- Research article
- Open Access
- Open Peer Review
Inhibition of interleukin-1beta-stimulated dedifferentiation of chondrocytes via controlled release of CrmA from hyaluronic acid-chitosan microspheres
© Ma et al.; licensee BioMed Central. 2015
- Received: 27 May 2014
- Accepted: 4 March 2015
- Published: 18 March 2015
The previous studies indicated that CrmA could ameliorate the interleukin-1β induced osteoarthritis. In this study, we investigated the controlled-released cytokine response modifier A (CrmA) from hyaluronic acid (HA)-chitosan (CS) microspheres to improve interleukin-1β (IL-1β)-stimulated dedifferentiation of chondrocytes.
A rat model of osteoarthritis (OA) in vitro was established using 10 ng/ml IL-1β as modulating and chondrocytes inducing agent. HA-CS-CrmA microspheres were added to the medium after IL-1β was co-cultured with freshly isolated rat chondrocytes for 48 hours. The chondrocytes viability and glycosaminoglycan (GAG) content were determined. The level of CrmA secreted was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The protein levels of type II collagen, aggrecan, collagen I and IL-1β were detected using western blotting analyses.
The CrmA release kinetics were characterized by an initial burst release, which was reduced to a linear release over ten days. The production of GAG and the expression of type II collagen, aggrecan significantly increased compared with the control group, while the expression of collagen I and IL-1β decreased.
This study demonstrated that HA-CS microspheres containing CrmA could attenuate the degeneration of articular cartilage by maintaining the phenotype of chondrocytes during culture expansion. The suppression of inflammatory cytokines activity within the joint might be one important mechanism of the action of the microspheres in the treatment of OA.
- Hyaluronic acid
Osteoarthritis (OA) is the most prevalent disease of articular joints. Pathophysiologic changes occur in OA cartilage due to the excessive expression of cartilage degrading proteinases, the resultant progressive breakdown of collagen fibers, and the degradation of proteoglycan, mainly aggrecan . IL-1β is considered to play an important role in the pathogenesis of OA, mainly because it can induce the resorption of proteoglycan and type II collagen [2,3]. Consequently, the inhibition of the IL-1β pathway presents a promising means of preventing cartilage degradation during OA pathogenesis. One of the major endogenous inhibitors of the IL-1 pathway is CrmA. CrmA can bond with IL-1β converting enzyme (ICE) (caspase-1) as a pseudosubstrate. This serpin can prevent the proteolytic activation of interleukin-1β, then block the cleavage of pro-IL-1β by ICE thereby suppressing an interleukin-1β response to infection and decreasing the secretion of IL-1β [4,5].
Chitosan (CS), a partially deacetylated derivative from chitin composed of D-gucosamine and N-acetylglucosamine, is structurally similar to GAGs. CS is widely used to elaborate different nanocarriers attributed to the capacity of the polymer to interact with the negatively charged cell surfaces . Many studies have shown its applications in drug, DNA delivery and tissue engineering because of its non-toxicity, biocompatibility and biodegradability [7-10].
Hyaluronic acid (HA) is another biocompatible anionic biopolymer used in a wide array of clinical application. HA is an abundant non-sulfated glycosaminoglycan component of synovial fluid and extracellular matrices and plays an important role in its function. It is involved in cell adhesion, morphogenesis, and inflammation regulation . In osteoarthritis, intra-articular injection of HA can improve the viscoelasticity of synovial fluid, augment the flow of joint fluid, normalize endogenous hyaluronate synthesis, inhibit hyaluronate degradation, reduce joint pain, and improve joint function [12-14]. In our previous study, our results have shown that HA could suppress chondrocyte apoptosis in IL-1β-induced osteoarthritis model in a dose-dependent way . Both of these can be considered as attractive materials for new biocompatible and biodegradable polymers.
In this study, we attempted to combine the virtues of CS and HA in the development of CrmA-loaded microspheres, and intended for attenuate the degeneration of articular cartilage. The interaction between these microspheres and chondrocytes will be investigated, and their potential for preventing OA chondrocytes dedifferentiation evaluated.
Chitosan (molecular weight:150 kDa, deacetylation:98%), Hyaluronic acid (molecular weight:500-730 kDa), sodium tripolyphosphate (STPP), and IL-1β ELISA kit were provided by Sigma-Aldrich. Cytokine response modifier A (CrmA) was purchaseded from PeproTech. Trypsinase, type II collagenase, DMEM/F12 Medium were purchased from Gibco. All the other chemicals used were of the highest available commercially grade.
Microsphere preparation and characterization
2 g of chitosan was dispersed into the acetic acid (100 mL) under vigorous stirring for 3 h at ambient temperature (below 20°C) to obtain a transparent chitosan solution (2% w/v), and the hyaluronic acid solution (0.1%, w/v) was obtained using the same method. Then, a desired amount of chitosan solution (10 ml) and hyaluronic acid fluid (5 ml) were immediately dispersed with vigorous stirring to obtain a stable mixture of HA-CS solution. A well-mixed suspension containing 100 mL of paraffin oils and 1 g of Span 80 was dispersed in a reactor and and stirred at 1000rmp for 1 h. 6 mL of HA-CS solution prepared was dropped into the suspension with a speed of 1 ml/min. The suspension in the vessel was stirred at the same speed and temperature for an additional 2 h. Next, 10 ml sTPP solution (10% w/v) was added and kept for an additional 1 h at the same stirring speed. After removing the supernatant liquid paraffin, HA-CS microspheres deposited in the bottom were collected. The microspheres were washed with alcohol and acetone several times to completely remove the residual paraffin oil and Span 80. Microspheres of CS-CrmA, HA-CS-CrmA were prepared. Finally, the microspheres were freeze-dried. The sizes and shapes of the microspheres were examined under a scanning electron microscope (SEM).
In vitro release profiles
Approximately 20 mg of microspheres, containing CrmA, was dispersed into a phosphate-buffered saline (PBS, PH = 7.4) solution containing 104 U/ml of lysozyme. Next, this solution was placed in a shaking water bath at 37°C at 135 rpm for various time periods, up to ten days. Periodically, the microsphere suspension was centrifuged to collect the supernatant for analysis, followed by the resuspension of microspheres in a fresh PBS containing lysozyme. Samples were assayed for CrmA concentration using ELISA kits (PeproTech, USA) according to the manufacturer’s protocols.
Chondrocytes isolation and culture conditions
Seven-day-old rats were obtained from the Experimental Animal Center of Wuhan University, China, and were fed under standard conditions (temperature:21 ± 1°C; humidity:55–60%) with food and water continuously available. The care and use of animals followed the recommendations and guidelines of the National Institutes of Health and were approved by the Wuhan University Animal Care and Use Committee.
The cartilage tissues were harvested under sterile conditions from the knees of seven-day-old rats. Then, the cartilage tissues were cut into small pieces (<1 mm3) and digested with 0.2% trypsin and 0.2% type II collagenase for 30 min and 2 hours respectively. After washing twice with DMEM, isolated chondrocytes were suspended in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C with 5% CO2. Cell viability was determined using cell viability analyzer (viability > 90%). Primary cells were maintained in monolayer culture throughout the study. After the cells reached confluence, the medium was changed to DMEM/F12 with 0.5% FBS and antibiotics for 6 hours. Then IL-1β (10 ng/ml) was added to the culture medium without rinse for a further 48 hours. The blank group was cultured in DMEM/F12 with 10% FBS without IL-1β, while the control group was only treated with IL-1β. Chondrocytes were divided into five groups cultured in DMEM/F12 containing 10%FBS without antibiotics and incubated for a period of 4 h: A. blank group, B. controls, C. chondrocytes cultured with CS microspheres, D. chondrocytes cultured with HA-CS microspheres, E. chondrocytes cultured with CS-CrmA microspheres, F. chondrocytes cultured with HA-CS-CrmA microspheres. There were 5 samples in each group and each experiment was repeated 5 times.
Determination of cell viability and GAG synthesis
After 72 hours of co-culture, the microspheres solution was discarded, and the chondrocytes were removed to a fresh media. After standardizing cell samples to one million in different groups using a cell counter, 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-dippphenyltetrazolium bromide (MTT) was added to chondrocytes incubated at 37°C with 5% CO2 for 4 h. The resulting formation was dissolved in dimethylsulphoxide and absorbance was measured at 570 nm with a microplate reader.
The GAG content in cell supernatants was assessed using Blyscan assay kit (Biocolor, UK), and the chondrocytes were normalized to one million cells in each group. Briefly, cell supernatants were digested enzymatically using proteinase K. Following digestion, a desired amount of supernatant was reacted with Blyscan dye for 30 min. GAG-dye precipitate was obtained by centrifugation and the resulting formation was dissolved in 1 ml dissociation reagent and incubated for 10 minutes. Finally, samples were transferred to a 96-well-plate and absorbance at 656 nm was measured on the spectrophotometer. A standard curve was derived from mixed-isomer shark chondroitin sulfate, and the GAG content was calculated.
Western blot for type II collagen, Aggrecan, type I collagen and IL-1β
Proteins were extracted from harvested chondrocytes. Protein concentrations were determined using a BCA Protein Assay Kit. After adjusting to equal amounts (50 ul protein per lane) of proteins, they were separated by sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked in phosphate buffered saline (PBS), pH 7.4, containing 5% nonfat dry milk, and incubated with anti-IL-1β, -type II collagen, Aggrecan, and -type I collagen, respectively. Then incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG), followed with visualization by the enhanced chemiluminescence kit.
SPSS 20.0 software was applied for data analysis. Data were presented as the means ± S.D. of five separate experiments. Each experimental condition was represented by triplicate wells, with replicates from each culture averaged and used as one value for analysis. Significant differences among the mean values of multiple groups were evaluated by one-way analysis of variance and Student-Newman-Keuls q-test. P < 0.05 was considered as being significant.
Characterization of microspheres
In vitro release profiles
Assay of cell survival rate
GAG contents in cell supernatants
Western blot for type II collagen, Aggrecan, collagen I and IL-1β
This study investigated that the feasibility of the novel microspheres containing CrmA as a drug release system to effectively interact with chondrocytes, produce therapeutic levels of ligand, ameliorate the dedifferentiation of chondrocytes. Our results demonstrated that cell viability, and ECM production such as GAGs and phenotype of the chondrocytes cultured with microspheres evolved normalization compared with OA group. Based on previous reports and our results, we reasonably deduce that the control-released CrmA from hyaluronic acid-chitosan microspheres has a great potential as a new way to protect chondrocyte in the process of OA.
In our study, one of the main factors affecting our results was the microsphere structure. Comparing the CS-CrmA group with HA-CS-CrmA group, it was notable that HA conjugation could enhance the ability of the microspheres to interact with the chondrocytes and to controlled-released drug. Collagen II, GAG and aggrecan seemed more prominent in HA-CS-CrmA microspheres. Moreover, collagen I and IL-1β downregulation had previously been shown in chondrocytes treated with HA-CS-CrmA microspheres. It has been reported that HA treatment may prevent the IL-1β-induced downregulation of collagen II and proteoglycan in OA chondrocytes by blocking collagenases, so as to decelerate the progression of OA . Meanwhile, the control-released action was demonstrated to last much longer in the presence of HA. More importantly, the efficiency of this release manner was improved by 12%. This may have been due to the high negative charges and the very highly swollen gel-like characteristic of HA . HA may promote controlled-release by loosening the CS-protein binding . In addition, HA is an adhesion modulator molecule, which can mediate the early stage of cell-substrate interaction . Furthermore, CD44 is known as a cell surface receptor for HA internalization and turnover [20,21]. CD44 is a transmembrane glycoprotein expressed in a variety of cell types in connective tissues and a major cell surface protein in chondrocytes, and is highly expressed in inflammatory conditions . This has been postulated to have a function as the principal receptor for HA. Anti-CD44 treatment using IM7 antibody has been reported to activate some intracellular signaling pathways that block the action of IL-1β . Therefore, the application of HA as a component of the microspheres might be a reasonable approach for enhancing the interaction with chondrocytes in OA. Simultaneously, the presence of HA could allow HA combining with CS through electrostatic interaction . It was observed that the size of the microspheres was strongly influenced when HA was included in the microspheres.
The protein expressions of collagenII, Aggrecan, collagenI and IL-1β
1.14 ± 0.07
1.26 ± 0.12
0.12 ± 0.03
0.30 ± 0.04
0.55 ± 0.12#
0.50 ± 0.06#
0.51 ± 0.10#
0.67 ± 0.02#
0.76 ± 0.03
0.75 ± 0.03
0.48 ± 0.04
0.59 ± 0.10
0.78 ± 0.08
0.76 ± 0.13
0.44 ± 0.06
0.57 ± 0.07
0.92 ± 0.05*
0.89 ± 0.08*
0.36 ± 0.08*
0.49 ± 0.11*
0.97 ± 0.04*
1.02 ± 0.05*
0.32 ± 0.11*
0.34 ± 0.06*
For the successful interaction with OA chondrocytes, we prepared the HA-CS microspheres containing CrmA. It was revealed that the sustained release of CrmA from HA-CS microspheres could attenuate the degeneration of articular cartilage by maintaining the phenotype of chondrocyte during culture expansion. The data derived from this study suggest great promise to utilize HA-CS microsphere as CrmA carriers for protection of the chondrocyte in the process of OA.
In conclusion, our previous study has demonstrated that HA could suppress chondrocyte apoptosis in IL-1β-induced osteoarthritis model in a dose-dependent way. In this study, for the first time, we found that the release kinetics of CrmA were characterized by an initial burst release, and the production of GAG and the expression of type II collagen, aggrecan significantly increased compared with the control group, while the expression of collagen I and IL-1β decreased. These results indicated that CrmA could attenuate the degeneration of articular cartilage by maintaining the phenotype of chondrocytes during culture expansion. The suppression of inflammatory cytokines activity within the joint might be one important mechanism of the clinical action of the microspheres in the treatment of OA.
This research was supported by the National Natural Science Foundation (81071494), the Natural Science Foundation of Hubei Province (2011CHB021) and Young Scientists Foundation of Health Department of Hubei Province (QJX2012-12) in China. There is no actual or potential conflict of interest including financial, personal or other relationships with other people or organizations.
- Hardingham T. Extracellular matrix and pathogenic mechanisms in osteoarthritis. Curr Rheumatol Rep. 2008;10:30–6.View ArticlePubMedGoogle Scholar
- Santangelo KS, Nuovo GJ, Bertone AL. In vivo reduction or blockade of interleukin-1β in primary osteoarthritis influences expression of mediators implicated in pathogenesis. Osteoarthritis Cartilage. 2012;20(12):1610–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Daheshia M, Yao JQ. The interleukin 1beta pathway in the pathogenesis of osteoarthritis. J Rheumatol. 2008;35(12):2306–12.View ArticlePubMedGoogle Scholar
- Fujino M, Kawasaki M, Funeshima N, Kitazawa Y, Kosuga M, Okabe K, et al. CrmA gene expression protects mice against concanavalin-A-induced hepatitis by inhibiting IL-18 secretion and hepatocyte apoptosis. Gene Ther. 2003;10(20):1781–90.View ArticlePubMedGoogle Scholar
- Ray CA et al. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell. 1992;69:597–604.View ArticlePubMedGoogle Scholar
- Borchard G, Lueben HL, de Boer AG, Verhoef JC, Lehr CM, Juginger HE. The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro. J Control Release. 1996;36:131–8.View ArticleGoogle Scholar
- Borchard G. Chitosans for gene delivery. Adv Drug Deliv Rev. 2001;52(2):145–50.View ArticlePubMedGoogle Scholar
- Niu L, Xu YC, Dai Z, Tang HQ. Gene therapy for type 1 diabetes mellitus in rats by gastrointestinal administration of chitosan nanoparticles containing human insulin gene. World J Gastroenterol. 2008;14(26):4209–15.View ArticlePubMedPubMed CentralGoogle Scholar
- Hasanova GI, Noriega SE, Mamedov TG, Guha Thakurta S, Turner JA, Subramanian A. The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds. J Tissue Eng Regen Med. 2011;5(10):815–22.View ArticlePubMedGoogle Scholar
- Breyner NM, Hell RC, Carvalho LR, Machado CB, Peixoto Filho IN, Valério P, et al. Effect of a three-dimensional chitosan porous scaffold on the differentiation of mesenchymal stem cells into chondrocytes. Cells Tissues Organs. 2010;191(2):119–28.View ArticlePubMedGoogle Scholar
- Forsey RW, Fisher J, Thompson J, Stone MH, Bell C, Ingham E. The effect of hyaluronic acid and phospholipid based lubricants on friction within a human cartilage damage model. Biomaterials. 2006;27(26):4581–90.View ArticlePubMedGoogle Scholar
- Yoo HS, Lee EA, Yoon JJ, et al. Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering. Biomaterials. 2005;26(14):1925–33.View ArticlePubMedGoogle Scholar
- Li Y, Nagira T, Tsuchiya T. The effect of hyaluronic acid on insulin secretion in HIT-T15 cells through the enhancement of gap-junctional intercellular communication. Biomaterials. 2006;27(8):1437–43.View ArticlePubMedGoogle Scholar
- Park YD, Tirelli N, Hubbell JA. Photopolymerized hyaluronic acid-based hydrogels and interpenetrating networks. Biomaterials. 2003;24(6):893–900.View ArticlePubMedGoogle Scholar
- Zhou P-H, Liu S-Q, Peng H. The effect of hyaluronic acid on IL-1beta-induced chondrocyte apoptosis in a rat model of osteoarthritis. J Orthop Res. 2008;26(12):1643–8.View ArticlePubMedGoogle Scholar
- Turlier V, Delalleau A, Casas C, Rouquier A, Bianchi P, Alvarez S, et al. Association between collagen production and mechanical stretching in dermal extracellular matrix: in vivo effect of cross-linked hyaluronic acid filler. A randomised, placebo-controlled study. J Dermatol Sci. 2013;69(3):187–94.View ArticlePubMedGoogle Scholar
- Schmitt C, Moitzi C, Bovay C, Rouvet M, Bovetto L, Donato L, et al. Interal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment. Soft Matter. 2010;6:4876–84.View ArticleGoogle Scholar
- Al-Qadi S, Alatorre-Meda M, Zaghloul EM, Taboada P, Remunán-López C. Chitosanhyaluronic acid nanoparticles for gene silencing: the role of hyaluronic acid on the nanoparticles’ formation and activity. Colloids Surf B Biointerfaces. 2013;103:615–23.View ArticlePubMedGoogle Scholar
- Ito T, Iida-Tanaka N, Niidome T, Kawano T, Kubo K, Yoshikawa K, et al. Hyaluronic acid and its derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation. J Control Release. 2006;112(3):382–8.View ArticlePubMedGoogle Scholar
- Khurana SS, Riehl TE, Moore BD, Fassan M, Rugge M, Romero-Gallo J, et al. The hyaluronic acid receptor CD44 coordinates normal and metaplastic gastric epithelial progenitor cell proliferation. J Biol Chem. 2013;288(22):16085–97.View ArticlePubMedPubMed CentralGoogle Scholar
- Yu M, Jambhrunkar S, Thorn P, Chen J, Gu W, Yu C. Hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells. Nanoscale. 2013;5(1):178–83.View ArticlePubMedGoogle Scholar
- Salter DM, Godolphin JL, Gourlay MS, Lawson MF, Hughes DE, Dunne E. Analysis of human articular chondrocyte CD44 isoform expression and function in health and disease. J Pathol. 1996;179(4):396–402.View ArticlePubMedGoogle Scholar
- Sohel M. Julovi, Tadashi Yasuda, Makoto Shimizu, Teruko Hiramitsu, Takashi nakamura, Inhibition of interleukin-1β-stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage. Arthritis Rheum. 2004;50(2):516–25.View ArticleGoogle Scholar
- Liu Y, Kong M, Cheng XJ, Wang QQ, Jiang LM, Chen XG. Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery. Carbohydr Polym. 2013;94(1):309–16.View ArticlePubMedGoogle Scholar
- Vickers SM, Squitieri LS, Spector M. Effects of cross-linking type II collagen-GAG scaffolds on chondrogenesis in vitro: dynamic pore reduction promotes cartilage formation. Tissue Eng. 2006;12:1345–55.View ArticlePubMedGoogle Scholar
- MacDonald G et al. Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis. J Exp Med. 1999;189:131–44.View ArticlePubMedPubMed CentralGoogle Scholar
- Gu Y et al. Activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme. Science. 1997;275:206–9.View ArticlePubMedGoogle Scholar
- Li P et al. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell. 1995;80:401–11.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.