous composite provides a potential promising alternative for load-bearing bone replacements.
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Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the contents and writing of the paper.
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Synthesis of calcium phosphate-zirconia scaffold and human endometrial adult stem cells for bone tissue engineering Aliakbar Alizadeh1,3, Fathollah Moztarzadeh2, Seyed Naser Ostad1, Mahmoud Azami1, Bita Geramizadeh3, Gholamreza Hatam4, Davood Bizari2, Seyed Mohammad Tavangar1, Mohammad Vasei1 & Jafar Ai1,5 1Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences,
Tehran, Iran, 2Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran, 3Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, 4Department of Parasitology, Shiraz University of Medical Sciences, Shiraz, Iran, and 5Brain and Spinal Injury Research Center, Imam Hospital, Tehran University of Medical Sciences, Tehran, Iran
a biocompatible inert scaffold to deliver the cells to the damaged region (Biazar et al. 2010, 2013a, Biazar and Heidari 2013a, 2013b, 2013c, 2014, Biazar 2013). Highly porous calcium phosphate-based bioceramic scaffolds have been widely investigated as three-dimensional (3D) templates for cell adhesion, proliferation, and differentiation promoting the bone regeneration. Their fragility, however, limits their clinical application especially for a large bone defect. Open pore structure of the scaffold enhances the new bone tissue formation, because the structure readily allows the nutrient supply, gas diffusion, and metabolic waste removal, which are important for cell survival and activity (Biazar et al. 2013b, 2014, Ai et al. 2012, Azami et al. 2012). Calcium phosphate is the first choice as a source material for bone reconstructive scaffolds because it is the main component of bone and is known for its excellent cellular and tissue affinity (Lee et al. 2007). Porous sintered hydroxyapatite (HAp), however, does not have sufficient mechanical properties (Jo et al. 2007). The compressive strength of pure HAp porous blocks synthesized in previous works is only 0.3 MPa, whereas that of trabecular bone is 12 MPa and that of cortical bone is 200 MPa (Mosekilde and Mosekilde 1986). Zirconia (ZrO2) would be a good additive material because it is a stable inorganic material with high biocompatibility and good mechanical properties. ZrO2 itself, however, does not have good cellular and tissue affinity. ZrO2 and calcium phosphate mixtures having porous structure were therefore prepared in efforts to obtain a nondegradable and bone reconstructive substrate that can enhance the bone regeneration as well (Masonis et al. 2004). The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. Human endometrium contains a low number of endometrial stem cells (ESCs) that seem to belong to the family of the mesenchymal stromal/stem cells (MSC). These cells are engaged in the monthly restructuring and
Abstract To address the hypothesis that using a zirconia (ZrO2)/ b-tricalcium phosphate (b-TCP) composite might improve both the mechanical properties and cellular compatibility of the porous material, we fabricated ZrO2/b-TCP composite scaffolds with different ZrO2/b-TCP ratios, and evaluated their physical and mechanical characteristics, also the effect of threedimensional (3D) culture (ZrO2/b-TCP scaffold) on the behavior of human endometrial stem cells. Results showed the porosity of a ZrO2/b-TCP scaffold can be adjusted from 65% to 84%, and the compressive strength of the scaffold increased from 4.95 to 6.25 MPa when the ZrO2 content increased from 30 to 50 wt%. The cell adhesion and proliferation in the ZrO2/b-TCP scaffold was greatly improved when ZrO2 decreased. Moreover, in vitro study showed that an osteoblasts-loaded ZrO2/b-TCP scaffold provided a suitable 3D environment for osteoblast survival and enhanced bone regeneration. We thus showed that a porous ZrO2/b-TCP composite scaffold has excellent mechanical properties, and cellular/tissue compatibility, and would be a promising substrate to achieve both bone reconstruction and regeneration needed during in vivo study for treatment of large bone defects. Keywords: β-tricalcium phosphate, bone tissue engineering, endometrial stem cells, porous scaffold, zirconia
Introduction Tissue engineering (TE) is defined as the combination of living cells and biocompatible scaffolds to generate a biologic substitute capable of sustaining itself and integrating with functional native tissue. In TE, there are many issues to consider in the creation of a functional, implantable replacement tissue. Most importantly, there must be an easily accessible, readily abundant cell source with the capacity to express the desired tissues’ phenotype, and
Correspondence: Jafar Ai, Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. Tel: ⫹ 982188991118. E-mail: [email protected] (Received 5 March 2014; accepted 26 March 2014)
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A. Alizadeh et al. (Sigma–Aldrich, USA) and β-TCP mixed at various wt% ratios (ZrO2.Y2O3/β-TCP: A1: 50/50, A2: 40/60 and A3: 30/70) in 20 ml of distilled water containing 0.02%wt of tripolyphosphate (TPP, Sigma–Aldrich, USA) and 0.04%wt carboxymethyl cellulose (CMC, Sigma–Aldrich, USA), and colloidal silica for 90 min. Porous scaffolds were fabricated by impregnating the struts of a polyurethane sponge (60 pores per inch) with the slurry. The sponge block was dipped into the slurry and compressed slightly to remove the excess slurry on the foam. After the sponge was dried at 60°C for 1 h, heated at thermal cycle 0–600°C to burn out the sponge block and binder, and then sintered at 1500°C for 4 h. Figure 1 shows designed scaffolds with different ratios.
Scaffold characterization Figure 1. Designed ZrO2/β-TCP scaffolds with different ratios.
remodeling of a human endometrium (Jabbour et al. 2006, Dimitrov et al. 2008, Gargett et al. 2007, Ai and Mehrabani 2010, Ai et al. 2013). Previous studies have shown the potential differentiation of the ESCs into mesoderm-derived cells, such as chondrogenic and osteoblastic lineages, when cultured in the appropriate induction media (Schwab et al. 2005, Kato et al. 2007). Since endometrial stromal cells (EnSC) are easy to isolate, expand rapidly without leading to major ethical and technical problems, and produce a higher overall clonogenicity, they have a unique potential as autologous therapeutic agents. Therefore, endometrium may be an alternative source of MSC-like cells for tissue engineering purposes, obtainable with no more morbidity than any other source of stem cells (Gargett 2006, Schwab and Gargett 2007, Patel et al. 2008). To evaluate the usefulness of the porous ZrO2/calcium phosphate composite material for bone tissue repair, in this study we investigated physical properties and ESCs compatibility of the material.
Materials and methods ZrO2/β-tricalcium phosphate (β-TCP) slurries were prepared by dispersing 80 g of powder consisting of (ZrO2)0.97(Y2O3)0.03
The morphology and pore size of the obtained scaffold were observed using a scanning electron microscope (SEM, Camscan MV2300, USA). X-ray diffraction analysis (XRD, PHILIPS PV 3710, the Netherlands) was carried out to identify the phase composition of sintered scaffolds with different ZrO2/HAp ratios. The porosity of the porous scaffolds was measured based on Archimedes’ principle (Jo et al. 2007, Mosekilde and Mosekilde 1986). The compressive strength of the porous scaffolds was measured by a mechanical tester (ZWICK/ROELL 2005, Germany) with a crosshead speed of 1 mm/min. Five samples were tested for each group, and the average and standard deviation were calculated.
Cell analyses Informed consent was obtained and approval was granted by the ethics committee of the University of Tehran of Medical Sciences (Iran). Stem cells isolation and culture were obtained from a biopsy samples of the endometrium collected during the proliferation phase of a fertile 20–26year-old woman who had not undergone a hormonal exogenous treatment during at least 3 months before surgery. ESC separation was done as follows: human endometrial tissue was digested into single-cell suspensions using Collagenase type 3 (300 mg/ml; Worthington Biochemical
Figure 2. XRD analyses for the scaffolds with different ratios.
Osteoconductivity of calcium phosphate-zirconia composite 3
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Figure 3. SEM image of the samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
Corporation, Freehold, NJ, USA) and mechanical digestion as previously described (Ai and Mehrabani 2010, Ai et al. 2013). Red blood cells were removed using Ficoll-Paque density gradient centrifugation (Pharmacia Biotechnology, Uppsala, Sweden). The cells were then plated in DMEM/F-12 supplemented with 10% FBS, 100 IU/ml penicillin (Invitrogenâ), and 100 IU/ml streptomycin (Invitrogenâ) in tissue culture flask (25 cm2), and incubated in 5% CO2 at 37°C. The culture medium used for the development was first changed every 3–4 days and regularly replaced twice a week thereafter. For evaluation of cell surface marker by flowcytometry, human endometrial stem cells (hESCs) were first trypsinized and counted. Tubes containing about 1 ⫻ 106 cells were incubated on rocking and centrifuged at 300 g for 6 min, and then 2% human serum added to the repository. The cell mixture was passed through a nylon mesh, 100 μl of the mixture was added to each tube with the following antibodies: anti-CD105, anti-CD73, anti-CD45, anti-CD146, anti-CD90, and anti-CD34 (all products Abcam). In addition, these tubes were incubated at 4°C in a dark room for 45 min. After the washing process, the cells were fixed in 100 μl of 1% paraformaldehyde. Finally, flowcytometric
analysis was performed. The ratios of fluorescence signals from scattered signals were calculated by the flowcytometer (Partec). Histograms were generated using the software WinDmi 2.9. The MTT assay is a rapid colorimetric method 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 serumfree 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 formazan training. After adding 200 μl of dimethylsulfoxide solution was used for the density (OD) measurement with optical absorbance detection microplate reader at 570 nm (Rayeto, USA). For osteoconductivity assay, 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 M dexamethasone, and 10 mM b-glycerophosphate (all from Sigma, St. Louis, MO). Osteogenesis was assessed by staining with alizarin red and 4,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich). Values are presented as mean ⫾ standard
Figure 4. SEM image of the cross section of the bridges for samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
Figure 5. EDAX image of distribution of zirconium and calcium from ZrO2.Y2O3/β-TCP in the samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
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A. Alizadeh et al. All data were analyzed by one-way ANOVA with Duncan’s multiple range tests (ANOVA; Duncan multiple range test, p ⬍ 0.05 and 0.01).
Results and discussion
Figure 6. The porosity and density diagram for samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
deviation unless otherwise noted. Statistical analysis was performed using Originâ 6.1 (OriginLab, Northampton, MA). Experimental results were expressed as means ⫾ SD. A1
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y = 56.761x + 0.1339 R2 = 0.9592
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The XRD profile of the scaffold sintered at 1500°C had β-TCP, and CaZrO3 peaks addition to the original ZrO2 peaks for all the samples (Figure 2). The partial transition of the tetragonal phase of ZrO2 to the cubic phase of ZrO2 was also detected in XRD profile of the samples. The porous structure of the synthesized scaffold altered according to concentrations of slurry containing the ceramic powders (Figure 3). The obtained scaffold with the highest porosity had interconnected pores (A1), but the scaffolds with lower porosity had pores with only limited interconnection (A3). Differences in porosity and pore size
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Characterization of synthesized scaffolds
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Figure 7. The stress-strain curves for samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
Figure 8. Flow cytometric analysis of isolated EnSC for mesenchymal stem cell markers (CD90, CD105, and CD44), haemopoietic marker (CD34 and CD133), endothelial marker (CD31) and ES cell marker (OCT4). As shown the isolated cells are positive for CD90, CD105, CD44, and OCT4 and are negative for CD31, CD34.
among samples are due to removal of water vapor and gas from the surface. Figure 4 shows cross-section of bridges between pores in all samples that reaches enough strength. In the sample A3, it is clear that the materials show ingredients well-formed together and its bridges show more strength than the other samples. Results from EDAX elemental analysis, show more distribution of zirconium and calcium from ZrO2/β-TCP in the A1 sample than other samples (Figure 5). Figure 6 shows the porosity (A1: 68 ⫾ 2.5%, A2: 78 ⫾ 1.5%, and A3: 84.2 ⫾ 2.8%) and density amounts (A1: 2.67 ⫾ 0.14%, A2: 2.17 ⫾ 0.32%, and A3: 1.85 ⫾ 0.28%) of scaffolds with different ratios of ZrO2.Y2O3/β-TCP. The compressive strength of 6.25 ⫾ 0.01 (A1), 5.65 ⫾ 0.23 (A2), and 4.95 ⫾ 0.94 (A3) MPa and elasticity modulus of 63 ⫾ 0.01 (A1), 54 ⫾ 0.23 (A2), and 48 ⫾ 0.94 (A3) MPa were obtained for scaffolds. Results show decreasing strength and increasing porosity for the samples when ZrO2 content decreased from 50% to 30% in the scaffold (Figure 7).
(E)
Cell results After plating for 24–48 h, some adherent cells appeared in the flask, and the cells were heterogeneous in appearance. Approximately 10 days later, these cells developed into many clusters, and could be used for sub-culturing. After 3 passages, human ESCs became relatively homogeneous in appearance, being relatively elongated or spindle-shaped The immunophenotype was based on the flowcytometric analysis of a subset of stem cell marker (OCT4), MSC markers (CD90, CD105 and CD44), haemopoietic markers (CD34 and CD133), and endothelial marker (CD31). The analysis showed that they were positive for CD90, CD105, and OCT4 and negative for CD31 and CD34 (Figure 8). Given the phenotypic, morphological, proliferative characteristics of the ESCs, we determined whether these cells were capable of differentiating into various lineages as can other stem cell types. Differentiation into osteocytes was demonstrated by culturing of EnSC using standard commercially available culture reagents and methodologies.
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Osteoconductivity of calcium phosphate-zirconia composite 5
genes relative expression ratio plot [ mean ±S.E. ] Figure 9. Osteogenic capacity under in vitro condition (A, B), immunocytochemistry for osteocyte special marker osteopontin (C) and osteocalsin (D) [Scale bar: 100 μm], evaluation of gene expression of critical genes (SPP1, ALP, SPARC) for osteoconductivity by Real time PCR in 3D culture condition (E).
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Figure 10. MTT result after 3 and 6 days co-culture ESC and ZrO2/ β-TCP composite. Y2O3/β-TCP in the samples with different ratios of ZrO2.Y2O3/β-TCP. A1: 50/50, A2: 40/60, and A3: 30/70.
EnSCs are easier to isolate and expand with less technical problems compared to bone marrow MSCs. Here we show that EnSCs can be differentiated into osteogenic cells as one of the most important issues in orthopedic surgery is associated with bone loss in traumas, infections, tumors, or congenital disorders (Figure 9B) and also show immunocytochemistry for osteocyte special marker osteopontin and osteocalcin (Figure 9C and D). Evaluation of gene expression of critical genes (SPP1, ALP, SPARC) for osteoconductivity has been performed by Real time PCR in 3D culture condition. The MTT results after 48 hours showed that 96% of hESCs are alive in the presence of ZrO2/β-TCP nanocomposite scaffold which confirms the biocompatibility of the prepared scaffold (Figure 10). At day 3 of culture, the cells on the surface of a scaffold composed of 30/70 ZrO2/β-TCP had a well-spread morphology but the cells on the surface of a scaffold composed of 50/50 ZrO2/β-TCP were less attached and remained spindle shaped. A plot of cell number against culture period showed that cell proliferation was significantly
higher in the scaffolds containing less than 50% ZrO2. The EnSC proliferation test and MTT assay results demonstrated that ZrO2 scaffolds have good biocompatibility and cell viability. Staining with osteoblasts specific markers showed that these cells expressed osteoblast cell markers (osteopontin and osteocalcin) 6 days after implantation of ESCs on the scaffold. These results indicate that the nanocomposite scaffolds is suitable for ESCs and their differentiation to osteoblast cells (Figure 11). SEM images of differentiated cells cultured in the scaffolds with different compositions are shown in Figure 12. The osteocyte cells adhesion and proliferation in the ZrO2/β-TCP scaffold was greatly improved in the samples with ratios of ZrO2.Y2O3/β-TCP: 30/70. The material chosen for a bone scaffold is very important because it must possess or be able to be modified to achieve adequate biocompatibility, porosity, pore size, surface texture, mechanical properties, and biodegradability. To date, ceramics and polymers from both natural and synthetic origins have been used to construct bone scaffolds for bone defects. However, most ceramics are not biodegradable, which presents a problem in bone restoration applications that require the scaffold material to degrade over time. This limits scaffold material choices to a small number of ceramics and biodegradable polymers. Ceramics, such as HAp, bioactive glasses, and calcium phosphates, have been widely used for bone regeneration and replacement (Oe et al. 2007). All three materials exhibit a bioactive and biocompatible behavior and have been traditionally used as coatings on metals for orthopedic/ dental implants but more recently have been proposed as filler material for bone defects and as bone scaffolds (Oe et al. 2007). These materials are unique in that they form a calcium-deficient (non-stoichiometric) apatite surface layer in the presence of biological fluids (Einhorn 1998a). This surface layer is thought to stimulate bioceramic-bone binding and, in some cases, promote new bone formation
Figure 11. Immunocytochemistry for the expression of osteoblast markers, 6 days after the implantation of differentiated cells on the scaffolds. Osteopontin (A: osteopontin specificity marker, B: 4,6-diamidino-2-phenylindole dihydrochloride, and C: negative control) and osteocalsin (D: osteocalcin specificity marker, E: 4,6-diamidino-2-phenylindole dihydrochloride, and F: negative control); Scale bar: 100 μm.
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Osteoconductivity of calcium phosphate-zirconia composite 7
Figure 12. SEM images of the culture ESC on ZrO2/β-TCP composite (Y2O3/β-TCP: 30/70) in different magnifications (A: 250X, B: 500X, C: 1000X, and D: 5000X).
(El-Ghannam 2005). Calcium phosphate based materials, in particular, have been widely investigated for use as bone replacement materials due to their biocompatibility and osteoconductivity (Chao and Inoue 2003, Khan et al. 2005, Fleming et al. 2000). Synthetic β-TCP is the two most common calcium phosphate materials used as hard-tissue replacements (Oe et al. 2007). Several studies have shown that using these two bioceramics as bone scaffolds, both seeded with and seeded without bone marrow cells, have yielded bone regeneration (Liao et al. 2004, Oest et al. 2007, Tanaka et al. 2010). Calcium phosphates are biocompatible and osteoconductive, although they have some drawbacks for use as bone scaffolds. These materials are brittle and have low mechanical stability, which limits their use in large defects that require load bearing (Einhorn 1998a). Furthermore, their dissolution and degradation rates are difficult to control in vivo (Einhorn 1998a). These factors could present a problem because if the scaffold degrades too fast, the mechanical stability would be compromised and could fail. In this study cell adherence ability was used to assess cell biocompatibility of the material. The results showed that ZrO2/β-TCP composites, especially the graded one, were more conducive to cell adhesion and proliferation. Significantly increased adhesion rate of graded composite may be due to many reasons. SEM images showed that the surface of the graded composite was porous with uniform pore diameter of 100–200 μm. XRD analysis showed that after ZrO2/β-TCP composite was sintered at 1500°C, new phases of β-Ca3(PO4)2, α-Ca3(PO4)2, and CaZrO3 were generated. The biocompatibility of composite materials was achieved, because after the high-temperature phosphate contacted with water or body fluid at 37°C, β-phase was formed as
well as Ca2⫹, HPO42⫺ were decomposed and deposited on the surface of bioceramic substrate. Over a period of time of contact, composition in bioceramic-based body changed slightly. ZrO2/β-TCP phase decreased and HAp increased. It is helpful for adsorbing serum protein and forming cell adhesion. We speculate that the adhesion of all the attachment-dependent cells on the biological scaffold material is enhanced. The increased proliferation rate of MSCs on the surface of ZrO2/β-TCP composites shows some characteristics of the material or components. Moreover, it is also due to the rough surface of substrates. Many studies have shown that the substrate surface roughness relates to the rate of cell proliferation (Einhorn 1998b, El-Ghannam 2005). Adherence enhancement may be another reason leading to increased proliferation rate. The adhesion enhancement of initially inoculated cell can result in a higher increase in cell density, thus affecting the biological behavior, such as proliferation rate.
Conclusions In this study, we have designed a new bioceramic composite consisting of porous calcium phosphate and strong load-bearing ZrO2 ceramic. Nanocomposite scaffold were fabricated via foam casting with different compositions. These scaffolds were sintered at 1500°C to obtain porous scaffolds. Porous ZrO2/β-TCP composite scaffold has excellent mechanical properties, supports cellular attachment and differentiation. We showed that ESCs differentiate into osteogenic cells using this scaffolds. This porous composite provides a potential promising alternative for load-bearing bone replacements.
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Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the contents and writing of the paper.
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