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Influence of hydrodynamic pressure on chondrogenic differentiation of human bone marrow mesenchymal stem cells cultured in perfusion system
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Influence of hydrodynamic pressure on chondrogenic differentiation of human bone marrow mesenchymal stem cells cultured in perfusion system Soheila Zamanluia,1, Leila Mohammadi Amirabadb, Masoud Soleimanic,d,∗∗, Shahab Faghihia,∗ a
Stem Cell and Regenerative Medicine Group, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161, Iran School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran c Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 111/14115, Iran d Nanotechnology and Tissue Engineering Department, Stem Cell Technology Research Center, Tehran 1997775555, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chondrocytes Hydrodynamic pressure Perfusion system Nanofibrous scaffold Mesenchymal stem cells
The natural conditions of chondrocytes in native cartilage including mechanical forces and surface topology could be simulated to enhance chondrogenesis. A perfusion system recapitulating the hydrodynamic pressure of cartilage tissue is designed. Mesenchymal stem cells (MSCs) are isolated and seeded on aligned nanofibrous PCL/ PLGA scaffolds that mimic the structure of superficial zone of articular cartilage. The cell-seeded scaffolds are placed into the perfusion bioreactor and exposed to chondrogenic differentiating medium. The chondrogenesis is then investigated by histological analysis and real time PCR for cartilage-specific genes. The highest expression levels of aggrecan and type II collagen are observed in the cells cultured in the presence of differentiating medium and mechanical stimulation. The expression level of type II collagen is higher than aggrecan in presence of differentiating medium and absence of mechanical stimulation. On the contrary, the expression ratio of aggrecan is higher than type II collagen in presence of mechanical stimulation and absence of differentiating medium. These results show the dominant role of mechanical stimulation and differentiating medium on upregulated expression of aggrecan and type II collagen, respectively. The application of mechanical stimulation upon cells-seeded scaffolds could mimic superficial zone of articular cartilage tissue and increase derivation of chondrocytes from MSCs.
1. Introduction Articular cartilage damages which are commonly originated from accidents, degenerative joint diseases and normal wear and tear are known as the main causes of musculoskeletal morbidity. Cartilage tissue has a limited capacity to regenerate because of the lack of innervation and blood vessels [1]. Therefore, designing a functional construct by tissue engineering approach could be a promising strategy to restore articular cartilage function. In order to produce a functional cartilage construct, the native articular cartilage setting for the cells should be recapitulated, including extracellular matrix (ECM) composition and mechanical-loaded environment [2]. Due to their high accessibility and capacity for cartilage differentiation, adipose and bone marrow mesenchymal stem cells (MSCs) could be an appropriate cell sources for cartilage tissue engineering.
Another important factor for cartilage tissue engineering is to mimic ECM using biocompatible scaffolds in vitro. It has been shown that surface topology, stiffness, and alignment of biological scaffold components could affect the proliferation and fate of cells [3,4]. Recently, electrospinning technique has enabled the fabrication of scaffolds with nano-scaled fibers providing high surface to volume ratio and mimicking the topology of ECM. The ECM of cartilage is primarily composed of type II collagen and negatively charged proteoglycans that hold water in the tissue. Although most of the cartilage tissue is made of water, it has enough strength to bear the loaded compressive forces. The scaffolds used in chondrogenesis should be highly porous to hold a great volume of water at the same time as having appropriate mechanical strength to be similar to the structure of cartilage tissue. Poly D, L-lactic-co-glycolic acid (PLGA) is a polyester giving high degree of porosity to the scaffolds, which has a tailored degradation rate and
∗ Corresponding author. Stem Cell and Regenerative Medicine Group, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14965/161, Iran. ∗∗ Corresponding author. Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 111/14115, Iran. E-mail addresses: [email protected] (M. Soleimani), [email protected], [email protected] (S. Faghihi). 1 Present address: Stem Cells and Cell Therapy Research Center, Tissue Engineering and Regenerative Medicine Institute, Tehran Central Branch, Islamic Azad University, Tehran, Iran.
https://doi.org/10.1016/j.biologicals.2018.04.004 Received 13 September 2017; Received in revised form 8 April 2018; Accepted 20 April 2018 1045-1056/ © 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Zamanlui, S., Biologicals (2018), https://doi.org/10.1016/j.biologicals.2018.04.004
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10 × 60 × 0.06 mm. The tensile testing of the fabricated scaffolds was performed by applying 0.5 kN force at 50 mm/min traverse speed using a mechanical test machine (Instron 5565 A).
biocompatibility. Previous studies have shown that the diameter for PLGA nanofibers in electrospun scaffolds is commonly in the range of 50–300 nm [5]. Therefore, due to the poor tensile strength of PLGA in comparison to cartilage tissue, it should be used along with other polymers. Polycaprolactone (PCL) is another biodegradable polymer that is commonly used for fabrication of scaffolds in tissue engineering which provides appropriate mechanical properties. Moreover, PCL could promote chondrocyte proliferation and maintain its phenotype [6]. However, owing to the poor hydrophilic trait of PCL and its slow degradation rate, it should be used with a more hydrophilic polymer. The scaffolds composed of PLGA and PCL could be a good candidate for cartilage tissue engineering given the capacity of PLGA to make a highly porous scaffold and the ability of PCL to offer appropriate tensile strength [7]. There is growing evidence that in vitro mechanical stress has an important role in sustaining and enhancing the chondrogenic phenotype. Recently, different types of devices and bioreactors have been manufactured to investigate the in vitro effects of mechanical forces on functional tissue formation. Many studies have shown that hydrodynamic loads could induce the deformation of MSC ECM and activate integrin signaling [8,9]. Subsequently, this hydrodynamic pressure over cartilage can improve chondrogenic differentiation via upregulation and downregulation of SOX-9 and IL-1β expression, respectively. This in turn will lead to the expression of proteoglycan, collagen type II, and cartilage-specified ECM [10]. On the other hand, the mechanical stimuli have shown to affect cartilage differentiation in various ways based on the types of scaffolds. For example, static and hydrodynamic pressures have revealed to provide diverse effects on the efficiency of cartilage differentiation [11–13]. In addition, perfusion culture systems could supply a steady gas exchange and nutrition supply into the center of engineered constructs and makes the cells proliferate and differentiate more effectively. The aim of this study was to investigate chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSC) cultured on the fabricated aligned nanofibrous scaffolds under mechanical forces of perfusion bioreactor. The composite PCL/PLGA aligned nanofibrous scaffolds were fabricated using electrospinning followed by chemical and mechanical characterization by FTIR, contact angle, SEM, and tensile analysis. Human bone marrow mesenchymal stem cells (hBMMSC) were seeded on the scaffolds and placed into the chamber of perfusion bioreactor and exposed to chondrogenic differentiating medium for 21 days. Ultimately, the chondrogenic activity of hBMMSCs were assessed by Real-Time PCR, Alcian blue, Safranin O staining and immunohistochemistry (IHC).
2.2.2. Contact angle and porosity measurements The hydrophilicity of the prepared scaffolds was measured using sessile drop method. For this purpose, the average value of contact angle was calculated by an optical bench type contact angle measuring system using four water droplets (Rame- Hart Instrument Company, USA). The apparent density of scaffold (A) (g/cm3) and its porosity (P) (%) was calculated from five separate samples using equations (1) and (2), respectively:
A=
P=1−
2.2.4. Morphological study The morphology of the surfaces of aligned scaffolds before and after cell culture was monitored by scanning electron microscopy (SEM). The scaffolds seeded with cells were washed with PBS and fixed with 2.5% glutaraldehyde for 40 min. The scaffolds were then dehydrated in ascending alcohol concentrations. Finally, the samples were left to dry at room temperature (RT) and were sputter-coated with gold. The accelerating voltage of scanning electron microscope (KYKY-EM3200, Madell Technology Corporation, CA, USA) was 24 kV. The average diameter of nanofiber was determined from at least 100 chosen fibers using image analysis software (Image J, NIH, USA) and the results were reported as mean ± SD. 2.3. Cell isolation and culture Human bone marrow mesenchymal stem cells (hBMMSCs) from seven healthy Caucasian females (18–25 years) were isolated and cultured according to previously described protocol [15]. In brief, the mononuclear cell layer was isolated using ficoll from bone marrow samples collected from the iliac bone (1.077 gl−1, sigma) at a ratio of 1:3. The study was approved by the Research Ethics Board of Taleqani Hospital and all the subjects participated in this study with informed consent. The isolated hBMMSCs were cultured in culture media [DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin)] in T25 flasks. For cell passage, the cells were treated with trypsin–EDTA solution (GIBCO) and centrifuged after washing with PBS.
PLGA/PCL composite nanofibrous scaffolds were fabricated using electrospinning process according to previously described protocol [4]. Briefly, 12% (w/v) PCL granule (Sigma) was dissolved in dimethylformamide (DMF)/chloroform (1:9). To prepare PLGA solution, 7% (w/v) PLGA was dissolved in DMF/chloroform solution (1:3). Aligned PLGA/PCL nanofibrous scaffolds were fabricated with PCL and PLGA solutions (50:50) having flow rates of 0.5 and 0.3 mL/h. The distances between needles of PCL and PLGA syringes and collector were adjusted at 24 and 16 cm, rotating collector speed was 2800 rpm and voltage kept at 24 kV. The fabricated scaffolds were treated with O2 plasma at 100 W for 30 s (Diener electronics, Germany) to increase the surface hydrophilicity.
2.4. MTT assay The viability of hBMMSCs on the fabricated scaffolds was assessed using MTT assay as previously described protocol [16]. The cells were seeded with an initial density of 3000 cells/cm2 in a 96-well culture plate with sterilized scaffolds. On day 1, 4, 7 and 14, the cell culture medium was discarded and the cells were incubated with 100 μl of MTT solution (500 μg/ml) (Sigma-Aldrich, Cat. R8755) for 3.5 h. Formazan crystals in the cells were dissolved in DMSO and the plates were analyzed using an ELISA plate reader at 570 nm.
2.2. Scaffolds characterization
cut
with
a
dimension
(2)
2.2.3. ATR-FTIR analysis ATR-FTIR spectroscopy was performed using Bruker Equinox 55 over the range of 500–4000 cm−1 to analyze the chemical content of PCL/PLGA hybrid fabricated scaffolds.
2.1. Scaffold fabrication
were
A × 100 A0
(1)
where A is the apparent density of each composite nanofibrous scaffolds and A0 is the bulk density of scaffold (g/cm3) [14].
2. Materials and methods
2.2.1. Mechanical properties The scaffold specimens
mass of scaffold(g) Area of scaffold(cm2) × Thickness of scaffolds(cm)
of 2
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Scheme 1. A representation of the bioreactor designed by Rhino ceros software showing dimensions (cm) of the bioreactor and a single chamber (A), connection of the bioreactor to a peristaltic pump in a CO2 incubator (B).
mesenchymal tissue (1 × 10–13 m4/N s). The temperature of the media in the reservoirs and 6-well plate in each chamber of bioreactor (7 mL) was repeatedly scanned every 15 min until it became stable. Similarly, the pH was measured continuously using a pH meter (Thermo Scientific, USA) until equilibrium.
2.5. Design of perfusion bioreactor A four-well bioreactor was designed to apply continuous perfusion using Rhino Ceros software as depicted in Scheme. 1A. The bioreactor was constructed of Plexiglas [Poly (methyl methacrylate) (PMMA)] and the chambers were carved with a CNC router (C.R. Onsrud, Inc., USA). Each chamber was connected to the outside with two stainless steel (316) ports at the top and bottom sides of each chamber. The dimension of each cylindrical chamber was 20 mm (diameter) × 25 mm (length) and a stainless-steel mesh with 20 mm (diameter) × 1 mm (length) was placed on the middle of each chamber (Scheme. 1A). The media was entered into the top of each chamber through a silicon tube which was connected to the reservoir. A tube discharged the medium from the bottom port of each chamber. The bioreactor was sterilized by autoclave before use. After seeding the cells in the bioreactor, it was placed in a CO2 incubator and connected to a peristaltic pump (Gilson-miniplus 3, USA) to circulate the media between the chambers and reservoirs (Scheme. 1B). The pressure differential across the construct was calculated as 2.06 × 107 MPa using the Darcy's Law equation [17].
ΔP =
2.6. Cartilage cell differentiation The punched PCL/PLGA hybrid nanofibrous scaffolds with a diameter of 2 cm were sterilized with 70% ethanol and UV exposure. After washing the scaffolds with phosphate buffered saline solution (PBS), they were incubated with DMEM supplemented with 10% FBS overnight. Then, the hBMMSCs (passage three) were seeded on the scaffolds at a density of 3 × 105 cells per cm2 in 12- well tissue culture plates. After 24 h, the scaffolds containing the cells were placed on the stainless-steel mesh located on the middle of each chamber of bioreactor. The chondrogenic differentiation medium [50 μg/mL ascorbic acid 2phosphate (Sigma), ITS + 6.25 μg/mL of insulin, transferrin, and selenous acid (Gibco) plus 10 ng/mL TGF-β1 (Peprotech), 100 nM dexamethasone (Sigma) in DMEM] was added to the reservoir and constant perfusion was conducted for 21 days with flow entry and exit through the top and bottom ports of each chamber. The flow speed was adjusted at 1 mL/min and the medium of reservoirs was exchanged every three days. The control cell group was placed in a CO2 incubator with static
ϑ × μ× Δx κ
Where, ν is the volumetric flow rate (1 mL/min), μ is the dynamic viscosity of medium at 37 °C (686.5 Pas), Δx is the thickness of the construct (30 μm), and κ is the constant of permeability for multipotent
Scheme 2. Presentation of time schedule for chondrogenic differentiation of hBMMSCs. 3
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Table 1 The list of primers used for real-time PCR analysis.
Table 2 The results of scaffold characterization.
Genes
Forward sequence 5'→ 3′
Reverse sequence 5'→ 3′
Product size (bp)
Sample
Contact angle (°)
Porosity (%)
Tensile strength (MPa)
Elongation (%)
aggrecan
TGT CAG ATA CCC CAT CCA C GGT CTT GGT GGA AAC TTT GCT ATG CCT GCC GTG TGA AC
CAT AAA AGA CCT CAC CCT CC GGT CCT TGC ATT ACT CCC AAC ATC TTC AAA CCT CCA TGA TG
85
A50 BP A50 AP Significant change BP & AP of PCL/PLGA
120 ± 2 25 ± 2.3 Yes
70 ± 3 74 ± 5 No
20.66 ± 0.42 19.8 ± 0.3 No
85 ± 3 86.4 ± 2 No
collagen II h-β-2 M
79 90
Abbreviations BP: Before O2 plasma treatment, AP: After O2 plasma treatment.
condition for 21 days (Scheme 2). 2.7. Conventional and quantitative PCR (qPCR) The cells were cultured on nanofibrous scaffolds for 21 days and then treated with RNX-plus (Cinnagen) to extract total RNA for reverse transcription by cDNA Synthesis Kit (Fermentas, Canada). Real-time PCR was performed using the SYBR premix Ex Taq II kit (Takara) on an Applied Biosystems 7900HT Fast Real-Time PCR System. The primer sequences of collagen-II, aggrecan, and β-2 microglobulin genes are listed in Table 1. The stability of internal control gene (β-2 microglobulin) was investigated using bestkeeper tools. For calculating the relative gene expression, Ct values of the target genes were normalized to β-2 microglobulin and the fold changes of expression were calculated with comparative ΔΔCt method. The cells cultured in plates with basal medium were used as the control group.
Fig. 2. The stress-strain curve of the aligned PCL/PLGA nanofibrous scaffold.
performed using one-way ANOVA to estimate significant difference between the experimental groups. Statistical differences were calculated using Tukey's multiple comparison test. P < 0.05 was considered as statistically significant.
2.8. Histology & immunofluorescence After 21 days incubation in differentiating media and perfusion bioreactor, the cryosection procedure was performed on the specimens that were embedded in optimal cutting temperature (OCT) compound. For histological analysis, the sections (4 μm diameter) were stained with hematoxylin and eosin (H&E), Alcian blue (pH = 1.0), and Safranin O. In addition, few more sections were immunostained for the detection of type II collagen using a rabbit anti-human Collagen-II primary antibody (Abcam) and horseradish peroxidase (HRP)-labeled secondary antibody (Abcam). The samples were then imaged using a light microscope (BX51 Olympus, USA).
3. Results and discussion 3.1. Characterization of the scaffolds 3.1.1. ATR-FTIR analysis ATR-FTIR spectroscopic analysis was performed on the scaffolds to determine the chemical composition of the electrospun nanofibrous scaffolds and verify that the hybrid PCL/PLGA was fabricated (Fig. 1). The main bands of PCL and PLGA were observed at 1730 and 1760 cm−1, which were attributed to stretching vibration of carboxyl (-COO-) and carbonyl (C=O) groups, respectively. These two peaks were also observed in PCL/PLGA hybrid nanofibrous scaffolds. The peaks at 1000–1300 cm−1 were correlated to C-O stretching vibration in both PCL and PLGA. Moreover, the two peaks of 2924 and
2.9. Statistical analysis P-value of expression ratios in real time PCR was analyzed by randomization tests and the standard error was calculated using the complex Taylor algorithm. Statistical analysis in other experiments was
Fig. 1. ATR-FTIR spectra of PLGA, PCL, and PCL/PLGA nanofibrous scaffold. 4
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Fig. 3. The SEM micrographs of PCL/PLGA scaffold with aligned nanofibers fabricated by electrospinning methods (A), SEM micrographs of MSC cells after 5 days (B) and 21 days (C) of culture on the scaffolds. Histogram represents the measurement of nanofibers diameter (D), the analysis of fiber angles in aligned fibrous scaffolds compared to their major axis (E).
cell attachment. It has been shown that increase of PLGA content compared to PCL could decrease the contact angle value and ultimately enhance the surface hydrophilicity. However, the amount of PLGA in the hybrid scaffolds cannot exceed a limit as it will weaken mechanical properties of the nanofibrous membranes. Moreover, it has been shown that the porosity value could be enhanced by using a ratio of 50:50 in PCL/PLGA scaffolds [4]. The results of porosity measurement of the scaffolds was revealed 70% which is very suitable for cartilage tissue engineering [19]. 3.1.3. Mechanical properties The tensile testing of aligned electrospun PCL/PLGA hybrid nanofibrous scaffolds showed typical non-linear stress–strain curve (Fig. 2). The mechanical properties of the scaffolds are depicted in Table 2. The results indicated that the mechanical properties of aligned nanofibrous membranes along the longitudinal direction were in the range of native superficial zone of articular cartilage which make it appropriate for cartilage differentiation [20]. The mechanical strength of the scaffolds were coming from PCL rather than PLGA which has poor mechanical properties [21].
Fig. 4. MTT assay for hBMMSCs cultured on plasma-treated aligned PCL/PLGA scaffolds. Cells were grown in 96-well plates and used as a control. Data are shown as mean ± SD.
2855 cm−1 were assigned to C-H bands in PCL and PLGA whereas the bands at 1480 and 1390 cm−1were related to C–H and –CH2 bending vibrations in aforementioned polymers. The results of FTIR spectra verified that the peaks observed in PCL and PLGA polymers were also detected in hybrid PCL/PLGA scaffolds. It is therefore believed that no chemical reaction was occurred between PCL and PLGA polymers in the composite scaffolds [18].
3.1.4. Morphological analysis SEM images were used to analyze the diameter, alignment, and morphology of fibers on the scaffolds (Fig. 3). Fig. 3A show the scaffold before cell seeding and figures of 3B and 3C show the scaffolds after 5 and 21 days of cell seeding. SEM micrographs of the scaffolds seeded with cells showed that the hBMMSCs attached and proliferated on the scaffolds. Moreover, the morphology of cells oriented in the direction of aligned nanofibers was observed (Fig. 3B and C). The fiber diameters were ranged between 60 and 1150 nm with the average diameter of 485.4 ± 67 nm (Fig. 3D). The alignment of nanofibers was identified using statistical analysis of the nanofiber angles in comparison to their major axis. The results showed that the angles of over 68% of the fibers in fibrous scaffolds were −30°- 30° compared to their major axis
3.1.2. Contact angle and porosity measurements The results of contact angle and porosity measurements of PCL/ PLGA nanofibrous scaffolds before and after plasma treatment are shown in Table 2. It was shown that plasma treatment enhanced hydrophilicity of the scaffold surfaces which is a favorable property for 5
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Fig. 5. The graphs showing the media temperature (A), pH of bioreactor and 6-well plate (control) over time (B).
the highest cell viability and proliferation of hBMMSCs has been observed on the scaffold with 50% PLGA [4]. 3.3. Bioreactor design The temperature measurements showed that the media in the plate was reached to 37 °C more quickly (60 min) than the bioreactor. The medium in the bioreactor took at least 90 min to equilibrate to the temperature of the incubator (Fig. 5A). The pH measurements showed steady pH values after 75 and 120 min in the chambers of bioreactor and wells of culture plate, respectively which is an indication for adequate gas exchange in the bioreactor system (Fig. 5B). 3.4. Quantitative PCR (qPCR)
Fig. 6. The expression levels of chondrogenic-specific genes (aggrecan and collagen II) in differentiated groups compared to the MSCs cultured in plates with basal medium (Control -).
The expression of cartilage specific genes (aggrecan, collagen II (was investigated by real time PCR at RNA level after 21-day culture of hBMMSC in the perfusion bioreactor (Fig. 6). The experimental groups were consisted of chemical group, mechanical group, mechanical–chemical group, control + (chondrocytes extracted from human articular cartilage), and control − (MSCs cultured in plate and basal medium) (Table 3). The higher expression of type II collagen in comparison to aggrecan was observed in the control + of articular cartilage. The matrix of cartilage is mainly composed of type II collagen providing mechanical strength for the tissue. Aggrecan is another ECM component of cartilage whose negative charges absorb the water molecules, offering compressive resistance to cartilage under mechanical loading. The results showed that the highest levels of aggrecan and collagen type II expression was observed in mechanical–chemical group which confirmed the synergistic positive effect of mechanical stimuli and chemical components on chondrogenesis. Matrix metalloproteinase 3 (MMP-3) is an enzyme that causes proteoglycans deterioration. It has been shown that mechanical stimulation would decrease MMP-3 expression which results in aggrecan increases in human chondrocytes [25]. Other studies have shown that TGF-β1 exerts a diphasic effect on chondrocyte differentiation [26]. A short exposure of TGF-β1 enhances Sox9 expression by activating the Smad2/3 pathway and Sox9 induces
(Fig. 3E). It is known that electrospinning of PLGA would lead to a smaller average diameter of fibers (60–300 nm) compared to PCL which is in agreement with our results [22,23]. However, PCL electrospun nanofibrous scaffolds revealed fiber diameters of 300–1150 nm which could provide an appropriate mechanical strength [24].
3.2. MTT assay The viability of hBMMSCs was measured on PCL/PLGA scaffolds after 1, 3, 5 and 9 days of cell seeding (Fig. 4). The results showed that the proliferation of cells on aligned nanofibrous scaffolds was higher than cell culture which was used as control. This shows that the fabricated scaffolds were not toxic for the cells and may enhance cell proliferation which is possibly due to the recapitulation of ECM by PCL/ PLGA nanofibrous scaffolds. A similar outcome of nanofibrous scaffolds on cell proliferation has been previously reported [16]. Moreover, it has been shown that scaffolds with higher PLGA ratio could enhance cell proliferation perhaps as the results of higher hydrophilicity and porosity. However, among the various scaffolds with different PLGA ratios, Table 3 A presentation of test groups used having different culture conditions. Factors
Mechanical stimulation
Aligned nanofibrous scaffolds
Differentiation medium
MSCs
Chondrocytes extracted from human articular cartilage
Groups Chem. Mech. Chem.-Mech Control − Control +
− + + − −
+ + + − +
+ − + − −
+ + + + −
− − − +
6
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Fig. 7. H&E, alcian blue, safranin O, and IHC (for Collagen type II) staining of the cells cultured on the scaffolds and in presence of chondrogenic medium (Chem. group), with application of mechanical stimuli in the perfusion bioreactor (Mech. group), with mechanical stimuli in the perfusion bioreactor and chondrogenic medium (Mech.–Chem. group), and chondrocytes extracted from human articular cartilage (Control +). Arrows in the images show the stained ECM (proteoglycan and glycosaminoglycans in alcian blue, safranin O staining and Collagen type II in IHC staining). The left right arrows in the pictures indicate the scaffolds.
matrix were look pink with a bluish feature on matrix of the constructs. To detect proteoglycans and glycosaminoglycans (GAGs) in the matrix, the constructs were stained using Safranin O in which the nuclei, cytoplasm, and matrix of cartilage-like construct were appeared orange toward red. Alcian blue was another cationic dye used to stain acidic polysacharides such as GAGs in cartilage-like constructs [29]. The experimental groups consisted of chemical group (the cells cultured on the scaffolds in chondrogenic medium), mechanical group (cells cultured on aligned PCL/PLGA nanofibrous scaffolds which were under mechanical stimuli in perfusion bioreactor), mechanical–chemical group (the same condition as mechanical group in chondrogenic medium), and articular cartilage (Control +). Here, the scaffolds after staining showed stained uniform fibers (in the Fig. 7 showed with right left arrows) and the extracellular molecules secreted from the cells are seen as sheets on the surface of the scaffolds. H&E staining results showed that most of cells were flattened on the scaffolds which were embedded in the bioreactor with chondrogenic medium (mechanical–chemical group) as compared to the chemical or mechanical groups. The flat cells are the most abundant cell type in superficial zone of cartilage tissue which has the highest mechanical strength [30]. Moreover, the Alcian blue and Safranin O staining showed that secreted proteoglycans and GAGs in the mechanical group make a thicker sheet on the scaffolds in comparison to chemical group. The Safranin O staining showed that the secreted proteoglycans even penetrate into the scaffolds. The higher expression of these molecules in mechanical group may be due to the positive effect of mechanical stimulation on proteoglycans expression [25]. IHC staining revealed that in the chemical
the expression of type II collagen. The second peak of type II collagen expression is observed during continuous administration of TGF-β1 which blocks the Smad2/3-mediated TGF-β signaling and causes Sox9 level decrease. This phenomenon is related to increased collagen expression and diminution of aggrecan expression [27,28]. Interestingly, this is in agreement with our results revealing that the expression of aggrecan was higher than type II collagen in mechanical group whereas the aggrecan expression was less than type II collagen in chemical group. 3.5. Histological study Membrane-like constructs were harvested from perfusion bioreactor and the static cell culture groups after 21-day culture period. Histological and immunohistochemical (IHC) studies were then carried out (Fig. 7). The cartilage matrix is primarily composed of type II collagen and negatively charged proteoglycans such as aggrecan having water absorption capacity and compressive resistance against mechanical loading. When the progenitor cells proliferate on the dish, they secrete proteoglycan in their microenvironment whereas type II collagen is absent in the extracellular space in the early stage of chondrogenesis. After a few days the chondrocyte colonies of superficial zone form and type II collagen is secreted into the ECM of the flattened shaped differentiated cells. Finally, proteoglycan molecules are expressed and released out while fibronectin glycoproteins disappear from the ECM. In this study, the morphology and distribution of chondrocytes were investigated using H&E staining in which the nuclei of the cells were observed blue-purple whereas the cytoplasm and 7
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group type II collagen make a ticker sheets on the scaffolds compared with mechanical group. This is possibly because of the direct effect of TGFβ on type II collagen expression [27,28]. However, the cells of mechanical–chemical group showed that secreted proteoglycans and type II collagen made not only a thick sheet on the scaffolds but also penetrated in the scaffolds. It is known that alignment of the fibers in the scaffold has a positive effect on chondrogenesis even more than scaffold composition [4]. These results demonstrated that scaffold alignment and the presence of chondrogenic medium accompanied with continuous perfusion could activate the expression of cartilage specific genes (aggrecan and type II collagen) through signaling pathways reported by Mauck et al. [12].
[9]
[10]
[11] [12]
[13]
[14]
4. Conclusion [15]
In this study a perfusion bioreactor was designed to induce hydrodynamic pressure and shear stress to mesenchymal stem cells cultured on the aligned nanofibrous PCL/PLGA scaffolds. The application of hydrodynamic pressure and shear stress on the aligned scaffolds was led to high chondrogenesis efficiency and provoked the secretion of cartilage-related ECM. The mechanical stimulation affected aggrecan secretion more than type II collagen whereas the chondrogenesis medium was influenced the secretion of type II collagen more significantly. The results of this study could be a useful asset in detection of parameters influencing in vitro chondrogenesis for cartilage generation and replacement therapy.
[16]
[17] [18] [19]
[20] [21]
Acknowledgments
[22]
The authors gratefully acknowledge Biotechnology and Stem Cell Technology Research Center and National Institute of Genetic Engineering for their financial support.
[23] [24]
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8
mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J Biomech 2003;36(8):1087–96. Salasznyk RM, et al. Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. Exp Cell Res 2007;313(1):22–37. Takahashi I, et al. Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells. J Cell Sci 1998;111(14):2067–76. Waldman SD, et al. Effect of biomechanical conditioning on cartilaginous tissue formation in vitro. J Bone Joint Surg Am 2003;85(suppl 2):101–5. Mauck RL, et al. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng 2000;122(3):252–60. Mizuno S, et al. Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture. J Cell Physiol 2002;193(3):319–27. Wang X, et al. High flux filtration medium based on nanofibrous substrate with hydrophilic nanocomposite coating. Environ Sci Technol 2005;39(19):7684–91. Yazdani SO,S, et al. Safety and possible outcome assessment of autologous Schwann cell and bone marrow mesenchymal stromal cell co-transplantation for treatment of patients with chronic spinal cord injury. Cytotherapy 2013;15(7):782–91. Mohammadi Amirabad L, et al. Enhanced cardiac differentiation of human cardiovascular disease patient-specific induced pluripotent stem cells by applying unidirectional electrical pulses using aligned electroactive nanofibrous scaffolds. ACS Appl Mater Interfaces 2017;9(8):6849–64. Shifrin EG, et al. Filtration equations and the Darcy Law. Am J Appl Math Stat 2014;2(3):143–9. Stuart B. Infrared spectroscopy. Wiley Online Library; 2005. Ateshian G, Wang H, Lai W. The role of interstitial fluid pressurization and surface porosities on the boundary friction of articular cartilage. J Tribol 1998;120(2):241–8. Fung YC, Biomechanics: mechanical properties of living tissues: Springer Science & Business Media. Broz M, VanderHart DL, Washburn N. Structure and mechanical properties of poly (D, L-lactic acid)/poly (Îμ-caprolactone) blends. Biomaterials 2003;24(23):4181–90. Franco RA, Nguyen TH, and Lee B-T. Preparation and characterization of electrospun PCL/PLGA membranes and chitosan/gelatin hydrogels for skin bioengineering applications. J Mater Sci Mater Med 22(10): p. 2207. Smith L, Ma P. Nano-fibrous scaffolds for tissue engineering. Colloids Surfaces B Biointerfaces 2004;39(3):125–31. Baker SR, et al., Determining the mechanical properties of electrospun poly-Îμ-caprolactone (PCL) nanofibers using AFM and a novel fiber anchoring technique. Mater Sci Eng C 59: p. 203–212. Millward-Sadler S, et al. Mechanotransduction via integrins and interleukin-4 results in altered aggrecan and matrix metalloproteinase 3 gene expression in normal, but not osteoarthritic, human articular chondrocytes. Arthritis Rheum 2000;43(9):2091–9. Baugé C, et al., Modulation of transforming growth factor beta signalling pathway genes by transforming growth factor beta in human osteoarthritic chondrocytes: involvement of Sp1 in both early and late response cells to transforming growth factor beta. Arthritis Res Ther 13(1): p. 1. Hellingman CA, et al., Smad signaling determines chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells: inhibition of Smad1/5/8P prevents terminal differentiation and calcification. Tissue Eng 17(7–8): p. 1157–1167. Davidson ENB, et al., Correction: increase in ALK1/ALK5 ratio as a cause for elevated MMP-13 expression in osteoarthritis in humans and mice. J Immunol 185(4): p. 2629–2629. Chen C-H., et al., Surface modification of polycaprolactone scaffolds fabricated via selective laser sintering for cartilage tissue engineering. Mater Sci Eng C 40: p. 389–397. Athanasiou KA, et al. Basic science of articular cartilage repair. Clin Sports Med 2001;20(2):223–47.
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Influence of hydrodynamic pressure on chondrogenic differentiation of human bone marrow mesenchymal stem cells cultured in perfusion system Soheila Zamanluia,1, Leila Mohammadi Amirabadb, Masoud Soleimanic,d,∗∗, Shahab Faghihia,∗ a
Stem Cell and Regenerative Medicine Group, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161, Iran School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran c Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 111/14115, Iran d Nanotechnology and Tissue Engineering Department, Stem Cell Technology Research Center, Tehran 1997775555, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chondrocytes Hydrodynamic pressure Perfusion system Nanofibrous scaffold Mesenchymal stem cells
The natural conditions of chondrocytes in native cartilage including mechanical forces and surface topology could be simulated to enhance chondrogenesis. A perfusion system recapitulating the hydrodynamic pressure of cartilage tissue is designed. Mesenchymal stem cells (MSCs) are isolated and seeded on aligned nanofibrous PCL/ PLGA scaffolds that mimic the structure of superficial zone of articular cartilage. The cell-seeded scaffolds are placed into the perfusion bioreactor and exposed to chondrogenic differentiating medium. The chondrogenesis is then investigated by histological analysis and real time PCR for cartilage-specific genes. The highest expression levels of aggrecan and type II collagen are observed in the cells cultured in the presence of differentiating medium and mechanical stimulation. The expression level of type II collagen is higher than aggrecan in presence of differentiating medium and absence of mechanical stimulation. On the contrary, the expression ratio of aggrecan is higher than type II collagen in presence of mechanical stimulation and absence of differentiating medium. These results show the dominant role of mechanical stimulation and differentiating medium on upregulated expression of aggrecan and type II collagen, respectively. The application of mechanical stimulation upon cells-seeded scaffolds could mimic superficial zone of articular cartilage tissue and increase derivation of chondrocytes from MSCs.
1. Introduction Articular cartilage damages which are commonly originated from accidents, degenerative joint diseases and normal wear and tear are known as the main causes of musculoskeletal morbidity. Cartilage tissue has a limited capacity to regenerate because of the lack of innervation and blood vessels [1]. Therefore, designing a functional construct by tissue engineering approach could be a promising strategy to restore articular cartilage function. In order to produce a functional cartilage construct, the native articular cartilage setting for the cells should be recapitulated, including extracellular matrix (ECM) composition and mechanical-loaded environment [2]. Due to their high accessibility and capacity for cartilage differentiation, adipose and bone marrow mesenchymal stem cells (MSCs) could be an appropriate cell sources for cartilage tissue engineering.
Another important factor for cartilage tissue engineering is to mimic ECM using biocompatible scaffolds in vitro. It has been shown that surface topology, stiffness, and alignment of biological scaffold components could affect the proliferation and fate of cells [3,4]. Recently, electrospinning technique has enabled the fabrication of scaffolds with nano-scaled fibers providing high surface to volume ratio and mimicking the topology of ECM. The ECM of cartilage is primarily composed of type II collagen and negatively charged proteoglycans that hold water in the tissue. Although most of the cartilage tissue is made of water, it has enough strength to bear the loaded compressive forces. The scaffolds used in chondrogenesis should be highly porous to hold a great volume of water at the same time as having appropriate mechanical strength to be similar to the structure of cartilage tissue. Poly D, L-lactic-co-glycolic acid (PLGA) is a polyester giving high degree of porosity to the scaffolds, which has a tailored degradation rate and
∗ Corresponding author. Stem Cell and Regenerative Medicine Group, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14965/161, Iran. ∗∗ Corresponding author. Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 111/14115, Iran. E-mail addresses: [email protected] (M. Soleimani), [email protected], [email protected] (S. Faghihi). 1 Present address: Stem Cells and Cell Therapy Research Center, Tissue Engineering and Regenerative Medicine Institute, Tehran Central Branch, Islamic Azad University, Tehran, Iran.
https://doi.org/10.1016/j.biologicals.2018.04.004 Received 13 September 2017; Received in revised form 8 April 2018; Accepted 20 April 2018 1045-1056/ © 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Zamanlui, S., Biologicals (2018), https://doi.org/10.1016/j.biologicals.2018.04.004
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10 × 60 × 0.06 mm. The tensile testing of the fabricated scaffolds was performed by applying 0.5 kN force at 50 mm/min traverse speed using a mechanical test machine (Instron 5565 A).
biocompatibility. Previous studies have shown that the diameter for PLGA nanofibers in electrospun scaffolds is commonly in the range of 50–300 nm [5]. Therefore, due to the poor tensile strength of PLGA in comparison to cartilage tissue, it should be used along with other polymers. Polycaprolactone (PCL) is another biodegradable polymer that is commonly used for fabrication of scaffolds in tissue engineering which provides appropriate mechanical properties. Moreover, PCL could promote chondrocyte proliferation and maintain its phenotype [6]. However, owing to the poor hydrophilic trait of PCL and its slow degradation rate, it should be used with a more hydrophilic polymer. The scaffolds composed of PLGA and PCL could be a good candidate for cartilage tissue engineering given the capacity of PLGA to make a highly porous scaffold and the ability of PCL to offer appropriate tensile strength [7]. There is growing evidence that in vitro mechanical stress has an important role in sustaining and enhancing the chondrogenic phenotype. Recently, different types of devices and bioreactors have been manufactured to investigate the in vitro effects of mechanical forces on functional tissue formation. Many studies have shown that hydrodynamic loads could induce the deformation of MSC ECM and activate integrin signaling [8,9]. Subsequently, this hydrodynamic pressure over cartilage can improve chondrogenic differentiation via upregulation and downregulation of SOX-9 and IL-1β expression, respectively. This in turn will lead to the expression of proteoglycan, collagen type II, and cartilage-specified ECM [10]. On the other hand, the mechanical stimuli have shown to affect cartilage differentiation in various ways based on the types of scaffolds. For example, static and hydrodynamic pressures have revealed to provide diverse effects on the efficiency of cartilage differentiation [11–13]. In addition, perfusion culture systems could supply a steady gas exchange and nutrition supply into the center of engineered constructs and makes the cells proliferate and differentiate more effectively. The aim of this study was to investigate chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSC) cultured on the fabricated aligned nanofibrous scaffolds under mechanical forces of perfusion bioreactor. The composite PCL/PLGA aligned nanofibrous scaffolds were fabricated using electrospinning followed by chemical and mechanical characterization by FTIR, contact angle, SEM, and tensile analysis. Human bone marrow mesenchymal stem cells (hBMMSC) were seeded on the scaffolds and placed into the chamber of perfusion bioreactor and exposed to chondrogenic differentiating medium for 21 days. Ultimately, the chondrogenic activity of hBMMSCs were assessed by Real-Time PCR, Alcian blue, Safranin O staining and immunohistochemistry (IHC).
2.2.2. Contact angle and porosity measurements The hydrophilicity of the prepared scaffolds was measured using sessile drop method. For this purpose, the average value of contact angle was calculated by an optical bench type contact angle measuring system using four water droplets (Rame- Hart Instrument Company, USA). The apparent density of scaffold (A) (g/cm3) and its porosity (P) (%) was calculated from five separate samples using equations (1) and (2), respectively:
A=
P=1−
2.2.4. Morphological study The morphology of the surfaces of aligned scaffolds before and after cell culture was monitored by scanning electron microscopy (SEM). The scaffolds seeded with cells were washed with PBS and fixed with 2.5% glutaraldehyde for 40 min. The scaffolds were then dehydrated in ascending alcohol concentrations. Finally, the samples were left to dry at room temperature (RT) and were sputter-coated with gold. The accelerating voltage of scanning electron microscope (KYKY-EM3200, Madell Technology Corporation, CA, USA) was 24 kV. The average diameter of nanofiber was determined from at least 100 chosen fibers using image analysis software (Image J, NIH, USA) and the results were reported as mean ± SD. 2.3. Cell isolation and culture Human bone marrow mesenchymal stem cells (hBMMSCs) from seven healthy Caucasian females (18–25 years) were isolated and cultured according to previously described protocol [15]. In brief, the mononuclear cell layer was isolated using ficoll from bone marrow samples collected from the iliac bone (1.077 gl−1, sigma) at a ratio of 1:3. The study was approved by the Research Ethics Board of Taleqani Hospital and all the subjects participated in this study with informed consent. The isolated hBMMSCs were cultured in culture media [DMEM supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin)] in T25 flasks. For cell passage, the cells were treated with trypsin–EDTA solution (GIBCO) and centrifuged after washing with PBS.
PLGA/PCL composite nanofibrous scaffolds were fabricated using electrospinning process according to previously described protocol [4]. Briefly, 12% (w/v) PCL granule (Sigma) was dissolved in dimethylformamide (DMF)/chloroform (1:9). To prepare PLGA solution, 7% (w/v) PLGA was dissolved in DMF/chloroform solution (1:3). Aligned PLGA/PCL nanofibrous scaffolds were fabricated with PCL and PLGA solutions (50:50) having flow rates of 0.5 and 0.3 mL/h. The distances between needles of PCL and PLGA syringes and collector were adjusted at 24 and 16 cm, rotating collector speed was 2800 rpm and voltage kept at 24 kV. The fabricated scaffolds were treated with O2 plasma at 100 W for 30 s (Diener electronics, Germany) to increase the surface hydrophilicity.
2.4. MTT assay The viability of hBMMSCs on the fabricated scaffolds was assessed using MTT assay as previously described protocol [16]. The cells were seeded with an initial density of 3000 cells/cm2 in a 96-well culture plate with sterilized scaffolds. On day 1, 4, 7 and 14, the cell culture medium was discarded and the cells were incubated with 100 μl of MTT solution (500 μg/ml) (Sigma-Aldrich, Cat. R8755) for 3.5 h. Formazan crystals in the cells were dissolved in DMSO and the plates were analyzed using an ELISA plate reader at 570 nm.
2.2. Scaffolds characterization
cut
with
a
dimension
(2)
2.2.3. ATR-FTIR analysis ATR-FTIR spectroscopy was performed using Bruker Equinox 55 over the range of 500–4000 cm−1 to analyze the chemical content of PCL/PLGA hybrid fabricated scaffolds.
2.1. Scaffold fabrication
were
A × 100 A0
(1)
where A is the apparent density of each composite nanofibrous scaffolds and A0 is the bulk density of scaffold (g/cm3) [14].
2. Materials and methods
2.2.1. Mechanical properties The scaffold specimens
mass of scaffold(g) Area of scaffold(cm2) × Thickness of scaffolds(cm)
of 2
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Scheme 1. A representation of the bioreactor designed by Rhino ceros software showing dimensions (cm) of the bioreactor and a single chamber (A), connection of the bioreactor to a peristaltic pump in a CO2 incubator (B).
mesenchymal tissue (1 × 10–13 m4/N s). The temperature of the media in the reservoirs and 6-well plate in each chamber of bioreactor (7 mL) was repeatedly scanned every 15 min until it became stable. Similarly, the pH was measured continuously using a pH meter (Thermo Scientific, USA) until equilibrium.
2.5. Design of perfusion bioreactor A four-well bioreactor was designed to apply continuous perfusion using Rhino Ceros software as depicted in Scheme. 1A. The bioreactor was constructed of Plexiglas [Poly (methyl methacrylate) (PMMA)] and the chambers were carved with a CNC router (C.R. Onsrud, Inc., USA). Each chamber was connected to the outside with two stainless steel (316) ports at the top and bottom sides of each chamber. The dimension of each cylindrical chamber was 20 mm (diameter) × 25 mm (length) and a stainless-steel mesh with 20 mm (diameter) × 1 mm (length) was placed on the middle of each chamber (Scheme. 1A). The media was entered into the top of each chamber through a silicon tube which was connected to the reservoir. A tube discharged the medium from the bottom port of each chamber. The bioreactor was sterilized by autoclave before use. After seeding the cells in the bioreactor, it was placed in a CO2 incubator and connected to a peristaltic pump (Gilson-miniplus 3, USA) to circulate the media between the chambers and reservoirs (Scheme. 1B). The pressure differential across the construct was calculated as 2.06 × 107 MPa using the Darcy's Law equation [17].
ΔP =
2.6. Cartilage cell differentiation The punched PCL/PLGA hybrid nanofibrous scaffolds with a diameter of 2 cm were sterilized with 70% ethanol and UV exposure. After washing the scaffolds with phosphate buffered saline solution (PBS), they were incubated with DMEM supplemented with 10% FBS overnight. Then, the hBMMSCs (passage three) were seeded on the scaffolds at a density of 3 × 105 cells per cm2 in 12- well tissue culture plates. After 24 h, the scaffolds containing the cells were placed on the stainless-steel mesh located on the middle of each chamber of bioreactor. The chondrogenic differentiation medium [50 μg/mL ascorbic acid 2phosphate (Sigma), ITS + 6.25 μg/mL of insulin, transferrin, and selenous acid (Gibco) plus 10 ng/mL TGF-β1 (Peprotech), 100 nM dexamethasone (Sigma) in DMEM] was added to the reservoir and constant perfusion was conducted for 21 days with flow entry and exit through the top and bottom ports of each chamber. The flow speed was adjusted at 1 mL/min and the medium of reservoirs was exchanged every three days. The control cell group was placed in a CO2 incubator with static
ϑ × μ× Δx κ
Where, ν is the volumetric flow rate (1 mL/min), μ is the dynamic viscosity of medium at 37 °C (686.5 Pas), Δx is the thickness of the construct (30 μm), and κ is the constant of permeability for multipotent
Scheme 2. Presentation of time schedule for chondrogenic differentiation of hBMMSCs. 3
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Table 1 The list of primers used for real-time PCR analysis.
Table 2 The results of scaffold characterization.
Genes
Forward sequence 5'→ 3′
Reverse sequence 5'→ 3′
Product size (bp)
Sample
Contact angle (°)
Porosity (%)
Tensile strength (MPa)
Elongation (%)
aggrecan
TGT CAG ATA CCC CAT CCA C GGT CTT GGT GGA AAC TTT GCT ATG CCT GCC GTG TGA AC
CAT AAA AGA CCT CAC CCT CC GGT CCT TGC ATT ACT CCC AAC ATC TTC AAA CCT CCA TGA TG
85
A50 BP A50 AP Significant change BP & AP of PCL/PLGA
120 ± 2 25 ± 2.3 Yes
70 ± 3 74 ± 5 No
20.66 ± 0.42 19.8 ± 0.3 No
85 ± 3 86.4 ± 2 No
collagen II h-β-2 M
79 90
Abbreviations BP: Before O2 plasma treatment, AP: After O2 plasma treatment.
condition for 21 days (Scheme 2). 2.7. Conventional and quantitative PCR (qPCR) The cells were cultured on nanofibrous scaffolds for 21 days and then treated with RNX-plus (Cinnagen) to extract total RNA for reverse transcription by cDNA Synthesis Kit (Fermentas, Canada). Real-time PCR was performed using the SYBR premix Ex Taq II kit (Takara) on an Applied Biosystems 7900HT Fast Real-Time PCR System. The primer sequences of collagen-II, aggrecan, and β-2 microglobulin genes are listed in Table 1. The stability of internal control gene (β-2 microglobulin) was investigated using bestkeeper tools. For calculating the relative gene expression, Ct values of the target genes were normalized to β-2 microglobulin and the fold changes of expression were calculated with comparative ΔΔCt method. The cells cultured in plates with basal medium were used as the control group.
Fig. 2. The stress-strain curve of the aligned PCL/PLGA nanofibrous scaffold.
performed using one-way ANOVA to estimate significant difference between the experimental groups. Statistical differences were calculated using Tukey's multiple comparison test. P < 0.05 was considered as statistically significant.
2.8. Histology & immunofluorescence After 21 days incubation in differentiating media and perfusion bioreactor, the cryosection procedure was performed on the specimens that were embedded in optimal cutting temperature (OCT) compound. For histological analysis, the sections (4 μm diameter) were stained with hematoxylin and eosin (H&E), Alcian blue (pH = 1.0), and Safranin O. In addition, few more sections were immunostained for the detection of type II collagen using a rabbit anti-human Collagen-II primary antibody (Abcam) and horseradish peroxidase (HRP)-labeled secondary antibody (Abcam). The samples were then imaged using a light microscope (BX51 Olympus, USA).
3. Results and discussion 3.1. Characterization of the scaffolds 3.1.1. ATR-FTIR analysis ATR-FTIR spectroscopic analysis was performed on the scaffolds to determine the chemical composition of the electrospun nanofibrous scaffolds and verify that the hybrid PCL/PLGA was fabricated (Fig. 1). The main bands of PCL and PLGA were observed at 1730 and 1760 cm−1, which were attributed to stretching vibration of carboxyl (-COO-) and carbonyl (C=O) groups, respectively. These two peaks were also observed in PCL/PLGA hybrid nanofibrous scaffolds. The peaks at 1000–1300 cm−1 were correlated to C-O stretching vibration in both PCL and PLGA. Moreover, the two peaks of 2924 and
2.9. Statistical analysis P-value of expression ratios in real time PCR was analyzed by randomization tests and the standard error was calculated using the complex Taylor algorithm. Statistical analysis in other experiments was
Fig. 1. ATR-FTIR spectra of PLGA, PCL, and PCL/PLGA nanofibrous scaffold. 4
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Fig. 3. The SEM micrographs of PCL/PLGA scaffold with aligned nanofibers fabricated by electrospinning methods (A), SEM micrographs of MSC cells after 5 days (B) and 21 days (C) of culture on the scaffolds. Histogram represents the measurement of nanofibers diameter (D), the analysis of fiber angles in aligned fibrous scaffolds compared to their major axis (E).
cell attachment. It has been shown that increase of PLGA content compared to PCL could decrease the contact angle value and ultimately enhance the surface hydrophilicity. However, the amount of PLGA in the hybrid scaffolds cannot exceed a limit as it will weaken mechanical properties of the nanofibrous membranes. Moreover, it has been shown that the porosity value could be enhanced by using a ratio of 50:50 in PCL/PLGA scaffolds [4]. The results of porosity measurement of the scaffolds was revealed 70% which is very suitable for cartilage tissue engineering [19]. 3.1.3. Mechanical properties The tensile testing of aligned electrospun PCL/PLGA hybrid nanofibrous scaffolds showed typical non-linear stress–strain curve (Fig. 2). The mechanical properties of the scaffolds are depicted in Table 2. The results indicated that the mechanical properties of aligned nanofibrous membranes along the longitudinal direction were in the range of native superficial zone of articular cartilage which make it appropriate for cartilage differentiation [20]. The mechanical strength of the scaffolds were coming from PCL rather than PLGA which has poor mechanical properties [21].
Fig. 4. MTT assay for hBMMSCs cultured on plasma-treated aligned PCL/PLGA scaffolds. Cells were grown in 96-well plates and used as a control. Data are shown as mean ± SD.
2855 cm−1 were assigned to C-H bands in PCL and PLGA whereas the bands at 1480 and 1390 cm−1were related to C–H and –CH2 bending vibrations in aforementioned polymers. The results of FTIR spectra verified that the peaks observed in PCL and PLGA polymers were also detected in hybrid PCL/PLGA scaffolds. It is therefore believed that no chemical reaction was occurred between PCL and PLGA polymers in the composite scaffolds [18].
3.1.4. Morphological analysis SEM images were used to analyze the diameter, alignment, and morphology of fibers on the scaffolds (Fig. 3). Fig. 3A show the scaffold before cell seeding and figures of 3B and 3C show the scaffolds after 5 and 21 days of cell seeding. SEM micrographs of the scaffolds seeded with cells showed that the hBMMSCs attached and proliferated on the scaffolds. Moreover, the morphology of cells oriented in the direction of aligned nanofibers was observed (Fig. 3B and C). The fiber diameters were ranged between 60 and 1150 nm with the average diameter of 485.4 ± 67 nm (Fig. 3D). The alignment of nanofibers was identified using statistical analysis of the nanofiber angles in comparison to their major axis. The results showed that the angles of over 68% of the fibers in fibrous scaffolds were −30°- 30° compared to their major axis
3.1.2. Contact angle and porosity measurements The results of contact angle and porosity measurements of PCL/ PLGA nanofibrous scaffolds before and after plasma treatment are shown in Table 2. It was shown that plasma treatment enhanced hydrophilicity of the scaffold surfaces which is a favorable property for 5
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Fig. 5. The graphs showing the media temperature (A), pH of bioreactor and 6-well plate (control) over time (B).
the highest cell viability and proliferation of hBMMSCs has been observed on the scaffold with 50% PLGA [4]. 3.3. Bioreactor design The temperature measurements showed that the media in the plate was reached to 37 °C more quickly (60 min) than the bioreactor. The medium in the bioreactor took at least 90 min to equilibrate to the temperature of the incubator (Fig. 5A). The pH measurements showed steady pH values after 75 and 120 min in the chambers of bioreactor and wells of culture plate, respectively which is an indication for adequate gas exchange in the bioreactor system (Fig. 5B). 3.4. Quantitative PCR (qPCR)
Fig. 6. The expression levels of chondrogenic-specific genes (aggrecan and collagen II) in differentiated groups compared to the MSCs cultured in plates with basal medium (Control -).
The expression of cartilage specific genes (aggrecan, collagen II (was investigated by real time PCR at RNA level after 21-day culture of hBMMSC in the perfusion bioreactor (Fig. 6). The experimental groups were consisted of chemical group, mechanical group, mechanical–chemical group, control + (chondrocytes extracted from human articular cartilage), and control − (MSCs cultured in plate and basal medium) (Table 3). The higher expression of type II collagen in comparison to aggrecan was observed in the control + of articular cartilage. The matrix of cartilage is mainly composed of type II collagen providing mechanical strength for the tissue. Aggrecan is another ECM component of cartilage whose negative charges absorb the water molecules, offering compressive resistance to cartilage under mechanical loading. The results showed that the highest levels of aggrecan and collagen type II expression was observed in mechanical–chemical group which confirmed the synergistic positive effect of mechanical stimuli and chemical components on chondrogenesis. Matrix metalloproteinase 3 (MMP-3) is an enzyme that causes proteoglycans deterioration. It has been shown that mechanical stimulation would decrease MMP-3 expression which results in aggrecan increases in human chondrocytes [25]. Other studies have shown that TGF-β1 exerts a diphasic effect on chondrocyte differentiation [26]. A short exposure of TGF-β1 enhances Sox9 expression by activating the Smad2/3 pathway and Sox9 induces
(Fig. 3E). It is known that electrospinning of PLGA would lead to a smaller average diameter of fibers (60–300 nm) compared to PCL which is in agreement with our results [22,23]. However, PCL electrospun nanofibrous scaffolds revealed fiber diameters of 300–1150 nm which could provide an appropriate mechanical strength [24].
3.2. MTT assay The viability of hBMMSCs was measured on PCL/PLGA scaffolds after 1, 3, 5 and 9 days of cell seeding (Fig. 4). The results showed that the proliferation of cells on aligned nanofibrous scaffolds was higher than cell culture which was used as control. This shows that the fabricated scaffolds were not toxic for the cells and may enhance cell proliferation which is possibly due to the recapitulation of ECM by PCL/ PLGA nanofibrous scaffolds. A similar outcome of nanofibrous scaffolds on cell proliferation has been previously reported [16]. Moreover, it has been shown that scaffolds with higher PLGA ratio could enhance cell proliferation perhaps as the results of higher hydrophilicity and porosity. However, among the various scaffolds with different PLGA ratios, Table 3 A presentation of test groups used having different culture conditions. Factors
Mechanical stimulation
Aligned nanofibrous scaffolds
Differentiation medium
MSCs
Chondrocytes extracted from human articular cartilage
Groups Chem. Mech. Chem.-Mech Control − Control +
− + + − −
+ + + − +
+ − + − −
+ + + + −
− − − +
6
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Fig. 7. H&E, alcian blue, safranin O, and IHC (for Collagen type II) staining of the cells cultured on the scaffolds and in presence of chondrogenic medium (Chem. group), with application of mechanical stimuli in the perfusion bioreactor (Mech. group), with mechanical stimuli in the perfusion bioreactor and chondrogenic medium (Mech.–Chem. group), and chondrocytes extracted from human articular cartilage (Control +). Arrows in the images show the stained ECM (proteoglycan and glycosaminoglycans in alcian blue, safranin O staining and Collagen type II in IHC staining). The left right arrows in the pictures indicate the scaffolds.
matrix were look pink with a bluish feature on matrix of the constructs. To detect proteoglycans and glycosaminoglycans (GAGs) in the matrix, the constructs were stained using Safranin O in which the nuclei, cytoplasm, and matrix of cartilage-like construct were appeared orange toward red. Alcian blue was another cationic dye used to stain acidic polysacharides such as GAGs in cartilage-like constructs [29]. The experimental groups consisted of chemical group (the cells cultured on the scaffolds in chondrogenic medium), mechanical group (cells cultured on aligned PCL/PLGA nanofibrous scaffolds which were under mechanical stimuli in perfusion bioreactor), mechanical–chemical group (the same condition as mechanical group in chondrogenic medium), and articular cartilage (Control +). Here, the scaffolds after staining showed stained uniform fibers (in the Fig. 7 showed with right left arrows) and the extracellular molecules secreted from the cells are seen as sheets on the surface of the scaffolds. H&E staining results showed that most of cells were flattened on the scaffolds which were embedded in the bioreactor with chondrogenic medium (mechanical–chemical group) as compared to the chemical or mechanical groups. The flat cells are the most abundant cell type in superficial zone of cartilage tissue which has the highest mechanical strength [30]. Moreover, the Alcian blue and Safranin O staining showed that secreted proteoglycans and GAGs in the mechanical group make a thicker sheet on the scaffolds in comparison to chemical group. The Safranin O staining showed that the secreted proteoglycans even penetrate into the scaffolds. The higher expression of these molecules in mechanical group may be due to the positive effect of mechanical stimulation on proteoglycans expression [25]. IHC staining revealed that in the chemical
the expression of type II collagen. The second peak of type II collagen expression is observed during continuous administration of TGF-β1 which blocks the Smad2/3-mediated TGF-β signaling and causes Sox9 level decrease. This phenomenon is related to increased collagen expression and diminution of aggrecan expression [27,28]. Interestingly, this is in agreement with our results revealing that the expression of aggrecan was higher than type II collagen in mechanical group whereas the aggrecan expression was less than type II collagen in chemical group. 3.5. Histological study Membrane-like constructs were harvested from perfusion bioreactor and the static cell culture groups after 21-day culture period. Histological and immunohistochemical (IHC) studies were then carried out (Fig. 7). The cartilage matrix is primarily composed of type II collagen and negatively charged proteoglycans such as aggrecan having water absorption capacity and compressive resistance against mechanical loading. When the progenitor cells proliferate on the dish, they secrete proteoglycan in their microenvironment whereas type II collagen is absent in the extracellular space in the early stage of chondrogenesis. After a few days the chondrocyte colonies of superficial zone form and type II collagen is secreted into the ECM of the flattened shaped differentiated cells. Finally, proteoglycan molecules are expressed and released out while fibronectin glycoproteins disappear from the ECM. In this study, the morphology and distribution of chondrocytes were investigated using H&E staining in which the nuclei of the cells were observed blue-purple whereas the cytoplasm and 7
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group type II collagen make a ticker sheets on the scaffolds compared with mechanical group. This is possibly because of the direct effect of TGFβ on type II collagen expression [27,28]. However, the cells of mechanical–chemical group showed that secreted proteoglycans and type II collagen made not only a thick sheet on the scaffolds but also penetrated in the scaffolds. It is known that alignment of the fibers in the scaffold has a positive effect on chondrogenesis even more than scaffold composition [4]. These results demonstrated that scaffold alignment and the presence of chondrogenic medium accompanied with continuous perfusion could activate the expression of cartilage specific genes (aggrecan and type II collagen) through signaling pathways reported by Mauck et al. [12].
[9]
[10]
[11] [12]
[13]
[14]
4. Conclusion [15]
In this study a perfusion bioreactor was designed to induce hydrodynamic pressure and shear stress to mesenchymal stem cells cultured on the aligned nanofibrous PCL/PLGA scaffolds. The application of hydrodynamic pressure and shear stress on the aligned scaffolds was led to high chondrogenesis efficiency and provoked the secretion of cartilage-related ECM. The mechanical stimulation affected aggrecan secretion more than type II collagen whereas the chondrogenesis medium was influenced the secretion of type II collagen more significantly. The results of this study could be a useful asset in detection of parameters influencing in vitro chondrogenesis for cartilage generation and replacement therapy.
[16]
[17] [18] [19]
[20] [21]
Acknowledgments
[22]
The authors gratefully acknowledge Biotechnology and Stem Cell Technology Research Center and National Institute of Genetic Engineering for their financial support.
[23] [24]
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