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Article Cite This: ACS Omega 2019, 4, 449−457
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Fabrication and in Vitro Evaluation of Nanocomposite Hydrogel Scaffolds Based on Gelatin/PCL−PEG−PCL for Cartilage Tissue Engineering Nahideh Asadi,†,‡ Effat Alizadeh,§,∥ Azizeh Rahmani Del Bakhshayesh,‡,§,⊥ Ebrahim Mostafavi,∇ Abolfazl Akbarzadeh,*,†,# and Soodabeh Davaran*,†,§ Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, ‡Student Research Committee, §Drug Applied Research Center, ∥Department of Biotechnology, Faculty of Advanced Medical Sciences, ⊥Department of Tissue Engineering, Faculty of Advanced Medical Sciences, and #Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz 15731, Iran ∇ Department of Chemical Engineering, Northeastern University, Boston, Massachusetts 02115, United States ACS Omega 2019.4:449-457. Downloaded from pubs.acs.org by 179.61.200.25 on 01/12/19. For personal use only.
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ABSTRACT: The self-repair of cartilage tissue is limited. Hydrogels with 3D hydrated polymers have properties that make them a suitable material for cartilage tissue engineering. In this study, a novel nanocomposite hydrogel based on gelatin/polycaprolactone−polyethylene glycol (Gel/PCEC−TGFβ1) was prepared and evaluated for the proliferation and chondrogenic differentiation of human mesenchymal stem cells derived from adipose tissue (hAD-MSCs). The porosity of the prepared scaffolds was characterized by scanning electron microscopy (SEM). Swelling behavior and mechanical properties of the prepared hydrogels were evaluated. h-AD-MSCs were cultured on each scaffold for 14 days, and cell attachment was investigated by SEM. The cell viability was studied using MTT assay. Toluidine blue staining was carried out to evaluate the synthesis of proteoglycan. Real-time PCR assay was used to monitor the expression of collagen II and aggrecan genes. The data showed that scaffolds support cell proliferation in comparison to control group. Histological analyses with toluidine blue staining exhibited the deposition of glycosaminoglycans (GAGs). Real-time PCR analysis also indicated that hydrogels, especially the nanocomposite hydrogel, showed the potential to express collagen II and aggrecan genes. Therefore, the Gel/PCEC−TGFβ1 nanocomposite hydrogels have the potential for the growth and differentiation of h-AD-MSCs and can be a promising biomaterial for cartilage tissue engineering.
1. INTRODUCTION Articular cartilage is a connective tissue that is responsible for load bearing in joints.1 Degeneration of hyaline cartilage in the articular joints leads to osteoarthritis (OA). It affects a large number of individuals, especially the aging population. The self-repair of cartilage tissue is limited because it is an avascular, alymphatic, and aneural tissue and is composed of low chondrocytes that is embedded within a dense extracellular matrix (ECM), so there are challenges in the regeneration of cartilage.2−4 Among the therapies for the repair of cartilage defects, tissue engineering has taken more consideration. This approach combines cells, scaffolds, and environmental factors such as growth factors for the repair of articular cartilage defects. The scaffold acts as an ECM that provides a threedimensional (3D) conformation for the cells.5−7 Hydrogels are 3D hydrated polymers that have a structure similar to the microenvironment of chondrocytes in vivo.8 They have a highly swollen and porous structure that is suitable for the diffusion of various solutes and nutrients.9 There are various methods for the creation of the polymeric networks of hydrogels such as chemical and physical crosslinking. In physically cross-linked hydrogels, physical inter© 2019 American Chemical Society
actions between the polymer chains preserve the 3D structure in aqueous environments. Changes in environmental conditions (e.g., temperature, pH, and ionic interactions) play a role in formation of physically cross-linked hydrogels. In chemically cross-linked hydrogels, the covalent bonds between polymer chains make stable gels. Each of these gels has some drawbacks; for example, in physically cross-linked hydrogels, the low mechanical properties is a downside. In chemically cross-linked gels, the residual of chemical cross-linkers, organic solvents, and the photoinitiator may cause cytotoxicity.10 Nanocomposite hydrogels, also known as hybrid hydrogels, are hydrated 3D polymeric networks that physically or covalently cross-linked with each other and/or with nanoparticles or nanostructures. Various nanostructures such as carbon-based nanomaterials, inorganic nanoparticles (e.g., hydroxyapatite and silica), and polymeric nanoparticles (e.g., polymer nanoparticles and dendrimers) are applied in nanocomposite hydrogels. These nanostructures physically or Received: September 30, 2018 Accepted: December 20, 2018 Published: January 7, 2019 449
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belongs to the methylene protons of −CH2CH2O− in PEG units. The strong peaks at 1.35, 1.6, 2.25, and 4 ppm are attributed to the α, β, γ, and δ methylene protons of the carbonyl group of PCL, respectively. 2.2. Size Distribution of the PCEC Nanoparticles. DLS technique was used to determine the particle size of nanoparticles. The results of size and zeta potential are shown in Figures 4 and 5, respectively. The PCEC nanoparticles are approximately 177 nm. The zeta potential value of the nanoparticles (pH 7.4) was −6.58 mV. 2.3. SEM Images of the PCEC Nanoparticles. The morphology and size of nanoparticles were studied by SEM (Figure 6). The average size of particles was approximately 100 nm. Also, nanoparticles have a spherical shape. 2.4. SEM Analysis of the Prepared Scaffolds. The morphology and pores of prepared scaffolds were studied using SEM. Images indicated that hydrogels have interconnected porous structures (Figure 7A,B). We used the freeze-drying method in the fabrication of porous hydrogel scaffolds. Porosity is an important factor in cell growth, because it provides an appropriate interaction of cells with the scaffold. There are various factors such as the temperature of the crosslinking and freeze-drying process that has an effect on the porosity of scaffolds. In a freeze-dried twice method, the ice crystals that formed in the second freeze drying can penetrate the walls of the pores, which create more tiny interconnecting pores where cells move more quickly in the scaffold.22 The average pore sizes of the Gel/PCEC−TGFβ1 and Gel hydrogels were 140.60 ± 49.69 and 263.19 ± 95.36 μm, respectively (Figure 7C). There was a significant difference between the Gel/PCEC−TGFβ1 and Gel hydrogels (P ≤ 0.05). The average pore size of the nanocomposite hydrogel was decreased by the addition of PCEC−TGFβ1 nanoparticles. These nanoparticles can fill the pores. Also, they aggregate by van der Waals force.23 The pore size of the scaffold is an important factor; it should be large enough for nutrients to diffuse and cells to migrate and also small enough for cells to have an adequate area for attachment. The optimal pore size for scaffold is dependent on the cell type.24 In previous studies, it was found that the pore size of the scaffold has an important role in the growth of the chondrocytes and ECM production.22 Also, there are reports suggesting that scaffolds in the size of 100−500 μm support chondrogenesis and the production of ECM.25 2.5. Swelling Behavior. The swellability of the scaffold is important because 70−85% of the weight of natural cartilage tissues is water.26 Absorption and retention of a large amount of water are basic properties of 3D polymeric networks such as hydrogels.25 This property is suitable for the absorption of body fluid and the diffusion of nutrients and metabolites, cell infiltration into the scaffolds, and cell adhesion.27 So, it is necessary to evaluate the swelling behavior of the scaffolds. To measure the swelling properties, the scaffolds were immersed in PBS and weighed at determined times (Figure 8). A higher swelling ratio was observed in Gel hydrogels compared to the Gel/PCEC−TGFβ1 (P ≤ 0.05). The addition of PCEC nanoparticles to the hydrogel decreased the swelling of the nanocomposite scaffold. Nanoparticles can act as filler and make a denser and tighter network that has smaller pores.26 The pore size of the scaffold affects the swelling behavior, and a larger pore size has more space for water absorption.28 In the present study, the Gel/PCEC−TGFβ1 scaffold has a smaller pore size in comparison to the gelatin hydrogel.
covalently interact with the polymeric chains to create novel properties in hydrogels.11 The nanoscale dimension can modify the limitations of the hydrogel. For instance, a high surface area-to-volume ratio improves the surface reactivity, bioavailability, release of the loaded bioactive agents and mechanical properties. Also, they influence the transport properties and lead to an effective delivery of therapeutic agents to target the cells because they can penetrate tissues via capillaries and epithelial lining.12−16 Gelatin is derived from collagen by hydrolysis, and its chemical composition is similar to collagen. It has low antigenicity and immunogenicity.17 The gelatin possesses a cell-attachable sequence, Arg-Gly-Asp (RGD), that has an important role in tissue engineering as a scaffold.18 Poly-ε-caprolactone (PCL) is a biodegradable and biocompatible polymer, so it is used as a carrier for controlled drug delivery systems and tissue engineering in many studies. This polymer is nontoxic and thus it can be an ideal material for tissue engineering applications.19,20 The aim of this study was to develop hydrogel scaffolds using gelatin and nanocomposite hydrogel scaffold based on gelatin and PCL−PEG− PCL (PCEC) nanoparticles that are loaded with transforming growth factor β1 (TGFβ1) and investigate their potential as scaffolds for cartilage tissue engineering. The scaffolds were studied by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). Moreover, the mechanical properties and swelling behavior of the scaffolds were evaluated. Human mesenchymal stem cells derived from adipose tissue (h-AD-MSCs) were utilized for the in vitro cell studies.
2. RESULTS AND DISCUSSION In the present study, hydrogel scaffolds based on gelatin and TGFβ1-loaded PCEC nanoparticles were fabricated and evaluated for cartilage tissue engineering by h-AD-MSCs. The prepared scaffolds are shown in Figure 1.
Figure 1. The fabricated hydrogels in this study.
2.1. Characterization of PCEC Copolymer. 2.1.1. FT-IR Analysis. FT-IR analysis provides information about functional groups in the structure of a molecule. In the present study, the chemical structure of PCEC copolymer was studied by FT-IR. Figure 2 shows the FT-IR spectra of the PCEC copolymer. The absorption bands at 1188 and 1246 cm−1 are attributed to the C−O−C stretching vibration and asymmetric C−O−C stretching vibration of PEG, respectively. The peaks at 1368, 1728, 2859, and 2943 cm−1 belong to the C−O and C−C stretching vibration, CO stretching vibration, symmetric CH2 stretching, and asymmetric CH2 stretching, respectively. The peak at 1728 cm−1 corresponds to the stretching vibrations of the ester carbonyl group. Terminal −OH group peak is at 3437 cm−1.21 2.1.2. 1H NMR. The 1H NMR spectra of the PCEC copolymer are shown in Figure 3. The sharp signal at 3.6 ppm 450
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Figure 2. FT-IR spectra of the PCEC copolymer.
Figure 3. 1H NMR of the PCEC copolymer.
stiffness and strength.15 Also, the existence of larger pores of the Gel hydrogel compared to Gel/PCEC−TGFβ1 hydrogel can lead to lower mechanical properties of the Gel scaffold. The Young’s modulus of cartilage is in the range of 0.45−0.80 MPa.30 The physiochemical properties of scaffolds were shown in Table 1. 2.7. Cell Culture Studies. 2.7.1. Cell Viability Test. h-ADMSCs were seeded onto the Gel/PCEC−TGFβ1 and Gel scaffolds, and cell proliferation was assessed after 4, 7, and 14 days of culturing. For the evaluation of cell proliferation in the scaffolds, MTT assay was used (Figure 10). All the prepared scaffolds induced the proliferation of h-AD-MSCs compared to
2.6. Mechanical Analysis. The mechanical properties of the scaffold are important factors because they affect cellular adhesion, proliferation, and signaling. The mechanical properties of the scaffolds were studied by compression mode.29 Figure 9 shows the compressive stress−strain curves of the scaffolds. The Young’s modulus of nanocomposite hydrogel and Gel scaffolds was calculated from the data obtained from the stress−strain curves. The Young’s modulus of Gel/PCEC− TGFβ1 hydrogel was not significantly different from that of Gel hydrogel. The addition of PCEC nanoparticles enhances the Young’s modulus of the hydrogel scaffold. Nanocomposite biomaterials provide superior mechanical properties and higher 451
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Gel/PCEC−TGFβ1 scaffold showed significantly (P ≤ 0.05) higher expression levels of aggrecan and collagen type II in comparison to the other scaffolds. Collagen II is the main collagen of cartilage tissue. Aggrecan is a major proteoglycan of the ECM that contains one core protein and GAGs binding to it. So, the evaluation of it reflects the overall GAG content.25
3. CONCLUSIONS In the present study, a nanocomposite hydrogel based on Gel/ PCEC−TGFβ1 and Gel hydrogel was successfully synthesized. The physicochemical properties of the scaffolds such as morphologies, pore size, swelling ratio, and mechanical characteristics were assessed. The prepared scaffolds showed a porous structure. The Gel scaffold showed a larger pore size (263.19 ± 95.36 μm) than the Gel/PCEC scaffold (140.60 ± 49.69 μm). Also, the addition of PCEC nanoparticles leads to a higher Young’s modulus of nanocomposite scaffold compared to the Gel scaffold. h-AD-MSCs were used for proliferation and differentiation studies. The biocompatibility and cell adhesion of the prepared scaffolds were evaluated by MTT assay and SEM. These studies showed good viability and proliferation of MSCs on the scaffolds with higher cell survival for the Gel/PCEC−TGFβ1 hydrogel. RT-PCR technique was applied for the investigation of the expression of cartilagespecific ECM genes such as collagen type II and aggrecan. The results indicated that the scaffolds stimulated the ECM production, which is useful for cartilage tissue engineering. On the basis of these results, in vivo studies are suggested for future examinations.
Figure 4. Size distribution of the PCEC nanoparticles.
the control group, and cell viability was over 80% in all scaffold groups. Cell viability in Gel/PCEC−TGFβ1 scaffolds was significantly higher than that in the control and Gel with free TGFβ1 groups (P ≤ 0.05). It seems that the PCEC nanoparticles loaded with TGFβ1 can release growth factors in a sustained manner and have more porosity, which leads to cell proliferation. It can be suggested that all the scaffolds are biocompatible with cells. 2.7.2. Cell Adhesion Study. The adhesion of h-AD-MSCs on the porous Gel/PCEC−TGFβ1 and Gel scaffolds after 14 days was investigated using SEM. As shown in the FESEM micrographs (Figure 11), cell-seeded hydrogels showed that MSCs adhered to the scaffolds and made a cell-to-cell connection. Also, the pores of scaffolds are filled with cells. It means that scaffolds are biocompatible with h-AD-MSCs. Hydrogels with a 3D structure act as an ECM and support for cell adhesion and proliferation. 2.7.3. Histological Study. The histological analysis was done after 19 days of culture period. The production of glycosaminoglycans in ECM was evaluated by toluidine blue staining.31 This staining showed the typical metachromasia (purple color) of articular cartilage.32 As shown in Figure 12, positive staining with toluidine blue was observed, which was homogeneously distributed throughout the Gel/PCEC− TGFβ1 and Gel hydrogels. The Gel/PCEC−TGFβ1 scaffold showed more GAG deposition as compared to Gel hydrogel. Control group showed weakly positive toluidine blue stain compared to scaffolds group. 2.7.4. Real-Time PCR Analysis. Real-time PCR (RT-PCR) analysis was done after 19 days of culture period to study the expression of chondrogenic specific genes such as collagen type II and aggrecan (Figure 13). According to the figure, the upregulation of the genes was observed in the hydrogels. The
4. MATERIALS AND METHODS 4.1. Materials. Gelation (porcine skin, type A), polyvinyl alcohol (PVA), stannous 2-ethyl hexanoate (stannous octoate, Sn(Oct)2), and toluidine blue were purchased from SigmaAldrich (USA). TGFβ1 was obtained from Abcam. εCaprolactone (ε-CL), PEG (Mw = 2000), glutaraldehyde (25% aqueous solution), and all the solvents were purchased from Merck Inc. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) was purchased from SigmaAldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin−EDTA were purchased from Gibco. 4.2. Synthesis of PCL−PEG2000−PCL (PCEC) Copolymer. PCEC triblock copolymer was synthesized by ringopening polymerization of ε-caprolactone in the presence of PEG2000 using stannous octoate as a catalyst.33 One gram of PEG2000 was heated at 90 °C for 20 min in a two-necked,
Figure 5. Zeta potential of the PCEC nanoparticles. 452
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Figure 6. SEM image of the PCEC nanoparticles.
Figure 9. The stress−strain curves of the Gel and the Gel/PCEC− TGFβ1 scaffolds.
Table 1. Physiochemical Properties of the Scaffolds
Figure 7. (A, B) SEM images of the (A) Gel/PCEC−TGFβ1 and (B) Gel hydrogels. (C) Average pore size of the scaffolds.
property
Gel/PCEC−TGFβ1
Gel
pore size (μm)
140.60 ± 49.69
263.19 ± 95.36
swelling ratio Young’s modulus (MP)
9.96 ± 0.33 0.65
15.5±0.2 0.57
Figure 10. In vitro cytotoxic effects of the Gel/PCEC−TGFβ1 and the Gel scaffolds on human MSCs. Figure 8. Swelling behavior of the scaffolds in PBS. 453
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Figure 11. SEM images of MSCs seeded on the (A) Gel/PCEC−TGFβ1 and (B) Gel scaffolds.
Figure 12. Toluidine blue staining for the evaluation of the synthesized GAGs in the hydrogels after 19 days of culture. (A) Gel/PCEC−TGFβ1 and (B) Gel hydrogels.
Figure 13. RT-PCR analysis of cartilage-related genes after 19 days of cell culture. The genes were normalized to GAPDH.
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round-bottom flask. Then, 2 mL of ε-CL and Sn(Oct)2 (1%, w/w) was added to the flask. After that, the temperature of the reaction was enhanced to 130 °C, and the mixture of the reaction was stirred for 10 h under nitrogen atmosphere. Next, the product was dissolved in dichloromethane and precipitated in cold diethyl ether. Finally, the copolymer was left to dry. 4.3. Preparation of the TGFβ1-Loaded PCEC Nanoparticles. For the encapsulation of TGFβ1 in PCEC nanoparticles, water-in-oil-in-water (w/o/w) system was used. Briefly, 200 mg of PCEC copolymer was dissolved in 4 mL of dichloromethane (oil phase). Then, 100 μL of TGFβ1 (1 μg/mL recombinant human TGFβ1/mL stock) was dispensed in the copolymer solution using an ultrasonicator (SYCLON, China) for 10 s (w1/o). This w1/o emulsion was added to 2 mL of aqueous solution of PVA (4%, w/v) and sonicated to form the w1/o/w2 emulsion. This emulsion was added into 50 mL of another PVA (0.3%, w/v) and was stirred for 3 h to evaporate its solvent. Finally, the resultant nanocapsules were frozen at −80 °C and lyophilized for 48 h.34,35 4.4. Preparation of the Hydrogel Scaffolds. Hydrogel scaffolds were prepared by chemical cross-linking of aqueous gelatin solution with glutaraldehyde as a cross-linker. Briefly, an aqueous solution of gelatin (5%, w/v) was prepared by dissolving gelatin in distilled water at 40 °C. Then, TGFβ1loaded PCEC nanoparticles (concentration of 10 ng/mL of TGFβ1), free TGFβ1 (10 ng/mL), and unloaded nanoparticles were added to the gelatin solution (in three different groups) and stirred. At the next step, glutaraldehyde solution (1%, v/v) was dropped into the mixtures and stirred for 10 min at 30 °C. The cross-linked scaffolds were frozen at −70 °C, and finally, they were lyophilized by a freeze dryer (Telstar) for 48 h. To block the remaining aldehyde groups of glutaraldehyde, scaffolds were immersed in 50 mM aqueous glycine solution and incubated at 37 °C for 1.5 h, rinsed three times with distilled water, and then freeze-dried for the second time.36,37 4.5. Characterization of the PCEC Nanoparticles. The chemical structure of the synthesized PCEC copolymer was studied by a Fourier transform infrared (FT-IR) spectrometer (Bruker, Germany). The sample was mixed with potassium bromide and pressed to form a tablet. The FT-IR spectra of the sample were analyzed in the range of 400−4000 cm−1. Also, the chemical structure of the synthesized PCEC copolymer was studied using 1H NMR (Bruker spectra spin 400 MHz). To determine the distribution of the particle size of nanoparticles, dynamic light scattering (Malvern) technique was used. The morphology and size of the nanoparticles were studied by scanning electron microscopy (MIRA3 FEG-SEM, Tescan, 30 kV voltage) after gold coating. 4.6. Morphology Analysis of the Prepared Hydrogels. The morphology and pore size of scaffolds were studied by FESEM. The average sizes of pores were determined with ImageJ software by random selection of at least 20 different pores. 4.7. Swelling Test. To investigate the swellability of the scaffolds, swelling studies were performed. Scaffolds were placed into PBS (pH = 7.4) and incubated at 37 °C for up to 72 h. Before the immersion of the scaffolds in PBS, the dry weight (Wd) of the scaffolds was determined. At certain times, the samples were taken out from the PBS, and surfaceadsorbed water was removed with a filter paper and weighted (Ww). The swelling ratio was determined using the following equation:27,38
swelling ratio =
(Ww − Wd) Wd
4.8. Mechanical Properties of the Scaffolds. For the evaluation of mechanical properties of the scaffolds, compression tests were used with a universal testing machine (AI-7000-M, Gotech Testing Machine Inc., Taiwan). Samples (cylinder shape: approximately 6 mm in diameter and 10 mm in height) were located on a test plate and compressed with a speed of 5 mm/min by a 25 N load cell at room temperature. The modulus was calculated from the initial linear slope of the stress−strain curve.27,39 4.9. Cell Culture Studies. 4.9.1. Preparation of Scaffolds for Cell Culture. For cell culture studies, scaffolds were placed in cell culture plates and immersed in a 70% ethanol solution for 30 min. After ethanol evaporation, scaffolds were rinsed three times with PBS solution to take out the residual ethanol. Then, the samples were immersed in DMEM and incubated at 37 °C for 4 days before cell seeding. Scaffolds in 12-well plates were seeded with h-AD-MSCs, which was cultured in DMEM containing 10% fetal bovine serum (FBS). For differentiation studies, after 48 h, scaffolds media were changed to chondrogenic media. The chondrogenic media contained DMEM, 2% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), 100 nM dexamethasone, 1X-ITS, bovine serum albumin (50 mg/mL), and ascorbic acid (50 μg/mL) and was incubated at 37 °C in a humid atmosphere with 5% CO2. Cell culture media were changed every 6 days. 4.9.2. Cell Proliferation Analysis by MTT Assay. Cells with a confluency of 80% were detached by trypsin (0.05% trypsin containing 1 mM EDTA) from a cell culture flask. We seeded 2 × 104 cells per scaffold. Culturing studies were performed in triplicate for every type of scaffolds. Cell proliferation analysis was studied with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) assay. This test shows the mitochondrial dehydrogenase activity of viable cells by spectrophotometry. At predetermined times, the old medium was removed, and 150 μL of MTT solution (2 mg/mL in DMEM) and 450 μL of sample medium were added to each well and incubated for 4 h at 37 °C. Then, the medium was removed, and 600 μL of dimethyl sulfoxide (DMSO) was added. The MTT was reduced by the mitochondrial dehydrogenase of normally metabolizing cells, and DMSO can dissolve the purple formazan crystals. The results were obtained with an ELISA reader (Awareness Technologies Stat Fax 2100 Microplate Reader) at 570 nm (reference wavelength of 690 nm). 4.9.3. Cell Adhesion. For cell attachment investigation, scaffolds were seeded with cells and observed by SEM. After cell culturing (14 days), scaffolds that have cells were rinsed two times with PBS, fixed in 2.5% glutaraldehyde for 30 min at 4 °C, dehydrated in a series of ethanol (50, 70, 90, and 96 %), and finally dried at room temperature. 4.9.4. Histological Study by Toluidine Blue Staining. For the assessment of ECM components of cartilage (glycosaminoglycans), toluidine blue staining was used.40 At the end of the experiment (19 days of culture), cell-seeded scaffolds were rinsed twice with PBS, fixed in paraformaldehyde 4%, dehydrated, and embedded in paraffin wax. The paraffinized scaffolds were cut by a microtome and placed on microscope slides. Samples were stained by toluidine blue. The stained slices were observed using a light microscope. 455
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(5) Jia, S.; Liu, L.; Pan, W.; Meng, G.; Duan, C.; Zhang, L.; et al. Oriented cartilage extracellular matrix-derived scaffold for cartilage tissue engineering. J. Biosci. Bioeng. 2012, 113, 647−653. (6) Cui, W.; Wang, Q.; Chen, G.; Zhou, S.; Chang, Q.; Zuo, Q.; et al. Repair of articular cartilage defects with tissue-engineered osteochondral composites in pigs. J. Biosci. Bioeng. 2011, 111, 493− 500. (7) Dikina, A. D.; Alt, D. S.; Herberg, S.; McMillan, A.; Strobel, H.A.; Zheng, Z.; et al. A Modular Strategy to Engineer Complex Tissues and Organs. Adv. Sci. 2018, 5, 1700402. (8) Li, X.; Zhang, J.; Kawazoe, N.; Chen, G. Fabrication of Highly Crosslinked Gelatin Hydrogel and Its Influence on Chondrocyte Proliferation and Phenotype. Polymers 2017, 9, 309. (9) Vega, S. L.; Kwon, M. Y.; Burdick, J. A. Recent advances in hydrogels for cartilage tissue engineering. Eur. Cells Mater. 2017, 33, 59. (10) Annabi, N.; Tamayol, A.; Uquillas, J. A.; Akbari, M.; Bertassoni, L. E.; Cha, C.; et al. 25th anniversary article: Rational design and applications of hydrogels in regenerative medicine. Adv. Mater. 2014, 26, 85−124. (11) Gaharwar, A. K.; Peppas, N. A.; Khademhosseini, A. Nanocomposite hydrogels for biomedical applications. Biotechnol. Bioeng. 2014, 111, 441−453. (12) Asadi, N.; Alizadeh, E.; Salehi, R.; Khalandi, B.; Davaran, S.; Akbarzadeh, A. Nanocomposite hydrogels for cartilage tissue engineering: a review. Artif. Cells, Nanomed., Biotechnol. 2018, 46, 465−471. (13) Asghari, F.; Samiei, M.; Adibkia, K.; Akbarzadeh, A.; Davaran, S. Biodegradable and biocompatible polymers for tissue engineering application: a review. Artif. Cells, Nanomed., Biotechnol. 2017, 45, 185−192. (14) Jayaraman, P.; Gandhimathi, C.; Venugopal, J. R.; Becker, D. L.; Ramakrishna, S.; Srinivasan, D. K. Controlled release of drugs in electrosprayed nanoparticles for bone tissue engineering. Adv. Drug Delivery Rev. 2015, 94, 77−95. (15) Eftekhari, H.; Jahandideh, A.; Asghari, A.; Akbarzadeh, A.; Hesaraki, S. Assessment of polycaprolacton (PCL) nanocomposite scaffold compared with hydroxyapatite (HA) on healing of segmental femur bone defect in rabbits. Artif. Cells, Nanomed., Biotechnol. 2017, 45, 961−968. (16) Liao, J.; Wang, B.; Huang, Y.; Qu, Y.; Peng, J.; Qian, Z. Injectable alginate hydrogel cross-linked by calcium gluconate-loaded porous microspheres for cartilage tissue engineering. ACS Omega. 2017, 2, 443−454. (17) Chen, S.; Zhang, Q.; Nakamoto, T.; Kawazoe, N.; Chen, G. Gelatin scaffolds with controlled pore structure and mechanical property for cartilage tissue engineering. Tissue Eng., Part C 2016, 22, 189−198. (18) Han, M.-E.; Kang, B. J.; Kim, S.-H.; Kim, H. D.; Hwang, N. S. Gelatin-based extracellular matrix cryogels for cartilage tissue engineering. J. Ind. Eng. Chem. 2017, 45, 421−429. (19) Dash, T. K.; Konkimalla, V. B. Poly-ε-caprolactone based formulations for drug delivery and tissue engineering: A review. J. Controlled Release 2012, 158, 15−33. (20) Rahmani Del Bakhshayesh, A.; Mostafavi, E.; Alizadeh, E.; Asadi, N.; Akbarzadeh, A.; Davaran, S. Fabrication of ThreeDimensional Scaffolds Based on Nano-biomimetic Collagen Hybrid Constructs for Skin Tissue Engineering. ACS Omega. 2018, 3, 8605− 8611. (21) Valizadeh, A.; Bakhtiary, M.; Akbarzadeh, A.; Salehi, R.; Frakhani, S. M.; Ebrahimi, O.; et al. Preparation and characterization of novel electrospun poly(ϵ-caprolactone)-based nanofibrous scaffolds. Artif. Cells, Nanomed., Biotechnol. 2016, 44, 504−509. (22) Lien, S.-M.; Ko, L.-Y.; Huang, T.-J. Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta Biomater. 2009, 5, 670−679. (23) Chern, M.-J.; Yang, L.-Y.; Shen, Y.-K.; Hung, J.-H. 3D scaffold with PCL combined biomedical ceramic materials for bone tissue regeneration. Int. J. Precis. Eng. Manuf. 2013, 14, 2201−2207.
4.9.5. RT-PCR Analysis. The specific genes of cartilage such as collagen type II and aggrecan were used for gene expression analysis.25 GAPDH was applied as the internal control (Table 2 lists the used primers). Total RNA was extracted from cellTable 2. Sequence of Primers for RT-PCR gene
sequence
collagen II
F: 5′-GACAATCTGGCTCCCAAC-3′ R: 5′-ACAGTCTTGCCCCACTTAC-3′ F: 5′-TGAGTCCTCAAGCCTCCTGT-3′ R: 5′-TGGTCTGCAGCAGTTGATTC-3′ F: 5′-ACAGTCAGCCGCATCTTCTT-3′ R: 5′-ACGACCAAATCCGTTGACTC-3′
aggrecan GAPDH
seeded scaffolds and control group by TRIzol reagent (Invitrogen) and spin columns (BIO BASIC) according to the manufacturer’s protocols. RNA samples were used for cDNA synthesis (Takara, cDNA Synthesis kit). RT-PCR was performed using SYBR Green Master Mix (APLIQON), forward primer, reverse primer, and cDNA to make a reaction volume of 12 μL. Reaction samples were evaluated in a RTPCR system (magnetic induction cycler; Bio Molecular Systems). All experiments were studied in triplicate. Results were analyzed using the 2−ΔΔCt method. 4.10. Statistical Analysis. For statistical analysis, t test and ANOVA were carried out to compare the different groups. For statistical significance, P < 0.05 was considered. All experiments were performed in triplicate.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (A.A.). *E-mail:
[email protected]. Tel./Fax: 984133341933 (S.D.). ORCID
Ebrahim Mostafavi: 0000-0003-3958-5002 Abolfazl Akbarzadeh: 0000-0001-9941-0357 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
Authors would like to thank Tabriz University of Medical Sciences for supporting this project (Grant no. 95/2-2/3). This research also was financially supported by Iran National Science Foundation (INSF).
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DOI: 10.1021/acsomega.8b02593 ACS Omega 2019, 4, 449−457
