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hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues
Materials Science and Engineering C 64 (2016) 173–182
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Mimicked cartilage scaffolds of silk fibroin/hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues Jirayut Jaipaew a, Piyanun Wangkulangkul a,b, Jirut Meesane a,⁎, Pritsana Raungrut c, Puttisak Puttawibul a,b a b c
Institute of Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, 15 Karnjanavanich Road, Hat Yai, Songkhla, Thailand 90110 Department of Surgery, Faculty of Medicine, Prince of Songkla University, 15 Karnjanavanich Road, Hat Yai, Songkhla, Thailand 90110 Department of Biomedical Science, Faculty of Medicine, Prince of Songkla University, 15 Karnjanavanich Road, Hat Yai, Songkhla, Thailand 90110
a r t i c l e
i n f o
Article history: Received 17 June 2015 Received in revised form 3 March 2016 Accepted 21 March 2016 Available online 26 March 2016 Keywords: Silk fibroin Hyaluronic acid Mimicked scaffold Cartilage tissue engineering
a b s t r a c t Osteoarthritis is a critical disease that comes from degeneration of cartilage tissue. In severe cases surgery is generally required. Tissue engineering using scaffolds with stem cell transplantation is an attractive approach and a challenge for orthopedic surgery. For sample preparation, silk fibroin (SF)/hyaluronic acid (HA) scaffolds in different ratios of SF/HA (w/w) (i.e., 100:0, 90:10, 80:20, and 70:30) were formed by freeze-drying. The morphological, mechanical, and physical clues were considered in this research. The morphological structure of the scaffolds was observed by scanning electron microscope. The mechanical and physical properties of the scaffolds were analyzed by compressive and swelling ratio testing, respectively. For the cell experiments, scaffolds were seeded and cultured with human umbilical cord-derived mesenchymal stem cells (HUMSCs). The cultured scaffolds were tested for cell viability, histochemistry, immunohistochemistry, and gene expression. The SF with HA scaffolds showed regular porous structures. Those scaffolds had a soft and elastic characteristic with a high swelling ratio and water uptake. The SF/HA scaffolds showed a spheroid structure of the cells in the porous structure particularly in the SF80 and SF70 scaffolds. Cells could express Col2a, Agg, and Sox9 which are markers for chondrogenesis. It could be deduced that SF/HA scaffolds showed significant clues for suitability in cartilage tissue engineering and in surgery for osteoarthritis. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Currently, there are many patients who suffer from osteoarthritis. Osteoarthritis is a disease that is the result of cartilage degeneration [1]. The cartilage degeneration leads to a defect of the chondral area. A chondral defect propagates into a large area of the cartilage. In a severe case of osteoarthritis, the patients have pain in their daily lives. In mild cases, the patients usually need to take medication [2,3]. On the other hand, in severe cases, the patients must have surgery by biomaterials replacement [4]. To create new biomaterials using an effective technique and novel technology for surgery is a challenging task for researchers and orthopedic surgeons. Therefore, to create an effective surgical approach for osteoarthritis was chosen for this research. Tissue engineering is an attractive method that uses the principle of science, engineering, and biomedicine to create novel methods for surgery [5]. Especially, to fabricate new biomaterials that have high effectiveness is interesting for cartilage tissue engineering in osteoarthritis [6]. For a small damaged area of the chondral area, active injectable ⁎ Corresponding author. E-mail address: [email protected] (J. Meesane).
http://dx.doi.org/10.1016/j.msec.2016.03.063 0928-4931/© 2016 Elsevier B.V. All rights reserved.
hydrogels with or without cells have been used to fill the damaged area [7]. Those hydrogels played an important role of inducing cartilage tissue regeneration [8]. On the other hand, for a large area, scaffolds with and without cells were used for cartilage tissue engineering in osteoarthritis [9]. Those scaffolds were put in the chondral defect areas for stability and to induce tissue regeneration. An interesting and attractive method is to use scaffolds with cells for cartilage tissue engineering, and especially to add stem cells into the scaffold and culture them until they differentiate into chondrocytes that would lead to cartilage regeneration. Differentiating stem cells in scaffolds as native cartilage tissue is an important step before transplanting the scaffolds into a chondral defect site of osteoarthritis. Therefore, to engineer scaffolds with stem cells and culture them as native tissue is a challenge to create a performance approach for osteoarthritis surgery. The mimic approach is attractive for tissue scaffold fabrication that can create the function and structure as native tissue. The mimic approach has been used often to create bio-functionalities for scaffolds [10]. Bio-functional scaffolds can act as potential materials to enhance tissue regeneration [11]. Furthermore, the mimic method was used to construct scaffolds as an extracellular matrix that acted as a native scaffold [12]. The constructed scaffolds based on the mimic approach could
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induce tissue regeneration [13,14]. Due to the attractiveness of the mimic approach, we selected this method to create performance scaffolds with stem cells for cartilage tissue engineering. Hyaluronic acid (HA) is a natural polymer in the human body particularly in bone joints. HA has antiabrasive and compressive-resistant properties in the bone joint areas. Furthermore, HA has the biofunction as an insoluble signal for tissue regeneration [15]. Due to the unique functionalities of HA, it has been used as a material for tissue engineering. For cartilage tissue engineering, HA was fabricated into an injectable hydrogel with or without encapsulated cells [16–18]. The injectable hydrogel was used to fill a defect or degenerated area of cartilage tissue. The important role of that hydrogel is to resist compressive forces and induce cartilage tissue regeneration. In this research, we selected HA to maintain the bio-functionality of the scaffold. However, because of its instability and biodegradation, HA was blended with other polymers to induce stability [19]. SF is a biomaterial used for various tissue engineering applications because it has good mechanical stability that can maintain the structure of tissue during regeneration [20]. For cartilage tissue engineering, SF was fabricated into scaffolds with and without modification [21,22]. Due to the interesting properties and mechanical stability of SF, we choose it to be the material for scaffolds combined with HA to improve the potential and performance for cartilage tissue engineering. Keeping in mind the critical challenge of cartilage degeneration, the advantages of HA and SF, and the attractiveness of the mimic approach, the performance scaffold was proposed for cartilage tissue engineering. We fabricated blended SF/HA into 3D porous scaffolds based on the mimic approach. An interesting report presented the salt leaching preparation with genipin crosslinking to combine SF with HA for cartilage scaffold fabrication. The report mainly demonstrated the molecular organization related to the physical performance of the SF scaffold [23]. The SF scaffolds were often incorporated with mesenchymal stem cells (MSCs) for cartilage tissue engineering [24]. This present research focused on the mimicked morphological, mechanical, and physical characteristics of an SF/HA scaffold incorporated with stem cells. The scaffolds incorporated with stem cells were considered to have the potential to induce chondrogenic differentiation. Therefore, as a novel approach in this research we created mimicked structural porous scaffolds that acted as the morphological, mechanical, and physical clues for stem cells to induce cartilage tissue engineering. Eventually, those clues with stem cells were engineered as native cartilage tissue for osteoarthritis surgery. 2. Materials and method
dissolved in the purified SF solutions. The blends were incubated under slight agitation with a magnetic stirrer for 1 h at room temperature in order to complete the dissolution of HA and to allow possible interactions between the two components. The total solid weights of SF and HA in the mixtures were controlled at 3% w/v. After complete mixing, the SF/HA blends were then reacted with 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) (5%w/w) for 15 min under mild agitation. Then the mixtures were left for 2 h at room temperature to allow the SF-HA to crosslink. Finally, 2 mL of crosslinked solution were added in each well of a 24-well polystyrene tissue culture plate (TCPS) and frozen at −20 °C overnight prior to lyophilization at −80 °C for 24 h. Additionally, the freeze-dried scaffolds were treated with 80% methanol to stabilize the scaffolds in aqueous solution [25] and also to sterilize the scaffolds. 2.3. Pore size measurement The pore sizes of the scaffolds in each group were analyzed from SEM images of freeze-dried scaffolds by ImageJ software (v. 1.48). The pore sizes of the scaffolds were determined by randomized sampling (n = 20) to calculate the average pore size. 2.4. Morphological structure observation of SF/HA scaffolds The scaffold morphologies and pore sizes were observed by scanning electron microscopy (SEM-JSM5800LV, JEOL, USA) at an operating voltage of 20 kV for scaffold imaging. In order to observe their structure, the scaffolds were broken vertically in liquid nitrogen and sputter coated with gold prior to investigation by SEM. 2.5. Mechanical properties testing of SF/HA scaffolds The cylinder shape scaffolds (n = 6) were also subjected to unconfined compression tests by using the Universal Testing Machine (Lloyd instruments, LRX-Plus, AMETEK Lloyd Instrument Ltd., Hampshire, UK) equipped with a 10 N capacity load cell. Tests were conducted at room temperature in PBS (wet state) at a constant compression rate of 2 mm min− 1. The compressive stress and strain were graphed by NEXYGEN software to measure the sample dimensions of the crosssectional area and sample height (measured automatically at 0.01 N preload). The compressive modulus and standard derivation were analyzed after testing. The compressive moduli were calculated from the slope between 5% and 10% strain of the compressive stress-strain curve. The values were reported as mean ± standard derivation (n = 6).
2.1. Preparation of regenerated SF 2.6. Swelling ratio and water uptake testing of SF/HA scaffolds Silk cocoons from Bombyx mori were provided by Queen Sirikit Sericulture Center, Narathiwat, Thailand. Regenerated SF solution was prepared as previously described [1]. Briefly, in the degumming process, raw silk cocoons were boiled twice in 0.25% (w/v) Na2CO3 (Merck or Sigma, 1 g degummed silk/100 mL) for 30 min in order to remove the sericin, waxes, and other impurities and then washed several times with deionized water. The air-dried degummed SF fibers were dissolved in 9.3 M LiBr (Merck 2 g/10 mL) for 4 h at 60 °C. The solution was centrifuged at 4000 rpm for 10 min to remove the insoluble residue and then the solution was dialyzed against deionized distilled water using a dialysis membrane (MW3500, SpectraPor®, USA) for 72 h. The concentration of the obtained SF was determined by Biuret assay and then adjusted to 6% w/v. 2.2. Preparation of blended SF/HA scaffolds HA (Sigma-Aldrich) was weighed according to the weight required for fabrication with the silk fibroin in SF/HA ratios (w/w) of 100:0 (SF100), 90:10 (SF90), 80:20 (SF80) and 70:30 (SF70) and then
The swelling ratios of the SF/HA scaffolds were also investigated as previously described [26]. In brief, the dry weight of the scaffolds (W0) was measured before submersion in PBS for 24 h at room temperature. After removal of excess PBS, the wet weights of the scaffolds (Wt) were measured. The swelling ratios, expressed as S = (Wt-Wo)/Wo, were then calculated where Wo is the initial weight of dried scaffold at time t = 0 and Wt is the weight of the hydrated scaffolds at 24 h. The water uptake was expressed as Wu = [(Wt-Wo)/Wo] × 100. The values were reported as mean ± standard deviation (n = 6). 2.7. Cell viability and fluorescence micrograph In this research, human umbilical cord-derived mesenchymal stem cells (HUMSCs) were used for the cell experiments. Human umbilical cords from both sexes were collected from full-term births after either cesarean section or normal vaginal delivery with informed consent which was approved by the Prince of Songkla University Ethics Committee for Human Research at Songklanagarind Hospital, Hat Yai, Songkhla,
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Thailand (EC.55–037-25-4-3). Human umbilical cord-derived mesenchymal stem cells (HUMSCs) were isolated as previous reported [27]. Cell viability of the HUMSCs in the constructed scaffolds was evaluated by fluorescence-based live-dead assay by using fluorescein diacetate (FDA, Sigma) and propidium iodide (PI, Sigma), which stained the viable and dead cells, respectively. At day 28 after chondrogenic induction, the scaffolds were incubated in DMEM-LG containing 8 μg/mL FDA and 20 μg/mL PI for 5 min at 37 °C in a 5% CO2 atmosphere. After removal of the staining solution, the scaffolds were rinsed with PBS and then incubated in a culture medium prior to monitor by fluorescence microscopy (Olympus IX71). The cell morphologies in the 3D matrices were also observed by fluorescence microscopy. Chondrogenic-induced HUMSCs-seeded scaffolds were fixed in buffered PBS (pH 7.4) containing 3.7% para-formaldehyde for 5 min and then permeabilized the cells using 0.1% Triton X-100 for 5 min at room temperature. After blocking nonspecific binding with 3% bovine serum albumin, actin filaments were labeled by incubating the cell-seeded scaffolds in 25 μg/ml fluorescein isothiocyanateconjugated phalloidin (FITC-phalloidin conjugate, Sigma-Aldrich, USA) solution in PBS for 20 min. The nuclei were counterstained with 2.5 μg/mL bisBenzimide H 33342 trihydrochloride for 5 min at room temperature. Fluorescence images of actin filaments and nuclei were acquired by using fluorescence microscopy. 2.8. Histochemistry and immunohistochemistry On day 28, cell-constructed scaffolds were fixed overnight in 3.7% paraformaldehyde in buffered PBS (pH 7.4) and then dehydrated in a gradient series of ethanol, cleared with xylene, and embedded in paraffin blocks. Five-mm paraffin embedded sections were stained with H&E for the observation of cell morphology in the scaffolds and alcian blue for the presence of sulfated glycosaminoglycans. For immunohistochemical analysis, mouse anti-human type II collagen monoclonal antibody and mouse anti-human aggrecan monoclonal antibody (all from EMD Millipore, Temecula, USA) were used as a primary antibody for the evaluation of the expression of collagen II and aggrecan. In brief, the sections from each scaffold were neutralized of their endogenous peroxidase activity by using Novocastra Peroxidase Block. Sections were blocked in 0.4% casein/PBS for 5 min at room temperature and reacted with primary antibodies (1:250) at 4 °C for 2 h. After post-primary incubation, immunohistochemical staining was performed using the Novolink™ polymer detection system (Leica Biosystems, Newcastle, UK) following the manufacturer's protocol. 2.9. RNA isolation, cDNA synthesis, and real time PCR Gene expression profiles of the HUMSCs constructed scaffolds were analyzed at days 7, 14, and 28 after chondrogenic induction. Constructed scaffolds were homogenized and RNA was extracted from the differentiated cells using a TRIzol® reagent (Life Technology, USA) following the manufacturer's instructions. Total RNA was reverse transcribed to cDNA by using the Superscript® III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with Oligo(dT)20 primers (Invitrogen, Carlsbad, CA, USA). Primers specific to human mRNA for collagen type II (Col2a), aggrecan (Agg), and Sry-type high mobility group box transcription factor 9 (Sox9) are shown in Table 1. Quantitative relative expression of each targeted gene was analyzed on a CFX96™ Real-Time PCR detection system (BioRad, USA) with EXPRESS SYBR GreenER qPCR supermix (Invitrogen, Carlsbad, CA, USA). The expression values of targeted genes (n = 6) were normalized to the endogenous expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH). 2.10. Statistical analysis All values in graphs and tables are presented as mean ± standard derivation. Six samples (n = 6) were used for mechanical testing,
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Table 1 Primers and their sequences for quantitative real-time PCR analysis. Gene
Primer-sequence
GAPDH Forword 5′-gagtcaaccgatttggtcgt-3′ Reverse 5′-ttgattttggagggatctcg-3′ Col2a Forword 5′-accccaatccagcaaacgtt-3′ Reverse 5′-atctggacgttggcagtgttg-3’ Agg Forword 5’-acagctggggacattagtgg-3′ Reverse 5′-gtggaatgcagaggtggttt-3’ Sox9 Forword 5’-gcggaggaagtcggtgaagaacgggca-3′ Reverse 5′-tgtgaccgggtgatcggcggg-3’
Product Accession size (bp) number 242
XM_005253678.1
151
NM_001844.4
189
NM_001135.3
786
NM_000346.3
swell testing, and testing for the expression of genes. The pore size measurements of the scaffolds were determined by randomized sampling (n = 20) to calculate the average pore size. The samples were measured and statistically compared by one-way ANOVA followed by the Tukey's HSD test. P-values b0.05 were considered statistically significant. 3. Results and discussion 3.1. Morphological structure of mimicked scaffolds Generally, when we consider cartilage tissue, the extracellular matrix shows a porous structure that acts as chambers for chondrocytes. The porous structure offers the morphological mechanical and physical clues for the regulation of cells into cartilage tissue [28–30]. Therefore, it is important to mimic the morphological structure and the mechanical and physical characteristics of the native extracellular matrix in cartilage tissue in creating performance scaffolds. The morphological structure, mechanical and physical characteristics were considered as clues for cartilage tissue engineering in this research. The SEM micrographs of the pure SF and SF/HA scaffolds with different blending ratios are shown in Fig. 1A-D. The results showed that as the HA content increased the pore thickness also increased markedly. The surface of the pores was smooth in SF100 and rougher as the percentage of HA increased. When the polymers are blended the morphological structure is often organized into a non-homogenous texture [31]. Therefore, the surface roughness of the pores came from the non-homogenous texture of blending between SF and HA. Interestingly, a previous report demonstrated that the surface roughness of the substrate could induce cell adhesion and proliferation that led to enhanced tissue engineering [32,33]. Notably, the SF90, SF80, and SF70 scaffolds showed continuous and regular porous walls, whereas the SF100 scaffold showed irregular porous walls that had some broken parts. The various ratios of SF to HA showed different morphological structures of the pores. These different morphological structures might offer the morphological clues of the scaffold for cartilage regeneration. 3.2. Pore size measurement After freeze drying, the pore sizes of the SF scaffolds were measured by ImageJ software (v. 1.48) as described in the materials and methods section. The results of the pore size measurements are shown in Fig. 2. The results demonstrated that the SF80 scaffold had a mean pore size (172.24 ± 20.23) that was significantly larger than the SF100 (146.06 ± 22.36), SF90 (145.15 ± 13.47), and SF70 (140.97 ± 25.89). The HA probably disturbed the organization of the SF molecules into a regular structure in the SF80. This could enhance the flexibility of the scaffold which led to an increased pore size [34], particularly in the SF80. There was no significant difference between the pore sizes of the SF100 and SF90 scaffolds. This indicates that the HA did not interfere with the organization of SF into a regular structure. The pore size of the SF70 scaffold was also smaller than the SF80 scaffold and had no significant difference compared with the SF100 and SF90 scaffolds. This demonstrated that the HA could induce molecular interaction of the SF [35].
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Fig. 1. Scaffold morphology observed by SEM after treatment with methanol: A. SF100; B. SF90; C. SF80; and D. SF70.
The molecular interaction was the cause to retard the extension of the pore size. Therefore, the pore size of the SF70 scaffold was smaller than the SF80 scaffold. Interestingly, the results showed a suitable pore size in the range of 90 to 300 μm for the chondrocytes [36]. A suitable pore size of the scaffolds is an important clue for tissue engineering.
Other than the structure and size of the pores, the mechanical characteristics are important clues for tissue regeneration [37,38]. SF scaffolds with and without HA were tested and the mechanical characteristics were analyzed in the wet condition as native tissue.
The mechanical properties of the scaffolds were tested after submersion in PBS and the results are shown in Fig. 3 and Table 2. The load and stress at the limitation of 40% strain were significantly reduced as the content of HA increased. The compressive modulus was also reduced as the content of HA increased. The decrease in the load, stress, and compressive modulus may occur from molecular hydrophilicity of HA that could induce softness of the scaffolds [39]. The results showed that all scaffolds reverted completely after load withdrawal. Interestingly, this demonstrated that the SF scaffolds with and without HA could maintain an elastic sponge behavior. The mechanical characteristics of SF and SF/HA scaffolds demonstrated that the SF scaffolds with HA showed softness and elasticity in
Fig. 2. Pore sizes of the SF scaffolds. *P b 0.05 was accepted as statistically significant.
Fig. 3. Stress-strain curves of the scaffolds: a) SF100; b) SF90; c) SF80; d) SF70.
3.3. Mechanical properties of mimicked scaffolds
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scaffolds showed performance as clues to regulate stem cells into microspheres of cell aggregation as chondrocytes in native cartilage.
Table 2 Mechanical properties of SF/HA scaffolds after swelling in PBS. Sample
Load at limit (N)
Stress at limit (kPa)
Compressive Modulus (kPa)
SF100 SF90 SF80 SF70
0.72 ± 0.02 0.41 ± 0.03 0.24 ± 0.01 0.13 ± 0.02
5.01 ± 0.16 2.65 ± 0.21 1.38 ± 0.08 0.59 ± 0.01
33.55 ± 3.85 9.04 ± 0.78 4.88 ± 0.17 2.21 ± 0.11
Values are average ± standard derivation (n = 6).
the wet condition. The softness and elasticity of SF/HA behaved similarly to the mechanical characteristics of cartilage tissue [40]. Thus, SF/HA showed suitable performance for cartilage tissue engineering. These mechanical characteristics of scaffolds might be important clues for cartilage regeneration. 3.4. Swelling and water uptake properties of mimicked scaffolds The water-binding capacity of scaffolds is an essential indicator for the performance of tissue engineering scaffolds. The water uptake of SF/HA scaffolds increased significantly as the content of HA increased (Table 3). These results suggested that the hydrophilicity of the scaffolds improved when adding HA. Generally, the hydrophilicity of HA improves the water uptake of the scaffolds. The swelling of silk based scaffolds in aqueous solutions increased as the HA content increased in mixtures (Table 3). Fundamentally, in cartilage tissue, the extracellular matrix shows high water uptake and swelling. Therefore, the water uptake and swelling properties might be a guide for cartilage regeneration. 3.5. HUMSC morphology and cell viability in SF/HA scaffolds Cell experiments were performed on SF with and without HA scaffolds to explain the morphological structure and the mechanical and physical characteristics. The attachment and morphology of HUMSCs under chondrogenic conditions within the scaffolds was observed by phalloidin staining. In the fourth week, HUMSCs in the SF80 and SF70 scaffolds were condensed to a spheroid shape (Fig. 4). However, the cells in the SF100 and SF90 scaffolds showed an elongated and flattened morphology that eventually spread throughout the scaffolds. HUMSCS viability during chondrogenesis in SF/HA scaffolds was detected by double staining with FDA and PI at day 28 after chondrogenic induction. Fluorescence micrographs showed that HUMSCS embedded in SF100 and SF90 under chondrogenic conditions displayed a distinct fibroblastic-liked morphology (Fig. 5). In contrast, the cells in the SF80 and SF70 scaffold aggregated as a microsphere in various sizes. The aggregation showed native cartilage tissue morphology. Live and dead staining revealed that most HUMSCs in all scaffold types were viable throughout the culture period. The viability of the cells was indicated by the present of green fluorescence light which resulted from the conversion of non-fluorescent FDA into the green fluorescent metabolite by the esterase enzymes of the living cells. Surprisingly, the dead signal of red nuclei staining by PI was not observed. This indicated that all fibroin-based scaffolds were non-toxic and suitable for cell cultivation for chondrogenic support. This indicated that the morphological structure and physical characteristics of the scaffolds acted as a clue to regulate stem cell into different shapes. In particular, the SF80 and SF70
Table 3 Swelling ratio and water uptake (%) of SF/HA scaffolds in PBS. Sample
Swelling ratio (S)
Water uptake (Wu) %
SF100 SF90 SF80 SF70
20.63 ± 1.85 25.53 ± 1.03 30.17 ± 1.27 40.07 ± 1.62
95.35 ± 0.43 96.22 ± 0.14 96.79 ± 0.13 97.56 ± 0.09
Value are average ± standard derivation (n = 6).
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3.6. Histological and immunohistological analyses of chondrogenesis in SF/HA scaffolds H&E and alcian blue histological stainings were performed in both pellet and scaffold culture experiments. Histological sections revealed the highly distributed typical spindle-shaped characteristics of HUMSCs in the SF100 and SF90 scaffolds; however, some characteristics of the cells embedded in each scaffold were quite different (Fig. 6). The cells in SF100 are homogenously distributed, but in SF90 the cells condensed in some areas. For the extracellular matrix production, the HUMSCs embedded in the SF90 produced considerably more extracellular matrix than those in the SF100 which were evaluated by the higher positive staining of eosin and alcian blue. With increased HA content, the spherical morphology of the cells was observed in both SF80 and SF70. The condensed spheroid showed the accumulation of extracellular matrix inside which was determined by H&E and alcian blue staining. These characteristics resembled the pellet culture of HUMSCs. The presence of chondrogenic-specific proteins including collagen type II and aggrecan in all cultures was determined by immunohistochemistry at day 28 after induction (Figs. 7 and 8). The results showed that both collagen type II and aggrecan were positively stained in all experiments suggesting that the HUMSCs underwent chondrogenic differentiation. Consistent with the staining of alcian blue, the immunostaining by antibodies against collagen type II and aggrecan in SF90 were more intense than SF100. Likewise, the spheroids in both SF80 and SF70 were also expressed in both collagen type II and aggrecan which resembled the results obtained from the pellet culture. The results obtained by immunohistochemistry were significantly consistent with that obtained by histochemical analysis of alcian blue staining. These results firmly demonstrated that scaffolds of SF with and without HA could induce HUMSCs to chondrogenic differentiation. Interestingly, the results obviously indicated that different porous structures and ratios of SF to HA could induce chondrogenic differentiation of HUMSCs into different forms. The structural porosity of SF80 and SF70 could induce chondrogenic differentiation of HUMSCs to form spheroids embedded in the pores as native cartilage tissue. The results indicated that the mechanical characteristics and swelling ratios were related to differentiation of the HUMSCs. Clearly, the mimicked porous scaffolds that showed soft characteristics and high swelling ratios acted as physical clues for chondrogenic differentiation. 3.7. Real-time PCR for the analysis of chondrogenesis in SF/HA scaffolds The expression of chondrogenic-specific genes including Col2a, Agg, and Sox9 were analyzed by time-course real-time PCR (Fig. 9). Scaffoldembedded cells cultured under chondrogenic conditions for 1, 2, and 3 weeks were investigated. The data indicated that all of the chondrogenic markers were significantly up-regulated in all scaffold types. The levels of gene expression for collagen type II, aggrecan, and Sry-type high-mobility group box transcription factor 9 increased with an increase in the HA content. The highest levels of Col2a, Agg, and Sox9 were detected on day 21 after chondrogenic induction. The results obtained by real-time PCR were consistence with the results obtained from immunohistochemical analysis. These results strongly confirmed the previous explanation in histological and immunological analyses that mimicked porous scaffolds acted as important clues to induce chondrogenic differentiation of HUMSCs. 4. Conclusions The mimicked cartilage scaffolds based on SF/HA were created and used with stem cells as a proposal for osteoarthritis surgery. The morphological mechanical and physical clues of the scaffolds were
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Fig. 4. Fluorescence micrographs of HUMSCs cultured on SF/HA scaffolds: A. SF100; B. SF90; C. SF80; and D. SF70 stained with FITC-phalloidin for actin filaments (green) and H33342 for nuclei (blue). Scale bars represent 100 μm.
Fig. 5. Cell viability during chondrogenesis. At day 28 after chondrogenic induction, the viability of HUMSCs in constructed scaffolds: A. SF100; B. SF90; C. SF80; and D. SF70 were evaluated by double-staining with FDA and PI. Green staining indicates viable cells. Scale bars represent 200 μm.
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Fig. 6. H&E and alcian blue staining: A. Chondrogenic differentiation of pellet cultured HUMSCs and HUMSCs embedded in SF/HA scaffolds evaluated by H&E staining at the fourth week after chondrogenic induction: C. SF100; E. SF90; G. SF80; and I. SF70, and alcian blue staining at the fourth week after initial induction: B. pellet cultured; D. SF100; F. SF90; H. SF80; and J. SF70. Scale bars represent 200 μm.
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Fig. 7. Immunohistochemical staining for the expression of collagen type II of induced HUMSCs. Chondrogenic differentiation of HUMSCs embedded in silk/HA scaffolds were evaluated by immunohistochemistry at the fourth week after chondrogenic induction: A. SF100; B. SF90; C. SF80; and D.SF70. Scale bars represent 500 μm.
Fig. 8. Immunohistological staining for the expression of aggrecan of induced HUMSCs. Chondrogenic differentiation of HUMSCs embedded in silk/HA scaffolds was evaluated by immunohistochemistry at the fourth week after chondrogenic induction: A. SF100; B. SF90; C. SF80; and D. SF70. Scale bars represent 500 μm.
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Acknowledgements This work was supported by the Academic Promotion and Development Unit (Grant No. 54-171-25-2-3), Faculty of Medicine, Prince of Songkla University, Hat Yai, 90110, Thailand. We also thank Queen Sirikit Sericulture Center, Narathiwat, Thailand for support in providing the silk fibroin in this research. References
Fig. 9. Relative normalized expression of chondrogenic-specific gene to GAPDH of HUMSCs embedded in SF/HA scaffolds at day 7, 14, and 21 after chondrogenic induction: A. Col2a; B. Agg; and C. Sox9. Values are average ± standard derivation (n = 6).
investigated. The results showed that the mimicked cartilage scaffolds that consisted of SF and HA were morphological, mechanical, and physical clues to regulate stem cells into chondrogenesis. Especially, the SF80 and SF70 scaffolds demonstrated predominant clues for chondrogenesis. The morphological structure of regulated stem cells in mimicked cartilage scaffolds showed a native cartilage tissue structure. It could be deduced that mimicked cartilage scaffolds based on SF/HA have suitable performance for cartilage tissue engineering and show promise for use in osteoarthritis surgery. Nevertheless, these scaffolds need in vivo testing to prove their performance for cartilage tissue engineering.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Mimicked cartilage scaffolds of silk fibroin/hyaluronic acid with stem cells for osteoarthritis surgery: Morphological, mechanical, and physical clues Jirayut Jaipaew a, Piyanun Wangkulangkul a,b, Jirut Meesane a,⁎, Pritsana Raungrut c, Puttisak Puttawibul a,b a b c
Institute of Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, 15 Karnjanavanich Road, Hat Yai, Songkhla, Thailand 90110 Department of Surgery, Faculty of Medicine, Prince of Songkla University, 15 Karnjanavanich Road, Hat Yai, Songkhla, Thailand 90110 Department of Biomedical Science, Faculty of Medicine, Prince of Songkla University, 15 Karnjanavanich Road, Hat Yai, Songkhla, Thailand 90110
a r t i c l e
i n f o
Article history: Received 17 June 2015 Received in revised form 3 March 2016 Accepted 21 March 2016 Available online 26 March 2016 Keywords: Silk fibroin Hyaluronic acid Mimicked scaffold Cartilage tissue engineering
a b s t r a c t Osteoarthritis is a critical disease that comes from degeneration of cartilage tissue. In severe cases surgery is generally required. Tissue engineering using scaffolds with stem cell transplantation is an attractive approach and a challenge for orthopedic surgery. For sample preparation, silk fibroin (SF)/hyaluronic acid (HA) scaffolds in different ratios of SF/HA (w/w) (i.e., 100:0, 90:10, 80:20, and 70:30) were formed by freeze-drying. The morphological, mechanical, and physical clues were considered in this research. The morphological structure of the scaffolds was observed by scanning electron microscope. The mechanical and physical properties of the scaffolds were analyzed by compressive and swelling ratio testing, respectively. For the cell experiments, scaffolds were seeded and cultured with human umbilical cord-derived mesenchymal stem cells (HUMSCs). The cultured scaffolds were tested for cell viability, histochemistry, immunohistochemistry, and gene expression. The SF with HA scaffolds showed regular porous structures. Those scaffolds had a soft and elastic characteristic with a high swelling ratio and water uptake. The SF/HA scaffolds showed a spheroid structure of the cells in the porous structure particularly in the SF80 and SF70 scaffolds. Cells could express Col2a, Agg, and Sox9 which are markers for chondrogenesis. It could be deduced that SF/HA scaffolds showed significant clues for suitability in cartilage tissue engineering and in surgery for osteoarthritis. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Currently, there are many patients who suffer from osteoarthritis. Osteoarthritis is a disease that is the result of cartilage degeneration [1]. The cartilage degeneration leads to a defect of the chondral area. A chondral defect propagates into a large area of the cartilage. In a severe case of osteoarthritis, the patients have pain in their daily lives. In mild cases, the patients usually need to take medication [2,3]. On the other hand, in severe cases, the patients must have surgery by biomaterials replacement [4]. To create new biomaterials using an effective technique and novel technology for surgery is a challenging task for researchers and orthopedic surgeons. Therefore, to create an effective surgical approach for osteoarthritis was chosen for this research. Tissue engineering is an attractive method that uses the principle of science, engineering, and biomedicine to create novel methods for surgery [5]. Especially, to fabricate new biomaterials that have high effectiveness is interesting for cartilage tissue engineering in osteoarthritis [6]. For a small damaged area of the chondral area, active injectable ⁎ Corresponding author. E-mail address: [email protected] (J. Meesane).
http://dx.doi.org/10.1016/j.msec.2016.03.063 0928-4931/© 2016 Elsevier B.V. All rights reserved.
hydrogels with or without cells have been used to fill the damaged area [7]. Those hydrogels played an important role of inducing cartilage tissue regeneration [8]. On the other hand, for a large area, scaffolds with and without cells were used for cartilage tissue engineering in osteoarthritis [9]. Those scaffolds were put in the chondral defect areas for stability and to induce tissue regeneration. An interesting and attractive method is to use scaffolds with cells for cartilage tissue engineering, and especially to add stem cells into the scaffold and culture them until they differentiate into chondrocytes that would lead to cartilage regeneration. Differentiating stem cells in scaffolds as native cartilage tissue is an important step before transplanting the scaffolds into a chondral defect site of osteoarthritis. Therefore, to engineer scaffolds with stem cells and culture them as native tissue is a challenge to create a performance approach for osteoarthritis surgery. The mimic approach is attractive for tissue scaffold fabrication that can create the function and structure as native tissue. The mimic approach has been used often to create bio-functionalities for scaffolds [10]. Bio-functional scaffolds can act as potential materials to enhance tissue regeneration [11]. Furthermore, the mimic method was used to construct scaffolds as an extracellular matrix that acted as a native scaffold [12]. The constructed scaffolds based on the mimic approach could
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induce tissue regeneration [13,14]. Due to the attractiveness of the mimic approach, we selected this method to create performance scaffolds with stem cells for cartilage tissue engineering. Hyaluronic acid (HA) is a natural polymer in the human body particularly in bone joints. HA has antiabrasive and compressive-resistant properties in the bone joint areas. Furthermore, HA has the biofunction as an insoluble signal for tissue regeneration [15]. Due to the unique functionalities of HA, it has been used as a material for tissue engineering. For cartilage tissue engineering, HA was fabricated into an injectable hydrogel with or without encapsulated cells [16–18]. The injectable hydrogel was used to fill a defect or degenerated area of cartilage tissue. The important role of that hydrogel is to resist compressive forces and induce cartilage tissue regeneration. In this research, we selected HA to maintain the bio-functionality of the scaffold. However, because of its instability and biodegradation, HA was blended with other polymers to induce stability [19]. SF is a biomaterial used for various tissue engineering applications because it has good mechanical stability that can maintain the structure of tissue during regeneration [20]. For cartilage tissue engineering, SF was fabricated into scaffolds with and without modification [21,22]. Due to the interesting properties and mechanical stability of SF, we choose it to be the material for scaffolds combined with HA to improve the potential and performance for cartilage tissue engineering. Keeping in mind the critical challenge of cartilage degeneration, the advantages of HA and SF, and the attractiveness of the mimic approach, the performance scaffold was proposed for cartilage tissue engineering. We fabricated blended SF/HA into 3D porous scaffolds based on the mimic approach. An interesting report presented the salt leaching preparation with genipin crosslinking to combine SF with HA for cartilage scaffold fabrication. The report mainly demonstrated the molecular organization related to the physical performance of the SF scaffold [23]. The SF scaffolds were often incorporated with mesenchymal stem cells (MSCs) for cartilage tissue engineering [24]. This present research focused on the mimicked morphological, mechanical, and physical characteristics of an SF/HA scaffold incorporated with stem cells. The scaffolds incorporated with stem cells were considered to have the potential to induce chondrogenic differentiation. Therefore, as a novel approach in this research we created mimicked structural porous scaffolds that acted as the morphological, mechanical, and physical clues for stem cells to induce cartilage tissue engineering. Eventually, those clues with stem cells were engineered as native cartilage tissue for osteoarthritis surgery. 2. Materials and method
dissolved in the purified SF solutions. The blends were incubated under slight agitation with a magnetic stirrer for 1 h at room temperature in order to complete the dissolution of HA and to allow possible interactions between the two components. The total solid weights of SF and HA in the mixtures were controlled at 3% w/v. After complete mixing, the SF/HA blends were then reacted with 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) (5%w/w) for 15 min under mild agitation. Then the mixtures were left for 2 h at room temperature to allow the SF-HA to crosslink. Finally, 2 mL of crosslinked solution were added in each well of a 24-well polystyrene tissue culture plate (TCPS) and frozen at −20 °C overnight prior to lyophilization at −80 °C for 24 h. Additionally, the freeze-dried scaffolds were treated with 80% methanol to stabilize the scaffolds in aqueous solution [25] and also to sterilize the scaffolds. 2.3. Pore size measurement The pore sizes of the scaffolds in each group were analyzed from SEM images of freeze-dried scaffolds by ImageJ software (v. 1.48). The pore sizes of the scaffolds were determined by randomized sampling (n = 20) to calculate the average pore size. 2.4. Morphological structure observation of SF/HA scaffolds The scaffold morphologies and pore sizes were observed by scanning electron microscopy (SEM-JSM5800LV, JEOL, USA) at an operating voltage of 20 kV for scaffold imaging. In order to observe their structure, the scaffolds were broken vertically in liquid nitrogen and sputter coated with gold prior to investigation by SEM. 2.5. Mechanical properties testing of SF/HA scaffolds The cylinder shape scaffolds (n = 6) were also subjected to unconfined compression tests by using the Universal Testing Machine (Lloyd instruments, LRX-Plus, AMETEK Lloyd Instrument Ltd., Hampshire, UK) equipped with a 10 N capacity load cell. Tests were conducted at room temperature in PBS (wet state) at a constant compression rate of 2 mm min− 1. The compressive stress and strain were graphed by NEXYGEN software to measure the sample dimensions of the crosssectional area and sample height (measured automatically at 0.01 N preload). The compressive modulus and standard derivation were analyzed after testing. The compressive moduli were calculated from the slope between 5% and 10% strain of the compressive stress-strain curve. The values were reported as mean ± standard derivation (n = 6).
2.1. Preparation of regenerated SF 2.6. Swelling ratio and water uptake testing of SF/HA scaffolds Silk cocoons from Bombyx mori were provided by Queen Sirikit Sericulture Center, Narathiwat, Thailand. Regenerated SF solution was prepared as previously described [1]. Briefly, in the degumming process, raw silk cocoons were boiled twice in 0.25% (w/v) Na2CO3 (Merck or Sigma, 1 g degummed silk/100 mL) for 30 min in order to remove the sericin, waxes, and other impurities and then washed several times with deionized water. The air-dried degummed SF fibers were dissolved in 9.3 M LiBr (Merck 2 g/10 mL) for 4 h at 60 °C. The solution was centrifuged at 4000 rpm for 10 min to remove the insoluble residue and then the solution was dialyzed against deionized distilled water using a dialysis membrane (MW3500, SpectraPor®, USA) for 72 h. The concentration of the obtained SF was determined by Biuret assay and then adjusted to 6% w/v. 2.2. Preparation of blended SF/HA scaffolds HA (Sigma-Aldrich) was weighed according to the weight required for fabrication with the silk fibroin in SF/HA ratios (w/w) of 100:0 (SF100), 90:10 (SF90), 80:20 (SF80) and 70:30 (SF70) and then
The swelling ratios of the SF/HA scaffolds were also investigated as previously described [26]. In brief, the dry weight of the scaffolds (W0) was measured before submersion in PBS for 24 h at room temperature. After removal of excess PBS, the wet weights of the scaffolds (Wt) were measured. The swelling ratios, expressed as S = (Wt-Wo)/Wo, were then calculated where Wo is the initial weight of dried scaffold at time t = 0 and Wt is the weight of the hydrated scaffolds at 24 h. The water uptake was expressed as Wu = [(Wt-Wo)/Wo] × 100. The values were reported as mean ± standard deviation (n = 6). 2.7. Cell viability and fluorescence micrograph In this research, human umbilical cord-derived mesenchymal stem cells (HUMSCs) were used for the cell experiments. Human umbilical cords from both sexes were collected from full-term births after either cesarean section or normal vaginal delivery with informed consent which was approved by the Prince of Songkla University Ethics Committee for Human Research at Songklanagarind Hospital, Hat Yai, Songkhla,
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Thailand (EC.55–037-25-4-3). Human umbilical cord-derived mesenchymal stem cells (HUMSCs) were isolated as previous reported [27]. Cell viability of the HUMSCs in the constructed scaffolds was evaluated by fluorescence-based live-dead assay by using fluorescein diacetate (FDA, Sigma) and propidium iodide (PI, Sigma), which stained the viable and dead cells, respectively. At day 28 after chondrogenic induction, the scaffolds were incubated in DMEM-LG containing 8 μg/mL FDA and 20 μg/mL PI for 5 min at 37 °C in a 5% CO2 atmosphere. After removal of the staining solution, the scaffolds were rinsed with PBS and then incubated in a culture medium prior to monitor by fluorescence microscopy (Olympus IX71). The cell morphologies in the 3D matrices were also observed by fluorescence microscopy. Chondrogenic-induced HUMSCs-seeded scaffolds were fixed in buffered PBS (pH 7.4) containing 3.7% para-formaldehyde for 5 min and then permeabilized the cells using 0.1% Triton X-100 for 5 min at room temperature. After blocking nonspecific binding with 3% bovine serum albumin, actin filaments were labeled by incubating the cell-seeded scaffolds in 25 μg/ml fluorescein isothiocyanateconjugated phalloidin (FITC-phalloidin conjugate, Sigma-Aldrich, USA) solution in PBS for 20 min. The nuclei were counterstained with 2.5 μg/mL bisBenzimide H 33342 trihydrochloride for 5 min at room temperature. Fluorescence images of actin filaments and nuclei were acquired by using fluorescence microscopy. 2.8. Histochemistry and immunohistochemistry On day 28, cell-constructed scaffolds were fixed overnight in 3.7% paraformaldehyde in buffered PBS (pH 7.4) and then dehydrated in a gradient series of ethanol, cleared with xylene, and embedded in paraffin blocks. Five-mm paraffin embedded sections were stained with H&E for the observation of cell morphology in the scaffolds and alcian blue for the presence of sulfated glycosaminoglycans. For immunohistochemical analysis, mouse anti-human type II collagen monoclonal antibody and mouse anti-human aggrecan monoclonal antibody (all from EMD Millipore, Temecula, USA) were used as a primary antibody for the evaluation of the expression of collagen II and aggrecan. In brief, the sections from each scaffold were neutralized of their endogenous peroxidase activity by using Novocastra Peroxidase Block. Sections were blocked in 0.4% casein/PBS for 5 min at room temperature and reacted with primary antibodies (1:250) at 4 °C for 2 h. After post-primary incubation, immunohistochemical staining was performed using the Novolink™ polymer detection system (Leica Biosystems, Newcastle, UK) following the manufacturer's protocol. 2.9. RNA isolation, cDNA synthesis, and real time PCR Gene expression profiles of the HUMSCs constructed scaffolds were analyzed at days 7, 14, and 28 after chondrogenic induction. Constructed scaffolds were homogenized and RNA was extracted from the differentiated cells using a TRIzol® reagent (Life Technology, USA) following the manufacturer's instructions. Total RNA was reverse transcribed to cDNA by using the Superscript® III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with Oligo(dT)20 primers (Invitrogen, Carlsbad, CA, USA). Primers specific to human mRNA for collagen type II (Col2a), aggrecan (Agg), and Sry-type high mobility group box transcription factor 9 (Sox9) are shown in Table 1. Quantitative relative expression of each targeted gene was analyzed on a CFX96™ Real-Time PCR detection system (BioRad, USA) with EXPRESS SYBR GreenER qPCR supermix (Invitrogen, Carlsbad, CA, USA). The expression values of targeted genes (n = 6) were normalized to the endogenous expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH). 2.10. Statistical analysis All values in graphs and tables are presented as mean ± standard derivation. Six samples (n = 6) were used for mechanical testing,
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Table 1 Primers and their sequences for quantitative real-time PCR analysis. Gene
Primer-sequence
GAPDH Forword 5′-gagtcaaccgatttggtcgt-3′ Reverse 5′-ttgattttggagggatctcg-3′ Col2a Forword 5′-accccaatccagcaaacgtt-3′ Reverse 5′-atctggacgttggcagtgttg-3’ Agg Forword 5’-acagctggggacattagtgg-3′ Reverse 5′-gtggaatgcagaggtggttt-3’ Sox9 Forword 5’-gcggaggaagtcggtgaagaacgggca-3′ Reverse 5′-tgtgaccgggtgatcggcggg-3’
Product Accession size (bp) number 242
XM_005253678.1
151
NM_001844.4
189
NM_001135.3
786
NM_000346.3
swell testing, and testing for the expression of genes. The pore size measurements of the scaffolds were determined by randomized sampling (n = 20) to calculate the average pore size. The samples were measured and statistically compared by one-way ANOVA followed by the Tukey's HSD test. P-values b0.05 were considered statistically significant. 3. Results and discussion 3.1. Morphological structure of mimicked scaffolds Generally, when we consider cartilage tissue, the extracellular matrix shows a porous structure that acts as chambers for chondrocytes. The porous structure offers the morphological mechanical and physical clues for the regulation of cells into cartilage tissue [28–30]. Therefore, it is important to mimic the morphological structure and the mechanical and physical characteristics of the native extracellular matrix in cartilage tissue in creating performance scaffolds. The morphological structure, mechanical and physical characteristics were considered as clues for cartilage tissue engineering in this research. The SEM micrographs of the pure SF and SF/HA scaffolds with different blending ratios are shown in Fig. 1A-D. The results showed that as the HA content increased the pore thickness also increased markedly. The surface of the pores was smooth in SF100 and rougher as the percentage of HA increased. When the polymers are blended the morphological structure is often organized into a non-homogenous texture [31]. Therefore, the surface roughness of the pores came from the non-homogenous texture of blending between SF and HA. Interestingly, a previous report demonstrated that the surface roughness of the substrate could induce cell adhesion and proliferation that led to enhanced tissue engineering [32,33]. Notably, the SF90, SF80, and SF70 scaffolds showed continuous and regular porous walls, whereas the SF100 scaffold showed irregular porous walls that had some broken parts. The various ratios of SF to HA showed different morphological structures of the pores. These different morphological structures might offer the morphological clues of the scaffold for cartilage regeneration. 3.2. Pore size measurement After freeze drying, the pore sizes of the SF scaffolds were measured by ImageJ software (v. 1.48) as described in the materials and methods section. The results of the pore size measurements are shown in Fig. 2. The results demonstrated that the SF80 scaffold had a mean pore size (172.24 ± 20.23) that was significantly larger than the SF100 (146.06 ± 22.36), SF90 (145.15 ± 13.47), and SF70 (140.97 ± 25.89). The HA probably disturbed the organization of the SF molecules into a regular structure in the SF80. This could enhance the flexibility of the scaffold which led to an increased pore size [34], particularly in the SF80. There was no significant difference between the pore sizes of the SF100 and SF90 scaffolds. This indicates that the HA did not interfere with the organization of SF into a regular structure. The pore size of the SF70 scaffold was also smaller than the SF80 scaffold and had no significant difference compared with the SF100 and SF90 scaffolds. This demonstrated that the HA could induce molecular interaction of the SF [35].
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Fig. 1. Scaffold morphology observed by SEM after treatment with methanol: A. SF100; B. SF90; C. SF80; and D. SF70.
The molecular interaction was the cause to retard the extension of the pore size. Therefore, the pore size of the SF70 scaffold was smaller than the SF80 scaffold. Interestingly, the results showed a suitable pore size in the range of 90 to 300 μm for the chondrocytes [36]. A suitable pore size of the scaffolds is an important clue for tissue engineering.
Other than the structure and size of the pores, the mechanical characteristics are important clues for tissue regeneration [37,38]. SF scaffolds with and without HA were tested and the mechanical characteristics were analyzed in the wet condition as native tissue.
The mechanical properties of the scaffolds were tested after submersion in PBS and the results are shown in Fig. 3 and Table 2. The load and stress at the limitation of 40% strain were significantly reduced as the content of HA increased. The compressive modulus was also reduced as the content of HA increased. The decrease in the load, stress, and compressive modulus may occur from molecular hydrophilicity of HA that could induce softness of the scaffolds [39]. The results showed that all scaffolds reverted completely after load withdrawal. Interestingly, this demonstrated that the SF scaffolds with and without HA could maintain an elastic sponge behavior. The mechanical characteristics of SF and SF/HA scaffolds demonstrated that the SF scaffolds with HA showed softness and elasticity in
Fig. 2. Pore sizes of the SF scaffolds. *P b 0.05 was accepted as statistically significant.
Fig. 3. Stress-strain curves of the scaffolds: a) SF100; b) SF90; c) SF80; d) SF70.
3.3. Mechanical properties of mimicked scaffolds
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scaffolds showed performance as clues to regulate stem cells into microspheres of cell aggregation as chondrocytes in native cartilage.
Table 2 Mechanical properties of SF/HA scaffolds after swelling in PBS. Sample
Load at limit (N)
Stress at limit (kPa)
Compressive Modulus (kPa)
SF100 SF90 SF80 SF70
0.72 ± 0.02 0.41 ± 0.03 0.24 ± 0.01 0.13 ± 0.02
5.01 ± 0.16 2.65 ± 0.21 1.38 ± 0.08 0.59 ± 0.01
33.55 ± 3.85 9.04 ± 0.78 4.88 ± 0.17 2.21 ± 0.11
Values are average ± standard derivation (n = 6).
the wet condition. The softness and elasticity of SF/HA behaved similarly to the mechanical characteristics of cartilage tissue [40]. Thus, SF/HA showed suitable performance for cartilage tissue engineering. These mechanical characteristics of scaffolds might be important clues for cartilage regeneration. 3.4. Swelling and water uptake properties of mimicked scaffolds The water-binding capacity of scaffolds is an essential indicator for the performance of tissue engineering scaffolds. The water uptake of SF/HA scaffolds increased significantly as the content of HA increased (Table 3). These results suggested that the hydrophilicity of the scaffolds improved when adding HA. Generally, the hydrophilicity of HA improves the water uptake of the scaffolds. The swelling of silk based scaffolds in aqueous solutions increased as the HA content increased in mixtures (Table 3). Fundamentally, in cartilage tissue, the extracellular matrix shows high water uptake and swelling. Therefore, the water uptake and swelling properties might be a guide for cartilage regeneration. 3.5. HUMSC morphology and cell viability in SF/HA scaffolds Cell experiments were performed on SF with and without HA scaffolds to explain the morphological structure and the mechanical and physical characteristics. The attachment and morphology of HUMSCs under chondrogenic conditions within the scaffolds was observed by phalloidin staining. In the fourth week, HUMSCs in the SF80 and SF70 scaffolds were condensed to a spheroid shape (Fig. 4). However, the cells in the SF100 and SF90 scaffolds showed an elongated and flattened morphology that eventually spread throughout the scaffolds. HUMSCS viability during chondrogenesis in SF/HA scaffolds was detected by double staining with FDA and PI at day 28 after chondrogenic induction. Fluorescence micrographs showed that HUMSCS embedded in SF100 and SF90 under chondrogenic conditions displayed a distinct fibroblastic-liked morphology (Fig. 5). In contrast, the cells in the SF80 and SF70 scaffold aggregated as a microsphere in various sizes. The aggregation showed native cartilage tissue morphology. Live and dead staining revealed that most HUMSCs in all scaffold types were viable throughout the culture period. The viability of the cells was indicated by the present of green fluorescence light which resulted from the conversion of non-fluorescent FDA into the green fluorescent metabolite by the esterase enzymes of the living cells. Surprisingly, the dead signal of red nuclei staining by PI was not observed. This indicated that all fibroin-based scaffolds were non-toxic and suitable for cell cultivation for chondrogenic support. This indicated that the morphological structure and physical characteristics of the scaffolds acted as a clue to regulate stem cell into different shapes. In particular, the SF80 and SF70
Table 3 Swelling ratio and water uptake (%) of SF/HA scaffolds in PBS. Sample
Swelling ratio (S)
Water uptake (Wu) %
SF100 SF90 SF80 SF70
20.63 ± 1.85 25.53 ± 1.03 30.17 ± 1.27 40.07 ± 1.62
95.35 ± 0.43 96.22 ± 0.14 96.79 ± 0.13 97.56 ± 0.09
Value are average ± standard derivation (n = 6).
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3.6. Histological and immunohistological analyses of chondrogenesis in SF/HA scaffolds H&E and alcian blue histological stainings were performed in both pellet and scaffold culture experiments. Histological sections revealed the highly distributed typical spindle-shaped characteristics of HUMSCs in the SF100 and SF90 scaffolds; however, some characteristics of the cells embedded in each scaffold were quite different (Fig. 6). The cells in SF100 are homogenously distributed, but in SF90 the cells condensed in some areas. For the extracellular matrix production, the HUMSCs embedded in the SF90 produced considerably more extracellular matrix than those in the SF100 which were evaluated by the higher positive staining of eosin and alcian blue. With increased HA content, the spherical morphology of the cells was observed in both SF80 and SF70. The condensed spheroid showed the accumulation of extracellular matrix inside which was determined by H&E and alcian blue staining. These characteristics resembled the pellet culture of HUMSCs. The presence of chondrogenic-specific proteins including collagen type II and aggrecan in all cultures was determined by immunohistochemistry at day 28 after induction (Figs. 7 and 8). The results showed that both collagen type II and aggrecan were positively stained in all experiments suggesting that the HUMSCs underwent chondrogenic differentiation. Consistent with the staining of alcian blue, the immunostaining by antibodies against collagen type II and aggrecan in SF90 were more intense than SF100. Likewise, the spheroids in both SF80 and SF70 were also expressed in both collagen type II and aggrecan which resembled the results obtained from the pellet culture. The results obtained by immunohistochemistry were significantly consistent with that obtained by histochemical analysis of alcian blue staining. These results firmly demonstrated that scaffolds of SF with and without HA could induce HUMSCs to chondrogenic differentiation. Interestingly, the results obviously indicated that different porous structures and ratios of SF to HA could induce chondrogenic differentiation of HUMSCs into different forms. The structural porosity of SF80 and SF70 could induce chondrogenic differentiation of HUMSCs to form spheroids embedded in the pores as native cartilage tissue. The results indicated that the mechanical characteristics and swelling ratios were related to differentiation of the HUMSCs. Clearly, the mimicked porous scaffolds that showed soft characteristics and high swelling ratios acted as physical clues for chondrogenic differentiation. 3.7. Real-time PCR for the analysis of chondrogenesis in SF/HA scaffolds The expression of chondrogenic-specific genes including Col2a, Agg, and Sox9 were analyzed by time-course real-time PCR (Fig. 9). Scaffoldembedded cells cultured under chondrogenic conditions for 1, 2, and 3 weeks were investigated. The data indicated that all of the chondrogenic markers were significantly up-regulated in all scaffold types. The levels of gene expression for collagen type II, aggrecan, and Sry-type high-mobility group box transcription factor 9 increased with an increase in the HA content. The highest levels of Col2a, Agg, and Sox9 were detected on day 21 after chondrogenic induction. The results obtained by real-time PCR were consistence with the results obtained from immunohistochemical analysis. These results strongly confirmed the previous explanation in histological and immunological analyses that mimicked porous scaffolds acted as important clues to induce chondrogenic differentiation of HUMSCs. 4. Conclusions The mimicked cartilage scaffolds based on SF/HA were created and used with stem cells as a proposal for osteoarthritis surgery. The morphological mechanical and physical clues of the scaffolds were
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Fig. 4. Fluorescence micrographs of HUMSCs cultured on SF/HA scaffolds: A. SF100; B. SF90; C. SF80; and D. SF70 stained with FITC-phalloidin for actin filaments (green) and H33342 for nuclei (blue). Scale bars represent 100 μm.
Fig. 5. Cell viability during chondrogenesis. At day 28 after chondrogenic induction, the viability of HUMSCs in constructed scaffolds: A. SF100; B. SF90; C. SF80; and D. SF70 were evaluated by double-staining with FDA and PI. Green staining indicates viable cells. Scale bars represent 200 μm.
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Fig. 6. H&E and alcian blue staining: A. Chondrogenic differentiation of pellet cultured HUMSCs and HUMSCs embedded in SF/HA scaffolds evaluated by H&E staining at the fourth week after chondrogenic induction: C. SF100; E. SF90; G. SF80; and I. SF70, and alcian blue staining at the fourth week after initial induction: B. pellet cultured; D. SF100; F. SF90; H. SF80; and J. SF70. Scale bars represent 200 μm.
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Fig. 7. Immunohistochemical staining for the expression of collagen type II of induced HUMSCs. Chondrogenic differentiation of HUMSCs embedded in silk/HA scaffolds were evaluated by immunohistochemistry at the fourth week after chondrogenic induction: A. SF100; B. SF90; C. SF80; and D.SF70. Scale bars represent 500 μm.
Fig. 8. Immunohistological staining for the expression of aggrecan of induced HUMSCs. Chondrogenic differentiation of HUMSCs embedded in silk/HA scaffolds was evaluated by immunohistochemistry at the fourth week after chondrogenic induction: A. SF100; B. SF90; C. SF80; and D. SF70. Scale bars represent 500 μm.
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Acknowledgements This work was supported by the Academic Promotion and Development Unit (Grant No. 54-171-25-2-3), Faculty of Medicine, Prince of Songkla University, Hat Yai, 90110, Thailand. We also thank Queen Sirikit Sericulture Center, Narathiwat, Thailand for support in providing the silk fibroin in this research. References
Fig. 9. Relative normalized expression of chondrogenic-specific gene to GAPDH of HUMSCs embedded in SF/HA scaffolds at day 7, 14, and 21 after chondrogenic induction: A. Col2a; B. Agg; and C. Sox9. Values are average ± standard derivation (n = 6).
investigated. The results showed that the mimicked cartilage scaffolds that consisted of SF and HA were morphological, mechanical, and physical clues to regulate stem cells into chondrogenesis. Especially, the SF80 and SF70 scaffolds demonstrated predominant clues for chondrogenesis. The morphological structure of regulated stem cells in mimicked cartilage scaffolds showed a native cartilage tissue structure. It could be deduced that mimicked cartilage scaffolds based on SF/HA have suitable performance for cartilage tissue engineering and show promise for use in osteoarthritis surgery. Nevertheless, these scaffolds need in vivo testing to prove their performance for cartilage tissue engineering.
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